Erythrocyte Cytoplasm Research Articles

Accompanying Hemoglobin Polymerization in Red Blood Cells in Patients with Sickle Cell Disease Using Fluorescence Lifetime Imaging.

In recent studies, it has been shown that fluorescence lifetime imaging (FLIM) may reveal intracellular structural details in unstained cytological preparations that are not revealed by standard staining procedures. The aim of our investigation was to examine whether FLIM images could reveal areas suggestive of polymerization in red blood cells (RBCs) of sickle cell disease (SCD) patients. We examined label-free blood films using auto-fluorescence FLIM images of 45 SCD patients and compared the results with those of 27 control persons without hematological disease. All control RBCs revealed homogeneous cytoplasm without any foci. Rounded non-sickled RBCs in SCD showed between zero and three small intensively fluorescent dots with higher lifetime values. In sickled RBCs, we found additionally larger irregularly shaped intensively fluorescent areas with increased FLIM values. These areas were interpreted as equivalent to polymerized hemoglobin. The rounded, non-sickled RBCs of SCD patients with homogeneous cytoplasm were not different from those of the erythrocytes of control patients in light microscopy. Yet, variables from the local binary pattern-transformed matrix of the FLIM values per pixel showed significant differences between non-sickled RBCs and those of control cells. In a linear discriminant analysis, using local binary pattern-transformed texture features (mean and entropy) of the erythrocyte cytoplasm of normal appearing cells, the final model could distinguish between SCD patients and control persons with an accuracy of 84.7% of the patients. When the classification was based on the examination of a single rounded erythrocyte, an accuracy of 68.5% was achieved. Employing the Linear Discriminant Analysis classifier method for machine learning, the accuracy was 68.1%. We believe that our study shows that FLIM is able to disclose the topography of the intracellular polymerization process of hemoglobin in sickle cell disease and that the images are compatible with the theory of the two-step nucleation. Furthermore, we think that the presented technique may be an interesting tool for the investigation of therapeutic inhibition of polymerization.

ЭЛЕКТРОННАЯ МИКРОСКОПИЯ ЭРИТРОЦИТОВ ПЕРИФЕРИЧЕСКОЙ КРОВИ КАК ПРЕДИКТ ОПРЕДЕЛЕНИЯ СТЕПЕНИ ТЯЖЕСТИ ОСТРОГО ПАНКРЕАТИТА

Background. Acute pancreatitis is one of the five most common diseases in urgent abdominal surgery. Recently, the methods of electron microscopy of erythrocytes as a predictor of determining the severity of acute pancreatitis have become firmly established in practice. Purpose. To compare the state of erythrocytes of healthy people with erythrocytes of patients with acute pancreatitis of different severity. Materials and methods. The study of peripheral blood erythrocytes of healthy people and patients with pancreatitis of varying severity was carried out. For comparison, the erythrocyte shape, cytoplasm state, as well as the content of potassium, sodium, calcium, nitrogen and oxygen were studied. Results. During the study, the correlation between the severity of acute pancreatitis course and changes in the shape, membrane and ionic composition of the peripheral blood erythrocyte cytoplasm was noted. Conclusions. According to the obtained changes in erythrocytes, it is possible to indirectly judge the processes occurring in the cells of the pancreas and, accordingly, to determine the severity of the acute pancreatitis course.

The Digestive Vacuole of the Malaria Parasite: A Specialized Lysosome.

The malaria parasite resides within erythrocytes during one stage of its life cycle. During this intraerythrocytic period, the parasite ingests the erythrocyte cytoplasm and digests approximately two-thirds of the host cell hemoglobin. This digestion occurs within a lysosome-like organelle called the digestive vacuole. Several proteases are localized to the digestive vacuole and these proteases sequentially breakdown hemoglobin into small peptides, dipeptides, and amino acids. The peptides are exported into the host cytoplasm via the chloroquine-resistance transporter and an amino acid transporter has also been identified on the digestive vacuole membrane. The environment of the digestive vacuole also provides appropriate conditions for the biocrystallization of toxic heme into non-toxic hemozoin by a poorly understood process. Hemozoin formation is an attribute of Plasmodium and Haemoproteus and is not exhibited by other intraerythrocytic protozoan parasites. The efficient degradation of hemoglobin and detoxification of heme likely plays a major role in the high level of replication exhibited by malaria parasites within erythrocytes. Unique features of the digestive vacuole and the critical importance of nutrient acquisition provide therapeutic targets for the treatment of malaria.

