A Complex Pattern of Gene Expression in Tissue Affected by Viperid Snake Envenoming: The Emerging Role of Autophagy-Related Genes.

Viperid snake venoms induce severe tissue damage, characterized by the direct toxic action of venom components, i.e., phospholipases A2 (PLA2s) and metalloproteinases (SVMPs), concomitantly with the onset of endogenous inflammatory processes, in an intricate scenario of tissue alterations. Understanding the expression of relevant genes in muscle tissue will provide valuable insights into the undergoing pathological and inflammatory processes. In this study, we have used the Nanostring technology to evaluate the patterns of gene expression in mouse skeletal muscle 1 h, 6 h, and 24 h after injection of the venoms of Bothrops asper and Daboia russelii, two medically relevant species in Latin America and Asia, respectively, with somewhat different clinical manifestations. The dose of venoms injected (30 µg) induced local pathological effects and inflammation in muscle tissue. We focused our analysis on genes related to extracellular matrix (ECM) metabolism, immune system, programmed cell death, and autophagy. The results revealed a complex pattern of expression of genes. Regarding ECM metabolism and regulation, up-regulated genes included proteinase inhibitor Serpine 1, thrombospondin 1, collagens 1A1 and 4A1 (at 1 h in the case of B. asper), TIMP1, MMP-3 (at 24 h), and lysil oxidase (LOX). In contrast, collagen chains 5A3 and 5A1 were down-regulated, especially at 6 h. Transforming growth factor ß (TGF-ß) and several genes related to myofibroblast regulation were also up-regulated, which might be related to the development of fibrosis. Several genes related to cytokine and chemokine synthesis and regulation and NFκB signaling were also up-regulated. Our observations show a variable expression of genes associated with programmed cell death and autophagy, thus revealing a hitherto unknown role of autophagy in tissue affected by snake venoms. These results provide clues to understanding the complex pattern of gene expression in tissue affected by viperid snake venoms, which likely impacts the final pathophysiology of damaged tissue in envenomings.

Periostin+ Stromal Cells Guide Lymphovascular Invasion by Cancer Cells.

Cancer cell dissemination to sentinel lymph nodes is associated with poor patient outcomes, particularly in breast cancer. The process by which cancer cells egress from 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 (CAF). The matricellular protein periostin can distinguish CAF subtypes in breast cancer and is associated with increased desmoplasia and disease recurrence in patients. However, as 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 labeling and ablation to lineage trace periostin+ cells and characterize their functions during tumor growth and metastasis. Periostin-expressing CAFs were spatially found at periductal and perivascular margins, were enriched at lymphatic vessel peripheries, and were differentially activated by highly metastatic cancer cells versus poorly metastatic counterparts. Surprisingly, genetically depleting periostin+ CAFs slightly accelerated primary tumor growth but impaired intratumoral collagen organization and inhibited lymphatic, but not lung, metastases. Periostin ablation in CAFs impaired their ability to deposit aligned collagen matrices and inhibited cancer cell invasion through collagen and across lymphatic endothelial cell monolayers. Thus, highly metastatic cancer cells mobilize periostin-expressing CAFs in the primary tumor site that promote collagen remodeling and collective cell invasion within lymphatic vessels and ultimately to sentinel lymph nodes. SIGNIFICANCE: Highly metastatic breast cancer cells activate a population of periostin-expressing CAFs that remodel the extracellular matrix to promote escape of cancer cells into lymphatic vessels and drive colonization of proximal lymph nodes.

Mapping the Immune Cell Microenvironment with Spatial Profiling in Muscle Tissue Injected with the Venom of Daboia russelii.

