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This site is about RV's and campers.
Travel trailer weight is a significant parameter if you are about to buy either a new or used one. The weight of trailer camper is generally mentioned by the manufacturer, but they also mention many other ratings which should not be overlooked. A trailer weight is not only the weight it carries when build, but will also include all other things you pack in. Their weight depends on length, and many other factors. Lets see some examples and draw conclusion on the weights of travel trailers or campers.
Many people buy travel trailer due to their cost and size. A travel trailer is very popular option in rv’s. One question that many people often have is how much do the travel trailers weigh. Travel trailer weight varies widely and on an average it is between 1000 lbs to 8800 lbs. Most of the popular travel trailer have lengths between 15 feet to 35 feet. The weight of travel trailer depends on the size or length. When understating the weight parameters in pounds there are multiple thing that you should understand rather then just going by the weight called out by the dealer in manual. In this post, we will see average weight of travel trailers and various other terminologies related to travel trailer weight.
Understanding the weigh of a travel trailer is important because you are supposed to pull it using a car or suv. A car will have certain limit and if your trailer exceeds that limit then that could be a problem. When you head out for camping, you end up packing lots of things and with a full gas tank plus a full water tank, will mean lots of weight. Thus, it becomes more important that you have fair idea of where the weight is heading. Apart from understanding the dry weight of travel trailer, get a fair idea of other weights related to it. A hitch should also be good enough to handle the overall weight. Also, with a rv, you are supposed to follow certain rules related to weight. Once you cross certain weight limit even the drivers license requirement change in some states.
One thing many people try to check is if they can find smaller travel trailer that have less weight. A smaller less weight travel trailer lets you pull it easily. We will also see how travel trailer weight varies by length. Once you have that idea, it can be helpful to determine the approximate weight based on length.
Main reason why people want to know about the weight of their travel trailer is to determine if their car or truck can tow the trailer or not. A travel trailer has length that ranges from 17 feet to 40 feet. More the length, more is the weight of travel trailer. A 15-18 feet travel trailer will obviously weigh less than a 30 feet one. That is to say, we can almost say that weight of the travel trailer is proportional to its length. This however, may not be always be correct as with new models, construction type material the weight is varies.
New year models released and built are less heavy and thus, we can also say weight is inversely proportional to year make of the travel trailer. On an average a 16 feet length travel trailer will have a unloaded weight of around 2200 lbs to around 3400 lbs. A 30 feet travel trailer can weigh around 7000 lbs to 7800 lbs.
This may however, be not true for all makes and brands, it will always vary. But, on an average you will find the numbers to be very close. A 20 feet travel trailer can be as heavy as 2700 lbs to 2900 lbs. With advances in the way travel trailers are manufactured, you will also find a 15 feet travel trailer weighing just above 1500 lbs unloaded or even an 13 feet long travel trailer weighing below 1200 pounds.
Travel trailer are smaller rv’s, they sleep 2-3 people at times and can also be lengthy about 40 feet long. A travel trailer weight also depends on the floor plan, kind of dinner built in, position of the bath, kitchen positioning, entry exit doors, window positioning, bed style (this things require changes, adjustments then lead to weight variations).
Lets see some examples of travel trailers along with their weight and length. This should give a fair idea of relation between length and weight of a travel trailer. These are some of the smaller travel trailer that are popular under the weight of 4000 pounds.
We can determine a fair idea of weight based on travel trailer length. But that may not be always true. A length cannot be the ultimate decider to figure out the exact weight of a travel trailer, as seen from examples a 20 feet long travel trailer (of not only different make, but even same brand) weighs differently. Thus, it also depends on the floor plan, amenities and many other things packaged in the trailer.
As we already saw a longer travel trailer will weigh much more and it is almost proportional to its length. A 40 feet travel trailer will have enough weight to make you think if the car you have, is good enough to pull it or not. Even a 25 feet or 30 feet long travel trailer is much heavier and you need to be sure about the capacity of your pulling car. Lets see some of the examples to understand better and draw some conclusion on length-weight relation for heavier travel trailers. Examples here in are also some of the best travel trailers you could buy in the range of 28 feet and 30 feet with weights under 5000 pounds and so on. While some are quoted just to see how the best and average travel trailers weigh in different segments of lengths.
A travel trailer weight or weight of any other rv need to be understood correctly. The numbers that you get from the manufacturer or dealer could be misleading and misunderstood. The weight details of various sized travel trailers that we saw are the unloaded weights. You basically need to do the math properly to see if the travel trailer weight is within limits for your car to pull.
Weight understanding in case of travel trailer is much more important than motorhomes which drives themselves (which is not the case for travel trailer or fifth wheel or pop up campers or any other towable trailers). Its important you understand the rv weight terminology. A rating provided by manufacturer cannot be changed. Normally weight and load are same when you talk weight of rv’s. Basically, a weight of rv puts load on rv tires, axles and hitches. This load is used to calculate various rating like GAWR (gross axle weight Rating), GCWR (gross combination weight Rating), GVWR (gross vehicle weight Rating). Lets, try to understand what exactly these things mean and how you should interpret values for these.
Gross Axle Weight Rating (GAWR) – This rating basically is the maximum load that the axle of the trailer can support. Trailer manufacturer provides this rating and it basically depends on axle, tire, wheels and the rating is based on the weakest link.
Unloaded Vehicle Weight (UVW) – This is the weight of the RV or travel trailer when it is fully unloaded without any extra things on it. Usually the weight that you see when it is at the dealer and not yet sold. The weights that we saw above were unloaded weights. Most times people are confused with this weight, and assume the unloaded weight as the final towing weight. But, in reality the weight increases when you actually pull the trailer with all the things packed in.
Gross Vehicle Weight Rating (GVWR) – This rating means the maximum weight that is allowed on the RV including all the things like the passengers, driver, water, fuel. The trailer and the towing vehicle will both have their own GVWR.
Gross combination Weight rating (GCWR) – Unlike GVWR, the GCWR is the combination rating for both the trailer and the towing vehicle. It gives the maximum combined limit for the tow vehicle and the travel trailer.
Cargo carrying capacity (CCCC) – This basically means the weight of the trailer without the optional accessories, equipment in the trailer.
Sleeping Capacity weight rating (SCWR) – This basically gives the maximum weight limit depending on the number of sleeping positions designated by manufacturer. Each included person is assumed to be around 154 lbs and thus, a 2 sleeping travel trailer will mean its SCWR will be 208 lbs and so on.
In case of travel trailer, the weight distribution is significant. An un-even weight distributed can be dangerous and the towing hitch and the trailer can be impacted. You need to make sure the overall weight is properly distributed. The idea is to make sure weight is properly distributed along all sides, front to back and from both right and left sides. Rv’s and travel trailer are originally designed with proper weight distribution, and when you tow the travel trailer you need to follow same when putting in more weight through other things. The weight of both the tow vehicle and travel trailer should be properly distributed. Technically speaking, you also need to make sure the Gross Vehicle weight Rating and gross combined vehicle weight rating is not crossed at any time.
To understand the proper rules of weight distribution, you can read the manufacturer provided manual for your travel trailer or camper. In most cases, the idea is to keep 60% of the weight ahead of the center of gravity of the travel trailer. To understand the correct weights of your travel trailer, its better to weigh it after it is fully loaded with all stuff.
An uneven distributed weight is notable with naked eyes, but may not always be visible hence its better to keep the weight within limits and properly distributed. One can also use weight distribution systems to make it properly distributed. Weight distribution systems work using spring bars which correct uneven weight distribution.
Lets try to understand how the weight of the travel trailer is affected. What all things put on that weight.? An unloaded weight will be less but when you head out finally, the weight of the overall travel trailer could go out of limits and that’s what we should try to limit.
Water – Camping need lots of water. And water adds weight to your travel trailer ? yes, do not underestimate the power of water weight. It weighs almost 8 lbs per gallon. A 50 gallon water in a travel trailer will mean you already have put on 400 lbs extra on travel trailer.
Construction material affects weight – A travel trailer construction material type is also responsible for its weight. A fiberglass made trailer generally weigh less as compared to aluminium ones. This however, should not be your problem as this will be included in the unloaded weight which is not in your hands anyways. But, understanding the material type of your travel trailer can be helpful as choosing the right one will mean your travel trailers unloaded weight itself is less.
Slide outs Increase weight – A slide out for your rv increases the overall weight. Its extra space but extra weight too. A slide out normally adds upto 1000 lbs on an average. Pop up are less heavy and that can be a good option as compared to slide outs if you are looking to reduce the overall weight on your travel trailer.
1. Get rid of extra things on your rv. Extra tools, extra equipment, unused personal belongings like shoes, clothes, books etc. Unused kitchen items, bedroom, bathroom accessories you don’t use more often should be kept out. All smaller things add up to put on those pounds which add-on to become a big number.
2. Weigh the travel trailer separately with car and without car. This gives great picture of what is happening and where the weight is distributed.
3. Get good weight distribution system to make sure weight is properly distributed.
6. Make sure tires are properly inflated. Maintain correct tire pressure for both travel trailer and car at all times.
7. Use aluminum in case you want to do any repairs or modifications to your travel trailer. Aluminium is light weight and thus will not put on extra weight on your travel trailer. Many times, people do patch work like filling in holes, cracks or structural changes to the travel trailer. All these smaller things should be done with materials that are less in weight and should not add on weights.
8. Get rid of things that are not in use for months or have become old. A ladder that you hardly need, an awning that is broken or things like kayak that you occasionally need. Remove things that you don’t use often or things of past that need replacement, just go ahead and replace them. This will not only save space but also reduce the unnecessary weight.
9. Get the sliding doors replaced with curtains. Doors that aren’t used for privacy and are in place just, can be removed and replaced with curtains or any other such fabrics used instead of heavy doors.
10. Replace old heavy things with new lightweight ones. Drawers, bins, shelving that are heavy and old, could be putting in weight unnecessarily and you should try and get things that are new and less in weight. You will find lots of innovative products specially designed for RV that are compact and lightweight.
A mini motorhome or a class C rv on an average weighs around 10,000 lbs to 14,000 lbs. That is almost double than an average weight of a travel trailer. Class C motorhome are also longer than travel trailers, they tend to be around 18 feet – 30 feet long. Class A motorhomes on the other hand are the heaviest ones, they weigh much more. On an average class A rv’s weighs anything between 15,000 lbs to 30,000 lbs. They are really heavy, and much more than a travel trailer. With so much weight they tend to intimidate you on road. Remember, with weight also the license requirement changes.
There is limit on weight up-to which you can drive using normal non commercial license but if the weight goes beyond certain limit then you need special license. The rule of weight is different for all states. Like in California, a weight more than 26,000 lbs for a non towable RV will need a class B Non commercial license. Most states have similar requirement but could be little different for some states (need to check with each states rules and regulations).
A travel trailer to be towed has a weight limitation of around 10,000 lbs in most states and beyond that you will need class B Non commercial license. Looking at these numbers of pound for travel trailer, most of the times you will be okay with the normal license unless you have that very big trailer weighing more than 10,000 lbs.
A travel trailer weight measurement is of much importance since it has the power to affect many other things in the camper trailer. Apart from getting to know just the unloaded weight of the trailer, knowing all the other ratings help in towing car-trailer system smooth. A travel trailer weighs less as compared to other bigger rv’s however it is more important to know the weight in case of travel trailers. A travel trailer weight unloaded should not be your only way of judging the capacity of your car to tow.
Understanding overall weight when fully loaded and with full water/fuel tanks is more necessary. Going through manufacturer manual, weighing your trailer often are best practices to follow. Keeping the travel trailer as light as possible can come good for your towing car or suv. Keeping the weight distribution proper is also important and proper hitches or distribution system can come handy. One should fully understand the towing capacity of the car and then manage the weight on travel trailer. Owning a very heavy trailer also needs special license requirements and thus better to make sure the weight is within limits.


