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Background: Lyme disease (LD) caused by Borrelia burgdorferi is the most prevalent tick-borne disease. There is evidence that vaccines based on tick proteins that promote tick transmission of B. burgdorferi could prevent LD. As Ixodes scapularis nymph tick bites are responsible for most LD cases, this study sought to identify nymph tick saliva proteins associated with B. burgdorferi transmission using LC-MS/MS. Tick saliva was collected using a non-invasive method of stimulating ticks (uninfected and infected: unfed, and every 12 h during feeding through 72 h, and fully-fed) to salivate into 2% pilocarpine-PBS for protein identification using LC-MS/MS. Results: We identified a combined 747 tick saliva proteins of uninfected and B. burgdorferi infected ticks that were classified into 25 functional categories: housekeeping-like (48%), unknown function (18%), protease inhibitors (9%), immune-related (6%), proteases (8%), extracellular matrix (7%), and small categories that account for <5% each. Notably, B. burgdorferi infected ticks secreted high number of saliva proteins (n=645) than uninfected ticks (n=376). Counter-intuitively, antimicrobial peptides, which function to block bacterial infection at tick feeding site were suppressed 23-85 folds in B. burgdorferi infected ticks. Similar to glycolysis enzymes being enhanced in mammalian cells exposed to B. burgdorferi : eight of the 10-glycolysis pathway enzymes were secreted at high abundance by B. burgdorferi infected ticks. Of significance, rabbits exposed to B. burgdorferi infected ticks acquired potent immunity that caused 40-60% mortality of B. burgdorferi infected ticks during the second infestation compared to 15-28% for the uninfected. This might be explained by ELISA data that show that high expression levels of immunogenic proteins in B. burgdorferi infected ticks. Conclusion: Data here suggest that B. burgdorferi infection modified protein content in tick saliva to promote its survival at the tick feeding site. For instance, enzymes; copper/zinc superoxide dismutase that led to production of H2O2 that is toxic to B. burgdorferi were suppressed, while, catalase and thioredoxin that neutralize H2O2, and pyruvate kinase which yields pyruvate that protects Bb from H2O2 killing were enhanced. We conclude data here is an important resource for discovery of effective antigens for a vaccine to prevent LD. [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]

The adaptation of hard ticks to feed for long periods is facilitated by the cement cone, which securely anchors the tick mouthparts onto host skin and protects the tick from being groomed off by the host. Thus, preventing tick cement deposition is an attractive target for the development of innovative tick control. We used LC-MS/MS sequencing to identify 160 Amblyomma americanum tick cement proteins that include glycine-rich proteins (GRP, 19%), protease inhibitors (12%), proteins of unknown function (11%), mucin (4%), detoxification, storage, and lipocalin at 1% each, and housekeeping proteins (50%). Spatiotemporal transcription analysis showing mRNA expression in multiple tick organs and transcript abundance increasing with feeding suggest that selected GRPs ( n  = 13) regulate multiple tick feeding functions, being classified as constitutively expressed (CE), feeding induced (FI), and up-regulated with feeding (UR). We show that transcription of CE GRPs is likely under the control of tick appetence associated factors in that mRNA abundance increased several thousand fold in 1 week old adult ticks, the time period that coincides with tick attainment of appetence. Given the high number of targets, we synthesized and injected unfed ticks with combinatorial (co) double stranded (ds)RNA and disrupted GRP mRNA in clusters according to similar transcription patterns: CE ( n  = 3), FI, ( n  = 4), and UR ( n  = 6) to streamline the work. Our data suggest that CE and FI GRPs are important for maintenance of the tick feeding site in that reddening and subsequent bleeding were observed around the mouthparts of CE and FI GRP co-dsRNA injected ticks during feeding. Furthermore, although not significantly different, indices for blood meal size and fecundity were apparently reduced in FI and UR ticks. We discuss our data with reference to A. americanum tick feeding physiology. [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]

