Erythropoiesis is marked by progressive changes in morphological, biochemical and mechanical properties of erythroid precursors to generate red blood cells (RBC). The earliest enucleated forms derived in this process, known as reticulocytes, are multi-lobular and spherical. As reticulocytes mature, they undergo a series of dynamic cytoskeletal re-arrangements and the expulsion of residual organelles, resulting in highly deformable biconcave RBCs (normocytes). To understand the significant, yet neglected proteome-wide changes associated with reticulocyte maturation, we undertook a quantitative proteomics approach. Immature reticulocytes (marked by the presence of surface transferrin receptor, CD71) and mature RBCs (devoid of CD71) were isolated from human cord blood using a magnetic separation procedure. After sub-fractionation into triton-extracted membrane proteins and luminal samples (isobaric tags for relative and absolute quantitation), quantitative mass spectrometry was conducted to identify more than 1800 proteins with good confidence and coverage. While most structural proteins (such as Spectrins, Ankyrin and Band 3) as well as surface glycoproteins were conserved, proteins associated with microtubule structures, such as Talin-1/2 and ß-Tubulin, were detected only in immature reticulocytes. Atomic force microscopy (AFM)-based imaging revealed an extended network of spectrin filaments in reticulocytes (with an average length of 48 nm), which shortened during reticulocyte maturation (average spectrin length of 41 nm in normocytes). The extended nature of cytoskeletal network may partly account for increased deformability and shape changes, as reticulocytes transform to normocytes.

Reticulocytes, the precursors of erythrocytes, undergo drastic alterations in cell size, shape, and deformability during maturation. Experimental evidence suggests that young reticulocytes are stiffer and less stable than their mature counterparts; however, the underlying mechanism is yet to be fully understood. Here, we develop a coarse-grained molecular-dynamics reticulocyte membrane model to elucidate how the membrane structure of reticulocytes contributes to their particular biomechanical properties and pathogenesis in blood diseases. First, we show that the extended cytoskeleton in the reticulocyte membrane is responsible for its increased shear modulus. Subsequently, we quantify the effect of weakened cytoskeleton on the stiffness and stability of reticulocytes, via which we demonstrate that the extended cytoskeleton along with reduced cytoskeleton connectivity leads to the seeming paradox that reticulocytes are stiffer and less stable than the mature erythrocytes. Our simulation results also suggest that membrane budding and the consequent vesiculation of reticulocytes can occur independently of the endocytosis-exocytosis pathway, and thus, it may serve as an additional means of removing unwanted membrane proteins from reticulocytes. Finally, we find that membrane budding is exacerbated when the cohesion between the lipid bilayer and the cytoskeleton is compromised, which is in accord with the clinical observations that erythrocytes start shedding membrane surface at the reticulocyte stage in hereditary spherocytosis. Taken together, our results quantify the stiffness and stability change of reticulocytes during their maturation and provide, to our knowledge, new insights into the pathogenesis of hereditary spherocytosis and malaria.

Malaria, caused by parasites of the species Plasmodium, is among the major life-threatening diseases to afflict humanity. The infectious cycle of Plasmodium is very complex involving distinct life stages and transitions characterized by cellular and molecular alterations. Therefore, novel single-cell technologies are warranted to extract details pertinent to Plasmodium-host cell interactions and underpinning biological transformations. Herein, we tested two emerging spectroscopic approaches: (a) Optical Photothermal Infrared spectroscopy and (b) Atomic Force Microscopy combined with infrared spectroscopy in contrast to (c) Fourier Transform InfraRed microspectroscopy, to investigate Plasmodium-infected erythrocytes. Chemical spatial distributions of selected bands and spectra captured using the three modalities for major macromolecules together with advantages and limitations of each method is presented here. These results indicate that O-PTIR and AFM-IR techniques can be explored for extracting sub-micron resolution molecular signatures within heterogeneous and dynamic samples such as Plasmodium-infected human RBCs.

Ferrocenyl phosphines targeting the digestive vacuole function of the malaria parasite, Plasmodium falciparum.

