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Ixodes scapularis is a blood-feeding obligate ectoparasite responsible for transmitting the Lyme disease (LD) agent, Borrelia burgdorferi. During the feeding process, I. scapularis injects B. burgdorferi into the host along with its saliva, facilitating the transmission and colonization of the LD agent. Tick calreticulin (CRT) is one of the earliest tick saliva proteins identified and is currently utilized as a biomarker for tick bites. Our recent findings revealed elevated levels of CRT in the saliva proteome of B. burgdorferi-infected I. scapularis nymphs compared to uninfected ticks. Differential precipitation of proteins (DiffPOP) and LC-MS/MS analyses were used to identify the interactions between Ixs (I. scapularis) CRT and human plasma proteins and further explore its potential role in shielding B. burgdorferi from complement killing. We observed that although yeast-expressed recombinant (r) IxsCRT binds to the C1 complex (C1q, C1r, and C1s), the activator of complement via the classical cascade, it did not inhibit the deposition of the membrane attack complex (MAC) via the classical pathway. Intriguingly, rIxsCRT binds intermediate complement proteins (C3, C5, and C9) and reduces MAC deposition through the lectin pathway. Despite the inhibition of MAC deposition in the lectin pathway, rIxsCRT did not protect a serum-sensitive B. burgdorferi strain (B314/pBBE22Luc) from complement-induced killing. As B. burgdorferi establishes a local dermal infection before disseminating to secondary organs, it is noteworthy that rIxsCRT promotes the replication of B. burgdorferi in culture. We hypothesize that rIxsCRT may contribute to the transmission and/or host colonization of B. burgdorferi by acting as a decoy activator of complement and by fostering B. burgdorferi replication at the transmission site. [ABSTRACT FROM AUTHOR]

Lyme disease (LD) caused by Borrelia burgdorferi is among the most important human vector borne diseases for which there is no effective prevention method. Identification of tick saliva transmission factors of the LD agent is needed before the highly advocated tick antigen-based vaccine could be developed. We previously reported the highly conserved Ixodes scapularis (Ixs) tick saliva serpin (S) 17 (IxsS17) was highly secreted by B. burgdorferi infected nymphs. Here, we show that IxsS17 promote tick feeding and enhances B. burgdorferi colonization of the host. We show that IxsS17 is not part of a redundant system, and its functional domain reactive center loop (RCL) is 100% conserved in all tick species. Yeast expressed recombinant (r) IxsS17 inhibits effector proteases of inflammation, blood clotting, and complement innate immune systems. Interestingly, differential precipitation analysis revealed novel functional insights that IxsS17 interacts with both effector proteases and regulatory protease inhibitors. For instance, rIxsS17 interacted with blood clotting proteases, fXII, fX, fXII, plasmin, and plasma kallikrein alongside blood clotting regulatory serpins (antithrombin III and heparin cofactor II). Similarly, rIxsS17 interacted with both complement system serine proteases, C1s, C2, and factor I and the regulatory serpin, plasma protease C1 inhibitor. Consistently, we validated that rIxsS17 dose dependently blocked deposition of the complement membrane attack complex via the lectin complement pathway and protected complement sensitive B. burgdorferi from complement-mediated killing. Likewise, co-inoculating C3H/HeN mice with rIxsS17 and B. burgdorferi significantly enhanced colonization of mouse heart and skin organs in a reverse dose dependent manner. Taken together, our data suggests an important role for IxsS17 in tick feeding and B. burgdorferi colonization of the host. Author summary: Ticks feed on animals and humans for their survival. During blood meal feeding, ticks inject saliva along with disease causative agents into the hosts. Here, we demonstrate that I. scapularis tick saliva protein, IxsS17 inhibits host innate immune system proteases and enhances B. burgdorferi colonization of the host. Recombinant IxsS17 (rIxsS17) inhibits blood clotting and inflammation systems serine proteases including pancreatic trypsin and trypsin IV (~100%), blood clotting factor Xa and XIa (~60–80%), plasmin and cathepsin G (~50%). Similarly, rIxsS17 interacts with complement system factors, C1s, C2 and factor I and blocks complement membrane attack complex via the lectin complement pathway by up to 97%. We found that, in the mouse model for Lyme disease, rIxsS17 significantly increases B. burgdorferi colonization of mouse heart and ear tissues by 5.7 and 2.3 times. Taken together, we conclude that IxsS17 is a key protein in tick feeding and B. burgdorferi colonization of the host, and thus, a potential target antigen for developing tick antigen-based vaccines against Lyme disease agent transmission. [ABSTRACT FROM AUTHOR]

