Sequence elements within the PEXEL motif and its downstream region modulate PTEX-dependent protein export in Plasmodium falciparum.

The parasite Plasmodium falciparum causes the most severe form of malaria and to invade and replicate in red blood cells (RBCs), it exports hundreds of proteins across the encasing parasitophorous vacuole membrane (PVM) into this host cell. The exported proteins help modify the RBC to support rapid parasite growth and avoidance of the human immune system. Most exported proteins possess a conserved Plasmodium export element (PEXEL) motif with the consensus RxLxE/D/Q amino acid sequence, which acts as a proteolytic cleavage recognition site within the parasite's endoplasmic reticulum (ER). Cleavage occurs after the P1 L residue and is thought to help release the protein from the ER so it can be putatively escorted by the HSP101 chaperone to the parasitophorous vacuole space surrounding the intraerythrocytic parasite. HSP101 and its cargo are then thought to assemble with the rest of a Plasmodium translocon for exported proteins (PTEX) complex, that then recognises the xE/D/Q capped N-terminus of the exported protein and translocates it across the vacuole membrane into the RBC compartment. Here, we present evidence that supports a dual role for the PEXEL's conserved P2 ' position E/Q/D residue, first, for plasmepsin V cleavage in the ER, and second, for efficient PTEX mediated export across the PVM into the RBC. We also present evidence that the downstream 'spacer' region separating the PEXEL motif from the folded functional region of the exported protein controls cargo interaction with PTEX as well. The spacer must be of a sufficient length and permissive amino acid composition to engage the HSP101 unfoldase component of PTEX to be efficiently translocated into the RBC compartment.

Sulfonylpiperazine compounds prevent Plasmodium falciparum invasion of red blood cells through interference with actin-1/profilin dynamics.

With emerging resistance to frontline treatments, it is vital that new antimalarial drugs are identified to target Plasmodium falciparum. We have recently described a compound, MMV020291, as a specific inhibitor of red blood cell (RBC) invasion, and have generated analogues with improved potency. Here, we generated resistance to MMV020291 and performed whole genome sequencing of 3 MMV020291-resistant populations. This revealed 3 nonsynonymous single nucleotide polymorphisms in 2 genes; 2 in profilin (N154Y, K124N) and a third one in actin-1 (M356L). Using CRISPR-Cas9, we engineered these mutations into wild-type parasites, which rendered them resistant to MMV020291. We demonstrate that MMV020291 reduces actin polymerisation that is required by the merozoite stage parasites to invade RBCs. Additionally, the series inhibits the actin-1-dependent process of apicoplast segregation, leading to a delayed death phenotype. In vitro cosedimentation experiments using recombinant P. falciparum proteins indicate that potent MMV020291 analogues disrupt the formation of filamentous actin in the presence of profilin. Altogether, this study identifies the first compound series interfering with the actin-1/profilin interaction in P. falciparum and paves the way for future antimalarial development against the highly dynamic process of actin polymerisation.

PTEX helps efficiently traffic haemoglobinases to the food vacuole in Plasmodium falciparum.

A key element of Plasmodium biology and pathogenesis is the trafficking of ~10% of the parasite proteome into the host red blood cell (RBC) it infects. To cross the parasite-encasing parasitophorous vacuole membrane, exported proteins utilise a channel-forming protein complex termed the Plasmodium translocon of exported proteins (PTEX). PTEX is obligatory for parasite survival, both in vitro and in vivo, suggesting that at least some exported proteins have essential metabolic functions. However, to date only one essential PTEX-dependent process, the new permeability pathways, has been described. To identify other essential PTEX-dependant proteins/processes, we conditionally knocked down the expression of one of its core components, PTEX150, and examined which pathways were affected. Surprisingly, the food vacuole mediated process of haemoglobin (Hb) digestion was substantially perturbed by PTEX150 knockdown. Using a range of transgenic parasite lines and approaches, we show that two major Hb proteases; falcipain 2a and plasmepsin II, interact with PTEX core components, implicating the translocon in the trafficking of Hb proteases. We propose a model where these proteases are translocated into the PV via PTEX in order to reach the cytostome, located at the parasite periphery, prior to food vacuole entry. This work offers a second mechanistic explanation for why PTEX function is essential for growth of the parasite within its host RBC.

