Morphological and Biochemical Characterization of Bovine Congenital Psudomyotonia

The Ca2+-ATPase of sarco(endo)plasmic reticulum (SERCA) is a protein of about 110 kDa member of the P-type ATPases family. SERCA pumps utilize the energy derived from the hydrolysis of a molecule of ATP to transport two Ca2+ ions across the Sarcoplasmic Reticulum (SR) membrane to decrease the Ca2+ concentration in the cytosol. SERCA isoform 1a (SERCA1a) is the mainly expressed isoform in adult fast-twitch muscle fibre and it is both structurally and functionally the best characterized member of the P-type ion translocating ATPases. An inherited muscle disorder defined as Bovine Congenital Pseudomyotonia (PMT) has been recently described in two important Italian cattle breeds Chianina and Romagnola (RMG) and, as a single case, in a Dutch improved Red&White (VRB) crossbred calf in the Netherlands. Clinically the disorder is characterized by an exercise-induced muscle contraction not associated to electromyogram alterations. Basing on the clinical signs, the explanation proposed for the delayed relaxation of muscle was a prolonged elevation in Ca2+ ion cytoplasmic concentration. Because the structure primary involved in the re-uptake of the Ca2+ ion into the SR after muscle-contraction is the ATPase pump, the more likely aetiological hypothesis was an involvement of SERCA1a protein. Indeed, DNA sequencing of affected animals provided evidence of mutations in ATP2A1 gene coding for SERCA1. So far, different point mutations have been described in the different breeds that displayed the disease. Bovine PMT has been well characterized at both genetic and biochemical levels in Chianina breed. The main objective of this project is to complete the characterization of the disease by mean of analysing from a morphological, histochemical and biochemical point of view the other PMT-cases diagnosed in RMG and VRB breeds. The final aims are to increase the knowledge on morphological and biochemical effects of the disease on muscle tissue, to evaluate phenotypic variability of the disease and to identify possible adaptive/compensatory mechanisms that could drive the future research to develop new therapeutic approaches. The interest on bovine PMT arises from its similarities with a rare human inherited muscular dysfunction named Brody disease. Indeed, PMT-affected Chianina cattle has been accepted as an interesting and useful non-conventional animal model for the study of this human pathological condition. The histopathological examinations on semimembranosus muscle biopsies from RMG and VRB affected animals did not show severe signs of muscle damage. Some pale degenerated fibres, filled or surrounded by macrophages, were identified within the tissue, although they were always accompanied by regenerative phenomena (small size fibres neonatal MHC-positive). Histochemical protocols (COX-SDH and PAS staining) did not reveal metabolic alterations or changes in glycogen distribution. Immunohistochemical investigations on SERCA1 expression showed that pathological cases had a decrease in immunoreactivity for SERCA1 antibody suggesting a lower presence of the protein. The latter finding was confirmed by biochemical analysis on microsomal fractions enriched in content of SR membranes. Immunoblotting proved that only SERCA1 protein had a reduction in quantity whereas nor junctional (RyR) nor non-junctional (SL and GP53) markers of SR showed any variance. The quantitative decrease was also associated with a lower activity of the protein demonstrated by Ca2+ATPase activity assay. Real Time RT-PCR assays demonstrated that mRNA for SERCA1 was normally expressed suggesting that the protein reduction was due to post-transcriptional events. Interestingly, the sections of the affected VRB subject showed an increased and more homogeneous stain for COX and SDH suggesting an augmentation in mitochondria content. This finding was reinforced by immunohistochemical and immunoblot surveys using an antibody against a protein of the mitochondrial outer membrane (Tom20). Moreover, microsomal fractions from VRB animal showed an increase in plasma membrane Ca2+ATPase (PMCA) when compare with controls. Finally in RMG and VRB affected subjects some fibres were immunoreactive for SERCA1 and phospholamban (PLB, regulator protein of SERCA2) or SERCA isoforms 1 and 2 suggesting an alteration in the classic mutually exclusive protein expression pattern (either SERCA1 or SERCA2 and PLB). The results of this research describe Bovine Congenital Pseudomyotonia in RGM and VRB as a muscular disease characterized by mild histopathological alterations and a sole decrease in SERCA1 content and activity, in full agreement with the studies on Chianina. All the analyses performed on VRB and RGM animals allow to obtain a complete picture of the PMT in these breeds and afford to define a general histopathological and biochemical case of PMT. In addition, for the first time compensatory mechanisms were demonstrated providing histochemical and biochemical evidences to support mitochondria and PMCA involvement in the Ca2+ homeostasis during the pathological condition caused by PMT mutation. Finally, the first evidence of co-expression of SERCA1, SERCA2 and PLB in affected animals, was an intriguing collateral finding that suggests a great potential plasticity of cattle muscle resembling the human muscle once again. A side project was developed in collaboration with the group of Prof. R. Smith and Dr. J. Dudhia of the Clinical Sciences and Services Department of the Royal Veterinary College (London). Two different pilot studies were carry out on extracellular matrix response to tendon injury in equine. The first had as object of study the cartilage oligomeric matrix protein (COMP) recently proposed as useful biomarker of tendinopathy. The aim of this project was to assess the level of fragmentation (index of alteration but also re-modelling) of COMP in healthy tendon tissue collected in sites well-known to be more frequently involved in lesions (predisposed to injury). The tendons analysed were the superficial digital flexor (SDFT) and the deep digital flexor (DDFT). Immunoblotting and densitometric analysis of total protein extracts showed an higher COMP fragmentation level in healthy predisposed sites when compare with non-predisposed sites. Moreover, for the first time the different patterns of COMP expression in SDFT and DDFT of young and old subjects were described. The second pilot study analysed the morphological aspect of tendon tissue affected by tear-type lesion. The objective was to evaluate the influence of the lesion on the tissue adjacent, far and remote to the injury site. The tendon was extremely altered close to the lesion while the other areas showed high cellularity and vascularity but normal matrix organization. Masson trichrome staining distinguished damaged tissue (blue) from healthy (red) running to become an intriguing tool for histological investigation. Immunohistochemistry showed an increased reactivity for COMP antibody in the areas more proximal to the tear reinforcing the studies that support a fundamental role of this protein in the matrix re-modelling. Increasing the knowledge of COMP distribution in SDFT and DDFT, and providing information of tendon response to tear lesion, these two pilot studies create a useful platform for future researches with possible clinical and therapeutic implication

