Egress Process Research Articles

A Structural Bioinformatics Study of Calcium Dependent Protein Kinase 6 (CDPK6) of Cryptosporidium Parvum for Identification of Novel Therapeutics

Cryptosporidium parvum is an obligate intracellular protozoan parasite that infects the intestinal tract of mammals and causes cryptosporidiosis. C. parvum oocysts are ingested by a host that reproduces sexually and asexually in the intestinal lining cells, resulting in diarrhea. Calcium Dependent Protein Kinases (CDPKs) are found in the apicomplexan, algae, and plants; however, they are reported in vertebrates and are regarded as excellent drug targets for pharmaceutical interventions. CDPKs of Cryptosporidium are probably involved in the regulation of invasion and egress process during the infection of the host cells. Calcium-dependent protein kinase6 (CDPK6) (cgd4_3330) is a hypothetical protein of the CDPKs family which was originated from the plants and also a few species of protozoa. These proteins regulate several biological processes of apicomplexan for example Cryptosporidium, Plasmodium, and Toxoplasma. In this study, an attempt is made to address the sequences similarity and phylogenetic study of CDPK6 among apicomplexans CDPKs family. Furthermore, the three-dimensional structure determination of the target protein was performed through a molecular modeling approach (MODELLER v9.21 software) followed by virtual screening (GOLD software suite & Glide software) of small molecule inhibitors from five different datasets. The best inhibitor Tres Cantos Antimalarial Set (TCAMS) _11730 reported a binding affinity of -8.2 kcal/mol towards the CDPK6 of C. parvum through molecular docking studies (AutoDock v4.2.6 software). Furthermore, the reliability and validation of the binding mode of the inhibitor were performed through a complex molecular dynamics simulation in an aqueous environment with GROMACS 2018.3 software for a time interval of 100 ns. The findings of the study advocate that the best inhibitor may be considered for industrial pharmaceutical research in future.

Proteomics Analysis Identifies IRSp53 and Fascin as Critical for PRV Egress and Direct Cell-Cell Transmission.

Pseudorabies virus (PRV) has been widely used as a live trans-synaptic tracer for mapping neuronal circuits. Systematically identifying mature PRV virion proteomes and defining co-purified host proteins are necessary to fully understand the detailed mechanism underlying PRV transmission processes. Here, a PRV virion purification strategy based on sorting with flow cytometry is developed and the mature extracellular and intracellular PRV virion proteomes using LC coupled with MS/MS are characterized. In addition to viral proteins, a large number of host proteins are also identified, including proteins related to actin cytoskeletal dynamics and membrane protrusion. How many of these host proteins are true virion components are unknown and the majority of these may not be. Through functional analysis, it is found that IRSp53 and fascin are critical for the egress process and play a role in direct cell-cell transmission. Moreover, it is shown that CDC42 and Rac1 are also involved in the production of mature extracellular virions. The results suggest that the formation of the filopodia-like cytoskeleton and the rearrangement of the membrane, which are both associated with IRSp53 and fascin, may be important for the transmission of viruses used in neuronal tracing.

Effect of hydrogen-induced surface steps on the nanomechanical behavior of a CoCrFeMnNi high-entropy alloy revealed by in-situ electrochemical nanoindentation

The effect of hydrogen on the nanomechanical properties of CoCrFeMnNi high-entropy alloy was investigated by in-situ electrochemical nanoindentation testing. The changes in surface morphology, elastic modulus, pop-in load, and hardness during hydrogen ingress and egress processes were systematically evaluated. The results show that hydrogen charging leads to the formation of irreversible slip lines accumulated as surface steps. Furthermore, the irreversible reduced pop-in load and elastic modulus, and reversible increased hardness are detected. In this paper, the mechanisms of hydrogen-induced surface steps together with their further influences on the nanomechanical properties are discussed in detail.

Host Cytoskeleton Remodeling throughout the Blood Stages of Plasmodium falciparum.

The asexual intraerythrocytic development of Plasmodium falciparum, causing the most severe form of human malaria, is marked by extensive host cell remodeling. Throughout the processes of invasion, intracellular development, and egress, the erythrocyte membrane skeleton is remodeled by the parasite as required for each specific developmental stage. The remodeling is facilitated by a plethora of exported parasite proteins, and the erythrocyte membrane skeleton is the interface of most of the observed interactions between the parasite and host cell proteins. Host cell remodeling has been extensively described and there is a vast body of information on protein export or the description of parasite-induced structures such as Maurer's clefts or knobs on the host cell surface. Here we specifically review the molecular level of each host cell-remodeling step at each stage of the intraerythrocytic development of P. falciparum We describe key events, such as invasion, knob formation, and egress, and identify the interactions between exported parasite proteins and the host cell cytoskeleton. We discuss each remodeling step with respect to time and specific requirement of the developing parasite to explain host cell remodeling in a stage-specific manner. Thus, we highlight the interaction with the host membrane skeleton as a key event in parasite survival.

