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Abstract The GDS(L)-like lipase from the Basidiomycota Pleurotus sapidus (PSA_Lip) was heterologously expressed using Trichoderma reesei with an activity of 350 U L−1. The isoelectric point of 5.0 was determined by isoelectric focusing. The novel PSA_Lip showed only 23.8–25.1%, 25.5%, 26.6% and 28.4% identity to the previously characterized GDSL-like enzymes phospholipase, plant lipase, acetylcholinesterase and acetylxylan esterase, from the carbohydrate esterase family 16, respectively. Therefore, the enzyme was purified from the culture supernatant and the catalytic properties and the substrate specificity of the enzyme were investigated using different assays to reveal its potential function. While no phospholipase, acetylcholinesterase and acetylxylan esterase activities were detected, studies on the hydrolysis of ferulic acid methyl ester (~ 8.3%) and feruloylated carbohydrate 5-O-transferuloyl-arabino-furanose (~ 0.8%) showed low conversions of these substrates. By investigating the hydrolytic activity towards p-nitrophenyl-(pNP)-esters with various chain-lengths, the highest activity was determined for medium chain-length pNP-octanoate at 65 °C and a pH value of 8, while almost no activity was detected for pNP-hexanoate. The enzyme is highly stable when stored at pH 10 and 4 °C for at least 7 days. Moreover, using consensus sequence analysis and homology modeling, we could demonstrate that the PSA_Lip does not contain the usual SGNH residues in the actives site, which are usually present in GDS(L)-like enzymes.

Summary: Macrophage migration inhibitory factor (MIF) is a pleiotropic protein with chemotactic, pro-inflammatory, and growth-promoting activities first discovered in mammals. In parasites, MIF homologs are involved in immune evasion and pathogenesis. Here, we present the first comprehensive analysis of an MIF protein from the devastating plant pathogen Magnaporthe oryzae (Mo). The fungal genome encodes a single MIF protein (MoMIF1) that, unlike the human homolog, harbors multiple low-complexity regions (LCRs) and is unique to Ascomycota. Following infection, MoMIF1 is expressed in the biotrophic phase of the fungus, and is strongly down-regulated during subsequent necrotrophic growth in leaves and roots. We show that MoMIF1 is secreted during plant infection, affects the production of the mycotoxin tenuazonic acid and inhibits plant cell death. Our results suggest that MoMIF1 is a novel key regulator of fungal virulence that maintains the balance between biotrophy and necrotrophy during the different phases of fungal infection.

Abstract Background Snake envenomation is a major neglected tropical disease, lacking data in many countries including Cyprus, a Mediterranean island inhabited by the medically important blunt-nosed viper (Macrovipera lebetina). Reviewing the 2013–2019 period, we present first-time epidemiological snakebite data in the Republic of Cyprus. Methods We obtained data on snake envenomation-related hospital admissions from the Ministry of Health, and population and rainfall data from the Statistical Service of Cyprus and Department of Meteorology websites. Human-viper conflict information was acquired from interviews with 12 representatives of Cypriot institutions. Results Between 2013 and 2019, 288 snake envenomation cases were admitted to public hospitals, averaging 41 people annually. The minimum was 29 cases (2017) and the maximum was 58 (2015). Snake envenomation incidence increased from 4.55 per 100,000 population (2013) to 6.84 (2015), but remained low since 2017 (3.49 in 2019). Between 2000 and 2018, the deaths of one man (73 years), and indirectly, one woman (77 years), were related to snake envenomation. While 266 cases (92%) happened between April and October (the blunt-nosed viper activity period), most envenomations occurred in September (cumulative for 2013–2019), with 88 cases (31%). Snakebite incidence peaked in the 60–69 years age group (9.19 per 100,000 population), and was higher in males (6.85) than in females (2.82). Of all admitted patients, 242 (84%) were discharged within 4 days. Mean hospital stay duration was 2.65 days, with one case of 13 days. Most patients were admitted to the general hospitals in Paphos (51%), Limassol (30%) and Nicosia (11%), which provide secondary healthcare, with the last one providing tertiary healthcare. Conclusions Snakebite-related deaths are very rare in the Republic of Cyprus. Most envenomation cases happened in late summer (September). Short hospital stays indicate mostly non-severe clinical courses. The hospital admission data suggest that snake envenomation risk is highest in Paphos district. The statistical data hint at males and middle- to older-aged people being at highest risk, whereas from our interview data we assume that outdoor workers are at higher risk than other occupational groups.

