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A possible role of superoxide dismutase 2 in the pathogenesis of parkinson disease
No full textParkinson disease is the second most common neurodegenerative disorder after Alzheimer disease. Less than the 5% of the cases are associated with recessive or dominant Mendelian inheritance. It is characterized by tremor at rest,
bradykinesia, postural instability and rigidity. The pathogenesis is still unknow but evidence suggests multifactorial mechanisms. In particular, mitochondrial dysfunction and oxidative stress are believed to have a central role in the pathology.
The hallmark of PD is the loss of dopaminergic neurons of substantia nigra pars compacta, hence dopamine may play an important role in the etiology due its metabolism. In fact, the cytosolic oxidation of dopamine may be very deleterious
to neurons. This neurotransmitter, once synthesized, is sequestered in acid vesicles. However, mitochondrial dysfunction, in particular the inhibition of the complex I, causes ATP depletion, which can perturb the H+-ATPase necessary to
drive the dopamine into vesicles. The consequence will be a redistribution of the neurotransmitter in cytoplasm, where it can auto-oxidize spontaneously at alkaline pH yielding reactive dopamine quinones (DAQs) and reactive oxygen species
(ROS) contributing to oxidative stress. DAQs are suggested to be involved in dopaminergic neurotoxicity because they can form adducts with cysteine residues exposed at the surface of proteins to generate 5-S-cysteinyl-DA. The covalent
modification of proteins could lead to the impairment of their functional properties.
The cells have different mechanisms to protect from oxidative stress: catalase, glutathione peroxidise and superoxide dismutase (SOD). The function of SOD enzymes consists in the dismutation of superoxide anions to hydrogen peroxide and molecular oxygen. Among the three different isoforms of SOD,
SOD2 is particularly important due its mitochondrial localization. Mitochondria are in fact the main source of superoxide anions.
The aim of this thesis is to investigate if SOD2 is a target of DAQs in vitro, the effects of this interaction on the enzymatic function of the protein and the residue/s target of DAQs.
The starting point was the cloning of the cDNA of the human SOD2 in the pET28a+ vector. Since E.coli present 3 different SOD that could potentially interfere with the purification, a second cloning was obtained, in which the cDNA was inserted downstream a cleavable His-tag. A protocol of SOD2 purification
was set up but a control purification, where the cells were transformed with empty vector, led to purify an endogenous SOD of E.coli demonstrating that a E.coli SOD co-purify with the human recombinant enzyme. To avoid the co-purification,
a protocol of purification was optimized for the protein with the His-tag leading to obtain 40-60 mg of protein per 1 liter of culture.
The interaction between SOD2 and DAQs was investigated using different biochemical and biophysical techniques. Dopamine oxidation was induced by the addition of tyrosinase, which prevented the formation of radical species normally
produced during dopamine auto-oxidation. It was verified that the protein did not interact with tyrosine residues of SOD2. The protein was incubated with dopamine at different ratio SOD2: dopamine in the presence of tyrosinase, following by UV-vis spectroscopy the formation of DAQs. The interaction of these products with the protein was studied by mass spectrometry analysis, which revealed the formations of new species whose molecular masses are compatible with the covalent modification of one cystiene residue with dopamine-o-quinone and with the indole 5,6 quinone. Other species were detected, however they were not attribuible to a specific DAQ, hence they might result from more complex interactions. Moreover, the presence of +287.8 Da product suggested the interaction of both the cystiene residues. SDS-PAGE analysis of the reaction products revealed the presence of the protein dimer and aggregates. Native-PAGE showed multiple bands and aggregates upon the interaction with DAQs. The multiple band profile was assigned to the combination of tetramers with monomers of SOD2 with different modifications (as demonstrated by mass analysis). This was supported by 2 dimensional gel, where at least 4 products of SOD2 with different isoelectric point appeared. Activity assay indicated inhibition of proten covalently modified by DAQs. Hence, pulse radiolysis measurements were performed to determine the kinetic parameters of the dismutation reaction.
Data are currently under processing.