Tick saliva serine protease inhibitors (serpins) facilitate tick blood meal feeding through inhibition of protease mediators of host defense pathways. We previously identified a highly conserved Amblyomma americanum serpin 19 that is characterised by its reactive center loop being 100% conserved in ixodid ticks. In this study, biochemical characterisation reveals that the ubiquitously transcribed A. americanum serpin 19 is an anti-coagulant protein, inhibiting the activity of five of the eight serine protease blood clotting factors. Pichia pastoris -expressed recombinant (r) A. americanum serpin 19 inhibits the enzyme activity of trypsin, plasmin and blood clotting factors (f) Xa and XIa, with stoichiometry of inhibition estimated at 5.1, 9.4, 23.8 and 28, respectively. Similar to typical inhibitory serpins, recombinant A. americanum serpin 19 forms irreversible complexes with trypsin, fXa and fXIa. At a higher molar excess of recombinant A. americanum serpin 19, fXIIa is inhibited by 82.5%, and thrombin (fIIa), fIXa, chymotrypsin and tryptase are inhibited moderately by 14–29%. In anti-hemostatic functional assays, recombinant A. americanum serpin 19 inhibits thrombin but not ADP and cathepsin G activated platelet aggregation, delays clotting in recalcification and thrombin time assays by up to 250 s, and up to 40 s in the activated partial thromboplastin time assay. Given A. americanum serpin 19 high cross-tick species conservation, and specific reactivity of recombinant A. americanum serpin 19 with antibodies to A. americanum tick saliva proteins, we conclude that recombinant A. americanum serpin 19 is a potential candidate for development of a universal tick vaccine. [ABSTRACT FROM AUTHOR]

Abstract: Important role of chemical interaction in flame extinction is numerically investigated in downstream interaction among lean (rich) and lean (rich) premixed as well as partially premixed H2- and CO-air flames. The strain rate varies from 30 to 5917 s−1 until interacting flames cannot be sustained anymore. Flame stability diagrams mapping lower and upper limit fuel concentrations for flame extinction as a function of strain rate are presented. Highly stretched interacting flames are survived only within two islands in the flame stability map where partially premixed mixture consists of rich H2-air flame, extremely lean CO-air flame, and a diffusion flame. Further increase in strain rate finally converges to two points. It is found that hydrogen penetrated from H2-air flame (even at lean flame condition) participates in CO oxidation vigorously due to the high diffusivity such that it modifies the slow main reaction route CO + O2 → CO2 + O into the fast cyclic reaction route involving CO + OH → CO2 + H. These chemical interactions force even rich extinction boundaries with deficient reactant Lewis numbers larger than unity to be slanted at high strain rate. Appreciable amount of hydrogen in the side of lean H2-air flame also oxidizes the CO penetrated from CO-air flame, and this reduces flame speed of the H2-air flame, leading to flame extinction. At extremely high strain rates, interacting flames are survived only by a partially premixed flame such that it consists of a very rich H2-air flame, an extremely lean CO-air flame, and a diffusion flame. In such a situation, both the weaker H2- and CO-air flames are parasite on the stronger diffusion flame such that it can lead to flame extinction in the situation of weakening the stronger diffusion flame. Important role of chemical interaction in flame extinction is discussed in detail. [Copyright &y& Elsevier]

Abstract: We previously identified a cross-tick species conserved tick feeding stimuli responsive Amblyomma americanum (Aam) AV422 gene. This study demonstrates that AamAV422 belongs to a novel group of arthropod proteins that is characterized by 14 cysteine amino acid residues: C23-X7/9-C33–X23/24-C58-X8-C67-X7-C75-X23-C99-X15-C115-X10-C126-X24/25/33-C150C151-X7-C159-X8-C168-X23/24-C192-X9/10-C202 predicted to form seven disulfide bonds. We show that AamAV422 protein is a ubiquitously expressed protein that is injected into the host within the first 24h of the tick attaching onto the host as revealed by Western blotting analyses of recombinant (r)AamAV422, tick saliva and dissected tick organ protein extracts using antibodies to 24 and 48h tick saliva proteins. Native AamAV422 is apparently involved with mediating tick anti-hemostasis and anti-complement functions in that rAamAV422 delayed plasma clotting time in a dose responsive manner by up to ∼160s, prevented platelet aggregation by up to ∼16% and caused ∼24% reduction in production of terminal complement complexes. Target validation analysis revealed that rAamAV422 is a potential candidate for a cocktail or multivalent tick vaccine preparation in that RNA interference (RNAi)-mediated silencing of AamAV422 mRNA caused a statistically significant (∼44%) reduction in tick engorgement weights, which is proxy for amounts of ingested blood. We speculate that AamAV422 is a potential target antigen for development of the highly desired universal tick vaccine in that consistent with high conservation among ticks, antibodies to 24h Ixodes scapularis tick saliva proteins specifically bound rAamAV422. We discuss data in this study in the context of advancing the biology of tick feeding physiology and discovery of potential target antigens for tick vaccine development. [Copyright &y& Elsevier]