Babesia ovis secreted antigen-1 is a diagnostic marker during the active Babesia ovis infections in sheep.

Ovine babesiosis caused by Babesia ovis is an economically significant disease. Recently, a few B. ovis-specific proteins, including recombinant B. ovis secreted antigen-1 (rBoSA1), have been identified. Immunological analyses revealed that rBoSA1 resides within the cytoplasm of infected erythrocytes and exhibits robust antigenic properties for detecting anti-B. ovis antibodies. This protein is released into the bloodstream during the parasite's development. It would be possible to diagnose active infections by detecting this secretory protein. For this purpose, a rBoSA1-specific polyclonal antibody-based sandwich ELISA was optimized in this study. Blood samples taken from the naturally (n: 100) and experimentally (n: 15) infected sheep were analyzed for the presence of native BoSA1. The results showed that native BoSA1 was detectable in 98% of naturally infected animals. There was a positive correlation between parasitemia level in microscopy and protein density in sandwich ELISA. Experimentally infected animals showed positive reactions from the first or second day of inoculations. However, experimental infections carried out by Rhipicephalus bursa ticks revealed the native BoSA1 was detectable from the 7th day of tick attachment when the parasite began to be seen microscopically. Sandwich ELISA was sensitive enough to detect rBoSA1 protein at a 1.52 ng/ml concentration. Additionally, no serological cross-reactivity was observed between animals infected with various piroplasm species, including Babesia bovis, B. bigemina, B. caballi, B. canis, B. gibsoni, Theileria equi, and T. annulata. Taken collectively, the findings show that the rBoSA1-specific polyclonal antibody-based sandwich ELISA can be successfully used to diagnose clinical B. ovis infections in sheep at the early stage.

Malignant human thyroid neoplasms associated with blood parasitic (haemosporidian) infection

A retrospective investigation of archival cytology specimens obtained and verified by a fine-needle aspiration biopsy from patients with follicular, papillary, and medullary human thyroid cancers revealed haemosporidian (blood parasitic) infection in thyrocytes (schizogony) and erythrocytes. The exoerythrocytic stage of infection is represented by finding microschizonts. Cytologic material was stained with RomanowskyGiemsa dye in medical laboratories. Original specimens were stained (re-stained) with Schiff reagent according to the Feulgen method to clarify location of thyrocyte DNA and hemosporidian pathogens, wherein fuchsine was incorporated into DNA molecules after they were hydrolyzed by hydrochloric acid to stain specimens into red-violet color. An intentionally unstained hemosporidian protoplasm during blood parasitic infection was observed as a light band around erythrocyte nuclei. In follicular thyroid cancer, thyrocyte Feulgen staining revealed nuclear DNA and parasitic DNA (haemosporidium nuclei) as punctate inclusions and rings diffusely distributed in the thyrocyte cytoplasm. The thyrocyte cytoplasm and nuclei were vacuolated, with thyrocyte nuclei being deformed, flattened, and displaced to the cell periphery. The erythrocytes contained haemosporidian nuclei (DNA). In papillary thyroid cancer, we were able to localize the nuclear DNA of thyrocytes and the parasitic DNA as punctate inclusions diffusely distributed in the thyrocyte cytoplasm. Two or more polymorphic nuclei may be positioned eccentrically in the hyperplastic cytoplasm. Haemosporidian microschizonts were found circumnuclearly in thyrocytes as well as an exoerythrocytic stage in the blood. In medullary thyroid cancer, the hyperplastic cytoplasm of thyrocytes contained eccentrically located nuclei (DNA) of thyrocytes and small haemosporidian nuclei (DNA), which may occupy the whole thyrocyte. There were thyrocytes with vacuolated cytoplasm and prominent nuclear polymorphism. The size of hyperplastic nuclei was several times larger than that of normal thyrocyte nuclei. The color of stained thyrocyte cytoplasmic and nuclear vacuoles was less red-violet compared with that of surrounding tissues, which potentially indicates the presence of parasitic DNA inside them. The intra-erythrocyte nuclear haemosporidian material of varying sizes in papillary and medullary cancers may evidence about various species and/or pathogen generation. Intracellular parasitism of haemosporidian infection in thyrocytes (schizogony) associated with three thyroid cancer types leads to marked thyrocyte cytoplasmic hyperplasia, cytoplasmic vacuolization, and nuclear vacuolization. Multinucleated thyrocytes with incomplete cytokinesis emerge. Nuclear deformation occurs, which leads to decreased nucleus size, flattening and displacement to the cell periphery, with high risk of DNA mutations and deletions in affected cells, reaching a neoplastic level.