Abstract Background The explosive radiation and diversification of the advanced snakes (superfamily Colubroidea) was associated with changes in all aspects of the shared venom system. Morphological changes included the partitioning of the mixed ancestral glands into two discrete glands devoted for production of venom or mucous respectively, as well as changes in the location, size and structural elements of the venom-delivering teeth. Evidence also exists for homology among venom gland toxins expressed across the advanced snakes. However, despite the evolutionary novelty of snake venoms, in-depth toxin molecular evolutionary history reconstructions have been mostly limited to those types present in only two front-fanged snake families, Elapidae and Viperidae. To have a broader understanding of toxins shared among extant snakes, here we first sequenced the transcriptomes of eight taxonomically diverse rear-fanged species and four key viperid species and analysed major toxin types shared across the advanced snakes. Results Transcriptomes were constructed for the following families and species: Colubridae - Helicops leopardinus, Heterodon nasicus, Rhabdophis subminiatus; Homalopsidae – Homalopsis buccata; Lamprophiidae - Malpolon monspessulanus, Psammophis schokari, Psammophis subtaeniatus, Rhamphiophis oxyrhynchus; and Viperidae – Bitis atropos, Pseudocerastes urarachnoides, Tropidolaeumus subannulatus, Vipera transcaucasiana. These sequences were combined with those from available databases of other species in order to facilitate a robust reconstruction of the molecular evolutionary history of the key toxin classes present in the venom of the last common ancestor of the advanced snakes, and thus present across the full diversity of colubroid snake venoms. In addition to differential rates of evolution in toxin classes between the snake lineages, these analyses revealed multiple instances of previously unknown instances of structural and functional convergences. Structural convergences included: the evolution of new cysteines to form heteromeric complexes, such as within kunitz peptides (the beta-bungarotoxin trait evolving on at least two occasions) and within SVMP enzymes (the P-IIId trait evolving on at least three occasions); and the C-terminal tail evolving on two separate occasions within the C-type natriuretic peptides, to create structural and functional analogues of the ANP/BNP tailed condition. Also shown was that the de novo evolution of new post-translationally liberated toxin families within the natriuretic peptide gene propeptide region occurred on at least five occasions, with novel functions ranging from induction of hypotension to post-synaptic neurotoxicity. Functional convergences included the following: multiple occasions of SVMP neofunctionalised in procoagulant venoms into activators of the clotting factors prothrombin and Factor X; multiple instances in procoagulant venoms where kunitz peptides were neofunctionalised into inhibitors of the clot destroying enzyme plasmin, thereby prolonging the half-life of the clots formed by the clotting activating enzymatic toxins; and multiple occasions of kunitz peptides neofunctionalised into neurotoxins acting on presynaptic targets, including twice just within Bungarus venoms. Conclusions We found novel convergences in both structural and functional evolution of snake toxins. These results provide a detailed roadmap for future work to elucidate predator–prey evolutionary arms races, ascertain differential clinical pathologies, as well as documenting rich biodiscovery resources for lead compounds in the drug design and discovery pipeline.

Cryptosporidium parvum is a zoonotic-relevant parasite belonging to the phylum Alveolata (subphylum Apicomplexa). One of the most zoonotic-relevant etiologies of cryptosporidiosis is the species C. parvum, infecting humans, cattle and wildlife. C. parvum-infected intestinal mucosa as well as host cells infected in vitro have not yet been the subject of extensive biochemical investigation. Efficient treatment options or vaccines against cryptosporidiosis are currently not available. Human cryptosporidiosis is currently known as a neglected poverty-related disease (PRD), being potentially fatal in young children or immunocompromised patients. In this study, we used a combination of atmospheric pressure scanning microprobe matrix-assisted laser desorption/ionization (AP-SMALDI) mass spectrometry imaging (MSI) and liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) to determine and locate molecular biomarkers in in vitro C. parvum-infected host cells as well as parasitized neonatal calf intestines. Sections of C. parvum-infected and non-infected host cell pellets and infected intestines were examined to determine potential biomarkers. Human ileocecal adenocarcinoma cells (HCT-8) were used as a suitable in vitro host cell system. More than a thousand different molecular signals were found in both positive- and negative-ion mode, which were significantly increased in C. parvum-infected material. A database search in combination with HPLC-MS/MS experiments was employed for the structural verification of markers. Our results demonstrate some overlap between the identified markers and data obtained from earlier studies on other apicomplexan parasites. Statistically relevant biomarkers were imaged in cell layers of C. parvum-infected and non-infected host cells with 5 µm pixel size and in bovine intestinal tissue with 10 µm pixel size. This allowed us to substantiate their relevance once again. Taken together, the present approach delivers novel metabolic insights on neglected cryptosporidiosis affecting mainly children in developing countries.

