Ventilatory Parameters in Obstetric Patients With COVID-19 and Impact of Delivery: A Multicenter Prospective Cohort Study.

BACKGROUND: Current evidence on obstetric patients requiring advanced ventilatory support and impact of delivery on ventilatory parameters is retrospective, scarce, and controversial. RESEARCH QUESTION: What are the ventilatory parameters for obstetric patients with COVID-19 and how does delivery impact them? What are the risk factors for invasive mechanical ventilation (IMV) and for maternal, fetal, and neonatal mortality? STUDY DESIGN AND METHODS: Prospective, multicenter, cohort study including pregnant and postpartum patients with COVID-19 requiring advanced ventilatory support in the ICU. RESULTS: Ninety-one patients were admitted to 21 ICUs at 29.2 ± 4.9 weeks; 63 patients (69%) delivered in ICU. Maximal ventilatory support was as follows: IMV, 69 patients (76%); high-flow nasal cannula, 20 patients (22%); and noninvasive mechanical ventilation, 2 patients (2%). Sequential Organ Failure Assessment during the first 24 h (SOFA24) score was the only risk factor for IMV (OR, 1.97; 95% CI, 1.29-2.99; P = .001). Respiratory parameters at IMV onset for pregnant patients were: mean ± SD plateau pressure (PP), 24.3 ± 4.5 cm H2O; mean ± SD driving pressure (DP), 12.5 ± 3.3 cm H2O; median static compliance (SC), 31 mL/cm H2O (interquartile range [IQR], 26-40 mL/cm H2O); and median Pao2 to Fio2 ratio, 142 (IQR, 110-176). Respiratory parameters before (< 2 h) and after (≤ 2 h and 24 h) delivery were, respectively: mean ± SD PP, 25.6 ± 6.6 cm H2O, 24 ± 6.7 cm H2O, and 24.6 ± 5.2 cm H2O (P = .59); mean ± SD DP, 13.6 ± 4.2 cm H2O, 12.9 ± 3.9 cm H2O, and 13 ± 4.4 cm H2O (P = .69); median SC, 28 mL/cm H2O (IQR, 22.5-39 mL/cm H2O), 30 mL/cm H2O (IQR, 24.5-44 mL/cm H2O), and 30 mL/cm H2O (IQR, 24.5-44 mL/cm H2O; P = .058); and Pao2 to Fio2 ratio, 134 (IQR, 100-230), 168 (IQR, 136-185), and 192 (IQR, 132-232.5; P = .022). Reasons for induced delivery were as follows: maternal, 43 of 71 patients (60.5%); maternal and fetal, 21 of 71 patients (29.5%); and fetal, 7 of 71 patients (9.9%). Fourteen patients (22.2%) continued pregnancy after ICU discharge. Risk factors for maternal mortality were BMI (OR, 1.10; 95% CI, 1.006-1.204; P = .037) and comorbidities (OR, 4.15; 95% CI, 1.212-14.20; P = .023). Risk factors for fetal or neonatal mortality were gestational age at delivery (OR, 0.67; 95% CI, 0.52-0.86; P = .002) and SOFA24 score (OR, 1.53; 95% CI, 1.13-2.08; P = .006). INTERPRETATION: Contrary to expectations, pregnant patient lung mechanics were similar to those of the general population with COVID-19 in the ICU. Delivery was induced mainly for maternal reasons, but did not change ventilatory parameters other than Pao2 to Fio2 ratio. SOFA24 score was the only risk factor for IMV. Maternal mortality was associated independently with BMI and comorbidities. Risk factors for fetal and neonatal mortality were SOFA24 score and gestational age at delivery.

Neuroinflammation in the pathogenesis of axonal Charcot-Marie-Tooth disease caused by lack of GDAP1.