To identify the site of DAQs interaction, the cysteine residues were mutagenized obtaining the following mutated proteins: C140A, C196A and C140A/C196A. Incubation of the mutants incubated C14 labeled dopamine demonstrated that the cysteine 196 was the primary target of DAQs. This result was confirmed also by mass spectrometry. The enzymatic activity of the mutant C140A upon interaction of dopamine proved to be lower in comparison with the unmodified protein.
A weakly positive radioactivite signal was found also in association with the mutant C196A and the C140A/C196A suggesting secondary site of DAQs interaction. Since two covalent DAQ-derivatives were found by mass spectrometry after the incubation of the mutant C140A with dopamine, the
existence of a second site of interaction probably another nucleophilic residue is suggested.
Structural changes of SOD2 upon DAQs modification suggested protein precipitation and a possible change in tertiary structure. HFEPR spectra did not show any significant changes in metal coordination.
The results presented in this thesis demonstrate that SOD2 is covalently modified by DAQs in vitro. This suggests a possible role of SOD2 in the PD. The idea is that SOD2 can be covalently modified by DAQs in mitochondria. This would increase the oxidative stress in mitochondria, due to the decrease of dismutase activity of the target protein, ultimately determining mitochondrial dysfunction and contributing to the neuron cell death.La malattia di Parkinson è la malattia neurologica degenerativa più diffusa dopo la malattia di Alzheimer. Meno del 5% dei casi presenta una correlazione genetica (ereditarietà autosomica dominante o recessiva). I sintomi principali caratteristici sono tremore a riposo, bradicinesia, instabilità posturale e rigidità.
L’eziologia rimane ancora sconosciuta ma evidenze perimentali suggeriscono che la malattia sia multifattoriale. In particolare due fattori, disfunzione mitocondriale e stress ossidativo, sembrano avere un ruolo chiave nella patogenesi.
La caratteristica della malattia di Parkinson è la perdita dei neuroni dopaminergici della substantia nigra pars compacta. Questo fa presupporre che la dopamina abbia un ruolo importante nell’eziologia attraverso il suo metabolismo.
Infatti, l’ossidazione citosolica della dopamina può contribuire alla tossicità neuronale. Questo neurotrasmettitore, una volta sintetizzato, in condizioni normali è sequestrato nelle vescicole sinaptiche. Tuttavia, la disfunzione mitocondriale, in particolare l’inibizione a livello del complesso I riscontrata nella malattia di Parkinson, porta a causare una diminuzione dei livelli di ATP. Questo può influenzare il trasporto di dopamina nelle vescicole che è ATP-dipendente. La conseguenza sarebbe una ridistribuzione del neurotrasmettitore nel citoplasma,
dove può auto-ossidarsi spontaneamente a pH alcalini producendo dopaminochinoni (DAQs) e specie reattive dell’ossigeno (ROS) che contribuiscono allo stress ossidativo. E’ stato suggerito che i DAQs possano essere coinvolti nella
neurotossicità dei neuroni dopaminergici. Questo perchè possono interagire con le cisteine esposte sulla superficie di proteine generando 5-S-cisteinil-DA e la loro modificazione covalente da parte dei DAQs può compromettere la funzionalità e stabilità di queste proteine.
Le cellule possiedono differenti meccanismi per proteggersi dallo stress ossidativo: catalasi, glutatione perossidasi e superossido dismutasi (SOD). La funzione delle SOD consiste nel dismutare l’anione superossido a perossido d’idrogeno e ossigeno molecolare. Tra le tre isoforme di SOD presenti nell’uomo, la SOD2 è particolarmente importante a causa della sua localizzazione all’interno del mitocondrio.
Lo scopo di questa tesi è quello di investigare se la SOD2 sia un target dei DAQs in vitro, gli effetti di questa interazione sull’attività enzimatica della proteina e il/l residuo/i target dei DAQs.
