Comprehensive study of proteasome inhibitors against Plasmodium falciparum laboratory strains and field isolates from Gabon

<p>Abstract</p> <p>Background</p> <p>The emergence and spread of <it>Plasmodium falciparum </it>resistance to almost all available antimalarial drugs necessitates the search for new chemotherapeutic compounds. The ubiquitin/proteasome system plays a major role in overall protein turnover, especially in fast dividing eukaryotic cells including plasmodia. Previous studies show that the 20S proteasome is expressed and catalytically active in plasmodia and treatment with proteasome inhibitors arrests parasite growth. This is the first comprehensive screening of proteasome inhibitors with different chemical modes of action against laboratory strains of <it>P. falciparum</it>. Subsequently, a selection of inhibitors was tested in field isolates from Lambaréné, Gabon.</p> <p>Methods</p> <p>Epoxomicin, YU101, YU102, MG132, MG115, Z-L₃-VS, Ada-Ahx₃-L₃-VS, lactacystin, bortezomib (Velcade^®), gliotoxin, PR11 and PR39 were tested and compared to chloroquine- and artesunate-activities in a standardized <it>in vitro </it>drug susceptibility assay against <it>P. falciparum </it>laboratory strains 3D7, D10 and Dd2. Freshly obtained field isolates from Lambaréné, Gabon, were used to measure the activity of chloroquine, artesunate, epoxomicin, MG132, lactacystin and bortezomib. Parasite growth was detected through histidine-rich protein 2 (HRP2) production. Raw data were fitted by a four-parameter logistic model and individual inhibitory concentrations (50%, 90%, and 99%) were calculated.</p> <p>Results</p> <p>Amongst all proteasome inhibitors tested, epoxomicin showed the highest activity in chloroquine-susceptible (IC50: 6.8 nM [3D7], 1.7 nM [D10]) and in chloroquine-resistant laboratory strains (IC50: 10.4 nM [Dd2]) as well as in field isolates (IC50: 8.5 nM). The comparator drug artesunate was even more active (IC50: 1.0 nM), whereas all strains were chloroquine-resistant (IC50: 113 nM).</p> <p>Conclusion</p> <p>The peptide α',β'-epoxyketone epoxomicin is highly active against <it>P. falciparum </it>regardless the grade of the parasite's chloroquine susceptibility. Therefore, inhibition of the proteasome is a highly promising strategy to develop new antimalarials. Epoxomicin can serve as a standard to compare new inhibitors with species-specific activity.</p

Highly tunable thiosulfonates as a novel class of cysteine protease inhibitors with anti-parasitic activity against Schistosoma mansoni

The development of a new class of cysteine protease inhibitors utilising the thiosulfonate moiety as an SH specific electrophile is described. This moiety has been introduced into suitable amino acid derived building blocks, which were incorporated into peptidic sequences leading to very potent i.e. sub micromolar inhibitors of the cysteine protease papain in the same range as the vinyl sulfone based inhibitor K11777. Therefore, their inhibitory effect on Schistosoma mansoni, a human blood parasite, that expresses several cysteine proteases, was evaluated. The homophenylalanine side chain containing compounds 27 - 30 and especially 36 showed promising activities compared with K11777 and warrant further investigations of these peptidic thiosulfonate inhibitors as new potential anti-parasitic compounds

Longitudinal Monitoring of Lactate in Hospitalized and Ambulatory COVID-19 Patients.

Hypoxemia is readily detectable by assessing SpO2 levels, and these are important in optimizing COVID-19 patient management. Hyperlactatemia is a marker of tissue hypoxia, particularly in patients with increased oxygen requirement and microvascular obstruction. We monitored peripheral venous lactate concentrations in hospitalized patients with moderate to severe COVID-19 (n = 18) and in mild ambulatory COVID-19 patients in home quarantine (n = 16). Whole blood lactate decreased significantly during the clinical course and recovery in hospitalized patients (P = 0.008). The blood lactate levels were significantly higher in hospitalized patients than ambulatory patients (day 1: hospitalized versus ambulatory patients P = 0.002; day 28: hospitalized versus ambulatory patients P = < 0.0001). Elevated lactate levels may be helpful in risk stratification, and serial monitoring of lactate may prove useful in the care of hospitalized COVID-19 patients

A review on innovative optical devices for the diagnosis of human soil-transmitted helminthiasis and schistosomiasis: from research and development to commercialization

