Leishmania genetic exchange is mediated by IgM natural antibodies.

Host factors that mediate Leishmania genetic exchange are not well defined. Here we demonstrate that natural IgM (IgMn)1-4 antibodies mediate parasite genetic exchange by inducing the transient formation of a spherical parasite clump that promotes parasite fusion and hybrid formation. We establish that IgMn from Leishmania-free animals binds to the surface of Leishmania parasites to induce significant changes in the expression of parasite transcripts and proteins. Leishmania binding to IgMn is partially lost after glycosidase treatment, although parasite surface phosphoglycans, including lipophosphoglycan, are not required for IgMn-induced parasite clumping. Notably, the transient formation of parasite clumps is essential for Leishmania hybridization in vitro. In vivo, we observed a 12-fold increase in hybrid formation in sand flies provided a second blood meal containing IgMn compared with controls. Furthermore, the generation of recombinant progeny from mating hybrids and parental lines were only observed in sand flies provided with IgMn. Both in vitro and in vivo IgM-induced Leishmania crosses resulted in full genome hybrids that show equal patterns of biparental contribution. Leishmania co-option of a host natural antibody to facilitate mating in the insect vector establishes a new paradigm of parasite-host-vector interdependence that contributes to parasite diversity and fitness by promoting genetic exchange.

A bispecific inhibitor of complement and coagulation blocks activation in complementopathy models via a novel mechanism.

Inhibitors of complement and coagulation are present in the saliva of a variety of blood-feeding arthropods that transmit parasitic and viral pathogens. Here, we describe the structure and mechanism of action of the sand fly salivary protein lufaxin, which inhibits the formation of the central alternative C3 convertase (C3bBb) and inhibits coagulation factor Xa (fXa). Surface plasmon resonance experiments show that lufaxin stabilizes the binding of serine protease factor B (FB) to C3b but does not detectably bind either C3b or FB alone. The crystal structure of the inhibitor reveals a novel all ß-sheet fold containing 2 domains. A structure of the lufaxin-C3bB complex obtained via cryo-electron microscopy (EM) shows that lufaxin binds via its N-terminal domain at an interface containing elements of both C3b and FB. By occupying this spot, the inhibitor locks FB into a closed conformation in which proteolytic activation of FB by FD cannot occur. C3bB-bound lufaxin binds fXa at a separate site in its C-terminal domain. In the cryo-EM structure of a C3bB-lufaxin-fXa complex, the inhibitor binds to both targets simultaneously, and lufaxin inhibits fXa through substrate-like binding of a C-terminal peptide at the active site as well as other interactions in this region. Lufaxin inhibits complement activation in ex vivo models of atypical hemolytic uremic syndrome (aHUS) and paroxysmal nocturnal hemoglobinuria (PNH) as well as thrombin generation in plasma, providing a rationale for the development of a bispecific inhibitor to treat complement-related diseases in which thrombosis is a prominent manifestation.

Comprehensive Proteomics Analysis of the Hemolymph Composition of Sugar-Fed Aedes aegypti Female and Male Mosquitoes.

In arthropods, hemolymph carries immune cells and solubilizes and transports nutrients, hormones, and other molecules that are involved in diverse physiological processes including immunity, metabolism, and reproduction. However, despite such physiological importance, little is known about its composition. We applied mass spectrometry-based label-free quantification approaches to study the proteome of hemolymph perfused from sugar-fed female and male Aedes aegypti mosquitoes. A total of 1403 proteins were identified, out of which 447 of them were predicted to be extracellular. In both sexes, almost half of these extracellular proteins were predicted to be involved in defense/immune response, and their relative abundances (based on their intensity-based absolute quantification, iBAQ) were 37.9 and 33.2%, respectively. Interestingly, among them, 102 serine proteases/serine protease-homologues were identified, with almost half of them containing CLIP regulatory domains. Moreover, proteins belonging to families classically described as chemoreceptors, such as odorant-binding proteins (OBPs) and chemosensory proteins (CSPs), were also highly abundant in the hemolymph of both sexes. Our data provide a comprehensive catalogue of A. aegypti hemolymph basal protein content, revealing numerous unexplored targets for future research on mosquito physiology and disease transmission. It also provides a reference for future studies on the effect of blood meal and infection on hemolymph composition.

