A Protein Misfolding Shaking Amplification-based method for the spontaneous generation of hundreds of bona fide prions.

Prion diseases are a group of rapidly progressing neurodegenerative disorders caused by the misfolding of the endogenous prion protein (PrPC) into a pathogenic form (PrPSc). This process, despite being the central event underlying these disorders, remains largely unknown at a molecular level, precluding the prediction of new potential outbreaks or interspecies transmission incidents. In this work, we present a method to generate bona fide recombinant prions de novo, allowing a comprehensive analysis of protein misfolding across a wide range of prion proteins from mammalian species. We study more than 380 different prion proteins from mammals and classify them according to their spontaneous misfolding propensity and their conformational variability. This study aims to address fundamental questions in the prion research field such as defining infectivity determinants, interspecies transmission barriers or the structural influence of specific amino acids and provide invaluable information for future diagnosis and therapy applications.

Understanding the key features of the spontaneous formation of bona fide prions through a novel methodology that enables their swift and consistent generation.

Among transmissible spongiform encephalopathies or prion diseases affecting humans, sporadic forms such as sporadic Creutzfeldt-Jakob disease are the vast majority. Unlike genetic or acquired forms of the disease, these idiopathic forms occur seemingly due to a random event of spontaneous misfolding of the cellular PrP (PrPC) into the pathogenic isoform (PrPSc). Currently, the molecular mechanisms that trigger and drive this event, which occurs in approximately one individual per million each year, remain completely unknown. Modelling this phenomenon in experimental settings is highly challenging due to its sporadic and rare occurrence. Previous attempts to model spontaneous prion misfolding in vitro have not been fully successful, as the spontaneous formation of prions is infrequent and stochastic, hindering the systematic study of the phenomenon. In this study, we present the first method that consistently induces spontaneous misfolding of recombinant PrP into bona fide prions within hours, providing unprecedented possibilities to investigate the mechanisms underlying sporadic prionopathies. By fine-tuning the Protein Misfolding Shaking Amplification method, which was initially developed to propagate recombinant prions, we have created a methodology that consistently produces spontaneously misfolded recombinant prions in 100% of the cases. Furthermore, this method gives rise to distinct strains and reveals the critical influence of charged surfaces in this process.

Estimating sequence diversity of prion protein gene (PRNP) in Portuguese populations of two cervid species: red deer and fallow deer.

Among the transmissible spongiform encephalopathies (TSEs), chronic wasting disease (CWD) in cervids is now a rising concern in wildlife within Europe, after the detection of the first case in Norway in 2016, in a wild reindeer and until June 2022 a total of 34 cases were described in Norway, Sweden and Finland. The definite diagnosis is post-mortem, performed in target areas of the brain and lymph nodes. Samples are first screened using a rapid test and, if positive, confirmed by immunohistochemistry and Western immunoblotting. The study of the genetics of the prion protein gene, PRNP, has been proved to be a valuable tool for determining the relative susceptibility to TSEs. In the present study, the exon 3 of PRNP gene of 143 samples from red deer (Cervus elaphus) and fallow deer (Dama dama) of Portugal was analysed. Three single nucleotide polymorphisms (SNPs) were found in red deer - codon A136A, codon T98A, codon Q226E - and no sequence variation was detected in fallow deer. The low genetic diversity found in our samples is compatible with previous studies in Europe. The comparison with results from North America suggests that the free-ranging deer from our study may present susceptibility to CWD, although lack of experimental data and the necessity of continuous survey are necessary to evaluate these populations.

Genomic Medicine in Periodontal Disease: Old Issue, New Insights.

Genetic variability is the main cause of phenotypic variation. Some variants may be associated with several diseases and can be used as risk biomarkers, identifying animals with higher susceptibility to develop the pathology. Genomic medicine uses this genetic information for risk calculation, clinical diagnosis and prognosis, allowing the implementation of more effective preventive strategies and/or personalized therapies. Periodontal disease (PD) is the inflammation of the periodontium induced mainly by bacterial plaque and is the leading cause of tooth loss. Microbial factors are responsible for the PD initiation; however, several studies support the genetic influence on the PD progression. The main purpose of the present publication is to highlight the main steps involved in the genomic medicine applied to veterinary patients, describing the flowchart from the characterization of the genetic variants to the identification of potential associations with specific clinical data. After investigating which genes might potentially be implicated in canine PD, the RANK gene, involved in the regulation of osteoclastogenesis, was selected to illustrate this approach. A case-control study was performed using DNA samples from a population of 90 dogs - 50 being healthy and 40 with PD. This analysis allowed for the discovery of four new intronic variations that were banked in GenBank (g.85A>G, g.151G>T, g.268A>G and g.492T>C). The results of this study are not intended to be applied exclusively to PD. On the contrary, this genetic information is intended to be used by other researchers as a foundation for the development of multiple applications in the veterinary clinical field.

