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Pathogen-mediated manipulation of arthropod microbiota to promote infection
Edited by Joao Pedra, University of Maryland School of Medicine, Baltimore, MD, and accepted by Editorial Board Member Carolina Barillas-Mury December 8, 2016 (received for review August 17, 2016)
The importance of arthropod microbiota in the capacity of pathogens (including malaria and flaviviruses, among others) to persist in vectors and cause infection is just beginning to be appreciated. The influence of pathogens, either directly or indirectly, to manipulate vector microbiota for their own benefit, has not been described. In this study, we demonstrate that a pathogen can use an arthropod molecule to alter vector microbiota and enhance infection. We believe that this work will help others consider that pathogens are not passive microbes when they enter the arthropod vector but actively influence vector gene expression that can manipulate the local environment (in this case the microbiota) and facilitate pathogen infection of the vector.
Arthropods transmit diverse infectious agents; however, the ways microbes influence their vector to enhance colonization are poorly understood. Ixodes scapularis ticks harbor numerous human pathogens, including Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis. We now demonstrate that A. phagocytophilum modifies the I. scapularis microbiota to more efficiently infect the tick. A. phagocytophilum induces ticks to express Ixodes scapularis antifreeze glycoprotein (iafgp), which encodes a protein with several properties, including the ability to alter bacterial biofilm formation. IAFGP thereby perturbs the tick gut microbiota, which influences the integrity of the peritrophic matrix and gut barrier—critical obstacles for Anaplasma colonization. Mechanistically, IAFGP binds the terminal d-alanine residue of the pentapeptide chain of bacterial peptidoglycan, resulting in altered permeability and the capacity of bacteria to form biofilms. These data elucidate the molecular mechanisms by which a human pathogen appropriates an arthropod antibacterial protein to alter the gut microbiota and more effectively colonize the vector.
Arthropods serve as vectors of numerous pathogens that cause important human and animal diseases. Many of these arthropods are blood feeders and transmit or acquire microorganisms during engorgement (1, 2). Arthropods harbor a diverse group of native microbes ranging from bacteria to fungi, and the microbiota play critical functions in vector physiology, nutrition, and digestion (3⇓⇓⇓⇓–8). Microbial communities within vectors have important roles in the ability of specific pathogens to colonize and persist within the arthropod, and also to be efficiently transmitted to the mammalian host (9⇓–11). In arthropods, the microbiota is associated with a biofilm, generated from diverse bacterial species. These microbial biofilms are necessary for establishing successful symbiotic relationships with their arthropod hosts (12⇓–14).
Ticks are obligate blood-sucking ectoparasites that host a variety of disease-causing pathogens, including Borrelia, Rickettsia, Anaplasma, Ehrlichia, Francisella, and Babesia (15). The diversity of microbial communities has been characterized in different ticks (16⇓⇓⇓⇓–21). The effect of tick microbiota on the transmission of human infectious diseases, and the molecular mechanisms underlying these processes, has only recently received attention. We have demonstrated the importance of the normal gut microbiota of the blacklegged tick, Ixodes scapularis, a major vector of the Lyme disease agent, Borrelia burgdorferi, in the effective colonization of the tick by the spirochete (10). Understanding the interactions between arthropod gut microbiota and the pathogens that ticks transmit, in both a positive and negative manner, may provide new strategies for controlling and preventing vector-borne diseases.
Anaplasma phagocytophilum is an obligate intracellular pathogen transmitted by I. scapularis. This pathogen causes human granulocytic anaplasmosis, an illness that can result in fever, headache, muscle pains, and pancytopenia. In the northeastern United States, A. phagocytophilum is primarily maintained within I. scapularis and Peromyscus leucopus, the white-footed mouse (22). Ixodes ticks acquire Anaplasma during the blood meal. Upon entering the tick, the bacterium rapidly migrates from the gut and establishes itself in the salivary glands, where it is transstadially maintained through different stages of the arthropod life cycle (23, 24). The molecular mechanisms that Anaplasma uses to colonize and penetrate the tick gut barrier are not known. We recently demonstrated that the I. scapularis antifreeze glycoprotein (IAFGP) inhibits bacterial biofilms (25). In this study, we delineate how A. phagocytophilum appropriates IAFGP to alter the tick gut microbiota by inhibiting biofilm development, thereby enabling this pathogen to efficiently colonize the tick vector.
Tick Microbiota Are Involved in A. phagocytophilum Colonization of the Gut.
We first examined whether A. phagocytophilum modulates the tick microbiota. Illumina sequencing of amplified 16S rDNA assessed the diversity of bacterial species in A. phagocytophilum-infected and uninfected nymphs. At the taxonomic rank of order, distinct differences were noted in the relative abundance of bacteria between uninfected and A. phagocytophilum-infected nymphal guts (SI Appendix, Fig. S1 A and B). Post analysis and screening (see SI Appendix, SI Materials and Methods and Tables S1 and S2) we observed eight bacterial genera including Acinetobacter, Pseudomonas, Rickettsia, Lysinibacillus, Cornybacterium, Staphylococcus, Enterococcus, and Delftia that were identified in both experimental groups, but at different relative levels of abundance (Fig. 1A). Enterococcus and Rickettsia were decreased in A. phagocytophilum-infected ticks, whereas Pseudomonas was increased in A. phagocytophilum-infected ticks, in comparison with uninfected ticks (Fig. 1B). Principal coordinate analysis of the distance of microbial communities showed a clear separation between uninfected and A. phagocytophilum-infected nymphs (Fig. 1C). These results show that A. phagocytophilum alters the relative composition of the tick microbiota when it infects the tick gut. To examine the role of tick microbiota in A. phagocytophilum colonization, clean nymphal ticks were fed on gentamicin-treated mice that had been infected with A. phagocytophilum. Gentamicin kills many bacteria but not A. phagocytophilum. Quantitative reverse transcription (qRT)-PCR analysis of the bacterial burden in ticks engorging on gentamicin-treated mice, that was demonstrated to possess an altered microbiome (SI Appendix, Figs. S1 C−E and S2), significantly increased A. phagocytophilum colonization, compared with control mice (Fig. 1 D and E). These results demonstrate that the gut microbiota plays an important role in A. phagocytophilum colonization of ticks.
The Peritrophic Matrix Is Essential for Colonization of the Tick by A. phagocytophilum.
The peritrophic matrix (PM) is a glycoprotein-rich layer that separates the epithelial cells from the tick gut lumen, and peritrophin is the major component of the PM. We determined whether the PM plays a role in A. phagocytophilum colonization of ticks. The presence of A. phagocytophilum influenced the expression of several peritrophin genes, as assessed by qRT-PCR. The expression levels of peritrophin-1, peritrophin-2, and peritrophin-4 were significantly decreased in A. phagocytophilum-infected nymphs compared with uninfected nymphs (Fig. 2 A, B, and D). In contrast, expression of peritrophin-3 and peritrophin-5 was not altered in A. phagocytophilum-infected ticks (Fig. 2 C and E). Nymphs that fed on A. phagocytophilum-infected mice showed a decrease in thickness of the PM compared with nymphs that were fed on control mice (Fig. 2 F and G). To further examine the role of the PM in A. phagocytophilum colonization, RNAi was used to silence peritrophin expression. A decrease in peritrophin expression, using a dsRNA mixture of peritrophin-1, peritrophin-2, and peritrophin-4 injected into nymphal ticks (Fig. 2H), significantly enhanced the colonization of A. phagocytophilum in the tick gut and salivary glands, compared with control nymphs (Fig. 2 I and J). These results demonstrate that A. phagocytophilum infection induces, via the host, changes in the gut barrier.
IAFGP Modulates the Gut PM.
IAFGP, a secreted I. scapularis antifreeze glycoprotein, was recently determined to inhibit bacterial biofilm formation (25). The expression of iafgp is induced by cold exposure and A. phagocytophilum infection (26). The qRT PCR demonstrated that iafgp is highly induced in the tick gut by the presence of A. phagocytophilum (26), and silencing iafgp expression by RNAi diminishes the A. phagocytophilum burden in tick guts (Fig. 3 A and B). These data suggest that IAFGP plays an important role in A. phagocytophilum colonization of ticks.
To further investigate the role of IAFGP, a peptide (P1) (PARKARAATAATAATAATAATAAT) derived from IAFGP, which retains the antibiofilm properties (25), was injected into tick guts, and those nymphs were then fed on A. phagocytophilum-infected mice. A scrambled P1 (sP1) peptide (AATAATATAAARRAAAAPTTAKTT) served as control (25). The A. phagocytophilum burden in P1-injected tick guts was significantly increased compared with sP1-injected ticks, and more A. phagocytophilum migrated to the salivary glands as a consequence (Fig. 3 C and D). Because the PM was identified to play a role in A. phagocytophilum infection of tick guts (Fig. 2 F−I), we examined whether P1 affected the tick gut PM. The qRT-PCR showed that expression of peritrophin-2 and peritophin-4, but not peritrophin-1, was decreased in P1-injected ticks compared with sP1-injected control ticks (Fig. 3 E−G). Periodic acid−Schiff staining of 72-h-fed, P1-injected nymphs showed decreased thickness of the PM compared with sP1-injected control nymphs (Fig. 3 H and I). Having previously demonstrated that alterations in the PM thickness compromises the barrier integrity of the PM (10), we similarly evaluated the improved permeability of the PM by introducing Fluorescein-conjugated 500,000 molecular weight (MW) dextran into the guts of P1- or sP1-injected 24-h-fed nymphs and examined the unfixed guts by confocal microscopy. In contrast to sP1-injected ticks, the 500,000 MW dextran was predominantly localized outside gut lumen, suggesting that P1 injection of ticks reduces the thickness of the PM, permitting the large-sized dextran beads to pass from the gut lumen through the PM and out of the gut (Fig. 3J). We propose that this improvement in A. phagocytophilum infection is attributed to the thinning of the PM, which potentially allows for improved access of the pathogen through the PM to the midgut epithelium and subsequently it's egress to the salivary glands. Cumulatively, these results indicate that IAFGP, or its peptide derivative P1, facilitates A. phagocytophilum colonization of the tick gut by indirectly modulating the PM.
IAFGP Alters the Tick Microbiota and Influences the Gut Barrier.