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Apicomplexan parasites employ a unique form of movement, termed gliding motility, in order to invade the host cell. This movement depends on the parasite’s actomyosin system, which is thought to generate the force during gliding. However, recent evidence questions the exact molecular role of this system, since mutants for core components of the gliding machinery, such as parasite actin or subunits of the MyoA-motor complex (the glideosome), remain motile and invasive, albeit at significantly reduced efficiencies. While compensatory mechanisms and unusual polymerisation kinetics of parasite actin have been evoked to explain these findings, the actomyosin system could also play a role distinct from force production during parasite movement.
In this study, we compared the phenotypes of different mutants for core components of the actomyosin system in Toxoplasma gondii to decipher their exact role during gliding motility and invasion. We found that, while some phenotypes (apicoplast segregation, host cell egress, dense granule motility) appeared early after induction of the act1 knockout and went to completion, a small percentage of the parasites remained capable of motility and invasion well past the point at which actin levels were undetectable. Those act1 conditional knockout (cKO) and mlc1 cKO that continue to move in 3D do so at speeds similar to wildtype parasites. However, these mutants are virtually unable to attach to a collagen-coated substrate under flow conditions, indicating an important role for the actomyosin system of T. gondii in the formation of attachment sites.
We demonstrate that parasite actin is essential during the lytic cycle and cannot be compensated by other molecules. Our data suggest a conventional polymerisation mechanism in vivo that depends on a critical concentration of G-actin. Importantly, we demonstrate that the actomyosin system of the parasite functions in attachment to the surface substrate, and not necessarily as force generator.
Apicomplexan parasites employ a unique form of movement, termed gliding motility, to disseminate and invade host cells. The current model for gliding motility is centred on the actomyosin system of the parasite in force generation during this process. According to the ‘linear motor’ model, micronemal transmembrane proteins, such as MIC2, are secreted at the apical pole of the parasite and act as force transmitters by interacting with the substrate surface and the actomyosin system, similar to the role of integrins in other motility systems . Accordingly, this model predicts a smooth actomyosin dependent rearwards translocation of the microneme-substrate complex, which results in forward gliding on the substrate or into the host cell . However, recent reverse genetic approaches demonstrated that parasites remain motile in the absence of what were thought to be key components of the motility machinery, including parasite actin [3–6]. In an attempt to reconcile these data, we proposed a novel, hypothetical model based on gelation/solation , where actin/myosin is required for force transmission rather than force production . This model predicts the generation of an osmotic gradient that results in membrane tension and retrograde membrane flow. In order to regulate membrane balance, the parasite then either sheds membrane at the posterior end, as previously observed , and/or recycles the membrane via endocytosis, as suggested for other systems [8, 9].
Many other eukaryotic cells move by crawling, a process powered by the controlled assembly and disassembly of the actin cytoskeleton in proximity to the plasma membrane [10, 11]. While it was previously assumed that the majority of the force for the retraction of the cell rear is powered by myosins, several alternative mechanisms can operate within the cell that also power motility [10, 11]. For example, the motility of Caenorhabditis elegans sperm occurs in the absence of any actin and therefore myosin activity ; instead, force is generated by controlled polymerisation of the major sperm protein. Furthermore, when tumour cells are confined within a microenvironment, motility depends on an osmotic engine and can be independent of actomyosin activity . Finally, migrating cells can employ various migration modes in response to their microenvironment , which allows them to move even in the absence of adhesive coupling . For example, crawling cells, such as Dictyostelium and human leukocytes, can swim efficiently when suspended in a viscous medium [16, 17], demonstrating that adhesion to a solid substrate is not always necessary for movement . It is thus possible that motility can be driven purely by surface membrane flow, which itself results from a secretory-endocytic cycle acting as a fluid drive from the anterior to the posterior end of the cell . In support of membrane flow, many motile cells show a distinct capping activity of surface ligands . Furthermore, surface capping depends on vesicular transport, and both on actin and microtubule function . In good agreement with these observations, numerous modulators of endocytic and secretory trafficking have been demonstrated to be key regulators of cell motility , leading to the hypothesis that membrane transport and retrograde flow during motility are rate-limiting for cell forward translocation .
In the case of apicomplexan parasites, a recent study demonstrated that retrograde membrane flow of malaria sporozoites occurs even at relatively high concentrations of the actin-disrupting drug Cytochalasin D (CD) . Furthermore, biophysical studies on malaria sporozoites have demonstrated the discrete, localised turnover of attachment sites that are not evenly translocated along the surface of the parasite . This involves the formation/disengagement of adhesion sites at the front and rear ends of the zoite, while the sporozoite undergoes a stretching phase. Interestingly, actin is important for the definition and release of the attachment sites , raising the possibility that apicomplexan motility may be similar to amoeboid-like crawling.
In T. gondii, an alternative explanation for the observed phenotypes for mutants of the actomyosin system is the possibility of functional redundancy of actomyosin components . While redundancies cannot be ruled out, in particular for myosins and micronemal proteins [3, 4], even the removal of ostensibly critical structural proteins does not completely block gliding motility or host cell invasion. For example, removal of the gliding-associated protein GAP45 results in the relocation of the remaining MyoA-complex components to the cytosol and severe disruption of the inner membrane complex (IMC), within which the motor complex is normally anchored [3, 25]. While these gap45 conditional knockout (cKO) parasites are completely blocked in host cell egress, they remain motile and can invade host cells . These surprising findings have led to a reassessment of the mechanisms underlying parasite motility and host-cell invasion, as recently reviewed . In fact, not only do the roles of the individual components need to be reanalysed, but also their orientation and organisation within the parasite .
Here, we show that, during motility, the actomyosin system of the parasite functions in attachment to the surface substrate, and may, therefore, act as force transmitter rather than a force generator. We provide evidence that retrograde membrane flow, which may play a role in force generation and motility, does not depend on an intact actomyosin system.
We previously reported that ACT1 is undetectable in act1 cKO parasites 72 h after rapamycin-induced removal of act1 . A subsequent study suggested that residual actin may, in fact, be present in some of these parasites as long as 5 days after rapamycin treatment . This led us to re-address the question of residual actin levels in the act1 cKO parasites, using additional antibodies and additional controls.
We began by comparing the specificity of different antibodies, from various labs, raised against apicomplexan actin (Additional file 1: Figure S1). While immunoblot analysis suggested that all antibodies are highly specific for T. gondii actin without recognising host cell actin (Additional file 1: Figure S1A), some of the tested antibodies demonstrated significant cross-reactivity within the parasites when used for immunofluorescence analysis, as previously reported . We performed IFAs at 4 days (96 h) and 8 days (192 h) after rapamycin induction (Fig. 1b, c and ). Notably, we found that the polyclonal antibodies employed previously to quantify actin levels in the act1 cKO parasites  showed a strong and persistent signal in proximity to the IMC of the parasite. This signal remained unchanged between 4 and 8 days after rapamycin treatment, even though the parasites are no longer expressing levels of actin above background within 4 days of induction (see below). Since this apparent cross-reactivity would interfere with accurate quantification of actin levels, we focused on two other antibodies that were highly specific and showed no cross-reactivity (Additional file 1: Figure S1).
Consistent with other studies, we found that actin levels in wildtype (wt) parasites were somewhat variable (Fig. 1a, b). Similar results were obtained with other housekeeping genes such as myosin light chain 1 (MLC1, Fig. 5a) and aldolase (not shown). Importantly, no overlap of actin levels could be observed between controls and act1 cKO parasites as early as 48 h after rapamycin-induced excision of act1 (Fig. 1a, b). At earlier time points (24–36 h) post induction, significant amounts of ACT1 were still detectable and protein levels were highly variable between vacuoles (Fig. 1a, b). Nevertheless, ACT1 levels were significantly reduced in comparison to control vacuoles. Importantly, 96 h after rapamycin induction, no ACT1 was detectable in YFP+ parasites. Lack of ACT1 detection by IFA correlates well with a recent study suggesting that no F-actin can be formed approximately 48 h after the removal of act1 , the inference being that G-actin levels fall below the critical concentration required for polymerisation.
In good agreement with this result, quantitative immunoblotting of act1 cKO parasites at different time points post-induction demonstrated the loss of all or nearly all of the ACT1 as early as 72 h post induction (Fig. 1c, d), confirming previous results .
In summary, our quantification demonstrates that actin is depleted in the act1 cKO parasites between 72 and 96 h post induction. Based on these observations, assays were performed in a time course up to 96 h after addition of rapamycin to adequately readdress the effects of ACT1 loss.
We previously demonstrated that parasite actin is required for apicoplast maintenance and host cell egress . These phenotypes were followed over time after act1 excision to estimate the time when G-actin levels are below the critical concentration in order to form F-actin. Significant numbers of act1 cKO parasites started to lose their apicoplasts as early as 24 h post-induction, indicating that once G-actin levels drop below a critical concentration, the apicoplast cannot be maintained during parasite replication (Fig. 2a, b). By 96 h after rapamycin induction of act1, we were unable to see any parasitophorous vacuoles that had a normal number of apicoplasts (Fig. 3b). Similar to apicoplast maintenance, host cell egress is significantly decreased as soon as 36 h post induction and was completely blocked 96 h post induction (Fig. 2c, d).