Acid phosphatase activity has been studied in hepatopancreas and foot tissues of the Indian apple snail, Pila globosa (Swainson), with reference to aestiva. tion and starvation. The enzyme activity in the tissues of control snails is higher in hepatopancreas, than in foot. The activity of acid phosphatase increased in hepatopancreas and decreased in foot during starvation while it decreased in both the tissues during aestivation. The significance of these findings is discussed.

Antimalarial drug discovery expands on targeted and phenotype-based screening of potential inhibitory molecules to ascertain overall efficacy, phenotypic characteristics and toxicity, prior to exploring pharmacological optimizations. Candidate inhibitors may have varying chemical properties, thereby requiring specific reconstitution conditions to ensure solubility, stability or bioavailability. Hence, a variety of solvents, buffers, detergents and stabilizers become part of antimalarial efficacy assays, all of which, above certain threshold could interfere with parasite viability, invasion or red blood cell properties leading to misinterpretation of the results. Despite their routine use across malaria research laboratories, there is no documentation on non-toxic range for common constituents including DMSO, glycerol, ethanol and methanol. We herein constructed a compatibility reference guide for 14 such chemicals and estimated their Permissible Limit against P. falciparum asexual stages at which viability and replication of parasites are not compromised. We also demonstrate that at the estimated Permissible Limit, red blood cells remain healthy and viable for infection by merozoites. Taken together, this dataset provides a valuable reference tool for the acceptable concentration range for common chemicals during in vitro antimalarial tests.

29Plasmodia are host-specific, both at the organism and cellular levels. During asexual 30 development, Plasmodium spp. infect cells of erythroid lineage, with an overall 31 propensity towards reticulocytes. This applies to even Plasmodium (P.) falciparum, the 32 most common causative agent of human malaria, implications of which remain 33 unexplored. Herein, for the first time, we characterize the developmental stages and 34 features of P. falciparum cultured in vitro in young reticulocytes (CD71 + ) in comparison to 35 standard normocyte (CD71 -) cultures. We demonstrate that there are notable differences 36 in the patterns of invasion, development and sensitivity to potent antimalarials (such as 37 artemisinin and dihydroartemisinin) for parasites residing in CD71 + reticulocytes. 38Through a transcriptomic approach, we report that P. falciparum parasites are able to 39 sense the host cell environment, and calibrate their metabolic and host cell remodelling 40 pathways through differential gene expression. These results form an exciting avenue on 41 which hitherto unexplored interactions between Plasmodium spp and different stages of 42 host red blood cells could be investigated in the broader contexts of drug resistance, 43 host tropism and zoonosis. 45Author Summary 46Parasites causing malaria infect red blood cells for development and proliferation during 47 asexual development. This asexual erythrocytic stage determines higher parasite 48 densities and eventual disease manifestation. Although the most virulent species of 49 Plasmodium infecting humans known as Plasmodium falciparum is able to infect red 50 blood cells of all ages, these parasites show a preference for younger blood cells. Of 51 note, the biochemical and biophysical properties of young and adult red blood cells vary 52 significantly. Herein, we undertook a comparative profiling of invasion process, parasite 53 development and drug response of Plasmoddium falciparum in two host cells: young red 54 blood cells (reticulocytes) and mature red blood cells (normocytes). We demonstrate that 55 P. falciparum infects human reticulocytes with higher affinity and demonstrate differential 56 3 sensitivity to drugs such as artemisinin while they reside within reticulocytes. 57Furthermore, we show that P. falciparum is able to detect differences in host 58 environment and adapt to it by changing the expression of genes required for host cell 59 remodelling. 61 Introduction 62Plasmodium infection and associated mortality remain an important concern to the 63 developing world with 218 million malaria cases and ~450,000 deaths annually 1 64 (https://www.who.int/malaria/publications/world-malaria-report-2018/en/). 65Widespread drug-resistance 2-3 and evolution of newer phenotypes; influenced by factors 66 such as changing availability and distribution of insect vector 4 , haematological 67 malignancies(1, 2) 5-6 (thalassemia, sickle cell anaemia, G6PD deficiency etc), providing 68 protective immunity to certain populations 7 , adversely impact malaria eradic...

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