In Ixodes ticks, the integrity of the PM is indirectly modulated by the gut microbiota, in part, by the normal microbiota's influence on the STAT pathway (10). Our previous study showed that IAFGP and P1 have antivirulence properties against diverse bacteria, and specific antibiofilm activity was observed using Staphylococcus aureus as our model organism (25). Based on these two lines of evidence, we hypothesized that IAFGP influences the tick gut microbiota, which results in alterations in the thickness and permeability of the PM.
Exopolysaccharides such as poly-N-acetylglucosamine (PNAG) are a major component of bacterial biofilms. PNAG is broadly distributed in diverse microbes and can be detected by a specific antibody (27). Having observed native biofilm formation in tick guts using a PNAG antibody (SI Appendix, Fig. S2A), we examined the presence of biofilms using the PNAG antibody in A. phagocytophilum-infected nymphal guts. The guts from A. phagocytophilum-infected and uninfected fed nymphs were collected, and PNAG was extracted and detected by immunoblot. The amount of detectable PNAG in A. phagocytophilum-infected nymphs was significantly decreased in comparison with uninfected nymphs (Fig. 4A). Moreover, endogenous IAFGP induced within A. phagocytophilum-infected nymphs was found to colocalize with these biofilms within tick guts, as detected by confocal microscopy (SI Appendix, Fig. S2B). These results suggest that A. phagocytophilum infection and induction of IAFGP within ticks influences native biofilm formation. To examine whether P1 can similarly influence biofilm formation in ticks, P1-injected guts from 96-h-fed nymphal ticks were collected. The amount of PNAG was significantly decreased in P1-injected nymphs compared with sP1-injected control ticks, which confirms that P1 can also modify biofilms in ticks (Fig. 4B). To visualize biofilms in the tick guts, we performed scanning electron microscopy (SEM). In normal tick guts, the commensal bacteria could form intact biofilm structures; however, A. phagocytophilum-infected tick guts showed disrupted biofilms (Fig. 4C, Top). Similarly, P1-injected tick guts showed a significant depreciation in biofilms compared with sP1-injected ticks (Fig. 4C, Bottom).
The microbiota of arthropods and mammals has long been associated with robust biofilms (12, 28). To further explore whether altering the biofilm has an effect on the whole tick gut microbial community, we compared the microbiota profiles of P1-injected and sP1-injected ticks. Bacteria of the genera Acinetobacter and Delftia were more abundant in P1-injected nymphs than sP1-injected nymphs, whereas Enterococcus and Rickettsia genera were significantly decreased in P1-injected nymphs (Fig. 4 D and E). Enterococci, as an example, are robust biofilm formers (29, 30). Microbiota and Enterococcus-specific qPCR analysis from individual and pooled samples further confirmed that the relative abundance of Enterococci was dramatically decreased in P1-injected tick guts compared with sP1-injected ticks (Fig. 4 F−H). The reduction of Enterococcus in P1-injected ticks could therefore contribute to the change in biofilm formation and, thus, the overall architecture of the gut microbiome. Because we previously observed that A. phagocytophilum-infected ticks were also decreased in Enterococci sp. (Fig. 1B and SI Appendix, Fig. S2 C−E), this would further suggest a role for IAFGP in manipulation of the tick microbiota and their associated biofilms.
IAFGP Binds to the d-alanine Residue of Bacterial Peptidoglycan.
As alterations in biofilm formation and the microbiota impacted Anaplasma colonization, we examined the mechanism by which IAFGP interferes with biofilm development. Enterococci, a genus known to produce tenacious biofilms, were consistently observed as being negatively impacted within ticks through A. phagocytophilum colonization or P1 injection (see SI Appendix, Fig. S2 C−E and Fig. 4 F−H). Because we had previously documented that binding of IAFGP's binding to S. aureus adversely impacted the bacterium's capacity to form biofilms (25), we similarly tested a panel of Enterococci species including Enterococci faecalis, Enterococci faecium, and vancomycin-resistant E. faecalis to bind either IAFGP or P1.
Each of these species has been represented in different arthropod populations including Rhipicephalus ticks, some of which are commonly known as cattle ticks (3, 31, 32). Recombinant GST-tagged IAFGP, but not GST alone, bound to each of the three species with varying affinities: Binding to E. faecalis was the strongest, followed by E. faecium and then vancomycin-resistant E. faecalis (Fig. 5A). Similar binding affinities were observed with P1 relative to sP1 (SI Appendix, Fig. S3A). Previous work showed that IAFGP binds to the bacterial surface and, more importantly, that both IAFGP and P1 bind S. aureus peptidoglycan in vitro (25) (SI Appendix, Fig. S3C). Binding of IAFGP or P1 to peptidoglycan extracted from each Enterococcus species was assessed by using a wheat germ agglutinin (WGA) antibody that specifically detects bacterial peptidoglycan. The strongest binding to biotinylated GST-tagged IAFGP or peptide P1 (immobilized to streptavidin beads) was detected with peptidoglycan muropeptides (associated fraction) extracted from E. faecalis, followed by E. faecium (Fig. 5B and SI Appendix, Fig. S3B). Minimal binding was noted with vancomycin-resistant E. faecalis (Fig. 5B and SI Appendix, Fig. S3B). Comparing the previously obtained results with S. aureus (25) and the current data from each of the three Enterococci species, we recognized that S. aureus and E. faecalis are very similar in their peptidoglycan structure (except for their cross-bridge) (33). We therefore postulated that IAFGP binds the pentapeptide chain of bacterial peptidoglycan at its terminal amino acid residues (Fig. 5C). E. faecium and vancomycin-resistant E. faecalis show greater variability in their stem peptides, with only 3% of E. faecium having the same terminal d-alanyl-d-alanine amino acids as S. aureus or E. faecalis (34); vancomycin-resistant E. faecalis replaces its terminal d-alanine residue with lactate, contributing to its vancomycin resistance.
Observing similar bacterial and peptidoglycan binding between S. aureus and E. faecalis, we determined the specific residues required for IAFGP or peptide binding using S. aureus as our model bacteria. S. aureus peptidoglycan grown in tryptic soy broth (TSB), typically shares a 3:2 ratio of d-alanyl-d-alanine as its terminal pentapeptide residues versus a single terminal d-alanine, resulting from the hydrolytic transpeptidation or carboxypeptidation reaction (Fig. 5D and SI Appendix, Fig. S4 and Table S4). Supplementing medium with an alternate d-amino acid such as d-serine causes S. aureus to incorporate the newly supplemented d-amino acid into the carboxyl terminus of the stem peptides (35). This results in the d-alanyl-d-serine terminating peptidoglycan stem residue in ∼75% of all muropeptides compared with only 6% of cells retaining the native d-alanyl-d-alanine (Fig. 5 C and D and SI Appendix, Fig. S4 B and C and Table S4). Ultra performance liquid chromatography (UPLC) analysis also revealed a significantly lower percentage of crosslinking between the glycan strands for d-serine and IAFGP cultured bacteria (SI Appendix, Fig. S4E).
Observing an overwhelming shift toward replacement of the terminal alanine residue with serine at the terminal muropeptide site with supplementation of 125 mM d-serine, we determined IAFGP binding to muropeptides grown from either medium, similar to what was previously done with the Enterococci (Fig. 5B and SI Appendix, Fig. S3B). Biotinylated GST-tagged IAFGP, but not biotinylated GST alone, successfully bound muropeptides (associated fraction) obtained from TSB medium, as detected by the WGA antibody (Fig. 5E). There was no signal detected by WGA antibody with muropeptides obtained from d-serine cultured cells. The WGA antibody, which recognizes the N-acetylglucosamine (NAG) sugar, was used because all of the muropeptides obtained from either TSB or d-serine cultures were associated with both the N-acetylmuramic acid (NAM) and NAG sugar residues (SI Appendix, Fig. S4C). To confirm this binding interaction between IAFGP and muropeptides with a terminal (fifth) d-alanine residue, we generated five amino acid synthetic pentapeptides with either d-alanine or d-serine as their terminal (fifth) residue. Using these biotinylated pentapeptides, bound to streptavidin beads, GST-tagged IAFGP, but not GST alone or the PBS buffer control, could be detected in the associated fraction of pentapeptides with d-alanine residue (Fig. 5F). This confirmed IAFGP binding to the muropeptides generated with the d-alanine terminal residue, but not to those with the d-serine residue. Histidine (His)-tagged P1, and not scramble peptide sP1 or PBS, was also found to similarly bind synthetic muropeptides with a terminal d-alanine residue (SI Appendix, Fig. S3D). Similarly, neither IAFGP nor P1 could bind E. faecalis or E. faecium whole bacteria cultured in media supplemented with125 mM d-serine (SI Appendix, Fig. S3 E and F).
Beyond the peptidoglycan layer, other cell wall glycopolymers including teichoic acids (TAs) decorate the surface of Gram-positive bacteria. TAs including wall teichoic acids (WTAs) and lipoteichoic acids (LTAs) are a constant motif of Gram-positive cell envelopes, and they form part of the fabric of the cell wall attaching either to the peptidoglycan or to membrane lipids, respectively (36). Both WTAs and LTAs are usually modified with d-alanine residues (37). To determine whether binding of IAFGP is specific for terminal d-alanine residues of peptidoglycan and not those of WTAs, we tested IAFGP and P1 binding to S. aureus mutants deficient in producing WTA (Fig. 5G and SI Appendix, Fig. S3G). IAFGP and P1 demonstrated consistent binding across wild-type and mutant S. aureus strains without any significant depreciation. In addition, there was no significant binding of IAFGP to LTA (SI Appendix, Fig. S3H). These data demonstrate a direct interaction between IAFGP (or P1) and the terminal d-alanine residue of the peptidoglycan chain of Gram-positive bacteria like S. aureus or E. faecalis. Gram-negative bacteria have been previously demonstrated to possess this same terminal d-alanine amino acid moiety (38, 39). However, multiple reasons including (i) access to peptidoglycan being limited, due to the presence of the outer membrane, and (ii) the efficient action of the DD-carboxypeptidase enzymes, that converts almost all of their pentapeptide (NAG-NAM-l-ala-d-glu-mDap-d-ala-d-ala) to tetrapeptides (NAG-NAM-l-ala-d-glu-mDap-d-ala), skews the binding specificity of either IAFGP or its peptide derivative P1 more toward Gram positives. These two major criteria notwithstanding, muropeptides from Gram-negative bacteria, as demonstrated by the Escherichia coli DV9000 mutant, would also be able to bind either IAFGP or P1 (SI Appendix, Fig. S5 A and B).
Alterations in Peptidoglycan Reduce S. aureus and E. faecalis Biofilm Formation.
Because IAFGP binds the terminal d-alanine of the peptidoglycan peptide stem, we investigated the relationship between this observation and the antibiofilm activity of IAFGP (25). Our studies in serine-supplemented medium provided a model system in which to study how S. aureus or E. faecalis behaves with an altered peptidoglycan. We established a connection between biofilm formation and peptidoglycan by performing in vitro biofilm assays with bacterial cultures grown in traditional rich medium (native peptidoglycan) versus medium supplemented with 125 mM d-serine (errant peptidoglycan) (Fig. 5C). S. aureus and E. faecalis cultures passaged in 125 mM d-serine showed a 75% decrease in biofilm formation (Fig. 6A). Supplementation with 125 mM d-alanine, the native residue at the carboxyl terminus, did not have a significant decrease in biofilms compared with medium alone or supplementation with d-serine (Fig. 6A). This result suggested that changing the terminal residue from alanine to serine resulted in the drastic decrease in biofilm formation. Growth and viability for each bacterium were unaffected under any of these treatment conditions (SI Appendix, Fig. S6).