Next, we analysed the capability of act1 cKO parasites to move by gliding motility and to invade the host cell at different time points after rapamycin induction (Fig. 2e, f). In good agreement with previous results , overall motility and host cell invasion were significantly reduced in the act1 cKO parasites, with less than approximately 25% being motile and capable of invading host cells compared to control parasites (Fig. 2e, f). This phenotype was observed as early as 48 h after rapamycin induction, at a time point when significant amounts of actin were still detectable (Fig. 1). Interestingly, and in striking contrast to the results with apicoplast inheritance and egress, gliding and invasion rates remained constant at 48–96 h post induction, suggesting that once actin levels fall below a critical concentration, these phenotypes are not further affected. The simplest explanation for this result is that apicoplast maintenance and egress are completely dependent on actin, whereas gliding and invasion can occur independent of actin, albeit at significantly reduced efficiencies.
A recent study demonstrated the essential role of actin and the unconventional myosin F (MyoF) in directed dense granule transport . To assess if potential, residual F-actin in act1 cKO parasites is capable of serving as a track for MyoF-dependent dense granule transport, dense granule motions were tracked at various times after actin excision. In good agreement with the results obtained for apicoplast replication, parasite egress, parasite gliding and invasion, we observed an approximately 50% reduction in the percentage of granules moving in a directed manner from 37% in control to 14% as early as 18 h post excision (Fig. 3a, b). At 36 and 72 h post excision, directed granule motion was completely ablated (Fig. 3b, c) given that the percentage of trajectories in each population was indistinguishable from simulated pure 2D diffusion analysed using the changepoint algorithm (See Methods for details) (Fig. 3b; hashed bars).
The observation of diminished granule movement soon after induction provides further evidence that apicomplexan actin in vivo requires a critical monomer concentration (G-actin) in order to form sufficient F-actin to serve as a substrate for myosin motors such as MyoF. We therefore conclude that act1 cKO parasites are a powerful tool to analyse actin-independent motility and invasion processes.
Intriguingly, the phenotypes described above differ significantly from the effects caused by incubating parasites in the presence of drugs used to modulate F-actin turnover, such as CD or Latrunculins. For example, treatment of intracellular parasites with CD results in the formation of huge residual bodies, where organelles such as the apicoplast or secretory organelles accumulate [30, 31], which is not observed in the case of the act1 cKO parasites .
Our finding that parasites remain motile in the absence of ACT1 further suggests that CD might act on an unknown target, a hypothesis supported by an earlier study in which CD resistant parasites were isolated that do not have any mutations in TgAct1 . To test this hypothesis, we generated a parasite line where endogenous actin has been replaced with a LoxP-flanked copy of act1 containing a mutation (Ala136Gly) that confers CD resistance  and can be excised in a DiCre-dependent manner (Fig. 4a). Consequently, these parasites only express CD resistant actin before removal of act1 CDr . We compared the CD sensitivity of this new parasite line, called act1 CDr cKO, to that of control (RH) parasites, CytDr parasites  and the act1 cKO parasites. Interestingly, the gliding rates of all parasite lines, including CytDr parasites, were significantly reduced at higher concentrations of CD (1–4 μM) and it appeared the ACT1-specific effect was limited to ≤ 0.5 μM CD (Fig. 4b). The observed reduction in motility of CytDr parasites is consistent with a previous study, demonstrating loss of motility in the presence of 1 μM CD . Importantly, in the absence of ACT1 or ACT1CDr, parasites remained sensitive to CD (Fig. 4b); in both cases, the gliding rate was reduced from approximately 20% in untreated parasites to 1% in the presence of high CD concentrations, indicating that CD was acting on (a) yet unidentified target(s) in a non-ACT1 specific manner in T. gondii. Conclusions drawn from experiments using high concentrations of CD should, therefore, be interpreted with caution. Next, we tested the parasites’ ability to glide in the presence of the F-actin polymerisation drug Jasplakinolide (Jas). Jas was previously found to block attachment and motility of P. berghei sporozoites . At lower concentrations, T. gondii tachyzoites are inhibited in both circular and helical gliding but exhibit a counter-clockwise twirling motion . In agreement with previous findings, we found that forcing polymerisation blocks motility in wt parasites, whereas no significant reduction is observed for the act1 cKO (Fig. 4c). We also tested latrunculins A and B, independent F-actin disrupting drugs that have been previously described to act on apicomplexan actin . Surprisingly, even at 1 μM, we were unable to observe any significant effect on gliding rates (Fig. 4d), demonstrating that latrunculins are not acting on Toxoplasma tachyzoite actin. This conclusion supports previous observations for Plasmodium berghei gliding motility . In addition, the PfACT1 crystal structure suggests that latrunculins should not effectively bind apicomplexan ACT1 .
In summary, the experiments presented above demonstrate that, in the absence of actin, less than 25% of parasites remain capable of gliding motility and host cell invasion and establish act1 cKO parasites as a valuable tool for understanding the role of actin in the invasion and gliding mechanisms of T. gondii. The observed phenotype for the act1 cKO parasites is very similar to the phenotype observed for a clonal knockout for myoA . We therefore re-addressed the role of myosins during gliding motility.
It was previously suggested that MyoC can compensate for MyoA motor function in myoA KO parasites [3, 39]. In addition, it has been shown that MyoA and MyoC can interact with the same light chain, MLC1, and expression of MyoC from a heterologous promoter demonstrated a certain degree of MyoC association with the parasite pellicle in the absence of MyoA .
In order to address if the localisation of MyoC at the IMC in the myoA KO parasites depends on the level of MyoC expression, we expressed a second copy of TyMyoC under the control of either a strong promoter, p5RT70 , or its own promoter, pMyoC, and compared its localisation with that of an endogenously tagged version (Fig. 5a).
While strong expression of MyoC results in efficient relocalisation from the parasite basal end to the periphery of the myoA KO parasites (Fig. 5a, b), as previously reported , expression of MyoC under its own promoter or endogenously-tagged MyoC showed only slight relocalisation, with the majority of the protein being correctly localised to the basal ring of the parasites. This demonstrates that the localisation of MyoC to the IMC is critically dependent on strong expression levels of the protein, which are not reached with native MyoC expression levels in the myoA KO parasite (Fig. 5b). We next analysed propagation of these parasite strains using plaque assays and found that the relative plaque size is larger in myoA KO parasites strongly overexpressing MyoC compared to parasites expressing MyoC under the control of pMyoC (Fig. 5c). This demonstrates that overexpression of MyoC can partially complement the phenotype of the myoA KO parasites (Fig. 5c). Since the localisation of MyoC to the IMC is apparently critical for complementation of the myoA KO parasites, we decided to analyse mlc1 cKO in more detail, given that MLC1 is thought to be required to anchor both MyoA and MyoC to the pellicle [3, 39].
We previously analysed the mlc1 cKO 96 h after rapamycin induction; based on qualitative immunofluorescence analysis, no MLC1 was detectable at this time point . To verify that MLC1 is depleted at 96 h, we quantified MLC1 protein levels by IFA, analogous to ACT1 quantification described above (Fig. 6a). We confirmed that, as early as 48 h after induction, no MLC1 can be detected in the cKO parasites; this was also confirmed using immunoblot analysis (Fig. 6a, b). We succeeded in keeping mlc1 cKO parasites in cell culture for several weeks by mechanically releasing parasites and enriching the culture using FACS, demonstrating that MLC1 is essential for egress but dispensable for the following rounds of invasion and replication (Fig. 6c).
Next, we were interested in the localisation of MyoA and MyoC in mlc1 cKO parasites. To determine this, we generated two independent lines for colocalization studies, one expressing Ty-tagged MyoA under the control of its endogenous promoter (pMyoA) and another with endogenously Ty-tagged MyoC (Fig. 6d–f). MyoA is completely undetectable at the parasite’s pellicle 72 h after rapamycin-induced excision of mlc1 (Fig. 6d, e), demonstrating that MLC1 is crucial for the localization and stability of MyoA. In contrast, MyoC remains present and shows a normal localization at the basal end of the parasite in the absence of MLC1 (Fig. 6f).
In summary, we have generated and validated important parasite strains that can be used to analyse the role of the actomyosin system during gliding motility and host cell invasion and to investigate alternative mechanisms for these essential processes.
We previously demonstrated that conditional mutants for act1, mlc1 and myoA invade the host cell via the tight junction, demonstrating that these parasites still use an active invasion mechanism and are not passively taken up by the host cell via phagocytosis . However, kinetic analysis of myoA KO parasites demonstrated that both gliding speed and penetration times are significantly reduced, validating a direct function of MyoA during these processes. We were interested how act1 cKO, mlc1 cKO and myoA KO would compare during gliding motility and invasion.
To date, characterisation of gliding motility mainly depends on trail deposition assays, which allow a good estimate of overall motility rates. However, motility rates can be reduced by several factors, such as reduced or increased parasite attachment to the surface, and does not allow a firm conclusion regarding the function of a protein during this process. We speculated that parasites depleted in crucial parts of the actomyosin system should move significantly differently in terms of gliding speed and/or average distance travelled.
Therefore, we performed time-lapse analysis of gliding parasites (Additional files 2–7: Movies S1–S6; Fig. 7a–c) to assess the ability of parasites to generate the necessary force for motility in the presence and absence of the actomyosin system of the parasite. We confirm that myoA KO parasites show significantly lower trajectory length (~7 μm, corresponding roughly to one parasite length) and an average speed of approximately 0.3 μm/sec, as reported previously . Intriguingly, in the case of act1 cKO and mlc1 cKO parasites both average trajectory length and speed are increased compared to myoA KO parasites (Fig. 7a, b). While helical motility was significantly affected, with shorter average trajectories and reduced speed, circular gliding motility appeared relatively normal for mlc1 cKO and act1 cKO parasites (Fig. 7a–c). In good agreement with the gliding data, we found that host cell invasion can occur rapidly in act1 cKO and mlc1 cKO parasites, though several slow invasion events can be observed (Additional files 8–11: Movies S7–10; Fig. 7d, e). As is the case for gliding motility, the invasion of myoA KO parasites appears to be more strongly affected, with slower invasion events compared to act1 cKO and mlc1 cKO. This demonstrates that the parasite is capable of producing the force for motility and invasion in the absence of the actomyosin motor and suggests that actomyosin may instead be required to coordinate the force, most likely by regulating attachment sites, as observed in Plasmodium sporozoites [22, 34] and other diverse motility systems .
