Dermonecrosis caused by a spitting cobra snakebite results from toxin potentiation and is prevented by the repurposed drug varespladib.

Snakebite envenoming is a neglected tropical disease that causes substantial mortality and morbidity globally. The venom of African spitting cobras often causes permanent injury via tissue-destructive dermonecrosis at the bite site, which is ineffectively treated by current antivenoms. To address this therapeutic gap, we identified the etiological venom toxins in Naja nigricollis venom responsible for causing local dermonecrosis. While cytotoxic three-finger toxins were primarily responsible for causing spitting cobra cytotoxicity in cultured keratinocytes, their potentiation by phospholipases A2 toxins was essential to cause dermonecrosis in vivo. This evidence of probable toxin synergism suggests that a single toxin-family inhibiting drug could prevent local envenoming. We show that local injection with the repurposed phospholipase A2-inhibiting drug varespladib significantly prevents local tissue damage caused by several spitting cobra venoms in murine models of envenoming. Our findings therefore provide a therapeutic strategy that may effectively prevent life-changing morbidity caused by snakebite in rural Africa.

Selected humanization of yeast U1 snRNP leads to global suppression of pre-mRNA splicing and mitochondrial dysfunction in the budding yeast.

The recognition of 5' splice site (5' ss) is one of the earliest steps of pre-mRNA splicing. To better understand the mechanism and regulation of 5' ss recognition, we selectively humanized components of the yeast U1 snRNP to reveal the function of these components in 5' ss recognition and splicing. We targeted U1C and Luc7, two proteins that interact with and stabilize the yeast U1 (yU1) snRNA and the 5' ss RNA duplex. We replaced the Zinc-Finger (ZnF) domain of yU1C with its human counterpart, which resulted in a cold-sensitive growth phenotype and moderate splicing defects. We next added an auxin-inducible degron to yLuc7 protein (to mimic the lack of Luc7Ls in human U1 snRNP) and found that Luc7-depleted yU1 snRNP resulted in the concomitant loss of PRP40 and Snu71 (two other essential yeast U1 snRNP proteins), and further biochemical analyses suggest a model of how these three proteins interact with each other in the U1 snRNP. The loss of these proteins resulted in a significant growth retardation accompanied by a global suppression of pre-mRNA splicing. The splicing suppression led to mitochondrial dysfunction as revealed by a release of Fe2+ into the growth medium and an induction of mitochondrial reactive oxygen species. Together, these observations indicate that the human U1C ZnF can substitute that of yeast, Luc7 is essential for the incorporation of the Luc7-Prp40-Snu71 trimer into yeast U1 snRNP, and splicing plays a major role in the regulation of mitochondrial function in yeast.

Assessing Target Specificity of the Small Molecule Inhibitor MARIMASTAT to Snake Venom Toxins: A Novel Application of Thermal Proteome Profiling.

New treatments that circumvent the pitfalls of traditional antivenom therapies are critical to address the problem of snakebite globally. Numerous snake venom toxin inhibitors have shown promising cross-species neutralization of medically significant venom toxins in vivo and in vitro. The development of high-throughput approaches for the screening of such inhibitors could accelerate their identification, testing, and implementation and thus holds exciting potential for improving the treatments and outcomes of snakebite envenomation worldwide. Energetics-based proteomic approaches, including thermal proteome profiling and proteome integral solubility alteration (PISA) assays, represent "deep proteomics" methods for high throughput, proteome-wide identification of drug targets and ligands. In the following study, we apply thermal proteome profiling and PISA methods to characterize the interactions between venom toxin proteoforms in Crotalus atrox (Western Diamondback Rattlesnake) and the snake venom metalloprotease (SVMP) inhibitor marimastat. We investigate its venom proteome-wide effects and characterize its interactions with specific SVMP proteoforms, as well as its potential targeting of non-SVMP venom toxin families. We also compare the performance of PISA thermal window and soluble supernatant with insoluble precipitate using two inhibitor concentrations, providing the first demonstration of the utility of a sensitive high-throughput PISA-based approach to assess the direct targets of small molecule inhibitors for snake venom.

IL7 in combination with radiotherapy stimulates a memory T-cell response to improve outcomes in HNSCC models.