Exploring the longitudinal expression dynamics of midguts in adult female Amblyomma americanum ticks.

BACKGROUND: Female ticks remain attached to their host for multiple days to complete a blood meal. This prolonged feeding period is accompanied by a significant increase in the tick's size and body weight, paralleled by noteworthy changes to the tick midgut. While the midgut is recognized for its established role in blood storage and processing, its importance extends to playing a crucial role in the acquisition, survival, and proliferation of pathogens. Despite this, our overall understanding of tick midgut biology is limited. RESULTS: Our transcriptome analysis identified 15,599 putative DNA coding sequences (CDS), which were classified into 26 functional groups. Dimensional and differential expression analyses revealed four primary transcriptional profiles corresponding to unfed, slow-feeding, transitory (from slow- to rapid-feeding), and rapid-feeding stages. Additionally, comparing the current dataset with previously deposited transcriptome from other tick species allowed the identification of commonly expressed transcripts across different feeding stages. CONCLUSION: Our findings provide a detailed temporal resolution of numerous metabolic pathways in the midgut of A. americanum adult females throughout the feeding process, highlighting the dynamic transcriptional regulation of the tick's midgut as feeding progresses. Furthermore, we identified conserved transcripts across three different tick species that exhibit similar expression patterns. This knowledge not only enhances our understanding of the physiological processes within the tick midgut but also opens up potential avenues for developing control methods that target multiple tick species.

Distinctive Amplitude-Integrated EEG Ictal Pattern and Targeted Therapy with Carbamazepine in KCNQ2 and KCNQ3 Neonatal Epilepsy: A Case Series.

BACKGROUND: Carbamazepine (CBZ) is effective in treating KCNQ2/3-related seizures, which may present with a distinctive amplitude-integrated electroencephalography (aEEG) pattern. OBJECTIVE: To assess how improved recognition of the distinctive aEEG ictal pattern associated with KCNQ2/3 variants has enabled early and effective targeted therapy with CBZ. METHODS: Retrospective descriptive study of five neonates with KCNQ2/3 pathogenic gene variants admitted at a level 3 neonatal intensive care unit (NICU) over an 8-year period. RESULTS: The distinctive ictal aEEG pattern was recognized in four neonates after an average of 61.5 hours (minimum 12 hours, maximum 120 hours) from the first electroclinical seizure and prompted the use of CBZ that was effective in all. The two most recently diagnosed patients could avoid polytherapy as they received CBZ as the first and second antiseizure medication, respectively. Three out of five patients with continuous normal voltage (CNV), sleep-wake cycling (SWC), and shorter postictal suppression had normal neurodevelopmental outcome. Regarding the remaining two infants, one was not trialed with CBZ and had a high seizure burden, both presented with a prolonged postictal suppression, no SWC, and had moderate-to-severe developmental delay. Genetic results became available after the neonatal period in all but one of the infants, who had a prenatal diagnosis. CONCLUSION: Recognition of the distinctive ictal aEEG pattern in the NICU allowed early and effective targeted therapy with CBZ in four neonates, well before genetic results became available. Furthermore, a CNV background pattern with SWC and short postictal suppression were associated with normal developmental outcomes.

Development and validation of a stability-indicating method, structural elucidation of new degradation products from misoprostol by LC-MS time-of-flight, and an ex vivo study of vaginal permeation.

Misoprostol (MSP) is commonly prescribed in obstetrics and gynecology clinical practice for labor induction, cervical ripening, first-trimester pregnancy termination, and the treatment of postpartum hemorrhage. Furthermore, there is a lack of comprehensive discussion evaluating how different commercially available formulations influence the overall efficacy of MSP, even though reports indicate issues with the quality of these formulations, particularly regarding stability and vaginal absorption processes. This study investigates the stability of MSP under acidic conditions and its in vitro permeation using swine vaginal mucosa. A forced degradation study was conducted using 0.2 M HCl, and a high-efficiency LC method was developed. Three degradation products were identified and characterized using electrospray ionization-high-resolution quadrupole-time-of-flight-MS, with respective m/z values of 391.2508, 405.2705, and 387.2259, respectively. These results suggest that the degradation mechanism involves dehydration of the ß-hydroxy ketone moiety, followed by isomerization to its most resonance-stable form and de-esterification. Finally, the in vitro permeation study revealed that the esterified form of MSP was unable to permeate the mucosa and required prior degradation for any component to be detected in the receptor fluid.

