Uncovering interactions between exported Plasmodium falciparum and human erythrocyte cytoskeleton proteins in the process of host cell remodeling

The protozoan parasite Plasmodium falciparum causes the most severe form of human malaria, an infectious tropical disease of global public health importance. Despite efforts and means to prevent or treat this disease, there are still over 200 million cases and almost half a million deaths annually attributed to P. falciparum. Transmitted to the human host by female Anopheles mosquitos serving as vector, the parasite eventually invades erythrocytes and starts asexual replication. This stage causes the clinical symptoms of malaria. The red blood cell is an interesting choice of a host cell for the intracellular parasite P. falciparum as it lacks a nucleus, protein transport machinery, and its nutrient channels are inactive. To survive within this host environment, the parasite therefore has to remodel its host cell. The extensive host cell remodelling of human erythrocytes during the course of P. falciparum infection is facilitated by a large number of proteins which the parasite exports into its host cell cytoplasm. The function of the majority of these proteins remains elusive. Existing data suggests that some of these exported parasite proteins target the host cytoskeleton and modulate its properties, as apparent in changed mechanical properties of the host cell. The aim of this project was to identify interactions between host cytoskeleton and exported parasite proteins and to create a protein interaction network of the remodeled cytoskeleton. Identifying the key players and essential interactions in the process of host cell remodelling will lead to the identification of new targets in the fight against the malaria parasite. To this end, a number of exported proteins belonging to the PHIST family were selected. All selected PHIST proteins were exported into the host cell with most of them localizing in proximity to the erythrocyte cytoskeleton or membrane. The promiscuous biotin ligase BirA* (BioID) was fused to these proteins and upon addition of biotin proteins in the proximity were labelled with biotin. This allowed the pull-down using streptavidin-beads and identification of potential interaction partners of these transgenic, exported proteins by mass spectrometry. Based on the results from this study and additional data from previous projects, I generated a network of potential protein-protein interactions at the erythrocyte cytoskeleton. A standard approach to verify potential protein interactions is to perform reverse protein pull-downs. Because erythrocytes lack a nucleus, the classical transgenic approach to add molecular tags to erythrocyte proteins or to modify them in any way is not possible. To circumvent this holdback and to facilitate immunoprecipitations with erythrocyte proteins as bait, I generated parasite lines which expressed and exported different tagged human cytoskeleton proteins. These transgenic human proteins were designed to be exported and to be soluble within the cytosol of the infected erythrocyte. It was expected that these proteins would bind to their putative endogenous parasite binding partners while these are transported to their final destination within the host cell. These transgenic human proteins can then be used for immunoprecipitations to identify these binding partners. I tested several export sequences and showed that each of them resulted in efficient export of the intracellular loop of band 3 (residues 1-379) and the full-length band 4.1. In both of these cell lines, the majority of the protein was soluble in the host cytosol. Due to time constraints, these cell lines could not be further analyzed in detail. While little is known about the function and role of exported proteins in host cell remodeling during asexual developmental stages, even less is known about these proteins and their functions during gametocyte development of P. falciparum. Until recently, it was difficult to obtain high numbers of gametocytes, making it difficult to study host cell remodeling in these stages. The availability of a transgenic cell line from the Voss lab at Swiss TPH, in which high sexual conversion rates can be induced, provides a great opportunity to study these interactions in gametocytes. Taking advantage of this cell line we characterized GEXP02, a member of the PHIST protein family which is expressed and exported in gametocytes. We confirmed the expression pattern and localized GEXP02 at the periphery of the gametocyte-infected erythrocyte. By immuno-precipitation and mass spectrometry we could identify cytoskeleton proteins as well as other exported proteins as potential interaction partners. Based on co-labelling of GEXP02 with PFI1780w and PF3D7_0424600, two other PHIST proteins, we could confirm these as likely interaction partners. In GEXP02 knock-out parasites, no obvious detrimental effect or phenotype could be observed in asexual parasites or during gametocyte development nor throughout the mosquito stages or in liver hepatocyte infectivity. Although no function could be assigned to this protein, our study is one of the first to characterize in great detail an exported protein in gametocytes and shows that the erythrocyte cytoskeleton is targeted by exported parasite proteins also during gametocyte development. Furthermore, within the context of this present study, I conducted two extensive literature reviews. In one review I collected information on the functionally elusive PHIST family in the genus Plasmodium. The review on the PHIST protein family presents an in-depth overview on this protein family. It acts as a reference work for quick, but detailed information on these proteins that are thought to be involved in cytoskeleton remodelling. The other review concerned protein-protein interactions involved in host cytoskeleton remodeling of P. falciparum. By combining pieces of existing information, new insights were gained in this review. I could show that each stage of the intraerythrocytic life cycle presents different challenges to the intracellular survival of the parasite. Consequently, P. falciparum remodels its host cell differently in the various stages to meet the specific needs. In summary, this thesis provides new insight into host cell remodeling by the malaria parasite, shows the importance of exported proteins in this process, and offers a new tool in the study of interactions between erythrocyte cytoskeleton and exported parasite proteins

Multidimensional phenotyping of the post-COVID-19 syndrome: A Swiss survey study.