Clinically approved head and neck squamous cell carcinoma (HNSCC) immunotherapies manipulate the immune checkpoint blockade (ICB) axis but have had limited success outside of recurrent/metastatic disease. Interleukin-7 (IL7) has been shown to be essential for effector T-cell survival, activation, and proliferation. Here, we show that IL7 in combination with radiotherapy (RT) is effective in activating CD8 + T-cells for reducing tumor growth. Our studies were conducted using both human papillomavirus related and unrelated orthotopic HNSCC murine models. Immune populations from the tumor, draining lymph nodes, and blood were compared between treatment groups and controls using flow cytometry, proteomics, immunofluorescence staining, and RNA sequencing. Treatment with RT and IL7 (RT + IL7) resulted in significant tumor growth reduction, high CD8 T-cell tumor infiltration, and increased proliferation of T-cell progenitors in the bone marrow. IL7 also expanded a memory-like subpopulation of CD8 T-cells. These results indicate that IL7 in combination with RT can serve as an effective immunotherapy strategy outside of the conventional ICB axis to drive the antitumor activity of CD8 T-cells.

Quantification of snake venom proteomes by mass spectrometry-considerations and perspectives.

The advent of soft ionization mass spectrometry-based proteomics in the 1990s led to the development of a new dimension in biology that conceptually allows for the integral analysis of whole proteomes. This transition from a reductionist to a global-integrative approach is conditioned to the capability of proteomic platforms to generate and analyze complete qualitative and quantitative proteomics data. Paradoxically, the underlying analytical technique, molecular mass spectrometry, is inherently nonquantitative. The turn of the century witnessed the development of analytical strategies to endow proteomics with the ability to quantify proteomes of model organisms in the sense of "an organism for which comprehensive molecular (genomic and/or transcriptomic) resources are available." This essay presents an overview of the strategies and the lights and shadows of the most popular quantification methods highlighting the common misuse of label-free approaches developed for model species' when applied to quantify the individual components of proteomes of nonmodel species (In this essay we use the term "non-model" organisms for species lacking comprehensive molecular (genomic and/or transcriptomic) resources, a circumstance that, as we detail in this review-essay, conditions the quantification of their proteomes.). We also point out the opportunity of combining elemental and molecular mass spectrometry systems into a hybrid instrumental configuration for the parallel identification and absolute quantification of venom proteomes. The successful application of this novel mass spectrometry configuration in snake venomics represents a proof-of-concept for a broader and more routine application of hybrid elemental/molecular mass spectrometry setups in other areas of the proteomics field, such as phosphoproteomics, metallomics, and in general in any biological process where a heteroatom (i.e., any atom other than C, H, O, N) forms integral part of its mechanism.

Snakes on a plain: biotic and abiotic factors determine venom compositional variation in a wide-ranging generalist rattlesnake.

BACKGROUND: Snake venoms are trophic adaptations that represent an ideal model to examine the evolutionary factors that shape polymorphic traits under strong natural selection. Venom compositional variation is substantial within and among venomous snake species. However, the forces shaping this phenotypic complexity, as well as the potential integrated roles of biotic and abiotic factors, have received little attention. Here, we investigate geographic variation in venom composition in a wide-ranging rattlesnake (Crotalus viridis viridis) and contextualize this variation by investigating dietary, phylogenetic, and environmental variables that covary with venom. RESULTS: Using shotgun proteomics, venom biochemical profiling, and lethality assays, we identify 2 distinct divergent phenotypes that characterize major axes of venom variation in this species: a myotoxin-rich phenotype and a snake venom metalloprotease (SVMP)-rich phenotype. We find that dietary availability and temperature-related abiotic factors are correlated with geographic trends in venom composition. CONCLUSIONS: Our findings highlight the potential for snake venoms to vary extensively within species, for this variation to be driven by biotic and abiotic factors, and for the importance of integrating biotic and abiotic variation for understanding complex trait evolution. Links between venom variation and variation in biotic and abiotic factors indicate that venom variation likely results from substantial geographic variation in selection regimes that determine the efficacy of venom phenotypes across populations and snake species. Our results highlight the cascading influence of abiotic factors on biotic factors that ultimately shape venom phenotype, providing evidence for a central role of local selection as a key driver of venom variation.

Zoster-Associated Prothrombotic Plasma Exosomes and Increased Stroke Risk.

