Plasmodium falciparum exploits CD44 as a coreceptor for erythrocyte invasion.

The malaria parasite Plasmodium falciparum invades and replicates asexually within human erythrocytes. CD44 expressed on erythrocytes was previously identified as an important host factor for P falciparum infection through a forward genetic screen, but little is known about its regulation or function in these cells, nor how it may be used by the parasite. We found that CD44 can be efficiently deleted from primary human hematopoietic stem cells using CRISPR/Cas9 genome editing, and that the efficiency of ex vivo erythropoiesis to enucleated cultured red blood cells (cRBCs) is not affected by lack of CD44. However, the rate of P falciparum invasion was reduced in CD44-null cRBCs relative to isogenic wild-type control cells, validating CD44 as an important host factor for this parasite. We identified 2 P falciparum invasion ligands as binding partners for CD44, erythrocyte binding antigen 175 (EBA-175) and EBA-140 and demonstrated that their ability to bind to human erythrocytes relies primarily on their canonical receptors, glycophorin A and glycophorin C, respectively. We further show that EBA-175 induces phosphorylation of erythrocyte cytoskeletal proteins in a CD44-dependent manner. Our findings support a model in which P falciparum exploits CD44 as a coreceptor during invasion of human erythrocytes, stimulating CD44-dependent phosphorylation of host cytoskeletal proteins that alter host cell deformability and facilitate parasite entry.

Plasmodium falciparum exploits CD44 as a co-receptor for erythrocyte invasion.

The malaria parasite Plasmodium falciparum invades and replicates asexually within human erythrocytes. CD44 expressed on erythrocytes was previously identified as an important host factor for P. falciparum infection through a forward genetic screen, but little is known about its regulation or function in these cells, nor how it may be utilized by the parasite. We found that CD44 can be efficiently deleted from primary human hematopoietic stem cells using CRISPR/Cas9 genome editing, and that the efficiency of ex-vivo erythropoiesis to enucleated cultured red blood cells (cRBCs) is not impacted by lack of CD44. However, the rate of P. falciparum invasion was substantially reduced in CD44-null cRBCs relative to isogenic wild-type (WT) control cells, validating CD44 as an important host factor for this parasite. We identified two P. falciparum invasion ligands as binding partners for CD44, Erythrocyte Binding Antigen-175 (EBA-175) and EBA-140, and demonstrated that their ability to bind to human erythrocytes relies primarily on their canonical receptors-glycophorin A and glycophorin C, respectively. We further show that EBA-175 induces phosphorylation of erythrocyte cytoskeletal proteins in a CD44-dependent manner. Our findings support a model where P. falciparum exploits CD44 as a co-receptor during invasion of human erythrocytes, stimulating CD44-dependent phosphorylation of host cytoskeletal proteins that alter host cell deformability and facilitate parasite entry.

Trisomy 21 induces pericentrosomal crowding delaying primary ciliogenesis and mouse cerebellar development.

Trisomy 21, the genetic cause of Down syndrome, disrupts primary cilia formation and function, in part through elevated Pericentrin, a centrosome protein encoded on chromosome 21. Yet how trisomy 21 and elevated Pericentrin disrupt cilia-related molecules and pathways, and the in vivo phenotypic relevance remain unclear. Utilizing ciliogenesis time course experiments combined with light microscopy and electron tomography, we reveal that chromosome 21 polyploidy elevates Pericentrin and microtubules away from the centrosome that corral MyosinVA and EHD1, delaying ciliary membrane delivery and mother centriole uncapping essential for ciliogenesis. If given enough time, trisomy 21 cells eventually ciliate, but these ciliated cells demonstrate persistent trafficking defects that reduce transition zone protein localization and decrease sonic hedgehog signaling in direct anticorrelation with Pericentrin levels. Consistent with cultured trisomy 21 cells, a mouse model of Down syndrome with elevated Pericentrin has fewer primary cilia in cerebellar granule neuron progenitors and thinner external granular layers at P4. Our work reveals that elevated Pericentrin from trisomy 21 disrupts multiple early steps of ciliogenesis and creates persistent trafficking defects in ciliated cells. This pericentrosomal crowding mechanism results in signaling deficiencies consistent with the neurological phenotypes found in individuals with Down syndrome.