Systematic Review of Emotion Detection with Computer Vision and Deep Learning.

Emotion recognition has become increasingly important in the field of Deep Learning (DL) and computer vision due to its broad applicability by using human-computer interaction (HCI) in areas such as psychology, healthcare, and entertainment. In this paper, we conduct a systematic review of facial and pose emotion recognition using DL and computer vision, analyzing and evaluating 77 papers from different sources under Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Our review covers several topics, including the scope and purpose of the studies, the methods employed, and the used datasets. The scope of this work is to conduct a systematic review of facial and pose emotion recognition using DL methods and computer vision. The studies were categorized based on a proposed taxonomy that describes the type of expressions used for emotion detection, the testing environment, the currently relevant DL methods, and the datasets used. The taxonomy of methods in our review includes Convolutional Neural Network (CNN), Faster Region-based Convolutional Neural Network (R-CNN), Vision Transformer (ViT), and "Other NNs", which are the most commonly used models in the analyzed studies, indicating their trendiness in the field. Hybrid and augmented models are not explicitly categorized within this taxonomy, but they are still important to the field. This review offers an understanding of state-of-the-art computer vision algorithms and datasets for emotion recognition through facial expressions and body poses, allowing researchers to understand its fundamental components and trends.

Ten years of directing seeding restoration in the Brazilian savanna: Lessons learned and the way forward.

Savannas and grasslands have lost almost 50% of their original cover worldwide. Therefore, the development of methods and information on open-canopy ecosystem restoration is urgent for the inclusion of these ecosystems into global and regional priorities. In the Brazilian savanna, the most diverse savanna in the world, restoration efforts focused on open ecosystems have been virtually absent, but have increased in the last 10 years. Such efforts are frequently threatened by invasive exotic grasses (IEG) that invade and dominate areas excluding native species, oftentimes aided by altered soil conditions. Long-term studies of savanna restoration trajectories are rare. In this study, we surveyed 22 savanna restoration areas established two to ten years before the study with similar restoration methods to assess their current status. We show that the current restoration methods are successful in establishing native species and allowing species turnover but they are threatened by IEG. Restoration success varies and is affected by soil conditions, IEG landscape cover and post-sowing weeding. Despite that, the simultaneous introduction of different plant functional groups allows turnover from fast to slow-growing plants. Establishing savanna native species is possible at an operational scale with current knowledge and techniques. However, native species establishment fails to prevent IEG reinfestation, which needs to be managed in restoration efforts in the Brazilian savanna.

Pre-harvest application of sodium nitroprusside enhances storage root quality in red beet cultivated under normal and drought conditions.

BACKGROUND: The role of nitric oxide (NO) in plant stress tolerance, as well as in increasing post-harvest quality, has been extensively demonstrated in several fruits and vegetable crops; however, the effects of its pre-harvest application on post-harvest quality are still poorly documented. Therefore, the pre-harvest application of NO in red beet (Beta vulgaris subsp. vulgaris) plants cultivated under well-watered and drought conditions was evaluated to assess whether it improves the post-harvest quality of their storage roots. Red beet plants cultivated under well-watered (80% of water holding capacity) or drought condition (15% of water holding capacity) were sprayed weekly with water (control) or 100 µmol L-1 sodium nitroprusside (SNP), an NO donor. Sixty-six days after sowing, red beet roots were harvested, and root yield, total sugar yield, reducing sugars, non-reducing sugars, proteins, lipids, root ashes, root moisture, soluble solids, titratable acidity, pH, vitamin C, total phenolics, total betalains, betacyanins, betaxanthins and antioxidant capacity were determined. RESULTS: While drought led to a reduction in root yield, sugars, lipids and titratable acidity, it increased phenolic compounds, betalains and the antioxidant capacity of beets. SNP reversed the negative effects of drought on sugar, lipid and organic acid contents and increased antioxidant capacity independent of stress. CONCLUSION: Pre-harvest SNP treatment reversed drought-induced yield reductions in beets, while boosting bioactive compounds and antioxidant capacity. It also enhanced vitamin C content independently, indicating its dual role in stress mitigation and beet quality improvement. Future research should explore other crops and stress conditions. © 2024 Society of Chemical Industry.

