Comparative Genomics Reveals Insights into the Divergent Evolution of Astigmatic Mites and Household Pest Adaptations

Highly diversified astigmatic mites comprise many medically important human household pests such as house dust mites causing roughly 1–2% of the allergic diseases globally; however, their evolutionary origin, diverse lifestyles including reversible parasitism and quick adaptation to rather new human household environments have not been illustrated at genomic level, which hamper the allergy prevention and our exploration of these household pests. Using six high-quality assembled and annotated genomes, this comparative genomics study not only refuted the monophyly of mites and ticks, but also thoroughly explored the divergence of Acariformes and the divergent evolution of astigmatic mites. In the monophyletic Acariformes, Prostigmata known as notorious plant pests first evolved, then rapidly evolving Astigmata diverged from soil oribatid mites. Within astigmatic mites, a wide range of gene families rapidly expanded via tandem gene duplications, including ionotropic glutamate receptors, triacylglycerol lipases, serine proteases and UDP glucuronosyltransferases (UGTs), which enriched their capacities of adapting to rapidly changing household environments. The gene diversification after tandem duplications provided plenty of genetic resources for their adaptations of sensing environmental signals, digestion, and detoxification. Whilst many gene decay events only occurred in the skin-burrowing parasitic mite Sarcoptes scabiei. Throughout the evolution of Acariformes, massive horizontal gene transfer events occurred in gene families such as UGTs and several important fungal cell wall lytic enzymes, which enable the detoxification and associated digestive functions and provide perfect drug targets for pest control. Our comparative study sheds light on the rapid divergent evolution of astigmatic mites from the divergence of Acariformes to their diversification and provides novel insights into the genetic adaptations and even control of human household pests.

Fasciola hepatica is refractory to complement killing by preventing attachment of mannose binding lectin (MBL) and inhibiting MBL-associated serine proteases (MASPs) with serpins

The complement system is a first-line innate host immune defence against invading pathogens. It is activated via three pathways, termed Classical, Lectin and Alternative, which are mediated by antibodies, carbohydrate arrays or microbial liposaccharides, respectively. The three complement pathways converge in the formation of C3-convertase followed by the assembly of a lethal pore-like structure, the membrane attack complex (MAC), on the pathogen surface. We found that the infectious stage of the helminth parasite Fasciola hepatica, the newly excysted juvenile (NEJ), is resistant to the damaging effects of complement. Despite being coated with mannosylated proteins, the main initiator of the Lectin pathway, the mannose binding lectin (MBL), does not bind to the surface of live NEJ. In addition, we found that recombinantly expressed serine protease inhibitors secreted by NEJ (rFhSrp1 and rFhSrp2) selectively prevent activation of the complement via the Lectin pathway. Our experiments demonstrate that rFhSrp1 and rFhSrp2 inhibit native and recombinant MBL-associated serine proteases (MASPs), impairing the primary step that mediates C3b and C4b deposition on the NEJ surface. Indeed, immunofluorescence studies show that MBL, C3b, C4b or MAC are not deposited on the surface of NEJ incubated in normal human serum. Taken together, our findings uncover new means by which a helminth parasite prevents the activation of the Lectin complement pathway to become refractory to killing via this host response, in spite of presenting an assortment of glycans on their surface.

Sperm Storage Costs Determine Survival and Immunocompetence in Newly Mated Queens of the Leaf-Cutting Ant Atta colombica

In the leaf-cutting ant Atta colombica, queens receive ejaculates from multiple males during one single mating event early in their lives. A queen’s fertility and fitness therefore depend on maximizing the number of sperm cells she can store and maintain inside her spermatheca. Previous studies implied significant physiological mating costs, either originating from energetic investments maximizing sperm survival, or from resolving sexual conflicts to terminate male-driven incapacitation of rival sperm via serine proteases found in seminal fluid. Here we conducted an artificial insemination experiment, which allowed us to distinguish between the effects of sperm and seminal fluid within the queen’s sexual tract on her survival and immunocompetence. We found significantly higher mortality in queens that we had inseminated with sperm, independently of whether seminal fluid was present or not. Additionally, after receiving sperm, heavier queens had a higher probability of survival compared to lightweight queens, and immunocompetence decreased disproportionally for queens that had lost weight during the experiment. These findings indicate that queens pay significant physiological costs for maintaining and storing sperm shortly after mating. On the other hand, the presence of seminal fluid within the queens’ sexual tract neither affected their survival nor their immunocompetence. This suggests that the energetic costs that queens incur shortly after mating are primarily due to investments in sperm maintenance and not costs of terminating conflicts between competing ejaculates. This outcome is consistent with the idea that sexually selected traits in social insects with permanent castes can evolve only when they do not affect survival or life-time fitness of queens in any significant way.

