Profiling invasive Plasmodium falciparum merozoites using an integrated omics approach

The symptoms of malaria are brought about by blood-stage parasites, which are established when merozoites invade human erythrocytes. Our understanding of the molecular events that underpin erythrocyte invasion remains hampered by the short-period of time that merozoites are invasive. To address this challenge, a Plasmodium falciparum gamma-irradiated long-lived merozoite (LLM) line was developed and investigated. Purified LLMs invaded erythrocytes by an increase of 10–300 fold compared to wild-type (WT) merozoites. Using an integrated omics approach, we investigated the basis for the phenotypic difference. Only a few single nucleotide polymorphisms within the P. falciparum genome were identified and only marginal differences were observed in the merozoite transcriptomes. By contrast, using label-free quantitative mass-spectrometry, a significant change in protein abundance was noted, of which 200 were proteins of unknown function. We determined the relative molar abundance of over 1100 proteins in LLMs and further characterized the major merozoite surface protein complex. A unique processed MSP1 intermediate was identified in LLM but not observed in WT suggesting that delayed processing may be important for the observed phenotype. This integrated approach has demonstrated the significant role of the merozoite proteome during erythrocyte invasion, while identifying numerous unknown proteins likely to be involved in invasion

Assessing the Effectiveness of In-Vehicle Highway Back-of-Queue Alerting System

This paper proposes an in-vehicle back-of-queue alerting system that is able to issue alerting messages to drivers on highways approaching traffic queues. A prototype system was implemented to deliver the in-vehicle alerting messages to drivers via an Android-based smartphone app. To assess its effectiveness, a set of test scenarios were designed and implemented on a state-of-the-art driving simulator. Subjects were recruited and their testing data was collected under two driver states (normal and distracted) and three alert types (no alerts, roadside alerts, and in-vehicle auditory alerts). The effectiveness was evaluated using three parameters of interest: 1) the minimum Time-to-Collision (mTTC), 2) the maximum deceleration, and 3) the maximum lateral acceleration. Statistical models were utilized to examine the usefulness and benefits of each alerting type. The results show that the in-vehicle auditory alert is the most effective way for delivering alerting messages to drivers. More specifically, it significantly increases the mTTC (30% longer than that of 'no warning') and decreases the maximum lateral acceleration (60% less than that of 'no warning'), which provides drivers with more reaction time and improves driving stability of their vehicles. The effects of driver distraction significantly decrease the efficiency of roadside traffic sign alert. More specifically, when the driver is distracted, the roadside traffic sign alert performs significantly worse in terms of mTTC compared with that of normal driving. This highlights the importance of the in-vehicle auditory alert when the driver is distracted

miR-182 and miR-10a Are Key Regulators of Treg Specialisation and Stability during Schistosome and Leishmania-associated Inflammation

A diverse suite of effector immune responses provide protection against various pathogens. However, the array of effector responses must be immunologically regulated to limit pathogen- and immune-associated damage. CD4+Foxp3+ regulatory T cells (Treg) calibrate immune responses; however, how Treg cells adapt to control different effector responses is unclear. To investigate the molecular mechanism of Treg diversity we used whole genome expression profiling and next generation small RNA sequencing of Treg cells isolated from type-1 or type-2 inflamed tissue following Leishmania major or Schistosoma mansoni infection, respectively. In-silico analyses identified two miRNA “regulatory hubs” miR-10a and miR-182 as critical miRNAs in Th1- or Th2-associated Treg cells, respectively. Functionally and mechanistically, in-vitro and in-vivo systems identified that an IL-12/IFNγ axis regulated miR-10a and its putative transcription factor, Creb. Importantly, reduced miR-10a in Th1-associated Treg cells was critical for Treg function and controlled a suite of genes preventing IFNγ production. In contrast, IL-4 regulated miR-182 and cMaf in Th2-associed Treg cells, which mitigated IL-2 secretion, in part through repression of IL2-promoting genes. Together, this study indicates that CD4+Foxp3+ cells can be shaped by local environmental factors, which orchestrate distinct miRNA pathways preserving Treg stability and suppressor function

Comparative Pathogenesis of Three Human and Zoonotic SARS-CoV Strains in Cynomolgus Macaques