Because IAFGP, P1, and now d-serine severely alter bacterial biofilms, we next investigated whether d-serine's antibiofilm phenotype could be observed within ticks. Microbiota and Enterococcus-specific qRT-PCR analysis for individual and pooled samples using d-serine-treated nymphal ticks further demonstrated that the relative abundance of Enterococci was dramatically decreased with d-serine compared with injection with control d-alanine (Fig. 6B and SI Appendix, Fig. S7 A–D). Visualization of tick gut biofilms by SEM showed a severe depreciation in biofilm-like structures, associated fibers, and matrix material within d-serine-treated ticks compared with d-alanine-treated ticks (Fig. 6C). To confirm that the changes within the microbiota and reductions in biofilms were attributed to the administered treatment—d-serine, P1 (Fig. 4), or gentamicin (Fig. 1)—IAFGP expression levels within these ticks were determined (SI Appendix, Fig. S8A). Our data indicate that alterations to the tick microbiota and deleterious effects on biofilms, previously observed with IAFGP through A. phagocytophilum acquisition, in these instances was not due to increased iafgp expression (SI Appendix, Fig. S8A). Moreover, in contrast to recent work (40), we have additionally ruled out the possibility of host-related factors like IFN-γ to induce expression of IAFGP or improved infection of A. phagocytophilum (SI Appendix, Fig. S8 B and C).
Because IAFGP or d-serine is able to bind or incorporate into peptidoglycan, thereby significantly affecting biofilms, we hypothesized that these treated cells would show a change in cellular morphology. Therefore, we examined physical differences in bacterial cell morphology using transmission electron microscopy. For this, S. aureus cells were grown in rich versus serine-supplemented media. S. aureus cells passaged in d-serine exhibited a more diffuse peptidoglycan layer with distinct ruffled features (Fig. 6D). This may be associated with the decrease in cross-linked peptidoglycan, as shown by UPLC (see SI Appendix, Fig. S4E). Interestingly, a similar morphological phenotype was exhibited by cells grown in 0.1 mg/mL of IAFGP but not by those grown in GST alone (Fig. 6D). Although neither IAFGP- nor GST-treated cells showed any difference in the peptidoglycan composition (SI Appendix, Fig. S4D), IAGFP-treated cells, similar to d-serine, were significantly reduced in their cross-linking (SI Appendix, Fig. S4E). The peptidoglycan-occupied area for cells grown with either d-serine or IAFGP was significantly higher compared with controls (SI Appendix, Fig. S9A). It is likely that this phenomenon extends beyond S. aureus, as we also observed biofilm inhibition by P1 in other Gram-positive bacteria. Biofilm biomass was dramatically reduced, in vitro, by two other Staphylococcus sp., Streptococcus mutans, and Corynebacterium pseudodiptheriticum in the presence of P1 (SI Appendix, Fig. S7E).
Biofilm inhibition by IAFGP or P1, mediated by peptidoglycan binding, is likely to be important in reducing the barrier for clearance of bacteria by the immune system, antimicrobial peptides, or antibiotics. We tested this hypothesis by determining the permeability of the peptidoglycan layer using a fluorescent WGA protein. S. aureus cells grown in d-serine or IAFGP showed significantly higher fluorescent intensities compared with TSB or other control growth conditions (Fig. 6E). The diffused peptidoglycan layer with IAFGP or d-serine (Fig. 6D and SI Appendix, Fig. S9A) would suggest better penetration by the fluorescent WGA. WGA intercalates through the layers of peptidoglycan to gain better access to their native binding site, the NAG sugar residues. Alternatively, however, the increase in fluorescence with IAFGP or d-serine could implicate more peptidoglycan within these respective treatment conditions. To differentiate between these two plausible hypotheses, we determined the permeability of the cell using the cell membrane as a binding target. Permeability past the peptidoglycan, with subsequent access to the cell membrane, was tested using the membrane-binding fluorescent dye, FM 4-64. Treatment with FM 4-64 revealed increased fluorescent signal intensities for cells cultured with IAFGP or d-serine compared with controls (SI Appendix, Fig. S9B), indicating an increase in permeability. These data demonstrate that alterations in peptidoglycan, mediated by d-serine incorporation or via IAFGP binding, inhibit biofilm formation and alter the peptidoglycan in a nonlethal manner, allowing for enhanced permeability.
A. phagocytophilum, an obligate intracellular pathogen, enters its tick host following a blood meal and rapidly penetrates the gut barrier and migrates to salivary glands, where it persists (41, 42). Gut commensal microbes play critical roles in enabling hosts to resist invading bacteria, and alterations of the microbial gut community of vectors can influence infection loads with pathogens (43). In our A. phagocytophilum−I. scapularis model, distinct differences were noted in the relative abundance and composition of the microbiota of ticks feeding on uninfected, or A. phagocytophilum-infected, mice, indicating that the presence of A. phagocytophilum alters the tick gut microbial community. Numerous factors including exposure to environmental bacteria, geographic location, tick species, gender, developmental status, and time since molting and feeding, among others, are expected to play an important role in contributing to the microbiome composition in ticks (10, 44, 45). Therefore, although the tick gut-associated microbes might not be conserved across different regions, with several published reports suggesting a large diversity (46⇓⇓–49), and others suggesting that inflated diversities are due to environmental contaminants (50), we used multiple criteria (see SI Appendix) to accurately assess the microbial composition of our laboratory-reared and murine host-fed I. scapularis nymphs. Based on our stringencies and exclusion criteria, we observed eight genera including Acinetobacter, Pseudomonas, Rickettsia, Lysinibacillus, Corynebacterium, Staphylococcus, Enterococcus, and Delftia that were consistently observed, independent of treatment condition. Comparing the unfed nymphal microbiome in our earlier work (10) to the fed nymphal microbiome in this work, genera such as Streptococcus [observed in some tick cohorts, although excluded from this main analysis (SI Appendix, Table S3)], Rickettsia, Pseudomonas, Acinetobacter, Staphylococcus, and Delftia are observed in both, albeit at different proportions. Genera such as Acidovorax, Brevundimonas, Commamonas, Stenotrophomonas, and Sphingobium were observed in the unfed nymphs but not in the fed nymphs, and genera such as Lysinibacillus, Corynebacteria, and Enterococcus were observed only in the fed nymphs. Some of these differences could be attributed to the fed versus unfed status of the nymphs or differences in the sequencing platforms used: 454 sequencing versus Illumina MiSeq platform for the 16S rDNA sequencing used in this study. Within the rigors of our experimental framework, we demonstrate that there are significant variations in the relative abundance of gut microbial genera during A. phagocytophilum colonization of ticks. Our paper unravels the potential mechanism by which these changes occur and invokes the tick protein, IAFGP. Additionally, we highlight the mechanism by which A. phagocytophilum coopts this tick protein to best potentiate its infection in ticks.
IAFGP, the I. scapularis antifreeze glycoprotein, is induced by ticks in the presence of A. phagocytophilum (26), and silencing iafgp impairs A. phagocytophilum colonization of the gut. Independently, we demonstrated IAFGP to inhibit biofilm formation among Gram-positive pathogens such as S. aureus (25). Because A. phagocytophilum colonization reduced the abundance of Gram-positive biofilm-forming species like the Enterococci, we hypothesized that IAFGP alters the tick microbiota by inhibiting bacterial biofilms in the gut. Biofilms have been demonstrated to serve important roles in facilitating pathogen colonization in arthropod intestines (12⇓–14). To elucidate the importance of IAFGP in shaping the tick gut microbiota, we injected the antibiofilm peptide, P1, derived from IAFGP (25), and showed that this improved A. phagocytophilum infection, in ticks. Exopolysaccharides such as PNAG, a major component of bacterial biofilms, are broadly distributed among diverse microbes (27). Using antibodies directed against PNAG as a proxy to gauge biofilms within the tick, P1-injected I. scapularis guts showed a marked reduction of biofilms, similar those with A. phagocytophilum infection. SEM confirmed this phenotype with normal tick guts (clean or sP1-injected) having thicker and well-connected matrix-like material with embedded bacteria such as cocci representing robust biofilms, whereas A. phagocytophilum-infected or P1-injected tick guts showed looser biofilms without connecting fibers or matrix-like representative material indicating reduced biofilms. In an effort to delineate the molecular mechanism of IAFGP expression and P1 injection adversely affecting biofilms, we sought to use tick gut commensal Enterococci, a species that was markedly reduced within our microbiome analysis during A. phagocytophilum infection or P1-treatment, and compare results with S. aureus, which also forms robust biofilms. Using three different Enterococci species (E. faecalis, vancomycin-resistant E. faecalis, and E. faecium), we identified a difference in the capacity of IAFGP and its peptide homolog to bind the individual species. Because IAFGP binds S. aureus peptidoglycan (25), we hypothesized that the variability in binding to the Enterococci could be attributed to the difference in the peptidoglycan composition. We now demonstrate that IAFGP binds to the terminal d-alanine residue of the bacterial peptidoglycan peptide moiety—common to E. faecalis and S. aureus.
d-amino acids are readily incorporated into bacterial peptidoglycan by replacing the terminal residue with the newly provided d-amino acid (35), and these types of alterations inhibit biofilm formation (51) including those by tick gut commensals like E. faecalis. Because IAFGP and P1 can bind the terminal d-alanine residue of bacterial peptidoglycan, use of compounds like d-serine that prevent biofilms and biochemically alter peptidoglycan provide an independent assessment of changes within the tick gut in an Anaplasma (IAFGP) free system. Similarities in the relative abundance of Enterococci within the tick gut, found by microbiome analysis or SEM imaging of tick gut biofilms when treated with d-serine, to those during A. phagocytophilum infection or P1 injection of tick guts support the mechanistic action of IAFGP's peptidoglycan binding activity on modulating the tick microbiota. d-amino acids interfere with the linking of biofilm adhesion proteins, and with cell wall remodeling (51⇓⇓–54). Therefore, we hypothesized that IAFGP binding to peptidoglycan could cause alterations in bacterial physiology and morphology that would reduce the ability of Gram-positive bacteria to form tenacious biofilms—adversely affecting the relative composition of the gut microbiota. IAFGP binding to peptidoglycan resulted in a more permeable peptidoglycan with significantly reduced cross-linking between the glycan strands; this is of particular importance because the increased permeability increases the sensitivity of bacteria to host immune defenses such as antimicrobial peptides and other toxins (55, 56), which could eventually cause a change in the composition of the tick guts microbial community.
