New Assays to Characterise Growth-Related Phenotypes of Plasmodium falciparum Reveal Variation in Density-Dependent Growth Inhibition between Parasite Lines

The growth phenotype of asexual blood stage malaria parasites can influence their virulence and also their ability to survive and achieve transmission to the next host, but there are few methods available to characterise parasite growth parameters in detail. We developed a new assay to measure growth rates at different starting parasitaemias in a 96-well format and applied it to characterise the growth of Plasmodium falciparum lines 3D7-A and 3D7-B, previously shown to have different invasion rates and to use different invasion pathways. Using this simple and accurate assay we found that 3D7-B is more sensitive to high initial parasitaemia than 3D7-A. This result indicates that different parasite lines show variation in their levels of density-dependent growth inhibition. We also developed a new assay to compare the duration of the asexual blood cycle between different parasite lines. The assay is based on the tight synchronisation of cultures to a 1 h parasite age window and the subsequent monitoring of schizont bursting and formation of new rings by flow cytometry. Using this assay we observed differences in the duration of the asexual blood cycle between parasite lines 3D7 and HB3. These two new assays will be useful to characterise variation in growth-related parameters and to identify growth phenotypes associated with the targeted deletion of specific genes or with particular genomic, transcriptomic or proteomic patterns. Furthermore, the identification of density-dependent growth inhibition as an intrinsic parasite property that varies between parasite lines expands the repertoire of measurable growth-related phenotypic traits that have the potential to influence the outcome of a malarial blood infection

Presence of Ceramidase Activity in Electronegative LDL

Electronegative low-density lipoprotein (LDL(−)) is a minor modified fraction of human plasma LDL with several atherogenic properties. Among them is increased bioactive lipid mediator content, such as lysophosphatidylcholine (LPC), non-esterified fatty acids (NEFA), ceramide (Cer), and sphingosine (Sph), which are related to the presence of some phospholipolytic activities, including platelet-activating factor acetylhydrolase (PAF-AH), phospholipase C (PLC), and sphingomyelinase (SMase), in LDL(−). However, these enzymes' activities do not explain the increased Sph content, which typically derives from Cer degradation. In the present study, we analyzed the putative presence of ceramidase (CDase) activity, which could explain the increased Sph content. Thin layer chromatography (TLC) and lipidomic analysis showed that Cer, Sph, and NEFA spontaneously increased in LDL(−) incubated alone at 37 °C, in contrast with native LDL(+). An inhibitor of neutral CDase prevented the formation of Sph and, in turn, increased Cer content in LDL(−). In addition, LDL(−) efficiently degraded fluorescently labeled Cer (NBD-Cer) to form Sph and NEFA. These observations defend the existence of the CDase-like activity's association with LDL(−). However, neither the proteomic analysis nor the Western blot detected the presence of an enzyme with known CDase activity. Further studies are thus warranted to define the origin of the CDase-like activity detected in LDL(−)

Presence of Ceramidase Activity in Electronegative LDL

Ceramide; SphingomyelinaseCeramida; EsfingomielinasaCeramida; EsfingomielinasaElectronegative low-density lipoprotein (LDL(−)) is a minor modified fraction of human plasma LDL with several atherogenic properties. Among them is increased bioactive lipid mediator content, such as lysophosphatidylcholine (LPC), non-esterified fatty acids (NEFA), ceramide (Cer), and sphingosine (Sph), which are related to the presence of some phospholipolytic activities, including platelet-activating factor acetylhydrolase (PAF-AH), phospholipase C (PLC), and sphingomyelinase (SMase), in LDL(−). However, these enzymes’ activities do not explain the increased Sph content, which typically derives from Cer degradation. In the present study, we analyzed the putative presence of ceramidase (CDase) activity, which could explain the increased Sph content. Thin layer chromatography (TLC) and lipidomic analysis showed that Cer, Sph, and NEFA spontaneously increased in LDL(−) incubated alone at 37 °C, in contrast with native LDL(+). An inhibitor of neutral CDase prevented the formation of Sph and, in turn, increased Cer content in LDL(−). In addition, LDL(−) efficiently degraded fluorescently labeled Cer (NBD-Cer) to form Sph and NEFA. These observations defend the existence of the CDase-like activity’s association with LDL(−). However, neither the proteomic analysis nor the Western blot detected the presence of an enzyme with known CDase activity. Further studies are thus warranted to define the origin of the CDase-like activity detected in LDL(−).This research was funded by grants PI13/00364, PI16/00471, FIS PI019/00421, and PI20/00334 from the Instituto de Salud Carlos III, Spanish Ministry of Health (co-financed by the European Regional Development Fund). N.P. is the recipient of FI20/00252 from Instituto de Salud Carlos III. This research was supported by CIBER (Consorcio Centro de Investigación Biomédica en Red) (CB07/08/0016), Instituto de Salud Carlos III, and Ministerio de Ciencia e Innovación and Unión Europea—European Regional Development Fund. CIBERDEM (CB07/08/0016) and CIBERCV (CB16/11/00257) are Instituto de Salud Carlos III Projects. A.A.-S. is member of RETICS INVICTUS PLUS (RD16/0019/0010, the Instituto de Salud Carlos III project). N.P., S.B., N.R., and J.L.S.-Q. are members of the Quality Research Group 2017-SGR-1149 from Generalitat de Catalunya and the Group of Vascular Biology of the Spanish Society of Atherosclerosis

