Gerstmann-Sträussler-Scheinker disease revisited: accumulation of covalently-linked multimers of internal prion protein fragments

Despite their phenotypic heterogeneity, most human prion diseases belong to two broadly defined groups: Creutzfeldt-Jakob disease (CJD) and Gerstmann-Sträussler-Scheinker disease (GSS). While the structural characteristics of the disease-related proteinase K-resistant prion protein (resPrPD) associated with the CJD group are fairly well established, many features of GSS-associated resPrPD are unclear. Electrophoretic profiles of resPrPD associated with GSS variants typically show 6-8 kDa bands corresponding to the internal PrP fragments as well as a variable number of higher molecular weight bands, the molecular nature of which has not been investigated. Here we have performed systematic studies of purified resPrPD species extracted from GSS cases with the A117V (GSSA117V) and F198S (GSSF198S) PrP gene mutations. The combined analysis based on epitope mapping, deglycosylation treatment and direct amino acid sequencing by mass spectrometry provided a conclusive evidence that high molecular weight resPrPD species seen in electrophoretic profiles represent covalently-linked multimers of the internal ~ 7 and ~ 8 kDa fragments. This finding reveals a mechanism of resPrPD aggregate formation that has not been previously established in prion diseases

A novel mechanism of phenotypic heterogeneity in Creutzfeldt-Jakob disease

One of remarkable features of sporadic Creutzfeldt-Jakob disease (sCJD) is the great phenotypic variability. Understanding the molecular basis of this variability has important implications for the development of therapeutic approaches. It is well established that, in many cases, phenotypic heterogeneity of sCJD is under control of two determinants: the genotype at the methionine (M)/valine (V) polymorphic codon 129 of the human prion protein gene and the type, 1 or 2, of the pathogenic and disease-related form of the prion protein, PrPD. However, this scenario fails to explain the existence of distinct heterozygous sCJDMV2 subtypes, where heterogeneity occurs without any variation of the 129 allotype and PrPD type. One of these subtypes, denoted sCJDMV2C, associated with PrPD type 2, is characterized by widespread spongiform degeneration of the cerebral cortex (C). The second variant, denoted sCJDMV2K, features prominent deposition of PrPD amyloid forming kuru type (K) plaques. Here we used a mass spectrometry based approach to test the hypothesis that phenotypic variability within the sCJDMV2 subtype is at least partly determined by the abundance of 129 M and 129 V polymorphic forms of proteinase K-resistant PrPD (resPrPD). Consistent with this hypothesis, our data demonstrated a strong correlation of the MV2C and MV2K phenotypes with the relative populations of protease-resistant forms of the pathogenic prion proteins, resPrPD-129 M and resPrPD-129 V, where resPrPD-129 M dominated in the sCJDMV2C variant and resPrPD-129 V in the sCJDMV2K variant. This finding suggests an important, previously unrecognized mechanism for phenotypic determination in human prion diseases

Detection of TDP-43 seeding activity in the olfactory mucosa from patients with frontotemporal dementia

Introduction: We assessed TAR DNA-binding protein 43 (TDP-43) seeding activity and aggregates detection in olfactory mucosa of patients with frontotemporal lobar degeneration with TDP-43-immunoreactive pathology (FTLD-TDP) by TDP-43 seeding amplification assay (TDP43-SAA) and immunocytochemical analysis. Methods: The TDP43-SAA was optimized using frontal cortex samples from 16 post mortem cases with FTLD-TDP, FTLD with tau inclusions, and controls. Subsequently, olfactory mucosa samples were collected from 17 patients with FTLD-TDP, 15 healthy controls, and three patients carrying MAPT variants. Results: TDP43-SAA discriminated with 100% accuracy post mortem cases presenting or lacking TDP-43 neuropathology. TDP-43 seeding activity was detectable in the olfactory mucosa, and 82.4% of patients with FTLD-TDP tested positive, whereas 86.7% of controls tested negative (P < 0.001). Two out of three patients with MAPT mutations tested negative. In TDP43-SAA positive samples, cytoplasmatic deposits of phosphorylated TDP-43 in the olfactory neural cells were detected. Discussion: TDP-43 aggregates can be detectable in olfactory mucosa, suggesting that TDP43-SAA might be useful for identifying and monitoring FTLD-TDP in living patients

Sporadic fatal insomnia in a young woman: A diagnostic challenge: Case Report

<p>Abstract</p> <p>Background</p> <p>Sporadic fatal insomnia (sFI) and fatal familial insomnia (FFI) are rare human prion diseases.</p> <p>Case Presentation</p> <p>We report a case of a 33-year-old female who died of a prion disease for whom the diagnosis of sFI or FFI was not considered clinically. Following death of this patient, an interview with a close family member indicated the patient's illness included a major change in her sleep pattern, corroborating the reported autopsy diagnosis of sFI. Genetic tests identified no prion protein (PrP) gene mutation, but neuropathological examination and molecular study showed protease-resistant PrP (PrP^res) in several brain regions and severe atrophy of the anterior-ventral and medial-dorsal thalamic nuclei similar to that described in FFI.</p> <p>Conclusions</p> <p>In patients with suspected prion disease, a characteristic change in sleep pattern can be an important clinical clue for identifying sFI or FFI; polysomnography (PSG), genetic analysis, and nuclear imaging may aid in diagnosis.</p