Comparative and functional genomics of the protozoan parasite Babesia divergens highlighting the invasion and egress processes.

Babesiosis is considered an emerging disease because its incidence has significantly increased in the last 30 years, providing evidence of the expanding range of this rare but potentially life-threatening zoonotic disease. Babesia divergens is a causative agent of babesiosis in humans and cattle in Europe. The recently sequenced genome of B. divergens revealed over 3,741 protein coding-genes and the 10.7-Mb high-quality draft become the first reference tool to study the genome structure of B. divergens. Now, by exploiting this sequence data and using new computational tools and assembly strategies, we have significantly improved the quality of the B. divergens genome. The new assembly shows better continuity and has a higher correspondence to B. bovis chromosomes. Moreover, we present a differential expression analysis using RNA sequencing of the two different stages of the asexual lifecycle of B. divergens: the free merozoite capable of invading erythrocytes and the intraerythrocytic parasite stage that remains within the erythrocyte until egress. Comparison of mRNA levels of both stages identified 1,441 differentially expressed genes. From these, around half were upregulated and the other half downregulated in the intraerythrocytic stage. Orthogonal validation by real-time quantitative reverse transcription PCR confirmed the differential expression. A moderately increased expression level of genes, putatively involved in the invasion and egress processes, were revealed in the intraerythrocytic stage compared with the free merozoite. On the basis of these results and in the absence of molecular models of invasion and egress for B. divergens, we have proposed the identified genes as putative molecular players in the invasion and egress processes. Our results contribute to an understanding of key parasitic strategies and pathogenesis and could be a valuable genomic resource to exploit for the design of diagnostic methods, drugs and vaccines to improve the control of babesiosis.

Ligand Pathways in Nuclear Receptors.

Nuclear receptors (NRs) are ligand-inducible transcription factors that play an essential role in a multitude of physiological processes as well as diseases, rendering them attractive drug targets. Crystal structures revealed the binding site of NRs to be buried in the core of the protein, with no obvious route for ligands to access this cavity. The process of ligand binding is known to be an often-neglected contributor to the efficacy of drug candidates and is thought to influence the selectivity and specificity of NRs. While experimental methods generally fail to highlight the dynamic processes of ligand access or egress on the atomistic scale, computational methods have provided fundamental insight into the pathways connecting the buried binding pocket to the surrounding environment. Methods based on molecular dynamics (MD) and Monte Carlo simulations have been applied to identify pathways and quantify their capability to transport ligands. Here, we systematically review findings of more than 20 years of research in the field, including the applied methodology and controversies. Further, we establish a unified nomenclature to describe the pathways with respect to their location relative to protein secondary structure elements and summarize findings relevant to drug design. Lastly, we discuss the effect of NR interaction partners such as coactivators and corepressors, as well as mutations on the pathways.

Bone marrow sinusoidal endothelium as a facilitator/regulator of cell egress from the bone marrow.

Despite more attention to cell migration from circulation into the bone marrow (BM), particularly homing of haematopoietic stem/progenitor cells, the process and mechanisms of cell mobilisation from the BM into the circulation remain largely underexplored. The process of cell mobilisation or transcellular cell migration from BM into the circulation (cell egress) is a crucial biological process in mammals as it is important to maintain homeostasis of various physiological functions including, but not limited to, the immune system, erythropoiesis, platelet release, and stem cell migration. The BM microvascular system composes of a monolayer of specialized endothelial cells, called sinusoidal endothelial cells (SECs). While it is very well evident that the process of cell egress occurs exclusively through BM SECs, there is a lack of systematic analyses addressing the extent of contribution of BM SECs to the process of cell egress from the BM. Therefore, this review aims to address the potential ability of BM SECs in regulating and/or facilitating the process of cell egress from BM. In this review, we address, firstly, the unique ultra-/structural and molecular features of BM SECs and discuss the possible biological interactions between BM SECs and various egressing cells in physiological conditions. Secondly, we propose the potential role of BM SECs in egress of leukemic cells from BM into the circulation. Finally, we discuss the potential role of BM SECs in homing of haematopoietic stem cells. Collectively, the current review suggests that the BM SECs may not be merely a neutral gatekeeper for cell intravasation and extravasation, but rather is a dynamic trafficking surveillance system, thereby the process of BM cell egress/mobilisation can be regulated.