Next, we employed a Matrigel-based 3D motility assay to characterise the motility phenotypes of the mutants generated  under more physiological conditions. In good agreement with the time-lapse analysis of parasites gliding in 2D , a significantly decreased proportion of the myoA KO parasites was able to move in Matrigel compared to the parental line (39.5 ± 2.5% for LoxPMyoA parental, n = 2795 trajectories analysed vs. 5.1 ± 0.9% for myoA KO parasites, n = 3825 trajectories analysed) (Fig. 8a, b). Of the myoA KO parasites that were classified as motile, there was also a significant reduction in mean trajectory displacement and mean instantaneous speed compared to the parental line. Overexpression of Ty-tagged MyoC driven by the p5RT70 promoter in the myoA KO parasites was able to restore the percentage of moving parasites to wt levels and produce a modest increase in the mean trajectory displacement but not instantaneous speed (Fig. 8a, b; see also Additional file 12: Figure S2 for a comparison of the trajectory displacement distributions for the three lines). When we extended the analysis to examine mlc1 cKO and act1 cKO parasites, the results likewise closely mirrored those of the 2D assay (Fig. 7) – excision of mlc1 or act1 significantly reduced the percentage of moving parasites and mean trajectory displacement, but did not affect the mean instantaneous speed compared to their respective parental lines (Fig. 8c–f). Of note, it appears that mean trajectory displacement is more impacted in a 3D environment than in 2D, with few mutant parasites showing a displacement of more than one parasite length (Additional file 12: Figure S2), perhaps because they have to overcome more resistance in order to move in the Matrigel.
To test the role of actin and the glideosome in attachment to surfaces, we employed flow chambers coated with collagen (Fig. 9). To this end, we tested the ability of the myoA KO, mlc1 cKO and act1 cKO parasites to withstand shear flow stress. While surface attachment of myoA KO parasites was similar to wt controls, both the mlc1 cKO and act1 cKO parasites were unable to promote efficient attachment to the surface and were removed even at very low shear flow rates (Fig. 9a–d). Only RH and the myoA KO can withstand shear forces up to 15 dyn/cm2, which is within the range of arterial shear stress . These results suggest that the reason many act1 cKO and mlc1 cKO parasites cannot initiate gliding (Fig. 7c–f) is that they cannot attach sufficiently to the surface. These data suggest that the actomyosin system is required to coordinate the formation and release of attachment sites observed in a variety of other motility systems , including Plasmodium sporozoites [22, 34].
Apicomplexan parasites are known to translocate beads from the apical to the basal end and such ‘capping activity’ has been directly implicated in the motility of many cell types, including Plasmodium sporozoites . Interestingly, treatment of sporozoites with CD had no significant effect on the speed of retrograde flow, while the force generated at the attached bead was reduced . This could also be explained by the reduced formation of attachment sites, meaning a failure in force transmission rather than production. We therefore wished to analyse if retrograde membrane flow can occur in absence of the actomyosin system.
We modified a bead translocation assay first developed by King et al. over 30 years ago . Incubation of parasites with latex nano-beads leads to efficient binding of beads along the surface of the parasites and, within 10 min, 51% of parasites translocated beads to the parasite’s basal end (Fig. 10a). We next tested the role of the actomyosin system in the process of bead translocation, using either pharmacological perturbation or parasite mutants. In these experiments, we classified parasites as either ‘unbound’, ‘bound-uncapped’ or ‘capped’ (Fig. 10a). When parasites were kept in Endo buffer, which is known to diminish microneme secretion and motility , we found that a significant number of parasites were incapable of binding and translocating beads (Fig. 10c). When we tested the role of the actomyosin system in bead translocation (Fig. 10b, c), no abrogation of bead translocation was observed, suggesting the presence of retrograde flow in absence of the actomyosin system. Interestingly, in the mlc1 cKO, myoA KO or parasites treated with CD, fewer beads appeared to bind to the parasite surface (Fig. 10c), confirming a role of the actomyosin system in attachment. Nevertheless, capping in the act1 cKO occurred normally compared to RH when we assume that capped beads must have firstly bound. Similarly, the addition of CD had only minor effects on capping in the wt parasites. Overall, this demonstrates that bead translocation, and therefore the establishment of retrograde membrane flow, can occur independently of the actomyosin system of the parasite.
In this study, we set out to reconcile currently conflicting interpretations regarding the role of the apicomplexan actomyosin system during gliding motility and host cell invasion . While there is no doubt about a critical role of the actomyosin system during motility and invasion, alternative models and mechanisms regarding the exact function of this system have been put forward based on biophysical, reverse genetic and inhibitor studies [3, 21, 22, 26, 38]. Indeed, several observations cannot be easily reconciled with the canonical linear motor model: (1) Biophysical studies on malaria sporozoites demonstrate the discrete, localised turnover of attachment sites that are not evenly translocated along the surface of the parasite . Adhesion sites are formed/disengaged at the front and rear ends of the zoite, while the sporozoite undergoes a stretching phase. Interestingly, actin is important for the definition and release of the attachment sites , raising the possibility that apicomplexan motility is similar to amoeboid-like crawling. (2) Eimeria sporozoites show different types of motility (bending and pivoting). While pivoting appeared substrate- and actin-dependent, bending showed resistance to the actin-disrupting drug Cytochalasin B . (3) A recent study demonstrates that retrograde membrane flow of malaria sporozoites occurs even at relatively high concentrations of the actin-disrupting drug CD . (4) Reverse genetic approaches demonstrated that parasites remain motile in the absence of molecules thought to be key components of the motor machinery, including parasite actin [3–6].
In the case of reverse genetic studies, other groups have suggested that the observed phenotypes are due either to the presence of redundancies within the repertoire of genes involved in motility and invasion  or to an unusually stable actin (encoded by a single copy gene ), which persists many days and parasite generations after gene excision and can polymerize via an unusual isodesmic process that does not require a critical monomer (G-actin) concentration [27, 48]. While isodesmic polymerisation has been demonstrated in vitro, its in vivo relevance is unclear . Previous polymerisation studies used G-actin produced in heterologous protein expression systems. However, a recent study demonstrated that apicomplexan actin is not properly folded when heterologously expressed due to differences in the chaperonin T-complex . Furthermore, a recent study suggests that Toxoplasma actin is capable of forming F-actin structures in vivo that depend on a critical monomer concentration and demonstrates that F-actin is involved in multiple essential processes during intracellular parasite growth such as daughter cell assembly, vacuole organisation and parasite egress . Using a conditional knockout for act1 it was demonstrated that no ACT1 filament formation can occur as early as 48 h after removal of act1 . Finally, although apicomplexans possess actin-like proteins, it is unlikely that they can compensate for ACT1 and form filaments [27, 51]. Here, we performed a careful quantification of actin levels at different time points in the act1 conditional knockout and find that actin is still detectable at 48 h, but fully depleted by 96 h after induction with rapamycin.
In good agreement with previous studies [3, 29], we observed essential roles of actin in apicoplast segregation, host cell egress and dense granule motility upon deletion of act1, while parasite motility and host cell invasion still occur, albeit at significantly reduced levels. Since these findings were in direct conflict with a number of studies that use actin-modulating drugs to probe for actin function, we also performed an analysis of the effects of the actin-disrupting drugs CD, LatA, and LatB, and the actin-stabilising drug Jas on wt and act1 cKO parasites. While the effect of Jas appears to be specific for actin, since treatment of act1 cKO parasites showed no effect of this drug, we find that CD is not uniquely specific for actin, since similar effects on parasite motility can be observed irrespective if wt, act1 cKO or parasites expressing ACT1CDr were analysed. While CD is certainly acting on parasite actin, as indicated by the significant differences between wt and CytDr parasites, we find that concentrations above 0.5 μM CD lead to significant, non-actin-dependent effects on parasite motility, confirming previous studies . In contrast, latA and latB, previously used to analyse the role of apicomplexan actin , appear to have no effect on tachyzoite actin, as noted previously. Together, these data indicate great care should be taken in choosing the correct concentration for actin-modulating drugs, especially when using CD, where an independent, unknown target(s) appears to be present in T. gondii .
Though all myosins are thought to depend on F-actin to function as a motor and, therefore, the act1 cKO likely reflects the cumulative phenotype of all myosins, we were interested in the extent of redundancy between MyoA and MyoC, since previous studies indicated that MyoC can complement a MyoA deficiency [3, 39]. While we confirm that MyoC can partially complement for MyoA function, this complementation appears to depend on overexpression and consequent localisation of MyoC at the IMC. Indeed, our localisation analysis of MyoC in wt and myoA KO parasites demonstrates that, under physiological conditions, no significant relocation of MyoC to the IMC can be detected. In contrast, strong overexpression leads to redistribution of MyoC to the IMC, as described previously . This redistribution appears to be also critical for functional complementation, leading to a higher ratio of motile parasites and host cell invasion. However, MyoC cannot compensate for MyoA function completely, since the parasites overexpressing MyoC remain as slow as uncomplemented myoA KO parasites. These results are also in good agreement with a recent study demonstrating that MyoA is the central myosin involved in host cell invasion and that, in the absence of MyoA, host cell membrane dynamics play an important role in invasion . Importantly, deletion of mlc1 results in a complete loss of the motor complex at the IMC, therefore representing a functional double KO for MyoA and MLC1. This mutant can be kept in cell culture indefinitely when the strong egress phenotype is bypassed by artificially releasing the parasites, followed by enrichment of the population using FACS, demonstrating that MLC1 is not essential for motility and host cell invasion per se. Indeed, mlc1 cKO parasites behave similarly to act1 cKO regarding motility and attachment to the surface.