Boost Camp’, a universal school-based transdiagnostic prevention program targeting adolescent emotion regulation; evaluating the effectiveness by a clustered RCT : a protocol paper

Abstract Background The transition from childhood into adolescence can be considered as a critical developmental period. Moreover, adolescence is associated with a decreased use of adaptive emotion regulation strategies and an increased use of maladaptive emotion regulation strategies increasing the risk of emotional problems. Targeting emotion regulation is therefore seen as an innovative prevention approach. The present study aims to evaluate the effectiveness of Boost camp, an innovative school-based prevention program targeting ER, on adolescents’ emotion regulation skills and emotional wellbeing. Also secondary outcomes and possible moderators will be included. Methods The aim is to reach 300 adolescents (16 class groups, 6 schools) in their first year of high school. A clustered Randomized Controlled Trial (RCT) with two conditions, intervention (n = 150) and control (n = 150), will be set up. Adolescents in the intervention condition will receive 14 lessons over the course of 2 days, followed by Booster sessions, and will be compared with adolescents in a non-intervention control group. The outcomes will be measured by self-report questionnaires at baseline, immediately after Boost camp, and at three and 6 months follow-up. Discussion Data-collection is planned to be completed in May 2018. Data-analyses will be finished the end of 2018. The presented paper describes the Boost camp program and the clustered RCT design to evaluate its effectiveness. It is expected that Boost camp will have beneficial effects. If found effective, Boost camp will have the potential to increase adolescent’s ER and well-being, and reduce the risk to become adults in need. The trials is registered on the 13th of June 2017 in ISRCTN registry [ISRCTN68235634]

Extracorporeal Membrane Oxygenation for Severe Acute Respiratory Distress Syndrome associated with COVID-19: An Emulated Target Trial Analysis.

RATIONALE: Whether COVID patients may benefit from extracorporeal membrane oxygenation (ECMO) compared with conventional invasive mechanical ventilation (IMV) remains unknown. OBJECTIVES: To estimate the effect of ECMO on 90-Day mortality vs IMV only Methods: Among 4,244 critically ill adult patients with COVID-19 included in a multicenter cohort study, we emulated a target trial comparing the treatment strategies of initiating ECMO vs. no ECMO within 7 days of IMV in patients with severe acute respiratory distress syndrome (PaO2/FiO2 <80 or PaCO2 ≥60 mmHg). We controlled for confounding using a multivariable Cox model based on predefined variables. MAIN RESULTS: 1,235 patients met the full eligibility criteria for the emulated trial, among whom 164 patients initiated ECMO. The ECMO strategy had a higher survival probability at Day-7 from the onset of eligibility criteria (87% vs 83%, risk difference: 4%, 95% CI 0;9%) which decreased during follow-up (survival at Day-90: 63% vs 65%, risk difference: -2%, 95% CI -10;5%). However, ECMO was associated with higher survival when performed in high-volume ECMO centers or in regions where a specific ECMO network organization was set up to handle high demand, and when initiated within the first 4 days of MV and in profoundly hypoxemic patients. CONCLUSIONS: In an emulated trial based on a nationwide COVID-19 cohort, we found differential survival over time of an ECMO compared with a no-ECMO strategy. However, ECMO was consistently associated with better outcomes when performed in high-volume centers and in regions with ECMO capacities specifically organized to handle high demand. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Fatty acid-binding proteins as markers of brain injury