Herpes zoster (HZ; shingles) caused by varicella zoster virus reactivation increases stroke risk for up to 1 year after HZ. The underlying mechanisms are unclear, however, the development of stroke distant from the site of zoster (eg, thoracic, lumbar, sacral) that can occur months after resolution of rash points to a long-lasting, virus-induced soluble factor (or factors) that can trigger thrombosis and/or vasculitis. Herein, we investigated the content and contributions of circulating plasma exosomes from HZ and non-HZ patient samples. Compared with non-HZ exosomes, HZ exosomes (1) contained proteins conferring a prothrombotic state to recipient cells and (2) activated platelets leading to the formation of platelet-leukocyte aggregates. Exosomes 3 months after HZ yielded similar results and also triggered cerebrovascular cells to secrete the proinflammatory cytokines, interleukin 6 and 8. These results can potentially change clinical practice through addition of antiplatelet agents for HZ and initiatives to increase HZ vaccine uptake to decrease stroke risk.

Decoding the molecular interplay in the central dogma: An overview of mass spectrometry-based methods to investigate protein-metabolite interactions.

With the emergence of next-generation nucleotide sequencing and mass spectrometry-based proteomics and metabolomics tools, we have comprehensive and scalable methods to analyze the genes, transcripts, proteins, and metabolites of a multitude of biological systems. Despite the fascinating new molecular insights at the genome, transcriptome, proteome and metabolome scale, we are still far from fully understanding cellular organization, cell cycles and biology at the molecular level. Significant advances in sensitivity and depth for both sequencing as well as mass spectrometry-based methods allow the analysis at the single cell and single molecule level. At the same time, new tools are emerging that enable the investigation of molecular interactions throughout the central dogma of molecular biology. In this review, we provide an overview of established and recently developed mass spectrometry-based tools to probe metabolite-protein interactions-from individual interaction pairs to interactions at the proteome-metabolome scale.

Mass Spectrometry-Based Atlas of Extracellular Matrix Proteins across 25 Mouse Organs.

The extracellular matrix (ECM) is a critical non-cellular component of multicellular organisms containing a variety of proteins, glycoproteins, and proteoglycans which have been implicated in a wide variety of essential biological processes, including development, wound healing, and aging. Due to low solubility, many ECM proteins have been underrepresented in previous proteomic datasets. Using an optimized three-step decellularization and ECM extraction method involving chaotrope extraction and digestion via hydroxylamine hydrochloride, we have generated coverage of the matrisome across 25 organs. We observe that the top 100 most abundant proteins from the ECM fractions of all tissues are generally present in all tissues, indicating that tissue matrices are principally composed of a shared set of ECM proteins. However, these proteins vary up to 4000-fold between tissues, resulting in highly unique matrix profiles even with the same primary set of proteins. A data reduction approach was used to reveal related networks of expressed ECM proteins across varying tissues, including basement membrane and collagen subtypes.

Characterization of decellularized left and right ventricular myocardial matrix hydrogels and their effects on cardiac progenitor cells.

Congenital heart defects are the leading cause of right heart failure in pediatric patients. Implantation of c-kit+ cardiac-derived progenitor cells (CPCs) is being clinically evaluated to treat the failing right ventricle (RV), but faces limitations due to reduced transplant cell survival, low engraftment rates, and low retention. These limitations have been exacerbated due to the nature of cell delivery (narrow needles) and the non-optimal recipient microenvironment (reactive oxygen species (ROS)). Extracellular matrix (ECM) hydrogels derived from porcine left ventricular (LV) myocardium have emerged as a potential therapy to treat the ischemic LV and have shown promise as a vehicle to deliver cells to injured myocardium. However, no studies have evaluated the combination of an injectable biomaterial, such as an ECM hydrogel, in combination with cell therapy for treating RV failure. In this study we characterized LV and RV myocardial matrix (MM) hydrogels and performed in vitro evaluations of their potential to enhance CPC delivery, including resistance to forces experienced during injection and exposure to ROS, as well as their potential to enhance angiogenic paracrine signaling. While physical properties of the two hydrogels are similar, the decellularized LV and RV have distinct protein signatures. Both materials were equally effective in protecting CPCs against needle forces and ROS. CPCs encapsulated in either the LV MM or RV MM exhibited similar enhanced potential for angiogenic paracrine signaling when compared to CPCs in collagen. The RV MM without cells, however, likewise improved tube formation, suggesting it should also be evaluated as a potential standalone treatment.

