Evolution and Biological Roles of Three-Finger Toxins in Snake Venoms

Snake venoms are complex mixtures of many enzymatic and non-enzymatic proteins, as well as small peptides. Several major venom protein superfamilies, including three-finger toxins, phospholipases A2, serine proteinases, metalloproteinases, proteinase inhibitors and lectins, are found in almost all snake venoms, from front-fanged viperids (vipers and pit vipers) and elapids (cobras, mambas, sea snakes, etc.) to rear-fanged colubrids. However, these proteins vary in abundance and functionality between species. Variation in snake venom composition is attributed to both differences in the expression levels of toxin encoding genes and occurrence of amino acid sequence polymorphisms. Documenting intraspecific venom variation has both clinical (antiserum development) and biological (predator and prey coevolution) implications. Venom is primarily a trophic adaptation and as such, the evolution and abundance of venom proteins relates directly to prey capture success and organism natural history. Without this biologically relevant perspective, proteomic and transcriptomic approaches could produce simply a list of proteins, peptides, and transcripts. It is therefore important to consider the presence and evolution of venom proteins in terms of their biological significance to the organism. Three-finger toxins (3FTx) comprise a particularly common venom protein superfamily that contributes significantly to differences in envenomation symptomology, toxicity, and overall venom composition. Three-finger toxins are non-enzymatic proteins that maintain a common molecular scaffold, and bind to different receptors/acceptors and exhibit a wide variety of biological effects. These toxins are the main lethal neurotoxins in some snake venoms and are currently the only known venom proteins associated with prey-specific toxicity. This dissertation has four major objectives: (i) to examine 3FTxs in front-fanged Elapidae and rear-fanged snake venoms for prey-specific toxicity, (ii) to examine differences in 3FTx expression within rear-fanged snake venom glands, (iii) to determine if mRNA transcripts obtained from crude venoms can be utilized for molecular evolutionary studies and venom proteomic studies, and (iv) to determine if a transcriptomic and proteomic integrated approach can more thoroughly characterize differences in rear-fanged snake venom composition. Three-finger toxins were isolated from the venom of the front-fanged Naja kaouthia (Family Elapidae; Monocled Cobra) and rear-fanged Spilotes (Pseustes) sulphureus (Family Colubridae; Amazon Puffing Snake) using chromatographic techniques, and toxicity assays were performed to evaluate prey specificity. Despite various 3FTxs being present in abundance within N. kaouthia venom, only one 3FTx (alpha-cobratoxin) demonstrated lethal toxicity (\u3c5 \u3eµg/g) toward both NSA mice (Mus musculus) and House Geckos (Hemidactylus frenatus). For P. sulphureus, the most abundant 3FTx (sulmotoxin A), a heterodimeric complex, displayed prey-specific toxicity towards House Geckos, and the second most abundant 3FTx (sulmotoxin B) displayed prey-specific toxicity towards mice. This demonstrates how a relatively simple venom with toxins dominated by one venom protein superfamily (3FTXs) can still allow for the targeting of a diversity of prey. Venom gland toxin transcriptomes and crude venom transcriptomes were obtained via individual transcripts with 3’RACE (Rapid Amplification of cDNA Ends) and next- generation sequencing to evaluate the abundance, diversity, and molecular evolution of 3FTxs. Venom protein gene expression within rear-fanged snake venom glands revealed trends towards either viper-like expression, dominated by snake venom metalloproteinases, or elapid-like expression, dominated by 3FTxs. For non-conventional 3FTxs transcripts within these glands and within crude venom, approximately 32% of 3FTx amino acid sites were under positive selection, and approximately 20% of sites were functionally critical and conserved. RNA isolated from crude venom demonstrated to be a successful approach to obtain venom protein transcripts for molecular evolutionary analyses, resulting in a novel approach without the need to sacrifice snakes for tissue. The use of a combined venom gland transcriptome with proteomic approaches aided in characterizing venom composition from previously unstudied rear-fanged snake venoms. This dissertation represents an important step in the incorporation of multiple high-throughput characterization methods and the addition of multiple assays to explore the biological roles of toxins, in particular 3FTxs, within these venoms