Together, this analysis suggests a role for the actomyosin system during parasite motility that is distinct from the canonical linear motor model. We hypothesise that, rather than being the force generator during motility, the actomyosin system is instead involved in the regulation of attachment sites that are required to transmit the motility force to the substrate. To address this hypothesis, we used the above mutants to analyse different parameters that are likely crucial for efficient parasite motility such as attachment strength and retrograde membrane flow. We found that most act1 cKO and mlc1 cKO parasites remain immobile. However, those that move do so at speeds similar to wt parasites, albeit with shorter trajectories, suggesting that the actomyosin system is required to initiate and maintain motility. In a 3D environment the motility defects are stronger than in a 2D environment, with most parasites showing a displacement of less than one parasite length (7 μm). In contrast, these mutants are virtually unable to attach, as measured under flow conditions, indicating an important role in the formation of attachment sites, as previously suggested . Unlike the mlc1 cKO and act1 cKO, myoA KO parasites show strong attachment to the surface, but significantly reduced speeds that cannot be compensated by overexpressing MyoC. We interpret these data to indicate that actin is required to form attachment sites, whereas MyoA might be required for the regulated release of these sites, leading to efficient force transmission during motility. A role for myosins in regulating adhesion and thereby traction force has recently been demonstrated . Here, the balance between myosin contraction and adhesion strength determines the frequency of motility initiation . A similar regulation of myosin contraction might occur in the case of apicomplexans in order to initiate motility and failure to do so could result in reduced motility rates. In this scenario, how is the motility and invasive force produced by the parasite? Previous studies implicated the generation of a retrograde membrane flow in parasite motility . Interestingly, the flow itself is relatively resistant to actin-modulating drugs. Here, we modified a bead translocation assay as an indicator of retrograde membrane flow  to analyse the role of ACT1, MLC1 and MyoA in establishing membrane flow. Surprisingly, while fewer beads bind to the surface of the parasite in the absence of these proteins, bead translocation itself appears to be not significantly affected, again indicating a role of the actomyosin system in the formation of attachment sites (in this case for latex beads), but not in bead translocation. Future experiments and tools will be required to carefully address the role of retrograde membrane flow in force production and its exact role, if any, during motility and invasion.
In conclusion, our study demonstrates the importance of the actomyosin system during motility and invasion. It is worth noting that, in physiological settings, this system is absolutely essential for parasite viability. In fact, in vivo experiments with myoA KO parasites demonstrate the complete inability of these parasites to establish an infection or to induce an adaptive immune response (our unpublished results). However, it is important to understand the molecular functions of the components of the gliding and invasion machinery in order to obtain a clear picture of how the individual components of this fascinating system work together to allow the parasite to move, invade into and egress from the host cell.
All primers used in this study are listed in Additional file 13: Table S1 and were synthesised from Eurofins (UK).
The act1 ORF containing the mutation C408G at the nucleotide level which confers the amino acid mutation of A136G was synthesised by GenScript (USA). Both the original LoxPAct1 geneswap vector  and the synthesised plasmid were digested with XmaI and PacI to replace act1 cDNA.
To generate p5RT70-Ty-MyoC, the myoC ORF was amplified from cDNA using the primers myoC ORF fw/rv. The fragment was cloned via AvrII and PacI into the parental plasmid p5RT70-Ty-KillerRed. To put myoC under its endogenous promotor a 1 kb fragment upstream of the start codon of myoC was amplified from genomic DNA using oligos pmyoC fw/rv and cloned using as template vector p5RT70-Ty-MyoC via KpnI and AvrII.
This vector was generated by cloning two 5’UTR fragments obtained by amplifying genomic DNA and a fragment of myoC gene using pBSSK + pSag1-Ble-Sag1 as a template vector. Fragment 1, pMyoC, was obtained by amplification of a 1 kb fragment upstream of the start codon of myoC, via HpaI-NotI sites using oligos pmyoC 2 fw/rv. Fragment 2, 5’UTR myoC, was obtained by amplifying a region of approximately 1.5 kb located 1 kb upstream of the myoC start codon. Amplification was done using primers 5’UTR myoC fw/rv and cloned via Kpn1-XhoI. Finally, a 2.2 kb fragment of myoC was amplified using primers myoC gene fw/rv with a Ty-tag included in the forward primer. Subsequently, cloning was performed using enzymes SpeI/NotI.
To express myoA under its endogenous promotor a 2 kb fragment upstream of the start codon of myoA was amplified from genomic DNA using oligos 5’UTR myoA fw/rv and cloned using as template vector p5RT70-Ty-MyoA via KpnI and EcoRI.
Human foreskin fibroblasts (HFFs) (SRC-1041, ATCC®) were grown on tissue culture-treated plastics and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% foetal bovine serum, 2 mM L-glutamine and 25 mg/mL gentamycin. Parasites were cultured on HFFs and maintained at 37 °C and 5% CO2. Cultured cells and parasites were regularly screened against mycoplasma contamination using the LookOut® Mycoplasma detection kit (Sigma) and cured with Mycoplasma Removal Agent (Bio-Rad) if necessary.
To generate stable expressing parasites, 1 × 107 of freshly lysed RH ∆hxgprt  or RH-DiCre ∆ku80  parasites were transfected with 20 μg DNA by AMAXA electroporation. Selection was based on mycophenolic acid and xanthine, as described previously , or bleomycin.
The conditional act1 KOCDr strain (RH-DiCre ∆ku80/endogenous act1::loxPact1 CDr loxP, referred to as loxPAct1 CDr ) was generated by replacing the act1 cassette in the LoxPAct1 geneswap vector  with a synthesised act1 cassette containing the mutation Ala 136 Gly . Integration after transfection was confirmed by PCR using primer sets (1-1’) for act1 ORF (gDNA vs. cDNA), 5’UTR integration and cre-mediated recombination with primer sets (2-2’) and 3’UTR integration (3-3’).
For stabilisation of p5RT70-Ty-MyoC, pMyoC-Ty-MyoC and pMyoA-Ty-MyoA, RH ∆hxgprt and myoA KO parasites were selected using bleomycin. The vector pBSSK + pSag1-Ble-Sag1 was co-transfected with each respective construct in the proportion (2:1), and selection was carried out as described previously .
act1 cKO and mlc1 cKO were obtained by addition of 50 nM and 100 nM of rapamycin to the parental lines, respectively. Strains were incubated for 4 h at 37 °C and 5% CO2, and cultured as described previously . To increase the population of these lines since they have an egress phenotype, act1 cKO and mlc1 cKO parasite media was swapped for DMEMcomplete supplemented with 2.5% dextran sulphate after 24 h to enrich egress mutants .
Immunofluorescence analysis was carried out as previously described . Briefly, parasites were allowed to invade and replicate in an HFF monolayer grown on glass coverslips. The intracellular parasites were fixed in 4% paraformaldehyde for 20 min at room temperature. Afterwards, coverslips were blocked and permeabilised in 2% BSA and 0.2% Triton X-100 in PBS for 20 min. The staining was performed using the indicated combinations of primary antibodies (Additional file 14: Table S2) for 1 h. Followed by the incubation with secondary AlexaFluor 350, AlexaFluor 488, AlexaFluor 594 or AlexaFluor 633 conjugated antibodies (1:3000, Invitrogen – Molecular Probes) for another 45 min, respectively.
Super-resolution SIM was carried out using an ELYRA PS.1 microscope (Zeiss). Images were acquired using a Plan Apochromat 63×, 1.4 NA oil immersion lens, recorded with a CoolSNAP HQ camera (Photometrics), and analysed using ZEN Black software (Zeiss) and ImageJ software.
Extracellular parasites were pelleted and then resuspended in RIPA buffer (50 mM Tris-HCl pH 8; 150 mM NaCl; 1% Triton X-100; 0.5% sodium deoxycholate; 0.1% SDS; 1 mM EDTA), incubation for 5 min on ice was used to lyse the cells. Afterwards, samples were centrifuged for 60 min at 14,000 rpm at 4 °C and laemmli buffer was added to the supernatant. A total of 5 × 106 parasites were loaded onto an SDS acrylamide gel while, for act1 cKO down-regulation, only 1 × 105 parasites were loaded. Western blotting was performed as described previously  using IRDye680RD or IRDye800RD (Li-Cor) secondary antibodies.
Proteins were detected using the Li-Cor Odyssey and quantified using infrared detection of the protein of interest on Image Studio 5.0 (Li-Cor).
Wt and parental LoxP strains were used as controls. For knockout lines, parasites were induced with rapamycin as described previously  and cultured for 24 h on HFFs prior to fixation. An IFA was conducted using the respective primary antibody; TgACT1 (Soldati, 1:100) or TgMLC1 (Soldati, 1:2000) and AlexaFlour 594 (Molecular Probes, 1:3000) as the secondary antibody. Images were obtained using a DeltaVision® Core microscope equipped with a CoolSNAP HQ2 CCD camera and were saved as single red and green channel files. Freeware CellProfiler 2.1.1 software (www.cellprofiler.org) was used to analyse and quantify fluorescence intensities. Greyscale images were imported to the program for analysis. The pipeline was adjusted to detect objects within the range of 5 to 40 pixels. Objects were identified using a global threshold strategy with a three class-Otsu threshold and weighted variances. Under these parameters, we identified in each image YFP-expressing parasites (generated by the DiCre cassette after correct gene excision) and the red coloured region of interest (corresponding to ACT1 and MLC1, respectively). The red signal was quantified on the basis of the total pixel occupied by the YFP expressing objects. The intensity of the red channel was measured for each object and exported to an Excel spreadsheet. The data obtained was filtered manually using area and integrated intensity parameters to include all parasites in a vacuole and exclude clustered vacuoles or random objects. Intensities from 60 vacuoles per time point were quantified and processed to remove the calculated internal background. The background fluorescence of the host cells was calculated using a YFP+ strain that was allowed to replicate under the same conditions as the tested parasites. However, no antibodies were added to the coverslips and, from this, the auto-fluorescence of the host was calculated and plotted as a horizontal dotted line.