Mutations in the GDAP1 mitochondrial outer membrane gene cause Charcot-Marie-Tooth (CMT) neuropathy. Reduction or absence of GDAP1 has been associated with abnormal changes in the mitochondrial morphology and dynamics, oxidative stress and changes in calcium homeostasis. Neuroinflammation has been described in rodent models of genetic demyelinating CMT neuropathies but not in CMT primarily associated with axonopathy. Inflammatory processes have also been related to mitochondrial changes and oxidative stress in central neurodegenerative disorders. Here we investigated the presence of neuroinflammation in the axonal neuropathy of the Gdap1-/- mice. We showed by transcriptome profile of spinal cord and the in vivo detection of activated phagocytes that the absence of GDAP1 is associated with upregulation of inflammatory pathways. We observed reactive gliosis in spinal cord with increase of the astroglia markers GFAP and S100B, and the microglia marker IBA1. Additionally, we found significant increase of inflammatory mediators such as TNF-α and pERK, and C1qa and C1qb proteins of the complement system. Importantly, we observed an increased expression of CD206 and CD86 as M2 and M1 microglia and macrophage response markers, respectively, in Gdap1-/- mice. These inflammatory changes were also associated with abnormal molecular changes in synapses. In summary, we demonstrate that inflammation in spinal cord and sciatic nerve, but not in brain and cerebellum, is part of the pathophysiology of axonal GDAP1-related CMT.

[An update on catheter-associated urinary tract infection. Inter-Society recommendations]. / Infección del tracto urinario asociada a sonda vesical. Actualización y recomendaciones intersociedades.

Representatives of the Argentine Society of Infectious Diseases (SADI) and the Argentine Society of Intensive Therapy (SATI) issued the present recommendations on diagnosis, treatment, and prevention of catheter associated urinary tract infection (CA-UTI). Articles published during 2006-2016 were analyzed in the light of experts' opinion and local data. This document aims to offer basic strategies to optimize the diagnosis based on clinical and microbiological criteria, provide guidance in empirical and targeted antibiotic schemes, and promote effective measures to reduce the risk of CA-UTI. The joint work of both societies highlights the experts' concern about the mismanagement of CA-UTI, which is associated to the indiscriminate use of antimicrobials, and the importance of improving daily practices of CA-UTI management. Through these recommendations, local guidelines are established to optimize the diagnosis, treatment and prevention of CAUTI in order to reduce morbimortality, days of hospitalization, costs and antibiotic resistance due to the misuse of antimicrobials.

Infección del tracto urinario asociada a sonda vesical: Actualización y recomendaciones intersociedades / An update on catheter-associated urinary tract infection. Inter-Society recommendations

Representantes de la Sociedad Argentina de Infectología (SADI) y la Sociedad Argentina de Terapia Intensiva (SATI) acordaron la elaboración de recomendaciones de diagnóstico, tratamiento y prevención de la infección del tracto urinario asociada a sonda vesical (ITU-SV). La metodología utilizada fue el análisis de la bibliografía publicada en 2006-2016, complementada con la opinión de expertos y datos epidemiológicos locales. En este documento se pretende ofrecer herramientas básicas de optimización de diagnóstico en base a criterios clínicos y microbiológicos, orientación en los esquemas antibióticos empíricos y dirigidos, y promover las medidas efectivas para reducir el riesgo de ITU-SV. Se destaca la preocupación por el control y tratamiento inadecuados de la ITU-SV, en particular el uso indiscriminado de antimicrobianos y la importancia de garantizar la mejora en las prácticas diarias. Se establecen pautas locales para mejorar la prevención, optimizar el diagnóstico y tratamiento de la ITU-SV, y así disminuir la morbimortalidad, los días de internación, los costos y la resistencia a antibióticos debidos al mal uso de los antimicrobianos.

Representatives of the Argentine Society of Infectious Diseases (SADI) and the Argentine Society of Intensive Therapy (SATI) issued the present recommendations on diagnosis, treatment, and prevention of catheter associated urinary tract infection (CA-UTI). Articles published during 2006-2016 were analyzed in the light of experts' opinion and local data. This document aims to offer basic strategies to optimize the diagnosis based on clinical and microbiological criteria, provide guidance in empirical and targeted antibiotic schemes, and promote effective measures to reduce the risk of CA-UTI. The joint work of both societies highlights the experts' concern about the mismanagement of CA-UTI, which is associated to the indiscriminate use of antimicrobials, and the importance of improving daily practices of CA-UTI management. Through these recommendations, local guidelines are established to optimize the diagnosis, treatment and prevention of CAUTI in order to reduce morbimortality, days of hospitalization, costs and antibiotic resistance due to the misuse of antimicrobials.

Levosimendan versus placebo in cardiac surgery: a systematic review and meta-analysis.