Abstract: Metal current collectors, offering a good connection between the active materials and the external circuit, is an important component in a rechargeable lithium ion battery. Some necessary thermal treatment in the battery fabrication and assembly procedure results in current collectors with some non-negligible reversible energy capacities; however, these energy capacities were negligible in the previous references. In this research, for the first time, we investigated the influence of the thermal treatment of current collectors (such as copper foil and stainless steel disk) on energy capacities. Our results indicate that different current collector materials have different thermal treatment effects on their electrochemical performance. The non-negligible capacity contribution is closely related to the treatment temperature. [Copyright &y& Elsevier]

Abstract: Current collectors are typically electrochemically inactive in lithium ion batteries. However, they might affect the whole cell performance once they are thermally treated during battery assembly procedure. The goal of this study is to investigate the heat treatment effect of Ni foam current collectors on the electrochemical performance in lithium ion batteries. Ni foams were thermally treated at different temperatures in air, and they were characterized in terms of morphology, structure and electrochemical performance in a half cell. SEM images showed that Ni foam surface became coarse with an increase of the heating temperature. Thermally treated Ni foams below 200°C had negligible capacity of about 0.025mAh, but above 300°C showed certain capacity up to 3.13mAh at 30th cycles due to the oxide layer formed by the thermal oxidation of the Ni foam. The results demonstrate that the thermal treatment of Ni foam current collector promotes the gradual oxidation on the Ni foam surface with nonnegligible capacity contribution. [Copyright &y& Elsevier]

The talA gene encoding transaldolase, the key enzyme in the nonoxidative pentose phosphate pathway, was amplified in a transformant Escherichia coli harboring the phbCAB operon to shift the metabolic flux of the hexose mono-phosphate shunt to the odd-ball biosynthesis pathway for poly-β-hydroxybutyrate overproduction. The PHB content in the transformant E. coli coharboring the phbCAB operon and talA gene increased from 28.2% to 52.3%, and the retarded cell growth was overcome. This increase seems to be mainly due to the concomitant supplies of the intermediates NADPH and acetyl-CoA, which are from the activated pentose phosphate pathway through the modulation of the talA gene and from the Embden–Meyerhof pathway. [Copyright &y& Elsevier]

A visualization study on the effect of forcing amplitude in tone-excited jet diffusion flames has been conducted. Visualization techniques are employed using optical schemes, which are a light scattering photography. Flame stability curve is attained according to Reynolds number and forcing amplitude at a fuel tube resonant frequency. Flame behavior is globally grouped into two from attached flame to blown-out flame according to forcing amplitude; one sticks the tradition flame behavior which has been observed in general jet diffusion flames and the other shows a variety of flame modes such as the flame of a feeble forcing amplitude where traditionally well-organized vortex motion evolves, a fat flame, an elongated flame, and an in-burning flame. Particular attention is focused on an elongation flame, which is associated with a turnabout phenomenon of vortex motion, and on a reversal of the direction of vortex roll-up. It is found that the flame length with forcing amplitude is the direct outcome of the evolution process of the formed inner flow structure. Especially the negative part of the acoustic cycle under the influence of a strong positive pressure gradient causes the shapes of the fuel stem and fuel branch part and even the direction of vortex roll-up to dramatically change. [Copyright &y& Elsevier]

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]

Ixodes scapularis long-term blood feeding behavior is facilitated by a tick secreted bio adhesive (tick cement) that attaches tick mouthparts to skin tissue and prevents the host from dislodging the attached tick. Understanding tick cement formation is highly sought after as its disruption will prevent tick feeding. This study describes proteins that form the inner core layer of I. scapularis tick cement as disrupting these proteins will likely stop formation of the outer cortical layer. The inner core cement layer completes formation by 24 h of tick attachment. Thus, we used laser-capture microdissection to isolate cement from cryosections of 6 h and 24 h tick attachment sites and to distinguish between early and late inner core cement proteins. LC–MS/MS analysis identified 138 tick cement proteins (TCPs) of which 37 and 35 were unique in cement of 6 and 24 h attached ticks respectively. We grouped TCPs in 14 functional categories: cuticular protein (16%), tick specific proteins of unknown function, cytoskeletal proteins, and enzymes (13% each), enzymes (10%), antioxidant, glycine rich, scaffolding, heat shock, histone, histamine binding, proteases and protease inhibitors, and miscellaneous (3–6% each). Gene ontology analysis confirm that TCPs are enriched for bio adhesive properties. Our data offer insights into tick cement bonding patterns and set the foundation for understanding the molecular basis of I. scapularis tick cement formation. [ABSTRACT FROM AUTHOR]

