scholarly journals Immunofluorescence staining protocol for STED nanoscopy of Plasmodium-infected red blood cells

2018 ◽  
Author(s):  
Ann-Kathrin Mehnert ◽  
Caroline Sophie Simon ◽  
Julien Guizetti
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Single Cells ◽  
Blood Stage ◽  
Immunofluorescence Staining ◽  
Cell Seeding ◽  
Cell Integrity ◽  
Nuclear Pores ◽  
Staining Protocol ◽  
Intranuclear Microtubules

AbstractImmunofluorescence staining is the key technique for visualizing organization of endogenous cellular structures in single cells. Labeling and imaging of blood stage Plasmodium falciparum has always been challenging since it is a small intracellular parasite. The gold standard for parasite immunofluorescence is fixation in suspension with addition of minute amounts of glutaraldehyde to the paraformaldehyde-based solution. While this maintains red blood cell integrity, it has been postulated that antigenicity of the parasite proteins was, if at all, only slightly reduced. Here we show the deleterious effect that even these small quantities of glutaraldehyde can have on immunofluorescence staining quality and present an alternative cell seeding protocol that allows fixation with only paraformaldehyde. The highly improved signal intensity and staining efficiency enabled us to carry out RescueSTED nanoscopy on microtubules and nuclear pores and describe their organization in greater detail throughout the blood stage cycle.Highlights- Omitting glutaraldehyde from fixative improves immunofluorescence staining- STED nanoscopy is readily applicable to infected red blood cells- Intranuclear microtubules are nucleated from distinct sites

scholarly journals Heterogeneous timing of asexual cycles in Plasmodium falciparum quantified by extended time-lapse microscopy

2018 ◽  
Author(s):  
Heungwon Park ◽  
Shuqiang Huang ◽  
Katelyn A. Walzer ◽  
Lingchong You ◽  
Jen-Tsan Ashley Chi ◽  
...  
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Single Cells ◽  
Time Lapse ◽  
Bimodal Distribution ◽  
Host Cells ◽  
Cycle Period ◽  
Human Red Blood Cells ◽  
Asexual Cycle

ABSTRACTMalarial fever arises from the synchronous bursting of human red blood cells by the Plasmodium parasite. The released parasites re-infect neighboring red blood cells and undergo another asexual cycle of differentiation and proliferation for 48 hours, before again bursting synchronously. The synchrony of bursting is lost during in vitro culturing of the parasite outside the human body, presumably because the asexual cycle is no longer entrained by host-specific circadian cues. Therefore, most in vitro malaria studies have relied on the artificial synchronization of the parasite population. However, much remains unknown about the degree of timing heterogeneity of asexual cycles and how artificial synchronization may affect this timing. Here, we combined time-lapse fluorescence microscopy and long-term culturing to follow single cells and directly measure the heterogeneous timing of in vitro asexual cycles. We first demonstrate that unsynchronized laboratory cultures are not fully asynchronous and the parasites exhibit a bimodal distribution in their first burst times. We then show that synchronized and unsynchronized cultures had similar asexual cycle periods, which indicates that artificial synchronization does not fundamentally perturb asexual cycle dynamics. Last, we demonstrate that sibling parasites descended from the same schizont exhibited significant variation in asexual cycle period, although smaller than the variation between non-siblings. The additional variance between non-siblings likely arises from the variable environments and/or developmental programs experienced in different host cells.

Hemoglobins in Single Chick Erythrocytes as Determined by a Differential Elution Procedure

1978 ◽  
Vol 33 (5-6) ◽  
pp. 330-336 ◽  
Author(s):  
Nicholas Zagris ◽  
Charles G. Melton
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Single Cells ◽  
Electrophoretic Analysis ◽  
Erythroid Cell ◽  
Cell Dynamics ◽  
Adult Chicken ◽  
Acid Buffer ◽  
Adult Hemoglobin

Abstract The switch from embryonic to adult hemoglobin (Hb) has been studied in vivo by a correlated cytological and electrophoretic analysis of circulating red blood cells from early, purely embryonic-Hb stages to purely adult-Hb stages including the adult chicken. It has been discovered, by using an acid buffer treatment that selectively elutes adult but not embryonic Hb from intact red blood cells, that embryonic and adult Hbs occur together in single cells, and that the switch occurs simultaneously in all cells. These results together with knowledge of the chick erythroid cell dynamics and ontogenetic titers indicate that the initiation of adult Hb synthesis occurs in the circulation in cells previously committed only to embryonic Hb synthesis.