The aim of this meta-analysis was to review all published randomized clinical trials comparing levosimendan versus placebo in patients undergoing cardiac surgery. PubMed, EMBASE and the Cochrane library database of clinical trials were searched for prospective randomized clinical trials investigating the perioperative use of levosimendan versus placebo in patients undergoing adult cardiac surgery from 1 May 2000 to 10 April 2017. Binary outcomes from individual studies were analysed to compute individual and pooled risk ratios (RRs) with pertinent 95% confidence intervals (CIs). Fourteen randomized clinical trials with a total of 2243 patients were included in this review. Overall meta-analysis results demonstrated that levosimendan was associated with a significant reduction in 30-day mortality (RR = 0.71, 95% CI = 0.53-0.95; P = 0.023). Subgroup analysis showed that this benefit was confined to the moderate and low ejection fraction studies (RR = 0.44, 95% CI = 0.27-0.70; P < 0.001), whereas no benefit was observed in the preserved ejection fraction studies (RR = 1.06, 95% CI = 0.72-1.56; P = 0.78). Levosimendan also reduced the risk of renal replacement therapy (RR = 0.66, 95% CI = 0.47-0.92; P = 0.015) and low cardiac output (RR = 0.40, 95% CI = 0.22-0.73; P = 0.003). No significant differences were detected, between the levosimendan group and the placebo group, in terms of risk of myocardial injury (RR = 0.90, 95% CI = 0.69-1.17; P = 0.44), intensive care unit stay (weighted mean differences = -0.57, 95% CI = -1.15 to 0.01; P = 0.055) and the use of ventricular assist device (RR = 0.42, 95% CI = 0.07-2.63; P = 0.35). In conclusion, levosimendan was associated with a reduced risk of mortality, renal replacement therapy and low cardiac output syndrome in patients undergoing cardiac surgery.

Lack of GDAP1 induces neuronal calcium and mitochondrial defects in a knockout mouse model of charcot-marie-tooth neuropathy.

Mutations in GDAP1, which encodes protein located in the mitochondrial outer membrane, cause axonal recessive (AR-CMT2), axonal dominant (CMT2K) and demyelinating recessive (CMT4A) forms of Charcot-Marie-Tooth (CMT) neuropathy. Loss of function recessive mutations in GDAP1 are associated with decreased mitochondrial fission activity, while dominant mutations result in impairment of mitochondrial fusion with increased production of reactive oxygen species and susceptibility to apoptotic stimuli. GDAP1 silencing in vitro reduces Ca2+ inflow through store-operated Ca2+ entry (SOCE) upon mobilization of endoplasmic reticulum (ER) Ca2+, likely in association with an abnormal distribution of the mitochondrial network. To investigate the functional consequences of lack of GDAP1 in vivo, we generated a Gdap1 knockout mouse. The affected animals presented abnormal motor behavior starting at the age of 3 months. Electrophysiological and biochemical studies confirmed the axonal nature of the neuropathy whereas histopathological studies over time showed progressive loss of motor neurons (MNs) in the anterior horn of the spinal cord and defects in neuromuscular junctions. Analyses of cultured embryonic MNs and adult dorsal root ganglia neurons from affected animals demonstrated large and defective mitochondria, changes in the ER cisternae, reduced acetylation of cytoskeletal α-tubulin and increased autophagy vesicles. Importantly, MNs showed reduced cytosolic calcium and SOCE response. The development and characterization of the GDAP1 neuropathy mice model thus revealed that some of the pathophysiological changes present in axonal recessive form of the GDAP1-related CMT might be the consequence of changes in the mitochondrial network biology and mitochondria-endoplasmic reticulum interaction leading to abnormalities in calcium homeostasis.

Neural and molecular features on Charcot-Marie-Tooth disease plasticity and therapy.

In the peripheral nervous system disorders plasticity is related to changes on the axon and Schwann cell biology, and the synaptic formations and connections, which could be also a focus for therapeutic research. Charcot-Marie-Tooth disease (CMT) represents a large group of inherited peripheral neuropathies that involve mainly both motor and sensory nerves and induce muscular atrophy and weakness. Genetic analysis has identified several pathways and molecular mechanisms involving myelin structure and proper nerve myelination, transcriptional regulation, protein turnover, vesicle trafficking, axonal transport and mitochondrial dynamics. These pathogenic mechanisms affect the continuous signaling and dialogue between the Schwann cell and the axon, having as final result the loss of myelin and nerve maintenance; however, some late onset axonal CMT neuropathies are a consequence of Schwann cell specific changes not affecting myelin. Comprehension of molecular pathways involved in Schwann cell-axonal interactions is likely not only to increase the understanding of nerve biology but also to identify the molecular targets and cell pathways to design novel therapeutic approaches for inherited neuropathies but also for most common peripheral neuropathies. These approaches should improve the plasticity of the synaptic connections at the neuromuscular junction and regenerate cell viability based on improving myelin and axon interaction.