INTRODUCTION Post-COVID-19 syndrome affects approximately 10-25% of people after a COVID-19 infection, irrespective of initial COVID-19 severity. The aim of this project was to assess the clinical characteristics, course, and prognosis of post-COVID-19 syndrome using a systematic multidimensional approach. PATIENTS AND METHODS An online survey of people with suspected and confirmed COVID-19 and post-COVID-19 syndrome, distributed via Swiss COVID-19 support groups, social media, and our post-COVID-19 consultation, was performed. A total of 8 post-infectious domains were assessed with 120 questions. Data were collected from October 15 to December 12, 2021, and 309 participants were included. Analysis of clinical phenomenology of post-COVID-19 syndrome was performed using comparative statistics. RESULTS The three most prevalent post-COVID-19 symptoms in our survey cohort were fatigue (288/309, 93.2%), pain including headache (218/309, 70.6%), and sleep-wake disturbances (mainly insomnia and excessive daytime sleepiness, 145/309, 46.9%). Post-COVID-19 syndrome had an impact on work ability, as more than half of the respondents (168/268, 62.7%) reported an inability to work, which lasted on average 26.6 weeks (95% CI 23.5-29.6, range 1-94, n = 168). Quality of life measured by WHO-5 Well-being Index was overall low in respondents with post-COVID-19 syndrome (mean, 95% CI 9.1 [8.5-9.8], range 1-25, n = 239). CONCLUSION Fatigue, pain, and sleep-wake disturbances were the main symptoms of the post-COVID-19 syndrome in our cohort and had an impact on the quality of life and ability to work in a majority of patients. However, survey respondents reported a significant reduction in symptoms over 12 months. Post-COVID-19 syndrome remains a significant challenge. Further studies to characterize this syndrome and to explore therapeutic options are therefore urgently needed

U-Sleep's resilience to AASM guidelines

AASM guidelines are the result of decades of efforts aiming at standardizing sleep scoring procedure, with the final goal of sharing a worldwide common methodology. The guidelines cover several aspects from the technical/digital specifications,e.g., recommended EEG derivations, to detailed sleep scoring rules accordingly to age. Automated sleep scoring systems have always largely exploited the standards as fundamental guidelines. In this context, deep learning has demonstrated better performance compared to classical machine learning. Our present work shows that a deep learning based sleep scoring algorithm may not need to fully exploit the clinical knowledge or to strictly adhere to the AASM guidelines. Specifically, we demonstrate that U-Sleep, a state-of-the-art sleep scoring algorithm, can be strong enough to solve the scoring task even using clinically non-recommended or non-conventional derivations, and with no need to exploit information about the chronological age of the subjects. We finally strengthen a well-known finding that using data from multiple data centers always results in a better performing model compared with training on a single cohort. Indeed, we show that this latter statement is still valid even by increasing the size and the heterogeneity of the single data cohort. In all our experiments we used 28528 polysomnography studies from 13 different clinical studies

Fatigue in Post-COVID-19 Syndrome: Clinical Phenomenology, Comorbidities and Association With Initial Course of COVID-19.

Introduction Post-COVID-19 syndrome affects approximately 10-25% of people suffering from COVID-19 infection, irrespective of initial COVID-19 severity. Fatigue is one of the major symptoms, occurring in 30-90% of people with post-COVID-19 syndrome. This study aims at describing factors associated with fatigue in people with Post-COVID-19 seen in our newly established Post-Covid clinic. Methods This retrospective single center study included 42 consecutive patients suffering from Post-COVID-19 syndrome treated at the Department of Neurology, University Hospital Bern, between 11/2020 and05/2021. Clinical phenomenology of Post-COVID-19 syndrome with a special focus on fatigue and risk factor identification was performed using Mann-Whitney U Test, Pearson Correlation, and Chi-Quadrat-Test. Results Fatigue (90.5%) was the most prevalent Post-COVID-19 symptom followed by depressive mood (52.4%) and sleep disturbance (47.6%). Fatigue was in mean severe (Fatigue severity scale (FSS) mean 5.5 points (95% Confidence interval (95CI) 5.1 - 5.9, range .9 - 6.9, n = 40), and it was unrelated to age, COVID-19 severity or sex. The only related factors with fatigue severity were daytime sleepiness and depressed mood. Conclusion Fatigue is the main symptom of the Post-COVID-19 syndrome in our cohort. Further studies describing this syndrome are needed to prepare the healthcare systems for the challenge of treating patients with Post-COVID-19 syndrome

Memory CD8+ T Cells Balance Pro- and Anti-inflammatory Activity by Reprogramming Cellular Acetate Handling at Sites of Infection.