scholarly journals Structurally conserved erythrocyte-binding domain in Plasmodium provides a versatile scaffold for alternate receptor engagement

2015 ◽  
Vol 113 (2) ◽  
pp. E191-E200 ◽  
Author(s):  
Jakub Gruszczyk ◽  
Nicholas T. Y. Lim ◽  
Alicia Arnott ◽  
Wen-Qiang He ◽  
Wang Nguitragool ◽  
...  
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Binding Protein ◽  
Structural Features ◽  
Binding Domain ◽  
Blood Stage ◽  
Sequence Alignments ◽  
Blood Stage Infection ◽  
Erythrocyte Binding ◽  
Stage Infection

Understanding how malaria parasites gain entry into human red blood cells is essential for developing strategies to stop blood stage infection. Plasmodium vivax preferentially invades reticulocytes, which are immature red blood cells. The organism has two erythrocyte-binding protein families: namely, the Duffy-binding protein (PvDBP) and the reticulocyte-binding protein (PvRBP) families. Several members of the PvRBP family bind reticulocytes, specifically suggesting a role in mediating host cell selectivity of P. vivax. Here, we present, to our knowledge, the first high-resolution crystal structure of an erythrocyte-binding domain from PvRBP2a, solved at 2.12 Å resolution. The monomeric molecule consists of 10 α-helices and one short β-hairpin, and, although the structural fold is similar to that of PfRh5—the essential invasion ligand in Plasmodium falciparum—its surface properties are distinct and provide a possible mechanism for recognition of alternate receptors. Sequence alignments of the crystallized fragment of PvRBP2a with other PvRBPs highlight the conserved placement of disulfide bonds. PvRBP2a binds mature red blood cells through recognition of an erythrocyte receptor that is neuraminidase- and chymotrypsin-resistant but trypsin-sensitive. By examining the patterns of sequence diversity within field isolates, we have identified and mapped polymorphic residues to the PvRBP2a structure. Using mutagenesis, we have also defined the critical residues required for erythrocyte binding. Characterization of the structural features that govern functional erythrocyte binding for the PvRBP family provides a framework for generating new tools that block P. vivax blood stage infection.

scholarly journals Biomechanics of Ex Vivo-Generated Red Blood Cells Investigated by Optical Tweezers and Digital Holographic Microscopy

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 552
Author(s):  
Claudia Bernecker ◽  
Maria Augusta R. B. F. Lima ◽  
Catalin D. Ciubotaru ◽  
Peter Schlenke ◽  
Isabel Dorn ◽  
...  
Keyword(s):  
Red Blood Cells ◽  
Optical Tweezers ◽  
Blood Cells ◽  
Ex Vivo ◽  
Single Cells ◽  
Membrane Lipid ◽  
Membrane Properties ◽  
Digital Holographic Microscopy ◽  
Holographic Microscopy

Ex vivo-generated red blood cells are a promising resource for future safe blood products, manufactured independently of voluntary blood donations. The physiological process of terminal maturation from spheroid reticulocytes to biconcave erythrocytes has not been accomplished yet. A better biomechanical characterization of cultured red blood cells (cRBCs) will be of utmost interest for manufacturer approval and therapeutic application. Here, we introduce a novel optical tweezer (OT) approach to measure the deformation and elasticity of single cells trapped away from the coverslip. To investigate membrane properties dependent on membrane lipid content, two culture conditions of cRBCs were investigated, cRBCPlasma with plasma and cRBCHPL supplemented with human platelet lysate. Biomechanical characterization of cells under optical forces proves the similar features of native RBCs and cRBCHPL, and different characteristics for cRBCPlasma. To confirm these results, we also applied a second technique, digital holographic microscopy (DHM), for cells laid on the surface. OT and DHM provided related results in terms of cell deformation and membrane fluctuations, allowing a reliable discrimination between cultured and native red blood cells. The two techniques are compared and discussed in terms of application and complementarity.