An IFA was performed in myoA KO and RH ΔKu80 lines expressing MyoC under different levels (p5RT70-Ty-MyoC, pMyoc-Ty-MyoC and endogenous tagged MyoC). Primary antibody against Ty-tag was used to localise MyoC and DAPI as a DNA marker. Images were obtained using ELYRA PS.1 microscope (Zeiss). All 3D SIM images were collected at the same exposure and resolution parameters. Images were obtained using Zen Black (Zeiss) and analysed using ImageJ. Vacuoles to be analysed were visually identified and traced using the drawing tool. Measurements obtained were area, integrated density and mean grey value. Background measurements were obtained by selecting a portion of the image with no signal. Data was plotted by using the corrected total cell fluorescence (CTCF) which was calculated using the following formula: [CTFC = Integrated density – (Area of selected cell × Mean fluorescence of background readings)] .
Was conducted as described previously . Briefly, 1 × 103 parasites were inoculated on a confluent layer of HFFs and incubated for 5 days, after which the HFFs were washed once with PBS and fixed with ice cold MeOH for 20 min. HFFs were stained with Giemsa with plaque area measured using Fiji software. Mean values of three independent experiments ± standard deviation were determined.
Gliding assays were performed as described before . Briefly, freshly lysed parasites were allowed to glide on FBS-coated glass slides for 30 min before they were fixed with 4% paraformaldehyde (PFA) and stained with α-SAG1 under non-permeabilising conditions. Mean values of three independent experiments ± standard error of the mean (SEM) were determined. Where actin-modulating drugs were used, parasites were pre-incubated for 10 min in the respective concentration before the start of the assay.
For the assay, 5 × 104 freshly lysed parasites were allowed to invade a confluent layer of HFFs for 1 h. Subsequently, five washing steps were performed for removal of extracellular parasites. Cells were then incubated for a further 24 h before fixation with 4% PFA. Afterwards, parasites were stained with the α-IMC1 antibody . The number of vacuoles in 15 fields of view was counted. Mean values of three independent experiments ± SEM were determined.
Egress assays were performed as described previously . Briefly, 5 × 104 parasites were grown on HFF monolayers for 36 h. Media was exchanged for pre-warmed, serum-free DMEM supplemented with 2 μM A23187 (in DMSO) to artificially induce egress. After 5 min, the cells were fixed with 4% PFA and stained with a-SAG1 antibody; 200 vacuoles were counted for their ability to egress out of the host cells. Mean values of three independent assays ± SEM were determined.
Prior to sorting, mlc1 cKO parasites were scratched, syringed through a 25-G needle and filtered through a 3-μm Millipore filter (Millipore Merck) for purification of the sample. An S3e™ Cell Sorter (Bio-Rad Laboratories, Inc.), equipped with 488, 561 and 640 nm lasers, was used to sort mlc1 cKO/YFP expressing parasites. Parasites were sorted into 5 mL tubes. The temperature for sample loading stage and collection area was set at 37 °C for culturing parasites and at 4 °C for western blot analysis. Before sorting parasites, the equipment was calibrated using ProLine™ universal calibration beads (Bio-Rad Laboratories, Inc.). Gates were adjusted to separate and collect YFP expressing population (mlc1 cKO). For the long-term culturing parasites experiment, sorting was performed in single cell mode in order to obtain the purest population possible. After sorting, parasites were cultured under standard conditions described above. For western blot analysis, sorting was performed in enrichment mode to obtain the highest number of parasites possible.
Time-lapse video microscopy was used to analyse the kinetics over a 2D surface similarly to as previously described . Briefly, Ibidi μ-dish35mm-high were coated in 100% FBS for 2 h at room temperature. Freshly egressed parasites were added to the dish. Time-lapse videos were taken with a 20× objective at one frame per second using a DeltaVision® Core microscope. Analysis was made using ImageJ wrMTrckr tracking plugin. For analysis, 20 parasites were tracked during both helical and circular trails with the corresponding distance travelled, average and maximum speeds determined. Mean values of three independent experiments ± standard deviation were determined.
Parasites contained in heavily infected HFFs were scratched and passed through a 26-G needle three times to release parasites artificially. Parasites were added to HFFs grown in Ibidi m-Dish35mm-high. Invasion events were observed after approximately 5 min when parasites had settled and penetration time was determined. Time-lapse videos were recorded at 40× objective at a rate of one frame per second using DeltaVision® Core microscope. Invasion events and time were manually tracked and calculated.
Tachyzoites were prepared and assayed as previously described . Briefly, the rapamycin-induced populations were harvested by syringe release of infected HFF monolayers using a 27-G needle, filtered through a 3-μm Nuclepore filter, and gently centrifuged at 1000 × g for 4 min. The tachyzoite pellet was washed and resuspended at a final concentration of 1–2 × 108 tachyzoites/mL in 3D Motility Media (1× Minimum Essential Medium lacking sodium bicarbonate, 1% (v/v) FBS, 10 mM HEPES pH 7.0 and 10 mM GlutaMAX L-alanyl-L-glutamine dipeptide) supplemented with 0.3 mg/mL Hoechst 33342. The tachyzoite suspension was then mixed with three volumes of 3D Motility Media and three volumes of Matrigel (BD Biosciences, San Jose, CA), pre-chilled on ice. Motility in Matrigel was imaged, tracked and processed using Imaris × 64 v. 7.6.1 software (Bitplane AG, Zurich, Switzerland), as previously described . Three independent biological replicates, each with three technical replicates, were performed. Parameters calculated from 3D motility assays were analysed using two-way ANOVA with Sidak’s multiple comparisons test and with the Kolmogorov–Smirnov test using GraphPad Prism v. 6.01 (La Jolla, CA). Under our imaging conditions, trajectory displacements less than 2 μm (i.e. slightly less than one-third the body length of a parasite (~7 μm)) cannot be distinguished from Brownian motion as measured empirically with heat-killed parasites , and so a cut-off of 2 μm was applied to exclude stationary parasites. Longer displacements were considered parasite-driven and used for more detailed analysis of motility parameters.
Ibidi live cell dishes (29 mm) were coated with 0.1% poly-L-lysine for 30 min and washed with MilliQ water. Fluorescent latex beads (FluoSpheres®, 0.04 μm, Invitrogen) were diluted with 5 μL in 400 μL Hanks Balanced Salt Solution + HEPES (25 mM) (described hereafter as H-H buffer) and sonicated twice for 2 min. After a short spin (10 s, 6000 × g), the supernatant was recovered and left on ice for 30 min before use. Parasites of interest were harvested (by scratch, syringe and filtration). Parasites were pelleted for 5 min at 3000 × g, washed and resuspended in cold 250 μL H-H buffer to achieve 107 parasites/mL. Parasites were then transferred to poly-L-lysine-coated dishes and left on ice for 20 min; 5 μL of diluted beads were added to 250 μL of H-H buffer and added to the parasites. Immediately, the dish was incubated at 37 °C for 15 min. The experiment was stopped by addition of 2 mL of 4% PFA and incubated at 4 °C for 10 min. The PFA was washed gently and parasite nuclei stained with Hoechst 0.01%. For time course, the parasites were fixed at different time points after the addition of the beads. For the drug and buffer assays, parasites were incubated for 10 min in the buffer of interest before their incubation on the coated dish. Bead dilution and the rest of the experiment were also made using the same buffer (Endo Buffer or H-H buffer + 0.5 μM CD).
For each experiment (n), an average of 1000 parasites were analysed. Total numbers of parasites, number of parasites without beads, with beads bound and with beads capped were quantified (e.g. For RH n1, a total of 1615 parasites were counted, within those, 63 were not interacting with the beads, 1062 had beads around them and 490 had capped beads). For each condition, experiments were performed in three independent experiments n = 3. Mean values of each interaction type for three independent experiments ± SEM were determined and compared to RH.
LoxP strains were induced to generate KOs as previously described in Egarter et al. . Fresh extracellular parasites (4 × 105 in total, consisting of approximately equal numbers of control and KO) were loaded into collagen IV coated fluidic chambers (Ibidi IB-80192) and allowed to attach at 37 °C for 20 min. PBS was pumped through the chamber using an ‘open loop flow’ microfluidic pump (KD Scientific Legato 200 syringe pump) system, similar to that described previously , to control flow rates and generate fluidic shear stress. In our setup, a flow rate of 1 mL/min achieves 3 dyn/cm2 shear stress at the surface of the channel. Flow at 0.1 mL/min (equivalent to 0.3 dyn/cm2) was used to remove all non-attached parasites. At each fluidic shear stress level, control and mutant parasites were counted from five fields of view per experiment. Parasite count after the 0.1 mL/min wash was taken as 100% of attached parasites. Counts at all other rates of flow were normalised to the 100%. Data collected was analysed using Excel to assess the significance of differences between control and mutants using Student t-test and further analysed using GraphPad Prism v. 6.01 software to display data as trends. Parasites in the chamber were monitored via a Zeiss Axio Vert.A1 microscope setup with a 40× objective combined with an AxioCam ICm1 camera and Zen capture software.
We would like to thank members of the Meissner lab for thoughtful discussions. We would also like to thank Prof. David Sibley (Washington Univeristy, St. Louis), Prof. Dominque Soldati-Favre (University of Geneva), Prof. Artur Scherf (Institut Pasteur, Paris), Dr. Jake Baum (Imperial College, London) and Dr. Maryse Lebrun (University of Montpellier) for generously providing antibodies and parasite strains.
This work was supported by an ERC-Starting grant (ERC-2012-StG 309255-EndoTox) and the Wellcome Trust 087582/Z/08/Z Senior Fellowship for MM, and US Public Health Service grant AI054961 for GEW. The Wellcome Trust Centre for Molecular Parasitology is supported by core funding from the Wellcome Trust (085349). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
All data generated or analysed during this study are included in this published article and its supplementary information files, or are available from the authors on reasonable request.
JW and FLB performed experiments analysed results and interpreted data of mutant parasites characterisation; participated in the writing, revision and edition of the manuscript. SG and GSP carried out and analysed retrograde flow and flow chamber experiments respectively. JML set up and analysed 3D motility experiments. AH designed and analysed experiments of dense granules motility. SE and NA generated and analysed the studied mutant lines. GW conceived the 3D motility project, designed the study, analysed results, interpreted data, and drafted and revised the article. SRN and DMW analysed dense granule motility data and revised the article. MM conceived the project, designed the study, analysed results, interpreted data, and wrote the first manuscript. All authors participated in the revision of the manuscripts and approved the final version.
Periz J, Whitelaw J, Harding C, Lemgruber L, Gras S, Reimer M, et al. Toxoplasma gondii establishes an extensive filamentous network consisting of stable F-actin during replication. bioRxiv. 2016. doi:https://doi.org/10.1101/066522.
Coleman BI, Gubbels MJ. A genetic screen to isolate Toxoplasma gondii host-cell egress mutants. J Vis Exp. 2012;60. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3350636/.