Pathological and inflammatory events in muscle after the injection of snake venoms vary in different regions of the affected tissue and at different time intervals. In order to study such heterogeneity in the immune cell microenvironment, a murine model of muscle necrosis based on the injection of the venom of Daboia russelii was used. Histological and immunohistochemical methods were utilized to identify areas in muscle tissue with a different extent of muscle cell damage, based on the presence of hypercontracted muscle cells, a landmark of necrosis, and on the immunostaining for desmin. A gradient of inflammatory cells (neutrophils and macrophages) was observed from heavily necrotic areas to less damaged and non-necrotic areas. GeoMx® Digital Spatial Profiler (NanoString, Seattle, WA, USA) was used for assessing the presence of markers of various immune cells by comparing high-desmin (nondamaged) and low-desmin (damaged) regions of muscle. Markers of monocytes, macrophages, M2 macrophages, dendritic cells, neutrophils, leukocyte adhesion and migration markers, and hematopoietic precursor cells showed higher levels in low-desmin regions, especially in samples collected 24 hr after venom injection, whereas several markers of lymphocytes did not. Moreover, apoptosis (BAD) and extracellular matrix (fibronectin) markers were also increased in low-desmin regions. Our findings reveal a hitherto-unknown picture of immune cell microheterogeneity in venom-injected muscle which greatly depends on the extent of muscle cell damage and the time lapse after venom injection.

Longitudinal Metabolomics and Lipidomics Analyses Reveal Alterations Associated with Envenoming by Bothrops asper and Daboia russelii in an Experimental Murine Model.

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 phospholipids. In addition, envenomed mice had higher levels of cortisol, heme, and some oxidative stress markers. The overall pattern of metabolic changes in envenomed mice bears similarities with the patterns described in several traumatic injuries, thus underscoring a metabolic response/adaptation to the injurious action of the venoms.

Phostensin enables lymphocyte integrin activation and population of peripheral lymphoid organs.

Rap1 GTPase drives assembly of the Mig-10/RIAM/Lamellipodin (MRL protein)-integrin-talin (MIT) complex that enables integrin-dependent lymphocyte functions. Here we used tandem affinity tag-based proteomics to isolate and analyze the MIT complex and reveal that Phostensin (Ptsn), a regulatory subunit of protein phosphatase 1, is a component of the complex. Ptsn mediates dephosphorylation of Rap1, thereby preserving the activity and membrane localization of Rap1 to stabilize the MIT complex. CRISPR/Cas9-induced deletion of PPP1R18, which encodes Ptsn, markedly suppresses integrin activation in Jurkat human T cells. We generated apparently healthy Ppp1r18-/- mice that manifest lymphocytosis and reduced population of peripheral lymphoid tissues ascribable, in part, to defective activation of integrins αLß2 and α4ß7. Ppp1r18-/- T cells exhibit reduced capacity to induce colitis in a murine adoptive transfer model. Thus, Ptsn enables lymphocyte integrin-mediated functions by dephosphorylating Rap1 to stabilize the MIT complex. As a consequence, loss of Ptsn ameliorates T cell-mediated colitis.

Observation of Bothrops atrox Snake Envenoming Blister Formation from Five Patients: Pathophysiological Insights.

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, and matrix metalloproteinase-9 (MMP-9), suggesting that the mechanisms by which blisters are formed includes the toxins very early in envenomation and continue even after antivenom treatment, due to the pro-inflammatory molecules generated by the toxins in the first moments after envenomings, indicating the need for local treatments with anti-inflammatory drugs plus toxin inhibitors to prevent the severity of the wounds.

Report of the 1st International Electronic Conference on Toxins (IECT2021), 16-31 January 2021.

The 1st International Electronic Conference on Toxins (IECT2021) was successfully held online by https://sciforum [...].

Observation of Bothrops atrox snake envenoming blister formation from five patients: pathophysiological insights

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, and matrix metalloproteinase-9 (MMP-9), suggesting that the mechanisms by which blisters are formed includes the toxins very early in envenomation and continue even after antivenom treatment, due to the pro-inflammatory molecules generated by the toxins in the first moments after envenomings, indicating the need for local treatments with anti-inflammatory drugs plus toxin inhibitors to prevent the severity of the wounds.

Loss of ADAM9 Leads to Modifications of the Extracellular Matrix Modulating Tumor Growth.