The Plasmodium falciparum parasitophorous vacuole protein P113 interacts with the parasite protein export machinery and maintains normal vacuole architecture.

Infection with Plasmodium falciparum parasites results in approximately 627,000 deaths from malaria annually. Key to the parasite's success is their ability to invade and subsequently grow within human erythrocytes. Parasite proteins involved in parasite invasion and proliferation are therefore intrinsically of great interest, as targeting these proteins could provide novel means of therapeutic intervention. One such protein is P113 which has been reported to be both an invasion protein and an intracellular protein located within the parasitophorous vacuole (PV). The PV is delimited by a membrane (PVM) across which a plethora of parasite-specific proteins are exported via the Plasmodium Translocon of Exported proteins (PTEX) into the erythrocyte to enact various immune evasion functions. To better understand the role of P113 we isolated its binding partners from in vitro cultures of P. falciparum. We detected interactions with the protein export machinery (PTEX and exported protein-interacting complex) and a variety of proteins that either transit through the PV or reside on the parasite plasma membrane. Genetic knockdown or partial deletion of P113 did not significantly reduce parasite growth or protein export but did disrupt the morphology of the PVM, suggesting that P113 may play a role in maintaining normal PVM architecture.

Property activity refinement of 2-anilino 4-amino substituted quinazolines as antimalarials with fast acting asexual parasite activity.

Malaria is a devastating disease caused by Plasmodium parasites. Emerging resistance against current antimalarial therapeutics has engendered the need to develop antimalarials with novel structural classes. We recently described the identification and initial optimization of the 2-anilino quinazoline antimalarial class. Here, we refine the physicochemical properties of this antimalarial class with the aim to improve aqueous solubility and metabolism and to reduce adverse promiscuity. We show the physicochemical properties of this class are intricately balanced with asexual parasite activity and human cell cytotoxicity. Structural modifications we have implemented improved LipE, aqueous solubility and in vitro metabolism while preserving fast acting P. falciparum asexual stage activity. The lead compounds demonstrated equipotent activity against P. knowlesi parasites and were not predisposed to resistance mechanisms of clinically used antimalarials. The optimized compounds exhibited modest activity against early-stage gametocytes, but no activity against pre-erythrocytic liver parasites. Confoundingly, the refined physicochemical properties installed in the compounds did not engender improved oral efficacy in a P. berghei mouse model of malaria compared to earlier studies on the 2-anilino quinazoline class. This study provides the framework for further development of this antimalarial class.

Disrupting assembly of the inner membrane complex blocks Plasmodium falciparum sexual stage development.

Transmission of malaria parasites relies on the formation of a specialized blood form called the gametocyte. Gametocytes of the human pathogen, Plasmodium falciparum, adopt a crescent shape. Their dramatic morphogenesis is driven by the assembly of a network of microtubules and an underpinning inner membrane complex (IMC). Using super-resolution optical and electron microscopies we define the ultrastructure of the IMC at different stages of gametocyte development. We characterize two new proteins of the gametocyte IMC, called PhIL1 and PIP1. Genetic disruption of PhIL1 or PIP1 ablates elongation and prevents formation of transmission-ready mature gametocytes. The maturation defect is accompanied by failure to form an enveloping IMC and a marked swelling of the digestive vacuole, suggesting PhIL1 and PIP1 are required for correct membrane trafficking. Using immunoprecipitation and mass spectrometry we reveal that PhIL1 interacts with known and new components of the gametocyte IMC.

Similar dynamics of intraapheresis autologous CD34+ recruitment and collection efficiency in patients undergoing mobilization with or without plerixafor.