The Role of Host Cytoskeleton in Flavivirus Infection.

The family of flaviviruses is one of the most medically important groups of emerging arthropod-borne viruses. Host cell cytoskeletons have been reported to have close contact with flaviviruses during virus entry, intracellular transport, replication, and egress process, although many detailed mechanisms are still unclear. This article provides a brief overview of the function of the most prominent flaviviruses-induced or -hijacked cytoskeletal structures including actin, microtubules and intermediate filaments, mainly focus on infection by dengue virus, Zika virus and West Nile virus. We suggest that virus interaction with host cytoskeleton to be an interesting area of future research.

Effect of hydrogen on nanomechanical properties in Fe-22Mn-0.6C TWIP steel revealed by in-situ electrochemical nanoindentation

In-situ electrochemical nanoindentation was applied to study the effect of hydrogen on the mechanical properties of Fe-22Mn-0.6C TWIP steel at nanoscale. Distinctive behaviors in three defined grain orientations: (001), (101), and (111) were investigated in a sequence of air, hydrogen ingress, and hydrogen egress processes. The obvious pop-in load drop caused by introducing hydrogen was analyzed using the classical dislocation theory in combination with the “Defactant” model, wherein hydrogen-enhanced homogeneous dislocation nucleation through the reduction of dislocation line energy and stacking fault energy were proposed as the reasons. The dependence of pop-in behaviors on crystallographic orientations was also discussed. Tabor relation-based models were applied to analyze the nanohardness increment, which was related to the hydrogen-enhanced lattice friction and the hydrogen-reduced plastic zone size. The different recovery behaviors of pop-in load and nanohardness during hydrogen egress were assessed according to the different amounts of residual hydrogen in the corresponding affected zone.

Kinetics of the invasion and egress processes of Babesia divergens, observed by time-lapse video microscopy

Based on confocal fluorescence and bright field video microscopy, we present detailed observations on the processes of invasion and egress of erythrocytes by the apicomplexan parasite Babesia divergens. Time-lapse images reveal numerous unexpected findings associated with the dynamics of B. divergens and its ability to manipulate the erythrocyte during both processes in its asexual cycle under in vitro conditions. Despite the speed at which these processes occur and the small size of the parasite, we capture infective merozoites moving vigorously and causing striking deformations in the erythrocyte’s plasma membrane during an active invasion. We also observed intraerythrocytic dynamic stages as paired pyriforms, double paired pyriforms, tetrads, unattached pyriform sister cells and multiple parasite stages resulting in the release of large numbers of merozoites over a short period. Of considerable interest is that time-lapse images reveal a novel mechanism of egress used by B. divergens to exit the human erythrocyte. The release occurs when B. divergens parasites establish contacts with the plasma membrane of the erythrocyte from within, before exiting the cell. Visualization and analysis of the images enabled us to obtain useful information and broaden our knowledge of complex and crucial events involved with parasitisation of human erythrocytes by B. divergens.

Electron microscopic characterization of nuclear egress in the sea urchin gastrula.

Nuclear egress, also referred to as nuclear envelope (NE) budding, is a process of transport in which vesicles containing molecular complexes or viral particles leave the nucleus through budding from the inner nuclear membrane (INM) to enter the perinuclear space. Following this event, the perinuclear vesicles (PNVs) fuse with the outer nuclear membrane (ONM), where they release their contents into the cytoplasm. Nuclear egress is thought to participate in many functions such as viral replication, cellular differentiation, and synaptic development. The molecular basis for nuclear egress is now beginning to be elucidated. Here, we observe in the sea urchin gastrula, using serial section transmission electron microscopy, strikingly abundant PNVs containing as yet unidentified granules that resemble the ribonucleoprotein complexes (RNPs) previously observed in similar types of PNVs. Some PNVs were observed in the process of fusion with the ONM where they appeared to release their contents into the cytoplasm. These vesicles were abundantly observed in all three presumptive germ layers. These findings indicate that nuclear egress is likely to be an important mechanism for nucleocytoplasmic transfer during sea urchin development. The sea urchin may be a useful model to characterize further and gain a better understanding of the process of nuclear egress.