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 ADAM9-/- fibroblast mono- and co-cultures with melanoma media. We show here for the first time that ADAM9 can release a collagen fragment. Still, it is not able to degrade collagen type I. However, the deletion of ADAM9 in fibroblasts resulted in reduced MMP-13 and -14 expression that may account for the reduced processing of collagen type I. Altogether, the data show that the ablation of ADAM9 in the host leads to the altered expression of peritumoral extracellular matrix proteins that generate a more favorable environment for melanoma cell growth. These data underscore the suppressive role of stromal expression of ADAM9 in tumor growth and call for a better understanding of how protease activities function in a cellular context for improved targeting.

Analysis of wound exudates reveals differences in the patterns of tissue damage and inflammation induced by the venoms of Daboia russelii and Bothrops asper in mice.

Clinical manifestations of envenomings by bites of the viperid snakes Bothrops asper and Daboia russelii show marked differences. Both venoms elicit the typical effects induced by viperid venoms (local tissue damage, bleeding, coagulopathies, shock). In addition, envenomings by D. russelii are characterized by a high incidence of acute kidney injury and by systemic capillary leak syndrome. The present investigation aimed to compare the local pathological and inflammatory events induced by the intramuscular injection of these venoms in a mouse model. B. asper venom induced stronger local hemorrhage, whereas D. russelii venom caused a higher extent of myonecrosis, and both venoms induced inflammation. Exudates collected from the site of tissue damage showed higher proteolytic activity in the case of samples from B. asper venom-treated mice. This activity was abrogated by antivenoms, indicating that it is the result of the action of venom proteinases. In addition, an increase in matrix metalloproteinases (MMPs) over time was detected in exudates induced by both venoms. Proteome analysis of exudates revealed higher abundance of extracellular matrix (ECM)-derived protein fragments in samples collected from B. asper venom-injected mice, whereas those from D. russelii venom-injected animals had higher amounts of intracellular proteins. Analysis of the subproteome of inflammatory mediators in exudates showed various patterns of change over time. Some mediators peaked at 180 min and decreased afterwards, whereas others increased and remained elevated during the 360 min observation period. Interestingly, various mediators (MIP-1α, MIP-1ß, KC, MIP-2, GM-CSF, VEGF, and LIX) increased and then decreased in the case of B. asper venom, while they remained elevated at 360 min in the case of D. russelii venom. Our findings show that these venoms induce a different pattern of local tissue damage and suggest that the venom of D. russelii induces a more sustained inflammatory reaction, an observation that may have implications for the pathophysiology of envenomings.

Proteomic Analysis of Human Blister Fluids Following Envenomation by Three Snake Species in India: Differential Markers for Venom Mechanisms of Action.

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 being associated with the species of snake involved, suggesting that either the proteomic profiles described reflect a general response to venom-induced tissue damage or more patient data sets will be required to observe significant differences. Finally, it is of interest that venom proteins were also identified in the blister fluids suggesting that this fluid may serve as a reservoir of venom biologically active proteins/toxins, and as such, may indicate the clinical value of removing blister fluid to attenuate further tissue damage.

The neurotoxic secreted phospholipase A2 from the Vipera a. ammodytes venom targets cytochrome c oxidase in neuronal mitochondria.

The ß-neurotoxic secreted phospholipases A2 (sPLA2s) block neuro-muscular transmission by poisoning nerve terminals. Damage inflicted by such sPLA2s (ß-ntx) on neuronal mitochondria is characteristic, very similar to that induced by structurally homologous endogenous group IIA sPLA2 when its activity is elevated, as, for example, in the early phase of Alzheimer's disease. Using ammodytoxin (Atx), the ß-ntx from the venom of the nose-horned viper (Vipera a. ammodytes), the sPLA2 receptor R25 has been detected in neuronal mitochondria. This receptor has been purified from porcine cerebral cortex mitochondria by a new Atx-affinity-based chromatographic procedure. Mass spectrometry analysis revealed R25 to be the subunit II of cytochrome c oxidase (CCOX), an essential constituent of the respiratory chain complex. CCOX was confirmed as being the first intracellular membrane receptor for sPLA2 by alternative Atx-affinity-labellings of purified CCOX, supported also by the encounter of Atx and CCOX in PC12 cells. This discovery suggests the explanation of the mechanism by which ß-ntx hinders production of ATP in poisoned nerve endings. It also provides a new insight into the potential function and dysfunction of endogenous GIIA sPLA2 in mitochondria.