Morphological and cytochemical characteristics of Varanus niloticus (Squamata, Varanidae) blood cells.

Varanus niloticus is a lizard residing within the Varanidae family. To date no studies detailing its blood morphology and characteristics have been conducted. This study used histologically stained blood and bone marrow samples to visualize the cells and their characteristics. The erythrocytes were nucleated, these nuclei were located in the middle of the elliptical cells. Hemoglobin filled the erythrocyte cytoplasm. Eosinophils were large cells with lobed nuclei and spherical acidophilic granules. Large granulocytes called heterophils were present and characterized by their fusiform/pleomorphic cytoplasmic granules. Small spherical granulocytes, known as basophils, presented with round, deeply stained metachromatic granules that gave the cytoplasm a dusty or cobblestoned appearance which was able to cover the nucleus, which in turn had an unusual shape. Thrombocytes ranged in shape from ellipsoidal to fusiform. They featured an elliptical, centrally located nucleus and a pale cytoplasm, with small vacuoles, and fine acidophilic granulation. The smallest variety of non-granular leukocytes was the lymphocytes. Their cytoplasm was sparse, finely granular, light blue, had tiny cytoplasmic projections, featuring a high nucleus: cytoplasm ratio. Larger and smaller sized populations of lymphocytes were distinguished, with the larger cells similar in size to azurophils. In general, the pleomorphic monocytes were the biggest mononuclear leucocytes, displaying cytoplasmic projections. Their nuclei were ovoid, kidney- or bean-shaped, with vacuolated and granular cytoplasms. Round cells were common among the monocytic azurophils, and they had a granular cytoplasm, and their nuclei were typically eccentric. The present research identifies the cell types and morphologies within the Varanus niloticus. HIGHLIGHTS: H&E, PAS, toluidine blue, methylene blue, and Safranin O stains provided morphological and morphometric descriptions of Varanus niloticus blood cells from blood smears and bone marrow. The Varanus niloticus had nucleated erythrocytes and white blood cells, mostly granulocytes (heterophils, eosinophils, and basophils) and mononuclear cells (azurophils, lymphocytes, and monocytes). Aquatic vertebrate Varanus niloticus had larger erythrocytes than terrestrial counterparts. Blood cell morphological and cytochemical features were similar to other reptilian species, with some species-specific differences, which likely accommodate differing environmental conditions. These results may help clinical researchers track the pathological conditions and support conservation of these wild animals.

FEATURES AND POSSIBILITIES OF BLOOD BIOIMPEDANCEMETRY

The bioimpedancemetry has long been used to determine some blood parameters used in laboratory diagnostics. Due to the specificity of blood as a liquid disperse substance, its electrical conductivity is directly related to the relative volume of the non-conductive phase, that is, erythrocytes, and therefore, the bioimpedancemetry method is used to determine hematocrit. The ambiguity of the results in determining the frequencies corresponding to the achievement of the limiting value of the high-frequency electrical conductivity of the blood, as well as the patterns that connect the high-frequency electrical conductivity of the blood with the electrical conductivity of the plasma and erythrocytes, demonstrate the urgent need for additional research. The purpose of the study was to determine the features of the frequency dependence of the blood bioimpedance modulus with the values of hematocrit and blood plasma bioimpedance modulus in order to establish the capabilities of the blood bioimpedancemetry method as a whole. The frequency dependence of its bioimpedance was determined on 16 blood samples from healthy donors. For each blood sample, the frequency dependence of the bioimpedance of whole blood was taken, then the plasma formed after erythrocyte sedimentation in a vertically installed test tube. The impedance modulus was measured in the frequency range from 10 kHz to 10 megahertz. The optimal frequency of 5 megahertz was determined, at which the limiting high-frequency value of blood electrical conductivity is reached, which makes it possible to determine the electrical conductivity of the erythrocyte cytoplasm. An expression is obtained for high-frequency electrical conductivity as a function of the electrical conductivity of plasma, cytoplasm and relative hematocrit. It is shown that the measurement of low-frequency (up to 100 kilohertz) and high-frequency values of blood electrical conductivity, as well as plasma electrical conductivity, also makes it possible to determine the hematocrit and the electrical conductivity of the erythrocyte cytoplasm. An equation was obtained that relates the hematocrit index to the ratio of high-frequency and low-frequency values of blood electrical conductivity, which makes it possible to determine the true hematocrit of human blood in vitro. Thus, the measurement of low-frequency and high-frequency electrical conductivity of blood and the measurement of electrical conductivity of plasma make it possible to determine not only the hematocrit index, but also the electrical conductivity of the cytoplasm of cells in one blood sample. The work proves the practical possibility of determining the complex of electrophysical parameters of a native blood sample while maintaining their real values.