BACKGROUND: Compared with growth factor (G) alone, the combination of G with plerixafor (G + P) increases peripheral blood CD34+ count (PB-CD34+) and improves CD34+ collection yield (yCD34+) in multiple myeloma and lymphoma patients undergoing autologous hematopoietic progenitor cell (AHPC) mobilization. It is unknown whether the improved yCD34+ with G + P results entirely from expansion of PB-CD34+ or also from increased intraapheresis CD34+ recruitment and collection efficiency. STUDY DESIGN AND METHODS: We retrospectively studied 192 patients who underwent AHPC mobilization and collection with G (n = 73) or G + P (n = 119) to compare the adjusted relative efficiency (aRE), the proportion of the circulating CD34+ pool that is captured for each blood volume processed. Additionally, in a prospective cohort of nine patients mobilizing with G and 11 with G + P, PB-CD34+ after leukapheresis allowed calculation of the recruitment coefficient (RC), proportion of the initial CD34+ pool recruited from the marrow into peripheral blood for each blood volume processed. RESULTS: There was no difference in aRE between G and G + P (0.50 vs. 0.46; p = 0.37) and no substantial decline in aRE with higher blood volumes processed in either group. RC was also not different between G and G + P (median, 0.39 and 0.38, respectively; p = 0.7). Prediction of yCD34+ was determined essentially by PB-CD34+ and not affected independently by plerixafor. CONCLUSION: Kinetics of intraapheresis CD34+ recruitment and collection is proportional to PB-CD34+ but not influenced further by plerixafor.

The delayed bloodstream clearance of Plasmodium falciparum parasites after M5717 treatment is attributable to the inability to modify their red blood cell hosts.

M5717 is a promising antimalarial drug under development that acts against multiple stages of the life cycle of Plasmodium parasites by inhibiting the translation elongation factor 2 (PfeEF2), thereby preventing protein synthesis. The parasite clearance profile after drug treatment in preclinical studies in mice, and clinical trials in humans showed a notable delayed clearance phenotype whereby parasite infected red blood cells (iRBCs) persisted in the bloodstream for a significant period before eventual clearance. In a normal P. falciparum infection iRBCs sequester in the deep circulation by cytoadherence, allowing them to avoid surveillance and clearance in the spleen. We found that M5717 blocks parasite modification of their host red blood cells (RBCs) by preventing synthesis of new exported proteins, rather than by directly blocking the export of these proteins into the RBC compartment. Using in vitro models, we demonstrated that M5717 treated ring/trophozoite stage iRBCs became less rigid, and cytoadhered less well compared to untreated iRBCs. This indicates that in vivo persistence of M5717 treated iRBCs in the bloodstream is likely due to reduced cytoadherence and splenic clearance.

Stress-related reduction of hippocampal subfield volumes in major depressive disorder: A 7-Tesla study.

Background: Major depressive disorder (MDD) is a prevalent health problem with complex pathophysiology that is not clearly understood. Prior work has implicated the hippocampus in MDD, but how hippocampal subfields influence or are affected by MDD requires further characterization with high-resolution data. This will help ascertain the accuracy and reproducibility of previous subfield findings in depression as well as correlate subfield volumes with MDD symptom scores. The objective of this study was to assess volumetric differences in hippocampal subfields between MDD patients globally and healthy controls (HC) as well as between a subset of treatment-resistant depression (TRD) patients and HC using automatic segmentation of hippocampal subfields (ASHS) software and ultra-high field MRI. Methods: Thirty-five MDD patients and 28 HC underwent imaging using 7-Tesla MRI. ASHS software was applied to the imaging data to perform automated hippocampal segmentation and provide volumetrics for analysis. An exploratory analysis was also performed on associations between symptom scores for diagnostic testing and hippocampal subfield volumes. Results: Compared to HC, MDD and TRD patients showed reduced right-hemisphere CA2/3 subfield volume (p = 0.01, η 2 = 0.31 and p = 0.3, η 2 = 0.44, respectively). Additionally, negative associations were found between subfield volumes and life-stressor checklist scores, including left CA1 (p = 0.041, f 2 = 0.419), left CA4/DG (p = 0.010, f 2 = 0.584), right subiculum total (p = 0.038, f 2 = 0.354), left hippocampus total (p = 0.015, f 2 = 0.134), and right hippocampus total (p = 0.034, f 2 = 0.110). Caution should be exercised in interpreting these results due to the small sample size and low power. Conclusion: Determining biomarkers for MDD and TRD pathophysiology through segmentation on high-resolution MRI data and understanding the effects of stress on these regions can enable better assessment of biological response to treatment selection and may elucidate the underlying mechanisms of depression.

Whole blood transcriptional signatures associated with rapid antidepressant response to ketamine in patients with treatment resistant depression.