Effects of IAFGP against the microbiome and specific genera are critical for optimal A. phagocytophilum infection of ticks. In an effort to tease out the specificity of IAFGP's or P1's binding, multiple factors were evaluated. During tick feeding, depending on the host and other biological factors, blood ingested can contain varying levels of free amino acids, including free d-alanine (nanomolar range) (57). Concentrations only greater than 100 mM of free d-alanine were able to outcompete IAFGP's binding to peptidoglycan and inhibit the antibiofilm properties of IAFGP or P1, highlighting IAFGP's or P1's strong binding efficiency to the terminal d-alanine residues of the bacterial peptidoglycan (SI Appendix, Fig. S10). Moreover, using synthetic variations of the IAFGP or P1 sequence (SI Appendix, Table S5), we could confirm the binding specificity of either protein or peptide to the terminal d-alanine residue of peptidoglycan and its inextricable link to inhibiting biofilms among Gram-positive bacteria (SI Appendix, Figs. S11 and S12). These data, taken cumulatively, not only confirm a level of specificity for IAFGP or P1, for Gram-positive bacterial peptidoglycan, that terminate in a d-alanine residue, but also in restricting biofilm formation. Although Gram-positive bacteria are adversely affected, Gram-negative bacteria like Pseudomonas sp. or E. coli neither bind IAFGP or P1 nor are inhibited in their biofilms on account of their internalized peptidoglycan and absence of the terminal (fifth) d-alanine residue on their peptidoglycan (SI Appendix, Fig. S5) (58⇓⇓–61). This is particularly pertinent because A. phagocytophilum is technically categorized as a Gram-negative organism. However, unique physical characteristics and previously determined molecular evidence suggest the absence of peptidoglycan from A. phagocytophilum (62⇓–64), which would nullify any effect IAFGP (or P1) would have on A. phagocytophilum. Our earlier work described the role for elevated levels of IAFGP, upon A. phagocytophilum infection of Ixodes ticks, to protect ticks from the cold environment (26). Therefore, IAFGP aids Anaplasma in two manners: by increasing the ability of ticks to survive in the winter and therefore indirectly increasing the chance of Anaplasma transmission to mice, and by altering the tick microbiomes and their associated biofilms, thereby enhancing its colonization within the tick vector.
Understanding the functional consequence of tick–microbiome interactions is fundamental to developing new paradigms to control tick-transmitted pathogens. We previously demonstrated that dysbiosis causes the tick gut microbiota to interact differently with epithelial cells and diminishes the activity of a global transcriptional regulator, STAT (10). STAT controls expression of different downstream target genes involved in immunity and cell repair, including peritrophin, the major component of the PM (10). Interestingly, we previously showed that A. phagocytophilum infection of Ixodes ticks reduces expression of stat, and that colonization is enhanced when stat is silenced (65); this suggests a role for the PM as a contributing factor during A. phagocytophilum infection of the tick. The glycan-rich PM layer separates the gut lumen from the epithelial cells and provides a protective barrier in arthropods against microbes and components of the incoming blood meal (66). Periodic acid-Schiff (PAS) staining of A. phagocytophilum-infected and P1-injected nymphal guts showed a significant decrease of the PM-like layer, which leads to increased PM permeability. We propose that the improvement in A. phagocytophilum infection is attributed to the thinning of the PM, which potentially allows for improved access of the pathogen through the PM to the midgut epithelium and, subsequently, it's egress to the salivary glands. One could hypothesize that, during A. phagocytophilum infection, a series of events that include (i) alterations in the microbiome and reduction in biofilms through IAFGP and (ii) the thinning of the PM through stat, barriers that would otherwise prevent effective contact with the midgut epithelium, are accommodated so as to allow for the strongest colonization of the tick. Although thinning of the PM and the consequent increased PM permeability is conducive to increased A. phagocytophilum colonization, B. burgdorferi colonization is jeopardized by a compromised PM barrier (10). B. burgdorferi migrates through the PM to colonize the epithelial cells, and persists in this location, where an intact PM protects the spirochete from the toxic components of the gut lumen. The contrasting influence of PM integrity on two tick-borne pathogens could potentially result in competition during a natural cocolonization setting. An earlier study by Levin and Fish (67) suggests interference between these two agents during transfer from coinfected mice to ticks. These observations might also explain, in part, the generally lower prevalence of A. phagocytophilum and B. burgdorferi-coinfected ticks (<1 to 6%) versus ticks coinfected with B. burgdorferi and Babesia microti (2 to 19%) (68). Nevertheless, further studies, using both laboratory and field coinfected ticks, will be required to delineate the role of microbiota on infections with multiple pathogens.
Defining the interactions between the tick (gut) and A. phagocytophilum colonization furthers our understanding of the key events that determine prevalence of this common tick-borne infectious disease. Although we have yet to elucidate how acquisition of this pathogen induces expression of IAFGP, our current findings highlight a functional link between IAFGP, the tick microbiota and biofilm formation, and the PM and reveal their functional consequence on A. phagocytophilum colonization. Similar to the gut microbiota of mammals, the microbiota associated with arthropods plays an important role in host development, pathogen resistance, nutrition, and physiology (69). Microbial symbionts can reduce vector competence, and specific gut microbiota play a protective role in Anopheles mosquitoes against Plasmodium colonization (70⇓⇓–73). Although knowledge about the function of microbiota in diverse arthropods is still limited, cross-talk between the arthropod, its gut microbiota, and incoming pathogens is likely to be important (9⇓–11, 69). This study demonstrates how a pathogen induces the arthropod to express a protein that directly inhibits bacterial biofilms, and that the effects of this protein on the tick microbiota enable the microbe to effectively colonize the vector. This paradigm—delineated with A. phagocytophilum and I. scapularis—is likely to be applicable to other vector-borne diseases.
Materials and Methods
All studies with mice were carried out following the animal protocol number 2014-07941 approved by Yale University's Institutional Animal Care and Use Committee (IACUC). The IACUC is governed by applicable Federal and State regulations, including those of the Animal Welfare Act (AWA), Public Health Service (PHS), NIH Office of Laboratory Animal Welfare Assurance Number A3230-01, and the United States Department of Agriculture (USDA), License and Registration Number 16-R-0001, and is guided by the US Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research and Training. Experimental details on Anaplasma infection of mice, microbiome sample preparation, Illumina pyrosequencing, sequence analysis and selection criteria, sectioning and staining of nymphs, bacterial strains and growth conditions, immunoblot analysis, tick RNA isolation, quantitative RT-PCR and microinjection, extraction and UPLC and MS/MS analysis of bacterial peptidoglycan, binding assays, biofilms assays, and microscopy can be found in SI Appendix. Details of primers used in this study can be found in SI Appendix, Table S6.
We thank Dr. Can Bruce for microbiota sequence analysis, Michael Schadt for excellent technical assistance with histology, Huihui Dong for technical assistance with assorted in vitro experiments, and Morven Graham (Yale School of Medicine, Center for Cellular and Molecular Imaging) for help with the microscopic imaging. We thank William Gray and Bradley Parry for critical input on the analysis strategy for the fluorescence microscopy. The S. aureus WTA mutants were provided by Dr. Andreas Peschel (University of Tubingen, Tubingen, Germany). This work was supported, in part, by a gift from the John Monsky and Jennifer Weis Monsky Lyme Disease Research Fund. The F.C. laboratory received funding support from Laboratory for Molecular Infection Medicine Sweden, Knut and Alice Wallenberg Foundation, Kempe and the Swedish Research Council (VR). A.K.Y. was supported by a Umeå Center for Microbial Research/VR postdoctoral position. This work was supported by National Institutes of Health Grant 41440. E.F. and C.J.-W. are investigators of the Howard Hughes Medical Institute.
↵1N.M.A. and L.L. contributed equally to this work.
- ↵2To whom correspondence may be addressed. Email: lei.liu.ll69{at}yale.edu or erol.fikrig{at}yale.edu.
Author contributions: N.M.A., L.L., F.C., C.J.-W., and E.F. designed research; N.M.A., L.L., B.L.J., A.K.Y., S.N., V.G., J.M.A., and F.C. performed research; K.K.J. and C.J.-W. contributed new reagents/analytic tools; N.M.A., L.L., B.L.J., A.K.Y., S.N., V.G., J.M.A., F.C., C.J.-W., and E.F. analyzed data; and N.M.A., L.L., S.N., C.J.-W., and E.F. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission. J.P. is a Guest Editor invited by the Editorial Board.
Data deposition: The sequences reported in this paper have been deposited in the NCBI Sequence Read Archive (SRA; accession no. PRJNA353730).
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1613422114/-/DCSupplemental.
Freely available online through the PNAS open access option.
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From the WorldNomads.com network
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Video hotspots and Coursera
Hi! I am creating a MOOC course for Coursera. It would be a video-based course and I would like to know if I can use the H5P hotspots feature in my videos so the students can interact with the video lectures. Reporting on completion of the tasks is not relevant to me. The most important feature is the ability to present interactive videos.
Hi Elegram,I'm not sure I
Hi Elegram,
I'm not sure I follow. Do you want to know if you can use H5P contents in Coursera? If so yes you can as long as they provide you the ability to insert HTML code which is the basis of the embed feature of H5Ps. I'm sorry I tried checking the site but I'm unable to create a course or something similar to it so I am unable to check.