Effect of hemoglobin hydration on the physical properties of erythrocyte cytoplasm and whole blood

Electrochemical impedance spectroscopy of whole blood and blood cells has great potential for assessing the health of patients. This potential has not been fully exploited for a detailed examination of the erythrocyte interior. The erythrocyte interior (cytoplasm) is often considered as continuous media. However, cytoplasm is a colloidal suspension of hydrated hemoglobin molecules. This study mainly focuses on the effect of hemoglobin hydration on the dielectric properties of erythrocyte cytoplasm and whole blood. The impedance spectra of separated cytoplasm and whole blood were measured at frequencies from 1 kHz to 110 MHz. The effective medium theory was used to analyze the experimental data. The cytoplasm was found to be best described as a colloid of hemoglobin cores surrounded by double hydration shells. The dielectric properties of the hemoglobin core, hydration shells and free intracellular fluid, as well as the thickness of the shells, were numerically determined from the impedance spectrum. An approach was developed to determine the intracellular fluid's viscosity and the density of bound water in the hydration shells. It shows that the hemoglobin hydration has a significant effect on the physical properties of blood. The detailed electrical properties of the cytoplasm can be detected from the impedance spectrum of whole blood.

A member of the tryptophan-rich protein family is required for efficient sequestration of Plasmodium berghei schizonts.

Protein export and host membrane remodeling are crucial for multiple Plasmodium species to establish a niche in infected hosts. To better understand the contribution of these processes to successful parasite infection in vivo, we sought to find and characterize protein components of the intraerythrocytic Plasmodium berghei-induced membrane structures (IBIS) that form in the cytoplasm of infected erythrocytes. We identified proteins that immunoprecipitate with IBIS1, a signature member of the IBIS in P. berghei-infected erythrocytes. In parallel, we also report our data describing proteins that co-precipitate with the PTEX (Plasmodium translocon of exported proteins) component EXP2. To validate our findings, we examined the location of three candidate IBIS1-interactors that are conserved across multiple Plasmodium species, and we found they localized to IBIS in infected red blood cells and two further colocalized with IBIS1 in the liver-stage parasitophorous vacuole membrane. Successful gene deletion revealed that these two tryptophan-rich domain-containing proteins, termed here IPIS2 and IPIS3 (for intraerythrocytic Plasmodium-induced membrane structures), are required for efficient blood-stage growth. Erythrocytes infected with IPIS2-deficient schizonts in particular fail to bind CD36 as efficiently as wild-type P. berghei-infected cells and therefore fail to effectively sequester out of the circulating blood. Our findings support the idea that intra-erythrocytic membrane compartments are required across species for alterations of the host erythrocyte that facilitate interactions of infected cells with host tissues.

Identification of Exported Plasmodium falciparum Proteins That Bind to the Erythrocyte Cytoskeleton.