Ketamine has rapid and sustained antidepressant effects in patients with treatment-resistant depression (TRD). However, the underlying mechanisms of action are not well understood. There is increasing evidence that TRD is associated with a pro-inflammatory state and that ketamine may inhibit inflammatory processes. We thus investigated whole blood transcriptional profiles related to TRD and gene expression changes associated with treatment response to ketamine. Whole blood was collected at baseline (21 healthy controls [HC], 26 patients with TRD) and then again in patients with TRD 24 hours following a single intravenous infusion of ketamine (0.5 mg/kg). We performed RNA-sequencing and analyzed (a) baseline transcriptional profiles between patients with TRD and HC, (b) responders vs. non-responders before ketamine treatment, and (c) gene expression signatures associated with clinical improvement. At baseline, patients with TRD compared to HC showed a gene expression signature indicative of interferon signaling pathway activation. Prior to ketamine administration, the metabotropic glutamate receptor gene GRM2 and the ionotropic glutamate receptor gene GRIN2D were upregulated in responders compared to non-responders. Response to ketamine was associated with a distinct transcriptional signature, however, we did not observe gene expression changes indicative of an anti-inflammatory effect. Future studies are needed to determine the role of the peripheral immune system in the antidepressant effect of ketamine.

Nuclear bodies formed by polyQ-ataxin-1 protein are liquid RNA/protein droplets with tunable dynamics.

A mutant form of the ataxin-1 protein with an expanded polyglutamine (polyQ) tract is the underlying cause of the inherited neurodegenerative disease spinocerebellar ataxia 1 (SCA1). In probing the biophysical features of the nuclear bodies (NBs) formed by polyQ-ataxin-1, we defined ataxin-1 NBs as spherical liquid protein/RNA droplets capable of rapid fusion. We observed dynamic exchange of the ataxin-1 protein into these NBs; notably, cell exposure to a pro-oxidant stress could trigger a transition to slower ataxin-1 exchange, typical of a hydrogel state, which no longer showed the same dependence on RNA or sensitivity to 1,6-hexanediol. Furthermore, we could alter ataxin-1 exchange dynamics either through modulating intracellular ATP levels, RNA helicase inhibition, or siRNA-mediated depletion of select RNA helicases. Collectively, these findings reveal the tunable dynamics of the liquid RNA/protein droplets formed by polyQ-ataxin-1.

Neural correlates of rumination in major depressive disorder: A brain network analysis.

Patients with major depressive disorder (MDD) exhibit higher levels of rumination, i.e., repetitive thinking patterns and exaggerated focus on negative states. Rumination is known to be associated with the cortical midline structures / default mode network (DMN) region activity, although the brain network topological organization underlying rumination remains unclear. Implementing a graph theoretical analysis based on ultra-high field 7-Tesla functional MRI data, we tested whether whole brain network connectivity hierarchies during resting state are associated with rumination in a dimensional manner across 20 patients with MDD and 20 healthy controls. Applying this data-driven approach we found a significant correlation between rumination tendency and connectivity strength degree of the right precuneus, a key node of the DMN. In order to interrogate this region further, we then applied the Dependency Network Analysis (DEPNA), a recently developed method used to quantify the connectivity influence of network nodes. This revealed that rumination was associated with lower connectivity influence of the left medial orbito-frontal cortex (MOFC) cortex on the right precuneus. Lastly, we used an information theory entropy measure that quantifies the cohesion of a network's correlation matrix. We show that subjects with higher rumination scores exhibit higher entropy levels within the DMN i.e. decreased overall connectivity within the DMN. These results emphasize the general DMN involvement during self-reflective processing related to maladaptive rumination in MDD. This work specifically highlights the impact of the MOFC on the precuneus, which might serve as a target for clinical neuromodulation treatment.

Ultra-High-Resolution Imaging of Amygdala Subnuclei Structural Connectivity in Major Depressive Disorder.