Retrospective analysis of the role of Ulinastatin in reducing mortality in severe pancreatitis in critical care unit in Nepal

Background: Acute pancreatitis sequelae require a multidisciplinary approach and ICU care. Ulinastatin is a serine proteases inhibitor that reduces inflammation by suppressing the infiltration of neutrophils and elastase release and inflammatory mediators that help improve clinical symptoms and reduce mortality. This study aims to evaluate the clinical utility of Ulinastatin. Methods: Fifty-two patients admitted to ICU with acute pancreatitis were divided into; Ulinastatin group who received a 3 to 5 days course of 200,000IU, and Control Group who didn’t receive ulinastatin. Pearson's Chi-square and Fisher's exact test were used and a p-value < 0.05 was considered statistically significant. Results: Mean age was lower among the Ulinastatin group at 43 years (p-Value 0.014) and Hepatic dysfunction was more among this group (p-value 0.04). Among new onset of organ dysfunction, only CVS dysfunction was significant among the Control group ( p-value 0.044) while respiratory function recovery (p-value 0.04) and coagulation profile improvement (p-value 0.017) was statistically significant among the Ulinastatin group. The mean duration of hospital stay was shorter among control group, 9.65 days vs 14 days, a p-value of 0.05and also the average duration of stay in MDICU was lower, 4 days vs 8.5 days, p-value 0.0044 in comparison to Ulinastatin group. Overall mortality incidence was 15.38%, 19% in Ulinastatin group vs 11.5% in Control group. Conclusion: This retrospective study is our experience in the use of Ulinastatin which has shown little efficacy in declining mortality and/or hospital stay duration though it helps prevent new organ dysfunctions.

The Plasminogen–Activator Plasmin System in Physiological and Pathophysiological Angiogenesis

Angiogenesis is a process associated with the migration and proliferation of endothelial cells (EC) to form new blood vessels. It is involved in various physiological and pathophysiological conditions and is controlled by a wide range of proangiogenic and antiangiogenic molecules. The plasminogen activator–plasmin system plays a major role in the extracellular matrix remodeling process necessary for angiogenesis. Urokinase/tissue-type plasminogen activators (uPA/tPA) convert plasminogen into the active enzyme plasmin, which in turn activates matrix metalloproteinases and degrades the extracellular matrix releasing growth factors and proangiogenic molecules such as the vascular endothelial growth factor (VEGF-A). The plasminogen activator inhibitor-1 (PAI-1) is the main inhibitor of uPA and tPA, thereby an inhibitor of pericellular proteolysis and intravascular fibrinolysis, respectively. Paradoxically, PAI-1, which is expressed by EC during angiogenesis, is elevated in several cancers and is found to promote angiogenesis by regulating plasmin-mediated proteolysis and by promoting cellular migration through vitronectin. The urokinase-type plasminogen activator receptor (uPAR) also induces EC cellular migration during angiogenesis via interacting with signaling partners. Understanding the molecular functions of the plasminogen activator plasmin system and targeting angiogenesis via blocking serine proteases or their interactions with other molecules is one of the major therapeutic strategies scientists have been attracted to in controlling tumor growth and other pathological conditions characterized by neovascularization.

De Novo Transcriptome Analysis of the Venom of Latrodectus geometricus with the Discovery of an Insect-Selective Na Channel Modulator

The brown widow spider, Latrodectus geometricus, is a predator of a variety of agricultural insects and is also hazardous for humans. Its venom is a true pharmacopeia representing neurotoxic peptides targeting the ion channels and/or receptors of both vertebrates and invertebrates. The lack of transcriptomic information, however, limits our knowledge of the diversity of components present in its venom. The purpose of this study was two-fold: (1) carry out a transcriptomic analysis of the venom, and (2) investigate the bioactivity of the venom using an electrophysiological bioassay. From 32,505 assembled transcripts, 8 toxin families were classified, and the ankyrin repeats (ANK), agatoxin, centipede toxin, ctenitoxin, lycotoxin, scorpion toxin-like, and SCP families were reported in the L. geometricus venom gland. The diversity of L. geometricus venom was also uncovered by the transcriptomics approach with the presence of defensins, chitinases, translationally controlled tumor proteins (TCTPs), leucine-rich proteins, serine proteases, and other important venom components. The venom was also chromatographically purified, and the activity contained in the fractions was investigated using an electrophysiological bioassay with the use of a voltage clamp on ion channels in order to find if the neurotoxic effects of the spider venom could be linked to a particular molecular target. The findings show that U24-ctenitoxin-Pn1a involves the inhibition of the insect sodium (Nav) channels, BgNav and DmNav. This study provides an overview of the molecular diversity of L. geometricus venom, which can be used as a reference for the venom of other spider species. The venom composition profile also increases our knowledge for the development of novel insecticides targeting voltage-gated sodium channels.