Consumption of edible insects and insect-based foods: A systematic review of sensory properties and evoked emotional response.

Low consumer acceptance of edible insects and insect-based products is one of the main barriers to the successful implementation of entomophagy in Western countries. This rejection is mainly caused by consumers' negative emotional responses, psychological/personality traits, and attitudes toward food choices. However, as the role of intrinsic product characteristics on such food choices has not been adequately studied, a systematic review was conducted following the PRISMA method, to analyze studies that have assessed hedonic evaluations, sensory profiling, or emotional responses to edible insects or insect-based products. The majority of studies performed with whole insects and insect flour highlight that insect-based products are more negatively evaluated than control products. Although the sensory properties of insects are affected by species and processing conditions, they are generally negative across sensory dimensions. In particular, insects and insect-based products are generally associated with odor and flavor/taste attributes that are related to old/spoiled food. These negative attributes can be linked to the fat fraction of edible insects, with insect oils being very negatively evaluated by consumers. On the other hand, defatted fractions and deodorized oils are not associated with these negative attributes, further supporting the hypothesis that the fat fraction is responsible for the negative odor and flavor/taste attributes. However, there is still a lack of studies assessing the sensory profile of edible insects and insect-based products, as well as consumers' emotional responses to their consumption. Future studies should focus on the effects of different processing conditions, products incorporating insect fractions (namely protein concentrates/isolates and defatted fractions), and evaluation by target consumer groups.

A deeper insight into the sialome of male and female Culex quinquefasciatus mosquitoes.

INTRODUCTION: During evolution, blood-feeding arthropods developed a complex salivary mixture that can interfere with host haemostatic and immune response, favoring blood acquisition and pathogen transmission. Therefore, a survey of the salivary gland contents can lead to the identification of molecules with potent pharmacological activity in addition to increase our understanding of the molecular mechanisms underlying the hematophagic behaviour of arthropods. The southern house mosquito, Culex quinquefasciatus, is a vector of several pathogenic agents, including viruses and filarial parasites that can affect humans and wild animals. RESULTS: Previously, a Sanger-based transcriptome of the salivary glands (sialome) of adult C. quinquefasciatus females was published based on the sequencing of 503 clones organized into 281 clusters. Here, we revisited the southern mosquito sialome using an Illumina-based RNA-sequencing approach of both male and female salivary glands. Our analysis resulted in the identification of 7,539 coding DNA sequences (CDS) that were functionally annotated into 25 classes, in addition to 159 long non-coding RNA (LncRNA). Additionally, comparison of male and female libraries allowed the identification of female-enriched transcripts that are potentially related to blood acquisition and/or pathogen transmission. CONCLUSION: Together, these findings represent an extended reference for the identification and characterization of the proteins containing relevant pharmacological activity in the salivary glands of C. quinquefasciatus mosquitoes.

Association between serum Vitamin D levels and asthma severity and control in children and adolescents.