Systemic vascular leakage induced in mice by Russell's viper venom from Pakistan.

Envenomings by some populations of the Russell's viper (Daboia russelii) are characterized by a systemic capillary leak syndrome (CLS) which causes hemoconcentration, and is associated with the severity of envenoming. We adapted a model of CLS in mice by assessing hemoconcentration. The venom of D. russelii from Pakistan, but not that of another viperid, Bothrops asper, induced hemoconcentration and an increment in vascular permeability, being devoid of hemorrhagic activity at the doses tested. These findings reveal a dichotomous pattern of vasculotoxicity in viperid snake venoms. This difference might depend on variations in venom composition, especially regarding metalloproteinases (SVMPs), which are low in Pakistani D. russelii and high in B. asper. Inhibition of SVMPs and phospholipases A2 in D. russelii venom did not abrogate hemoconcentration. An hemoconcentration-inducing fraction was obtained by chromatography, which contains vascular endothelial growth factor (VEGF), a known potent inducer of increment in vascular permeability. Exudates collected from tissue injected with venom also induced hemoconcentration, and the effect was inhibited by antivenom. However, the amount of venom in exudate required to induce the effect is low, as compared with venom dissolved in saline solution, hence suggesting that endogenous proteins present in the exudate, probably inflammatory mediators, potentiate the effect.

Metalloproteinases in disease: identification of biomarkers of tissue damage through proteomics.

INTRODUCTION: Metalloproteinases play key roles in health and disease, by generating novel proteoforms with variable structure and function. Areas covered: This review focuses on the role of endogenous [a Disintegrin and Metalloproteinase (ADAMs), ADAMs with thrombospondin motifs (ADAMTS), and matrix metalloproteinases (MMPs)] and exogenous metalloproteinases in various disease conditions, and describes the application of mass spectrometry-based proteomics to detect qualitative and quantitative changes in protein profiles in tissues and body fluids in disease. Emphasis is placed on the proteomic analysis of exudates collected from affected tissues, including methods that enrich newly generated protein fragments derived from proteolysis in cells, stroma, or extracellular matrix. The use of proteomic analysis of exudates in the study of the local tissue damage induced by metalloproteinases derived from viperid snake venoms is discussed, particularly in relation to extracellular matrix degradation and to the overall pathology of these envenomings. Expert commentary: The information provided by these proteomics approaches is paving the way for the identification of biomarkers based on particular proteolytic signatures associated with different pathologies. Together with other methodological approaches, a comprehensive view of the mechanisms and dynamics of diseases can be achieved. Such basis of knowledge allows for the design of novel diagnostic and therapeutic approaches within the frame of 'precision' or 'personalized' medicine.

Combining discovery and targeted proteomics reveals a prognostic signature in oral cancer.

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 lymph node metastasis. In summary, we identify a robust signature, which may enhance prognostic decisions in OSCC and better guide treatment to reduce tumor recurrence or lymph node metastasis.

Unresolved issues in the understanding of the pathogenesis of local tissue damage induced by snake venoms.

Snakebite envenoming by viperid species, and by some elapids, is characterized by a complex pattern of tissue damage at the anatomical site of venom injection. In severe cases, tissue destruction may be so extensive as to lead to permanent sequelae, with serious pathophysiological, social and psychological consequences. Significant advances have been performed in the study of venom-induced tissue damage, including identification and characterization of the toxins involved, insights into the mechanisms of action of venoms and toxins, and study of tissue responses to venom-induced injury. Nevertheless, much remains to be known and understood on the pathogenesis of these alterations. This review focuses on some of the pending issues in the topic of snake venom-induced local tissue damage. The traditional 'reductionist' approach, which has predominated in the study of snake venoms and their actions, needs to be complemented by more integrative and holistic perspectives aimed at capturing the complexity of these pathological alterations. Future advances in the study of these topics will certainly pave the way for innovative therapeutic interventions, with the goal of reducing the impact of this aspect of snakebite envenoming.

