Red blood cells parasitized by Plasmodium falciparum can be distinguished from uninfected cells and characterized on the basis of reduced deformability. To enable improved and simplified analysis, we developed a microfluidic device to measure red blood cell deformability using precisely controlled pressure. Individual red blood cells are deformed through multiple funnel-shaped constrictions with openings ranging from 5 down to 1 μm. Precisely controlled pressures are generated on-chip using a microfluidic circuit that attenuates an externally applied pressure by a factor of 100. The pressures required to squeeze each cell through the constriction are used as a readout to determine the intrinsic stiffness of each cell. Using this method, parasitized cells from ring through schizont stages were shown to be 1.5 to 200 times stiffer than uninfected cells. The measured deformability values of uninfected and parasitized cells showed clearly distinct distributions, demonstrating the potential of using this technique to study the pathophysiology of this disease, and the effect of potential drugs.
SummaryOngoing human infections with highly pathogenic avian H5N1 viruses and the emergence of the pandemic swine-origin influenza viruses (IV) highlight the permanent threat elicited by these pathogens. Occurrence of resistant seasonal and pandemic strains against the currently licensed antiviral medications points to the urgent need for new and amply available anti-influenza drugs. The recently identified virus-supportive function of the cellular IKK/NF-kB signalling pathway suggests this signalling module as a potential target for antiviral intervention. We characterized the NF-kB inhibitor SC75741 as a broad and efficient blocker of IV replication in non-toxic concentrations. The underlying molecular mechanism of SC75741 action involves impaired DNA binding of the NF-kB subunit p65, resulting in reduced expression of cytokines, chemokines, and pro-apoptotic factors, subsequent inhibition of caspase activation and block of caspase-mediated nuclear export of viral ribonucleoproteins. SC75741 reduces viral replication and H5N1-induced IL-6 and IP-10 expression in the lung of infected mice. Besides its virustatic effect the drug suppresses virus-induced overproduction of cytokines and chemokines, suggesting that it might prevent hypercytokinemia that is discussed to be an important pathogenicity determinant of highly pathogenic IV. Importantly the drug exhibits a high barrier for development of resistant virus variants. Thus, SC75741-derived drugs may serve as broadly non-toxic anti-influenza agents.
Highly pathogenic avian influenza viruses (HPAIVs) cause severe disease in humans. Still, the basis for their increased pathogenesis remains unclear. Additionally, the high morbidity in the younger population stays inexplicable, and the recent pandemic H1N1v outbreak in 2009 demonstrated the urgent need for a better understanding about influenza virus infection. In the present study, we demonstrated that HPAIV infection of mice not only led to lung destruction but also to functional damage of the thymus. Moreover, respiratory dendritic cells in the lung functioned as targets for HPAIV infection being able to transport infectious virus from the lung into the thymus. The pandemic H1N1 influenza virus was able to infect respiratory dendritic cells without a proper transport to the thymus. The strong interference of HPAIV with the immune system is especially devastating for the host and can lead to lymphopenia. In summary, from our data, we conclude that highly pathogenic influenza viruses are able to reach the thymus via dendritic cells and to interfere with T lymphocyte development. Moreover, this exceptional mechanism might not only be found in influenza virus infection, but also might be the reason for the increased immune evasion of some new emerging pathogens.
BackgroundThe Plasmodium falciparum multidrug resistance 1 transporter, PfMDR1, contains five amino acid polymorphisms that are suggested to be involved in altered drug transport from the parasite’s cytosol into the digestive vacuole (DV). Transport of a substrate into another intracellular compartment influences drug availability at its site of action, therefore making the parasite more susceptible or resistant to a drug. Fluo-4 is a known fluorescent substrate that can be used as a molecular tool to investigate transport dynamics of PfMDR1 in many parasite strains.MethodsSix P. falciparum strains with varying PfMDR1 mutations were loaded with Fluo-4 AM. Accumulation of the fluorophore in the DV was measured using confocal microscopy. The role of a key amino acid mutation was verified using selected parasite clones with point mutations at PfMDR1 amino acid position 1042. Equal expression of PfMDR1 was confirmed by Western blot.ResultsFluo-4 was transported by PfMDR1 into the DV of most drug-sensitive and -resistant parasites. Asparagine at PfMDR1 amino acid position 1042 was crucial for Fluo-4 transport, while the N1042D substitution abolished Fluo-4 transport. Competition studies of Fluo-4 with chloroquine, quinine and mefloquine were performed on parasites harbouring asparagine at position 1042. A distinct Fluo-4 transport inhibition pattern for each tested anti-malarial drug was observed in parasite strains of different genetic background.ConclusionThis study demonstrates that Fluo-4 can be used to investigate PfMDR1 transport dynamics in both drug-sensitive and -resistant parasites. Furthermore, direct evidence of altered Fluo-4 transport in PfMDR1 is linked to a single amino acid mutation in the substrate binding pocket. This system offers a great tool to investigate the role of substrate transport by PfMDR1 and the mutations necessary to support transport, which would lead to new insights for the development of novel anti-malarial drugs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-0791-3) contains supplementary material, which is available to authorized users.
During H5N1 influenza virus infection, proinflammatory cytokines are markedly elevated in the lungs of infected hosts. The significance of this dysregulated cytokine response in H5N1-mediated pathogenesis remains to be determined. To investigate the influence of hypercytokinemia, or "cytokine storm," a transgenic mouse technology was used. The classical NF-B pathway regulates the induction of most proinflammatory cytokines. Deletion of the p50 subunit leads to a markedly reduced expression of the NF-B-regulated cytokines and chemokines. Here we show that H5N1 influenza virus infection of this transgenic mouse model resulted in a lack of hypercytokinemia but not in altered pathogenesis.
Mass releases of sterilized male insects, in the frame of sterile insect technique programs, have helped suppress insect pest populations since the 1950s. In the major horticultural pests Bactrocera dorsalis, Ceratitis capitata, and Zeugodacus cucurbitae, a key phenotype white pupae (wp) has been used for decades to selectively remove females before releases, yet the gene responsible remained unknown. Here, we use classical and modern genetic approaches to identify and functionally characterize causal wp− mutations in these distantly related fruit fly species. We find that the wp phenotype is produced by parallel mutations in a single, conserved gene. CRISPR/Cas9-mediated knockout of the wp gene leads to the rapid generation of white pupae strains in C. capitata and B. tryoni. The conserved phenotype and independent nature of wp− mutations suggest this technique can provide a generic approach to produce sexing strains in other major medical and agricultural insect pests.
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