Background: Heart failure (HF) is a global health burden, and its management in the emergency department (ED) is important. This study aimed to evaluate the association between focused cardiac ultrasound (FoCUS) and early administration of diuretics in patients with acute HF admitted to the ED.Methods: This retrospective observational study was conducted at a tertiary academic hospital. Patients with acute HF patients who were admitted to the ED and receiving intravenous medication between January 2018 and December 2019 were enrolled. The main exposure was a FoCUS examination performed within 2 h of ED triage. The primary outcome was the time to furosemide administration.Results: Of 1154 patients with acute HF, 787 were included in the study, with 116 of them having undergone FoCUS. The time to furosemide was significantly shorter in the FoCUS group (median time (q1-q3), 112 min; range, 65-163 min) compared to the non-FoCUS group (median time, 131 min; range, 71-229 min). In the multivariable logistic regression analysis adjusting for age, sex, chief complaint, mode of arrival, triage level, shock status, and desaturation at triage, early administration of furosemide within 2 h from triage was significantly higher in the FoCUS group (adjusted odds ratio, 1.63; 95% confidence intervals, 1.04-2.55) than in the non-FoCUS group.Conclusions: Early administration of intravenous furosemide was associated with FoCUS examination in patients with acute HF admitted to the ED. An early screening protocol could be useful for improving levels in clinical practice at EDs. [ABSTRACT FROM AUTHOR]

This study has comprehensively analyzed the ventilation system of an indoor coal storage shed using computational fluid dynamics (CFD). In addition, the effects of the plan to improve the ventilation system were investigated by synthesizing the results. First, the velocity of inlet wind entering through the natural ventilation system was measured. Then, the concentration of carbon monoxide inside the coal storage shed was measured at the spontaneous combustion of coal. The boundary conditions were set using the measurement results. The characteristics of carbon monoxide concentration, ventilation volume, and behavior of scattering dust were analyzed. According to the CFD analysis results, the upper recirculation strength increased as the flow rate of external air increased. The flow-stagnation area occurred on the center wall. In particular, the concentration of carbon monoxide was high in the flow-stagnation area. When the inflow velocity was 2.0 m/s, a risk of 30 ppm or more occurred in the area near the second-floor workspace and central retaining wall. When ventilation dropped sharply, coal dust emissions decreased to 14.5%. Thus, ventilation must be secured by installing a natural ventilation system, in case spontaneous ignition occurs in many cells or the ventilation sharply decreases. Finally, in order to improve the ventilation system, the effect on the additional installation of natural ventilation and the use of mobile blowers was analyzed. Finally, in order to improve the ventilation system, the effect on the additional installation of natural ventilation and the use of mobile blowers was analyzed. As a result of the analysis, we concluded that using a mobile blower is more effective than a method of additionally installing a natural ventilation device. Carbon monoxide may be locally diluted, and ventilation volume additionally secured. [ABSTRACT FROM AUTHOR]

