Disintegrins extracted from totonacan rattlesnake (Crotalus totonacus) venom and their anti-adhesive and anti-migration effects on MDA-MB-231 and HMEC-1 cells

Disintegrins are low molecular weight cysteine-rich proteins (4–14 kDa) that are isolated mainly from viperid snake venom. Due to their potential as lead compounds for binding and blocking integrin receptors, snake venom disintegrins have become one of the most studied venom protein families. The aim of this study was to obtain disintegrins from C. totonacus venom and evaluate their capability to bind and block integrin receptors. The C. totonacus disintegrin fraction (totonacin) represents two disintegrin isoforms obtained from C. totonacus venom. These disintegrins showed extracellular-matrix (ECM) protein adhesion and migration inhibitory effects on MDA-MB-231 and HMEC-1 cells. Totonacin (3 μM) inhibited MDA-MB-231 cell adhesion to the ECM proteins, fibronectin, vitronectin, and laminin by 31.2, 44.0, and 32.1, respectively. Adhesion inhibition to fibronectin, vitronectin, and laminin observed on HMEC-1 cells was 42.8, 60.8, and 51%, respectively. In addition, totonacin (3 μM) significantly inhibited MDA-MB-231 and HMEC-1 cell migration (41.4 and 48.3%, respectively). Totonacin showed more potent cell adhesion inhibitory activity toward vitronectin in both cell lines. These results suggest a major affinity of totonacin toward αVβ3, α8β1, αVβ5, αVβ1, and αIIbβ3 integrins. In addition, the inhibitory effect observed on MDA-MB-231 and HMEC-1 cell migration reinforces the evidence of an interaction between these disintegrins and αVβ3 integrin, which plays a key role in migration and angiogenesis

Tissue Localization and Extracellular Matrix Degradation by PI, PII and PIII Snake Venom Metalloproteinases: Clues on the Mechanisms of Venom-Induced Hemorrhage

20 páginas, 4 figuras, 3 tablas y 7 tablas en material suplementario.Snake venom hemorrhagic metalloproteinases (SVMPs) of the PI, PII and PIII classes were compared in terms of tissue localization and their ability to hydrolyze basement membrane components in vivo, as well as by a proteomics analysis of exudates collected in tissue injected with these enzymes. Immunohistochemical analyses of co-localization of these SVMPs with type IV collagen revealed that PII and PIII enzymes co-localized with type IV collagen in capillaries, arterioles and post-capillary venules to a higher extent than PI SVMP, which showed a more widespread distribution in the tissue. The patterns of hydrolysis by these three SVMPs of laminin, type VI collagen and nidogen in vivo greatly differ, whereas the three enzymes showed a similar pattern of degradation of type IV collagen, supporting the concept that hydrolysis of this component is critical for the destabilization of microvessel structure leading to hemorrhage. Proteomic analysis of wound exudate revealed similarities and differences between the action of the three SVMPs. Higher extent of proteolysis was observed for the PI enzyme regarding several extracellular matrix components and fibrinogen, whereas exudates from mice injected with PII and PIII SVMPs had higher amounts of some intracellular proteins. Our results provide novel clues for understanding the mechanisms by which SVMPs induce damage to the microvasculature and generate hemorrhage.This work was performed in partial fulfillment of the requirements for the PhD degree for Cristina Herrera at Universidad de Costa Rica.Peer reviewe

Toward Annealing Stable Molybdenum Oxide Based Hole Selective Contacts For Silicon Photovoltaics

Molybdenum oxide MoOX combines a high work function with broadband optical transparency. Sandwiched between a hydrogenated intrinsic amorphous silicon passivation layer and a transparent conductive oxide, this material allows a highly efficient hole selective front contact stack for crystalline silicon solar cells. However, hole extraction from the Si wafer and transport through this stack degrades upon annealing at 190 C, which is needed to cure the screen printed Ag metallization applied to typical Si solar cells. Here, we show that effusion of hydrogen from the adjacent layers is a likely cause for this degradation, highlighting the need for hydrogen lean passivation layers when using such metal oxide based carrier selective contacts. Pre MoOX deposition annealing of the passivating a Si H layer is shown to be a straightforward approach to manufacturing MoOX based devices with high fill factors using screen printed metallization cured at 190

Effects of PI and PIII Snake Venom Haemorrhagic Metalloproteinases on the Microvasculature: A Confocal Microscopy Study on the Mouse Cremaster Muscle