Persistente organiske forbindelser og effekter på utviklings- og funksjonsprosesser i mus

Initial exposure to persistent organic pollutants (POPs) occurs by placental and lactational transfer during fetal and neonatal life and continues during the adult stage through ingestion, inhalation and dermal absorption. Many POPs have functional groups that resemble endogenous molecules rendering them with the potential for endocrine disruption during sensitive periods of organ development and function. Thus, adverse health effects may arise from exposure to POPs such as cancer and dysfunction of the endocrine, immune, reproductive, developmental or neurological systems. Mice are extensively used in research related to human health and disease as they are small animals with short generation time and high genetic similarity to humans. In addition, transgenic and knockout technology has led to the development of murine models with high phenotypic similarity to human disease. However, several differences between humans and mice are present in the absorption, distribution, metabolism, excretion, sensitivity and susceptibility to POPs. Furthermore, attention has recently shifted from assessing the toxicity of single compounds or well-defined chemical mixtures to evaluating large and environmentally relevant mixtures of POPs. The present study utilized two mouse models (CD-1 and A/J Min) and two exposure regimes (dietary or perinatal) to explore the effects of a human relevant mixture of POPs on female mammary gland development and ovarian folliculogenesis, liver morphology and function, and colorectal cancer development, intestinal microbiota and metabolome. The mixture composition was based on chemicals present in Scandinavian food products and the individual compound concentrations were adjusted to 0x (Control), 5 000x (Low) or 100 000x (High) human estimated daily intake levels for the general Scandinavian population. As shown previously, the present study also demonstrated gestational and lactational transfer of POPs from mothers to offspring. Furthermore, the results showed absorption, distribution, accumulation and persistence of POPs in murine tissues. The Low dose resulted in human relevant concentrations (for some chemicals) and can be considered at least partly human relevant in both its composition and concentration. Perinatal exposure to the mixture restricted mammary gland development and led to a premature arrest of gland growth in female CD-1 mice. In addition, it decreased ovarian follicle maturation and possibly also increased follicle atresia. Together this indicated potential endocrine disruption. Furthermore, the mixture caused persistent hepatocellular hypertrophy in CD-1 mice, but not in A/J Min mice. Thus, a strain-dependent difference in hepatic sensitivity was illustrated and, together with the induction of cytochrome P450 enzymes, indicated that the mixture may cause hepatotoxicity in sensitive strains. The two exposure regimes, dietary and perinatal, resulted in contradictory effects on colorectal carcinogenesis in A/J Min mice. Dietary exposure moderately increased cancer development while perinatal exposure reduced carcinogenesis. The increase was synergistically enhanced when combined with one injection of azoxymethane. Interestingly, perinatal POP exposure modulated the biochemical and microbial environment of the intestine possibly to reduce colorectal carcinogenesis in the predetermined cancer model. In conclusion, the human relevant mixture of POPs affected several developmental and functional processes in mice. The results of the present study can facilitate future mechanistic investigations into how human