Biology subjects

Yanik, S.

Publications and source records attributed to Yanik, S..

3 recordsLinked to original sources

bioRxiv2024

Application of machine learning in a rodent malaria model for rapid, accurate, and consistent parasite counts

Rodent malaria models serve as important preclinical antimalarial and vaccine testing tools. Evaluating treatment outcomes in these models often requires manually counting parasite-infected red blood cells (iRBCs), a time-consuming process, which can be inconsistent between individuals and labs. We have developed an easy-to-use machine learning (ML)-based software, Malaria Screener R, to expedite and standardize such studies by automating the counting of Plasmodium iRBCs in rodents. This software can process Giemsa-stained blood smear images captured by any camera-equipped microscope. It features an intuitive graphical user interface that facilitates image processing and visualization of the results. The software has been developed as a desktop application that processes images on standard Windows and Mac OS computers. A previous ML model created by the authors designed to count P. falciparum-infected human RBCs did not perform well counting Plasmodium-infected mouse RBCs. We leveraged that model by loading the pre-trained weights and training the algorithm with newly collected data to target P. yoelii and P. berghei mouse iRBCs. This new model reliably measured both P. yoelii and P. berghei parasitemia (R2 = 0.9916). Additional rounds of training data to incorporate variances due to length of Giemsa staining, microscopes etc, have produced a generalizable model, meeting WHO Competency Level 1 for the sub-category of parasite counting using independent microscopes. Reliable, automated analyses of blood-stage parasitemia will facilitate rapid and consistent evaluation of novel vaccines and antimalarials across labs in an easily accessible in vivo malaria model.

microbiology↗

bioRxiv2024

Plasmodium RON11 triggers biogenesis of the merozoite rhoptry pair and is essential for erythrocyte invasion

Malaria is a global and deadly human disease caused by the apicomplexan parasites of the genus Plasmodium. Parasite proliferation within human red blood cells (RBC) is associated with the clinical manifestations of the disease. This asexual expansion within human RBCs, begins with the invasion of RBCs by P. falciparum, which is mediated by the secretion of effectors from two specialized club-shaped secretory organelles in merozoite-stage parasites known as rhoptries. We investigated the function of the Rhoptry Neck Protein 11 (RON11), which contains seven transmembrane domains and calcium-binding EF-hand domains. We generated conditional mutants of the P. falciparum RON11. Knockdown of RON11 inhibits parasite growth by preventing merozoite invasion. The loss of RON11 did not lead to any defects in processing of rhoptry proteins but instead led to a decrease in the amount of rhoptry proteins. We utilized ultrastructure expansion microscopy (U-ExM) to determine the effect of RON11 knockdown on rhoptry biogenesis. Surprisingly, in the absence of RON11, fully developed merozoites had only one rhoptry each. The single rhoptry in RON11 deficient merozoites were morphologically typical with a bulb and a neck oriented into the apical polar ring. Moreover, rhoptry proteins are trafficked accurately to the single rhoptry in RON11 deficient parasites. These data show that in the absence of RON11, the first rhoptry is generated during schizogony but upon the start of cytokinesis, the second rhoptry never forms. Interestingly, these single-rhoptry merozoites were able to attach to host RBCs but are unable to invade RBCs. Instead, RON11 deficient merozoites continue to engage with RBC for prolonged periods eventually resulting in echinocytosis, a result of secreting the contents from the single rhoptry into the RBC. Together, our data show that RON11 triggers the de novo biogenesis of the second rhoptry and functions in RBC invasion.

cell biology↗

bioRxiv2021

Engineering CAR-NK cells to secrete IL15 sustains their anti-AML functionality, but is associated with systemic toxicities

BackgroundThe prognosis of patients with recurrent/refractory acute myelogenous leukemia (AML) remains poor and cell-based immunotherapies hold promise to improve outcomes. NK cells can elicit an anti-leukemic response via a repertoire of activating receptors that bind AML surface ligands. NK cell adoptive transfer is safe but thus far has shown limited anti-AML efficacy. Here, we aimed to overcome this limitation by engineering NK cells to express chimeric antigen receptors (CARs) to boost their anti-AML activity, and interleukin-15 (IL15) to enhance their persistence. MethodsWe characterized in detail NK cell populations expressing a panel of AML (CD123)-specific CARs and/or IL15 in vitro and in AML xenograft models. ResultsCARs with 2B4.{zeta} or 4-1BB.{zeta} signaling domains demonstrated greater cell surface expression and endowed NK cells with improved anti-AML activity in vitro. Initial in vivo testing revealed that only 2B4.{zeta} CAR-NK cells had improved anti-AML activity in comparison to untransduced (UTD) and 4-1BB.{zeta} CAR-NK cells. However, the benefit was transient due to limited CAR-NK cell persistence. Transgenic expression of secretory (s)IL15 in 2B4.{zeta} CAR and UTD NK cells improved their effector function in the setting of chronic antigen simulation in vitro. Multiparameter flow analysis after chronic antigen exposure identified the expansion of unique NK cell subsets. 2B4.{zeta}/sIL15 CAR and sIL15 NK cells maintained an overall activated NK cell phenotype. This was confirmed by transcriptomic analysis, which revealed a highly proliferative and activated signature in these NK cell groups. In vivo, 2B4.{zeta}/sIL15 CAR-NK cells had potent anti-AML activity in one model, while 2B4.{zeta}/sIL15 CAR and sIL15 NK cells induced lethal toxicity in a second model. ConclusionTransgenic expression of CD123-CARs and sIL15 enabled NK cells to function in the setting of chronic antigen exposure but was associated with systemic toxicities. Thus, our study provides the impetus to explore inducible and controllable expression systems to provide cytokine signals to AML-specific CAR-NK cells before embarking on early phase clinical testing. O_FIG O_LINKSMALLFIG WIDTH=161 HEIGHT=200 SRC="FIGDIR/small/461509v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@4d09eaorg.highwire.dtl.DTLVardef@1c61411org.highwire.dtl.DTLVardef@14f8097org.highwire.dtl.DTLVardef@8375ac_HPS_FORMAT_FIGEXP M_FIG C_FIG In BriefSecretion of IL15 from anti-CD123.2B4.CD3{zeta} CAR-NK cells enhances their activation and killing potential against Acute Myelogenous Leukemia, but carries the risk of severe systemic toxicity.

immunology↗