Clinical Features, Cardiovascular Risk Profile, and Therapeutic Trajectories of Patients with Type 2 Diabetes Candidate for Oral Semaglutide Therapy in the Italian Specialist Care

Introduction: This study aimed to address therapeutic inertia in the management of type 2 diabetes (T2D) by investigating the potential of early treatment with oral semaglutide. Methods: A cross-sectional survey was conducted between October 2021 and April 2022 among specialists treating individuals with T2D. A scientific committee designed a data collection form covering demographics, cardiovascular risk, glucose control metrics, ongoing therapies, and physician judgments on treatment appropriateness. Participants completed anonymous patient questionnaires reflecting routine clinical encounters. The preferred therapeutic regimen for each patient was also identified. Results: The analysis was conducted on 4449 patients initiating oral semaglutide. The population had a relatively short disease duration (42%  60% of patients, and more often than sitagliptin or empagliflozin. Conclusion: The study supports the potential of early implementation of oral semaglutide as a strategy to overcome therapeutic inertia and enhance T2D management

Morphological and Biochemical Characterization of Bovine Congenital Psudomyotonia

The Ca2+-ATPase of sarco(endo)plasmic reticulum (SERCA) is a protein of about 110 kDa member of the P-type ATPases family. SERCA pumps utilize the energy derived from the hydrolysis of a molecule of ATP to transport two Ca2+ ions across the Sarcoplasmic Reticulum (SR) membrane to decrease the Ca2+ concentration in the cytosol. SERCA isoform 1a (SERCA1a) is the mainly expressed isoform in adult fast-twitch muscle fibre and it is both structurally and functionally the best characterized member of the P-type ion translocating ATPases. An inherited muscle disorder defined as Bovine Congenital Pseudomyotonia (PMT) has been recently described in two important Italian cattle breeds Chianina and Romagnola (RMG) and, as a single case, in a Dutch improved Red&White (VRB) crossbred calf in the Netherlands. Clinically the disorder is characterized by an exercise-induced muscle contraction not associated to electromyogram alterations. Basing on the clinical signs, the explanation proposed for the delayed relaxation of muscle was a prolonged elevation in Ca2+ ion cytoplasmic concentration. Because the structure primary involved in the re-uptake of the Ca2+ ion into the SR after muscle-contraction is the ATPase pump, the more likely aetiological hypothesis was an involvement of SERCA1a protein. Indeed, DNA sequencing of affected animals provided evidence of mutations in ATP2A1 gene coding for SERCA1. So far, different point mutations have been described in the different breeds that displayed the disease. Bovine PMT has been well characterized at both genetic and biochemical levels in Chianina breed. The main objective of this project is to complete the characterization of the disease by mean of analysing from a morphological, histochemical and biochemical point of view the other PMT-cases diagnosed in RMG and VRB breeds. The final aims are to increase the knowledge on morphological and biochemical effects of the disease on muscle tissue, to evaluate phenotypic variability of the disease and to identify possible adaptive/compensatory mechanisms that could drive the future research to develop new therapeutic approaches. The interest on bovine PMT arises from its similarities with a rare human inherited muscular dysfunction named Brody disease. Indeed, PMT-affected Chianina cattle has been accepted as an interesting and useful non-conventional animal model for the study of this human pathological condition. The histopathological examinations on semimembranosus muscle biopsies from RMG and VRB affected animals did not show severe signs of muscle damage. Some pale degenerated fibres, filled or surrounded by macrophages, were identified within the tissue, although they were always accompanied by regenerative phenomena (small size fibres neonatal