BACKGROUND: Major depressive disorder (MDD) is an increasingly common and disabling illness. As the amygdala has been reported to have pathological involvement in mood disorders, we aimed to investigate for the first time potential changes to structural connectivity of individual amygdala subnuclei in MDD using ultra-high-field 7T diffusion magnetic resonance imaging. METHODS: Twenty-four patients with MDD (11 women) and 24 age-matched healthy control participants (7 women) underwent diffusion-weighted imaging with a 1.05-mm isotropic resolution at 7T. Amygdala nuclei regions of interest were obtained through automated segmentation of 0.69-mm resolution T1-weighted images and 0.35-mm resolution T2-weighted images. Probabilistic tractography was performed on all subjects, with random seeding at each amygdala nucleus. RESULTS: The right lateral, basal, central, and centrocortical amygdala nuclei exhibited significantly increased connection density to the rest of the brain, whereas the left medial nucleus demonstrated significantly lower connection density (false discovery rate p < .05). Increased connection density in the right lateral and basal nuclei was driven by the stria terminalis, and the significant difference in the right central nucleus was driven by the uncinate fasciculus. Decreased connection density at the left medial nucleus did not appear to be driven by any individual white matter tract. CONCLUSIONS: By exploiting ultra-high-resolution magnetic resonance imaging, structural hyperconnectivity was demonstrated involving the amygdaloid nuclei in the right hemisphere in MDD. To a lesser extent, impairment of subnuclei connectivity was shown in the left hemisphere.

Ultra-high field MRI reveals mood-related circuit disturbances in depression: a comparison between 3-Tesla and 7-Tesla.

Ultra-high field 7-Tesla (7 T) MRI has the potential to advance our understanding of neuropsychiatric disorders, including major depressive disorder (MDD). To date, few studies have quantified the advantage of resting state functional MRI (fMRI) at 7 T compared to 3-Tesla (3 T). We conducted a series of experiments that demonstrate the improvement in temporal signal-to-noise ratio (TSNR) of a multi-echo multi-band fMRI protocol with ultra-high field 7 T MRI, compared to a similar protocol using 3 T MRI in healthy controls (HC). We also directly tested the enhancement in ultra-high field 7 T fMRI signal power by examining the ventral tegmental area (VTA), a small midbrain structure that is critical to the expected neuropathology of MDD but difficult to discern with standard 3 T MRI. We demonstrate up to 300% improvement in TSNR and resting state functional connectivity coefficients provided by ultra-high field 7 T fMRI compared to 3 T, indicating enhanced power for detection of functional neural architecture. A multi-echo based acquisition protocol and signal denoising pipeline afforded greater gain in signal power compared to classic acquisition and denoising pipelines. Furthermore, ultra-high field fMRI revealed mood-related neurocircuit disturbances in patients with MDD compared to HC, which were not detectable with 3 T fMRI. Ultra-high field 7 T fMRI may provide an effective tool for studying functional neural architecture relevant to MDD and other neuropsychiatric disorders.

Electrochemical measurement of membrane cholesterol correlates with CFTR function and is HDAC6-dependent.

BACKGROUND: Previous studies have demonstrated that CF epithelial cells exhibit increased cholesterol content at the plasma membrane compared to wild type controls as measured by electrochemical methods. Microtubule dysregulation that impacts intracellular transport has also been identified in CF cells and is reversible with histone deacetylase 6 (HDAC6) inhibition, a regulator of tubulin acetylation. The hypothesis of this study is that increased membrane cholesterol content in CF cells is dependent on HDAC6 regulation. METHODS: Electrochemical measurement of membrane cholesterol in mouse trachea and in primary human CF bronchial epithelial cells is used to monitor CFTR correction and manipulation of cholesterol processing by HDAC6 inhibition. RESULTS: Data demonstrate that induction of Cftr expression in an inducible CF mouse model restores tubulin acetylation levels and normalizes membrane cholesterol content. To test the relationship between tubulin acetylation, membrane cholesterol levels were measured in a CF mouse model depleted of Hdac6 expression (CF/HDA). CF/HDA mouse trachea have WT membrane cholesterol levels while CF mice have approximately two-fold increase in membrane cholesterol compared to WT consistent with previous studies. Pharmacological inhibition of HDAC6 in primary human CF bronchial epithelial cells also reduces membrane cholesterol levels. CONCLUSIONS: This study demonstrates that elevated membrane cholesterol in CF epithelium is regulated by HDAC6 function and that the electrochemical measure of membrane cholesterol correlates with both genetic and pharmacological CFTR correction.

Hippocampal subfield-specific connectivity findings in major depressive disorder: A 7 Tesla diffusion MRI study.