Human and Bacterial Toll-Interleukin Receptor Domains Exhibit Distinct Dynamic Features and Functions.

Toll-interleukin receptor (TIR) domains have emerged as critical players involved in innate immune signaling in humans but are also expressed as potential virulence factors within multiple pathogenic bacteria. However, there has been a shortage of structural studies aimed at elucidating atomic resolution details with respect to their interactions, potentially owing to their dynamic nature. Here, we used a combination of biophysical and biochemical studies to reveal the dynamic behavior and functional interactions of a panel of both bacterial TIR-containing proteins and mammalian receptor TIR domains. Regarding dynamics, all three bacterial TIR domains studied here exhibited an inherent exchange that led to severe resonance line-broadening, revealing their intrinsic dynamic nature on the intermediate NMR timescale. In contrast, the three mammalian TIR domains studied here exhibited a range in terms of their dynamic exchange that spans multiple timescales. Functionally, only the bacterial TIR domains were catalytic towards the cleavage of NAD+, despite the conservation of the catalytic nucleophile on human TIR domains. Our development of NMR-based catalytic assays allowed us to further identify differences in product formation for gram-positive versus gram-negative bacterial TIR domains. Differences in oligomeric interactions were also revealed, whereby bacterial TIR domains self-associated solely through their attached coil-coil domains, in contrast to the mammalian TIR domains that formed homodimers and heterodimers through reactive cysteines. Finally, we provide the first atomic-resolution studies of a bacterial coil-coil domain and provide the first atomic model of the TIR domain from a human anti-inflammatory IL-1R8 protein that undergoes a slow inherent exchange.

Integration of transcriptomic and proteomic approaches for snake venom profiling.

INTRODUCTION: Snake venoms contain many protein and peptide isoforms with high levels of sequence variation, even within a single species. AREAS COVERED: In this review, we highlight several examples, from both published and unpublished work in our lab, demonstrating how a combined venom gland transcriptome and proteome methodology allows for comprehensive characterization of venoms, including those from understudied rear-fanged snake species, and we provide recommendations for using these approaches. EXPERT OPINION: When characterizing venoms, peptide mass fingerprinting using databases built predominately from protein sequences originating from model organisms can be disadvantageous, especially when the intention is to document protein diversity. Therefore, the use of species-specific venom gland transcriptomes corrects for the absence of these unique peptide sequences in databases. The integration of transcriptomics and proteomics improves the accuracy of either approach alone for venom profiling.

Phenotypic and functional variation in venom and venom resistance of two sympatric rattlesnakes and their prey.

Predator-prey interactions often lead to the coevolution of adaptations associated with avoiding predation and, for predators, overcoming those defences. Antagonistic coevolutionary relationships are often not simple interactions between a single predator and prey but rather a complex web of interactions between multiple coexisting species. Coevolution between venomous rattlesnakes and small mammals has led to physiological venom resistance in several mammalian taxa. In general, viperid venoms contain large quantities of snake venom metalloproteinase toxins (SVMPs), which are inactivated by SVMP inhibitors expressed in resistant mammals. We explored variation in venom chemistry, SVMP expression, and SVMP resistance across four co-distributed species (California Ground Squirrels, Bryant's Woodrats, Southern Pacific Rattlesnakes, and Red Diamond Rattlesnakes) collected from four different populations in Southern California. Our aim was to understand phenotypic and functional variation in venom and venom resistance in order to compare coevolutionary dynamics of a system involving two sympatric predator-prey pairs to past studies that have focused on single pairs. Proteomic analysis of venoms indicated that these rattlesnakes express different phenotypes when in sympatry, with Red Diamonds expressing more typical viperid venom (with a diversity of SVMPs) and Southern Pacifics expressing a more atypical venom with a broader range of non-enzymatic toxins. We also found that although blood sera from both mammals were generally able to inhibit SVMPs from both rattlesnake species, inhibition depended strongly on the snake population, with snakes from one geographic site expressing SVMPs to which few mammals were resistant. Additionally, we found that Red Diamond venom, rather than woodrat resistance, was locally adapted. Our findings highlight the complexity of coevolutionary relationships between multiple predators and prey that exhibit similar offensive and defensive strategies in sympatry.