Plasmodium proteins are exported to the erythrocyte cytoplasm to create an environment that supports parasite replication. Although hundreds of proteins are predicted to be exported through Plasmodium export element (PEXEL)-dependent and -independent mechanisms, the functions of exported proteins are largely uncharacterized. In this study, we used a biochemical screening approach to identify putative exported P. falciparum proteins that bound to inside-out vesicles prepared from erythrocytes. Out of 69 P. falciparum PEXEL-motif proteins tested, 18 bound to inside-out vesicles (IOVs) in two or more independent assays. Using co-affinity purifications followed by mass spectrometry, pairwise co-purification experiments, and the split-luciferase assay, we identified 31 putative protein–protein interactions between erythrocyte cytoskeletal proteins and predicted exported P. falciparum proteins. We further showed that PF3D7_1401600 binds to the spectrin-binding domain of erythrocyte ankyrin via its MESA erythrocyte cytoskeleton binding (MEC) motif and to the N-terminal domains of ankyrin and 4.1R through a fragment that required an intact Plasmodium helical interspersed sub-telomeric (PHIST) domain. Introduction of PF3D7_1401600 into erythrocyte ghosts increased retention in the microsphiltration assay, consistent with previous data that reported a reduction of rigidity in red blood cells infected with PF3D7_1401600-deficient parasites.

RhopH2 and RhopH3 export enables assembly of the RhopH complex on P. falciparum-infected erythrocyte membranes

RhopH complexes consists of Clag3, RhopH2 and RhopH3 and are essential for growth of Plasmodium falciparum inside infected erythrocytes. Proteins are released from rhoptry organelles during merozoite invasion and trafficked to the surface of infected erythrocytes and enable uptake of nutrients. RhopH3, unlike other RhopH proteins, is required for parasite invasion, suggesting some cellular processes RhopH proteins function as single players rather than a complex. We show the RhopH complex has not formed during merozoite invasion. Clag3 is directly released into the host cell cytoplasm, whilst RhopH2 and RhopH3 are released into the nascent parasitophorous vacuole. Export of RhopH2 and RhopH3 from the parasitophorous vacuole into the infected erythrocyte cytoplasm enables assembly of Clag3/RhopH2/RhopH3 complexes and incorporation into the host cell membrane concomitant with activation of nutrient uptake. This suggests compartmentalisation prevents premature channel assembly before intact complex is assembled at the host cell membrane.

Prior Incubation as a Tool to Overcome Understaining of Leishman Stain: A Cross-sectional Study

Introduction: A well stained peripheral smear is an integral part in the diagnosis and management of many clinicohematologic conditions. Leishman stain is recommended universally by the International Committee for Standardisation in Haematology (ICSH). However, a commonly encountered problem in tropical countries, especially during the rainy season, is under staining by Leishman stain as a result of interference by the atmospheric moisture. Hence, incubation of slides and/or buffer prior to standard Leishman staining protocol could bring out best staining qualities. Aim: To assess the effects of prior incubation of slides and/or buffer on standard Leishman staining technique. Materials and Methods: The cross-sectional study was conducted over a period of 8 weeks in the Central Laboratory, Kempegowda Institute of Medical Sciences, Bangalore in the months of October 2020 to November 2020. Blood samples were received in the central laboratory for routine haematological investigations. Total of 100 samples of left over non haemolysed Dipotassium Ethylenediaminetetraacetic Acid (K2-EDTA) anticoagulated blood irrespective of age, gender and cell counts were included in the study. Slides were stained following prior incubation of slides and/or buffer at 37° C and compared with the standard staining technique. The staining characteristics of nucleus, cytoplasm and granules of White Blood Cells (WBCs), platelets and erythrocytes were observed. One-way Analysis of Variance (ANOVA) test and p-value of <0.05 was considered statistically significant and higher f value (>104.35152) rejected the null hypothesis. Results: Prior incubation of slides and/or buffer resulted in better stained smears which were found to be statistically significant (p-value<0.05) as compared to standard Leishman staining technique. Conclusion: Minor modifications when applied to standard Leishman staining technique such as prior incubation of slides and/ or buffer at 37° Centigrade produced best staining characteristics to overcome the effects of humidity which normally interfere with staining of slides.

Intraerythrocytic Mycoplasma-like organism diagnosed ultrastructurally as an agent of anaemia in laboratory-reared cyprinid hybrids.

A study targeting the etiology of severe anaemia that sporadically occurred in laboratory-bred cyprinid hybrids resulted in a diagnosis of a Mycoplasma-like organism selectively invading the cytoplasm of erythrocytes. Despite the fact that there was a concurrent yeast infection in moribund anaemic hybrids, the primary role in the development of anaemia was assigned to the Mycoplasma-like organism due to its regular occurrence in erythrocytes of both the moribund hybrids and hybrids that were free of yeast infection yet showed early to advanced symptoms of the disease. Novel data on the Mycoplasma-like organism's cytoskeleton were obtained from ultrathin sections of affected erythrocytes. An ultrastructural study of the concurrent yeast infection in moribund hybrids manifesting the most advanced anaemia revealed the presence of Titan cells in ascitic fluid. The original findings presented in this study underline the diagnostic relevance of transmission electron microscopy in the research of similar infections.

