Moving on: How malaria parasites exit the liver.

An essential step in the life cycle of malaria parasites is their egress from hepatocytes, which enables the transition from the asymptomatic liver stage to the pathogenic blood stage of infection. To exit the liver, Plasmodium parasites first disrupt the parasitophorous vacuole membrane that surrounds them during their intracellular replication. Subsequently, parasite-filled structures called merosomes emerge from the infected cell. Shrouded by host plasma membrane, like in a Trojan horse, parasites enter the vasculature undetected by the host immune system and travel to the lung where merosomes rupture, parasites are released, and the blood infection stage begins. This complex, multi-step process must be carefully orchestrated by the parasite and requires extensive manipulation of the infected host cell. This review aims to outline the known signaling pathways that trigger exit, highlight Plasmodium proteins that contribute to the release of liver-stage merozoites, and summarize the accompanying changes to the hepatic host cell.

Cryptosporidium parvum Elongation Factor 1α Participates in the Formation of Base Structure at the Infection Site During Invasion.

BACKGROUND: Cryptosporidium is a genus of apicomplexan parasites, the causative agents of cryptosporidiosis in humans and/or animals. Although most apicomplexans parasitize within the host cell cytosols, Cryptosporidium resides on top of host cells, but it is embraced by a double-layer parasitophorous vacuole membrane derived from host cell. There is an electron-dense band to separate the parasite from host cell cytoplasm, making it as an intracellular but extracytoplasmic parasite. However, little is known on the molecular machinery at the host cell-parasite interface. METHODS: Cryptosporidium parvum at various developmental stages were obtained by infecting HCT-8 cells cultured in vitro. Immunofluorescence assay was used to detect CpEF1α with a polyclonal antibody and host cell F-actin with rhodamine-phalloidin. Recombinant CpEF1α protein was used to evaluate its effect on the invasion by the parasite. RESULTS: We discovered that a C parvum translation elongation factor 1α (CpEF1α) was discharged from the invading sporozoites into host cells, forming a crescent-shaped patch that fully resembles the electron-dense band. At the same time, host cell F-actin aggregated to form a globular-shaped plug beneath the CpEF1α patch. The CpEF1α patch remained for most of the time but became weakened and dissolved upon the completion of the invasion process. In addition, recombinant CpEF1α protein could effectively interfere the invasion of sporozoites into host cells. CONCLUSIONS: CpEF1α plays a role in the parasite invasion by participating in the formation of electron-dense band at the base of the parasite infection site.

Video-Based Pulse Rate Variability Measurement Using Periodic Variance Maximization and Adaptive Two-Window Peak Detection.

Many previous studies have shown that the remote photoplethysmography (rPPG) can measure the Heart Rate (HR) signal with very high accuracy. The remote measurement of the Pulse Rate Variability (PRV) signal is also possible, but this is much more complicated because it is then necessary to detect the peaks on the temporal rPPG signal, which is usually quite noisy and has a lower temporal resolution than PPG signals obtained by contact equipment. Since the PRV signal is vital for various applications such as remote recognition of stress and emotion, the improvement of PRV measurement by rPPG is a critical task. Contact based PRV measurement has already been investigated, but the research on remotely measured PRV is very limited. In this paper, we propose to use the Periodic Variance Maximization (PVM) method to extract the rPPG signal and event-related Two-Window algorithm to improve the peak detection for PRV measurement. We have made several contributions. Firstly, we show that the newly proposed PVM method and Two-Window algorithm can be used for PRV measurement in the non-contact scenario. Secondly, we propose a method to adaptively determine the parameters of the Two-Window method. Thirdly, we compare the algorithm with other attempts for improving the non-contact PRV measurement such as the Slope Sum Function (SSF) method and the Local Maximum method. We calculated several features and compared the accuracy based on the ground truth provided by contact equipment. Our experiments showed that this algorithm performed the best of all the algorithms.

In silico study of M18 aspartyl amino peptidase (M18AAP) of Plasmodium vivax as an antimalarial drug target.