Ticks inject serine protease inhibitors (serpins) into their feeding sites to evade serine protease-mediated host defenses against tick-feeding. This study describes two highly identitical (97%) but functionally different Amblyomma americanum tick saliva serpins (AAS41 and 46) that are secreted at the inception of tick-feeding. We show that AAS41, which encodes a leucine at the P1 site inhibits inflammation system proteases: chymase (SI = 3.23, Ka = 5.6 ± 3.7X103M−1 s−1) and α-chymotrypsin (SI = 3.18, Ka = 1.6 ± 4.1X104M−1 s−1), while AAS46, which encodes threonine has no inhibitory activity. Similary, rAAS41 inhibits rMCP-1 purified from rat peritonuem derived mast cells. Consistently, rAAS41 inhibits chymase-mediated inflammation induced by compound 48/80 in rat paw edema and vascular permeability models. Native AAS41/46 proteins are among tick saliva immunogens that provoke anti-tick immunity in repeatedly infested animals as revealed by specific reactivity with tick immune sera. Of significance, native AAS41/46 play critical tick-feeding functions in that RNAi-mediated silencing caused ticks to ingest significantly less blood. Importantly, monospecific antibodies to rAAS41 blocked inhibitory functions of rAAS41, suggesting potential for design of vaccine antigens that provokes immunity to neutralize functions of this protein at the tick-feeding site. We discuss our findings with reference to tick-feeding physiology and discovery of effective tick vaccine antigens. • Amblyomma americanum secretes two highly identical (97% identity) serpins (AAS 41 and 46) that have different functions • Amblyomma americanum utilizes AAS41 to block host inflammation defense by disrupting chymase and chymotrypsin functions • Functions of AAS 41 and 46 are critical to tick feeding success as revealed by RNAi mediated silencing • Both AAS 41 and 46 are among immunogens that provoke anti-tick immunity in repeatedly infested rabbits. [ABSTRACT FROM AUTHOR]

Amblyomma americanum ticks transmit more than a third of human tick-borne disease (TBD) agents in the United States. Tick saliva proteins are critical to success of ticks as vectors of TBD agents, and thus might serve as targets in tick antigen-based vaccines to prevent TBD infections. We describe a systems biology approach to identify, by LC-MS/MS, saliva proteins (tick = 1182, rabbit = 335) that A. americanum ticks likely inject into the host every 24 h during the first 8 days of feeding, and towards the end of feeding. Searching against entries in GenBank grouped tick and rabbit proteins into 27 and 25 functional categories. Aside from housekeeping-like proteins, majority of tick saliva proteins belong to the tick-specific (no homology to non-tick organisms: 32%), protease inhibitors (13%), proteases (8%), glycine-rich proteins (6%) and lipocalins (4%) categories. Global secretion dynamics analysis suggests that majority (74%) of proteins in this study are associated with regulating initial tick feeding functions and transmission of pathogens as they are secreted within 24–48 h of tick attachment. Comparative analysis of the A. americanum tick saliva proteome to five other tick saliva proteomes identified 284 conserved tick saliva proteins: we speculate that these regulate critical tick feeding functions and might serve as tick vaccine antigens. We discuss our findings in the context of understanding A. americanum tick feeding physiology as a means through which we can find effective targets for a vaccine against tick feeding. Author summary: The lone star tick, Amblyomma americanum, is a medically important species in US that transmits 5 of the 16 reported tick-borne disease agents. Most recently, bites of this tick were associated with red meat allergies in humans. Vaccination of animals against tick feeding has been shown to be a sustainable and an effective alternative to current acaricide based tick control method which has several limitations. The pre-requisite to tick vaccine development is to understand the molecular basis of tick feeding physiology. Toward this goal, this study has identified proteins that A. americanum ticks inject into the host at different phases of its feeding cycle. This data set has identified proteins that A. americanum inject into the host within 24–48 h of feeding before it starts to transmit pathogens. Of high importance, we identified 284 proteins that are present in saliva of other tick species, which we suspect regulate important role(s) in tick feeding success and might represent rich source target antigens for a tick vaccine. Overall, this study provides a foundation to understand the molecular mechanisms regulating tick feeding physiology. [ABSTRACT FROM AUTHOR]