Adenovirus Co-Opts Neutrophilic Inflammation to Enhance Transduction of Epithelial Cells

Human adenoviruses (HAdV) cause a variety of infections in human hosts, from self-limited upper respiratory tract infections in otherwise healthy people to fulminant pneumonia and death in immunocompromised patients. Many HAdV enter polarized epithelial cells by using the primary receptor, the Coxsackievirus and adenovirus receptor (CAR). Recently published data demonstrate that a potent neutrophil (PMN) chemoattractant, interleukin-8 (IL-8), stimulates airway epithelial cells to increase expression of the apical isoform of CAR (CAREx8), which results in increased epithelial HAdV type 5 (HAdV5) infection. However, the mechanism for PMN-enhanced epithelial HAdV5 transduction remains unclear. In this manuscript, the molecular mechanisms behind PMN mediated enhancement of epithelial HAdV5 transduction are characterized using an MDCK cell line that stably expresses human CAREx8 under a doxycycline inducible promoter (MDCK-CAREx8 cells). Contrary to our hypothesis, PMN exposure does not enhance HAdV5 entry by increasing CAREx8 expression nor through activation of non-specific epithelial endocytic pathways. Instead, PMN serine proteases are responsible for PMN-mediated enhancement of HAdV5 transduction in MDCK-CAREx8 cells. This is evidenced by reduced transduction upon inhibition of PMN serine proteases and increased transduction upon exposure to exogenous human neutrophil elastase (HNE). Furthermore, HNE exposure activates epithelial autophagic flux, which, even when triggered through other mechanisms, results in a similar enhancement of epithelial HAdV5 transduction. Inhibition of F-actin with cytochalasin D partially attenuates PMN mediated enhancement of HAdV transduction. Taken together, these findings suggest that HAdV5 can leverage innate immune responses to establish infections.

Targeting Proteinase Activated Receptor-4 Reduces Mechanonociception During the Acute Inflammatory Phase but not the Chronic Neuropathic Phase of Osteoarthritis in Rats

Serine proteases are elevated in arthritic joints where they can cleave protease activated receptors (PARs) to modulate pain and inflammation. Activation of protease-activated receptor 4 (PAR4) has been implicated in inflammatory joint pain. Whether PAR4 is involved in osteoarthritis (OA) pain has not yet been explored. The aim of this study was to compare the role of PAR4 in modulating early versus late stage OA pain using two models of OA viz. monoiodoacetate (MIA) and medial meniscal transection (MMT). G-ratio calculation and electron microscopy analysis revealed saphenous nerve demyelination and structural damage during late stage but not early OA in both models. Using immunohistochemistry, neuronal expression of PAR4 was higher in early versus late OA. Systemic administration of the PAR4 antagonist pepducin P4pal10 reduced both secondary allodynia (von Frey hair algesiometry) and joint nociceptor firing (single unit recordings) in MMT and MIA animals compared to vehicle-treated animals in early OA. The PAR4 antagonist was ineffective at altering pain or joint afferent firing in post-inflammatory OA. During the acute phase of the models, joint inflammation as determined by laser speckle contrast analysis and intravital microscopy could be partially blocked by pepducin P4pal10. Compared to late-stage disease, inflammatory cytokines were elevated in early MIA and MMT rats. These findings suggest that PAR4 may be a viable target to treat the pain of early onset OA or during episodic inflammatory flares.

A rare CTSC mutation in Papillon-Lefèvre Syndrome results in abolished serine protease activity and reduced NET formation but otherwise normal neutrophil function

Papillon-Lefèvre Syndrome (PLS) is an autosomal recessive monogenic disease caused by loss-of-function mutations in the CTSC gene, thus preventing the synthesis of the protease Cathepsin C (CTSC) in a proteolytically active form. CTSC is responsible for the activation of the pro-forms of the neutrophil serine proteases (NSPs; Elastase, Proteinase 3 and Cathepsin G), suggesting its involvement in a variety of neutrophil functions. In PLS neutrophils, the lack of CTSC protease activity leads to inactivity of the NSPs. Clinically, PLS is characterized by an early, typically pre-pubertal, onset of severe periodontal pathology and palmoplantar hyperkeratosis. However, PLS is not considered an immune deficiency as patients do not typically suffer from recurrent and severe (bacterial and fungal) infections. In this study we investigated an unusual CTSC mutation in two siblings with PLS, a 503A>G substitution in exon 4 of the CTSC gene, expected to result in an amino acid replacement from tyrosine to cysteine at position 168 of the CTSC protein. Both patients bearing this mutation presented with pronounced periodontal pathology. The characteristics and functions of neutrophils from patients homozygous for the 503A>G CTSC mutation were compared to another previously described PLS mutation (755A>T), and a small cohort of healthy volunteers. Neutrophil lysates from patients with the 503A>G substitution lacked CTSC protein and did not display any CTSC or NSP activity, yet neutrophil counts, morphology, priming, chemotaxis, radical production, and regulation of apoptosis were without any overt signs of alteration. However, NET formation upon PMA-stimulation was found to be severely depressed, but not abolished, in PLS neutrophils.