The malaria merozoite, forty years on

Parasitology ◽  
2009 ◽  
Vol 136 (12) ◽  
pp. 1435-1444 ◽  
Author(s):  
L. H. BANNISTER ◽  
G. H. MITCHELL
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Blood Stage ◽  
Malaria Parasites ◽  
Research Attention ◽  
Considerable Body

SUMMARYThe invasive blood stage of malaria parasites, merozoites, are complex entities specialized for the capture and entry of red blood cells. Their potential for vaccination and other anti-malaria strategies have attracted much research attention over the last 40 years, and there is now a considerable body of data relating to their biology. In this article some of the major advances over this period and remaining challenges are reviewed.

scholarly journals Single Cell Analysis of Stored Red Blood Cells Using Ultra-High Throughput Holographic Cytometry

Cells ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 2455
Author(s):  
Han-Sang Park ◽  
Hillel Price ◽  
Silvia Ceballos ◽  
Jen-Tsan Chi ◽  
Adam Wax
Keyword(s):  
Red Blood Cells ◽  
High Throughput ◽  
Blood Cells ◽  
Storage Time ◽  
Single Cells ◽  
Machine Learning Algorithms ◽  
Training Data ◽  
Microfluidic Channels ◽  
Phase Imaging ◽  
Time Points

Holographic cytometry is introduced as an ultra-high throughput implementation of quantitative phase imaging of single cells flowing through parallel microfluidic channels. Here, the approach was applied for characterizing the morphology of individual red blood cells during storage under regular blood bank conditions. Samples from five blood donors were examined, over 100,000 cells examined for each, at three time points. The approach allows high-throughput phase imaging of a large number of cells, greatly extending our ability to study cellular phenotypes using individual cell images. Holographic cytology images can provide measurements of multiple physical traits of the cells, including optical volume and area, which are observed to consistently change over the storage time. In addition, the large volume of cell imaging data can serve as training data for machine-learning algorithms. For the study here, logistic regression was used to classify the cells according to the storage time points. The analysis showed that at least 5000 cells are needed to ensure accuracy of the classifiers. Overall, results showed the potential of holographic cytometry as a diagnostic tool.

scholarly journals Loss of complement regulatory proteins on red blood cells in mild malarial anaemia and in Plasmodium falciparum induced blood-stage infection

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
Damian A. Oyong ◽  
Jessica R. Loughland ◽  
Arya SheelaNair ◽  
Dean Andrew ◽  
Fabian D. L. Rivera ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Blood Stage ◽  
Regulatory Proteins ◽  
Complement Regulatory Proteins ◽  
Blood Stage Infection ◽  
Low Levels ◽  
Stage Infection ◽  
Blood Stage Malaria

Abstract Background Anaemia is a major consequence of malaria, caused by the removal of both infected and uninfected red blood cells (RBCs) from the circulation. Complement activation and reduced expression of complement regulatory proteins (CRPs) on RBCs are an important pathogenic mechanism in severe malarial anaemia in both Plasmodium falciparum and Plasmodium vivax infection. However, little is known about loss of CRPs on RBCs during mild malarial anaemia and in low-density infection. Methods The expression of CRP CR1, CD55, CD59, and the phagocytic regulator CD47, on uninfected normocytes and reticulocytes were assessed in individuals from two study populations: (1) P. falciparum and P. vivax-infected patients from a low transmission setting in Sabah, Malaysia; and, (2) malaria-naïve volunteers undergoing P. falciparum induced blood-stage malaria (IBSM). For clinical infections, individuals were categorized into anaemia severity categories based on haemoglobin levels. For IBSM, associations between CRPs and haemoglobin level were investigated. Results CRP expression on RBC was lower in Malaysian individuals with P. falciparum and P. vivax mild malarial anaemia compared to healthy controls. CRP expression was also reduced on RBCs from volunteers during IBSM. Reduction occurred on normocytes and reticulocytes. However, there was no significant association between reduced CRPs and haemoglobin during IBSM. Conclusions Removal of CRPs occurs on both RBCs and reticulocytes during Plasmodium infection even in mild malarial anaemia and at low levels of parasitaemia.

scholarly journals Learning-based screening of hematologic disorders using quantitative phase imaging of individual red blood cells