Recombinant expression of mutants of the Frankenstein disintegrin, RTS-ocellatusin. Evidence for the independent origin of RGD and KTS/RTS disintegrins.

The requirements to transform a short disintegrin of the RGD clade into an RTS disintegrin, were investigated through the generation of recombinant mutants of ocellatusin in which the RGD tripeptide was substituted for RTS in different positions along the integrin-specificity loop. Any attempt to create an active integrin α(1)ß(1) inhibitory motif within the specificity loop of ocellatusin was unsuccessful. Replacing the whole RGD-loop of ocellatusin by the RTS-loop of jerdostatin was neither sufficient for confering α(1)ß(1) binding specificity to this ocellatusin-RTS Frankenstein(2) mutant. Factors other than the integrin-binding loop sequence per se are thus required to transform a disintegrin scaffold from the RGD clade into another scaffold from the RTS/KTS clade. Moreover, our results provide evidences, that the RTS/KTS short disintegrins have potentially been recruited into the venom gland of Eurasian vipers independently from the canonical neofunctionalization pathway of the RGD disintegrins. PCR-amplifications of jerdostatin-like sequences from a number of taxa across reptiles, including snakes (Crotalinae, Viperinae, and Elapidae taxa) and lizards (Lacertidae and Iguanidae) clearly showed that genes coding for RTS/KTS disintegrins existed long before the split of Lacertidae and Iguania, thus predating the recruitment of the SVMP precursors of disintegrins, providing strong support for the view of an independent evolutionary history of the RTS/KTS and the RGD clades of short disintegrins.

Profiling the venom gland transcriptomes of Costa Rican snakes by 454 pyrosequencing.

BACKGROUND: A long term research goal of venomics, of applied importance for improving current antivenom therapy, but also for drug discovery, is to understand the pharmacological potential of venoms. Individually or combined, proteomic and transcriptomic studies have demonstrated their feasibility to explore in depth the molecular diversity of venoms. In the absence of genome sequence, transcriptomes represent also valuable searchable databases for proteomic projects. RESULTS: The venom gland transcriptomes of 8 Costa Rican taxa from 5 genera (Crotalus, Bothrops, Atropoides, Cerrophidion, and Bothriechis) of pitvipers were investigated using high-throughput 454 pyrosequencing. 100,394 out of 330,010 masked reads produced significant hits in the available databases. 5.165,220 nucleotides (8.27%) were masked by RepeatMasker, the vast majority of which corresponding to class I (retroelements) and class II (DNA transposons) mobile elements. BLAST hits included 79,991 matches to entries of the taxonomic suborder Serpentes, of which 62,433 displayed similarity to documented venom proteins. Strong discrepancies between the transcriptome-computed and the proteome-gathered toxin compositions were obvious at first sight. Although the reasons underlaying this discrepancy are elusive, since no clear trend within or between species is apparent, the data indicate that individual mRNA species may be translationally controlled in a species-dependent manner. The minimum number of genes from each toxin family transcribed into the venom gland transcriptome of each species was calculated from multiple alignments of reads matched to a full-length reference sequence of each toxin family. Reads encoding ORF regions of Kazal-type inhibitor-like proteins were uniquely found in Bothriechis schlegelii and B. lateralis transcriptomes, suggesting a genus-specific recruitment event during the early-Middle Miocene. A transcriptome-based cladogram supports the large divergence between A. mexicanus and A. picadoi, and a closer kinship between A. mexicanus and C. godmani. CONCLUSIONS: Our comparative next-generation sequencing (NGS) analysis reveals taxon-specific trends governing the formulation of the venom arsenal. Knowledge of the venom proteome provides hints on the translation efficiency of toxin-coding transcripts, contributing thereby to a more accurate interpretation of the transcriptome. The application of NGS to the analysis of snake venom transcriptomes, may represent the tool for opening the door to systems venomics.

Recombinant expression in human cells of active integrin alpha 1 beta 1-blocking RTS-disintegrin jerdostatin.