MHC-positive). Histochemical protocols (COX-SDH and PAS staining) did not reveal metabolic alterations or changes in glycogen distribution. Immunohistochemical investigations on SERCA1 expression showed that pathological cases had a decrease in immunoreactivity for SERCA1 antibody suggesting a lower presence of the protein. The latter finding was confirmed by biochemical analysis on microsomal fractions enriched in content of SR membranes. Immunoblotting proved that only SERCA1 protein had a reduction in quantity whereas nor junctional (RyR) nor non-junctional (SL and GP53) markers of SR showed any variance. The quantitative decrease was also associated with a lower activity of the protein demonstrated by Ca2+ATPase activity assay. Real Time RT-PCR assays demonstrated that mRNA for SERCA1 was normally expressed suggesting that the protein reduction was due to post-transcriptional events. Interestingly, the sections of the affected VRB subject showed an increased and more homogeneous stain for COX and SDH suggesting an augmentation in mitochondria content. This finding was reinforced by immunohistochemical and immunoblot surveys using an antibody against a protein of the mitochondrial outer membrane (Tom20). Moreover, microsomal fractions from VRB animal showed an increase in plasma membrane Ca2+ATPase (PMCA) when compare with controls. Finally in RMG and VRB affected subjects some fibres were immunoreactive for SERCA1 and phospholamban (PLB, regulator protein of SERCA2) or SERCA isoforms 1 and 2 suggesting an alteration in the classic mutually exclusive protein expression pattern (either SERCA1 or SERCA2 and PLB). The results of this research describe Bovine Congenital Pseudomyotonia in RGM and VRB as a muscular disease characterized by mild histopathological alterations and a sole decrease in SERCA1 content and activity, in full agreement with the studies on Chianina. All the analyses performed on VRB and RGM animals allow to obtain a complete picture of the PMT in these breeds and afford to define a general histopathological and biochemical case of PMT. In addition, for the first time compensatory mechanisms were demonstrated providing histochemical and biochemical evidences to support mitochondria and PMCA involvement in the Ca2+ homeostasis during the pathological condition caused by PMT mutation. Finally, the first evidence of co-expression of SERCA1, SERCA2 and PLB in affected animals, was an intriguing collateral finding that suggests a great potential plasticity of cattle muscle resembling the human muscle once again. A side project was developed in collaboration with the group of Prof. R. Smith and Dr. J. Dudhia of the Clinical Sciences and Services Department of the Royal Veterinary College (London). Two different pilot studies were carry out on extracellular matrix response to tendon injury in equine. The first had as object of study the cartilage oligomeric matrix protein (COMP) recently proposed as useful biomarker of tendinopathy. The aim of this project was to assess the level of fragmentation (index of alteration but also re-modelling) of COMP in healthy tendon tissue collected in sites well-known to be more frequently involved in lesions (predisposed to injury). The tendons analysed were the superficial digital flexor (SDFT) and the deep digital flexor (DDFT). Immunoblotting and densitometric analysis of total protein extracts showed an higher COMP fragmentation level in healthy predisposed sites when compare with non-predisposed sites. Moreover, for the first time the different patterns of COMP expression in SDFT and DDFT of young and old subjects were described. The second pilot study analysed the morphological aspect of tendon tissue affected by tear-type lesion. The objective was to evaluate the influence of the lesion on the tissue adjacent, far and remote to the injury site. The tendon was extremely altered close to the lesion while the other areas showed high cellularity and vascularity but normal matrix organization. Masson trichrome staining distinguished damaged tissue (blue) from