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Papers by Jay Fox

Toxins, Apr 9, 2021

The 1st International Electronic Conference on Toxins (IECT2021) was successfully held online by ... more The 1st International Electronic Conference on Toxins (IECT2021) was successfully held online by from 16 to 31 January 2021. The mission of this conference is to provide a platform for scientists working on toxins from all organisms to present the latest concepts under research on these toxins and for all to compare and contrast the actions of toxins. The potential uses of toxins for the benefit of science, as well as for humankind, are key concepts up for discussion. IECT2021 is led by Toxins Editor-in-Chief Prof. Dr. Jay Fox (University of Virginia, USA) and includes six section chairs from the editorial board of Toxins: Dr. Bryan Grieg Fry (University of Queensland, Australia; in charge of the contributions related to animal venoms), Prof. Dr. Sarah De De Saege (Ghent University, Belgium; in charge of the contributions related to mycotoxins), Prof. Dr. Michel R. Popoff (Institut Pasteur, France; in charge of the contributions related to bacterial toxins), Prof. Dr. Joachim Jankowski (University Hospital RWTH, Germany; in charge of the contributions related to uremic toxins), Prof. Dr. Mary Fletcher (University of Queensland, Australia; in charge of the contributions related to plant toxins), and Dr. Panagiota Katikou (Ministry of Rural Development and Food, Greece; in charge of the contributions related to marine and freshwater toxins). More than 400 researchers participated in the conference in the form of paper/poster submissions. Four online sessions were successfully held during the conference, where 17 speakers gave speeches on their latest research and around 300 researchers watched the sessions online via Zoom or YouTube.

Biochemical Journal, Jun 1, 1990

Glycogen synthase from Saccharomyces cerevisiae was purified to homogeneity. The enzyme showed a ... more Glycogen synthase from Saccharomyces cerevisiae was purified to homogeneity. The enzyme showed a subunit molecular mass of 80 kDa. The holoenzyme appears to be a tetramer. Antibodies developed against purified yeast glycogen synthase inactivated the enzyme in yeast extracts and allowed the detection of the protein in Western blots. Amino acid analysis showed that the enzyme is very rich in glutamate and/or glutamine residues. The N-terminal sequence (11 amino acid residues) was determined. In addition, selected tryptic-digest peptides were purified by reverse-phase h.p.l.c. and submitted to gas-phase sequencing. Up to eight sequences (79 amino acid residues) could be aligned with the human muscle enzyme sequence. Levels of identity range between 37 and 100 %, indicating that, although human and yeast glycogen synthases probably share some conserved regions, significant differences in their primary structure should be expected.

Toxins

Longitudinal metabolomics and lipidomics analyses were carried out on the blood plasma of mice in... more Longitudinal metabolomics and lipidomics analyses were carried out on the blood plasma of mice injected intramuscularly with venoms of the viperid species Bothrops asper or Daboia russelii. Blood samples were collected 1, 3, 6, and 24 h after venom injection, and a control group of non-envenomed mice was included. Significant perturbations in metabolomics and lipidomics were observed at 1, 3, and 6 h, while values returned close to those of control mice by 24 h, hence reflecting a transient pattern of metabolic disturbance. Both venoms induced significant changes in amino acids, as well as in several purines and pyrimidines, and in some metabolites of the tricarboxylic acid cycle. KEGG analysis of metabolic pathways that showed those with the greatest change included aminoacyl tRNA synthesis and amino acid biosynthesis and metabolism pathways. With regard to lipid metabolism, there was an increase in triglycerides and some acyl carnitines and a concomitant drop in the levels of some...

Cancer cell dissemination to the sentinel lymph node associates with poor patient outcomes, parti... more Cancer cell dissemination to the sentinel lymph node associates with poor patient outcomes, particularly in breast cancers. How cancer cells egress the primary tumor upon interfacing with the lymphatic vasculature is complex and driven by dynamic interactions between cancer cells and stromal cells including cancer associated fibroblasts (CAFs). The matricellular protein periostin can distinguish CAF subtypes in breast cancer and is associated with increased desmoplasia and disease recurrence in patients. However, since periostin is secreted, periostin-expressing CAFs are difficult to characterize in situ, limiting our understanding of their specific contribution to cancer progression. Here, we used in vivo genetic labelling and ablation to lineage trace periostin+ cells and characterize their function(s) during tumor growth and metastasis. We report that periostin-expressing CAFs are spatially found at periductal and perivascular margins, are enriched at lymphatic vessel peripheries...