Jerdostatin, an RTS short disintegrin cloned from Protobothrops jerdonii and recombinantly produced in Escherichia coli, is a potent and specific antagonist of the alpha(1)beta(1) integrin. Jerdostatin selectively blocked the adhesion of alpha(1)beta(1)-K562 cell to collagens I and IV in vitro and angiogenesis in vivo. Here we report the recombinant production of jerdostatin in a mammalian cell system, a prerequisite for developing a conditional transgenic mouse to investigate the effect of systemic expression of jerdostatin on tumor development. For proper export of jerdostatin, a secretion leader sequence was engineered at the protein's N-terminus. A FLAG epitope was also included at the N-terminus of the mature disintegrin to facilitate its isolation and characterization of recombinant jerdostatin (rJerd). This pRc-CMV/FLAG-rJerd construct was transiently expressed in HEK-293 cells and was efficiently secreted into the culture medium. rJerd bound to recombinant soluble alpha(1)beta(1) integrin in a saturable and cation-independent manner. Soluble rJerd also inhibited the binding of alpha(1)beta(1) integrin to the CB3 fragment of collagen IV in a dose-dependent manner (IC(50) 570 nM). Mammalian cell-expressed jerdostatin disrupted the adhesion of RuGli cells to collagen IV. Our results highlight pRc-CMV/FLAG-rJerd as a suitable construct for expressing soluble active alpha(1)beta(1)-blocking jerdostatin in a mammalian cell system.

Isolation of an acidic phospholipase A2 from the venom of the snake Bothrops asper of Costa Rica: biochemical and toxicological characterization.

Phospholipases A(2) (PLA(2)) are major components of snake venoms, exerting a variety of relevant toxic actions such as neurotoxicity and myotoxicity, among others. Since the majority of toxic PLA(2)s are basic proteins, acidic isoforms and their possible roles in venoms are less understood. In this study, an acidic enzyme (BaspPLA(2)-II) was isolated from the venom of Bothrops asper (Pacific region of Costa Rica) and characterized. BaspPLA(2)-II is monomeric, with a mass of 14,212 +/- 6 Da and a pI of 4.9. Its complete sequence of 124 amino acids was deduced through cDNA and protein sequencing, showing that it belongs to the Asp49 group of catalytically active enzymes. In vivo and in vitro assays demonstrated that BaspPLA(2)-II, in contrast to the basic Asp49 counterparts present in the same venom, lacks myotoxic, cytotoxic, and anticoagulant activities. BaspPLA(2)-II also differed from other acidic PLA(2)s described in Bothrops spp. venoms, as it did not show hypotensive and anti-platelet aggregation activities. Furthermore, this enzyme was not lethal to mice at intravenous doses up to 100 microg (5.9 microg/g), indicating its lack of neurotoxic activity. The only toxic effect recorded in vivo was a moderate induction of local edema. Therefore, the toxicological characteristics of BaspPLA(2)-II suggest that it does not play a key role in the pathophysiology of envenomings by B. asper, and that its purpose might be restricted to digestive functions. Immunochemical analyses using antibodies raised against BaspPLA(2)-II revealed that acidic and basic PLA(2)s form two different antigenic groups in B. asper venom.

Combined snake venomics and venom gland transcriptomic analysis of the ocellated carpet viper, Echis ocellatus.