Il punto di partenza è stato il clonaggio del cDNA della SOD2 umana nel vettore pET28a+. Dato che E.coli possiede tre differenti SOD che potrebbero interferire con la purificazione, un secondo clonaggio è stato effettuato, clonando l’inserto a valle di un histidine-tag. È stato messo a punto un protocollo di
purificazione della SOD2 ma la purificazione di controllo, dove le cellule erano state trasformate con il vettore senza l’inserto, ha portato alla purificazione di una SOD endogena di coli. Questo ha dimostrato che una SOD di coli co-purificava con l’enzima umano ricombinante. Per evitare questo, un protocollo di purificazione è stato ottimizzato con l’His-tag ottenendo 40-60 mg di proteina da un litro di coltura.
L’interazione tra la SOD2 e i DAQs è stata indagata utilizzando differenti tecniche biochimiche e biofisiche. L’ossidazione della dopamina è stata indotta dall’aggiunta di tirosinasi, che previene la formazione di specie radicaliche normalmente prodotte durante la sua auto-ossidazione. È stato verificato che la tirosinasi non interagisce con i residui tirosinici della proteina. La SOD2 è stata incubata con differenti rapporti SOD2:dopamina in presenza di tirosinasi, seguendo la formazione dei DAQs con spettroscopia UV-visibile. L’interazione di questi prodotti con la proteina è stata studiata tramite spettrometria di massa, che ha rilevato la formazione di nuove specie le cui masse erano compatibili con la modificazione covalente di una cisteina con il dopamino-o-chinone e con l’indolo 5,6 chinone. Altre specie sono state identificate ma non è stato possibile attribuirle ad uno specifico chinone, quindi queste potrebbero essere il risultato di interazioni più complesse. Inoltre, la presenza di una doppia modifica ha suggerito la possibile interazione di entrambe le cisteine della proteina. Analisi su SDS-PAGE dei prodotti di reazione ha mostrato la presenza di dimeri di proteina e aggregati.
Native-PAGE ha rilevato un pattern di bande multiple e aggregati, dopo interazione con i chinoni. Il profilo delle bande multiple è stato attribuito alla combinazione monomeri di SOD2 con differenti modifiche (come dimostrato dalle analisi di spettrometria di massa) associati a formare tetrameri. Questo è
supportato dal profilo nel gel bidimensionale, dove sono presenti 4 prodotti di SOD2 con differente punto isoelettrico. Il saggio enzimatico ha rilevato che la proteina modificata ovalentemente dai DAQs risulta essere inibita. Misure di radiolisi pulsata sono state effettuate per determinare i parametri cinetici della reazione di dismutazione.
Per identificare i siti di interazione dei DAQs, le cisteine sono state mutagenizzate in alanine ottenendo i seguenti mutanti: C140A, C196A e C140A/C196A. L’incubazione dei mutanti con C14-dopamina ha dimostrato che il target primario dei DAQs è la cisteina 196. Questo risultato è stato confermato anche da analisi di spettrometria di massa. L’attività enzimatica del mutante 140 dopo interazione con DAQs risulta essere minore rispetto alla proteina non modificata.
Un debole segnale di radioattività è stato trovato anche in associazione con il mutante 196 e il doppio. Ciò suggerisce la presenze di un sito di interazione con i chinoni secondario. Dato che due modificazioni dovute a DAQs sono state trovate incubando il mutante C140A, si deduce che il secondo sito di interazione coinvolga un altro residuo nucleofilo.
Cambiamenti strutturali della SOD2 suggeriscono precipitazione della proteina un possibile cambiamento della struttura terziaria. Inoltre modificazioni rilevanti nella coordinazione del metallo non sono state rilevate mediante HFEPR.
I risultati presentati in questa tesi dimostrano che la SOD2 è un target dei DAQs in vitro suggerendo l’idea che la SOD2 possa essere coinvolta nella malattia di Parkinson. Una volta modificata dai DAQs, lo stress ossidativo aumenterebbe nei mitocondri, a causa della inibizione dell’attività dismutasica
dell’enzima, determinando disfunzione mitocondriale e contribuendo alla morte neuronale
Editorial: New trends in osteoarthritis treatment
Full text linkOsteoarthritis (OA) is the most common type of arthritis affecting millions of persons
worldwide (1, 2). It is a complex and multifactorial disease that could affect any joint, but
particularly the knee, hip and hands. All the joint tissues are involved, including synovial
membrane, subchondral bone, infrapatellar fat pad, subchondral bone, and especially
cartilage, which undergoes several changes impacting its biomechanical behavior (3–5).