Proceedings of 1st International Electronic Conference on Toxins, 2021

Toxins, 2021

In the Brazilian Amazon, Bothrops atrox snakebites are frequent, and patients develop tissue dama... more In the Brazilian Amazon, Bothrops atrox snakebites are frequent, and patients develop tissue damage with blisters sometimes observed in the proximity of the wound. Antivenoms do not seem to impact blister formation, raising questions regarding the mechanisms underlying blister formation. Here, we launched a clinical and laboratory-based study including five patients who followed and were treated by the standard clinical protocols. Blister fluids were collected for proteomic analyses and molecular assessment of the presence of venom and antivenom. Although this was a small patient sample, there appeared to be a correlation between the time of blister appearance (shorter) and the amount of venom present in the serum (higher). Of particular interest was the biochemical identification of both venom and antivenom in all blister fluids. From the proteomic analysis of the blister fluids, all were observed to be a rich source of damage-associated molecular patterns (DAMPs), immunomodulators...

Molecular & Cellular Proteomics, 2021

A multi-omics strategy was used to map the proteome, miRNA, metabolome, and lipidome of EVs deriv... more A multi-omics strategy was used to map the proteome, miRNA, metabolome, and lipidome of EVs derived from human primary tumor (SCC-9) cells and matched lymph node metastatic (LN1) cells. Differentially abundant molecules associated with the metastatic phenotype were enriched for key processes and pathways. An integrative analysis revealed 11 'hub proteins' that are correlated with reduced survival and tumor aggressiveness in patients with cancer according to public databases. These EV molecules are candidates as prognostic markers in oral cancer. Highlights • Proteomic, miRNA, metabolomic, and lipidomic profiles were mapped in oral cancer EVs. • The molecular profile of EVs was associated with the lymph node metastatic phenotype. • A multi-omics integrative analysis revealed 11 highly connected 'hub proteins.' • 'Hub proteins' from EVs are candidates as prognostic markers in oral cancer.

Journal of Biological Chemistry, 1989

Digestion wlth endoproteinase LysC 900yg of HId was Incubated wlth l 8 p g of Digestion with cnym... more Digestion wlth endoproteinase LysC 900yg of HId was Incubated wlth l 8 p g of Digestion with cnymotrypsin 1 mg of Ht-d was dlssoIYed m 1 ml of 6M guanld#ne-HCI. dlalyred agalnst 10 mM Tris-CI pH 8 5 then incubated wlth 20 y g of chymotrypsln A4 lor I hr. The lr8cubatlon was continued for a further 22 hr with another 20 e g of chymotrypstn Peptldes were separated on a Plerce C8 COlUm" Dlgestlon at arginine residues Lys~ne residues 01 Ht-d were mod8fled r , t h CltracOniC anhydride accordlng lo Rwman el al. (1984). the proteln was then digested with trypsin (1-3 0 , w w) for 20 hat 37% Peptides were separated on a PLRP-300 column. room temperature The peptides were separated on a Brownlee C8 column. Peptlde 53.89 Was heated In 0.1% TFA at 100°C lor 2 hrs. The peptides from thlS U#gestIon were separated On a BtOWnlee c 8 Column Limited acid hydrolysis HId was digested Wlth cyanogen bromlde m 70% formic acld for 48 hr at Cyanogen bromide digestion Cyanogen bromide digestion was performed as described by Gross (1967). usmg 1 ml of 70% formic acid with an excess of cyanogen bromide. Digestion with HI-e 1 mg of Ht-d was incubated wlth 30 y g of tit-e in 1.1 M urea. 50 mM Tris-CI PH 8, I m~ c a u 2 for 24 hr at 37% The peptides were separated on a Pterce C8 column.

Biomolecules, 2020

ADAM9 is a metalloproteinase strongly expressed at the tumor-stroma border by both tumor and stro... more ADAM9 is a metalloproteinase strongly expressed at the tumor-stroma border by both tumor and stromal cells. We previously showed that the host deletion of ADAM9 leads to enhanced growth of grafted B16F1 melanoma cells by a mechanism mediated by TIMP1 and the TNF-α/sTNFR1 pathway. This study aimed to dissect the structural modifications in the tumor microenvironment due to the stromal expression of ADAM9 during melanoma progression. We performed proteomic analysis of peritumoral areas of ADAM9 deleted mice and identified the altered expression of several matrix proteins. These include decorin, collagen type XIV, fibronectin, and collagen type I. Analysis of these matrices in the matrix producing cells of the dermis, fibroblasts, showed that ADAM9−/− and wild type fibroblasts synthesize and secreted almost comparable amounts of decorin. Conversely, collagen type I expression was moderately, but not significantly, decreased at the transcriptional level, and the protein increased in ADA...