Diagnosis of soil-transmitted helminth (STH) and schistosome infections relies largely on conventional microscopy which has limited sensitivity, requires highly trained personnel and is error-prone. Rapid advances in miniaturization of optical systems, sensors and proces-sors have enhanced research and development of digital and automated microscopes suitable for the detection of these diseases in resource-limited settings. While some studies have reported proof-of-principle results, others have evaluated the performance of working proto-types in field settings. The extensive commercialization of these innovative devices has, how-ever, not yet been achieved. This review provides an overview of recent publications (2010- 2022) on innovative field applicable optical devices which can be used for the diagnosis of STH and schistosome infections. Using an adapted technology readiness level (TRL) scale tak -ing into account the WHO target product profile (TPP) for these diseases, the developmental stages of the devices were ranked to determine the readiness for practical applications in field settings. From the reviewed 18 articles, 19 innovative optical devices were identified and ranked. Almost all of the devices (85%) were ranked with a TRL score below 8 indicating that, most of the devices are not ready for commercialization and field use. The potential lim-itations of these innovative devices were discussed. We believe that the outcome of this review can guide the end-to-end development of automated digital microscopes aligned with the WHO TPP for the diagnosis of STH and schistosome infections in resource-limited settings.Host-parasite interactio

Calmodulin-like proteins localized to the conoid regulate motility and cell invasion by Toxoplasma gondii

Toxoplasma gondii contains an expanded number of calmodulin (CaM)-like proteins whose functions are poorly understood. Using a combination of CRISPR/Cas9-mediated gene editing and a plant-like auxin-induced degron (AID) system, we examined the roles of three apically localized CaMs. CaM1 and CaM2 were individually dispensable, but loss of both resulted in a synthetic lethal phenotype. CaM3 was refractory to deletion, suggesting it is essential. Consistent with this prediction auxin-induced degradation of CaM3 blocked growth. Phenotypic analysis revealed that all three CaMs contribute to parasite motility, invasion, and egress from host cells, and that they act downstream of microneme and rhoptry secretion. Super-resolution microscopy localized all three CaMs to the conoid where they overlap with myosin H (MyoH), a motor protein that is required for invasion. Biotinylation using BirA fusions with the CaMs labeled a number of apical proteins including MyoH and its light chain MLC7, suggesting they may interact. Consistent with this hypothesis, disruption of MyoH led to degradation of CaM3, or redistribution of CaM1 and CaM2. Collectively, our findings suggest these CaMs may interact with MyoH to control motility and cell invasion

Hemolysis Is Associated with Low Reticulocyte Production Index and Predicts Blood Transfusion in Severe Malarial Anemia

Background: Falciparum Malaria, an infectious disease caused by the apicomplexan parasite Plasmodium falciparum, is among the leading causes of death and morbidity attributable to infectious diseases worldwide. In Gabon, Central Africa, one out of four inpatients have severe malarial anemia (SMA), a life-threatening complication if left untreated. Emerging drug resistant parasites might aggravate the situation. This case control study investigates biomarkers of enhanced hemolysis in hospitalized children with either SMA or mild malaria (MM). Methods and Findings: Ninety-one children were included, thereof 39 SMA patients. Strict inclusion criteria were chosen to exclude other causes of anemia. At diagnosis, erythrophagocytosis (a direct marker for extravascular hemolysis, EVH) was enhanced in SMA compared to MM patients (5.0 arbitrary units (AU) (interquartile range (IR): 2.2–9.6) vs. 2.1 AU (IR: 1.3–3.9), p<0.01). Furthermore, indirect markers for EVH, (i.e. serum neopterin levels, spleen size enlargement and monocyte pigment) were significantly increased in SMA patients. Markers for erythrocyte ageing, such as CD35 (complement receptor 1), CD55 (decay acceleration factor) and phosphatidylserine exposure (annexin-V-binding) were investigated by flow cytometry. In SMA patients, levels of CD35 and CD55 on the red blood cell surface were decreased and erythrocyte removal markers were increased when compared to MM or reconvalescent patients. Additionally, intravascular hemolysis (IVH) was quantified using several indirect markers (LDH, alpha-HBDH, haptoglobin and hemopexin), which all showed elevated IVH in SMA. The presence of both IVH and EVH predicted the need for blood transfusion during antimalarial treatment (odds ratio 61.5, 95% confidence interval (CI): 8.9–427). Interestingly, this subpopulation is characterized by a significantly lowered reticulocyte production index (RPI, p<0.05). Conclusions: Our results show the multifactorial pathophysiology of SMA, whereby EVH and IVH play a particularly important role. We propose a model where removal of infected and non-infected erythrocytes of all ages (including reticulocytes) by EVH and IVH is a main mechanism of SMA. Further studies are underway to investigate the mechanism and extent of reticulocyte removal to identify possible interventions to reduce the risk of SMA development

A call to caution when hydroxychloroquine is given to elderly COVID-19 patients.