industry_corpus2: Biopharmaceuticals


text: The online brokerage business burst on the scene in the late 90s, as at-home traders were lured to try their hand at winning big in the stock market. These days, investors are inundated with the message that they shouldn't try stock picking, and that they should engage in passive, low cost strategies instead. So how has the online brokerage business adapted? Chris Larkin, Senior Vice President of Trading at E*Trade, explains.

industry_corpus2: Finance & Economics


text:
Le Steinala is located in Bergheim. Guests can enjoy the free WiFi. The apartment is composed of 1 separate bedroom, a living room, a fully equipped kitchen with a dishwasher and oven, and 1 bathroom. A flat-screen TV is available. Colmar is 17 km from the apartment, while Riquewihr is 8 km away. Strasbourg International Airport is 57 km from the property.
This apartment has a dishwasher, electric kettle and kitchenware.
Please inform Le Steinala in advance of your expected arrival time. You can use the Special Requests box when booking, or contact the property directly with the contact details provided in your confirmation. This property will not accommodate hen, stag or similar parties.

industry_corpus2: Hospitality & Catering


text:
yummylicious, one of my favourites but instead of keeping the wings, i keep the breasts , hehe!
Beautiful, easy and delicious !!!!
Mmm, rice porridge is such a comforting dish. Yours looks so tasty.
Chicken porridge is really absolutely delicious!
Such a filling and inviting chicken porridge..
Oh you have me at chicken porridge! Looks so delicious!
Your porridge looks so comforting and filling and delicious. I bet it's extremely tasty. And isn't our sister clever. I love the bowl!
Whoops, I meant isn't YOUR sister clever, lol.
Congee! I love it! I've only ever had it plain, but no doubt the chicken would be a great flavour enhancer.
I am not familiar with this dish, except by name. It looks delicious, I would be surprised by the flavor however, expecting sweet! It is always interesting to learn of other ways the same foods are prepared. Love that you served such a favorite in your special bowl!
Thank you for visiting! Chicken porridge is comfort food alright. We can have it for breakfast, lunch and even supper! A definite all-time favourite! Have a lovely day, ladies!
Thank you for stopping by. I hope that you will drop by again. Have a nice day!
I loved this dish because I like chiken and my family too. Your recipes are always very special and tasty. Kisses.
Your Chicken Porridge looks so delicious in that special soup bowl. The soup looks very light and yet very comforting, I will try it very soon. Thank you so much for bringing it to Full Plate Thursday and please come back!
Se ve una sopa delicosa,con la sustancia del pollo y más rica es servida en esos bowl muy bonitos,abrazos.
What a comforting bowl of porridge! I'd love to come home to that! Delicious.
This is always my number one comfort food...simple and delicious!
bestest!! ooh la la! slurp slurpp!
This looks so good!! I think I will try to make it. All your recipes sound delicious!!


text:
After dinner the other night, we left the dishes piled high and leftovers sitting on the counter so we could sneak out back to look at our bees. It was a beautiful, sunny evening with very little breeze- a perfect evening for taking a peek. Jamey and I donned our bee-protection- a hat with veil for me, a long-sleeved shirt with hood and veil for Jamey, and a smoker for us all.
We already knew that both packages were doing well. The queens were laying eggs and both colonies were already given more room to build comb so they could store their pollen and nectar. This evening we wanted to see if they had taken all the sugar syrup we made available and if they needed additional room.
Both hives drained their syrup so we added more. If there is plenty of nectar available in nature, bees prefer the real stuff and will ignore the syrup. The fact that they took it all told us that they were glad for the extra stores. We'd prefer that we didn't need to supplement in this way but after losing so many bees this past winter, we're apt to spoil them.
We saw plenty of brood, pollen and nectar in both hives.
Both hives were building new comb but the top bar colony built more. We added several empty bars to the top bar hive to let them continue building out. The Langstroth colony built a little but still has plenty of room in the top super so nothing was added to this hive.
Sometimes bees build comb in places that are inconvenient for us- like between frames, making it hard for us to do inspections. The comb needs scraping off with a hive tool.
Our hives are at the edge of a small wooded area with several black locust trees. This particular evening, the scent of their blooms was intoxicating. Honey bees love black locust nectar- something we didn't put together when we placed our hives.
They (and other native pollinators) also love clover. I wish we had fields and fields of these beautiful flowers.
You photos are amazing! The photos of the bees at the flowers are absolutely BEAUTIFUL!!
Please tell me you are eating those locust blossoms! They are SO delicious, but only good right around now for you folks (Ours aren't out until early June).
We use the fruit batter recipe in the Joy of Cooking, and dip the blossoms in, fry them in a few inches of oil, and then dust with powdered sugar. Don't eat the fibrous end of the stem. :-). They taste similar to how they smell- completely intoxicating, similar to what rosewater or orange blossom water does for middle eastern recipes.
Why not do some cover crops of clover? It looks like you have crimson clover and the bees really like that. Our winters are to cold here usually for the crimson. My daughter and some of her cousins, all farmers, have taken over a hive operation of a retiring older couple and they have been building up patches of bee-friendly crops.
Hi, there! The clover was an attempt at a cover crop but only a small patch came up for some reason. We'll try again for sure:-).
We've been wanting to start keeping bees!! I'm fairly certain that we live in the same area - I wanted to know if you would be willing to tell me where you get your bees, and which hives are best for starting up? Love these pictures!

industry_corpus2: Agriculture & Fisheries


text:
Gaylord National provides an experience you'll find nowhere else. From a luxurious spa to creative dining, inviting swimming pool to historic attractions, you'll find activities every family member will enjoy.
Relax and Rejuvenate in our Spa. Your health, wellness and relaxation are the focus of Relâche, a world-class spa & salon. Our spa features 12 luxurious treatment rooms, including a couples treatment room overlooking the Potomac River and a beautiful co-ed Riverview Relaxation Lounge. Call (301) 965-4400 to make your appointment.
Some of DC’s most exciting tours depart from Gaylord National, including trips to Washington’s major memorials and monuments and DC@Night, Washington’s only downtown night tour.
Visit historic and cultural attractions in Washington, D.C., conveniently located minutes away.
Find unique boutiques, stores and retail shops all under one roof.
The Atrium Fountain is a majestic display of lights, sound and dancing water that shoots 60-feet high. Gaze at the fountain from the Lower Atrium, or enjoy a bird’s-eye view while enjoying a cocktail at Belvedere Lobby Bar. See the fountain perform shows every night in our Atrium at 6pm, 7pm, 8pm, 9pm. Show times subject to change.
Guests love relaxing in our signature atriums framing dramatic views of the Potomac River, leading to DC’s famous monuments and museums. You won’t believe what we have under one roof!

industry_corpus2: Travel & Geography


text:
Internal Mini Blinds | AMICA Windows and Doors Inc.
Simple and beautiful describes our Custom Internal Mini Blind collection. With blinds built into the glass, you get controlled privacy with a maintenance-free mini blind. Our Internal Mini Blind collection features all the standard doorlite and sidelite sizes, and comes standard with the raise, lower and tilt feature. Refined luxury at its best.

industry_corpus2: Real Estate & Construction


text: This paper proposes a course for ethnography in design that problematizes the implied authenticity of “people out there,” and rather favors a performative worldview where people, things and business opportunities are continuously and reciprocally in the making, and where anthropological analysis is only one competence among others relevant for understanding how this making unfolds. In contrast to perpetuating the “real people” discourse that often masks the analytic work of the anthropologist relegating the role of the ethnographer to that of data collector (Nafus and Anderson 2006), this paper advocates a performative ethnography that relocates the inescapable creative aspects of analysis from the anthropologist's solitary working office into a collaborative project space. The authors have explored the use of video clips, descriptions and quotes detached from their “real” context, not to claim how it really is out there, but to subject them to a range of diverse competencies, each with different interests in making sense of them. Hereby the realness of the ethnographic fragments lie as much in their ability to prompt meaningful re-interpretations here-and-now as in how precisely they correspond to the imagined real world out there-and-then. We propose that it is precisely the investment of one self and one's own desires and agendas that lifts an ethnographic field inquiry out of its everydayness and into something of value to further-reaching processes of change and development of attractive alternatives.

industry_corpus2: Subject Education


text:
When Gov. Jerry Brown signed SB 350 into law earlier this month, he was praised for putting California at the forefront of energy and climate policy. But if the governor wants to make an even bigger impact on the environment, he should turn his attention to the meat and dairy industries and their devastating impact on global warming.
The U.N. singled out both in a sweeping study that called for the world to shift to plant-based diets. It found that agriculture, particularly meat and dairy production, accounts for 19 percent of global greenhouse-gas emissions, 38 percent of land use, and 70 percent of freshwater consumption. Given the state’s water crisis, that last statistic raises a red flag. So does this one: It can take more than 2,400 gallons of water to produce one pound of beef. By contrast, 25 gallons are needed for a pound of wheat.
Roughly 80 percent of California’s water is used for agricultural purposes. It has the nation’s fourth-largest cattle herd (5.2 million). It is the largest milk-producing state.
That is not sustainable. But an environmentally friendly, plant-based diet is. A study by Loma Linda University found that vegans have a greenhouse-gas footprint that is nearly 42 percent smaller than that of meat eaters.
-- Craig Shapiro - PETA Foundation, Norfolk, Virg.