OBJECTIVE: To assess the association of serum Vitamin D (vitD) levels with asthma control and severity in children and adolescents in different seasons of the year. METHOD: Longitudinal, prospective study with 7- to 17-year-old children and adolescents diagnosed with asthma. All participants underwent two assessments conducted in opposite seasons of the year which included a clinical assessment, a questionnaire for classification of asthma control (Asthma Control Test), spirometry, and blood collection to measure serum vitD levels. RESULTS: In total, 141 individuals with asthma were evaluated. The mean vitD was lower in females (p = 0.006) and sunlight exposure appears not to be an influencing factor for vitD levels. We found no differences in mean vitD of patients with controlled and uncontrolled asthma (p = 0.703; p = 0.956). However, the severe asthma group had lower mean Vitamin D than the mild/moderate asthma group for both assessments (p = 0.013; p = 0.032). In the first assessment, the group with vitD insufficiency had a higher prevalence of severe asthma (p = 0.015). Vitamin D was positively correlated with FEV1 in both assessments (p = 0.008; p = 0.006) and with FEF25-75% in the first assessment (p = 0.038). CONCLUSION: In a tropical climate zone, there is no evidence of association between seasonality and serum vitD levels or between serum vitD levels and asthma control in children and adolescents. However, vitD and lung function were positively correlated and the group with vitD insufficiency had a higher prevalence of severe asthma.

A Pilot Randomized Controlled Trial (RCT) of Acceptance and Commitment Therapy Versus Cognitive Behavioral Therapy for Chronic Insomnia.

OBJECTIVE: To compare the effectiveness of protocols for acceptance and commitment therapy for insomnia (ACT-I) and cognitive behavioral therapy for insomnia (CBT-I) in adults. METHOD: Participants were 37 adults (74.3% women; M = 43.7 years, SD = 10.7) with chronic insomnia who were randomized to 6 weekly group sessions consisting of ACT-I (n = 19) or CBT-I (n = 18). The primary outcome measures were based on the Insomnia Severity Index (ISI) total score, a measure of insomnia complaints, and included the proportions of treatment responders (defined as a change in score of 8 points or more) and remitters (defined as a final score below 8). RESULTS: Both treatment modalities significantly reduced insomnia severity. Post-treatment, the proportion of treatment responders was higher in the CBT-I than the ACT-I (64.7% vs. 50.0%, respectively) group and six months later, ACT-I made further improvements whereas CBT-I had a reduced treatment response (58.8% vs. 55.6%, respectively). CBT-I was associated with a higher proportion of insomnia remission at post treatment. CONCLUSIONS: Both CBT-I and ACT-I are effective, with a higher proportion of insomnia remitters in CBT-I post-treatment. The different change trajectories for the two therapy groups provide insights into behavioral change via a cognitive versus contextual approach.

Back pain in children and adolescents: a cross-sectional study.

PURPOSE: Back pain is a growing problem worldwide, not only in adults but also in children. Therefore, it is becoming increasingly important to investigate and understand the factors that influence the early onset of back pain. The aim of this study was to determine the prevalence of back pain in children and adolescents and to identify predisposing risk factors and protective factors. METHODS: A cross-sectional study was conducted between October and December 2019 in schools from northern Portugal, evaluating 1463 students aged 9 to 19 years, of both genders. The instruments used were the Spinal Mouse® to assess posture, the Inbody 230® to assess body composition, an online questionnaire to characterize the sample and back pain, and the FITescola® battery test to access physical fitness. RESULTS: Half of the subjects experienced back pain at least once in their lifetime. The most frequently mentioned were lumbar spine and thoracic spine, mostly with mild or moderate pain intensities. Age, female gender, percent body fat, prolonged smartphone and computer use, hyperkyphosis, and the lateral global spine tilt to the left side are all factors with higher relative risk of back pain. Practicing physical activity or sports regularly and video games have a protective effect. CONCLUSION: The prevalence of back pain in children and adolescents is very high: The study enhances the case for protective factors such as physical activity habits or video games while reinforcing risk factors such as percent body fat, prolonged smartphone or computer use, and posture.

A Model for Cognitive Personalization of Microtask Design.