Abstract: The lipase-catalyzed production of optically active (S)-flurbiprofen was carried out in a dispersion reaction-system induced by chiral succinyl β-cyclodextrin (suβ-CD). The optimal reaction conditions were 500mM (R,S)-flurbiprofen ethyl ester ((R,S)-FEE), 600 units of Candida rugosa lipase per 1mmol of (R,S)-FEE, and 1000mM suβ-CD at 37°C for 72h. An extremely high enantiomeric excess of 0.98 and conversion yield of 0.48 were achieved in the dispersed aqueous phase reaction system containing chiral suβ-CD added as a dispenser and chiral selector. The inclusion complex formability of the immiscible substrate (S)- and (R)-form of FEE with suβ-CD was compared using a phase-solubility diagram, DSC, and 1H NMR. (S)-Isomer formed a more stable and selective inclusion complex with chiral suβ-CD. It was hydrolyzed much more selectively by lipase from C. rugosa, due to the selective structural modification through inclusion complexation with chiral suβ-CD. [Copyright &y& Elsevier]

Cyclodextrin (CD), a doughnut-shaped molecule having a hydrophilic surface and hydrophobic interior, can form inclusion complexes with various hydrophobic guest molecules inducing them into the emulsion state. The CD-induced emulsion reaction system was applied to the esterification between the hydrophobic oleic acid as the carboxylic acid and n-butanol as the alcohol catalyzed by lipase. The rate and yield were substantially increased in the CD-induced emulsion reaction system. The optimal reaction condition and the kinetic constants of the esterification reaction were determined. The enhancing mechanism was investigated through comparing the microscopic observation of the reaction mixtures, the inclusion complex formability, and the distribution of substrates, product and lipase at aqueous phase and CD-induced water immiscible phase in the CD-induced emulsion reaction system. The increase seems to have been caused by three reasons, the increased accessibility of lipase due to the emulsion formation, the shift of the equilibrium to the product formation side through the decrement of the affinity of butyl oleate with lipase, and the activation of lipase adsorbed on interface of oil-droplets in CD-induced water immiscible phase. [Copyright &y& Elsevier]

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]

Abstract: Numerical study on the interaction between methane–air and syngas–air premixed flames is conducted according to equivalence ratio and global strain rate in detailed chemistry. This study targets at understanding of an interacting combustion system as an alternative retrofit concept where one can modify the existing facilities minimally in industrial and power plant burners in order to reduce the emission of carbon dioxide. It is seen that methane premixed flame interacting with syngas premixed flame can be sustained even over the rich flammable limit of single methane premixed flame. The inspection of detailed flame structure such as the distributions of major species and chain carrier radicals, flame separation distance, spatial flow velocity, and spatial distribution of the rate of production and consumption of CH4, H2, and CO is also conducted to depict the flame interactions. The importance of global strain rate and thus the flame separation distance in the enhanced burning of methane premixed flame is also stressed through the inspection of the consumption rates of H2, CO, and CH4. Anomalous phenomena such as the migration of premixed flame cross the stagnation plane and the nearly constant flame separation distance are also discussed. [Copyright &y& Elsevier]

Abstract: Numerical study is conducted to understand the impact of fuel composition and flame radiation in flame structure and their oxidation process in H2/CO synthetic gas diffusion flame with and without CO2 dilution. The models of Sun et al. and David et al., which have been well known to be best-fitted for H2/CO synthetic mixture flames, are evaluated for H2/CO synthetic mixture flames diluted with CO2. Effects of radiative heat loss to flame characteristics are also examined in terms of syngas mixture composition. Importantly contributing reaction steps to heat release rate are compared for the synthetic gas mixture flames of high contents of H2 and CO, individually, with and without CO2 dilution. The modification of the oxidation pathways is also addressed. [Copyright &y& Elsevier]

Abstract: Experiments in low-strain-rate methane–air counterflow diffusion flames diluted with nitrogen have been conducted to study flame extinction behavior and edge flame oscillation in which flame length is less than the burner diameter and thus lateral conductive heat loss, in addition to radiative loss, could be high at low global strain rates. The critical mole fraction at flame extinction is examined in terms of velocity ratio and global strain rate. Onset conditions of the edge flame oscillation and the relevant modes are also provided with global strain rate and nitrogen mole fraction in the fuel stream or in terms of fuel Lewis number. It is observed that flame length is intimately relevant to lateral heat loss, and this affects flame extinction and edge flame oscillation considerably. Lateral heat loss causes flame oscillation even at fuel Lewis number less than unity. Edge flame oscillations, which result from the advancing and retreating edge flame motion of the outer flame edge of low-strain-rate flames, are categorized into three modes: a growing, a decaying, and a harmonic-oscillation mode. A flame stability map based on the flame oscillation modes is also provided for low-strain-rate flames. The important contribution of lateral heat loss even to edge flame oscillation is clarified finally. [Copyright &y& Elsevier]