2018 ◽  
Author(s):  
Geon Kim ◽  
YoungJu Jo ◽  
Hyungjoo Cho ◽  
Hyun-seok Min ◽  
YongKeun Park
Keyword(s):  
Machine Learning ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Single Cells ◽  
Deficiency Anemia ◽  
High Dimensional ◽  
Phase Imaging ◽  
Label Free ◽  
Quantitative Phase Imaging ◽  
Quantitative Phase

We present a rapid and label-free method for hematologic screening for diseases and syndromes, utilizing quantitative phase imaging (QPI) and machine learning. We aim to establish an efficient blood examination framework that does not suffer from the drawbacks of conventional blood assays, which are incapable of profiling single cells or using labeling procedures. Our method involves the synergistic employment of QPI and machine learning. The high-dimensional refractive index information arising from the QPI-based profiling of single red blood cells is processed to screen for diseases and syndromes using machine learning, which can utilize high-dimensional data beyond the human level. Accurate screening for iron-deficiency anemia, reticulocytosis, hereditary spherocytosis, and diabetes mellitus is demonstrated (>99% accuracy) using the proposed method. Furthermore, we highlight the synergy between QPI and machine learning in the proposed method by analyzing the performance of the method.

scholarly journals Using Plasmodium knowlesi as a model for screening Plasmodium vivax blood-stage malaria vaccine targets reveals new candidates

2020 ◽  
Author(s):  
Duncan N. Ndegwa ◽  
Jessica B. Hostetler ◽  
Alejandro Marin-Menendez ◽  
Theo Sanderson ◽  
Kioko Mwikali ◽  
...  
Keyword(s):  
Red Blood Cells ◽  
Plasmodium Vivax ◽  
Blood Cells ◽  
Culture System ◽  
Parasite Species ◽  
Vaccine Development ◽  
Blood Stage ◽  
Effective Vaccine ◽  
Human Red Blood Cells

ABSTRACTPlasmodium vivax is responsible for the majority of malaria cases outside Africa. Unlike P. falciparum, the P. vivax life-cycle includes a dormant liver stage, the hypnozoite, which can cause infection in the absence of mosquito transmission. An effective vaccine against P. vivax blood stages would limit symptoms and pathology from such recurrent infections, and therefore could play a critical role in the control of this species. Vaccine development in P. vivax, however, lags considerably behind P. falciparum, which has many identified targets with several having transitioned to Phase II testing. By contrast only one P. vivax blood-stage vaccine candidate based on the Duffy Binding Protein (PvDBP), has reached Phase Ia, in large part because the lack of a continuous in vitro culture system for P. vivax limits systematic screening of new candidates. We used the close phylogenetic relationship between P. vivax and P. knowlesi, for which an in vitro culture system in human erythrocytes exists, to test the scalability of systematic reverse vaccinology to identify and prioritise P. vivax blood-stage targets. A panel of P. vivax proteins predicted to function in erythrocyte invasion were expressed as full-length recombinant ectodomains in a mammalian expression system. Eight of these antigens were used to generate polyclonal antibodies, which were screened for their ability to recognize orthologous proteins in P. knowlesi. These antibodies were then tested for inhibition of growth and invasion of both wild type P. knowlesi and chimeric P. knowlesi lines modified using CRISPR/Cas9 to exchange P. knowlesi genes with their P. vivax orthologues. Candidates that induced antibodies that inhibited invasion to a similar level as PvDBP were identified, confirming the utility of P. knowlesi as a model for P. vivax vaccine development and prioritizing antigens for further follow up.AUTHOR SUMMARYMalaria parasites cause disease after invading human red blood cells, implying that a vaccine that interrupts this process could play a significant role in malaria control. Multiple Plasmodium parasite species can cause malaria in humans, and most malaria outside Africa is caused by Plasmodium vivax. There is currently no effective vaccine against the blood stage of any malaria parasite, and progress in P. vivax vaccine development has been particularly hampered because this parasite species cannot be cultured for prolonged periods of time in the lab. We explored whether a related species, P. knowlesi, which can be propagated in human red blood cells in vitro, can be used to screen for potential P. vivax vaccine targets. We raised antibodies against selected P. vivax proteins and testedtheir ability to recognize and prevent P. knowlesi parasites from invading human red blood cells, thereby identifying multiple novel vaccine candidates.

Sign in / Sign up

Export Citation Format

Share Document