Feeding and transmission of tick-borne disease (TBD) agents by ticks are facilitated by tick saliva proteins (TSP). Thus, defining functional roles of TSPs in tick evasion is expected to reveal potential targets in tick-antigen based vaccines to prevent TBD infections. This study describes two types of Amblyomma americanum TSPs: those that are similar to LPS activate macrophage (MΦ) to express pro-inflammation (PI) markers and another set that suppresses PI marker expression by activated MΦ. We show that similar to LPS, three recombinant (r) A. americanum insulin-like growth factor binding-related proteins (rAamIGFBP-rP1, rAamIGFBP-rP6S, and rAamIGFBP-rP6L), hereafter designated as PI-rTSPs, stimulated both PBMC -derived MΦ and mice RAW 267.4 MΦ to express PI co-stimulatory markers, CD40, CD80, and CD86 and cytokines, TNFα, IL-1, and IL-6. In contrast, two A. americanum tick saliva serine protease inhibitors (serpins), AAS27 and AAS41, hereafter designated as anti-inflammatory (AI) rTSPs, on their own did not affect MΦ function or suppress expression of PI markers, but enhanced expression of AI cytokines (IL-10 and TGFβ) in MΦ that were pre-activated by LPS or PI-rTSPs. Mice paw edema test demonstrated that in vitro validated PI- and AI-rTSPs are functional in vivo since injection of HEK293-expressed PI-rTSPs (individually or as a cocktail) induced edema comparable to carrageenan-induced edema and was characterized by upregulation of CD40, CD80, CD86, TNF-α, IL-1, IL-6, and chemokines: CXCL1, CCL2, CCL3, CCL5, and CCL11, whereas the AI-rTSPs (individually and cocktail) were suppressive. We propose that the tick may utilize countervailing PI and AI TSPs to regulate evasion of host immune defenses whereby TSPs such as rAamIGFBP-rPs activate host immune cells and proteins such as AAS27 and AAS41 suppress the activated immune cells. Author summary: Several studies have documented immuno-suppressive activities in whole tick saliva and salivary gland protein extracts. We have made contribution toward understanding the molecular basis of tick feeding, as we have described functions of defined tick saliva immuno-modulatory proteins. We have shown that A. americanum injects two groups of functionally opposed tick saliva proteins: those that could counter-intuitively be characterized as pro-host defense, and those that are expected to have anti-host immune defense functions. Based on our data, we propose that the tick evades host defense using countervailing pro- and anti- inflammatory proteins in which the pro-host defense tick saliva proteins stimulate host immune cells such as macrophages, and the anti-host defense tick saliva proteins suppress functions of the activated immune cells. [ABSTRACT FROM AUTHOR]

Ticks successfully feed and transmit pathogens by injecting pharmacological compounds in saliva to thwart host defenses. We have previously used LC-MS/MS to identify proteins that are present in saliva of unfed Amblyomma americanum ticks that were exposed to different hosts. Here we show that A. americanum serine protease inhibitor (serpin) 27 (AAS27) is an immunogenic saliva protein that is injected into the host within the first day of tick feeding and is an anti-inflammatory protein that might act by blocking plasmin and trypsin functions. Although AAS27 is injected into the host throughout tick feeding, qRT-PCR and western blotting analyses indicate that the respective transcript and protein are present in high amounts within the first 24 h of tick feeding. Biochemical screening of Pichia pastoris-expressed recombinant (r) AAS27 against mammalian proteases related to host defense shows it is an inhibitor of trypsin and plasmin, with stoichiometry of inhibition indices of 3.5 and 3.8, respectively. Consistent with typical inhibitory serpins, rAAS27 formed heat- and SDS-stable irreversible complexes with both proteases. We further demonstrate that rAAS27 inhibits trypsin with ka of 6.46 ± 1.24 x 104 M-1 s-1, comparable to serpins of other tick species. We show that native AAS27 is part of the repertoire of proteins responsible for the inhibitory activity against trypsin in crude tick saliva. AAS27 is likely utilized by the tick to evade the hosts inflammation defense since rAAS27 blocks both formalin and compound 48/80-induced inflammation in rats. Tick immune sera of rabbits that had acquired resistance against tick feeding following repeated infestations with A. americanum or Ixodes scapularis ticks reacts with rAAS27. Of significant interest, antibody to rAAS27 blocks this serpin inhibitory functions. Taken together, we conclude that AAS27 is an anti-inflammatory protein secreted into the host during feeding and may represent a potential candidate for development of an anti-tick vaccine. [ABSTRACT FROM AUTHOR]