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After securing hundreds of publicity features for my clients over the years, I can tell you one media feature can create a new world of opportunities for you. Getting featured in any media is a great time to Continue Reading
In honor of Martin Luther King Day, ICY PR is giving 3 tips on how to brand your Non-Profit organization in order to increase impact, visibility and serve. "Everybody can be great. Because anybody can serve." Continue Reading


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- $1 Items
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- Arizona Gold Treks Mining Geology Placer Prospecting
Product Description
Arizona gold Treks
Ronald S. Wielgus
Another great book by this author leading you to gold in areas of Arizona where many others haven't traveled. Author has mined in Arizona for years and shares his knowledge of places to go that you won't find anywhere else.
Happy Hunting!


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Wyoming Public Service Commission
Employee Spotlight
Lori Brand
Staff Attorney
Lori L. Brand is the Assistant Secretary and Staff Attorney with the Wyoming Public Service Commission where she has been employed since January 2012. Lori graduated with a Bachelor of Science in Social Science from the University of Wyoming in 1997. She received her J.D., graduating with honors, from UW College of Law in 2000 and was awarded the Order of the Coif. Lori was Managing Editor of the Land and Water Law Review 1999-2000 and worked for the Albany County Attorney's office during its prosecution of the Matthew Shepherd case. Following graduation, she served as a law clerk for then Chief Justice Larry Lehman of the Wyoming Supreme Court. In 2018, Lori received a Master of Public Administration from the University of Wyoming.
Before opening her own firm in 2007, Lori practiced with Gay Woodhouse Law Office P.C. and at Rothgerber, Johnson & Lyons LLP. Lori is licensed to practice law in Wyoming and Colorado and is admitted to all Wyoming and Colorado Courts, Federal District Court for the District of Wyoming, and the United States Supreme Court. She is a member of NARUC's National Conference of Regulatory Attorneys, NARUC's subcommittee on Rate Design, and the Wyoming Energy Bar and recently passed 10 years of service with the State of Wyoming.
Lori enjoys being engaged in her community and currently serves on Boards for the Cheyenne After-Hours Rotary Club, and Wyoming nonprofits Wyoming Children's Legal Center, Family Promise and Pathfinder. Recently during its 50th Anniversary Gala, Lori received an award from the Wyoming Trial Lawyers' Association in recognition for her years of service as the Editor in Chief of the Coffeehouse Magazine. Lori is married to Ray and they have a grown son Steven and an ornery tabby cat named Hobbes. They will be celebrating their 30th Anniversary in October 2019 with a trip to Italy and a Mediterranean cruise.
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It's nice to be able to sit down to write this post, nearly 3/4 of the way through my 12 Week Challenge, and be in a positive frame of mind. It probably helps that I'm sitting in a sunny window of a cosy cafe waiting for my breakfast to arrive – scrambled eggs with avocado, sausage and no toast. And a latte with full fat milk – don't mind if I do! Sorry if I sound like a skite, but it sure does feel nice knowing that I got up early this morning, went for a run and did some hill sprints and body weight exercise and was home by 9am ready to start the day.
This has been a good week (oh, you could tell?) I think the conclusions I came to last week – to chill out a bit, up the carbs, and relax on trying to implement the Naturopath's advice, were the right ones. I also deliberately messed with my usual routine. So I had an awesome PT session on Monday night and backed that up with a gym workout on Wednesday night and Friday night. Friday night! That's for drinking and dancing, right? Not necessarily 😉 Tuesday and Thursday I did nothing but lots of walking – which is in itself a bit of a feat considering how foul our weather has been in Melbourne this week.
I also crafted a pretty yummy menu for myself, and enjoyed it so much I'm going to repeat it almost exactly this week. I had a sausage curry with veggies for dinner three nights last week and while it was yummy, it left me with the munchies an hour or so later, so this week I'm going to be smarter with the calories. Sausages can be a bit of a treat but they are fatty and the protein really isn't enough to justify the calories. Tuesday night I had the munchies after dinner and ended up eating a rice cake with peanut butter and going over my calories by about 150, which I was pretty cross about. It's not much, but it's the principle of 'failing' which threatens to derail me every time. It was only the next day I looked back at my food diary and realised I'd stuffed up and hadn't actually eaten the cauliflower mashed with butter, so it's no wonder I was still peckish after dinner and craving something fatty.
Saturday night I experimented a bit with some vegan, gluten free and sugar free recipes. Only a year or so ago I would have laughed at that and thought, 'well what the hell is left if you take all that out'? The answer is – plenty! I think it's really exciting that there's a whole new world of food possibilities opening up to us, and it's become such a natural default position now to look for these healthier alternatives to old favourites. I'll blog separately about what I made. There was nothing low carb about the meal, but it was fun to get creative in the kitchen.
I'm ⅔ of the way through this challenge and I've already reconciled myself to the fact that I'm not going to meet the goal I originally set myself of 3% fat loss. However I did cheat the other night and tested my body fat at the gym and found it was down 1.4% so that's pretty exciting! If I can achieve a 2% fat loss overall, I'll be happy, since I know (and you now know too if you've been reading along!!) that I've really only stuck to the plan for probably only 6 of the 9 weeks I've been at this so far. I also cheated a couple of weeks ago and tested my body fat and found to my despair that it had gone UP apparently to 31% (!!!) which made absolutely no sense to me considering I felt leaner and the other measurements I'd taken had been encouraging. So I came home feeling like a failure and jumped online to google the accuracy of the bioimpedance machine and found that a +/- 2% margin for error needs to be expected, and one of the possible reasons is levels of hydration. So this time, Wednesday, I made sure I drank plenty of water in the afternoon as well as the morning and the result I think was more accurate.
So! This week the plan looks much like last week:
Breakfast: egg, fetta and spinach scramble with sweet potato
Lunch: Crunchy asian salad with smoked chicken
Dinner: Either ½ a chicken breast or 150gm beef stirfried with a pile of veggies, chili, oyster sauce, garlic and ginger.
This menu gives me a perfect macronutrient split: 20% carbs, and 40% each fat and protein. Total of about 1700 calories which is fine for exercise days.
PT as usual on Monday night (last week we got up to 50kg deadlifts which I'm happy about considering it had been a while since we'd done any proper dead-lifting). And as with last week, I'll go to the gym basically whenever I feel like it, and hopefully that's at least 3 times. I also need to try and get in at least one run in addition to the weekend one, as I'm not going to make quick enough progress going only once a week.
I may have mentioned that I'm leaving Australia in October to go travel the world (or a fair portion of it), and I'd dearly love to be comfortably running a 5K again before I leave, as this is something I can likely do just about anywhere in the world I happen to be!
So that's the plan, I feel good about it. Fingers crossed there are no self-sabotage meltdowns this week!
For previous 12 Week Challenge updates click here.