Effect of Hydrogen on Nanomechanical Properties in Fe-22mn-0.6c TWIP Steel Revealed by In-Situ Electrochemical Nanoindentation

In-situ electrochemical nanoindentation was applied to study the effect of hydrogen on the mechanical properties of Fe-22Mn-0.6C TWIP steel at the nanoscale. Distinctive behaviors in three defined grain orientations: (001), (101), and (111) were investigated in a sequence of air, hydrogen ingress, and hydrogen egress processes. The obvious pop-in load drop caused by introducing hydrogen was analyzed using the classical dislocation theory in combination with the Defactant model, wherein hydrogen-enhanced homogenous dislocation nucleation through the reduction of the dislocation line energy and the stacking fault energy were proposed as the reasons. The dependence of pop-in behaviors on the crystallographic orientations was also discussed. Tabor relation-based models were applied to analyze the nanohardness increment, which was related to the hydrogen-enhanced lattice friction and the hydrogen-reduced plastic zone size. The different recovery behaviors of the pop-in load and nanohardness during hydrogen egress were assessed according to the different amounts of residual hydrogen in the corresponding affected zone.

Fire Emergency Evacuation Simulation of a shopping mall using Fire Dynamic Simulator (FDS)

Fire accident in a shopping mall, garments factory and other labor intensive industries nowadays has become a common incident in Bangladesh and poses a great threat to life, facilities and economy of our country. In this work, fire and evacuation simulation was performed for a single stored shopping complex utilizing computational fluid dynamic techniques. Fire Dynamic Simulator with evacuation (FDS+Evac) software was used to simulate a shopping mall fire and study the effects of fire on the emergency egress process of people. The shopping mall of area 64 m2 comprises of seven rooms with a pool fire at the center of the mall is modeled for simulation. The total evacuation time (TET) for a fixed population density were estimated with the change of heat release rate, soot yield, soot density and the design pattern or geometry of shopping mall. The evacuation of agents in different time and different design pattern of the mall has been assessed using the data obtained from the simulation. FDS+Evac provides an integrating platform where the interaction between fire growth and evacuees can be taken into account by simultaneous simulation allowing a full coupling of the fire conditions and human behavior. This makes FDS is an effective tool for simulating large and high density crowds where the movement dynamics of evacuees is affected by the crowd pressure. Full scale fire experiment is often quite difficult to study the fine and crowds evacuation behavior. This paper illustrates a promising application of fire dynamic simulator (FDS+Evac) for fire and evacuation modeling to predict the total evacuation time.Journal of Chemical Engineering, Vol. 30, No. 1, 2017: 32-36

Exploitation of a newly-identified entry pathway into the malaria parasite-infected erythrocyte to inhibit parasite egress

While many parasites develop within host cells to avoid antibody responses and to utilize host cytoplasmic resources, elaborate egress processes have evolved to minimize the time between escaping and invading the next cell. In human erythrocytes, malaria parasites perforate their enclosing erythrocyte membrane shortly before egress. Here, we show that these pores clearly function as an entry pathway into infected erythrocytes for compounds that inhibit parasite egress. The natural glycosaminoglycan heparin surprisingly inhibited malaria parasite egress, trapping merozoites within infected erythrocytes. Labeled heparin neither bound to nor translocated through the intact erythrocyte membrane during parasite development, but fluxed into erythrocytes at the last minute of the parasite lifecycle. This short encounter was sufficient to significantly inhibit parasite egress and dispersion. Heparin blocks egress by interacting with both the surface of intra-erythrocytic merozoites and the inner aspect of erythrocyte membranes, preventing the rupture of infected erythrocytes but not parasitophorous vacuoles, and independently interfering with merozoite disaggregation. Since this action of heparin recapitulates that of neutralizing antibodies, membrane perforation presents a brief opportunity for a new strategy to inhibit parasite egress and replication.

A structural analysis of the natural egress of Toxoplasma gondii

Previous studies have analysed the process of Toxoplasma gondii egress with the aid of inducers, such as calcium ionophores. Although calcium transients have been successful in triggering T. gondii egress, the structural panorama of “natural” and artificial events should match. The present study approaches the natural egress of this parasite using super-resolution and electron microscopy and reveals lytic and non-lytic events of individual egress; this corroborates the use of calcium ionophore as a reliable tool to trigger parasite egress. Altogether, our data suggest that different signalling routes can converge to similar structural aspects in natural and induced egress.

Recent buzz in malaria research.

In this Commentary, we highlight the latest findings in three active areas of malaria research: Plasmodium biology; host response; and malaria control, prevention and treatment.

A Computational Study of the Station Nightclub Fire Accounting for Social Relationships

Using agent-based modeling, this study presents the results of a computational study of social relationships among more than four hundreds evacuees in The Station Nightclub building in Rhode Island. The fire occurred on the night of February 20, 2003 and resulted in 100 fatalities. After summarizing and calibrating the computational method used, parametric studies are conducted to quantitatively investigate the influences of the presence of social relationships and familiarity of the building floor plan on the death and injury tolls. It is demonstrated that the proposed model has the ability to reasonably handle the complex social relationships and group behaviors present during egress. The simulations quantify how intimate social affiliations delay the overall egress process and show the extent by which lack of knowledge of a building floor plan limits exit choices and adversely affects the number of safe evacuations.