The severe acute respiratory syndrome (SARS) epidemic was characterized by increased pathogenicity in the elderly due to an early exacerbated innate host response. SARS-CoV is a zoonotic pathogen that entered the human population through an intermediate host like the palm civet. To prevent future introductions of zoonotic SARS-CoV strains and subsequent transmission into the human population, heterologous disease models are needed to test the efficacy of vaccines and therapeutics against both late human and zoonotic isolates. Here we show that both human and zoonotic SARS-CoV strains can infect cynomolgus macaques and resulted in radiological as well as histopathological changes similar to those seen in mild human cases. Viral replication was higher in animals infected with a late human phase isolate compared to a zoonotic isolate. While there were significant differences in the number of host genes differentially regulated during the host responses between the three SARS-CoV strains, the top pathways and functions were similar and only apparent early during infection with the majority of genes associated with interferon signaling pathways. This study characterizes critical disease models in the evaluation and licensure of therapeutic strategies against SARS-CoV for human use

Increased circulation time of Plasmodium falciparum underlies persistent asymptomatic infection in the dry season

The dry season is a major challenge for Plasmodium falciparum parasites in many malaria endemic regions, where water availability limits mosquito vectors to only part of the year. How P. falciparum bridges two transmission seasons months apart, without being cleared by the human host or compromising host survival, is poorly understood. Here we show that low levels of P. falciparum parasites persist in the blood of asymptomatic Malian individuals during the 5- to 6-month dry season, rarely causing symptoms and minimally affecting the host immune response. Parasites isolated during the dry season are transcriptionally distinct from those of individuals with febrile malaria in the transmission season, coinciding with longer circulation within each replicative cycle of parasitized erythrocytes without adhering to the vascular endothelium. Low parasite levels during the dry season are not due to impaired replication but rather to increased splenic clearance of longer-circulating infected erythrocytes, which likely maintain parasitemias below clinical and immunological radar. We propose that P. falciparum virulence in areas of seasonal malaria transmission is regulated so that the parasite decreases its endothelial binding capacity, allowing increased splenic clearance and enabling several months of subclinical parasite persistence

Increased Apoptosis in the Host By Guanidine Hydrochloride Treatment of Infectious Pancreatic Necrosis Virus

Apoptosis is an orchestrated type of cell death, characterized early by phosphatidylserine flipping to the outer leaflet of the membrane, followed by cell shrinkage and cell blebbing. DNA isolates from apoptotic cells typically present a laddering effect on agarose gels. Recent publications indicate the early protein products of some viruses are able to repress apoptosis within their host. Particles or empty capsids that produce inefficient or no protein products will allow host cells to undergo apoptosis. Empty capsids are mass produced by some viruses or can be created from wild type virus by nucleic acid extrusion. This process has been done with Bovine Herpes Virus (BHV) using guanidine hydrochloride (GuHC1) to extrude the DNA and leave the capsids intact. These \u27created\u27 empty capsids were used to demonstrate that attachment of the empty capsid induced apoptosis. Different strains of Reovirus, a naked double stranded RNA virus similar to Infectious Pancreatic Necrosis Virus (IPNV), have also been treated with GuHCI to show inactivation of ơ1 protein, responsible for haemagglutinin, without disrupting other capsid proteins. However, the researchers did not investigate different types of cytopathic effects. IPNV is primarily known to be a necrotic virus, not an apoptotic virus. Therefore, IPNV was treated with GuHC1 to test the effects it had on viral activity and apoptosis in Chinook Salmon Embryo cells (CHSE-214). Standard research methods of apoptotic detection were used: vital dye exclusion, fluorescent microscopy and DNA laddering in electrophoretic gels. The vital dye staining showed a significant decrease in viral activity post treatment with 4M GuHC1. Fluorescent microscopy results indicated examples of increased phosphatidylserine (PS) flip to the outside membrane in 4M GuHC1 treated virus versus wild type virus infected cells. DNA laddering in cells was observed in 4M GuHC1 treated viral samples compared to controls of positive apoptotic and positive necrotic samples. These results indicate that 4M GuHC1 treatment of IPNV will decrease viral activity and increase apoptosis in CHSE-214 cells, suggesting a viral product, which normally blocks apoptosis, is not operative

Proteomics Analysis of Amyloid and Nonamyloid Prion Disease Phenotypes Reveals Both Common and Divergent Mechanisms of Neuropathogenesis

Prion diseases are a heterogeneous group of neurodegenerative disorders affecting various mammals including humans. Prion diseases are characterized by a misfolding of the host-encoded prion protein (PrP^C) into a pathological isoform termed PrP^Sc. In wild-type mice, PrP^C is attached to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor and PrP^Sc typically accumulates in diffuse nonamyloid deposits with gray matter spongiosis. By contrast, when mice lacking the GPI anchor are infected with the same prion inoculum, PrP^Sc accumulates in dense perivascular amyloid plaques with little or no gray matter spongiosis. In order to evaluate whether different host biochemical pathways were implicated in these two phenotypically distinct prion disease models, we utilized a proteomics approach. In both models, infected mice displayed evidence of a neuroinflammatory response and complement activation. Proteins involved in cell death and calcium homeostasis were also identified in both phenotypes. However, mitochondrial pathways of apoptosis were implicated only in the nonamyloid form, whereas metal binding and synaptic vesicle transport were more disrupted in the amyloid phenotype. Thus, following infection with a single prion strain, PrP^C anchoring to the plasma membrane correlated not only with the type of PrP^Sc deposition but also with unique biochemical pathways associated with pathogenesis