Impact of the Identification Strategy on the Reproducibility of the DDA and DIA Results.

Data-independent acquisition (DIA) is a promising technique for the proteomic analysis of complex protein samples. A number of studies have claimed that DIA experiments are more reproducible than data-dependent acquisition (DDA), but these claims are unsubstantiated since different data analysis methods are used in the two methods. Data analysis in most DIA workflows depends on spectral library searches, whereas DDA typically employs sequence database searches. In this study, we examined the reproducibility of the DIA and DDA results using both sequence database and spectral library search. The comparison was first performed using a cell lysate and then extended to an interactome study. Protein overlap among the technical replicates in both DDA and DIA experiments was 30% higher with library-based identifications than with sequence database identifications. The reproducibility of quantification was also improved with library search compared to database search, with the mean of the coefficient of variation decreasing more than 30% and a reduction in the number of missing values of more than 35%. Our results show that regardless of the acquisition method, higher identification and quantification reproducibility is observed when library search was used.

EThcD and 213 nm UVPD for Top-Down Analysis of Bovine Seminal Plasma Proteoforms on Electrophoretic and Chromatographic Time Frames.

Seminal plasma is a critical and complex fluid that carries sperm to eggs to initiate the fertilization process. Here, we present a top-down mass spectrometry (TDMS) strategy for identifying proteins and posttranslational modifications (PTMs) in bovine seminal plasma. In this study, proteins were separated using sheathless capillary zone electrophoresis (CZE)-MS and reversed-phase liquid chromatography (LC)-MS, and then fragmented using electron-transfer/higher-energy collisional dissociation (EThcD) and 213 nm ultraviolet photodissociation (213 nm UVPD) to provide more comprehensive information about the proteomic landscape of this biological fluid. Four hundred and seventeen proteoforms were identified by sheathless CZE-MS, and one hundred and seventy-two species were unique to this method. LC-MS identified 3090 proteoforms, including 1707 unique species. All identifications were within ±10 ppm (mass error) and with a P-Score ≤1 × 10-04. Pooling results (triplicate measurements) from sheathless CZE-MS and LC-MS resulted in the identification of 1433 proteoforms (EThcD) and 2151 proteoforms (213 nm UVPD) with 612 species unique for EThcD and 1021 for 213 nm UVPD. The average sequence coverage was found to be higher for EThcD (28%) than for 213 nm UVPD (23%). The use of sheathless CZE-MS and LC-MS with EThcD and 213 nm UVPD provided complementary protein profiling and proteoform data that were more comprehensive than those of either method alone.

Venom Composition in a Phenotypically Variable Pit Viper ( Trimeresurus insularis) across the Lesser Sunda Archipelago.

The genus Trimeresurus comprises a group of venomous pitvipers endemic to Southeast Asia and the Pacific Islands. Of these, Trimeresurus insularis, the White-lipped Island Pitviper, is a nocturnal, arboreal species that occurs on nearly every major island of the Lesser Sunda archipelago. In the current study, venom phenotypic characteristics of T. insularis sampled from eight Lesser Sunda Islands (Flores, Lembata, Lombok, Pantar, Sumba, Sumbawa, Timor, and Wetar) were evaluated via SDS-PAGE, enzymatic activity assays, fibrinogenolytic assays, gelatin zymography, and RP-HPLC, and the Sumbawa sample was characterized by venomic analysis. For additional comparative analyses, venoms were also examined from several species in the Trimeresurus complex, including T. borneensis, T. gramineus, T. puniceus, T. purpureomaculatus, T. stejnegeri, and Protobothrops flavoviridis. Despite the geographical isolation, T. insularis venoms from all eight islands demonstrated remarkable similarities in gel electrophoretic profiles and RP-HPLC patterns, and all populations had protein bands in the mass ranges of phosphodiesterases (PDE), l-amino acid oxidases (LAAO), P-III snake venom metalloproteinases (SVMP), serine proteases, cysteine-rich secretory proteins (CRISP), phospholipases A2 (PLA2), and C-type lectins. An exception was observed in the Lombok sample, which lacked protein bands in the mass range of serine protease and CRISP. Venomic analysis of the Sumbawa venom also identified these protein families, in addition to several proteins of lesser abundance (<1%), including glutaminyl cyclase, aminopeptidase, PLA2 inhibitor, phospholipase B, cobra venom factor, 5'-nucleotidase, vascular endothelial growth factor, and hyaluronidase. All T. insularis venoms exhibited similarities in thrombin-like and PDE activities, while significant differences were observed for LAAO, SVMP, and kallikrein-like activities, though these differences were only observed for a few islands. Slight but noticeable differences were also observed with fibrinogen and gelatin digestion activities. Trimeresurus insularis venoms exhibited overall similarity to the other Trimeresurus complex species examined, with the exception of P. flavoviridis venom, which showed the greatest overall differentiation. Western blot analysis revealed that all major T. insularis venom proteins were recognized by Green Pitviper ( T. albolabris) antivenom, and reactivity was also seen with most venom proteins of the other Trimeresurus species, but incomplete antivenom-venom recognition was observed against P. flavoviridis venom proteins. These results demonstrate significant conservation in the venom composition of T. insularis across the Lesser Sunda archipelago relative to the other Trimeresurus species examined.

