This protocol describes an improved northern blot method that enhances detection of small RNA molecules (<40 nt) including regulatory species such as microRNA (miRNA), short-interfering RNA (siRNA) and Piwi-interacting RNA. Northern blot analysis involves the separation of RNA molecules by denaturing gel electrophoresis followed by transfer and cross-linking of the separated molecules to nylon membrane. RNA of interest is then detected by hybridization with labeled complementary nucleic acid probes. We have replaced conventional UV-cross-linking of RNA to nylon membranes with a novel, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)-mediated, chemical cross-linking step that enhances detection of small RNA by up to 50-fold. This requires no specialized equipment, is relatively inexpensive and is technically straightforward. Northern blotting can be done in 2 d, but detection of a specific RNA can vary from minutes to days. Although chemical cross-linking takes longer (15 min to 2 h) than UV cross-linking, improved sensitivity means shorter periods of exposure are required to detect signal after hybridization.

The northern blot, or RNA gel blot, is a widely used method for the discovery, validation and expression analysis of small regulatory RNA such as small interfering RNA (siRNA), microRNA (miRNA) and piwi-interacting RNA (piRNA). Although it is straightforward and quantitative, the main disadvantage of a northern blot is that it detects such RNA less sensitively than most other approaches. We found that the standard dose of UV used in northern blots was not the most efficient at immobilizing small RNA of 20–40 nt on nylon membranes. However, increasing the dose of UV reduced the detection of miRNA by hybridization in northern blotting experiments. We discovered that using the soluble carbodiimide, EDC, to cross-link RNA to nylon membranes greatly improved the detection of small RNA by hybridization. Compared to standard UV cross-linking procedures, EDC cross-linking provided a 25–50-fold increase in the sensitivity of detection of siRNA from plants and miRNA or piRNA from mammalian cells. All types of hybridization probes tested benefited from the new cross-linking procedure. Cross-linking was dependent on a terminal phosphate and so, should be applicable to other related categories of small RNA.

BackgroundApicomplexan 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.ResultsIn 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.ConclusionWe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-016-0343-5) contains supplementary material, which is available to authorized users.

Apicomplexan parasites are thought to actively invade the host cell by gliding motility. This movement is powered by the parasite's own actomyosin system, and depends on the regulated polymerisation and depolymerisation of actin to generate the force for gliding and host cell penetration. Recent studies demonstrated that Toxoplasma gondii can invade the host cell in the absence of several core components of the invasion machinery, such as the motor protein myosin A (MyoA), the microneme proteins MIC2 and AMA1 and actin, indicating the presence of alternative invasion mechanisms. Here the roles of MyoA, MLC1, GAP45 and Act1, core components of the gliding machinery, are re-dissected in detail. Although important roles of these components for gliding motility and host cell invasion are verified, mutant parasites remain invasive and do not show a block of gliding motility, suggesting that other mechanisms must be in place to enable the parasite to move and invade the host cell. A novel, hypothetical model for parasite gliding motility and invasion is presented based on osmotic forces generated in the cytosol of the parasite that are converted into motility.

Ultrasonics offers the possibility of developing sophisticated fluid manipulation tools in lab-on-a-chip technologies. Here we demonstrate the ability to shape ultrasonic fields by using phononic lattices, patterned on a disposable chip, to carry out the complex sequence of fluidic manipulations required to detect the rodent malaria parasite Plasmodium berghei in blood. To illustrate the different tools that are available to us, we used acoustic fields to produce the required rotational vortices that mechanically lyse both the red blood cells and the parasitic cells present in a drop of blood. This procedure was followed by the amplification of parasitic genomic sequences using different acoustic fields and frequencies to heat the sample and perform a real-time PCR amplification. The system does not require the use of lytic reagents nor enrichment steps, making it suitable for further integration into lab-ona-chip point-of-care devices. This acoustic sample preparation and PCR enables us to detect ca. 30 parasites in a microliter-sized blood sample, which is the same order of magnitude in sensitivity as lab-based PCR tests. Unlike other lab-on-a-chip methods, where the sample moves through channels, here we use our ability to shape the acoustic fields in a frequency-dependent manner to provide different analytical functions. The methods also provide a clear route toward the integration of PCR to detect pathogens in a single handheld system. phononic crystal | surface acoustic waves | nucleic acid amplification test | mechanical cell lysis A coustic waves contain a mechanical energy that can be used to manipulate fluids, cells, and samples (1). A range of ultrasonic transducers have previously been developed, including those using surface acoustic wave (SAW) devices, as a practical solution to actuate fluids on microfluidic chips (2, 3). SAWs have the advantage that, despite using low powers, the energy is concentrated at the interface between the fluid and the substrate, enabling a range of fluid manipulations on a chip. Despite this ability to implement low power microfluidics, one potential disadvantage of using a SAW chip is the relatively high cost of the piezoelectric wafer. In an alternative configuration, the SAW can be coupled into a disposable superstrate (Fig. 1A) placed on the surface of the piezoelectric chip (4, 5), thus providing a low cost technology.Using such superstrates, we have recently demonstrated an alternative and improved method for performing complex fluid manipulations in which the ultrasonic waves are coupled into phononic lattices. Importantly, the functionality of such phononic structures is dependent upon the acoustic frequency. By using phononics to locally shape the acoustic fields and by switching between different ultrasonic wavelengths, we have designed tools capable of enabling different fluid manipulations on the disposable superstrate (5-7).In this paper we show the implementation of nucleic acid based tests (NATs) on a microfluidic chip to demonstrate the potential of phon...

