How to Overcome a Snail? Identification of Putative Neurotoxins of Snail-Feeding Firefly Larvae (Coleoptera: Lampyridae, Lampyris noctiluca).

The larvae of some lampyrid beetles are highly specialized predators of snails. They have been observed to climb on the shells of their prey and use this exposed position to bite and inject secretions potentially originating from the midgut. Besides serving the purpose of extra-oral digestion (EOD), injected compounds also seem to have a paralyzing effect. Up to now, the toxins causing this paralyzing activity have not been identified. In the current study, we provide a first compositional analysis of the midgut secretion from lampyrid larvae, with a focus on identifying putative neurotoxins causing the observed paralyzing effect. For this purpose, we utilized a combined proteo-transcriptomic approach to characterize the compounds present in the midgut secretion of larval stages of Lampyris noctiluca. In terms of the absolute numbers of identified compounds, the midgut secretion is dominated by hydrolyzing enzymes comprising peptidases, carboxylesterases, and glycosidases. However, when considering expression levels, a few rather short cysteine-rich peptides exceed all other compounds. Some of these compounds show moderate similarity to putative neurotoxins identified in the venom of other arthropods and could be responsible for paralyzing effects. In addition to these potential toxins, we provide a list of peptides typical of the midgut secretion of L. noctiluca, supplemented by the corresponding precursor sequences.

Equipped for Sexual Stings? Male-Specific Venom Peptides in Euscorpius italicus.

In the animal kingdom, intraspecific variation occurs, for example, between populations, different life stages, and sexes. For venomous animals, this can involve differences in their venom composition. In cases where venom is utilized in the context of mating, the differences in composition might be driven by sexual selection. In this regard, the genus Euscorpius is a promising group for further research, as some of these scorpions exhibit a distinct sexual dimorphism and are known to perform a sexual sting during mating. However, the venom composition of this genus remains largely unexplored. Here, we demonstrate that Euscorpius italicus exhibits a male-specific venom composition, and we identify a large fraction of the substances involved. The sex specificity of venom peptides was first determined by analyzing the presence/absence patterns of ion signals in MALDI-TOF mass spectra of venom samples from both sexes and juveniles. Subsequently, a proteo-transcriptomic analysis provided sequence information on the relevant venom peptides and their corresponding precursors. As a result, we show that several potential toxin precursors are down-regulated in male venom glands, possibly to reduce toxic effects caused to females during the sexual sting. We have identified the precursor of one of the most prominent male-specific venom peptides, which may be an ideal candidate for activity tests in future studies. In addition to the description of male-specific features in the venom of E. italicus, this study also includes a general survey of venom precursors in this species.

Antimicrobial, Insecticidal and Cytotoxic Activity of Linear Venom Peptides from the Pseudoscorpion Chelifer cancroides.

Linear cationic venom peptides are antimicrobial peptides (AMPs) that exert their effects by damaging cell membranes. These peptides can be highly specific, and for some, a significant therapeutic value was proposed, in particular for treatment of bacterial infections. A prolific source of novel AMPs are arthropod venoms, especially those of hitherto neglected groups such as pseudoscorpions. In this study, we describe for the first time pharmacological effects of AMPs discovered in pseudoscorpion venom. We examined the antimicrobial, cytotoxic, and insecticidal activity of full-length Checacin1, a major component of the Chelifer cancroides venom, and three truncated forms of this peptide. The antimicrobial tests revealed a potent inhibitory activity of Checacin1 against several bacteria and fungi, including methicillin resistant Staphylococcus aureus (MRSA) and even Gram-negative pathogens. All peptides reduced survival rates of aphids, with Checacin1 and the C-terminally truncated Checacin11-21 exhibiting effects comparable to Spinosad, a commercially used pesticide. Cytotoxic effects on mammalian cells were observed mainly for the full-length Checacin1. All tested peptides might be potential candidates for developing lead structures for aphid pest treatment. However, as these peptides were not yet tested on other insects, aphid specificity has not been proven. The N- and C-terminal fragments of Checacin1 are less potent against aphids but exhibit no cytotoxicity on mammalian cells at the tested concentration of 100 µM.

Magnetic resonance imaging of cerebrospinal fluid outflow after low-rate lateral ventricle infusion in mice.