Scrapie at Abattoir: Monitoring, Control, and Differential Diagnosis of Wasting Conditions during Meat Inspection.

Wasting disease in small ruminants is frequently detected at slaughterhouses. The wasting disorder is manifested by the deterioration of the nutritional and physiological state of the animal indicated by thinness, emaciation, and cachexia. Evidence of emaciation and cachexia, alone, are pathological conditions leading to carcass condemnation during an inspection. Several diseases are associated with a wasting condition, including scrapie, pseudotuberculosis, tuberculosis, paratuberculosis, Maedi Visna, and tumor diseases. On the other hand, parasitic diseases, nutrition disorders, exposure or ingestion of toxins, metabolic conditions, inadequate nutrition due to poor teeth, or poor alimentary diet are conditions contributing to poor body condition. Classical and atypical scrapie is naturally occurring transmissible spongiform encephalopathies in small ruminants. The etiological agent for each one is prions. However, each of these scrapie types is epidemiologically, pathologically, and biochemically different. Though atypical scrapie occurs at low incidence, it is consistently prevalent in the small ruminant population. Hence, it is advisable to include differential diagnosis of this disease, from other possibilities, as a cause of wasting conditions detected during meat inspection at the abattoir. This manuscript is a review of the measures in force at the abattoir for scrapie control, focusing on the differential diagnosis of gross lesions related to wasting conditions detected in small ruminants during meat inspection.

Neuropathology of Animal Prion Diseases.

Transmissible Spongiform Encephalopathies (TSEs) or prion diseases are a fatal group of infectious, inherited and spontaneous neurodegenerative diseases affecting human and animals. They are caused by the conversion of cellular prion protein (PrPC) into a misfolded pathological isoform (PrPSc or prion- proteinaceous infectious particle) that self-propagates by conformational conversion of PrPC. Yet by an unknown mechanism, PrPC can fold into different PrPSc conformers that may result in different prion strains that display specific disease phenotype (incubation time, clinical signs and lesion profile). Although the pathways for neurodegeneration as well as the involvement of brain inflammation in these diseases are not well understood, the spongiform changes, neuronal loss, gliosis and accumulation of PrPSc are the characteristic neuropathological lesions. Scrapie affecting small ruminants was the first identified TSE and has been considered the archetype of prion diseases, though atypical and new animal prion diseases continue to emerge highlighting the importance to investigate the lesion profile in naturally affected animals. In this report, we review the neuropathology and the neuroinflammation of animal prion diseases in natural hosts from scrapie, going through the zoonotic bovine spongiform encephalopathy (BSE), the chronic wasting disease (CWD) to the newly identified camel prion disease (CPD).

Toll-like receptor 9 gene in Periodontal Disease - A promising biomarker.

Periodontal Disease is an infectious and inflammatory disorder triggered mainly by periodontopathogenic bacteria, however, as a multifactorial disease, several factors modulate its progression, namely, genetic factors. Toll-like receptors (TLR) recognize molecular patterns present in pathogens and trigger an immune response against them. Thus, sequences variants in TLR genes seem to have the potential to modify the predisposition to Periodontal Disease and its progression. Based on this fact, TLR9 gene were analysed in a case-control study. DNA was obtained from 90 dogs (50 control and 40 cases) and a fragment of TLR9 gene was amplified by PCR and sequenced. The variants were identified by comparison with the dog wild type sequences. Our results suggest that rs375556098 and rs201959275 polymorphisms in the TLR9 gene are good candidates to become biomarkers of the canine predisposition to Periodontal Disease. It's important to notice that these polymorphic sites exist in Human exactly in the same codon. Since the dog is the best animal model to replicate the pathophysiological mechanisms of human Periodontal Disease, these results can potentially be extrapolated to humans.