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Our Best Fortnite Skins List features the top rated skins from our community! These are the highest vote getting outfits in Fortnite, and if you disagree with the result you can rate them yourself and change the rankings!
The skins in Fortnite have been a big part of the game's success! While most games of this nature tend to be first person and you can barely see yourself, this game is in third person so you are always looking at your cosmetics. That means the skin you have on is a lot more important, and you can personalize it in a variety of ways.
The list below features the best skins that have been rated by our community! If you want to influence the rankings of this list, you can rate all of the skins via our Fortnite Skins Gallery.


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For this Grand Rounds, I chose David C. Kibbe & Joseph C. Kvedar's article, "Building a Research Agenda for Participatory Medicine" (JoPM, Vol. 1, 2009). I will highlight two of their "ready-to-go" research questions: What is the role of coaching in sparking and supporting increased participation over time? What can we learn from research on […]
found on the net
e-Patient Connections 2010–Susannah Fox
Kevin Kruse and his team have put together another incredible event in Philadelphia: e-Patient Connections 2010. Follow the tweets by searching for #epatcon or read the excellent summaries being written in real-time by Leigh Householder and Seth Quillin on the blog What's Your Digital IQ? Definitely watch the curtain-raiser video, starring our own e-Patient Dave: […]
The Power of Mobile (Video)
The video of my Mayo Transform 2010 speech, The Power of Mobile, is now up on the conference site as well as on YouTube. It was an honor to be part of this event. Many thanks to David Rosenman and his team for inviting me!
Living N=1–Susannah Fox
If you haven't listened to the Patient Voices series on The New York Times site, let me be the first to recommend it. I spend quite a bit of time writing up survey data, working with moderately large respondent pools (N=2,253 is the number of people who completed my last health survey; N=609 is the […]
Gov 2.0 Expo: Health Geek Guide
The cross-disciplinary smorgasbord that is Gov 2.0 Expo will be held this week in DC. The agenda is packed with nerdy temptations (danah boyd! Anil Dash! Tim Berners-Lee!) but here are my can't-miss sessions.
E-Patient Erin Proves a Point–Susannah Fox
Erin Turner recently wrote: When I arrived at the Mayo Clinic in Rochester, Minnesota a few weeks ago, I was asked: "Who referred you to us?" My answer was not traditional: "Twitter." (Read her full story on the SpectrumScience blog. And take that, 2.0 doubters!)


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TYSONS CORNER, VA, Nov. 10, 2017 — Boeing (NYSE: BA) has collaborated with the Missile Defense Agency to load the 44th interceptor onto the Ground-Based Midcourse Defense system located at Fort Greely in Alaska, ExecutiveBiz reported Wednesday.
The company said Tuesday the installation marks the completion of a Defense Department goal to increase the interceptor inventory to 44 by the end of 2017 in efforts to bolster the U.S. missile defense posture.
About Executive Mosaic: Founded in 2002, Executive Mosaic is a leadership organization and media company. Executive Mosaic offers highly coveted executive events, breaking business news on the Government Contracting industry, and delivers robust and reliable content through seven influential websites and four consequential E-newswires. Headquarters are located in Tysons Corner, VA. www.executivemosaic.com
David Smith
(703) 226-7002


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Everything You Ought To Know Regarding Dental Care
Do you dislike what your teeth look like? Do you have a crooked and stained smile? Do you wish to remedy it? Luckily, with these easy tips you will be able to get straight, healthy, white teeth. Here, you'll find tips to help.
If you are concerned about going to the dentist, then do some research on different dentists near your home. Check out online reviews and find a dentist who is well-liked and able to make patients feel comfortable. This will really make you feel more comfortable during your visits.
In order to clean your teeth in the best way, make sure you are using a soft-bristle toothbrush that is the perfect size for your mouth. To prevent the brush from growing bacteria, air dry it after using it. Set it in a toothbrush holder or cup to keep it upright so air can circulate.
Go to the dentist every six months. If you go to regularly scheduled dental visits, you can stop most problems from forming on your teeth. At these visits your dentist will check for tooth decay and tartar buildup.
You can strengthen your teeth with fluoride supplements. Fluoride can really help if you struggle to keep your teeth white, or you have gum sensitivity. Yellow spots will appear on your teeth if you take too much fluoride. If you are having trouble with it, take fluoride out of your diet until this stops happening.
Go see the dentist twice per year, at least. Over time, this allows you to maintain a brighter smile with strong, healthy teeth and gums. The dentist is trained in identifying problems early on and can guide you in the right direction. Without the proper dental treatment, your dental problems can become severe.
Are you afraid that your brushing sessions aren't long enough for effectively eliminating plaque build-up? Consider using mouthwash or a disclosing tablet. The product should be used before your begin to brush. You will instantly see the areas of your teeth that you missed, because the plaque and food will show up as a different color. It is important to have sufficient time to brush your teeth thoroughly after using these products. This is definitely something that you should only use when you have nowhere to go.
Read the label on your toothpaste. The toothpaste you use should contain fluoride. Most toothpastes also contain abrasive chemicals designed to make your teeth whiter. If the toothpaste is to harsh, try to find one with less of these chemical abrasives.
Be sure that you're brushing your tongue. Although it is often neglected, the tongue must be cleaned in addition to the teeth. There are all kinds of bacteria on the tongue. This can contribute to bad breath and tooth decay.
Now that you've read this information, you should know more about how to better your teeth. As long as you put these tips to use in your life, you will soon have a smile that can light up a room like you have always wanted. Work towards the great smile that you've been wanting.…


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1 Saunders Copse
In January 2019, Builder Sky started a new project, involving a full Refurbishment of the house what is included new wet-room and widening the kitchen. Builder Sky, as it will allow us to demonstrate the broad range, quality and professionalism of the construction services we offer. As with all our projects, Builder Sky will do its best to ensure the highest levels of customer satisfaction over time.