The anatomical routes for the clearance of cerebrospinal fluid (CSF) remain incompletely understood. However, recent evidence has given strong support for routes leading to lymphatic vessels. A current debate centers upon the routes through which CSF can access lymphatics, with evidence emerging for either direct routes to meningeal lymphatics or along cranial nerves to reach lymphatics outside the skull. Here, a method was established to infuse contrast agent into the ventricles using indwelling cannulae during imaging of mice at 2 and 12 months of age by magnetic resonance imaging. As expected, a substantial decline in overall CSF turnover was found with aging. Quantifications demonstrated that the bulk of the contrast agent flowed from the ventricles to the subarachnoid space in the basal cisterns. Comparatively little contrast agent signal was found at the dorsal aspect of the skull. The imaging dynamics from the 2 cohorts revealed that the contrast agent was cleared from the cranium through the cribriform plate to the nasopharyngeal lymphatics. On decalcified sections, we confirmed that fluorescently labeled ovalbumin drained through the cribriform plate and could be found within lymphatics surrounding the nasopharynx. In conclusion, routes leading to nasopharyngeal lymphatics appear to be a major efflux pathway for cranial CSF.

A Pseudoscorpion's Promising Pinch: The venom of Chelifer cancroides contains a rich source of novel compounds.

With pedipalps modified for venom injection, some pseudoscorpions possess a unique venom delivery system, which evolved independently from those of other arachnids like scorpions and spiders. Up to now, only a few studies have been focused on pseudoscorpion venom, which either identified a small fraction of venom compounds, or were based on solely transcriptomic approaches. Only one study addressed the bioactivity of pseudoscorpion venom. Here, we expand existing knowledge about pseudoscorpion venom by providing a comprehensive proteomic and transcriptomic analysis of the venom of Chelifer cancroides. We identified the first putative genuine toxins in the venom of C. cancroides and we showed that a large fraction of the venom comprises novel compounds. In addition, we tested the activity of the venom at specific ion channels for the first time. These tests demonstrate that the venom of C. cancroides causes inhibition of a voltage-gated insect potassium channel (Shaker IR) and modulates the inactivation process of voltage-gated sodium channels from Varroa destructor. For one of the smallest venomous animals ever studied, today's toolkits enabled a comprehensive venom analysis. This is demonstrated by allocating our identified venom compounds to more than half of the prominent ion signals in MALDI-TOF mass spectra of venom samples. The present study is a starting point for understanding the complex composition and activity of pseudoscorpion venom and provides a potential rich source of bioactive compounds useable for basic research and industrial application.

Substituting emergency services: primary care vs. hospital care.

Overcrowding in emergency departments (EDs) is inefficient, especially if it is caused by inappropriate visits for which primary care physicians could be equally effective as a hospital ED. Our paper investigates the extent to which both ambulatory ED visits and inpatient ED admissions are substitutes for primary care emergency services (PCES) in Germany. We use extensive longitudinal data and fixed effects models. Moreover, we add interaction terms to investigate the influence of various determinants on the strength of the substitution. Our results show significant substitution between PCES and ambulatory ED visits. Regarding the determinants, we find the largest substitution for younger patients. The more accessible the hospital ED is, the significantly larger the substitution. Moreover, substitution is larger among better-educated patients. For inpatient ED admission, we find significant substitution that is eight times smaller than the substitution for ambulatory ED visits. With regard to the determinants, we find the strongest substitution for non-urgent, short-stay admission and elderly patients. Countries with no gate-keeping system (such as Germany) have difficulties redirecting the patients streaming to EDs. Our estimated elasticities can help policy makers to resolve this issue, as our findings indicate where incentivizing the utilization of PCES is particularly effective.

Demand-side determinants of rising hospital admissions in Germany: the role of ageing.

In this study, we investigated the relationship between changes in demand-side determinants and changes in hospital admissions. We used longitudinal market-wide data, including a novel detailed measure of population morbidity. To assess the effect of ageing, we interacted age with shifts in the population structure for both the surviving population and the population in their last year of life. We used fixed effects models and addressed the endogeneity of morbidity with instrumental variables. We found that changes in morbidity had the largest impact on changes in hospital admissions. Changes in the size of the surviving population had the second largest impact, which differed substantially across the age spectrum. There was a large response in admissions to changes in the size of the population aged 60-79 years. The end-of-life effect had the smallest impact and began to play a greater role only in the population aged 80 years and older. In many studies, end of life presumably approximates high morbidity. Our results demonstrated robustness in several tests. We performed estimations in separate major diagnostic categories and included changes in personal preferences. We argue that the determinants included in our estimations capture the vast majority of change on the demand side. Taken together, our findings provide evidence that these determinants explain one-fifth of changes in hospital admissions.