An intensive, structured, mobile devices-based healthcare intervention to optimize the lipid-lowering therapy improves lipid control after an acute coronary syndrome

AimsDespite the evidence, lipid-lowering treatment (LLT) in secondary prevention remains insufficient, and a low percentage of patients achieve the recommended LDL cholesterol (LDLc) levels by the guidelines. We aimed to evaluate the efficacy of an intensive, mobile devices-based healthcare lipid-lowering intervention after hospital discharge in patients hospitalized for acute coronary syndrome (ACS).Methods and resultsAmbiespective register in which a mobile devices-based healthcare intervention including periodic follow-up, serial lipid level controls, and optimization of lipid-lowering therapy, if appropriate, was assessed in terms of serum lipid-level control at 12 weeks after discharge. A total of 497 patients, of which 462 (93%) correctly adhered to the optimization protocol, were included in the analysis. At the end of the optimization period, 327 (70.7%) patients had LDLc levels ≤ 70 mg/dL. 40% of patients in the LDLc ≤ 70 mg/dL group were upgraded to very-high intensity lipid-lowering ability therapy vs. 60.7% in the LDLc > 70 mg/dL group, p < 0.001. Overall, 38.5% of patients had at least a change in their LLT. Side effects were relatively infrequent (10.7%). At 1-year follow-up, LDLc levels were measured by the primary care physician in 342 (68.8%) of the whole cohort of 497 patients. In this group, 71.1% of patients had LDLc levels ≤ 70 mg/dL.ConclusionAn intensive, structured, mobile devices-based healthcare intervention after an ACS is associated with more than 70% of patients reaching the LDLc levels recommended by the clinical guidelines. In patients with LDLc measured at 1-year follow-up, 71.1% had LDLc levels ≤ 70 mg/dL

An intensive, structured, mobile devices-based healthcare intervention to optimize the lipid-lowering therapy improves lipid control after an acute coronary syndrome

Despite the evidence, lipid-lowering treatment (LLT) in secondary prevention remains insufficient, and a low percentage of patients achieve the recommended LDL cholesterol (LDLc) levels by the guidelines. We aimed to evaluate the efficacy of an intensive, mobile devices-based healthcare lipid-lowering intervention after hospital discharge in patients hospitalized for acute coronary syndrome (ACS). Ambiespective register in which a mobile devices-based healthcare intervention including periodic follow-up, serial lipid level controls, and optimization of lipid-lowering therapy, if appropriate, was assessed in terms of serum lipid-level control at 12 weeks after discharge. A total of 497 patients, of which 462 (93%) correctly adhered to the optimization protocol, were included in the analysis. At the end of the optimization period, 327 (70.7%) patients had LDLc levels ≤ 70 mg/dL. 40% of patients in the LDLc ≤ 70 mg/dL group were upgraded to very-high intensity lipid-lowering ability therapy vs. 60.7% in the LDLc > 70 mg/dL group, p < 0.001. Overall, 38.5% of patients had at least a change in their LLT. Side effects were relatively infrequent (10.7%). At 1-year follow-up, LDLc levels were measured by the primary care physician in 342 (68.8%) of the whole cohort of 497 patients. In this group, 71.1% of patients had LDLc levels ≤ 70 mg/dL. An intensive, structured, mobile devices-based healthcare intervention after an ACS is associated with more than 70% of patients reaching the LDLc levels recommended by the clinical guidelines. In patients with LDLc measured at 1-year follow-up, 71.1% had LDLc levels ≤ 70 mg/dL

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Novel intragenic deletion within the FXN gene in a patient with typical phenotype of Friedreich ataxia: may be more prevalent than we think?