Le Fatty Acid-Binding Proteins (FABPS) sono proteine intracellulari in grado di legare gli acidi grassi a catena lunga con elevata affinit\ue0, regolandone il trasporto all\u2019interno delle cellule. Nell\u2019uomo sono state finora identificate 9 diverse isoforme, ciascuna con un diverso profilo di espressione a livello di tessuti. La proteina heart-type FABP (H-FABP) da anni \ue8 considerata uno tra i pi\uf9 sensibili ed efficienti marcatori di danno cerebrale e al miocardio, ma la sua elevata espressione in differenti tessuti non la rende un marker specifico di un particolare disordine. Un\u2019altra isoforma espressa nel cervello, chiamata BFABP (brain-type FABP), potrebbe invece rappresentare un marcatore specifico di danno cerebrale, essendo la sua espressione limitata al tessuto nervoso. Questo lavoro ha avuto come scopo la generazione di anticorpi in grado di riconoscere la proteina B-FABP in modo specifico e selettivo. Il progetto si \ue8 articolato in due fasi. Nella prima parte del lavoro la proteina ricombinante B-FABP \ue8 stata espressa in E. coli e purificata mediante due successive cromatografie. Essa \ue8 stata quindi inoculata in conigli New Zealand e in topi BalbC, ottenendo dopo diverso tempo 2 antisieri e 8 diverse cellule ibridoma. Gli anticorpi presenti nel siero e nel surnatante delle cellule ibridoma in coltura hanno evidenziato diversi gradi di affinit\ue0 nei confronti delle due isoforme, BFABP e H-FABP; solamente una cellula ibridoma \ue8 stata in grado di produrre un anticorpo specifico e selettivo per la B-FABP ma purtroppo i successivi passaggi di subclonaggio della cellula hanno evidenziato una progressiva diminuzione dell\u2019attivit\ue0 anticorpale, non permettendo l\u2019utilizzo di questo reagente per l\u2019individuazione della proteina stessa in campioni biologici. Nella seconda parte del lavoro si \ue8 quindi cercato di analizzare e superare le problematiche riscontrate nella prima fase mediante un nuovo approccio che, con l\u2019ausilio di software specifici, ha mirato all\u2019identificazione di due regioni potenzialmente antigeniche della proteina stessa. Due peptidi sintetici, di sequenza identica alle regioni identificate, sono stati inoculati in conigli New Zealand con l\u2019ottenimento di due anticorpi policlonali, pAb 2979/2980 e pAb 2981/2982. Questi anticorpi si sono rivelati specifici e selettivi verso l\u2019isoforma B-FABP e non hanno evidenziato reazioni di cross-reazione verso H-FABP. Si pu\uf2 quindi affermare come l\u2019approccio sperimentale utilizzato abbia contribuito al raggiungimento dell\u2019obiettivo, la produzione di un anticorpo specifico per B-FABP umana. Questo risultato suggerisce possibili scenari nell\u2019utilizzo futuro degli anticorpi, in tecniche ELISA e western blot, per valutare se la proteina B-FABP possa essere considerata un marcatore ideale, specifico di danno neuronale, in disordini a diversa eziologia (ischemica, infettiva o degenerativa).Fatty acid\u2013binding proteins (FABPs) are abundant intracellular proteins that bind long-chain fatty acids with high affinity. 9 different FABPs, with tissuespecific distribution, have been identified so far. The primary role of all the FABP family members is regulation of fatty acid uptake and intracellular transport. Heart-type FABP (H-FABP) is considered one of the most sensible and efficient markers of heart and brain damage, but to date the cross-reactivity limits its specific use. Brain-type FABP (B-FABP) might be considered as a good marker of brain damage, being expressed only in the nervous tissue. The aim of this work was to generate a diagnostic reagent specific for human B-FABP, not cross-reacting with H-FABP. The work has been articulated in two parts: in the first one, recombinant protein B-FABP was expressed in E. coli cells and purified by two subsequent chromatographies. The protein was then utilized as immunogen in New Zealand rabbits and BalbC mice, obtaining two antisera and 8 different hybridoma cells. The antibodies in the rabbit serum and in culture supernatant of hybridoma cells displayed different degrees of affinity for B-FABP and H-FABP; only one hybridoma cell was able to produce antibodies specific and selective for brain-type FABP, but the antibody low-titer level and the activity decrease after subsequent subcloning steps affected its application on B-FABP detection in biological fluids. To overcome the limits encountered in the first part of work, a computer-assisted approach on B-FABP was employed in order to identify the potentially most antigenic regions of the protein. Two synthetic peptides were produced with the selected sequences and inoculated in rabbits. Two polyclonal antibodies were obtained, pAb 2979/2980 and pAb 2981/2982. They showed specific and selective reactivity for human B-FABP, without crossreactions with H-FABP. Taken together, our experimental approach was effective for the generation of specific \u3b1-B-FABP antibodies; these results suggest the antibodies obtained could be utilized in ELISA and immunoblot analyses in order to value B-FABP as marker of neurological disorders with ischemic, infective and degenerative etiology

Social group membership does not modulate automatic imitation in a contrastive multi-agent paradigm

A key prediction of motivational theories of automatic imitation is that people imitate in-group over out-group members. However, research on this topic has provided mixed results. Here, we investigate the possibility that social group modulations emerge only when people can directly compare in- and out-group. To this end, we conducted three experiments in which we measured automatic imitation of two simultaneously shown hands: one in-group and one out-group hand. Our general hypothesis was that the in-group hand would be imitated more than the out-group hand. However, even though both explicit and implicit manipulation checks showed that we succeeded in manipulating participants’ feelings of group membership, we did not find support for the predicted influence of group membership on automatic imitation. In contrast to motivational theories, this suggests that group membership does not influence who we do or do not imitate, not even in a contrastive multi-agent paradigm