Plasmodium vivax (Pv) is the second most malaria causing pathogen among Plasmodium species. M18 aspartic aminopeptidase (M18AAP) protein is a single gene copy present in Plasmodium. This protein is functional at the terminal stage of hemoglobin degradation of host and completes the hydrolysis process which makes it an important target for new chemotherapeutics. No experimental and structural study on M18AAP protein of P. vivax is reported till today. This paper advocates the application of multiple computational approaches like protein model prediction, ligand-based 3D QSAR study, pharmacophore, structure-based virtual screening and molecular docking simulation for identification of potent lead molecules against the enzyme. The 3D QSAR model was developed using known bioactive compounds against the PvM18AAP protein which statistically signify the k-NN model with q^2 = 0.7654. The study reports a lead molecule from ligand-centric approach with good binding affinity and possessing lowest docking score. The findings will be helpful for in-vivo and in-vitro validations and development of potent anti-malarial molecules against the drug resistant strains of malaria parasite.

Chelonian perivitelline membrane-bound sperm detection: A new breeding management tool.

Perivitelline membrane (PVM)-bound sperm detection has recently been incorporated into avian breeding programs to assess egg fertility, confirm successful copulation, and to evaluate male reproductive status and pair compatibility. Due to the similarities between avian and chelonian egg structure and development, and because fertility determination in chelonian eggs lacking embryonic growth is equally challenging, PVM-bound sperm detection may also be a promising tool for the reproductive management of turtles and tortoises. This study is the first to successfully demonstrate the use of PVM-bound sperm detection in chelonian eggs. Recovered membranes were stained with Hoechst 33342 and examined for sperm presence using fluorescence microscopy. Sperm were positively identified for up to 206 days post-oviposition, following storage, diapause, and/or incubation, in 52 opportunistically collected eggs representing 12 species. However, advanced microbial infection frequently hindered the ability to detect membrane-bound sperm. Fertile Centrochelys sulcata, Manouria emys, and Stigmochelys pardalis eggs were used to evaluate the impact of incubation and storage on the ability to detect sperm. Storage at -20°C or in formalin were found to be the best methods for egg preservation prior to sperm detection. Additionally, sperm-derived mtDNA was isolated and PCR amplified from Astrochelys radiata, C. sulcata, and S. pardalis eggs. PVM-bound sperm detection has the potential to substantially improve studies of artificial incubation and sperm storage, and could be used to evaluate the success of artificial insemination in chelonian species. Mitochondrial DNA from PVM-bound sperm has applications for parentage analysis, the study of sperm competition, and potentially species identification.

VirulenceFinder for Enterococcus faecium and Enterococcus lactis: an enhanced database for detection of putative virulence markers by using whole-genome sequencing data.

Enterococcus faecium (Efm) is a leading cause of hospital-associated (HA) infections, often enriched in putative virulence markers (PVMs). Recently, the Efm clade B was assigned as Enterococcus lactis (Elts), which usually lack HA-Efm infection markers. Available databases for extracting PVM are incomplete and/or present an intermix of genes from Efm and Enterococcus faecalis, with distinct virulence profiles. In this study, we constructed a new database containing 27 PVMs [acm, scm, sgrA, ecbA, fnm, sagA, hylEfm, ptsD, orf1481, fms15, fms21-fms20 (pili gene cluster 1, PGC-1), fms14-fms17-fms13 (PGC-2), empA-empB-empC (PGC-3), fms11-fms19-fms16 (PGC-4), ccpA, bepA, gls20-glsB1, and gls33-glsB] from nine reference genomes (seven Efm + two Elts). The database was validated against these reference genomes and further evaluated using a collection of well-characterized Efm (n = 43) and Elts (n = 7) control strains, by assessing PVM presence/absence and its variants together with a genomic phylogeny constructed as single-nucleotide polymorphisms. We found a high concordance between the phylogeny and in silico findings of the PVM, with Elts clustering separately and mostly carrying Elts-specific PVM gene variants. Based on our validation results, we recommend using the database with raw reads instead of assemblies to avoid missing gene variants. This newly constructed database of 27 PVMs will enable a more comprehensive characterization of Efm and Elts based on WGS data. The developed database exhibits scalability and boasts a range of applications in public health, including diagnostics, outbreak investigations, and epidemiological studies. It can be further used in risk assessment for distinguishing between safe and unsafe enterococci.IMPORTANCEThe newly constructed database, consisting of 27 putative virulence markers, is highly scalable and serves as a valuable resource for the comprehensive characterization of these closely related species using WGS data. It holds significant potential for various public health applications, including hospital outbreak investigations, surveillance, and risk assessment for probiotics and feed additives.