Background Friedreich ataxia is the most common inherited ataxia in Europe and is mainly caused by biallelic pathogenic expansions of the GAA trinucleotide repeat in intron 1 of the FXN gene that lead to a decrease in frataxin protein levels. Rarely, affected individuals carry either a large intragenic deletion or whole-gene deletion of FXN on one allele and a full-penetrance expanded GAA repeat on the other allele.Case presentation We report here a patient that presented the typical clinical features of FRDA and genetic analysis of FXN intron 1 led to the assumption that the patient carried the common biallelic expansion. Subsequently, parental sample testing led to the identification of a novel intragenic deletion involving the 5'UTR upstream region and exons 1 and 2 of the FXN gene by MLPA.Conclusions With this case, we want to raise awareness about the potentially higher prevalence of intragenic deletions and underline the essential role of parental sample testing in providing accurate genetic counselling

Prognostic Utility of a New Risk Stratification Protocol for Secondary Prevention in Patients Attending Cardiac Rehabilitation

Several risk scores have been used to predict risk after an acute coronary syndrome (ACS), but none of these risk scores include functional class. The aim was to assess the predictive value of risk stratification (RS), including functional class, and how cardiac rehabilitation (CR) changed RS. Two hundred and thirty-eight patients with ACS from an ambispective observational registry were stratified as low (L) and no-low (NL) risk and classified according to exercise compliance; low risk and exercise (L-E), low risk and control (no exercise) (L-C), no-low risk and exercise (NL-E), and no-low risk and control (NL-C). The primary endpoint was cardiac rehospitalization. Multivariable analysis was performed to identify variables independently associated with the primary endpoint. The L group included 56.7% of patients. The primary endpoint was higher in the NL group (18.4% vs. 4.4%, p < 0.001). After adjustment for age, sex, diabetes, and exercise in multivariable analysis, HR (95% CI) was 3.83 (1.51-9.68) for cardiac rehospitalization. For RS and exercise, the prognosis varied: the L-E group had a cardiac rehospitalization rate of 2.5% compared to 26.1% in the NL-C group (p < 0.001). Completing exercise training was associated with reclassification to low-risk, associated with a better outcome. This easy-to-calculate risk score offers robust prognostic information. No-exercise groups were independently associated with the worst outcomes. Exercise-based CR program changed RS, improving classification and prognosis

West Nile Virus Lineage 2 Spreads Westwards in Europe and Overwinters in North-Eastern Spain (2017–2020)

West Nile virus lineage 2 (WNV-L2) emerged in Europe in 2004; since then, it has spread across the continent, causing outbreaks in humans and animals. During 2017 and 2020, WNV-L2 was detected and isolated from four northern goshawks in two provinces of Catalonia (north-eastern Spain). In order to characterise the first Spanish WNV-L2 isolates and elucidate the potential overwintering of the virus in this Mediterranean region, complete genome sequencing, phylogenetic analyses, and a study of phenotypic characterisation were performed. Our results showed that these Spanish isolates belonged to the central-southern WNV-L2 clade. In more detail, they were related to the Lombardy cluster that emerged in Italy in 2013 and has been able to spread westwards, causing outbreaks in France (2018) and Spain (2017 and 2020). Phenotypic characterisation performed in vitro showed that these isolates presented characteristics corresponding to strains of moderate to high virulence. All these findings evidence that these WNV-L2 strains have been able to circulate and overwinter in the region, and are pathogenic, at least in northern goshawks, which seem to be very susceptible to WNV infection and may be good indicators of WNV-L2 circulation. Due to the increasing number of human and animal cases in Europe in the last years, this zoonotic flavivirus should be kept under extensive surveillance, following a One-Health approach.info:eu-repo/semantics/publishedVersio