Inflammation and hemostasis are part of the host's first line of defense to tick feeding. These systems are in part serine protease mediated and are tightly controlled by their endogenous inhibitors, in the serpin superfamily (serine protease inhibitors). From this perspective ticks are thought to use serpins to evade host defenses during feeding. The cattle tick Rhipicephalus microplus encodes at least 24 serpins, of which RmS-3, RmS-6, and RmS-17 were previously identified in saliva of this tick. In this study, we screened inhibitor functions of these three saliva serpins against a panel of 16 proteases across the mammalian defense pathway. Our data confirm that Pichia pastoris -expressed rRmS-3, rRmS-6, and rRmS-17 are likely inhibitors of pro-inflammatory and pro-coagulant proteases. We show that rRmS-3 inhibited chymotrypsin and cathepsin G with stoichiometry of inhibition (SI) indices of 1.8 and 2.0, and pancreatic elastase with SI higher than 10. Likewise, rRmS-6 inhibited trypsin with SI of 2.6, chymotrypsin, factor Xa, factor XIa, and plasmin with SI higher than 10, while rRmS-17 inhibited trypsin, cathepsin G, chymotrypsin, plasmin, and factor XIa with SI of 1.6, 2.6, 2.7, 3.4, and 9.0, respectively. Additionally, we observed the formation of irreversible complexes between rRmS-3 and chymotrypsin, rRmS-6/rRmS-17 and trypsin, and rRmS-3/rRmS-17 and cathepsin G, which is consistent with typical mechanism of inhibitory serpins. In blood clotting assays, rRmS-17 delayed plasma clotting by 60 s in recalcification time assay, while rRmS-3 and rRmS-6 did not have any effect. Consistent with inhibitor function profiling data, 2.0 μM rRmS-3 and rRmS-17 inhibited cathepsin G-activated platelet aggregation in a dose-responsive manner by up to 96% and 95% respectively. Of significant interest, polyclonal antibodies blocked inhibitory functions of the three serpins. Also notable, antibodies to Amblyomma americanum , Ixodes scapularis , and Rhipicephalus sanguineus tick saliva proteins cross-reacted with the three R. microplus saliva serpins, suggesting the potential of these proteins as candidates for universal anti-tick vaccines. [ABSTRACT FROM AUTHOR]

Ixodes scapularis is the most medically important tick species and transmits five of the 14 reportable human tick borne disease (TBD) agents in the USA. This study describes LC-MS/MS identification of 582 tick- and 83 rabbit proteins in saliva of I. scapularis ticks that fed for 24, 48, 72, 96, and 120 h, as well as engorged but not detached (BD), and spontaneously detached (SD). The 582 tick proteins include proteases (5.7%), protease inhibitors (7.4%), unknown function proteins (22%), immunity/antimicrobial (2.6%), lipocalin (3.1%), heme/iron binding (2.6%), extracellular matrix/ cell adhesion (2.2%), oxidant metabolism/ detoxification (6%), transporter/ receptor related (3.2%), cytoskeletal (5.5%), and housekeeping-like (39.7%). Notable observations include: (i) tick saliva proteins of unknown function accounting for >33% of total protein content, (ii) 79% of proteases are metalloproteases, (iii) 13% (76/582) of proteins in this study were found in saliva of other tick species and, (iv) ticks apparently selectively inject functionally similar but unique proteins every 24 h, which we speculate is the tick's antigenic variation equivalent strategy to protect important tick feeding functions from host immune system. The host immune responses to proteins present in 24 h I. scapularis saliva will not be effective at later feeding stages. Rabbit proteins identified in our study suggest the tick's strategic use of host proteins to modulate the feeding site. Notably fibrinogen, which is central to blood clotting and wound healing, was detected in high abundance in BD and SD saliva, when the tick is preparing to terminate feeding and detach from the host. A remarkable tick adaptation is that the feeding lesion is completely healed when the tick detaches from the host. Does the tick concentrate fibrinogen at the feeding site to aide in promoting healing of the feeding lesion? Overall, these data provide broad insight into molecular mechanisms regulating different tick feeding phases. These data set the foundation for in depth I. scapularis tick feeding physiology and TBD transmission studies. [ABSTRACT FROM AUTHOR]