The precise mechanisms by which Snake Venom Metalloproteinases (SVMPs) disrupt the microvasculature and cause haemorrhage have not been completely elucidated, and novel in vivo models are needed. In the present study, we compared the effects induced by BaP1, a PI SVMP isolated from Bothrops asper venom, and CsH1, a PIII SVMP from Crotalus simus venom, on cremaster muscle microvasculature by topical application of the toxins on isolated tissue (i.e., ex vivo model), and by intra-scrotal administration of the toxins (i.e., in vivo model). The whole tissue was fixed and immunostained to visualize the three components of blood vessels by confocal microscopy. In the ex vivo model, BaP1 was able to degrade type IV collagen and laminin from the BM of microvessels. Moreover, both SVMPs degraded type IV collagen from the BM in capillaries to a higher extent than in PCV and arterioles. CsH1 had a stronger effect on type IV collagen than BaP1. In the in vivo model, the effect of BaP1 on type IV collagen was widespread to the BM of arterioles and PCV. On the other hand, BaP1 was able to disrupt the endothelial barrier in PCV and to increase vascular permeability. Moreover, this toxin increased the size of gaps between pericytes in PCV and created new gaps between smooth muscle cells in arterioles in ex vivo conditions. These effects were not observed in the case of CsH1. In conclusion, our findings demonstrate that both SVMPs degrade type IV collagen from the BM in capillaries in vivo. Moreover, while the action of CsH1 is more directed to the BM of microvessels, the effects of BaP1 are widespread to other microvascular components. This study provides new insights in the mechanism of haemorrhage and other pathological effects induced by these toxins

Muscle Tissue Damage Induced by the Venom of Bothrops asper: Identification of Early and Late Pathological Events through Proteomic Analysis

Citation: Herrera C, Macêdo JKA, Feoli A, Escalante T, Rucavado A, Gutiérrez JM, et al. (2016) Muscle Tissue Damage Induced by the Venom of Bothrops asper: Identification of Early and Late Pathological Events through Proteomic Analysis. PLoS Negl Trop Dis 10(4): e0004599. doi:10.1371/journal. pntd.0004599The time-course of the pathological effects induced by the venom of the snake Bothrops asper in muscle tissue was investigated by a combination of histology, proteomic analysis of exudates collected in the vicinity of damaged muscle, and immunodetection of extracellular matrix proteins in exudates. Proteomic assay of exudates has become an excellent new methodological tool to detect key biomarkers of tissue alterations for a more integrative perspective of snake venom-induced pathology. The time-course analysis of the intracellular proteins showed an early presence of cytosolic and mitochondrial proteins in exudates, while cytoskeletal proteins increased later on. This underscores the rapid cytotoxic effect of venom, especially in muscle fibers, due to the action of myotoxic phospholipases A2, followed by the action of proteinases in the cytoskeleton of damaged muscle fibers. Similarly, the early presence of basement membrane (BM) and other extracellular matrix (ECM) proteins in exudates reflects the rapid microvascular damage and hemorrhage induced by snake venom metalloproteinases. The presence of fragments of type IV collagen and perlecan one hour after envenoming suggests that hydrolysis of these mechanically/structurally-relevant BM components plays a key role in the genesis of hemorrhage. On the other hand, the increment of some ECM proteins in the exudate at later time intervals is likely a consequence of the action of endogenous matrix metalloproteinases (MMPs) or of de novo synthesis of ECM proteins during tissue remodeling as part of the inflammatory reaction. Our results offer relevant insights for a more integrative and systematic understanding of the time-course dynamics of muscle tissue damage induced by B. asper venom and possibly other viperid venoms.Universidad de Costa Rica/[741-B4-660]/UCR/Costa RicaUniversidad de Costa Rica/[741-B6-125]/UCR/Costa RicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

Brucella abortus Uses a Stealthy Strategy to Avoid Activation of the Innate Immune System during the Onset of Infection

To unravel the strategy by which Brucella abortus establishes chronic infections, we explored its early interaction with innate immunity. Methodology/Principal Findings Brucella did not induce proinflammatory responses as demonstrated by the absence of leukocyte recruitment, humoral or cellular blood changes in mice. Brucella hampered neutrophil (PMN) function and PMN depletion did not influence the course of infection. Brucella barely induced proinflammatory cytokines and consumed complement, and was strongly resistant to bactericidal peptides, PMN extracts and serum. Brucella LPS (BrLPS), NH-polysaccharides, cyclic glucans, outer membrane fragments or disrupted bacterial cells displayed low biological activity in mice and cells. The lack of proinflammatory responses was not due to conspicuous inhibitory mechanisms mediated by the invading Brucella or its products. When activated 24 h post-infection macrophages did not kill Brucella, indicating that the replication niche was not fusiogenic with lysosomes. Brucella intracellular replication did not interrupt the cell cycle or caused cytotoxicity in WT, TLR4 and TLR2 knockout cells. TNF-α-induction was TLR4- and TLR2-dependent for live but not for killed B. abortus. However, intracellular replication in TLR4, TLR2 and TLR4/2 knockout cells was not altered and the infection course and anti-Brucella immunity development upon BrLPS injection was unaffected in TLR4 mutant mice. Conclusion/Significance We propose that Brucella has developed a stealth strategy through PAMPs reduction, modification and hiding, ensuring by this manner low stimulatory activity and toxicity for cells. This strategy allows Brucella to reach its replication niche before activation of antimicrobial mechanisms by adaptive immunity. This model is consistent with clinical profiles observed in humans and natural hosts at the onset of infection and could be valid for those intracellular pathogens phylogenetically related to Brucella that also cause long lasting infections