These changes lead to swelling, pain, and difficulty in joint movement, thus impacting
quality of lif
Giant cell reparative granuloma of the scapula: report of a case and literature review
Full text linkGiant cell reparative granulomas (GCRGs) are non-neoplastic inflammatory lesions, usually observed in the maxilla, mandible or small bones of the hands and feet. These lesions present a wide range of morphology and the misinterpretation with other giant cell lesions can often occur. We report the case of a 47-year-old woman with GCRG in the left scapula, presenting some uncommon features: the location (scapula) and age at presentation, the lack of underlying bone disease such as Paget's disease or fibrous dysplasia, the large aggressive expansile aspect of the lesion. This was a therapeutic study, level IV (case series with no or a historical control group)
Metastatic lesion from clear cell renal carcinoma after 40 years and a review of the literature
Full text linkRenal cancer carcinoma (RCC) is among the ten most common cancer in the worldwide affecting more frequently men 1. Clear cell renal cancer carcinoma (ccRCC) is the most frequent subtype and accounts for the majority of renal cancer death 1. The treatment depends on the tumor characteristics and staging at diagnosis. If RCC is localized, the treatment consists of surgery (partial or total nephrectomy) followed by surveillance, while if it is disseminated, surgery (if possible) should be followed by single/multiple target therapy or immunotherap
Musculoskeletal Diseases: From Molecular Basis to Therapy
Full text linkMusculoskeletal diseases (MSDs) comprise a plethora of different disorders (more than 150 conditions) affecting the locomotor system. Importantly, they are associated with significant morbidity and disability, impacting the quality of life of patients. The most common MSDs are osteoarthritis (OA), low-back pain (LBP), neck pain (NP), rheumatoid arthritis (RA), and gout [1]. A recent analysis of the Global Burden of Disease published in 2022 estimated that
approximately 1.71 billion people globally are affected by musculoskeletal disorders [2]. Although MSDs are widespread and the number of affected individuals is expected to increase as the population ages, MSD research has received little attention, likely because MSDs are rarely fatal and are assumed to be irreversible pathologies [1,2]. Thus, a better understanding of the etiology, biomarkers, as well as new and more effective therapeutic treatments, are needed. In this context, the purpose of this Special Issue, entitled “Musculoskeletal Diseases: From Molecular Basis to Therapy”, is to report on advances in pathophysiological mechanisms, the identification of biomarkers, and preclinical and clinical therapeutic approaches
to MSDs
Musculoskeletal Diseases: From Molecular Basis to Therapy (Volume II)
Full text linkMusculoskeletal diseases (MSDs) are among the leading causes of disability worldwide, placing a significant burden on healthcare systems and reducing patients’ quality of life [...
Mechanical insights into fat pads: a comparative study of infrapatellar and suprapatellar fat pads in osteoarthritis
No full textObjective: Osteoarthritis (OA) is the most common musculoskeletal disorder, primarily affecting knee joints and causing pain and disability. The infrapatellar (IFP) and the suprapatellar (SFP) fat pad are knee adipose tissues that play essential mechanical roles during articular activity but are also sources of adipokines and cytokines, contributing to OA progression. For this reason, this work aims to provide new insights into IFP and SFP implications in knee OA. Materials and methods: IFP and SFP tissue mechanical properties were studied through compression, indentation and shear mechanical tests performed on samples collected from patients who underwent total knee arthroplasty surgery due to end-stage OA. The energy loss, peak stress, and initial and final elastic moduli were calculated from the unconfined compression tests. The time-dependent response, evaluated in terms of equilibrium relative stiffness, was computed from stress-relaxation loading conditions. Considering shear tests, they provided strain-energy dissipation density, peak shear stress, and the shear moduli. Results: Experimental results showed the typical adipose tissue mechanics features: non-linear stiffening with strain and time-dependent response. Experimental results showed that OA IFP is stiffer than OA SFP, indeed IFP final compression elastic modulus was greater than the SFP (84.43 kPa vs 35.54 kPa respectively) (p = 0.042). Regarding the viscoelastic properties they were comparable: the equilibrium relative stiffness was reported as 0.13 for IFP and 0.11 for SFP (p = 0.026). Conclusions: These outcomes provide new insights into the OA influence on knee mechanics and lay the basis for developing computational tools to improve knee prosthesis design
The challenging management of a delayed union midshaft clavicle fracture complicated by an acute pseudoaneurysm of the subclavian artery in a superelderly diabetic patient
Full text linkClavicle fractures are among the most common fractures, accounting for 2.6–4% of all adult fractures and for 35–44% of those of the shoulder girdle. Up to 80% occur at the midshaft. Nowadays, the majority of these fractures tend to be treated non-operatively, even when displaced, using an arm supporting sling or a “figure-of-eight” bandage, with good clinical outcomes and an acceptable rate of nonunion.