OBJECTIVE: Diffusion magnetic resonance imaging (dMRI) enables non-invasive characterization of white matter (WM) structures in vivo. Prior studies suggest that certain WM tracts may be affected in major depressive disorder (MDD), however, hippocampal subfield-specific dMRI measures have not yet been explored in MDD. We use 7 Tesla dMRI to investigate differences in hippocampal subfield connectivity of MDD patients. METHODS: Eighteen MDD patients and eighteen matched healthy volunteers underwent 7 Tesla MRI. The hippocampal formations were segmented by subfields and tractography was performed to determine streamline count (SC), fractional anisotropy (FA), and mean diffusivity (MD) in patients and controls. Significant subfield connectivity measures were also correlated with age at depression onset. RESULTS: Compared with controls, MDD patients exhibited reduced SC in the molecular layer of the left dentate gyrus (p < 0.001). SC count in the left dentate gyrus was shown to positively correlate with age at disease onset (p < 0.05). Increased MD was observed in streamlines emanating from both the left (p = 0.0001) and right (p < 0.001) fimbriae in MDD patients. CONCLUSIONS: Increased MD of tracts in the fimbriae suggests compromised neuronal membranes in the major hippocampal output gate. Reduced SC of the dentate gyri indexes a disruption of normal cellular processes such as neurogenesis. These findings may have significant implications for identifying biomarkers of MDD and elucidating the neurobiological underpinnings of depression.

Improved Adherence Rates and Clinical Outcomes of an Integrated, Closed-Loop, Pharmacist-Led Oral Chemotherapy Management Program.

PURPOSE: To address the growing use of oral anticancer therapy, an integrated, closed-loop, pharmacist-led oral chemotherapy management program was created within an academic medical center. METHODS: An integrated, closed-loop, pharmacy-led oral chemotherapy management program was established. From September 2014 until June 2015, demographic information, rates of adherence, patient understanding of treatment, pharmacist interventions, patient and provider satisfaction, and molecular response rates in patients with chronic myeloid leukemia (CML) were collected. RESULTS: After full implementation, 107 patients were enrolled in our oral chemotherapy management program from September 2014 until June 2015. All patients were educated before starting oral chemotherapy, and using pre- and postassessment tests, comprehension of oral chemotherapy treatment increased from 43% to 95%. Patient-reported adherence was 86% and 94.7% for the GI/breast and malignant hematology patient populations, respectively, and these were validated with medication possession ratio, revealing adherence rates of 85% and 93.9% for the GI/breast and malignant hematology patient populations, respectively. A total of 350 encounters with a clinical pharmacist and 318 adverse effects were reported, which led to 235 interventions. This program led to a higher major molecular response rate (83%) in our CML population compared with published clinical trials (average major molecular response rates, 40% and 60% with 1- and 2-year follow-up, respectively). CONCLUSION: An innovative model was developed and resulted in improved patient knowledge regarding oral chemotherapy, improved adherence rates that exceeded nationally established thresholds, and superior major molecular response outcomes for patients with CML compared with published literature. As a result, this model has produced the gold standard in managing patients receiving oral chemotherapy.

An exported protein-interacting complex involved in the trafficking of virulence determinants in Plasmodium-infected erythrocytes.

The malaria parasite, Plasmodium falciparum, displays the P. falciparum erythrocyte membrane protein 1 (PfEMP1) on the surface of infected red blood cells (RBCs). We here examine the physical organization of PfEMP1 trafficking intermediates in infected RBCs and determine interacting partners using an epitope-tagged minimal construct (PfEMP1B). We show that parasitophorous vacuole (PV)-located PfEMP1B interacts with components of the PTEX (Plasmodium Translocon of EXported proteins) as well as a novel protein complex, EPIC (Exported Protein-Interacting Complex). Within the RBC cytoplasm PfEMP1B interacts with components of the Maurer's clefts and the RBC chaperonin complex. We define the EPIC interactome and, using an inducible knockdown approach, show that depletion of one of its components, the parasitophorous vacuolar protein-1 (PV1), results in altered knob morphology, reduced cell rigidity and decreased binding to CD36. Accordingly, we show that deletion of the Plasmodium berghei homologue of PV1 is associated with attenuation of parasite virulence in vivo.