Venom collection and analysis in the pseudoscorpion Chelifer cancroides (Pseudoscorpiones: Cheliferidae).

Pseudoscorpions are very small arthropods with almost worldwide distribution. They possess a unique venom delivery system in the chelal hands of their pedipalps that has evolved independently from that of scorpions and spiders. Studies on the venom composition of pseudoscorpions are very rare. Recently, the potential venom composition of the pseudoscorpion Synsphyronus apimelus Harvey, 1987 (Pseudoscorpiones: Garypidae) has been studied by transcriptome analysis. However, a proteome analysis of venom to identify the genuine venom compounds of pseudoscorpions has not yet been performed. In our study, we have developed a non-invasive approach for extracting minute amounts of venom, which for the first time allowed collecting pure venom samples of pseudoscorpions with minimal contaminations and high reproducibility. These experiments first required a morphological investigation of the venom delivery system with a focus on the role of the lamina defensor in the release of venom. Likely, the venom delivery system of pseudoscorpions has a mechanism that prevents the release of venom if the prey is not successfully penetrated by a venom tooth. Electrical stimulation of a gland-containing chelal hand in combination with a mechanical stimulation of the lamina defensor at the base of the venom tooth resulted in an average of 5 nl of collected venom. The utility of the method was then validated by repeated venom extractions and subsequent analysis of the venom composition using MALDI-TOF mass fingerprinting. Subsequent proteomics analysis in combination with transcriptome analyses of chelal hand tissue has identified the first genuine venom compounds of pseudoscorpions with putative antimicrobial peptides. For our experiments, we used the house pseudoscorpion Chelifer cancroides (Linnaeus, 1758) (Pseudoscorpiones: Cheliferidae).

Classification of hospital admissions into emergency and elective care: a machine learning approach.

Rising admissions from emergency departments (EDs) to hospitals are a primary concern for many healthcare systems. The issue of how to differentiate urgent admissions from non-urgent or even elective admissions is crucial. We aim to develop a model for classifying inpatient admissions based on a patient's primary diagnosis as either emergency care or elective care and predicting urgency as a numerical value. We use supervised machine learning techniques and train the model with physician-expert judgments. Our model is accurate (96%) and has a high area under the ROC curve (>.99). We provide the first comprehensive classification and urgency categorization for inpatient emergency and elective care. This model assigns urgency values to every relevant diagnosis in the ICD catalog, and these values are easily applicable to existing hospital data. Our findings may provide a basis for policy makers to create incentives for hospitals to reduce the number of inappropriate ED admissions.

Mode of action of closthioamide: the first member of the polythioamide class of bacterial DNA gyrase inhibitors.

OBJECTIVES: The spread of MDR bacteria represents a serious threat to human society and novel antibiotic drugs, preferably from new chemical classes, are urgently needed. Closthioamide was isolated from the strictly anaerobic bacterium Clostridium cellulolyticum and belongs to a new class of natural products, the polythioamides. Here, we investigated the antimicrobial activity and mechanism of action of closthioamide. METHODS: For assessing the antimicrobial activity of closthioamide, MIC values and killing kinetics were determined. To identify its target pathway, whole-cell-based assays were used including analysis of macromolecular synthesis and recording the susceptibility profile of a library of clones with down-regulated potential target genes. Subsequently, the inhibitory effect of closthioamide on the activity of isolated target enzymes, e.g. DNA gyrase and topoisomerase IV, was evaluated. RESULTS: Closthioamide had broad-spectrum activity against Gram-positive bacteria. Notably, closthioamide was very potent against MRSA and VRE strains. Closthioamide impaired DNA replication and inhibited DNA gyrase activity, in particular the ATPase function of gyrase and of topoisomerase IV, whereas there was little effect on the cleavage-rejoining function. Closthioamide also inhibited the relaxation activity of DNA gyrase, which does not require ATP hydrolysis, and thus may allosterically rather than directly interfere with the ATPase activity of gyrase. Cross-resistance to ciprofloxacin and novobiocin could not be detected in experimental mutants and clinical isolates. CONCLUSIONS: Closthioamide, a member of an unprecedented class of antibiotics, is a potent inhibitor of bacterial DNA gyrase; however, its molecular mechanism differs from that of the quinolones and aminocoumarins.