The comprehensive identification of the proteome content from a white wine (cv. Silvaner) is described here for the first time. The wine protein composition isolated from a representative wine sample (250 L) was identified via mass spectrometry (MS)-based proteomics following in-solution and in-gel digestion methods after being submitted to size exclusion chromatographic (SEC) fractionation to gain a comprehensive insight into proteins that survive the vinification processes. In total, we identified 154 characterized (with described functional information) or so far uncharacterized proteins, mainly from Vitis vinifera L. and Saccharomyces cerevisiae. With the complementarity of the two-step purification, the digestion techniques and the high-resolution (HR)-MS analyses provided a high-score identification of proteins from low to high abundance. These proteins can be valuable for future authentication of wines by tracing proteins derived from a specific cultivar or winemaking process. The proteomics approach presented herein may also be generally helpful to understand which proteins are important for the organoleptic properties and stability of wines.

Thermolabile grape berry proteins such as thaumatin-like proteins (TLPs) and chitinases (CHIs) promote haze formation in bottled wines if not properly fined. As a natural grapevine pest, the spotted-wing fly Drosophila suzukii is a promising source of peptidases that break down grape berry proteins because the larvae develop and feed inside mature berries. Therefore, we produced recombinant TLP and CHI as model thermolabile wine haze proteins and applied a peptidomics strategy to investigate whether D. suzukii larval peptidases were able to digest them under acidic conditions (pH 3.5), which are typically found in winemaking practices. The activity of the novel peptidases was confirmed by mass spectrometry, and cleavage sites within the wine haze proteins were visualized in 3D protein models. The combination of recombinant haze proteins and peptidomics provides a valuable screening tool to identify optimal peptidases suitable for clarification processes in the winemaking industry.

Envenoming by Macrovipera lebetina subspecies causes severe life-threatening difficulties for people living in North Africa and the Middle East. To better understand the pathophysiology of envenoming and improve patient management, knowledge about the venom components of the subspecies is essential. Here, the venom proteomes of Macrovipera lebetina lebetina from Cyprus and Macrovipera lebetina cernovi from Iran were characterized using RP-HPLC separation of the crude venom proteins, SDS-PAGE of fractionated proteins, and LC-MS/MS of peptides obtained from in-gel tryptic digestion of protein bands. Moreover, we also used high-resolution shot-gun proteomics to gain more reliable identification, where the whole venom proteomes were subjected directly to in-solution digestion before LC-HR-MS/MS. The data revealed that both venoms consisted of at least 18 protein families, of which snake venom Zn2+-dependent metalloprotease (SVMP), serine protease, disintegrin, phospholipase A2, C-type lectin-like, and L-amino acid oxidase, together accounted for more than 80% of the venoms’ protein contents. Although the two viper venoms shared mostly similar protein classes, the relative occurrences of these toxins were different in each snake subspecies. For instance, P-I class of SVMP toxins were found to be more abundant than P-III class in the venoms of M. l. cernovi compared to M. l. lebetina, which gives hints at a more potent myonecrotic effect and minor systemic hemorrhage following envenoming by M. l. cernovi than M. l. lebetina. Moreover, single-shot proteomics also revealed many proteins with low abundance (M. lebetina subspecies, particularly regarding toxin families associated with envenoming pathogenesis and those hard-detected protein classes expressed in trace amounts.

Cross-linking net aggregates of thermolabile thaumatin-like proteins (TLPs) and chitinases (CHIs) are the primary source of haze in white wines. Although bentonite fining is still routinely used in winemaking, alternative methods to selectively remove haze proteins without affecting wine organoleptic properties are needed. The availability of pure TLPs and CHIs would facilitate the research for the identification of such technological advances. Therefore, we proposed the usage of recombinant TLP (rTLP) and CHI (rCHI), expressed by Komagataella phaffii, as haze-protein models, since they showed similar characteristics (aggregation potential, melting point, functionality, glycosylation levels and bentonite adsorption) to the native-haze proteins from Vitis vinifera. Hence, rTLP and rCHI can be applied to study haze formation mechanisms on a molecular level and to explore alternative fining methods by screening proteolytic enzymes and ideal adsorptive resins.