Serum acetate increases upon systemic infection. Acutely, assimilation of acetate expands the capacity of memory CD8+ T cells to produce IFN-γ. Whether acetate modulates memory CD8+ T cell metabolism and function during pathogen re-encounter remains unexplored. Here we show that at sites of infection, high acetate concentrations are being reached, yet memory CD8+ T cells shut down the acetate assimilating enzymes ACSS1 and ACSS2. Acetate, being thus largely excluded from incorporation into cellular metabolic pathways, now had different effects, namely (1) directly activating glutaminase, thereby augmenting glutaminolysis, cellular respiration, and survival, and (2) suppressing TCR-triggered calcium flux, and consequently cell activation and effector cell function. In vivo, high acetate abundance at sites of infection improved pathogen clearance while reducing immunopathology. This indicates that, during different stages of the immune response, the same metabolite-acetate-induces distinct immunometabolic programs within the same cell type

The Swiss Primary Hypersomnolence and Narcolepsy Cohort study (SPHYNCS): Study protocol for a prospective, multicentre cohort observational study.

Narcolepsy type 1 (NT1) is a disorder with well-established markers and a suspected autoimmune aetiology. Conversely, the narcoleptic borderland (NBL) disorders, including narcolepsy type 2, idiopathic hypersomnia, insufficient sleep syndrome and hypersomnia associated with a psychiatric disorder, lack well-defined markers and remain controversial in terms of aetiology, diagnosis and management. The Swiss Primary Hypersomnolence and Narcolepsy Cohort Study (SPHYNCS) is a comprehensive multicentre cohort study, which will investigate the clinical picture, pathophysiology and long-term course of NT1 and the NBL. The primary aim is to validate new and reappraise well-known markers for the characterization of the NBL, facilitating the diagnostic process. Seven Swiss sleep centres, belonging to the Swiss Narcolepsy Network (SNaNe), joined the study and will prospectively enrol over 500 patients with recent onset of excessive daytime sleepiness (EDS), hypersomnia or a suspected central disorder of hypersomnolence (CDH) during a 3-year recruitment phase. Healthy controls and patients with EDS due to severe sleep-disordered breathing, improving after therapy, will represent two control groups of over 50 patients each. Clinical and electrophysiological (polysomnography, multiple sleep latency test, maintenance of wakefulness test) information, and information on psychomotor vigilance and a sustained attention to response task, actigraphy and wearable devices (long-term monitoring), and responses to questionnaires will be collected at baseline and after 6, 12, 24 and 36 months. Potential disease markers will be searched for in blood, cerebrospinal fluid and stool. Analyses will include quantitative hypocretin measurements, proteomics/peptidomics, and immunological, genetic and microbiota studies. SPHYNCS will increase our understanding of CDH and the relationship between NT1 and the NBL. The identification of new disease markers is expected to lead to better and earlier diagnosis, better prognosis and personalized management of CDH

The Swiss Primary Hypersomnolence and Narcolepsy Cohort study (SPHYNCS): Study protocol for a prospective, multicentre cohort observational study

Narcolepsy type 1 (NT1) is a disorder with well-established markers and a suspected autoimmune aetiology. Conversely, the narcoleptic borderland (NBL) disorders, including narcolepsy type 2, idiopathic hypersomnia, insufficient sleep syndrome and hypersomnia associated with a psychiatric disorder, lack well-defined markers and remain controversial in terms of aetiology, diagnosis and management. The Swiss Primary Hypersomnolence and Narcolepsy Cohort Study (SPHYNCS) is a comprehensive multicentre cohort study, which will investigate the clinical picture, pathophysiology and long-term course of NT1 and the NBL. The primary aim is to validate new and reappraise well-known markers for the characterization of the NBL, facilitating the diagnostic process. Seven Swiss sleep centres, belonging to the Swiss Narcolepsy Network (SNaNe), joined the study and will prospectively enrol over 500 patients with recent onset of excessive daytime sleepiness (EDS), hypersomnia or a suspected central disorder of hypersomnolence (CDH) during a 3-year recruitment phase. Healthy controls and patients with EDS due to severe sleep-disordered breathing, improving after therapy, will represent two control groups of over 50 patients each. Clinical and electrophysiological (polysomnography, multiple sleep latency test, maintenance of wakefulness test) information, and information on psychomotor vigilance and a sustained attention to response task, actigraphy and wearable devices (long-term monitoring), and responses to questionnaires will be collected at baseline and after 6, 12, 24 and 36 months. Potential disease markers will be searched for in blood, cerebrospinal fluid and stool. Analyses will include quantitative hypocretin measurements, proteomics/peptidomics, and immunological, genetic and microbiota studies. SPHYNCS will increase our understanding of CDH and the relationship between NT1 and the NBL. The identification of new disease markers is expected to lead to better and earlier diagnosis, better prognosis and personalized management of CDH