GRA47 is important for the morphology and permeability of the parasitophorous vacuole in Toxoplasma gondii.

Establishing an intact intracellular parasitophorous vacuole (PV) that enables efficient nutrient uptake and protein trafficking is essential for the survival and proliferation of Toxoplasma gondii. Although the PV membrane (PVM)-localized dense granule protein 17 (GRA17) and GRA23 mediate the permeability of the PVM to small molecules, including nutrient uptake and excretion of metabolic by-products, the molecular mechanism by which T. gondii acquires nutrients remains unclear. In this study, we showed that the secreted protein GRA47 contributed to normal PV morphology, PVM permeability to small molecules, growth, and virulence in T. gondii. Co-immunoprecipitation analysis demonstrated potential interaction of GRA47 with GRA72, and the loss of GRA72 affected PV morphology, parasite growth and infectivity. To investigate the biological relationship among GRA47, GRA72, GRA17 and GRA23, attempts were made to construct strains with double gene deletion and overexpressing strains. Only Δgra23Δgra72 was successfully constructed. This strain exhibited a significant increase in the proportion of aberrant PVs compared with the Δgra23 strain. Overexpressing one of the three related GRAs partially rescued PVs with aberrant morphology in Δgra47, Δgra72 and Δgra17, while the expression of the Plasmodium falciparum PVM protein PfExp2, an ortholog of GRA17 and GRA23, fully rescued the PV morphological defect in all three Δgra strains. These results suggest that these GRA proteins may not be functionally redundant but rather work in different ways to regulate nutrient acquisition. These findings highlight the versatility of the nutrient uptake mechanisms in T. gondii, which may contribute to the parasite's remarkable ability to grow in different cellular niches in a very broad range of hosts.

Two Toxoplasma gondii putative pore-forming proteins, GRA47 and GRA72, influence small molecule permeability of the parasitophorous vacuole.

Toxoplasma gondii, a medically important intracellular parasite, uses GRA proteins secreted from dense granule organelles to mediate nutrient flux across the parasitophorous vacuole membrane (PVM). GRA17 and GRA23 are known pore-forming proteins on the PVM involved in this process, but the roles of additional proteins have remained largely uncharacterized. We recently identified GRA72 as synthetically lethal with GRA17. Deleting GRA72 produced similar phenotypes to Δgra17 parasites, and computational predictions suggested it forms a pore. To understand how GRA72 functions, we performed immunoprecipitation experiments and identified GRA47 as an interactor of GRA72. Deletion of GRA47 resulted in an aberrant "bubble vacuole" morphology with reduced small molecule permeability, mirroring the phenotype observed in GRA17 and GRA72 knockouts. Structural predictions indicated that GRA47 and GRA72 form heptameric and hexameric pores, respectively, with conserved histidine residues lining the pore. Mutational analysis highlighted the critical role of these histidines for protein functionality. Validation through electrophysiology confirmed alterations in membrane conductance, corroborating their pore-forming capabilities. Furthermore, Δgra47 parasites and parasites expressing GRA47 with a histidine mutation had reduced in vitro proliferation and attenuated virulence in mice. Our findings show the important roles of GRA47 and GRA72 in regulating PVM permeability, thereby expanding the repertoire of potential therapeutic targets against Toxoplasma infections. IMPORTANCE: Toxoplasma gondii is a parasite that poses significant health risks to those with impaired immunity. It replicates inside host cells shielded by the PVM, which controls nutrient and waste exchange with the host. GRA72, previously identified as essential in the absence of the GRA17 nutrient channel, is implicated in forming an alternative nutrient channel. Here we found that GRA47 associates with GRA72 and is also important for the PVM's permeability to small molecules. Removal of GRA47 leads to distorted vacuoles and impairs small molecule transport across the PVM, resembling the effects of GRA17 and GRA72 deletions. Structural models suggest GRA47 and GRA72 form distinct pore structures, with a pore-lining histidine critical to their function. Toxoplasma strains lacking GRA47 or those with a histidine mutation have impaired growth and reduced virulence in mice, highlighting these proteins as potential targets for new treatments against toxoplasmosis.