The functional characterisation of essential genes in apicomplexan parasites, such as Toxoplasma gondii or Plasmodium falciparum, relies on conditional mutagenesis systems. Here we present a novel strategy based on U1 snRNP-mediated gene silencing. U1 snRNP is critical in pre-mRNA splicing by defining the exon-intron boundaries. When a U1 recognition site is placed into the 3’-terminal exon or adjacent to the termination codon, pre-mRNA is cleaved at the 3’-end and degraded, leading to an efficient knockdown of the gene of interest (GOI). Here we describe a simple method that combines endogenous tagging with DiCre-mediated positioning of U1 recognition sites adjacent to the termination codon of the GOI which leads to a conditional knockdown of the GOI upon rapamycin-induction. Specific knockdown mutants of the reporter gene GFP and several endogenous genes of T. gondii including the clathrin heavy chain gene 1 (chc1), the vacuolar protein sorting gene 26 (vps26), and the dynamin-related protein C gene (drpC) were silenced using this approach and demonstrate the potential of this technology. We also discuss advantages and disadvantages of this method in comparison to other technologies in more detail.

Micronemal proteins of the thrombospondin-related anonymous Background: protein (TRAP) family are believed to play essential roles during gliding motility and host cell invasion by apicomplexan parasites, and currently represent major vaccine candidates against , the causative agent Plasmodium falciparum of malaria. However, recent evidence suggests that they play multiple and different roles than previously assumed. Here, we analyse a null mutant for MIC2, the TRAP homolog in . We performed a Toxoplasma gondii Methods: careful analysis of parasite motility in a 3D-environment, attachment under shear stress conditions, host cell invasion and virulence. We in vivoResults: verified the role of MIC2 in efficient surface attachment, but were unable to identify any direct function of MIC2 in sustaining gliding motility or host cell invasion once initiated. Furthermore, we find that deletion of causes a mic2 slightly delayed infection leading only to mild attenuation of virulence; in vivo, like with wildtype parasites, inoculation with even low numbers of KO mic2 parasites causes lethal disease in mice. However, deletion of causes mic2 delayed host cell egress , possibly via disrupted signal transduction in vitro pathways.We confirm a critical role of MIC2 in parasite Conclusions: attachment to the surface, leading to reduced parasite motility and host cell invasion. However, MIC2 appears to not be critical for gliding motility or host cell invasion, since parasite speed during these processes is unaffected. Furthermore, deletion of MIC2 leads only to slight attenuation of the parasite. Amendments from Version 1In response to the reviewers comments, we modified figure 5 and 6 (error bars, size bars have been added). We also provide more information regarding the in vivo experiments and tuned down the interpretation regarding virulence of mic2KO parasites. Furthermore, supplementary information for the RNAseq analysis has been added. New data added: RH FPKM, MIC2 KO FPKM and AMA1 FPKM, contain the FPKM of the three triplicates for all Toxoplasma genes for RH, mic2 KO and ama1 KO strains. MIC2KO-RH and AMA1KO-RH total results obtained after Cutdiff analysis between RH and mutants. The addition of these data will allow to access the whole RNA sequencing results and perform independent analysis.

The obligate intracellular parasites Toxoplasma gondii and Plasmodium spp. invade host cells by injecting a protein complex into the membrane of the targeted cell that bridges the two cells through the assembly of a ring‐like junction. This circular junction stretches while the parasites apply a traction force to pass through, a step that typically concurs with transient constriction of the parasite body. Here we analyse F‐actin dynamics during host cell invasion. Super‐resolution microscopy and real‐time imaging highlighted an F‐actin pool at the apex of pre‐invading parasite, an F‐actin ring at the junction area during invasion but also networks of perinuclear and posteriorly localised F‐actin. Mutant parasites with dysfunctional acto‐myosin showed significant decrease of junctional and perinuclear F‐actin and are coincidently affected in nuclear passage through the junction. We propose that the F‐actin machinery eases nuclear passage by stabilising the junction and pushing the nucleus through the constriction. Our analysis suggests that the junction opposes resistance to the passage of the parasite's nucleus and provides the first evidence for a dual contribution of actin‐forces during host cell invasion by apicomplexan parasites.