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Feather Race Track Scarf
The Feather Weaves combine silk in the warp and yak wool in the weft and it takes an advanced weaver to find the subtle balance between the yak and silk fibres and maintain an always perfect edge. There are currently only three weavers at the Norlha Atelier who can create this scarf.
Combining many shades and intricate details on close inspection.
Styled with: Samten Cotton Shirt
Composition: 50% Yak Khullu, 50% Silk
Size: 35 x 170cm
Weight: 60 grams
Finishing: Rolled Edge
Origin: Handwoven at Norlha's Atelier on the Tibetan Plateau


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Material Super soft dying pure cotton
|Kameez/Kurta--2.5mtr Cotton
|Salwar/pant--2.4-2.5 mtr Cotton
|Dupatta/Chunni--2.3-2.5 mtr mulmul
|Occasion--Regular / Daily / Casual wear, Office wear, Summer wear NOTE:
Color and Texture may have slight variation. This happens because of photography and your device color setting. Can do soft hand wash at home in cold water. For precaution dip into salty water for few time and dry in soft sunlight or under shades. The color may bleed in case of natural dyes. Imperfections in hand printing may be noticed, and color may fate or bleed due to traditional dyeing and printing process employed.
Ready stock will be delivered in 2-3 days.Custom made stock will be delivered in 1-2 weeks of time. For urgent delivery ,kindly ping us with your Order ID at +91-89032 60653


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If you've been keeping up with my blog this summer, you knew I was out in Northern California interning for a small non-profit. After my internship I was asked to blog for InternMatch.com . As a little something different, I thought I would share my post here. If you are actively looking for an internship, job or advice, check out InternMatch.
Big company or small? Knowing the kind of environment you will work best in can make applying to jobs a lot less overwhelming. If you've never considered working at a small company, here are 5 things that I've learned after spending the summer working for a small non-profit.
1. You may be the only intern.
For some people, this can be scary. You don't have built in "friends" in your work environment that you commiserate with. On the plus side, you are much more likely to develop meaningful relationships with other employees. These people have been in the workforce for a few years so they lots of contacts in their industry. They already know their way around the office and are likely to become mentor figures.
2. You're likely to get cross-divisional experience.
Not sure what you want to do yet? Small companies may be the best atmosphere for you. Since the company divisions contain a lot less people, you will probably get asked to help with something outside of what you were hired for. This provides you the opportunity to network with other employees and learn more about how the company runs.
3. Easier lines of communication.
I worked in social media and our marketing department consisted of 4 people (including me). If I had a question on a project I could go directly to the Director of Marketing. Many interns at bigger companies struggle with communication. Sometimes they have to wait days for answers to questions or approval on projects since they have to go through so many more people.
4. You become a team player.
Many items on this list can depend on the industry or culture, but if you are selected to be an intern at a small company it is because they think you will fit in to their team. In my experience, I felt much more like a regular employee. I was invited to meetings, on calls, and asked to share my research. I felt like I was adding value with what I was doing instead of just being a cog in the wheel.
5. More face time.
Bigger companies tend to have employees located all over the world, so you spend a lot of time pinging and emailing someone you will never meet. I loved having face time with all of the employees on a regular basis. This was especially important with the higher ups. The executive director knows my name, where I go to school, and what I did last weekend. Bigger companies just aren't able to do this.
For me, the biggest challenge was not having other interns. It was my first time in the area so it was hard to make friends outside of work. I ended up becoming better friends with my roommate than I expected and taking lots of recommendations from my co-workers on stuff to do in the area.
I am very happy with my experience at a small company. I wanted to get the most out of my internship and my small company allowed me to learn and network from a group of passionate and intelligent people. Because it was so small, I was able to learn something from everyone there. Many students go into internship hunting thinking that they need an experience from a large and recognizable company. I've found that it doesn't matter as much about where you work, but it matters more about what you learned not just in the industry, but about yourself. If any of the above 5 things interests you, take a look at some of the smaller companies on InternMatch.


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Robin MacArthur
Robin MacArthur is a writer, musician and mother who lives in a self-built house in Southern Vermont on the land where she was born. She is the author of HALF WILD: STORIES, (Ecco, August 2016), and the editor of CONTEMPORARY VERMONT FICTION (Green Writers Press, 2014). She is one half of the musical duo Red Heart the Ticker, alongside her husband, Tyler Gibbons. She received an MFA in Fiction from Vermont College of Fine Arts and a BA from Brown University, and two creation grants from the Vermont Arts Council and the National Endowment for the Arts. When not writing, she spends her time prying rocks out of unruly garden soil, picking blackberries and raspberries outside her back door, and traipsing through woods with her big-hearted and half-wild children.
**Signed and Personalized copies are available. Please indicate signature or personalization preferences in "order comments". Shipping times will vary for signed/personalized copies.


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April 2, 2018
March 22, 2018
Today we took our youngster, Robin, out for his first visit to Heartwood Forest. In the picture, taken by Becky riding Mac, I am ponying Robin whilst riding Apple.
Ponying is the practice of leading one horse while riding another. It is used as a method to exercise hors...
We have an exciting new equine recruit to the Riding School team. Coco - a 16.3hh Irish Sports Horse. Coco is 10 years old and is brown and white. In equine terms we refer to this as skewbald.
Coco has been leading a leisurely life for some months now so we are working...
Meet Mac! Mac is a 13hh coloured cob. He is currently the youngest member of the team. Mac joined Sandridgebury in December as a newly started 4 year old. We have slowly but surely continued with his education to enable him to become a valued member of the Riding Schoo...
March 18, 2018
All our horses are checked daily to make sure they are in tip top shape. Here is a wonderful picture of Mouse, one of our mares, showing lots of signs of good health.
Mouse is showing;
Correct weight
Shiny coat
Bright eyes
Relaxed attitude
Other sign...
December 10, 2017
Winter is always a busy time for the staff looking after the Riding School horses and never more so than when it snows!
As the snow falls, it covers the grass which means the horses need to be given a grass replacement. We use haylage which is grown on Sandridgebury F...
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I'm busy working on my blog posts. Watch this space!
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I'm busy working on my blog posts. Watch this space!


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Also known as: pigeon toe, intoeing.
In-toeing is a fairly common condition among toddlers in which the toes point inward rather than straight ahead. In many cases, it doesn't cause any problems in toddlers and corrects itself as the child grows. Other cases of in-toeing may require medical attention.
In many cases, the problems that lead to in-toeing first occur in the uterus due to the baby's position. A twisted femur, shinbone or feet are the common causes of in-toeing.
In some cases, the condition causes no symptoms other than the physical anomaly of the toes being pointed inward as the child stands, walks or runs. Other children may be clumsy or fall frequently due to the condition.
Most cases of in-toeing correct themselves over time as the child grows. In the rare instances where the symptoms persist into adolescence, surgery may be required to correct the problems with the feet and legs and straighten the toes.
Reviewed by: Avi C Baitner, MD
This page was last updated on: 1/29/2019 3:21:13 PM