healthy (red) running to become an intriguing tool for histological investigation. Immunohistochemistry showed an increased reactivity for COMP antibody in the areas more proximal to the tear reinforcing the studies that support a fundamental role of this protein in the matrix re-modelling. Increasing the knowledge of COMP distribution in SDFT and DDFT, and providing information of tendon response to tear lesion, these two pilot studies create a useful platform for future researches with possible clinical and therapeutic implication.La Ca2+-ATPasi del reticolo sarco(endo)plasmatico (SERCA) è una proteina di circa 110 kDa membro della famiglia delle P-type ATPasi. Le SERCA utilizzano l'energia ricavata dall’idrolisi di una molecola di ATP per trasportare due ioni Ca2+ attraverso la membrana del reticolo sarcoplasmatico (SR) e diminuire così la concentrazione di Ca2+ nel citosol. La SERCA1a è l'isoforma espressa prevalentemente dalle fibre muscolari fast nell’adulto ed è strutturalmente e funzionalmente la meglio caratterizzata tra tutte le ATPasi. Un disturbo muscolare ereditario definito come Pseudomiotonia Congenita Bovina (PMT) è stato recentemente descritto in due importanti razze bovine da carne italiane Chianina e Romagnola (RMG) e, come caso isolato, in un incrocio olandese (Dutch improved Red&White -VRB-). La malattia è caratterizzata clinicamente da una contrazione muscolare indotta da esercizio ma non associata ad alterazioni elettromiografiche. La causa attribuita al ritardato rilassamento muscolare è stata una prolungata permanenza del Ca2+ nel citoplasma. Poiché la struttura primariamente coinvolta nel re-uptake del Ca2+ all’interno del SR dopo la contrazione muscolare è la pompa ATPasi, è stato proposto un coinvolgimento della proteine SERCA1a. Il sequenziamento del DNA di animali affetti ha dimostrato la presenza di mutazioni nel gene ATP2A1 che codifica appunto la SERCA1. Ad oggi diverse mutazioni puntiformi sono state descritte nelle varie razze che manifestano la malattia. La PMT è stato caratterizzata a livello genetico e biochimico nella razza Chianina. L'obiettivo principale di questo progetto è quello di completare la caratterizzazione della malattia attraverso l’analisi morfologica, istochimica e biochimica dei casi di PMT descritti nelle razze RMG e VRB. Scopo ultimo è quello di aumentare le conoscenze sugli effetti morfologici e biochimici della malattia sul tessuto muscolare, valutare la variabilità fenotipica della malattia e individuare eventuali meccanismi di adattamento o compensatori che potrebbero suggerire nuovi approcci terapeutici alle ricerche future. L'interesse per la PMT deriva dalla sua stretta somiglianza con una rara disfunzione muscolare ereditaria dell’uomo chiamata malattia di Brody. Il bovino di razza Chianina affetto da PMT è stato di fatto accettato come un interessante e utile modello animale non convenzionale per lo studio di questa condizione patologica umana. L’esame istopatologico delle biopsie muscolari ottenute dai soggetti RMG e VRB non hanno mostrato alterazioni particolarmente severe. Sono state individuate alcune fibre in degenerazione sempre accompagnate da fenomeni di rigenerazione (piccole fibre positive per la MHC neonatale). I protocolli istochimici (COX-SDH e PAS) non hanno evidenziato alterazioni metaboliche o cambiamenti nella distribuzione del glicogeno. Le indagini immunoistochimiche sull'espressione SERCA1 hanno mostrato una diminuita immunoreattività nei casi patologici suggerendo una minore presenza della proteina. Le analisi biochimiche effettuate sulle frazioni microsomiali hanno confermato la sola riduzione in contenuto di SERCA1, mentre né il RyR (marker del SR giunzionale) né SL e GP53 (marker del SR non-giunzionale) hanno mostrato alcuna variazione. Saggi di attività per la Ca2+ -ATPasi hanno associato una minore attività della proteina alla diminuzione quantitativa della stessa, mentre la Real Time RT-PCR ha dimostrato che l’mRNA della SERCA1 è espresso normalmente suggerendo che la riduzione della proteina avviene