Adaptive Evolution of the Venom-Targeted vWF Protein in Opossums that Eat Pitvipers

The rapid evolution of venom toxin genes is often explained as the result of a biochemical arms race between venomous animals and their prey. However, it is not clear that an arms race analogy is appropriate in this context because there is no published evidence for rapid evolution in genes that might confer toxin resistance among routinely envenomed species. Here we report such evidence from an unusual predator-prey relationship between opossums (Marsupialia: Didelphidae) and pitvipers (Serpentes: Crotalinae). In particular, we found high ratios of replacement to silent substitutions in the gene encoding von Willebrand Factor (vWF), a venom-targeted hemostatic blood protein, in a clade of opossums known to eat pitvipers and to be resistant to their hemorrhagic venom. Observed amino-acid substitutions in venom-resistant opossums include changes in net charge and hydrophobicity that are hypothesized to weaken the bond between vWF and one of its toxic snake-venom ligands, the C-type lectin-like protein botrocetin. Our results provide the first example of rapid adaptive evolution in any venom-targeted molecule, and they support the notion that an evolutionary arms race might be driving the rapid evolution of snake venoms. However, in the arms race implied by our results, venomous snakes are prey, and their venom has a correspondingly defensive function in addition to its usual trophic role

Highly Conductive and Broadband Transparent Zr-Doped In2O3 as Front Electrode for Solar Cells

Broadband transparent and highly conducting electrodes are key to avoid parasitic absorption and electrical losses in solar cells. Here, we propose zirconium-doped indium oxide (IO:Zr) as a transparent electrode intrinsically meeting both requirements and demonstrate its application as the front electrode in silicon heterojunction (SHJ) solar cells. The exceptional properties of this material rely on the combination of high-doping and high electron mobilities, achieving with this a wide optical band gap (3.5–4 eV), low free carrier absorption, and high lateral conductivity. A single film of IO:Zr has an electron mobility of 100 cm2/Vcs with a carrier density of 2.5–3 × 1020 cm–3, resulting in a sheet resistance of around 25 Ω/sq for 100-nm-thick films. Their implementation as a front electrode in SHJ solar cells results in an important gain in current density as compared to the standardly used Sn-doped indium oxide. This is due to reduced parasitic absorption in both, the UV and IR, as confirmed by external quantum efficiency measurements. SHJ devices with the optimized IO:Zr front electrode, resulting in current densities of 40 mA/cm2, a fill factor of 80%, and a conversion efficiency of 23.4%

Brucella abortus uses a stealthy strategy to avoid activation of the innate immune system during the onset of infection

To unravel the strategy by which Brucella abortus establishes chronic infections, we explored its early interaction with innate immunity. Methodology/Principal Findings Brucella did not induce proinflammatory responses as demonstrated by the absence of leukocyte recruitment, humoral or cellular blood changes in mice. Brucella hampered neutrophil (PMN) function and PMN depletion did not influence the course of infection. Brucella barely induced proinflammatory cytokines and consumed complement, and was strongly resistant to bactericidal peptides, PMN extracts and serum. Brucella LPS (BrLPS), NH-polysaccharides, cyclic glucans, outer membrane fragments or disrupted bacterial cells displayed low biological activity in mice and cells. The lack of proinflammatory responses was not due to conspicuous inhibitory mechanisms mediated by the invading Brucella or its products. When activated 24 h post-infection macrophages did not kill Brucella, indicating that the replication niche was not fusiogenic with lysosomes. Brucella intracellular replication did not interrupt the cell cycle or caused cytotoxicity in WT, TLR4 and TLR2 knockout cells. TNF-α-induction was TLR4- and TLR2-dependent for live but not for killed B. abortus. However, intracellular replication in TLR4, TLR2 and TLR4/2 knockout cells was not altered and the infection course and anti-Brucella immunity development upon BrLPS injection was unaffected in TLR4 mutant mice. Conclusion/Significance We propose that Brucella has developed a stealth strategy through PAMPs reduction, modification and hiding, ensuring by this manner low stimulatory activity and toxicity for cells. This strategy allows Brucella to reach its replication niche before activation of antimicrobial mechanisms by adaptive immunity. This model is consistent with clinical profiles observed in humans and natural hosts at the onset of infection and could be valid for those intracellular pathogens phylogenetically related to Brucella that also cause long lasting infections