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Reclusive eccentric Stillman is back with another dissection of a well known Diablo 2 character. This week, the question is: who do you suppose is the best at castrating the the Oblivion Knights of Act 4? Could it be a specific cookie cutter build or maybe a new character you are trying right now? No, it's actaully Blizzard. Nothing in the world can hurt them as much as patch 1.13. Click through below for the full story on Chaos runs post patch 1.13.
Oblivious Knights
When Blizzard plugged the dreaded Oblivion Knights back into the bnet matrix, something went…splat, or crunch, or ?Uuuuhhhngh.? The Oblivion Knights once had a whole repertoire of curses. Now, they can never cast Lower Resist or Iron Maiden, and Decrepify often looks like a splash of new paint on your character's thought cloud but has no other effect. It is as if the most powerful and intelligent monster in the game went in for some rhinoplasty and got a lobotomy instead. And we all know when this broken bad guy will get fixed and restored to his former glory…
Never. Why, that would be like taking Enigma away.
Welcome to happy land, fellow Chaos runners. The Chaos Sanctuary was the most efficiently packed, high treasure class slaughterhouse in the game next to cows, but with better experience gains. You could go in there and use all you know to exploit the general bad AI we all know and love. Now it is…somehow much easier. It seems the Oblivion Knights' younger brothers, the Abyss Knights, on the river bed just outside of the Chaos Sanctuary are the real threat now. At least they have that glitchy thing going on where a mob of them will sometimes nudge a bit…but really they are firing INVISIBLE shots at you in unison! The Oblivion Knights' current status makes you wonder what the Player Test Realm was for if Diablo's greatest defense system is left behind as quivering jelly.
Oblivion Knights are still pretty awesome just for being what they are. These undead warrior mages with Necromancer skills and standard issue splint armor show off the finest of the Diablo world's unique characters. There is nothing quite like Oblivion Knights, whereas in WoW, it seems the the whole player character selection is copy n' pasted from Dungeons and Dragons. Even better is how Oblivion Knights made life hell for us, especially if you relied on cold damage as your main punch. With 180 cold resistance, these royal guards bleed liquid nitrogen.
One weird thing, however, is how they often cast curses all at once even though only one curse can be active at a time. They are like girls cat fighting over who gets to stare at me the most. If I were there, I would jump in and tell them to kiss and make out?I mean kiss and make up. Long story short, Oblivion Knights should not make fools of themselves by overriding each others curses.
But this cheap AI could be forgiven. After all, it is inside the Chaos Sanctuary, so we half expect the chaos of many curses competing with each other. What no one expected, though, was the loss of three of their strongest curses. Let's see what this scholarly bespectacled mouse thinks of the situation.
And what happened to Infector of Souls? He is mentally retarded after 1.13 got its hands on him. You now have to run up almost nose to nose with him before he and his mob know of your existence.
Man, could this game get any easier? Look at this madness:
This is just after a few dozen Chaos runs. So it's infinite respecs for everyone at no real cost. Dammit Diablo, I thought we were friends. Instead of dropping something that could be valuable, he now must make room to carry garbage red powder every few games. How many of these gimmicks do we need on top of the pointless Standard of Heros, easy-to-get keys, organs identical to quest items, and the annihilus items that reward soj duping?
Looking on the bright side, at least this will drive godly items to become even more paramount now that your character's build is truly and forever irrelevant. But that just means magic find gear is even more required and you are even more of an idiot and outcast for not using it. Yet, most agree that any magic find beyond 250-300 is kind of useless, so nothing really changes. Hmm, what else? Anyone below level 90 is 'low level.' This noob bar might rise to 95 by mid season.
I know most of us yearn for more patch work, and we should be grateful for some of the much needed fixes the latest patch provided, but be careful what you wish for. It seems the game only gets less challenging and more laughable and exploitable with every patch. It's like watching Earnest goes to Hell where Earnest makes Hell a more lovable, friendly place.
[Do not click the yellow text. That movie does not exist. Clicking it will send you to the Golden Girls eating cheesecake. Or was that the Enigma link??]
So while Infector of Souls watches Viagra commercials ever so keenly, we will be picking off his thugs in that 'divide and conquer' approach. Of course, it will be like dividing and conquering retarded blind school children overdosing on amphetamines, but all is fair in love and war (whatever that means). And while Oblivion Knights fire off a chain of Bone Spirits that still actually hurt, we will pat them on the back…with a Grief Phase Blade. Did I mention high runes seem to drop more freqently in Chaos Sanctuary than mid runes? See you all there!
Rest in pieces, Oblivion Knights. And gimme your stuff.
Opinions expressed in columns and guest articles are those of their authors, and not necessarily those of Diii.net.
Stillman's Slab is where all Diablo characters are dissected and examined piece by piece. It is written by Nicholas Stillman to reintroduce Diablo series topics in a new light or put forth novel themes that have not been fully explored in the forums. Slurry collected from the centrifuge will always contain something new and unheard of at the time of publication. Post your comments below or directly.


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The logistics and transport industry finally has a lean supply chain solution to improve tracking technology.
For the first time you have real-time confirmation of the opening of the package by the end customer.
This new technology has a name STICK TRACKR
or just try to find out more
We are proud to present the first IOT tracker
for less than 1$ !
Track your shipments worldwide in real time with Sticktrackr!
Sticktrackr, is a small PCB measuring 7×2 cm and with a thickness of less than 5mm. It can be slipped inside any packages and its record breaking autonomy allows you to have a traceability during 15 days!
Combined with a magnetic proximity or a photosensitive sensor it will allow you to know when the customer opens his parcel!
Locate and manage your goods!
Track your equipment, find out if your equipment is still in your warehouse or if it is being used by a user. Follow your equipment and finally ensure that it was sent back to you.
Manage your production!
In order to control your production you need to know your stock level throughout the downstream chain of your supply chain. Thanks to sticktrackr you will be able to know in real time the inventory of your products and their location.


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The giant leaves and flowers of the lotus are mythical in their beauty. You can grow these amazing, large water plants from seed. The process will require patience, but that is just another lesson to be learned from these plants that steeped are in spiritual wisdom and historic significance. Once full-grown, your lotus plant will feature bold leaves with a single, giant bloom.
[Originally Posted by ehow.com ]


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We are always happy to hear from talented new people who want to join our team.
If you think you could help us make our company even better, send us your CV and a cover letter using our open application form.
Learn more about our company, our history and where we are today.
Get to know the people you could soon be calling your colleagues.


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Knowing mortgage rate trends and tracking accurate mortgage interest rate forecasts are important to the home buyer, seller and lender. Being on "the right side of the market" can enhance the profitability of a home sale and mortgage loan — or the affordability of a new mortgage or refinance. But because of its complexity, understanding mortgage rate trends may be as much art as science.
Mortgage rate trends
Interest rate analysts, economists, mortgage brokers and lenders all keep a close eye on mortgage rate trends. Of course, prospective home buyers do, too. The dynamics of a moving bond market, the U.S. Federal Reserve's asset purchases, domestic and foreign economies and housing demand all affect mortgage rate trends.
Moreover, a secondary market for mortgage-backed securities also impacts mortgage rate trends. Mortgage lenders often sell their mortgages to investors who then package the assets with many other loans to create mortgage-backed securities. When demand for mortgage-backed securities is high, mortgage lenders are more willing to underwrite mortgages and supply increases. This activity pushes mortgage rates lower in the long term. The converse is true when demand for mortgage-backed securities is low.
Mortgage rate forecast
As mortgage rates change incrementally throughout the day, lenders must actively manage their loan portfolio risk and profitability. The price of goods and services throughout the economy are an important factor in any mortgage rate forecast, upon which lenders make their pricing decisions. Whether these prices rise or fall — economists use the term inflation — influences the movement of the bond market, which in turn drives mortgage rates.
If inflation is expected to rise, mortgage rates will generally move higher as well. As inflation, or the threat of inflation falls, so do mortgage rates.
Fine-tuning the forecast
Unemployment, housing starts, application volume, loan payment delinquencies and the city of a loan's origination also impact mortgage rate trends and forecasts. And the home loan interest rate you receive will ultimately depend on personal data, including the amount you wish to borrow, your credit history and the dollar value of debt you carry.
Whether you are a first time home buyer or seeking a mortgage refinance, learn more about how to get the best mortgage rates for your situation in NerdWallet's mortgage guide.
Image via Shutterstock.


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We have already mentioned the load loss benefits of avocado oil, so it should come as no surprise that the mothership has its own fats-blasting properties. You're more more likely to grab fruits and veggies over much less-wholesome choices if they're able to eat and in plain sight.
In a 2015 examine in the journal Scientific Nutrition , researchers gave 117 sufferers with metabolic syndrome both dietary supplements of curcumin—the energetic ingredient in turmeric—or a placebo. Bloomberg stories that researchers at the Mayo Clinic found that standing burns about 54 calories over a six-hour day, and although that might not sound like a lot, those energy accumulate rapidly.
A 2016 research discovered that garlic powder reduces physique weight and fats mass amongst folks with non-alcoholi...Read More


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Starting A Bar and Restaurant Business, Some Basic Furnishing Needs
Furniture in Fashion Blog
Furniture in Fashion Blog
Furniture in Fashion Blog
So you want to start a bar along with a restaurant business but don't know the requirements furniture stores ? This article will help you. There are many different types of furnishings that can be put up in your business area. First of all think of the amount of space that you have, and even your budget. Decide whether you want fixed furnishings or do you want them movable. These important decisions can be made once you've made up your mind on the type of business that you're starting. Chances are, if you're reading this article, you will not be a starter so we start off directly on the types.
There should very obviously be a bar. It does not have to be a traditional brass bar. It can be of a contemporary design as well. What really matters here is the locality of the bar and the space that it is covering. If yours is a restaurant then the bar should be bigger, but if it's a dance club, then the bar needs to have lesser space and more in length so as to acquire the corner of the dance floor. Another option is my making booths. Although they are not a very wise consumption of space, they look great in placement. Banquettes are those soft sofas that line up the wall. These are one of the most frequently placed furnishing. Bar stools are also very common. These can be movable or fixed depending on your requirement. Fixed ones are around the bar itself. Movable are available in 3 sizes. They are tougher and give place for a rougher use.
The tables can be fixed however; movable tables will give you an option for frequent change of layout. The bad table has to be long and should have a lot of storage space. Sometimes the bar tables are usually as tall as the bar itself. There are some furnishings that come in bundles of the stool and the table so you don't have to go through the trouble of matching them.