INTRODUCTION: Hydroxychloroquine use in COVID-19 patients was widespread and uncontrolled until recently. Patients vulnerable to severe COVID-19 are at risk for hydroxychloroquine interactions with comorbidities and co-medications contributing to detrimental, including fatal adverse treatment effects. METHODS: This is a retrospective survey of health conditions and co-medications of COVID-19 patients who were pre-screened for enrolment into a randomized, double-blind, placebo-controlled hydroxychloroquine multicenter trial. RESULTS: Our survey involved 305 patients (median age 71 (IQR: 59-81) years). The majority of patients (N = 279, 92%) considered for inclusion into the clinical trial were not eligible mainly due to safety concerns caused by health conditions or co-medications. Most common were QT prolonging drugs (N = 188, 62%) and hematologic/hemato-oncologic diseases (N = 39, 13%) which prohibited the administration of hydroxychloroquine in our clinical trial. Additionally, 165 (54%) patients had health conditions and 167 (55%) were on co-medications that did not prohibit hydroxychloroquine treatment but had a risk of adverse interactions with hydroxychloroquine. Most common were diabetes (N = 86, 28%), renal insufficiency (N = 69, 23%) and heart failure (N = 58, 19%). CONCLUSION: The majority of hospitalized COVID-19 patients had health conditions or took co-medications precluding safe treatment with hydroxychloroquine. Therefore, especially in elderly, hydroxychloroquine should be administered with extreme caution and only in clinical trials

Modelování volatility devizových kurzů

Import 22/07/2015The aim of this Diploma thesis is modelling and in-sample forecasting volatility of selected exchange rates using linear and nonlinear conditional heteroskedasticity models. The main aim of the thesis is supported by two partial aims. The first partial aim is to compare whether linear or nonlinear models are more efficient for modelling conditional heteroskedasticity for exchange rates. The second partial aim is to assess the suitability of estimated models to predict volatility. Tested data are time series of daily exchange rates modified to time series of daily logarithmic returns of Slovenian tolar, Cyprus pound, Slovakian koruna and Latvian lat against Euro. Observed period is divided into three parts in order to model volatility of all exchange rates in different relations of domestic countries to European Union. Thesis is divided into six parts including Introduction and Conclusion. Second and third chapter is theoretical and methodological, while the fourth and fifth chapter is practical and empirical.Cílem Diplomové práce je modelování volatility a ve výběru provedená predikce vybraných měnových kurzů za využití lineárních a nelineárních modelů podmíněné heteroskedasticity. Hlavní cíl práce je dále rozdělen na dva dílčí cíle. První z dílčích cílů se věnuje otázce, zda jsou k modelování měnových kurzů podmíněné heteroskedasticity vhodnější lineární nebo nelineární modely. Druhý dílčí cíl zohledňuje, zda jsou odhadnuté modely vhodné k predikování volatility. Testovaná data jsou časové řady denních měnových kurzů upravené na časové řady denních logaritmovaných výnosů Slovinského tolaru, Kyperské libry, Slovenské koruny a Lotyšského latu vůči Euru. Sledovaná časová perioda je rozdělena na tři části za účelem modelovat volatilitu všech měnových kurzů při různém vztahu domácí země vůči Evropské Unii. Práce je rozdělena na šest částí včetně úvodu a závěru. Druhá a třetí kapitola jsou teoretické a metodologické, zatímco čtvrtá a pátá kapitola jsou praktické a empirické.154 - Katedra financívýborn

A cluster randomized controlled trial for assessing POC-CCA test based praziquantel treatment for schistosomiasis control in pregnant women and their young children: study protocol of the freeBILy clinical trial in Madagascar.

BACKGROUND: Mass drug administration (MDA) of praziquantel is one of the main control measures against human schistosomiasis. Although there are claims for including pregnant women, infants and children under the age of 5 years in high-endemic regions in MDA campaigns, they are usually not treated without a diagnosis. Diagnostic tools identifying infections at the primary health care centre (PHCC) level could therefore help to integrate these vulnerable groups into control programmes. freeBILy (fast and reliable easy-to-use-diagnostics for eliminating bilharzia in young children and mothers) is an international consortium focused on implementing and evaluating new schistosomiasis diagnostic strategies. In Madagascar, the study aims to determine the effectiveness of a test-based schistosomiasis treatment (TBST) strategy for pregnant women and their infants and children up until the age of 2 years. METHODS: A two-armed, cluster-randomized, controlled phase III trial including 5200 women and their offspring assesses the impact of TBST on child growth and maternal haemoglobin in areas of medium to high endemicity of Schistosoma mansoni. The participants are being tested with the point of care-circulating cathodic antigen (POC-CCA) test, a commercially available urine-based non-invasive rapid diagnostic test for schistosomiasis. In the intervention arm, a POC-CCA-TBST strategy is offered to women during pregnancy and 9 months after delivery, for their infants at 9 months of age. In the control arm, study visit procedures are the same, but without the POC-CCA-TBST procedure. All participants are being offered the POC-CCA-TBST 24 months after delivery. This trial is being integrated into the routine maternal and child primary health care programmes at 40 different PHCC in Madagascar's highlands. The purpose of the trial is to assess the effectiveness of the POC-CCA-TBST for controlling schistosomiasis in young children and mothers. DISCUSSION: This trial assesses a strategy to integrate pregnant women and their children under the age of 2 years into schistosomiasis control programmes using rapid diagnostic tests. It includes local capacity building for clinical trials and large-scale intervention research. TRIAL REGISTRATION: Pan-African Clinical Trial Register PACTR201905784271304. Retrospectively registered on 15 May 2019