post-trascrizione. Un’interessante osservazione è stata che le sezioni del soggetto VRB mostravano una maggiore e più omogenea colorazione per COX e SDH suggerendo un aumento in contenuto di mitocondri. Quest'ultimo risultato è stato rafforzato da indagini di immunoistochimica e immunoblot utilizzando un anticorpo contro una proteina della membrana mitocondriale esterna (Tom20). Inoltre, frazioni microsomiali ottenute dal soggetto VRB hanno mostrato un aumento in contenuto della Ca2+-ATPasi della membrana plasmatica (PMCA). Infine, alcune fibre dei soggetti analizzati in questo studio hanno mostrato immunoreattività per SERCA1 e fosfolambano (PLB, proteina regolatrice della SERCA2) o per entrambe le isoforme 1 e 2 suggerendo un'alterazione nel classico pattern di espressione esclusivo delle tre proteine. I risultati di questa ricerca descrivono la Pseudomyotonia Congenita Bovina nei soggetti RGM e VRB come una malattia muscolare caratterizzata da alterazioni istopatologiche lievi e una diminuzione del contenuto e dell'attività della SERCA1, sovrapponibile al quadro descritto nella Chianina. Le analisi effettuate su VRB e RGM hanno permesso di ottenere un’immagine dettagliata della PMT in queste razze e, insieme ai dati sulla Chianina, ha permesso di definire un quadro istopatologico e biochimico generale della PMT. Inoltre, per la prima volta sono stati dimostrati meccanismi compensatori fornendo prove istochimiche e biochimiche a sostegno di un coinvolgimento dei mitocondri e della PMCA nell’omeostasi del Ca2+ in situazioni patologiche come la PMT. Infine, il ritrovamento di fibre che co-esprimevano SERCA1, SERCA2 e PLB è stato un risultato collaterale interessante che suggerisce una elevata plasticità del muscolo bovino che lo avvicina ancora una volta al muscolo umano. Un progetto parallelo è stato sviluppato in collaborazione con il gruppo del Prof. R. Smith e del Dr. J. Dudhia del Clinical Sciences and Services Department of the Royal Veterinary College (London). Due diversi studi pilota sono stati effettuati per valutare la risposta della matrice extracellulare alla lesione tendinea nel cavallo. Il primo ha avuto come oggetto di studio la cartilage oligomeric matrix protein (COMP), recentemente proposta come biomarker di tendinopatia. Lo scopo di questo progetto è stato quello di valutare il livello di frammentazione (indice di alterazione, ma anche rimodellamento) di COMP nel tessuto tendineo sano raccolti in siti noti per essere più frequentemente coinvolti nelle lesioni (predisposti a lesione). I tendini analizzati sono stati il flessore digitale superficiale (SDFT) e il flessore digitale profondo (DDFT). L’immunoblot e l’analisi densitometrica di estratti totali di proteine hanno mostrato un più alto livello di frammentazione della COMP in siti sani ma predisposti a lesione quando confrontati con i siti non predisposti. Inoltre, per la prima volta sono stati descritti i diversi pattern di espressione della COMP in SDFT e DDFT di soggetti giovani e vecchi. Il secondo studio pilota ha analizzato l'aspetto morfologico del tessuto tendineo affetto da lacerazione (tear-type lesion). L'obiettivo è stato quello di valutare l'influenza della lesione sul tessuto adiacente, lontano e remoto al sito della lesione. Il tendine era estremamente alterato vicino alla lesione mentre nelle altre aree rimanevano un’alta cellularità e vascolarizzazione ma l’organizzazione della matrice migliorava notevolmente. La tricromica di Masson distingueva il tessuto danneggiato (blu) da quello sano (rosso) proponendosi come un interessante strumento nelle indagini istologiche. L'immunoistochimica ha mostrato un aumento della marcatura per COMP nelle aree più vicine allo strappo rafforzando gli studi che propongono questa proteina come fondamentale nel rimodellamento della matrice. Aumentando le conoscenze sulla distribuzione della COMP in SDFT e DDFT, e fornendo dati sulla risposta tissutale del tendine lacerato, questi due studi pilota hanno creato una piattaforma utile per le future ricerche con possibili implicazioni cliniche e terapeutiche