Scorpion venom is a complex secretory mixture of components with potential biological and physiological properties that attracted many researchers due to promising applications from clinical and pharmacological perspectives. In this study, we investigated the venom of the Iranian scorpion Hottentotta saulcyi (Simon, 1880) by applying mass-spectrometry-based proteomic and lipidomic approaches to assess the diversity of components present in the venom. The data revealed that the venom’s proteome composition is largely dominated by Na+- and K+-channel-impairing toxic peptides, following the enzymatic and non-enzymatic protein families, e.g., angiotensin-converting enzyme, serine protease, metalloprotease, hyaluronidase, carboxypeptidase, and cysteine-rich secretory peptide. Furthermore, lipids comprise ~1.2% of the dry weight of the crude venom. Phospholipids, ether-phospholipids, oxidized-phospholipids, triacylglycerol, cardiolipins, very-long-chain sphingomyelins, and ceramides were the most intensely detected lipid species in the scorpion venom, may acting either independently or synergistically during the envenomation alongside proteins and peptides. The results provide detailed information on the chemical makeup of the venom, helping to improve our understanding of biological molecules present in it, leading to a better insight of the medical significance of the venom, and improving the medical care of patients suffering from scorpion accidents in the relevant regions such as Iran, Iraq, Turkey, and Afghanistan.

Venoms are a rich source of potential lead compounds for drug discovery, and descriptive studies of venom form the first phase of the biodiscovery process. In this study, we investigated the pharmacological potential of crude Pseudocerastes and Eristicophis snake venoms in haematological disorders and cancer treatment. We assessed their antithrombotic potential using fibrinogen thromboelastography, fibrinogen gels with and without protease inhibitors, and colourimetric fibrinolysis assays. These assays indicated that the anticoagulant properties of the venoms are likely induced by the hydrolysis of phospholipids and by selective fibrinogenolysis. Furthermore, while most fibrinogenolysis occurred by the direct activity of snake venom metalloproteases and serine proteases, modest evidence indicated that fibrinogenolytic activity may also be mediated by selective venom phospholipases and an inhibitory venom-derived serine protease. We also found that the Pseudocerastes venoms significantly reduced the viability of human melanoma (MM96L) cells by more than 80%, while it had almost no effect on the healthy neonatal foreskin fibroblasts (NFF) as determined by viability assays. The bioactive properties of these venoms suggest that they contain a number of toxins suitable for downstream pharmacological development as candidates for antithrombotic or anticancer agents.

Snakes of the genera Pseudocerastes and Eristicophis (Viperidae: Viperinae) are known as the desert vipers due to their association with the arid environments of the Middle East. These species have received limited research attention and little is known about their venom or ecology. In this study, a comprehensive analysis of desert viper venoms was conducted by visualising the venom proteomes via gel electrophoresis and assessing the crude venoms for their cytotoxic, haemotoxic, and neurotoxic properties. Plasmas sourced from human, toad, and chicken were used as models to assess possible prey-linked venom activity. The venoms demonstrated substantial divergence in composition and bioactivity across all experiments. Pseudocerastes urarachnoides venom activated human coagulation factors X and prothrombin and demonstrated potent procoagulant activity in human, toad, and chicken plasmas, in stark contrast to the potent neurotoxic venom of P. fieldi. The venom of E. macmahonii also induced coagulation, though this did not appear to be via the activation of factor X or prothrombin. The coagulant properties of P. fieldi and P. persicus venoms varied among plasmas, demonstrating strong anticoagulant activity in the amphibian and human plasmas but no significant effect in that of bird. This is conjectured to reflect prey-specific toxin activity, though further ecological studies are required to confirm any dietary associations. This study reinforces the notion that phylogenetic relatedness of snakes cannot readily predict venom protein composition or function. The significant venom variation between these species raises serious concerns regarding antivenom paraspecificity. Future assessment of antivenom is crucial.

The Persian Gulf is known as the westernmost distribution limit for sea snakes, except for Hydrophis platurus (Linnaeus, 1766) that reaches southeastern Africa. Previous identification guides for sea snakes of the Persian Gulf and its adjacent waters in the Gulf of Oman were based on old data and confined mostly to written descriptions. Therefore, a series of field surveys were carried out in 2013 and 2014 through Iranian coastal waters of both gulfs to provide a comprehensive sampling of sea snakes in the area. This paper presents an illustrated and updated checklist and identification tool for sea snakes in the Persian Gulf and Gulf of Oman, which are based on new material and a review of the literature. This checklist includes ten species of marine hydrophiines, of which one, Microcephalophis cantoris (Günther, 1864), is a new record for the area. All specimens examined herein are deposited and available at the Zoological Museum of Shahid Bahonar University of Kerman, Kerman province, Iran.