Effect of Hemoglobin Hydration on the Physical Properties of Erythrocyte Cytoplasm and Whole Blood

Effect of Hemoglobin Hydration on the Physical Properties of Erythrocyte Cytoplasm and Whole Blood

Оптичний контроль межі розподілу між поверхнею золота та зразками клітин крові

The optical properties of blood (spectra of the extinction coefficient, k, refractive index, n, etc.) carry important diagnostic information and are usually monitored using bulk samples. In this work, attention is drawn to the interface between the blood volume and the surface of glass or thin gold films on it, where the refractive index may differ from the bulk one. We draw attention to the relationship between two effects – SPR and TIR. It is shown that if the named effects are measured for two different external media 0 and 1 with different refractive indices, then the values of the angles SPR and TIR will be linearly related by the empirical formula SPR1=SPR0+TIR1- TIR0)*K, where the coefficient K depends on the thickness of the transition layer di between the surface and the volume of the liquid medium (suspension). Numerical calculation of K (di) for gold films shows that K = 1.6 at di = 0 and monotonically decreases to 0.01 with an increase in di to 300 nm (and further to 0). Measurement of the angular dependences of reflection, R(), on (1) 100% hematocrit blood samples, (2) hemolyzed samples and (3) washed erythrocytes with dilutions with a buffer solution. It was shown that all samples exhibit a minimum SPR, but the TIR angle can be measured only for blood samples with destroyed membranes (hemolyzed), buffer solution and plasma. The n-value for hemolyzed blood is 1.3505, which is indicative of a low hemoglobin content in the sample. At the same time, di for a sample of 100% hematocrit was 60-105 nm, which indicates a strong deformation of erythrocytes in the form of polyhedrocytes and their dense packing after centrifugation. Washing the cells with a buffer increases di to 280 nm and more and practically eliminates blood cells from the SPR sensitivity region. The reason for this may be that in the blood of 100% hematocrit, erythrocytes are in the form of polyhedrocytes tightly adhering to the gold surface, while as a result of washing and diluting with a buffer solution, the cells relax back into discocytes. As a result, the containing hemoglobin erythrocyte cytoplasm moves away from the surface at a distance di> 300 nm into the suspension volume and leaves the area of the enhanced plasmon-polariton field.

Role of Liquid-Liquid Phase Transitions in Mechanism of Erythrocyte Protection During Cooling with CRIHBT-115 Cryopreservative Agent

The presence in the system of critical liquid-liquid phase transition (PT) by the mechanism, resulting in formation of dispersion system, namely high-concentrated emulsion, has been established here during cooling when using polarized light microscopy and fixation of critical opalescence phenomenon in erythrocyte concentrate with glycerol-containing cryopreservative agent, designed at the Central Research Institute of Haematology and Blood Transfusion (Russia) (CRIHBT-115 ). The studied cryobiological system displayed no signs of crystallization. A phase behaviour of cryopreservative and supernatant has been studied during cooling-warming cycle. Changes in the volume of cryopreservative and erythrocyte concentrate were comparatively and qualitatively evaluated during cooling. The mechanism of protective action of cryopreservation solution has been determined. The similarity between physical and chemical processes during cooling-warming of erythrocyte cytoplasm and garlic meristem cells (germinal plant tissue) when entering cold anabiosis has been established.

Unique Endomembrane Systems and Virulence in Pathogenic Protozoa.