Epidemiological and phylogenetic analysis of institutional mouse parvoviruses.

Mouse parvoviruses (MPVs) are small, single-stranded, 5 kb DNA viruses that are subclinical and endemic in many laboratory mouse colonies. MPVs cause more distinctive deleterious effects in immune-compromised or genetically-engineered mice than immuno-competent mice. At the University of Louisville (U of L), there was an unexpected increase of MPV sero-positivity for MPV infections in mouse colonies between January 2006 and February 2007, resulting in strategic husbandry changes aimed at controlling MPV spread throughout the animal facility. To investigate these MPVs, VP2 genes of seven MPVs were cloned and sequenced from eight documented incidences by PCR technology. The mutations in these VP2 genes were compared to those found at the Genbank database (NCBI; http://www.ncbi.nlm.nih.gov) and an intra-institutional phylogenetic tree for MPV infections at U of L was constructed. We discovered that the seven MPV isolates were different from those in Genbank and were not identical to each other. These MPVs were designated MPV-UL1 to 7; none of them were minute virus of mice (MVMs). Four isolates could be classified as MPV1, one was classified as MPV2, and two were defined as novel types with less than 96% and 94% homology with existing MPV types. Considering that all seven isolates had mutations in their VP2 genes and no mutations were observed in VP2 genes of MPV during a four-month time period of incubation, we concluded that all seven MPVs isolated at U of L between 2006 and 2007 probably originated from different sources. Serological survey for MPV infections verified that each MPV outbreak was controlled without further contamination within the institution.

Roles of the RON3 C-terminal fragment in erythrocyte invasion and blood-stage parasite proliferation in Plasmodium falciparum.

Plasmodium species cause malaria, and in the instance of Plasmodium falciparum is responsible for a societal burden of over 600,000 deaths annually. The symptoms and pathology of malaria are due to intraerythocytic parasites. Erythrocyte invasion is mediated by the parasite merozoite stage, and is accompanied by the formation of a parasitophorous vacuolar membrane (PVM), within which the parasite develops. The merozoite apical rhoptry organelle contains various proteins that contribute to erythrocyte attachment and invasion. RON3, a rhoptry bulb membrane protein, undergoes protein processing and is discharged into the PVM during invasion. RON3-deficient parasites fail to develop beyond the intraerythrocytic ring stage, and protein export into erythrocytes by the Plasmodium translocon of exported proteins (PTEX) apparatus is abrogated, as well as glucose uptake into parasites. It is known that truncated N- and C-terminal RON3 fragments are present in rhoptries, but it is unclear which RON3 fragments contribute to protein export by PTEX and glucose uptake through the PVM. To investigate and distinguish the roles of the RON3 C-terminal fragment at distinct developmental stages, we used a C-terminus tag for conditional and post-translational control. We demonstrated that RON3 is essential for blood-stage parasite survival, and knockdown of RON3 C-terminal fragment expression from the early schizont stage induces a defect in erythrocyte invasion and the subsequent development of ring stage parasites. Protein processing of full-length RON3 was partially inhibited in the schizont stage, and the RON3 C-terminal fragment was abolished in subsequent ring-stage parasites compared to the RON3 N-terminal fragment. Protein export and glucose uptake were abrogated specifically in the late ring stage. Plasmodial surface anion channel (PSAC) activity was partially retained, facilitating small molecule traffic across the erythrocyte membrane. The knockdown of the RON3 C-terminal fragment after erythrocyte invasion did not alter parasite growth. These data suggest that the RON3 C-terminal fragment participates in erythrocyte invasion and serves an essential role in the progression of ring-stage parasite growth by the establishment of the nutrient-permeable channel in the PVM, accompanying the transport of ring-stage parasite protein from the plasma membrane to the PVM.

Study on Modelling Method of Resilient Mat Used under Floating Slab Track.