RNA interference-mediated gene silencing was shown to be an efficient tool for validation of targets that may become anti-tick vaccine components. Here, we demonstrate the application of this approach in the validation of components of molecular signaling cascades, such as the Protein Kinase B (AKT) / Glycogen Synthase Kinase (GSK) axis during tick embryogenesis. It was shown that heptane and hypochlorite treatment of tick eggs can remove wax, affecting corium integrity and but not embryo development. Evidence of AKT and GSK dsRNA delivery into de-waxed eggs of via electroporation is provided. Primers designed to amplify part of the dsRNA delivered into the electroporated eggs dsRNA confirmed its entry in eggs. In addition, it was shown that electroporation is able to deliver the fluorescent stain, 4',6-diamidino-2-phenylindole (DAPI). To confirm gene silencing, a second set of primers was designed outside the dsRNA sequence of target gene. In this assay, the suppression of AKT and GSK transcripts (approximately 50% reduction in both genes) was demonstrated in 7-day-old eggs. Interestingly, silencing of GSK in 7-day-old eggs caused 25% reduction in hatching. Additionally, the effect of silencing AKT and GSK on embryo energy metabolism was evaluated. As expected, knockdown of AKT, which down regulates GSK, the suppressor of glycogen synthesis, decreased glycogen content in electroporated eggs. These data demonstrate that electroporation of de-waxed R. microplus eggs could be used for gene silencing in tick embryos, and improve the knowledge about arthropod embryogenesis. [ABSTRACT FROM AUTHOR]

Full article available online at Healio.com/Orthopedics Foreign body reactions may occur in patients who receive bioabsorbable implants during orthopedic surgery for fractures and ligament repair. The authors describe a 34-year-old man who presented with a palpable tender mass on the lateral aspect of the left knee of 1 month's duration. He underwent posterior cruciate ligament and posterolateral corner reconstruction 3 years earlier. Physical examination showed a 1×1-cm soft, nontender mass without localized warmth on the lateral epicondyle of the distal femur. Magnetic resonance imaging showed a broken screw fragment surrounded by a cyst-like mass. Under general anesthesia, the surgeon excised the screw fragment and the fibrotic mass, enclosing it in the subcutaneous tissue at the lateral epicondyle, the site at which a poly-L-lactic acid bioabsorbable screw had been inserted to fix the graft for posterolateral corner reconstruction. Histologic evaluation showed a foreign body reaction to the degraded screw particles. To the authors' knowledge, this report is the first description of a patient presenting with a delayed foreign body reaction to a broken poly-L-lactic acid bioabsorbable screw at the lateral femoral epicondyle after posterolateral corner reconstruction. Because delayed foreign body reactions can occur at any site of poly-L-lactic acid bioabsorbable screw insertion, care should be taken to avoid screw protrusion during ligament reconstruction because it can lead to screw breakage and delayed foreign body reaction. [Orthopedics. 2014; 37(12):e1129-e1132.] [ABSTRACT FROM AUTHOR]

Background and Objectives: We aimed to analyze the morphology of the common femoral artery (CFA) and common femoral vein (CFV) and the anatomical relationship between the two blood vessels, and to investigate the factors that influence the size of these blood vessels. Materials and Methods: This retrospective study included 584 patients who underwent abdominal and pelvic computed tomography from 1 February to 28 February 2021. We measured the vessels at three regions on both lower extremities (inguinal ligament, distal vessel bifurcation, midpoint) and analyzed and classified the degree of overlap between the CFA and CFV into three types, as well as the factors affecting vessel size. Results: After comparing the femoral vessels according to location, it was confirmed that the CFA and CFV were larger distally than proximally on both sides (p < 0.001). The degree of overlap increased distally (p < 0.001) but was less at the middle (p < 0.001) and distal (p = 0.011) regions on the right side. It was found that the size of CFA and CFV were related to age, sex, and body mass index (BMI) and that malignancy also affects the CFA size. Conclusions: The morphology of the CFA and CFV was conical and increased distally. The degree of overlap between the two blood vessels also increased distally but was less on the right than on the left. Age, sex, and BMI are significant factors affecting the sizes of the CFA and CFV, and malignancy is associated with the CFA size. [ABSTRACT FROM AUTHOR]