Host cytoskeleton remodeling throughout the blood stages of Plasmodium falciparum

SUMMARYThe asexual intraerythrocytic development of; Plasmodium falciparum; , causing the most severe form of human malaria, is marked by extensive host cell remodeling. Throughout the processes of invasion, intracellular development, and egress, the erythrocyte membrane skeleton is remodeled by the parasite as required for each specific developmental stage. The remodeling is facilitated by a plethora of exported parasite proteins, and the erythrocyte membrane skeleton is the interface of most of the observed interactions between the parasite and host cell proteins. Host cell remodeling has been extensively described and there is a vast body of information on protein export or the description of parasite-induced structures such as Maurer's clefts or knobs on the host cell surface. Here we specifically review the molecular level of each host cell-remodeling step at each stage of the intraerythrocytic development of; P. falciparum; We describe key events, such as invasion, knob formation, and egress, and identify the interactions between exported parasite proteins and the host cell cytoskeleton. We discuss each remodeling step with respect to time and specific requirement of the developing parasite to explain host cell remodeling in a stage-specific manner. Thus, we highlight the interaction with the host membrane skeleton as a key event in parasite survival

Plasmodium Helical Interspersed Subtelomeric (PHIST) proteins, at the Center of Host Cell Remodeling

During the asexual cycle, Plasmodium falciparum extensively remodels the human erythrocyte to make it a suitable host cell. A large number of exported proteins facilitate this remodeling process, which causes erythrocytes to become more rigid, cytoadherent, and permeable for nutrients and metabolic products. Among the exported proteins, a family of 89 proteins, called the Plasmodium helical interspersed subtelomeric (PHIST) protein family, has been identified. While also found in other Plasmodium species, the PHIST family is greatly expanded in P. falciparum. Although a decade has passed since their first description, to date, most PHIST proteins remain uncharacterized and are of unknown function and localization within the host cell, and there are few data on their interactions with other host or parasite proteins. However, over the past few years, PHIST proteins have been mentioned in the literature at an increasing rate owing to their presence at various localizations within the infected erythrocyte. Expression of PHIST proteins has been implicated in molecular and cellular processes such as the surface display of PfEMP1, gametocytogenesis, changes in cell rigidity, and also cerebral and pregnancy-associated malaria. Thus, we conclude that PHIST proteins are central to host cell remodeling, but despite their obvious importance in pathology, PHIST proteins seem to be understudied. Here we review current knowledge, shed light on the definition of PHIST proteins, and discuss these proteins with respect to their localization and probable function. We take into consideration interaction studies, microarray analyses, or data from blood samples from naturally infected patients to combine all available information on this protein family

Plasmodium Helical Interspersed Subtelomeric (PHIST) proteins, at the Center of Host Cell Remodeling

During the asexual cycle, Plasmodium falciparum extensively remodels the human erythrocyte to make it a suitable host cell. A large number of exported proteins facilitate this remodeling process, which causes erythrocytes to become more rigid, cytoadherent, and permeable for nutrients and metabolic products. Among the exported proteins, a family of 89 proteins, called the Plasmodium helical interspersed subtelomeric (PHIST) protein family, has been identified. While also found in other Plasmodium species, the PHIST family is greatly expanded in P. falciparum. Although a decade has passed since their first description, to date, most PHIST proteins remain uncharacterized and are of unknown function and localization within the host cell, and there are few data on their interactions with other host or parasite proteins. However, over the past few years, PHIST proteins have been mentioned in the literature at an increasing rate owing to their presence at various localizations within the infected erythrocyte. Expression of PHIST proteins has been implicated in molecular and cellular processes such as the surface display of PfEMP1, gametocytogenesis, changes in cell rigidity, and also cerebral and pregnancy-associated malaria. Thus, we conclude that PHIST proteins are central to host cell remodeling, but despite their obvious importance in pathology, PHIST proteins seem to be understudied. Here we review current knowledge, shed light on the definition of PHIST proteins, and discuss these proteins with respect to their localization and probable function. We take into consideration interaction studies, microarray analyses, or data from blood samples from naturally infected patients to combine all available information on this protein family