Kelvin's model is widely used to simulate the dynamic characteristic of a resilient mat under a slab track. To develop an effective calculation model for a resilient mat using a solid element, a three-parameter viscoelasticity model (3PVM) was employed. With the help of the user-defined material mechanical behavior, the proposed model was implemented in software ABAQUS. To validate the model, a laboratory test was performed on a slab track with a resilient mat. Then, a finite element model of the track-tunnel-soil system was built. The calculation results using the 3PVM was compared with those using Kelvin's model and the test results. The results indicate that the 3PVM can better reflect the dynamic characteristics of resilient mat than Kelvin's model, especially over 10 Hz. Compared with the test results, the 3PVM has an average error of 2.7 dB and a max error of 7.9 dB at 5 Hz.

GRA47 and GRA72 are Toxoplasma gondii pore-forming proteins that influence small molecule permeability of the parasitophorous vacuole.

Toxoplasma gondii, a medically important intracellular parasite, uses GRA proteins, secreted from dense granule organelles, to mediate nutrient flux across the parasitophorous vacuole membrane (PVM). GRA17 and GRA23 are known pore-forming proteins on the PVM involved in this process, but the roles of additional proteins have remained largely uncharacterized. We recently identified GRA72 as synthetically lethal with GRA17. Deleting GRA72 produced similar phenotypes to Δgra17 parasites, and computational predictions suggested it forms a pore. To understand how GRA72 functions we performed immunoprecipitation experiments and identified GRA47 as an interactor of GRA72. Deletion of GRA47 resulted in an aberrant 'bubble vacuole' morphology with reduced small molecule permeability, mirroring the phenotype observed in GRA17 and GRA72 knockouts. Structural predictions indicated that GRA47 and GRA72 form heptameric and hexameric pores, respectively, with conserved histidine residues lining the pore. Mutational analysis highlighted the critical role of these histidines for protein functionality. Validation through electrophysiology confirmed alterations in membrane conductance, corroborating their pore-forming capabilities. Furthermore, Δgra47 parasites and parasites expressing GRA47 with a histidine mutation had reduced in vitro proliferation and attenuated virulence in mice. Our findings show the important roles of GRA47 and GRA72 in regulating PVM permeability, thereby expanding the repertoire of potential therapeutic targets against Toxoplasma infections.

Analysis of Degenerative and Isthmic Lumbar Spondylolisthesis from the Difference of Pelvic Parameters and the Degree of Degeneration through Imaging Data.

BACKGROUND: In previous studies, many imaging analyses have been conducted to explore the changes in the intervertebral disc degeneration (DD), facet joint osteoarthritis (FJOA), L4 inclination angle (L4IA), pelvis-related parameters, lumbar lordosis (LL), and paravertebral muscle (PVM) in the occurrence and development of degenerative spinal diseases via measuring the X-ray, CT, and MRI data of clinical patients. However, few studies have quantitatively investigated the pelvic parameters and the degree of spine degeneration in patients with degenerative lumbar spondylolisthesis (DLS) and isthmic lumbar spondylolisthesis (ILS). This study discusses the changes in the imaging parameters of DLS, ILS, and a control group; explores the correlation between different measurement parameters; and discusses their risk factors. METHODS: We evaluated 164 patients with single L4-L5 grade 1 level degenerative lumbar spondylolisthesis (DLS group), 161 patients with single L4-L5 grade 1 level isthmic lumbar spondylolisthesis (ILS group), and 164 patients with non-specific back pain (control group). The grades of DD and FJOA as well as the percentage of the fat infiltration area (%FIA) of multifidus muscle (MM) at the L4-L5 level were measured via CT and MRI. Lumbar lordosis (LL), pelvic incidence (PI), pelvic tilt (PT), the L4 inclination angle (L4IA), and sacral slope (SS) were measured via X-ray film, and the differences among the DLS group, ILS group, and control group were analyzed. Furthermore, the risk factors related to the incidences of the DLS and ILS groups were discussed. RESULTS: First, the pelvis-related parameters of DLS and ILS patients were 51.91 ± 12.23 and 53.28 ± 11.12, respectively, while those of the control group were 40.13 ± 8.72 (p1 < 0.001, p2 < 0.001). Lumbar lordosis (LL) in DLS patients (39.34 ± 8.57) was significantly lower than in the control group (44.40 ± 11.79, p < 0.001). On the contrary, lumbar lordosis (LL) in the ILS group (55.16 ± 12.31) was significantly higher than in the control group (44.40 ± 11.79, p < 0.001). Secondly, the three groups of patients were characterized by significant variations in the L4 inclination angle (L4IA), disc degeneration (DD), facet joint osteoarthritis (FJOA), pelvis-related parameters, and paravertebral muscle (PVM) (p < 0.05). Finally, logistic regression suggests that the L4IA, FJOA, and PT may be risk factors for the occurrence of DLS, and the occurrence of ILS is correlated with the L4IA, FJOA, DD, PT, and LL. CONCLUSIONS: Compared with the control group, there are changes in pelvic parameters, the L4IA, LL, DD, FJOA, and PVM in DLS and ILS patients, and the degree is different. The parameters within the same group are related to each other, and DLS and ILS have different risk factors. The mechanical stability of the spine is affected by the parameter and angle changes, which may be of great significance for explaining the cause of spondylolisthesis, evaluating the health of the lumbar spine, and guiding the lifestyles of patients.