Citoarchitettura e caratteristiche neurochimiche dell'archicorteccia del tursiope (Tursiops truncatus)

SOno state analizzate la Citoarchitettura e la morfologia cellulare della formazione ippocampale di tursiope (tursiops truncatus) con colorazione di NISS1. Tramite metodo immunoenzimatico è stata caratterizzata la distribuzione delle proteine leganti il calcio (Parvalbumina Calbindina e Calretinina) e la presenza e organizzazione della popolazione Astrogliale

Natural mutation in bovine Sarco(endo)plasmatic Reticulum Ca2+ATPase1 (SERCA1): histological and biochemical aspect in the muscle fibers

Congenital Pseudomyotonia (PMT) is an inherited muscular disease affecting bovine species. Clinically it is characterized by an exercise-induced muscle contraction. The aetiology is related to a prolonged high level of cytosolic free Ca2+ ion in muscle fibers. Genetic analysis has provided evidence of mutations in ATP2A1 gene coding for Sarco(endo)plasmic Reticulum Ca2+-ATPase, isoform1 (SERCA1) a membrane protein involved in re-uptake of calcium from cytosol into sarcoplasmic reticulum [1]. The clinical symptoms and genetic correlations make bovine PMT the true counterpart of human Brody\u2019s disease [2,3,4]. SERCA1 has been largely investigated in structure, domains and functional mechanisms [5,6]. Mutant cDNAs were expressed in cell culture and SERCA1-null mice has been created to investigate the importance of this protein and the implication of the different domains. It has been demonstrated how a single substitution of Arginine 560 (in mouse and rabbit) in domain N leads to a severe alteration of the protein conformation with a substantial reduction of its functionality [7,8]. Our group is working on PMT and interestingly a pathological case, a Dutch cross-breed calf, has shown a natural mutation in ATP2A1 gene leading to a substitution of Arginine 559, corresponding to Arginine 560 in mouse and rabbit. This spontaneous mutation produce a situation similar to the experimental one. Some preliminary histological and biochemical results have been published in a case report [9]. Here we present new histological analysis and early results in the study of the bovine natural mutation in heterologous system