On the contrary, operative treatment is commonly performed in cases of open fractures, skin tenting with the potential for progression to open fracture, “floating shoulder,” and associated acute neurovascular injuries. Despite the proximity of the clavicle to the subclavian vessels, vascular complications in closed clavicle fractures are uncommon, with an incidence of 0.4% [2]. Nevertheless, their prompt diagnosis and proper knowledge is essential because of the high morbidity and mortality rates associated. This report describes the clinical, diagnostic, and therapeutic approaches to address an acute subclavian artery pseudoaneurysm caused by a closed displaced clavicle fracture, complicated by delayed union, in a comorbid octogenarian patient
Predictors of early failure of the cannulated screw system in patients, 65 years and older, with non-displaced femoral neck fractures
Full text linkBackground: Hip fractures represent the most common injury and the main cause of morbidity and mortality among patients 65 years and older. About 20% of all femoral neck fractures (FNFs) are non-displaced or valgus impacted, for which internal fixation with the cannulated screws system (CSS) is indicated. Aims: The aim of this study was to identify predictors of early failure of CSS. Methods: Patients with non-displaced FNFs (Garden type I and II) treated operatively using the CSS were enrolled. Their characteristics, Pauwels angle, and posterior tilt were assessed and related with outcomes. The primary outcome was fixation failure within 6 months. Results: 259 patients were included with a mean age of 81.44 years. Most patients were female with ASA 3. The majority of fractures were classified as Garden I and Pauwels I. On average, Pauwels angle was 27°, while posterior tilt was 12°. A linear correlation between Pauwels angle and posterior tilt was found; the failure rate was 9.7%. Using the adjusted Cox competing risk regression analysis, posterior tilt was found to be independently associated with failure rate (sub-distribution hazard ratio or SHR 1.14 [95% CI 1.05–1.24], p = 0.0020). A posterior tilt greater than 18° resulted predictive of failure. The 1-year mortality rate was 12%. Conclusions: Non-displaced Garden type II fractures, Pauwels type II or III fractures, and a posterior tilt greater than 18° represent radiographic predictors of CSS early failure in the elderly. Level of evidence: Level IV, retrospective cohort study
Girolamo Fabrici d'Acquapendente and the Oplomochlion: the several applications of an effective rehabilitation tool
Full text linkGirolamo Fabrici d'Acquapendente (1533-1619) was an Italian anatomist, surgeon and physiologist and a protagonist of the scientific revolution of the Renaissance. He made anatomy a scientific discipline and is justly considered a precursor of modern orthopaedics. He invented and used several external corrective devices for the treatment of congenital and acquired deformities of the limbs and spinal column, especially those following tubercular infection and rickets, torticollis, vertebral caries kyphosis, scoliosis, and rachitic deformities of the leg, but also congenital dislocation of the hip and congenital club-foot. He ascribed the pathogenesis of the equinovarus supinated foot to the position taken by the foot of the fetus during intrauterine life. The Oplomochlion, shown in the Operationes chirurgicae and attributed to Fabrici, is actually a collection of very diverse orthotic, prosthetic and surgical metal instruments invented by Fabrici and arranged with a demonstrative purpose and a topographic criterion, as if on an exhibition dummy
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