Journal of Investigative Dermatology, 2019

Cutaneous Squamous Cell Carcinoma (cSCC) is the most common and fastest-increasing cancer with me... more Cutaneous Squamous Cell Carcinoma (cSCC) is the most common and fastest-increasing cancer with metastatic potential. Long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) are novel regulators of gene expression and little is known about their altered expression in cSCC. To elucidate cSCC associated coding and non-coding transcriptomic changes, we performed RNA-seq of 9 cSCCs and 7 healthy skin samples. Differential expression analysis was performed by edgeR. CircRNAs were identified using the find_circ and CIRCexplorer pipelines. Altered expression of 5,352 protein-coding genes, 908 lncRNAs and 55 circular RNAs was identified. Targets of 519 transcription factors were enriched among DEGs, 105 of which displayed altered level in cSCCs, including fundamental regulators of skin development and cSCC malignancy (MYC, RELA, ETS1, TP63). Pathways related to cell cycle, apoptosis, inflammation and epidermal differentiation were enriched. In addition to known oncogenic lncRNAs (PVT1, LUCAT1, CASC9), a set of skin-specific lncRNAs were also dysregulated. A global downregulation of circRNAs was observed in cSCC, and novel circRNAs, including skin-enriched circ_IFFO2 and circ_POF1B, were identified. Results from the RNA-seq were further validated in an expanded cohort using NanoString nCounter assays. In conclusion, through deep sequencing approach we have identified a reference set of coding and non-coding transcripts with altered expression in cSCC, which represent potential therapeutic targets or biomarkers.

Toxins, 2019

Skin blistering as a result of snakebite envenomation is characteristic of some bites, however li... more Skin blistering as a result of snakebite envenomation is characteristic of some bites, however little is known regarding the mechanism of blister formation or the composition of the blister fluid. In order to investigate if blister fluid proteomes from humans suffering snakebite envenomation could provide insights on the pathophysiology of these skin alterations, blister fluid was collected from six patients upon presentation at a clinic in India bitten by three species of snakes, Daboia russelii (3), Hypnale hypnale (2), or Naja naja (1). Standard clinical data were recorded throughout the treatment. Approximately 805 proteins were identified in blister fluids using proteomic analyses. Informatics analyses of the proteomes identified the top biological response categories as: platelet degranulation, innate immune response, receptor-mediated endocytosis, complement activation, and blood coagulation. Hierarchical clustering did not show a clear segregation of patients’ proteomes bein...

The EMBO Journal, 1991

Recombinant mouse nidogen and two fragments were produced in mammalian cells and purified from cu... more Recombinant mouse nidogen and two fragments were produced in mammalian cells and purified from culture medium without resorting to denaturing conditions. The truncated products were fragments Nd-I (positions 1-905) comprising the N-terminal globule and rod-like domain and Nd-I1 corresponding mainly to the Cterminal globule (position 906-1217). Recombinant nidogen was indistinguishable from authentic nidogen obtained by guanidine dissociation from tumor tissue with respect to size, N-terminal sequence, CD spectra and immunochemical properties. They differed in protease stability and shape indicating that the N-terminal domain of the more native, recombinant protein consists of two globules connected by a flexible segment. This established a new model for the shape of nidogen consisting of three globes of variable mass (31-56 kDa) connected by either a rod-like or a thin segment. Recombinant nidogen forned stable complexes (Kd < 1 nM) with laminin and collagen IV in binding assays with soluble and immobilized ligands and as shown by electron microscopy. Inhibition assays demonstrated different binding sites on nidogen for both ligands with different specificities. This was confirmed in studies with fragment Nd-I binding to collagen IV and fragment Nd-I1 binding to laminin fragment P1. In addition, recombinant nidogen but not Nd-I was able to bridge between laniinin or P1 and collagen IV. Formation of such ternary complexes implicates a similar role for nidogen in the supramolecular organization of basement membranes.