Fast-twitch skeletal muscle fiber adaptation to SERCA1 deficiency in a Dutch Improved Red and White calf pseudomyotonia case.

Missense mutations in ATP2A1 gene, encoding SERCA1 protein, cause a muscle disorder designed as congenital pseudomyotonia (PMT) in Chianina and Romagnola cattle or congenital muscular dystonia1 (CMD1) in Belgian Blue cattle. Although PMT is not life-threatening, CMD1 affected calves usually die within a few weeks of age as a result of respiratory complication. We have recently described a muscular disorder in a double muscle Dutch Improved Red and White cross-breed calf. Mutation analysis revealed an ATP2A1 mutation identical to that described in CMD1, even though clinical phenotype was quite similar to that of PMT. Here, we provide evidence for a deficiency of mutated SERCA1 in PMT affected muscles of Dutch Improved Red and White calf, but not of its mRNA. The reduced expression of SERCA1 is selective and not compensated by the SERCA2 isoform. By contrast, pathological muscles are characterized by a broad distribution of mitochondrial markers in all fiber types, not related to intrinsic features of double muscle phenotype and by an increased expression of sarcolemmal calcium extrusion pump. Calcium removal mechanisms, operating in muscle fibers as compensatory response aimed at lowering excessive cytoplasmic calcium concentration caused by SERCA1 deficiency, could explain the difference in severity of clinical signs

ERAD plays a critical role in three severe muscle diseases.

The key pathogenetic event of an extremely heterogeneous group of genetic diseases, collectively called Unfolded Protein Diseases (UPDs), is the presence of gene mutations that cause either unfolding or misfolding of a coded protein. This usually leads to either toxic gain of function, because of the presence of protein aggregates, or loss of function because of the premature disposal of the mutated, but potentially functional, protein by the cell\u2019s quality control system (QCS). Type 2D Limb Girdle Muscular Dystrophy (LGMD-2D), a severe myopathy due to defect of the SGCA gene, encoding \u3b1-sarcoglycan (\u3b1-SG), has been recently included in the list of UPDs. \u3b1-SG is a type I membrane protein forming a tetrameric complex together with \u3b2-, \u3b3- and \u3b4-SG in the sarcolemma of striated muscle. We have demonstrated that, mutated \u3b1-SG is intercepted by the ER quality control, retrotranslocated into the cytosol, polyubiquitylated and degraded by the proteasome. Loss of the protein has dramatic consequences also on the other complex subunits, which are rapidly eliminated, although perfectly normal. Two additional UPDs have been considered to affect striated muscles, such as cattle congenital pseudomyotonia (PMT) and recessive Cathecolaminergic Polymorphic Ventricular Tachycardia (CPVT). PMT is due to defects in the ATP2A1 gene coding for sarco(endo)plasmic reticulum Ca2+-ATPase isoform 1 (SERCA1). We have recently demonstrated that cattle PMT is the true counterpart of human Brody\u2019s disease. CPVT has been linked to mutations in CASQ2 and TRDN genes, encoding calsequestrin 2 and triadin, respectively. Despite showing largely different clinical phenotypes both share a common feature, i.e., normal levels of the mutated transcripts and almost undetectable levels of the coded products. Our evidence shows that such proteins, being misfolded, are intercepted by the QCS and degraded by the ubiquitin-proteasome system. Relevant for possible therapeutical treatments, we demonstrate that usually protein mutants retain their function. Importantly, we show that some \u3b1-SG and SERCA1 mutants can be rescued by manipulating their degradative pathways, thus providing the basis for new potential therapeutical strategies

Pseudomyotonia in Romagnola cattle caused by novel ATP2A1 mutations.