relevant chemical mixtures may affect biological development and function.Eksponering for persistente organiske forbindelser (POPs) starter i foster- og neonatalstadiet igjennom morkaken og brystmelk, og fortsetter videre igjennom matinntak, innånding og dermal absorpsjon. Mange POPs har funksjonelle grupper som etterligner endogene molekyler, noe som gir kjemikaliene et potensiale for endokrine forstyrrelser i løpet av sensitive perioder for organutvikling og funksjon. Alvorlige helseeffekter kan dermed oppstå som følge av eksponering, slik som kreft og dysfunksjon av ulike kroppsfunksjoner. Mus er mye brukt i forskning på human helse og sykdommer fordi de er små dyr med kort generasjonstid og har høy genetisk likhet til mennesker. I tillegg har transgen- og knockoutteknologi ført til utvikling av musemodeller med høy fenotypisk likhet til humane sykdommer. Likevel finnes det flere ulikheter mellom mennesker og mus, blant annet når det gjelder absorpsjon, distribusjon, metabolisme og ekskresjon av POPs. Videre har oppmerksomheten skiftet fra å studere toksisiteten av enkeltstoffer eller veldefinerte mikser av kjemikalier til å undersøke store og miljørelevante sammensetninger av POPs. I denne avhandlingen ble det brukt to musemodeller (CD-1 og A/J Min) og to eksponeringsregimer (diett og perinatal) til å undersøke effektene av en humanrelevant miks av POPs på hunnlig brystutvikling og utvikling av follikler i ovariene, levermorfologi og funksjon. Forekomst av tykktarmskreft, sammensetning av tarmmikrobiota og metabolom i tarminnhold og tarmvev ble også undersøkt. Miksen hadde en sammensetning av stoffer basert på kjemikalier som er tilstede i Skandinaviske matprodukter og konsentrasjonene av de individuelle stoffene ble justert til 0x (Kontroll), 5 000x (Lav) eller 100 000x (Høy) nivået av estimert humant daglig inntak for den generelle Skandinaviske populasjonen. I likhet med tidligere studier viste også dette studiet at POPs overføres fra mor til barn igjennom graviditet og amming. Videre viste resultatene absorpsjon, distribusjon, akkumulasjon og persistens av POPs i vev fra mus. Lav eksponeringsdose resulterte i konsentrasjoner (for noen av kjemikaliene) som var humanrelevante og kan delvis vurderes til å være humanrelevant i både sin komposisjon og konsentrasjon. Perinatal eksponering for miksen begrenset brystutviklingen og førte til en prematur stans i kjertelveksten i CD-1 hunnmus. Videre førte eksponeringen til en reduksjon i follikkelmodningen og mulig også en økning av follikkelatresi i ovariene. Til sammen indikerte dette en mulig endokrin forstyrrelse. Miksen førte også til persistent hypertrofi av leverceller i CD-1 mus, men ikke i A/J Min mus. Dette tydet på forskjeller i leversensitivitet mellom musemodeller og, sammen med en induksjon av cytokrom P450 enzymer, indikerte dette at miksen kunne føre til levertoksisitet i høysensitive mus. De to eksponeringsregimene, igjennom diett og perinatal overføring, førte til motstridende effekter på tarmkreft i A/J Min mus. Eksponering igjennom dietten førte til en moderat økning av kreft, mens perinatal eksponering reduserte karsinogenesen. Økningen ble synergistisk forsterket i kombinasjon med én injeksjon av azoxymetan. Perinatal POP-eksponering modulerte det biokjemiske og mikrobielle miljøet i tarmen, noe som mulig reduserte tarmkreftutviklingen i den forutbestemte kreftmodellen. Vi konkluderer med at den humanrelevante miksen av POPs påvirket utviklingen og funksjonen til flere kroppsfunksjoner i mus. Disse resultatene gir et godt grunnlag for videre forskning knyttet til hvordan humanrelevante blandinger av POPs kan påvirke utvikling og helse hos dyr og mennesker