The study of data quality in crowdsourcing campaigns is currently a prominent research topic, given the diverse range of participants involved. A potential solution to enhancing data quality processes in crowdsourcing is cognitive personalization, which involves appropriately adapting or assigning tasks based on a crowd worker's cognitive profile. There are two common methods for assessing a crowd worker's cognitive profile: administering online cognitive tests, and inferring behavior from task fingerprinting based on user interaction log events. This article presents the findings of a study that investigated the complementarity of both approaches in a microtask scenario, focusing on personalizing task design. The study involved 134 unique crowd workers recruited from a crowdsourcing marketplace. The main objective was to examine how the administration of cognitive ability tests can be used to allocate crowd workers to microtasks with varying levels of difficulty, including the development of a deep learning model. Another goal was to investigate if task fingerprinting can be used to allocate crowd workers to different microtasks in a personalized manner. The results indicated that both objectives were accomplished, validating the usage of cognitive tests and task fingerprinting as effective mechanisms for microtask personalization, including the development of a deep learning model with 95% accuracy in predicting the accuracy of the microtasks. While we achieved an accuracy of 95%, it is important to note that the small dataset size may have limited the model's performance.

Molecular Interplay between Non-Host Resistance, Pathogens and Basal Immunity as a Background for Fatal Yellowing in Oil Palm (Elaeis guineensis Jacq.) Plants.

An oil palm (Elaeis guineensis Jacq.) bud rod disorder of unknown etiology, named Fatal Yellowing (FY) disease, is regarded as one of the top constraints with respect to the growth of the palm oil industry in Brazil. FY etiology has been a challenge embraced by several research groups in plant pathology throughout the last 50 years in Brazil, with no success in completing Koch's postulates. Most recently, the hypothesis of having an abiotic stressor as the initial cause of FY has gained ground, and oxygen deficiency (hypoxia) damaging the root system has become a candidate for stress. Here, a comprehensive, large-scale, single- and multi-omics integration analysis of the metabolome and transcriptome profiles on the leaves of oil palm plants contrasting in terms of FY symptomatology-asymptomatic and symptomatic-and collected in two distinct seasons-dry and rainy-is reported. The changes observed in the physicochemical attributes of the soil and the chemical attributes and metabolome profiles of the leaves did not allow the discrimination of plants which were asymptomatic or symptomatic for this disease, not even in the rainy season, when the soil became waterlogged. However, the multi-omics integration analysis of enzymes and metabolites differentially expressed in asymptomatic and/or symptomatic plants in the rainy season compared to the dry season allowed the identification of the metabolic pathways most affected by the changes in the environment, opening an opportunity for additional characterization of the role of hypoxia in FY symptom intensification. Finally, the initial analysis of a set of 56 proteins/genes differentially expressed in symptomatic plants compared to the asymptomatic ones, independent of the season, has presented pieces of evidence suggesting that breaks in the non-host resistance to non-adapted pathogens and the basal immunity to adapted pathogens, caused by the anaerobic conditions experienced by the plants, might be linked to the onset of this disease. This set of genes might offer the opportunity to develop biomarkers for selecting oil palm plants resistant to this disease and to help pave the way to employing strategies to keep the safety barriers raised and strong.

Results from Portugal's 2022 report card on physical activity for children and youth.

Background: The purpose of this study is to update results of Portuguese's Report Card on Physical activity (PA) for Children and Adolescents. Methods: The grades were assigned by results derived from the PA and Fitness in Portugal 2021 Portuguese Report Card and corresponds to the third report for the Portuguese children and adolescents. It includes indicators of PA and sedentary behavior (SB) that are common to the GLOBAL matrix 4.0: Overall Physical Activity, Organized Sport and Physical Activity, Active Play, Active Transportation, Sedentary Behaviors, Family and Peers, School, Community and the Environment, Government and Physical Fitness. The search focused on published national evidence/data sources (academia, NGO, governmental) from end 2018 onwards excluding data obtained during the covid-19 pandemic. Results: The grades were assigned as follows: Overall PA (D-), Organized Sport Participation (C-), Active Play (D+), Active Transportation (D-), Sedentary behaviors (C+), Physical Fitness (C), Family and Peers (B), School (A), Community and Environment (B), and Government (B). Conclusion: In line with previous Portuguese Report Cards, a large proportion of Portuguese children and adolescents are not sufficiently active nor fit enough setting urgency for effective strategies. Particular attention should be given to Active play, Active transport and Organized Sports Participation has their grades have decreased. Some actions in selected indicators as Governmental and policy seems promising however results weren't seen yet. Despite the strong support of schools with mandatory curricula in PE no correspondent change is observed in fitness or PA, so more research is needed to find why.