BACKGROUND Bovine congenital pseudomyotonia (PMT) is an impairment of muscle relaxation induced by exercise preventing animals from performing rapid movements. Forms of recessively inherited PMT have been described in different cattle breeds caused by two independent mutations in ATP2A1 encoding a skeletal-muscle Ca2+-ATPase (SERCA1). We observed symptoms of congenital PMT in four related Romagnola beef cattle from Italy and evaluated SERCA1 activity and scanned ATP2A1 for possible causative mutations. RESULTS We obtained four PMT affected Romagnola cattle and noted striking clinical similarities to the previously described PMT cases in other cattle breeds. The affected animals had a reduced SERCA1 activity in the sarcoplasmic reticulum. A single affected animal was homozygous for a novel complex variant in ATP2A1 exon 8 (c.[632 G>T; 857 G>T]). Three out of four cases were compound heterozygous for the newly identified exon 8 variant and the exon 6 variant c.491 G>A(p. Arg146Gly), which has previously been shown to cause PMT in Chianina cattle. Pedigree analysis showed that the exon 8 double mutation event dates back to at least 1978. Both nucleotide substitutions are predicted to alter the SERCA1 amino acid sequence (p.[(Gly211Val; Gly284Val)]), affect highly conserved residues, in particular the actuator domain of SERCA1. CONCLUSION Clinical, biochemical and DNA analyses confirmed the initial hypothesis. We provide functional and genetic evidence that one novel and one previously described ATP2A1 mutation lead to a reduced SERCA1 activity in skeletal muscles and pseudomyotonia in affected Romagnola cattle. Selection against these mutations can now be used to eliminate the mutant alleles from the Romagnola breed

Farmers’ attitudes towards antimicrobial use and awareness of antimicrobial resistance: a comparative study among turkey and rabbit farmers

We conducted a survey of attitudes towards antimicrobial use and awareness of antimicrobial resistance among turkey and rabbit farmers (N = 117 and N = 41, respectively) in Italy’s utmost turkey- and rabbit-producing region. We found either similarities or significant differences between these two livestock sectors. Most farmers of both groups (72% of turkey farmers vs 76% of rabbit farmers) reported that antimicrobials are properly used in their farms. Almost three-quarters of the farmers reported that antimicrobials solve the health problem treated for. However, 47% of turkey farmers and 78% of rabbit farmers reported that antimicrobial use could be decreased, with a 20–30% reduction being the most frequently chosen range. Genetic improvement was reported to be the main factor able to reduce antimicrobial use in turkeys, whereas improvements in feed quality and microclimate were the main factors for rabbits. Most farmers reported that high antimicrobial use may affect the quality of meat products and be hazardous to human health, but they also reported that antimicrobial resistance is mainly related to antimicrobial use in humans. In conclusion, turkey and rabbit farmers have a generally positive opinion on veterinary antimicrobial use, but also low levels of awareness of the negative impact on public health. Economic and structural factors of rabbit production industry may explain the differences observed. Farm veterinarians will be crucial to support farmers’ education and the expected transition to lower antimicrobial use while maintaining high animal health/welfare standards.Highlights 47% of turkey vs. 78% of rabbit farmers thought that antimicrobial use can be decreased A 20–30% reduction of antimicrobials was the most frequently supposed range Turkey and rabbit farmers showed a low level of awareness of the negative impact of antimicrobial usage in their farms on human healt