Virulence in pathogenic protozoa is often tied to secretory processes such as the expression of adhesins on parasite surfaces or the secretion of proteases to assisted in tissue invasion and other proteins to avoid the immune system. This review is a broad overview of the endomembrane systems of pathogenic protozoa with a focus on Giardia, Trichomonas, Entamoeba, kinetoplastids, and apicomplexans. The focus is on unique features of these protozoa and how these features relate to virulence. In general, the basic elements of the endocytic and exocytic pathways are present in all protozoa. Some of these elements, especially the endosomal compartments, have been repurposed by the various species and quite often the repurposing is associated with virulence. The Apicomplexa exhibit the most unique endomembrane systems. This includes unique secretory organelles that play a central role in interactions between parasite and host and are involved in the invasion of host cells. Furthermore, as intracellular parasites, the apicomplexans extensively modify their host cells through the secretion of proteins and other material into the host cell. This includes a unique targeting motif for proteins destined for the host cell. Most notable among the apicomplexans is the malaria parasite, which extensively modifies and exports numerous proteins into the host erythrocyte. These modifications of the host erythrocyte include the formation of unique membranes and structures in the host erythrocyte cytoplasm and on the erythrocyte membrane. The transport of parasite proteins to the host erythrocyte involves several unique mechanisms and components, as well as the generation of compartments within the erythrocyte that participate in extraparasite trafficking.

The Novel Zoonotic Pathogen, Anaplasma capra, Infects Human Erythrocytes, HL-60, and TF-1 Cells In Vitro.

Anaplasma capra, a species of the family Anaplasmataceae, is zoonotic tick-borne obligate intracellular bacteria. There have been no reports of human infection with this pathogen since 2015. Therefore, the zoonotic characteristics of A. capra need to be further studied. To verify the ability of A. capra to infect human cells, A. capra were inoculated in human erythrocytes, HL-60, and TF-1 cell lines in vitro. Cell smears were taken after inoculation, using Giemsa staining, transmission electron microscope (TEM), chromogenic in situ hybridization and immunocytochemistry for detection. In the Giemsa staining, many dark colored corpuscles or purple granules were seen in the inoculated erythrocytes, HL-60, and TF-1 cells. The results of chromogenic in situ hybridization show that there were brown precipitates on the surface of most erythrocytes. Immunocytochemistry results show many dark brown vacuolar structures or corpuscles in the cytoplasm of erythrocytes, HL-60, and TF-1 cell lines. The A. capra morulae were seen in the cytoplasm of both HL-60 and TF-1 in TEM, and their diameter was about 295–518 nm. Both dense-cored (DC) and reticulate cell (RC) form morulae could be seen. This study confirmed the ability of A. capra to infect human erythrocytes, HL-60, and TF-1. This study is of profound significance in further verifying the zoonotic characteristics of the pathogen and for establishing an in vitro cultivation model.

Creative interior design by Plasmodium falciparum: Lipid metabolism and the parasite's secret chamber.

Malaria parasites conceal themselves within host erythrocytes and establish a necessary logistics system through the three-membrane layered structures of these cells. To establish this system, lipid metabolism is needed for the de novo synthesis of lipids and the recycling of extracellular lipids and erythrocyte lipid components. Cholesterol supply depends on its uptake from the extracellular environment and erythrocyte cytoplasm, but phospholipids can be synthesized on their own. This differential production of lipid species creates unique modifications in the lipid profile of parasitized erythrocytes, which in turn may influence the biophysical and/or mechanical properties of organelles and vesicles and communication among them. Variations in local membrane properties possibly influence the transportation of various molecules such as parasite-derived proteins, because efficiencies in secretion, vesicle fusion and budding are partly determined by the lipid profiles. Comprehensive understanding of the parasite's lipid metabolism and the biophysics of lipid membranes provides fundamental knowledge about these pathogenic organisms and could lead to new anti-malarials.

Trafficking and Association of Plasmodium falciparum MC-2TM with the Maurer's Clefts.

In this study, we investigated stage specific expression, trafficking, solubility and topology of endogenous PfMC-2TM in P. falciparum (3D7) infected erythrocytes. Following Brefeldin A (BFA) treatment of parasites, PfMC-2TM traffic was evaluated using immunofluorescence with antibodies reactive with PfMC-2TM. PfMC-2TM is sensitive to BFA treatment and permeabilization of infected erythrocytes with streptolysin O (SLO) and saponin, showed that the N and C-termini of PfMC-2TM are exposed to the erythrocyte cytoplasm with the central portion of the protein protected in the MC membranes. PfMC-2TM was expressed as early as 4 h post invasion (hpi), was tightly colocalized with REX-1 and trafficked to the erythrocyte membrane without a change in solubility. PfMC-2TM associated with the MC and infected erythrocyte membrane and was resistant to extraction with alkaline sodium carbonate, suggestive of protein-lipid interactions with membranes of the MC and erythrocyte. PfMC-2TM is an additional marker of the nascent MCs.