Nature communications, Sep 5, 2018

Different regions of oral squamous cell carcinoma (OSCC) have particular histopathological and mo... more Different regions of oral squamous cell carcinoma (OSCC) have particular histopathological and molecular characteristics limiting the standard tumor-node-metastasis prognosis classification. Therefore, defining biological signatures that allow assessing the prognostic outcomes for OSCC patients would be of great clinical significance. Using histopathology-guided discovery proteomics, we analyze neoplastic islands and stroma from the invasive tumor front (ITF) and inner tumor to identify differentially expressed proteins. Potential signature proteins are prioritized and further investigated by immunohistochemistry (IHC) and targeted proteomics. IHC indicates low expression of cystatin-B in neoplastic islands from the ITF as an independent marker for local recurrence. Targeted proteomics analysis of the prioritized proteins in saliva, combined with machine-learning methods, highlights a peptide-based signature as the most powerful predictor to distinguish patients with and without lym...

mBio, Jan 10, 2016

Bacillus anthracis is killed by the interferon-inducible, ELR(-) CXC chemokine CXCL10. Previous s... more Bacillus anthracis is killed by the interferon-inducible, ELR(-) CXC chemokine CXCL10. Previous studies showed that disruption of the gene encoding FtsX, a conserved membrane component of the ATP-binding cassette transporter-like complex FtsE/X, resulted in resistance to CXCL10. FtsX exhibits some sequence similarity to the mammalian CXCL10 receptor, CXCR3, suggesting that the CXCL10 N-terminal region that interacts with CXCR3 may also interact with FtsX. A C-terminal truncated CXCL10 was tested to determine if the FtsX-dependent antimicrobial activity is associated with the CXCR3-interacting N terminus. The truncated CXCL10 exhibited antimicrobial activity against the B. anthracis parent strain but not the ΔftsX mutant, which supports a key role for the CXCL10 N terminus. Mutations in FtsE, the conserved ATP-binding protein of the FtsE/X complex, resulted in resistance to both CXCL10 and truncated CXCL10, indicating that both FtsX and FtsE are important. Higher concentrations of CX...

Venom Genomics and Proteomics, 2016

Snake venom metalloproteases (SVMP) are a key group of enzymes abundant in Viperidae venoms. Stru... more Snake venom metalloproteases (SVMP) are a key group of enzymes abundant in Viperidae venoms. Structurally, secreted SVMPs are typically organized into three main groups based on the presence or absence of domains: PI-which contains only a metalloproteinase domain; PII-includes also a disintegrin domain; and PIII-in addition to the first two domains, possesses a cysteine-rich domain. Diverse functions have been described to this group of proteases including their well-known hemorrhagic activity. Fibrin(ogen)olysis, prothrombin activation, interaction and lysis of von Willebrand factor, cytotoxicity, obstruction of angiogenesis, interference with platelet aggregation, myotoxicity, and proinflammatory

Oncotarget, Jan 30, 2015

Targeted proteomics has flourished as the method of choice for prospecting for and validating pot... more Targeted proteomics has flourished as the method of choice for prospecting for and validating potential candidate biomarkers in many diseases. However, challenges still remain due to the lack of standardized routines that can prioritize a limited number of proteins to be further validated in human samples. To help researchers identify candidate biomarkers that best characterize their samples under study, a well-designed integrative analysis pipeline, comprising MS-based discovery, feature selection methods, clustering techniques, bioinformatic analyses and targeted approaches was performed using discovery-based proteomic data from the secretomes of three classes of human cell lines (carcinoma, melanoma and non-cancerous). Three feature selection algorithms, namely, Beta-binomial, Nearest Shrunken Centroids (NSC), and Support Vector Machine-Recursive Features Elimination (SVM-RFE), indicated a panel of 137 candidate biomarkers for carcinoma and 271 for melanoma, which were differenti...

Current Protein & Peptide Science, 2015

Integrins regulate diverse functions in cancer pathology and in tumor cell development and contri... more Integrins regulate diverse functions in cancer pathology and in tumor cell development and contribute to important processes such as cell shape, survival, proliferation, transcription, angiogenesis, migration, and invasion. A number of snake venom proteins have the ability to interact with integrins. Among these are the disintegrins, a family of small, non-enzymatic, and cysteine-rich proteins found in the venom of numerous snake families. The venom proteins may have a potential role in terms of novel therapeutic leads for cancer treatment. Disintegrin can target specific integrins and as such it is conceivable that they could interfere in important processes involved in carcinogenesis, tumor growth, invasion and migration. Herein we present a survey of studies involving the use of snake venom disintegrins for cancer detection and treatment. The aim of this review is to highlight the relationship of integrins with cancer and to present examples as to how certain disintegrins can detect and affect biological processes related to cancer. This in turn will illustrate the great potential of these molecules for cancer research. Furthermore, we also outline several new approaches being created to address problems commonly associated with the clinical application of peptide-based drugs such as instability, immunogenicity, and availability.