Apoptosis induction in human breast cancer (MCF-7) cells by a novel venom L-amino acid oxidase (Rusvinoxidase) is independent of its enzymatic activity and is accompanied by caspase-7 activation and reactive oxygen species production.

We report the elucidation of a mechanism of apoptosis induction in breast cancer (MCF-7) cells by an L-amino acid oxidase (LAAO), Rusvinoxidase, purified from the venom of Daboia russelii russelii. Peptide mass fingerprinting analysis of Rusvinoxidase, an acidic monomeric glycoprotein with a mass of ~57 kDa, confirmed its identity as snake venom LAAO. The enzymatic activity of Rusvinoxidase was completely abolished after two cycles of freezing and thawing; however, its cytotoxicity toward MCF-7 cells remained unaffected. Dose- and time-dependent induction of apoptosis by Rusvinoxidase on MCF-7 cells was evident from changes in cell morphology, cell membrane integrity, shrinkage of cells and apoptotic body formation accompanied by DNA fragmentation. Rusvinoxidase induced apoptosis in MCF-7 cells by both the extrinsic (death-receptor) and intrinsic (mitochondrial) signaling pathways. The former pathway of apoptosis operated through activation of caspase-8 that subsequently activated caspase-7 but not caspase-3. Rusvinoxidase-induced intrinsic pathway of apoptosis was accompanied by a time-dependent depolarization of the mitochondrial membrane through the generation of reactive oxygen species, followed by a decrease in cellular glutathione content and catalase activity, and down-regulation of expression of anti-apoptotic proteins Bcl-XL and heat-shock proteins (HSP-90 and HSP-70). Rusvinoxidase treatment resulted in increase of the pro-apoptotic protein Bax, subsequently leading to the release of cytochrome c from mitochondria to the cytosol and activating caspase-9, which in turn stimulated effector caspase-7. Rusvinoxidase at a dose of 4 mg/kg was non-toxic in mice, indicating that it may be useful as a model for the development of peptide-based anticancer drugs.

Molecular basis for prey relocation in viperid snakes.