Announcing the 2018 Toxins Travel Awards for Post-Doctoral Fellows.

This year we enjoyed a large number of very highy meritorious applications for our annual Toxins Travel Awards.[...].

Understanding the Snake Venom Metalloproteinases: An Interview with Jay Fox and José María Gutiérrez.

Jay W. Fox and José María Gutiérrez recently finished editing a Special Issue on the topic "Snake Venom Metalloproteinases" in Toxins. The Special Issue covers a wide range of topics, including the molecular evolution and structure of snake venom metalloproteinases (SVMPs), the mechanisms involved in the generation of diversity of SVMPs, the mechanism of action of SVMPs, and their role in the pathophysiology of envenomings, with implications for improving the therapy of envenomings. In this interview, we discussed with Jay W. Fox and José María Gutiérrez their research on the SVMPs and their perspectives on the future trends and challenges for studying snake venoms.

Identification of actin beta-like 2 (ACTBL2) as novel, upregulated protein in colorectal cancer.

Early diagnosis of colorectal cancer (CRC) can be of value for increasing the survival rate of patients. Recently, proteomic strategies to identify markers for the diagnosis of cancer at an early stage have been employed with noteworthy results. To extend these studies, we utilized two dimensional gel electrophoresis and mass spectrometry for expression profiling of proteins extracted from the freshly frozen human colorectal cancer tissue specimens and the comparable regions of adjacent normal mucosa (serving as controls). Four gel spots were determined to be differentially stained between the tumor and the control samples on a consistent basis. Following mass spectrometric analysis of these spots, six proteins were identified; five of these had previously been reported to be associated with colorectal cancer. One protein actin beta-like 2 (ACTBL2), not linked with colorectal cancer in the earlier reports, was however found to be at higher abundance in colorectal tumor samples both by proteomics and immunohistochemistry analysis. Thus ACTBL2 association and differential upregulation in colorectal cancer is novel, and as such may contribute to our understanding of the colorectal carcinogenesis and potentially serve a function in developing markers for colorectal cancer. BIOLOGICAL SIGNIFICANCE: Colorectal cancer (CRC) is a major cause of death world-wide and good markers for early detection are lacking. In this study we conducted a proteomic analysis of tumor vs. normal tissue. We corroborated the finding of a number of previously identified proteins associated with CRC and more importantly identified a novel protein, ACTBL2, which we demonstrated to be upregulated in CRC. As additional proteins associated with CRC are identified the potential for developing panels of markers may be realized with better outcomes in early cancer detection.

Viperid Envenomation Wound Exudate Contributes to Increased Vascular Permeability via a DAMPs/TLR-4 Mediated Pathway.

Viperid snakebite envenomation is characterized by inflammatory events including increase in vascular permeability. A copious exudate is generated in tissue injected with venom, whose proteomics analysis has provided insights into the mechanisms of venom-induced tissue damage. Hereby it is reported that wound exudate itself has the ability to induce increase in vascular permeability in the skin of mice. Proteomics analysis of exudate revealed the presence of cytokines and chemokines, together with abundant damage associated molecular pattern molecules (DAMPs) resulting from both proteolysis of extracellular matrix and cellular lysis. Moreover, significant differences in the amounts of cytokines/chemokines and DAMPs were detected between exudates collected 1 h and 24 h after envenomation, thus highlighting a complex temporal dynamic in the composition of exudate. Pretreatment of mice with Eritoran, an antagonist of Toll-like receptor 4 (TLR4), significantly reduced the exudate-induced increase in vascular permeability, thus suggesting that DAMPs might be acting through this receptor. It is hypothesized that an "Envenomation-induced DAMPs cycle of tissue damage" may be operating in viperid snakebite envenomation through which venom-induced tissue damage generates a variety of DAMPs which may further expand tissue alterations.