Techniques in Protein Chemistry, 1994

I. INTRODUCTION The Association of Biomolecular Resource Facilities (ABRF) exists to serve the an... more I. INTRODUCTION The Association of Biomolecular Resource Facilities (ABRF) exists to serve the analytical and synthetic needs of laboratories which study and use proteins, nucleic acids and other macromolecules. Part of ABRF's mission is educational, assisting member laboratories to achieve and maintain the highest quality of services possible. The ABRF Amino Acid Analysis Research Committee has carried out annual studies since 1989 [1-6] as a means for members to readily assess their performance relative to other core facilities. The aim of this year's study was to evaluate amino acid analysis methods for identification and quantitation of posttranslationally modified residues with emphasis on phosphorylated amino acids (Ser, Thr, and Tyr) and on hydroxyproline. II. MATERIALS AND METHODS A. Sample Preparation and Distribution The 1993 ABRF amino acid analysis test sample (ABRF-93AAA) was a 30 residue synthetic peptide with the following sequence and four intended phosphorylation sites: L(pS)G(pY)A(pT)F(pS)(hP)LDTQSLLKIYNTPLSVIISDK. This peptide was designed to provide both phosphorylated and unmodified Ser, Thr and Tyr. In addition, a particularly slow cleaving bond (Ile-Ile) was included to provide a hydrolysis challenge. The peptide was synthesized by the University of Michigan Protein and Carbohydrate Facility on a preloaded Wang resin using Fmoc chemistry. The residues to be phosphorylated were introduced with free sidechains. The resin bound peptide was base treated and then globally phosphorylated (7), using a five-fold molar excess of di-t-butyl phosphoramidate in dry Nmethylpyrrolidone (NMP). Oxidation was with t-butylhydroperoxide in NMP.

Protein Expression and Purification, 2011

Nonstructural 3ABC protein of foot and mouth disease virus (FMDV) was widely used to differentiat... more Nonstructural 3ABC protein of foot and mouth disease virus (FMDV) was widely used to differentiate vaccinated from natural FMDV-infected animals. 3ABC is a polyprotein which is auto-processed to 3A, three copies of 3B and 3C(pro) by 3C(pro) protease. The 3ABC gene was cloned and expressed in Escherichia coli as native or mutated 3ABC (mu3ABC) forms. Cysteine residues 142 and 163 of the catalytic triad within the 3C(pro) of mu3ABC were changed to serine and glycine, respectively, to inhibit its protease activity. Both native and mutated 3ABC ORFs were cloned into BamHI and HindIII restriction sites of an expression vector, pQE80L. The expression of the recombinant native 3ABC and mu3ABC genes in E. coli BL21 was induced with 0.2mM isopropyl-beta-d-thiogalactopyranoside at 37 °C for 5h. SDS-PAGE and Western blot analysis revealed that the full length 3ABC was present in the lysate from mu3ABC but not native 3ABC transformed cells. The recombinant mu3ABC was expressed mainly in the inclusion body and presented as monomer and dimer. In addition, the mu3ABC reacted strongly with a convalescent serum from a natural FMDV-infected cattle but very weakly with a serum from vaccinated cattle. This study clearly demonstrates that successful expression of the full length 3ABC occurs only when the protease active sites within the 3C(pro) were completely abolished. This information would accelerate in house development of the 3ABC-based diagnostic test that can distinguish between vaccinated and FMDV-infected animals.

Proceedings of the National Academy of Sciences, 1994

The structure of the metalloproteinase and hemorrhagic toxin atrolysin C form d (EC 3.4.24.42), f... more The structure of the metalloproteinase and hemorrhagic toxin atrolysin C form d (EC 3.4.24.42), from the venom of the western diamondback rattlesnake Crotalus atrox, has been determined to atomic resolution by x-ray crystallographic methods. This study illuminates the nature of inhibitor binding with natural (< Glu-Asn-Trp, where < Glu is pyroglutamic acid) and synthetic (SCH 47890) ligands. The primary specificity pocket is exceptionally deep; the nature of inhibitor and productive substrate binding is discussed. Insights gained from the study of these complexes facilitate the design of potential drugs to treat diseases where matrix metalloproteinases have been implicated, e.g., arthritis and tumor metastasis.