BACKGROUND: Vertebrate predators use a broad arsenal of behaviors and weaponry for overcoming fractious and potentially dangerous prey. A unique array of predatory strategies occur among snakes, ranging from mechanical modes of constriction and jaw-holding in non-venomous snakes, to a chemical means, venom, for quickly dispatching prey. However, even among venomous snakes, different prey handling strategies are utilized, varying from the strike-and-hold behaviors exhibited by highly toxic elapid snakes to the rapid strike-and-release envenomation seen in viperid snakes. For vipers, this mode of envenomation represents a minimal risk predatory strategy by permitting little contact with or retaliation from prey, but it adds the additional task of relocating envenomated prey which has wandered from the attack site. This task is further confounded by trails of other unstruck conspecific or heterospecific prey. Despite decades of behavioral study, researchers still do not know the molecular mechanism which allows for prey relocation. RESULTS: During behavioral discrimination trials (vomeronasal responsiveness) to euthanized mice injected with size-fractionated venom, Crotalus atrox responded significantly to only one protein peak. Assays for enzymes common in rattlesnake venoms, such as exonuclease, L-amino acid oxidase, metalloproteinase, thrombin-like and kallikrein-like serine proteases and phospholipase A(2), showed that vomeronasal responsiveness was not dependent on enzymatic activity. Using mass spectrometry and N-terminal sequencing, we identified the proteins responsible for envenomated prey discrimination as the non-enzymatic disintegrins crotatroxin 1 and 2. Our results demonstrate a novel and critical biological role for venom disintegrins far beyond their well-established role in disruption of cell-cell and cell-extracellular matrix interactions. CONCLUSIONS: These findings reveal the evolutionary significance of free disintegrins in venoms as the molecular mechanism in vipers allowing for effective relocation of envenomated prey. The presence of free disintegrins in turn has led to evolution of a major behavioral adaptation (strike-and-release), characteristic of only rattlesnakes and other vipers, which exploits and refines the efficiency of a pre-existing chemical means of predation and a highly sensitive olfaction system. This system of a predator chemically tagging prey represents a novel trend in the coevolution of predator-prey relationships.

Altered metabolism and DAM-signatures in female brains and microglia with aging.

Despite Alzheimer's disease (AD) disproportionately affecting women, the mechanisms remain elusive. In AD, microglia undergo 'metabolic reprogramming', which contributes to microglial dysfunction and AD pathology. However, how sex and age contribute to metabolic reprogramming in microglia is understudied. Here, we use metabolic imaging, transcriptomics, and metabolic assays to probe age- and sex-associated changes in brain and microglial metabolism. Glycolytic and oxidative metabolism in the whole brain was determined using Fluorescence Lifetime Imaging Microscopy (FLIM). Young female brains appeared less glycolytic than male brains, but with aging, the female brain became 'male-like.' Transcriptomic analysis revealed increased expression of disease-associated microglia (DAM) genes (e.g., ApoE, Trem2, LPL), and genes involved in glycolysis and oxidative metabolism in microglia from aged females compared to males. To determine whether estrogen can alter the expression of these genes, BV-2 microglia-like cell lines, which abundantly express DAM genes, were supplemented with 17ß-estradiol (E2). E2 supplementation resulted in reduced expression of DAM genes, reduced lipid and cholesterol transport, and substrate-dependent changes in glycolysis and oxidative metabolism. Consistent with the notion that E2 may suppress DAM-associated factors, LPL activity was elevated in the brains of aged female mice. Similarly, DAM gene and protein expression was higher in monocyte-derived microglia-like (MDMi) cells derived from middle-aged females compared to age-matched males and was responsive to E2 supplementation. FLIM analysis of MDMi from young and middle-aged females revealed reduced oxidative metabolism and FAD+ with age. Overall, our findings show that altered metabolism defines age-associated changes in female microglia and suggest that estrogen may inhibit the expression and activity of DAM-associated factors, which may contribute to increased AD risk, especially in post-menopausal women.

Diverse gene regulatory mechanisms alter rattlesnake venom gene expression at fine evolutionary scales.

Understanding and predicting the relationships between genotype and phenotype is often challenging, largely due to the complex nature of eukaryotic gene regulation. A step towards this goal is to map how phenotypic diversity evolves through genomic changes that modify gene regulatory interactions. Using the Prairie Rattlesnake (Crotalus viridis) and related species, we integrate mRNA-seq, proteomic, ATAC-seq and whole genome resequencing data to understand how specific evolutionary modifications to gene regulatory network components produce differences in venom gene expression. Through comparisons within and between species, we find a remarkably high degree of gene expression and regulatory network variation across even a shallow level of evolutionary divergence. We use these data to test hypotheses about the roles of specific trans-factors and cis-regulatory elements, how these roles may vary across venom genes and gene families, and how variation in regulatory systems drive diversity in venom phenotypes. Our results illustrate that differences in chromatin and genotype at regulatory elements play major roles in modulating expression. However, we also find that enhancer deletions, differences in transcription-factor expression, and variation in activity of the insulator protein CTCF also likely impact venom phenotypes. Our findings provide insight into the diversity and gene-specificity of gene regulatory features and highlight the value of comparative studies to link gene regulatory network variation to phenotypic variation.