Snakebite in Africa causes thousands of deaths annually and considerable permanent physical disability. The saw-scaled viper, Echis ocellatus, represents the single most medically important snake species in West Africa. To provide a detailed compositional analysis of the venom of E. ocellatus for designing novel toxin-specific immunotherapy and to delineate sequence structure-function relationships of individual toxins, we characterised the venom proteome and the venom gland transcriptome. Whole E. ocellatus venom was fractionated by reverse-phase HPLC, followed by analysis of each chromatographic fraction using a combination of SDS-PAGE, N-terminal sequencing, MALDI-TOF mass fingerprinting, and CID-MS/MS of tryptic peptides. This analysis identified around 35 distinct proteins of molecular masses in the range of 5.5-110 kDa belonging to 8 different toxin families (disintegrin, DC-fragment, phospholipase A(2), cysteine-rich secretory protein, serine proteinase, C-type lectin, l-amino acid oxidase, and Zn(2+)-dependent metalloprotease). Comparison of the toxin composition of E. ocellatus venom determined using a proteomic approach, with the predicted proteome derived from assembly of 1000 EST sequences from a E. ocellatus venom gland cDNA library, shows some differences. Most notably, peptides derived from 26% of the venom proteins could not be ascribed an exact match in the transcriptome. Similarly, 64 (67%) out of the 95 putative toxin clusters reported in the transcriptome did not match to peptides detected in the venom proteome. These data suggest that the final composition of venom is influenced by transcriptional and post-translational mechanisms that may be more complex than previously appreciated. This, in turn, emphasises the value of combining proteomic and transcriptomic approaches to acquire a more complete understanding of the precise composition of snake venom, than would be gleaned from using one analysis alone. From a clinical perspective, the large amount of SVMPs (66.5% of the total venom proteins) is consistent with the haemorrhagic pathology associated with E. ocellatus envenoming. More significantly, whilst the proteomic analysis confirms the majority of these metalloproteinases (58%) belong to the SVMP PIII class, MS/MS derived peptide sequencing also demonstrates a major constituent (32%) of E. ocellatus venom is a PIV-SVMP with a quaternary structure comprising a 48 kDa (Q2UXQ4 or Q2UXQ5) PIII-SVMP subunit, and two 14-16 kDa C-type lectin-like domains [EOC_00087 and EOC_00124] which display similarity to echicetin alpha [P81017] and beta [P81996] subunits.

Evolution of snake venom disintegrins by positive Darwinian selection.

PII-disintegrins, cysteine-rich polypeptides broadly distributed in the venoms of geographically diverse species of vipers and rattlesnakes, antagonize the adhesive functions of beta(1) and beta(3) integrin receptors. PII-disintegrins evolved in Viperidae by neofunctionalization of disintegrin-like domains of duplicated PIII-snake venom hemorrhagic metalloproteinase (SVMP) genes recruited into the venom proteome before the radiation of the advanced snakes. Minimization of the gene (loss of introns and coding regions) and the protein structures (successive loss of disulfide bonds) underpins the postduplication divergence of disintegrins. However, little is known about the underlying genetic mechanisms that have generated the structural and functional diversity among disintegrins. Phylogenetic inference and maximum likelihood-based codon substitution approaches were used to analyze the evolution of the disintegrin family. The topology of the phylogenetic tree does not parallel that of the species tree. This incongruence is consistent with that expected for a multigene family undergoing a birth-and-death process in which the appearance and disappearance of loci are being driven by selection. Cysteine and buried residues appear to be under strong purifying selection due to their role in maintaining the active conformation of disintegrins. Divergence of disintegrins is strongly influenced by positive Darwinian selection causing accelerated rate of substitution in a substantial proportion of surface-exposed disintegrin residues. Global and lineage-specific sites evolving under diversifying selection were identified. Several sites are located within the integrin-binding loop and the C-terminal tail, two regions that form a conformational functional epitope. Arginine-glycine-aspartic acid (RGD) was inferred to represent the ancestral integrin-recognition motif, which emerged from the subgroup of PIII-SVMPs bearing the RDECD sequence. The most parsimonious nucleotide substitution model required for the emergence of all known disintegrin's integrin inhibitory motifs from an ancestral RGD sequence involves a minimum of three mutations. The adaptive advantage of the emergence of motifs targeting beta(1) integrins and the role of positively selected sites located within nonfunctional disintegrin regions appear to be difficult to rationalize in the context of a predator-prey arms race. Perhaps, this represents a consequence of the neofunctionalization potential of the disintegrin domain, a feature that may underlie its recruitment into the venom proteome followed by its successful transformation into a toxin.

Snake venomics. Strategy and applications.

Snake bites can be deadly, but the venoms also contain components of medical and biotechnological value. The proteomic characterization of snake venom proteomes, snake venomics, has thus a number of potential benefits for basic research, clinical diagnosis, and development of new research tools and drugs of potential clinical use. Snake venomics is also relevant for a deep understanding of the evolution and the biological effects of the venoms, and to generate immunization protocols to elicit toxin-specific antibodies with greater specificity and effectiveness than conventional systems. Our snake venomics approach starts with the fractionation of the crude venom by reverse-phase HPLC, followed by the initial characterization of each protein fraction by combination of N-terminal sequencing, SDS-PAGE, and mass spectrometric determination of the molecular masses and the cysteine (SH and S--S) content. Protein fractions showing a single electrophoretic band, molecular mass, and N-terminal sequence can be straightforwardly assigned by BLAST analysis to a known protein family. On the other hand, protein fractions showing heterogeneous or blocked N-termini are analyzed by SDS-PAGE and the bands of interest subjected to automated reduction, carbamidomethylation, and in-gel tryptic digestion. The resulting tryptic peptides are then analyzed by MALDI-TOF mass fingerprinting followed by amino acid sequence determination of selected doubly and triply charged peptide ions by collision-induced dissociation tandem mass spectrometry. The combined strategy allows us to assign unambiguously all the isolated venom toxins representing over 0.05% of the total venom proteins to known protein families. Protocols and applications of snake venomics are reviewed and discussed.