Snakebite Therapeutics Based on Endogenous Inhibitors from Vipers

Venomous snakebite is a major human health issue in many countries and has been categorized as a neglected tropical disease by the World Health Organization. Venomous snakes have evolved to produce venom, which is a complex mixture of toxic proteins and peptides, both enzymatic and nonenzymatic in nature. In this current era of high-throughput technologies, venomics projects, which include genome, transcriptome, and proteome analyses of various venomous species, have been conducted to characterize divergent venom phenotypes and the evolution of venom-related genes. Additionally, venomics can also inform about mechanisms of toxin production, storage, and delivery. Venomics can guide antivenom and therapeutic strategies against envenomations and identify new toxin-derived drugs/tools. One potentially promising drug development direction is the use of endogenous inhibitors present in snake venom glands and serum that could be useful for snakebite therapeutics. These inhibitors suppress the activity of venom proteases, enzymatic proteins responsible for the irreversible damage from snakebite. This book chapter will focus on insights from venomous snake adaptations, such as the evolution of venom proteases to generate diverse activities and snake natural resistance to inhibit activity, and how this information can inform and have applications in the treatment of venomous snakebite

総会抄録

<p><b>Aligned Middle American Rattlesnake (<i>Crotalus simus tzabcan</i>) C-type lectins (A) and serine proteases (B).</b> A) Four unique venom-based C-type lectin transcripts (asterisks) were identified for <i>C</i>. <i>s</i>. <i>tzabcan</i> and aligned to other crotaline species. Identical nucleotide sequences are shaded and corresponding GenBank accession numbers are as follows: Crotalus_adamanteus (AEJ31974.1), Deinagkistrodon_acutus (AAM22790.1), Crotalus_d_terrificus (Q719L8.1), and Crotalus_o_helleri (AEU60004.1). B) Venom-based serine proteases cDNA sequences (asterisks) were also obtained from <i>C</i>. <i>s</i>. <i>tzabcan</i> and were aligned with toxins from several other species; identical nucleotide sequences are shaded, and the catalytic triad composed of Ser195, Asp102, and His57 associated with thrombin-like activity in snake venom serine proteases are identified (arrowheads). Isoform 3 from <i>C</i>. <i>s</i>. <i>tzabcan</i> is a partial sequence. GenBank accession numbers are as follows: Agkistrodon_p_leucostoma (HQ270466.1), Bothrops_asper (DQ247724.1), Crotalus_d_terrificus7 (EU360954.1), Crotalus_d_terrificus4 (EU360952.1), Crotalus_d_terrificus3 (EU360951.1), Crotalus_d_durissus (DQ164401.1), Sistrurus_c_edwardsi (DQ464239.1), Trimeresurus_mucrosquamatus (X83225.1), Crotalus_adamanteus (HQ414118.1), Calloselasma_rhodostoma (L07308.1), Deinagkistrodon_acutus (AY861382.1), Trimeresurus_stejnegeri (AF545575.1), and Crotalus_atrox (AF227153.1).</p

Comparison of Results from Life Cycle Assessment when Using Predicted and Real-life Data for an Anaerobic Digestion Plant

Increased separation of food waste driven by the European Circular Economy Action Plan will create a need for establishing new treatment plants that can turn waste resources into high value products. This paper presents a real example of how life cycle assessment was used as decision support when establishing a new anaerobic digestion plant in Norway. The aim of the paper is to investigate the accuracy of the results of the predictive study performed before the plant was built by comparing them with results from the operation of the plant. The comparative assessment revealed that both the burdens and avoided emissions were underestimated. The production volume of upgraded biogas as a fuel for transport was higher than anticipated, resulting in a higher benefit than expected associated with substitution of diesel. Conversely, the impacts from transport and from the anaerobic digestion process were higher than forecasted. In total the net results from the predictive study were within an acceptable range compared with results based on real data

Local non-food yeast protein in pig production–environmental impacts and land use efficiency

publishedVersio

Niemann–Pick Type C2 Proteins in Aedes aegypti: Molecular Modelling and Prediction of Their Structure–Function Relationships

Aedes aegypti is a major vector that transmits arboviruses through the saliva injected into the host. Salivary proteins help in uninterrupted blood intake and enhance the transmission of pathogens. We studied Niemann–Pick Type C2 (NPC2) proteins, a superfamily of saliva proteins that play an important role in arbovirus infections. In vertebrates, a single conserved gene encodes for the NPC2 protein that functions in cholesterol trafficking. Arthropods, in contrast, have several genes that encode divergent NPC2 proteins. We compared the sequences of 20 A. aegypti NPC2 proteins to the cholesterol-binding residues of human and bovine, and fatty-acid-binding residues of ant NPC2 protein. We identified four mosquito NPC2 proteins as potential sterol-binding proteins. Two of these proteins (AAEL006854 and/or AAEL020314) may play a key role in ecdysteroid biosynthesis and moulting. We also identified one mosquito NPC2 protein as a potential fatty-acid-binding protein. Through molecular modelling, we predicted the structures of the potential sterol- and fatty-acid-binding proteins and compared them to the reference proteins