Are Prayer-Based Interventions Effective Pain Management Options? A Systematic Review and Meta-analysis of Randomized Controlled Trials.

This review examined the effects of private and communal participatory prayer on pain. Nine databases were searched. Six randomized controlled trials were included. For private prayer, medium to large effects emerged for 67% to 69% of between-group comparisons; participants in the prayer condition reported lower pain intensity (0.59 < d < 26.17; 4 studies) and higher pain tolerance (0.70 < d < 1.05; 1 study). Pre- to post-intervention comparisons yielded medium to large effects (0.76 < d < 1.67; 2 studies); pain intensity decreased. Although firm conclusions cannot be made because meta-analysis was based on only two studies, the analysis suggested prayer might reduce pain intensity (SMD = - 2.63, 95% CI [- 3.11, - 2.14], I = 0%). (PROSPERO: CRD42020221733).

Salivary complement inhibitors from mosquitoes: Structure and mechanism of action.

Inhibition of the alternative pathway (AP) of complement by saliva from Anopheles mosquitoes facilitates feeding by blocking production of the anaphylatoxins C3a and C5a, which activate mast cells leading to plasma extravasation, pain, and itching. We have previously shown that albicin, a member of the SG7 protein family from An. Albimanus, blocks the AP by binding to and inhibiting the function of the C3 convertase, C3bBb. Here we show that SG7.AF, the albicin homolog from An. freeborni, has a similar potency to albicin but is more active in the presence of properdin, a plasma protein that acts to stabilize C3bBb. Conversely, albicin is highly active in the absence or presence of properdin. Albicin and SG7.AF stabilize the C3bBb complex in a form that accumulates on surface plasmon resonance (SPR) surfaces coated with properdin, but SG7.AF binds with lower affinity than albicin. Albicin induces oligomerization of the complex in solution, suggesting that it is oligomerization that leads to stabilization on SPR surfaces. Anophensin, the albicin ortholog from An. stephensi, is only weakly active as an inhibitor of the AP, suggesting that the SG7 family may play a different functional role in this species and other species of the subgenus Cellia, containing the major malaria vectors in Africa and Asia. Crystal structures of albicin and SG7.AF reveal a novel four-helix bundle arrangement that is stabilized by an N-terminal hydrogen bonding network. These structures provide insight into the SG7 family and related mosquito salivary proteins including the platelet-inhibitory 30 kDa family.

Identification of a substrate-like cleavage-resistant thrombin inhibitor from the saliva of the flea Xenopsylla cheopis.

The salivary glands of the flea Xenopsylla cheopis, a vector of the plague bacterium, Yersinia pestis, express proteins and peptides thought to target the hemostatic and inflammatory systems of its mammalian hosts. Past transcriptomic analyses of salivary gland tissue revealed the presence of two similar peptides (XC-42 and XC-43) having no extensive similarities to any other deposited sequences. Here we show that these peptides specifically inhibit coagulation of plasma and the amidolytic activity of α-thrombin. XC-43, the smaller of the two peptides, is a fast, tight-binding inhibitor of thrombin with a dissociation constant of less than 10 pM. XC-42 exhibits similar selectivity as well as kinetic and binding properties. The crystal structure of XC-43 in complex with thrombin shows that despite its substrate-like binding mode, XC-43 is not detectably cleaved by thrombin and that it interacts with the thrombin surface from the enzyme catalytic site through the fibrinogen-binding exosite I. The low rate of hydrolysis was verified in solution experiments with XC-43, which show the substrate to be largely intact after 2 h of incubation with thrombin at 37 °C. The low rate of XC-43 cleavage by thrombin may be attributable to specific changes in the catalytic triad observable in the crystal structure of the complex or to extensive interactions in the prime sites that may stabilize the binding of cleavage products. Based on the increased arterial occlusion time, tail bleeding time, and blood coagulation parameters in rat models of thrombosis XC-43 could be valuable as an anticoagulant.