Loss of introns along the evolutionary diversification pathway of snake venom disintegrins evidenced by sequence analysis of genomic DNA from Macrovipera lebetina transmediterranea and Echis ocellatus.

Analysis of cDNAs from Macrovipera lebetina transmediterranea (Mlt) and Echis ocellatus (Eo) venom gland libraries encoding disintegrins argued strongly for a common ancestry of the messengers of short disintegrins and those for precursors of dimeric disintegrin chains. We now report the sequence analysis of disintegrin-coding genes from these two vipers. Genomic DNAs for dimeric disintegrin subunits Ml_G1 and Ml_G2 (Mlt) and Eo_D3 (Eo) contain single 1-kb introns exhibiting the 5'-GTAAG (donor)/3'-AG (acceptor) consensus intron splicing signature. On the other hand, the short RTS-disintegrins Ml_G3 (Mlt) and Eo_RTS (Eo) and the short RGD-disintegrin ocellatusin (Eo) are transcribed from intronless genomic DNA sequences, indicating that the evolutionary pathway leading to the emergence of short disintegrins involved the removal of all intronic sequences. The insertion position of the intron within Ml_G1, Ml_G2, and Eo_D3 is conserved in the genes for vertebrate ADAM (A disintegrin and metalloproteinase) protein disintegrin-like domains and within the gene for the medium-size snake disintegrins halystatins 2 and 3. However, a comparative analysis of currently available disintegrin(-like) genes outlines the view that a minimization of both the gene organization and the protein structure underlies the evolution of the snake venom disintegrin family.

Molecular cloning of Echis ocellatus disintegrins reveals non-venom-secreted proteins and a pathway for the evolution of ocellatusin.

We report the cloning and sequence analysis of Echis ocellatus cDNAs coding for dimeric disintegrin subunits and for the short disintegrin ocellatusin. All the dimeric disintegrin subunit messengers belong to the short-coding class, indicating that short messengers may be more widely distributed than previously thought. Mass spectrometric analysis of the HPLC-separated venom proteins was performed to characterize the dimeric disintegrins expressed in the venom proteome. In addition to previously reported EO4 and EO5 heterodimers, a novel dimeric disintegrin containing RGD- and KGD-bearing subunits was identified. However, a WGD-containing polypeptide encoded by clone Eo1-1 was not detected in the venom, suggesting the occurrence of larger genomic than proteomic diversity, which could represent part of a non-venom-secreted reservoir of disintegrin that may eventually acquire physiological relevance for the snake upon changes of ecological niches and prey habits. On the other hand, the realization of the existence of two distinct messengers coding for the short disintegrin ocellatusin reveals key events of the evolutionary emergence of the short disintegrin ocellatusin from a short-coding dimeric disintegrin precursor by two nucleotide mutations.

Molecular cloning of disintegrin-like transcript BA-5A from a Bitis arietans venom gland cDNA library: a putative intermediate in the evolution of the long-chain disintegrin bitistatin.

We report the cloning and sequence analysis of BA-5A from a venom gland cDNA library of the puff adder, Bitis arietans, that encodes a novel ECD-disintegrin-like domain. BA-5A is a unique PII disintegrin. It contains the 16 cysteine residues that are conserved in all known disintegrin-like domains of ADAM proteins and snake venom metalloproteinases but lacks the cysteine-rich domain. These features suggest that BA-5A may represent an intermediate in the evolutionary pathway of the long disintegrin bitistatin and that removal of the cysteine-rich domain and loss of the PIII-specific disulfide bond were separate events along the structural diversification pathway of disintegrins, the former predating the latter. The protein family composition of the Bitis arietans venom, as determined by combination of reversed-phase HPLC and proteomic analysis, was as follows: Zn(2+)-metalloproteinase (38.5%), serine proteinase (19.5%), disintegrin (17.8%), C-type lectin-like (13.2%), PLA(2) (4.3%), Kunitz-type inhibitor (4.1%), cystatin (1.7%), and unknown (0.9%). BA-5A could not be detected in the venom proteome of Bitis arietans. The occurrence of this very low-abundance (< 0.05%) or nonexpressed disintegrin transcript indicates a hitherto unrecognized structural diversity of this protein family. Whether BA-5A plays a physiological role or represents an orphan protein which could eventually evolve a role in the adaptation of snakes to changing ecological niches and prey habits deserves further investigation.

