Effect of snake venom procoagulants on snake plasma: implications for the coagulation cascade of snakes.

Several snake venoms contain proteinases that activate zymogens in the coagulation cascade and thus exhibit their procoagulant effects. While most procoagulant proteinases from snake venoms are dissimilar to coagulation factors, Group D (trocarin, notecarin) and C (pseutarin) prothrombin activators are structural and functional homologues of factor Xa and the prothrombinase complex, respectively. We examined the effect of these and other procoagulants from snake venoms as well as mammalian and snake thromboplastins on the coagulation of plasmas of Notechis scutatus, Pseudonaja textilis (both procoagulant venoms), Python reticulatus (non-venomous) and Crotalus atrox (non-procoagulant venom) snakes. The results indicate that the intrinsic pathway seems to be weak or absent only in venomous snakes, while the extrinsic pathway is fully functional in all snakes. Python and Crotalus plasmas have extrinsic pathways similar to that in mammals. In contrast, although Notechis and Pseudonaja plasmas were clotted by a Group C activator, they failed to clot upon the addition of factor Xa and Group D activators. The mechanism of this resistance is still elusive.     

Change in Explanatory Flexibility and Explanatory Style in Cognitive Therapy and its Components

The current study represents a secondary analysis of the dismantling study of cognitive therapy of depression originally conducted by Jacobson et al. (J Consult Clin Psychol 64:295–304, 1996). New analyses examined the role of explanatory flexibility and explanatory style in the recovery from depression. Results indicated that BA treatment responders, but not AT or CT participants evidenced significant improvement in explanatory flexibility, whereas patients from all three study arms, irrespective of responder status demonstrated improvements in explanatory style. Improvement in explanatory flexibility was associated with decreases in symptoms of depression for CT, but not BA or AT, participants. Further, the combination of high explanatory flexibility and low explanatory style conferred maximal protection over relapse. These results suggest that explanatory flexibility is a viable candidate as a process associated with treatment gains in CT. In addition, the results suggest that important cognitive change is possible without an explicit, deliberate focus on the part of the therapist.     

Panoramic view of a superfamily of phosphatases through substrate profiling

Large-scale activity profiling of enzyme superfamilies provides information about cellular functions as well as the intrinsic binding capabilities of conserved folds. Herein, the functional space of the ubiquitous haloalkanoate dehalogenase superfamily (HADSF) was revealed by screening a customized substrate library against >200 enzymes from representative prokaryotic species, enabling inferred annotation of ∼35% of the HADSF. An extremely high level of substrate ambiguity was revealed, with the majority of HADSF enzymes using more than five substrates. Substrate profiling allowed assignment of function to previously unannotated enzymes with known structure, uncovered potential new pathways, and identified iso-functional orthologs from evolutionarily distant taxonomic groups. Intriguingly, the HADSF subfamily having the least structural elaboration of the Rossmann fold catalytic domain was the most specific, consistent with the concept that domain insertions drive the evolution of new functions and that the broad specificity observed in HADSF may be a relic of this process.Since the first genomes were sequenced, there has been an exponential increase in the number of protein sequences deposited into databases worldwide. At the time of this writing the UniProtKB/TrEMBL database contains over 32 million protein sequences. Although this increase in sequence data has dramatically enhanced our understanding of the genomic organization of organisms, as the number of protein sequences grows, the proportion of firm functional assignments diminishes. Traditionally, methods of functional annotation involve comparing sequence identity between experimentally characterized proteins and newly sequenced ones, typically via BLAST (1). In cases where significant sequence similarity cannot be ascertained, proteins are annotated as “hypothetical” or “putative.” Moreover, the decrease in sequence identity leads to an increased uncertainty in functional assignment, especially as the phylogenetic distance between organisms grows, limiting iso-functional ortholog discovery.As the number of newly sequenced genomes grows larger, more protein sequences are likely to be misannotated, oftentimes resulting in the propagation of incorrect functional annotation across newly identified sequences. To tackle the problem of unannotated or misannotated proteins, newer methods for computational assignment have been created with varying degrees of success (2). Although these methods outperform historical methods, continued improvement is necessary to ensure accurate annotation of function (2). A greater swath of functional space can be covered by screening substrates in a high-throughput manner on multiple enzymes from a family (3, 4). Family-wide substrate profiling offers a data-rich resource. The use of sparse screening of sequence space and a diversified library permits the determination of substrate specificity profiles to provide a family-wide view of the range of substrates and insight into the structure of the prototypical substrate. Where structures are available, correlation between substrate range and structural determinants of specificity can be achieved. In addition, the approach has utility in genomic annotation (inferred function), iso-functional ortholog assignment, and the assignment of in vitro substrate profiles to orphaned PDB entries (enzymes with structure but no function, or SNFs). Here we report the application of in vitro high-throughput functional screening of metabolites and related compounds at the superfamily level. We use as an example prokaryotic members of the haloalkanoic acid dehalogenase superfamily (HADSF), a diverse superfamily of enzymes (5) that catalyze a wide range of reactions involving the formation of a covalent intermediate with an active-site aspartate. Reactions catalyzed by this superfamily include dehalogenation (6) as well as Mg²⁺-dependent phosphoryltransfer, although the vast majority (∼99%) are phosphotransferases (7). Members of the HADSF share a Rossmannoid fold “core” domain that contains the phosphoryl transfer site (8, 9) and a “cap” domain that provides substrate specificity determinants (10). There are three major types of caps in the HADSF (C0, C1, and C2A/C2B; see SI Appendix, Fig. S1) based on size, position of insert within the Rossmann fold, and overall topology (7). At the time of writing, the HADSF is known to comprise over 120,000 members across the three domains of life with at most 3% associated with an EC identifier (11).In this study, the functional space of the HADSF was sampled by screening a customized substrate library of 167 compounds against over 200 enzymes from numerous prokaryotic species. The study revealed that a large number of family members show a broad substrate range, with the majority of the HADSF enzymes reacting with five or more substrates. Thus, widespread promiscuity is not incompatible with participation in cell metabolism and may be advantageous to the evolution of new enzyme activities. The activity profiling when applied to putative iso-functional orthologs allowed us to infer annotation for ∼35% of the HADSF. Intriguingly, the HADSF subfamily with the least structural elaboration of the core catalytic Rossmann fold was the most specific with respect to substrate range and number, implying that domain insertions drive the evolution of new functions and that the broad specificity observed in the HADSF may be a relic of this process.     

The Metastatic Spine Disease Multidisciplinary Working Group Algorithms

The Metastatic Spine Disease Multidisciplinary Working Group consists of medical and radiation oncologists, surgeons, and interventional radiologists from multiple comprehensive cancer centers who have developed evidence- and expert opinion-based algorithms for managing metastatic spine disease. The purpose of these algorithms is to facilitate interdisciplinary referrals by providing physicians with straightforward recommendations regarding the use of available treatment options, including emerging modalities such as stereotactic body radiation therapy and percutaneous tumor ablation. This consensus document details the evidence supporting the Working Group algorithms and includes illustrative cases to demonstrate how the algorithms may be applied.

Implications for Practice:

The Metastatic Spine Disease Multidisciplinary Working Group algorithms can facilitate interdisciplinary referrals by providing physicians with straightforward recommendations regarding available treatment options, including emerging modalities such as stereotactic body radiation therapy and percutaneous tumor ablation.