Interindividual Spread of Herpesviruses.

Interindividual spread of herpesviruses is essential for the virus life cycle and maintenance in host populations. For most herpesviruses, the virus-host relationship is close, having coevolved over millions of years resulting in comparatively high species specificity. The mechanisms governing interindividual spread or horizontal transmission are very complex, involving conserved herpesviral and cellular proteins during the attachment, entry, replication, and egress processes of infection. Also likely, specific herpesviruses have evolved unique viral and cellular interactions during cospeciation that are dependent on their relationship. Multiple steps are required for interindividual spread including virus assembly in infected cells; release into the environment, followed by virus attachment; and entry into new hosts. Should any of these steps be compromised, transmission is rendered impossible. This review will focus mainly on the natural virus-host model of Marek's disease virus (MDV) in chickens in order to delineate important steps during interindividual spread.

Proteomic Analysis of the Plasmodium berghei Gametocyte Egressome and Vesicular bioID of Osmiophilic Body Proteins Identifies Merozoite TRAP-like Protein (MTRAP) as an Essential Factor for Parasite Transmission

Malaria transmission from an infected host to the mosquito vector requires the uptake of intraerythrocytic sexual precursor cells into the mosquito midgut. For the release of mature extracellular gametes two membrane barriers-the parasite parasitophorous vacuole membrane and the host red blood cell membrane-need to be dissolved. Membrane lysis occurs after the release of proteins from specialized secretory vesicles including osmiophilic bodies. In this study we conducted proteomic analyses of the P. berghei gametocyte egressome and developed a vesicular bioID approach to identify hitherto unknown proteins with a potential function in gametocyte egress. This first Plasmodium gametocyte egressome includes the proteins released by the parasite during the lysis of the parasitophorous vacuole membrane and red blood cell membrane. BioID of the osmiophilic body protein MDV1/PEG3 revealed a vesicular proteome of these gametocyte-specific secretory vesicles. Fluorescent protein tagging and gene deletion approaches were employed to validate and identify a set of novel factors essential for this lysis and egress process. Our study provides the first in vivo bioID for a rodent malaria parasite and together with the first Plasmodium gametocyte egressome identifies MTRAP as a novel factor essential for mosquito transmission. Our data provide an important resource for proteins potentially involved in a key step of gametogenesis.

Effects of nanoparticle size and surface charge on uptake into rat alveolar epithelial cells

Physicochemical properties of nanoparticles, including size, composition and surface charge, are likely to affect nanoparticle interactions with biological cells/tissues. We reported previously that uptake of polystyrene nanoparticles (PNP) from apical fluid into primary cultured rat alveolar epithelial cell monolayers (RAECM) takes place in a manner dependent on exposure time and dose. Classical endocytosis pathways do not appear to play a role in the uptake of PNP, consistent with a ‘diffusional’ process. We also reported that autophagy is involved in cellular processing of PNP, in that accumulation of PNP in lysosomes occurs via autophagic flux. Lysosomal exocytosis of PNP (modulated by intracellular [Ca2+]), takes place in parallel with a slower ‘diffusional’ egress process. In this study, we investigated the effects of nanoparticle size and surface charge on uptake into RAECM grown on Anopore filters. RAECM were exposed apically to 80 μg/mL carboxylated or amidinated 20, 100 or 200 nm PNP (with near‐infrared fluorescent label) and intracellular [PNP] was monitored over 24 hr using live cell imaging. Results showed that intracellular [PNP] reached a plateau of ~0.6 mg/mL after ≥12 hr of apical exposure to both 20 nm carboxylated or amidinated [PNP] at 80 μg/mL. As PNP size increased from 20 to 100 to 200 nm, steady state intracellular [PNP] decreased from ~0.6 to ~0.3 to ~0.1 μg/mL, respectively, after ≥12 hr. These data indicate that, for RAECM, PNP uptake is strongly dependent on PNP size, while the role of surface charge appears to be less important. Additional factors (e.g., nanoparticle‐associated protein corona) might alter the charge effect. In general, understanding the role of physicochemical properties of nanoparticles in cellular interactions will lead to improved nanoparticle design for protection from cellular injury and approaches to therapeutic utilization in drug/gene delivery.Support or Funding InformationFunding: NIH; Hastings and Whittier Foundations.