Synchronisation of Plasmodium falciparum and P. knowlesi In Vitro Cultures Using a Highly Specific Protein Kinase Inhibitor.

Synchronisation of Plasmodium cultures is essential to investigate the complexities of time-dependent events associated with the asexual blood stage of the malaria parasite life cycle. Here we describe a procedure using ML10, a highly specific inhibitor of the parasite cyclic GMP-dependent protein kinase (PKG), to attain high synchronicity of Plasmodium falciparum and P. knowlesi asexual blood-stage cultures and to obtain high levels of arrested mature schizonts as well as viable released merozoites. Additionally, we describe how to use ML10 to improve the transfection efficiency of P. falciparum parasites and also how to derive the half maximal effective concentration (EC50) of ML10 in other P. falciparum laboratory lines and clinical isolates.

The parasitophorous vacuole nutrient channel is critical for drug access in malaria parasites and modulates the artemisinin resistance fitness cost.

Intraerythrocytic malaria parasites proliferate bounded by a parasitophorous vacuolar membrane (PVM). The PVM contains nutrient permeable channels (NPCs) conductive to small molecules, but their relevance for parasite growth for individual metabolites is largely untested. Here we show that growth-relevant levels of major carbon and energy sources pass through the NPCs. Moreover, we find that NPCs are a gate for several antimalarial drugs, highlighting their permeability properties as a critical factor for drug design. Looking into NPC-dependent amino acid transport, we find that amino acid shortage is a reason for the fitness cost in artemisinin-resistant (ARTR) parasites and provide evidence that NPC upregulation to increase amino acids acquisition is a mechanism of ARTR parasites in vitro and in human infections to compensate this fitness cost. Hence, the NPCs are important for nutrient and drug access and reveal amino acid deprivation as a critical constraint in ARTR parasites.

Discovery of New Microneme Proteins in Cryptosporidium parvum and Implication of the Roles of a Rhomboid Membrane Protein (CpROM1) in Host-Parasite Interaction.

Apicomplexan parasites possess several unique secretory organelles, including rhoptries, micronemes, and dense granules, which play critical roles in the invasion of host cells. The molecular content of these organelles and their biological roles have been well-studied in Toxoplasma and Plasmodium, but are underappreciated in Cryptosporidium, which contains many parasites of medical and veterinary importance. Only four proteins have previously been identified or proposed to be located in micronemes, one of which, GP900, was confirmed using immunogold electron microscopy (IEM) to be present in the micronemes of intracellular merozoites. Here, we report on the discovery of four new microneme proteins (MICs) in the sporozoites of the zoonotic species C. parvum, identified using immunofluorescence assay (IFA). These proteins are encoded by cgd3_980, cgd1_3550, cgd1_3680, and cgd2_1590. The presence of the protein encoded by cgd3_980 in sporozoite micronemes was further confirmed using IEM. Cgd3_980 encodes one of the three C. parvum rhomboid peptidases (ROMs) and is, thus, designated CpROM1. IEM also confirmed the presence of CpROM1 in the micronemes of intracellular merozoites, parasitophorous vacuole membranes (PVM), and feeder organelles (FO). CpROM1 was enriched in the pellicles and concentrated at the host cell-parasite interface during the invasion of sporozoites and its subsequent transformation into trophozoites. CpROM1 transcript levels were also higher in oocysts and excysted sporozoites than in the intracellular parasite stages. These observations indicate that CpROM1, an intramembrane peptidase with membrane proteolytic activity, is involved in host-parasite interactions, including invasion and proteostasis of PVM and FO.