Human cells typically have 23 pairs of structures known as chromosomes. Each chromosome contains a unique set of genes which provide the instructions needed to make proteins and other essential molecules found in the body. Individuals with Down syndrome have an extra copy of chromosome 21. This genetic alteration is known as trisomy 21 and affects many different organs in the body, leading to various medical conditions including intellectual disability, heart defects, and immune deficiencies. A recent study showed that cells from individuals with Down syndrome had defects in forming primary cilia ­ structures on the surface of cells which work as signaling hubs to control how cells grow and develop. These cilia defects were in large part due to excess levels of a protein known as Pericentrin, which is encoded by a gene found on chromosome 21. But it is unclear how Pericentrin disrupts cilia assembly, and how this may contribute to the medical conditions observed in individuals with Down syndrome. To address these questions, Jewett et al. studied human cells that had been engineered to have trisomy 21. The experiments found that trisomy 21 led to higher levels of Pericentrin and altered the way molecules were organized at the sites where primary cilia form. This caused the components required to build and maintain the primary cilium to become trapped in the wrong locations. The trisomy 21 cells were eventually able to rearrange the molecules and build a primary cilium, but it took them twice as long as cells with 23 pairs of chromosomes and their primary cilium did not properly work. Further experiments were then conducted on mice that had been engineered to have an extra copy of a portion of genes on human chromosome 21, including the gene for Pericentrin. Jewett et al. found that these mice assembled cilia later and had defects in cilia signaling, similar to the human trisomy 21 cells. This resulted in mild abnormalities in brain development that were consistent with what occurs in individuals with Down syndrome. These findings suggest that the elevated levels of Pericentrin in trisomy 21 causes changes in cilia formation and function which, in turn, may alter how the mouse brain develops. Further studies will be required to find out whether defects in primary cilia may contribute to other medical conditions observed in individuals with Down syndrome.

RAB19 Directs Cortical Remodeling and Membrane Growth for Primary Ciliogenesis.

Primary cilia are sensory organelles that utilize the compartmentalization of membrane and cytoplasm to communicate signaling events, and yet, how the formation of a cilium is coordinated with reorganization of the cortical membrane and cytoskeleton is unclear. Using polarized epithelia, we find that cortical actin clearing and apical membrane partitioning occur where the centrosome resides at the cell surface prior to ciliation. RAB19, a previously uncharacterized RAB, associates with the RAB-GAP TBC1D4 and the HOPS-tethering complex to coordinate cortical clearing and ciliary membrane growth, which is essential for ciliogenesis. This RAB19-directed pathway is not exclusive to polarized epithelia, as RAB19 loss in nonpolarized cell types blocks ciliogenesis with a docked ciliary vesicle. Remarkably, inhibiting actomyosin contractility can substitute for the function of the RAB19 complex and restore ciliogenesis in knockout cells. Together, this work provides a mechanistic understanding behind a cytoskeletal clearing and membrane partitioning step required for ciliogenesis.

Resource Allocation and Outpatient Appointment Scheduling Using Simulation Optimization.

This paper studies the real-life problems of outpatient clinics having the multiple objectives of minimizing resource overtime, patient waiting time, and waiting area congestion. In the clinic, there are several patient classes, each of which follows different treatment procedure flow paths through a multiphase and multiserver queuing system with scarce staff and limited space. We incorporate the stochastic factors for the probabilities of the patients being diverted into different flow paths, patient punctuality, arrival times, procedure duration, and the number of accompanied visitors. We present a novel two-stage simulation-based heuristic algorithm to assess various tactical and operational decisions for optimizing the multiple objectives. In stage I, we search for a resource allocation plan, and in stage II, we determine a block appointment schedule by patient class and a service discipline for the daily operational level. We also explore the effects of the separate strategies and their integration to identify the best possible combination. The computational experiments are designed on the basis of data from a study of an ophthalmology clinic in a public hospital. Results show that our approach significantly mitigates the undesirable outcomes by integrating the strategies and increasing the resource flexibility at the bottleneck procedures without adding resources.

An erythroid enhancer of BCL11A subject to genetic variation determines fetal hemoglobin level.

Genome-wide association studies (GWASs) have ascertained numerous trait-associated common genetic variants, frequently localized to regulatory DNA. We found that common genetic variation at BCL11A associated with fetal hemoglobin (HbF) level lies in noncoding sequences decorated by an erythroid enhancer chromatin signature. Fine-mapping uncovers a motif-disrupting common variant associated with reduced transcription factor (TF) binding, modestly diminished BCL11A expression, and elevated HbF. The surrounding sequences function in vivo as a developmental stage-specific, lineage-restricted enhancer. Genome engineering reveals the enhancer is required in erythroid but not B-lymphoid cells for BCL11A expression. These findings illustrate how GWASs may expose functional variants of modest impact within causal elements essential for appropriate gene expression. We propose the GWAS-marked BCL11A enhancer represents an attractive target for therapeutic genome engineering for the ß-hemoglobinopathies.