cDNA cloning and functional expression of jerdostatin, a novel RTS-disintegrin from Trimeresurus jerdonii and a specific antagonist of the alpha1beta1 integrin.

Jerdostatin represents a novel RTS-containing short disintegrin cloned by reverse transcriptase-PCR from the venom gland mRNA of the Chinese Jerdons pit viper Trimeresurus jerdonii. The jerdostatins precursor cDNA contained a 333-bp open reading frame encoding a signal peptide, a pre-peptide, and a 43-amino acid disintegrin domain, whose amino acid sequence displayed 80% identity with that of the KTS-disintegrins obtustatin and viperistatin. The jerdostatin cDNA structure represents the first complete open reading frame of a short disintegrin and points to the emergence of jerdostatin from a short-coding gene. The different residues between jerdostatin and obtustatin/viperistatin are segregated within the integrin-recognition loop and the C-terminal tail. Native jerdostatin (r-jerdostatin-R21) and a R21K mutant (r-jerdostatin-K21) were produced in Escherichia coli. In each case, two conformers were isolated. One-dimensional (1)H NMR showed that conformers 1 and 2 of r-jerdostatin-R21 represent, respectively, well folded and unfolded proteins. The two conformers of the wild-type and the R21K mutant inhibited the adhesion of alpha(1)-K562 cells to collagen IV with IC(50) values of 180 and 703 nm, respectively. The IC(50) values of conformers 2 of r-jerdostatin-R21 and r-jerdostatin-K21 were, respectively, 5.95 and 12.5 microm. Neither r-jerdostatin-R21 nor r-jerdostatin-K21 showed inhibitory activity toward other integrins, including alpha(IIb)beta(3), alpha(v)beta(3), alpha(2)beta(1), alpha(5)beta(1), alpha(4)beta(1), alpha(6)beta(1), and alpha(9)beta(1) up to a concentration of 24 mum. Although the RTS motif appears to be more potent than KTS inhibiting the alpha(1)beta(1) integrin, r-jerdostatin-R21 is less active than the KTS-disintegrins, strongly suggesting that substitutions outside the integrin-binding motif and/or C-terminal proteolytic processing are responsible for the decreased inhibitory activity.

Snake venom disintegrins: evolution of structure and function.

Disintegrins represent a family of polypeptides present in the venoms of various vipers that selectively block the function of integrin receptors. Here, we review our current view and hypothesis on the emergence and the structural and functional diversification of disintegrins by accelerated evolution and the selective loss of disulfide bonds of duplicated genes. Research on disintegrins is relevant for understanding the biology of viper venom toxins, but also provides information on new structural determinants involved in integrin recognition that may be useful in basic and clinical research. The role of the composition, conformation, and dynamics of the integrin inhibitory loop acting in concert with the C-terminal tail in determining the selective inhibition of integrin receptors is discussed.

Snake venomics: characterization of protein families in Sistrurus barbouri venom by cysteine mapping, N-terminal sequencing, and tandem mass spectrometry analysis.

The protein composition of the crude venom of Sistrurus barbouri was analyzed by two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins were separated by reversed phase high-performance liquid chromatography and characterized by N-terminal sequence analysis. The molecular mass and number of cysteine residues of the purified proteins were determined by matrix-associated laser desorption/ionization-time of flight mass spectrometry. Selected protein bands were subjected to in-gel tryptic digestion and peptide mass fingerprinting. Analysis of the tandem mass spectrometry spectra of selected doubly-charged peptide ions was done by collision-induced dissociation in a quadrupole-linear ion trap instrument. Our results show that the venom proteome of the pigmy rattlesnake S. barbouri is composed of proteins belonging to a few protein families, which can be structurally characterized by their disulfide bond contents.