Proteomics Analysis of Amyloid and Nonamyloid Prion Disease Phenotypes Reveals Both Common and Divergent Mechanisms of Neuropathogenesis

Prion diseases are a heterogeneous group of neurodegenerative disorders affecting various mammals including humans. Prion diseases are characterized by a misfolding of the host-encoded prion protein (PrP^C) into a pathological isoform termed PrP^Sc. In wild-type mice, PrP^C is attached to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor and PrP^Sc typically accumulates in diffuse nonamyloid deposits with gray matter spongiosis. By contrast, when mice lacking the GPI anchor are infected with the same prion inoculum, PrP^Sc accumulates in dense perivascular amyloid plaques with little or no gray matter spongiosis. In order to evaluate whether different host biochemical pathways were implicated in these two phenotypically distinct prion disease models, we utilized a proteomics approach. In both models, infected mice displayed evidence of a neuroinflammatory response and complement activation. Proteins involved in cell death and calcium homeostasis were also identified in both phenotypes. However, mitochondrial pathways of apoptosis were implicated only in the nonamyloid form, whereas metal binding and synaptic vesicle transport were more disrupted in the amyloid phenotype. Thus, following infection with a single prion strain, PrP^C anchoring to the plasma membrane correlated not only with the type of PrP^Sc deposition but also with unique biochemical pathways associated with pathogenesis

Proteomics Analysis of Amyloid and Nonamyloid Prion Disease Phenotypes Reveals Both Common and Divergent Mechanisms of Neuropathogenesis

Prion diseases are a heterogeneous group of neurodegenerative disorders affecting various mammals including humans. Prion diseases are characterized by a misfolding of the host-encoded prion protein (PrP^C) into a pathological isoform termed PrP^Sc. In wild-type mice, PrP^C is attached to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor and PrP^Sc typically accumulates in diffuse nonamyloid deposits with gray matter spongiosis. By contrast, when mice lacking the GPI anchor are infected with the same prion inoculum, PrP^Sc accumulates in dense perivascular amyloid plaques with little or no gray matter spongiosis. In order to evaluate whether different host biochemical pathways were implicated in these two phenotypically distinct prion disease models, we utilized a proteomics approach. In both models, infected mice displayed evidence of a neuroinflammatory response and complement activation. Proteins involved in cell death and calcium homeostasis were also identified in both phenotypes. However, mitochondrial pathways of apoptosis were implicated only in the nonamyloid form, whereas metal binding and synaptic vesicle transport were more disrupted in the amyloid phenotype. Thus, following infection with a single prion strain, PrP^C anchoring to the plasma membrane correlated not only with the type of PrP^Sc deposition but also with unique biochemical pathways associated with pathogenesis

Proteomics Analysis of Amyloid and Nonamyloid Prion Disease Phenotypes Reveals Both Common and Divergent Mechanisms of Neuropathogenesis

Prion diseases are a heterogeneous group of neurodegenerative disorders affecting various mammals including humans. Prion diseases are characterized by a misfolding of the host-encoded prion protein (PrP^C) into a pathological isoform termed PrP^Sc. In wild-type mice, PrP^C is attached to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor and PrP^Sc typically accumulates in diffuse nonamyloid deposits with gray matter spongiosis. By contrast, when mice lacking the GPI anchor are infected with the same prion inoculum, PrP^Sc accumulates in dense perivascular amyloid plaques with little or no gray matter spongiosis. In order to evaluate whether different host biochemical pathways were implicated in these two phenotypically distinct prion disease models, we utilized a proteomics approach. In both models, infected mice displayed evidence of a neuroinflammatory response and complement activation. Proteins involved in cell death and calcium homeostasis were also identified in both phenotypes. However, mitochondrial pathways of apoptosis were implicated only in the nonamyloid form, whereas metal binding and synaptic vesicle transport were more disrupted in the amyloid phenotype. Thus, following infection with a single prion strain, PrP^C anchoring to the plasma membrane correlated not only with the type of PrP^Sc deposition but also with unique biochemical pathways associated with pathogenesis