text:
At Burlington, NC Cottman, located at Burlington, NC, our technicians are eminently familiar with the unique characteristics of today's diesel transmissions. We know how they work, and we know how to fix them when they don't.
And, just as with gas-fueled cars and trucks, today's diesel engines and transmissions are controlled by computer systems. At Burlington, NC Cottman, we have access to the most up-to-date diagnostic information and equipment for those computer control systems, allowing us to analyze and identify problems that can affect your transmission.
So, regardless of the engine — diesel or gas — at Burlington, NC Cottman, we'll diagnose your transmission problem right... the first time. And you can be sure that you'll never pay for a transmission unless you really need a transmission. That's our promise to you.
If you have a question about your diesel transmission or would like to set up an appointment for our free transmission checkup, call Burlington, NC Cottman right now at 336-794-6167.

industry_corpus2: Automobiles


text:
Dog Literally Scarred Stiff by Trip to the Veterinarian!
The Doctors are joined by veterinarian Dr. Arvid Edward to weigh in on viral pet videos including the case of a dog who was actually paralyzed by fear during a trip to the veterinarian. Dr. Edward shares how owners can help a pet who is fearful of the vet.


text:
Over the last few years, a few areas of my politics have become more conservative. I am pro-death penalty. I am anti-welfare. I am pro-personal responsibility. I am for a system that penalizes attorneys who repeatedly present frivolous cases to government funded courts. So I’ve felt an introduction of some libertarianism in my politics.
I have some areas that I am staunchy liberal though: I am pro decriminalization of marijuana and several other drugs, I am pro net neutrality, I am pro choice, and I cannot fathom why gay marriage should be prohibited, let alone introduce discrimination into the constitution. I am also extremely anti-war in the Middle East.
So today I took a political test on vajoe.com to find out which candidate best matches my current beliefs, and lo and behold, I guess I am a very typical democrat. Every Democrat matches me closer than every Republican, and, as I expected, Ron Paul matches me best from the GOP – since he’s barely even a Republican, more of a libertarian himself.
It’s also worth nothing that the “conservative” swing I’ve felt is more of a traditional conservative from the 60’s than the kind we see today. Barry Goldwater would vomit at what passes for a conservative today.

industry_corpus2: Politics & Administration


text:
One of the cool features of SAP SQL Anywhere, on-demand edition is that you can run multiple sever versions all within the same cloud. For example, it is possible to run both SQL Anywhere 12 and SQL Anywhere 16 servers at the same time, even on the same machine (Note that this is different from running multiple database versions at the same time, but you can do that too).
When you create a SQL Anywhere, on-demand edition cloud it will only have one server version available. To use a different server version, you will have to install the new server software into your cloud. This quick tutorial shows the steps required to add SQL Anywhere 12 support to an existing SP5 cloud.
Create a SQL Anywhere, on-demand edition SP5 cloud. If you don’t have a cloud, you can create one using the developer edition. The default server included in SP5 is SQL Anywhere 16.0.0.1824. Open the cloud console and click on the Cloud Software link. You will see there is only one server version currently installed.
We will need to download the new server version from the SAP website. Browse the list of available server versions, and download the install package for the server version that you want to add to your cloud. For this demo, I am downloading the SQL Anywhere 12.0.1.4086 for Windows package.
The install package must be added to the cloud before it can be installed. Click Events -> Run new task.
Enter the full path to the install package you downloaded in Step 2. Click Next. This will add the install package to the cloud.
Click on the Cloud Software tab and confirm that new server version has been added to the cloud.
Although the new servers version has been added to the cloud, it has not yet been installed. The install package only has to be added to the cloud once, but it also must be installed on every host on which you want to run that server version. This allows you to limit certain server versions to only specific hosts. Fortunately, the cloud console makes it easy to install the software on any host. Select the new cloud version and click Install.
Enter the list of hosts on which you want the new server version installed.
The cloud console will coordinate the installation of the new server version on all of the target hosts. Once the install is complete, you will see that the new server version appears in the list of Installed Cloud Software.
When creating a new server, you will now be able to select the new server version.

industry_corpus2: Computing & Telecommunications


text:
Harlaxton faculty, staff, and administration are dedicated to creating a supportive and caring community so that all of our students consider Harlaxton their second home. We understand, however, that our students will not reach Harlaxton Manor and enter through our magnificent front door without the support and care of parents, family, friends, and others from home.
Both the US and UK Harlaxton teams are readily available to provide information about our program, address any concerns that may arise, and answer any other questions you may have.
If you have general questions about the Harlaxton program, the application process, or the pre-departure process, you can contact the US Harlaxton office at 812-488-1040 or email harlaxton@evansville.edu.
If you have questions while your student is at the manor, you can call +44 1476 403000 or email enquiries@harlaxton.ac.uk.


text:
Note: the only reason I made .thumbnail span background-color: silver was to see if anything at all would show up in IE7.
Note: I removed most of the repetitive rows for space purposes.
This is my first post here, I look forward to any advice on this problem. Thanks in advance.
It was the table. For some reason the table negated the image from showing upon hover. I removed the table - works like a charm now.


text:
There are many forms of bonds to fit various business circumstances, such as performance bonds; contract bonds; or bid bonds. A surety bond is essentially a risk transfer mechanism. The bonding company (surety) provides the governing authorities (obligee) a guarantee that the construction contractor (principal) will live up to contract obligations. The government often requires surety bonds for construction companies competing for government contracts. Private firms or general contractors may also require a surety bond when accepting bids from sub-contractors.
Swiftbonds LLC are the industry leading experts in all forms of construction bonds.
A surety bond may be required to resolve disputes by paying a claim in case the contractor does not fulfill their obligations. Should a claim be made for a failure to live up to the contract provisions, the construction contractor will be required to pay back the surety company and may have difficulty obtaining future bonds. For more information on surety bonds, visit the Small Business Administration website: (sba: https://www.sba.gov/surety-bonds).
Bond capacity is the dollar amount (or bond line) a contractor may be approved for. The single limit is the largest amount a single bond may be written for on each job. The total limit is the aggregate sum of all outstanding bonds a contractor can carry at the same time.
Most companies distribute surety bonds through an agency or brokerage. In return the contractor pays a rate based on the size of the contract. At Swiftbonds LLC, we earned a reputation for providing the lowest possible rates. While many agents can be found locally and online, they must be licensed to operate in the contractor's state. As a professional bond producer we can guide the contractor in the bonding process, helping to establish a relationship with a surety company. We assist in managing surety capacity for the contractor. As a professional bond producer, we can offer business advice and expertise on related issues.
As brokers, we gain understanding of the contractor's business. Then guide the company in meeting the submission requirements of the surety company best matched to their needs and profile. All surety companies have various guidelines of their own and will not be suitable for all contractors.
Once the bond producer gathers all the required information, it is submitted to a surety bond underwriter who determines whether the contractor is capable of completing the project in question, such as required equipment or labor, and whether they have the means to pay back the bond in case of a claim. This is often called the prequalification process. This may take time as the bond underwriter analyzes the bond-seeker, and the bond producer may called upon for further information or clarification.
When searching where to buy a surety bond, construction firms can be confident that Swiftbonds has checked with every state to verify that the surety company is properly licensed to issue bonds in the contractor's state. We do everything possible to verify the reputation and integrity of the proposed surety company before documents are signed.
Swiftbonds has a demonstrable track record of taking personal interest in our client construction contractors, and over the years we have come to possess an in-depth understanding of the trade, particularly regarding accounting, finance, and familiarity with contracts and contract law.
As a highly professional bond producer we represent a vital link between the construction contractor and the surety bond company, and we can be counted on to maintain a helpful and professional relationship with both.


text:
Kent, Lia. PhD thesis. Justice Seidauk Mai (Justice  is yet to come): Rethinking the dynamics of transitional justice in  East Timor. Submitted August 2010, University of Melbourne.
This  thesis analyses the grounds of separation between the official claims  made for the UN-sponsored transitional justice process in East Timor and  community discontent. It does this by analysing fieldwork findings –  including interviews with survivors of East Timor’s conflict – and  engaging with critical and ethnographic scholarship on transitional  justice, in order to examine the interplay between different narratives  of justice circulating in East Timor in the post-referendum period.  These narratives emanate from three separate, yet intersecting, spheres:  the international, the national and the local.
The  thesis begins by examining narratives of retributive and restorative  justice promoted in the international sphere, through the UN’s  transitional justice ‘tool-kit’. These narratives emphasise the  importance of individual accountability, and the therapeutic value of  truth-telling, to respond to the violence of the past and foster East  Timor’s transition from conflict to peace. It then discusses the  contrasting narratives promoted in the national sphere, by East Timor’s  political elite, which stress collective and forward-looking ideas of  social justice as the best way to move on from the violent past.  Finally, it considers narratives generated within the local sphere,  promoted by East Timorese survivors. It shows that local narratives,  which are informed by survivors’ personal – and often continuing –  experiences of injustice, both contest and draw upon national and international narratives and are transforming them in new ways.
By  charting the intricate interactions between these narratives, the  thesis shows that the UN-sponsored transitional justice process has been  unable to provide a definitive ‘justice’ response that marks a rupture  between the past and future. Rather, it has contributed to an ongoing,  locally grounded, conversation about how best to ‘deal with the past.’  The thesis shows that the official transitional justice process has  produced new forms of political engagement and is transforming local  justice struggles in unexpected ways. By bringing to the fore the  dynamism of these developments in East Timor, the thesis argues that  transitional justice might best be understood as a continuing and  productive interaction between international, national and local actors.
5.4        Recent Developments: the Leadership’s Responses to Chega!

industry_corpus2: Law & Justice