Cryptosporidium parvum gp40/15 Is Associated with the Parasitophorous Vacuole Membrane and Is a Potential Vaccine Target.

Cryptosporidium parvum is a zoonotic intracellular protozoan responsible for the diarrheal illness cryptosporidiosis in humans and animals. Although a number of zoite surface proteins are known to be expressed during, and believed to be involved in, attachment and invasion of host cells, the molecular mechanisms by which C. parvum invades the host epithelial cells are not well understood. In the present study, we investigated the gene expression patterns, protein localization in developmental stages in culture, and in vitro neutralization characteristics of Cpgp40/15 and Cpgp40. Indirect immunofluorescence assay showed that Cpgp40/15 is associated with the parasitophorous vacuole membrane (PVM) during intracellular development. Both anti-gp40/15 and anti-gp40 antibodies demonstrated the ability to neutralize C. parvum infection in vitro. Further studies are needed to fully understand the specific role and functional mechanism of Cpgp40/15 (or gp40/15 complex) in the invasion of the host or in the PVM and to determine the feasibility of gp40/15 as a vaccine candidate for cryptosporidiosis in vivo.

The Contribution of Neutrophils to the Pathogenesis of RSV Bronchiolitis.

Acute viral bronchiolitis causes significant mortality in the developing world, is the number one cause of infant hospitalisation in the developed world, and is associated with the later development of chronic lung diseases such as asthma. A vaccine against respiratory syncytial virus (RSV), the leading cause of viral bronchiolitis in infancy, remains elusive, and hence new therapeutic modalities are needed to limit disease severity. However, much remains unknown about the underlying pathogenic mechanisms. Neutrophilic inflammation is the predominant phenotype observed in infants with both mild and severe disease, however, a clear understanding of the beneficial and deleterious effects of neutrophils is lacking. In this review, we describe the multifaceted roles of neutrophils in host defence and antiviral immunity, consider their contribution to bronchiolitis pathogenesis, and discuss whether new approaches that target neutrophil effector functions will be suitable for treating severe RSV bronchiolitis.

Pest categorisation of potato virus M (non-EU isolates).

Following a request from the EU Commission, the Panel on Plant Health has addressed the pest categorisation of non-EU isolates of potato virus M (PVM). The information currently available on geographical distribution, biology, epidemiology, potential entry pathways, potential additional impact compared to the current situation in the EU and availability of control measures of non-EU isolates of PVM has been evaluated with regard to the criteria to qualify as a potential Union quarantine pest. Because non-EU isolates of PVM are absent from the EU, they do not meet one of the requirements to be regulated as a regulated non-quarantine pest (RNQP) (presence in the EU); as a consequence, the Panel decided not to evaluate the other RNQP criteria for these isolates. Populations of PVM can be subdivided into two strains: the ordinary strain (PVM-O) is present in the EU, while the divergent strain (PVM-D) is absent from the EU or considered to have at most a limited distribution in the EU. Non-EU isolates of PVM-O are not expected to have an additional impact in the EU compared to EU isolates and therefore do not meet the corresponding criterion to qualify as a potential Union quarantine pest. The Panel is unable to conclude on the potential impact of non-EU PVM-D isolates in the EU territory, but PVM-D isolates meet all the other criteria to qualify as a potential Union quarantine pest.

GRA proteins of Toxoplasma gondii: maintenance of host-parasite interactions across the parasitophorous vacuolar membrane.

The dense granule of Toxoplasma gondii is a secretory vesicular organelle of which the proteins participate in the modification of the parasitophorous vacuole (PV) and PV membrane for the maintenance of intracellular parasitism in almost all nucleated host cells. In this review, the archives on the research of GRA proteins are reviewed on the foci of finding GRA proteins, characterizing molecular aspects, usefulness in diagnostic antigen, and vaccine trials in addition to some functions in host-parasite interactions.