SDH assembly in S. cerevisiae: focus on the functional role of SDH6

La succinato deidrogenasi (SDH) o complesso II è un enzima chiave del ciclo di Krebs e della catena respiratoria mitocondriale. E’ una ferro zolfo flavo proteina che catalizza l’ossidazione del succinato a fumarato con relativo passaggio degli elettroni dal FADH2 attraverso i tre centri Fe-S e infine all’ubichinone che viene ridotto ad ubichinolo. L’enzima, ubiquitario in tutte le specie, è un eterotetramero, di 124 kDa, composto da quattro sub-unità che sono codificate da altrettanti geni nucleari, SDHA-D nei mammiferi (corrispondenti ai geni SDH1-4 in lievito). E’ noto che difetti nelle diverse subunità dell’enzima sono associate a patologie differenti in relazione a quale gene è mutato. Infatti mentre mutazioni germinali in SDHA sono associate ad una patologia neurodegenerativa, la Sindrome di Leigh, mutazioni a carico di SDHB, SDHC, SDHD predispongono alla formazione di forme tumorali quali paragangliomi o feocromocitomi. Sebbene la struttura e la funzione del complesso SDH siano note da anni, poco si sa circa il suo assemblaggio. Solo recentemente sono stati descritti due nuovi fattori, SDHAF1(SDH6 in lievito) e SDHAF2 (SDH5) che hanno un ruolo evolutivamente conservato e dedicato nell’assemblaggio del complesso II. Anche mutazioni a carico dei geni che codificano per questi due fattori di assemblaggio sono associate a patologie diverse: disturbi mitocondriali e cancro. Il primo assemblatore specifico per la succinato deidrogenasi, Sdh6p, Sdhaf1p nell’uomo, è stato scoperto in seguito ad uno studio, compiuto presso l’Istituto Neurologico C.Besta dal gruppo di ricerca del Dott. M. Zeviani, su bambini affetti da leucoencefalopatia progressiva, associata ad una significativa diminuzione sia di attività enzimatica SDH che dei livelli di enzima SDH, supportato da studi eseguiti in lievito (Ghezzi et al., 2009). L’analisi di sequenza ha individuato due mutazioni missenso R55P e G57R in una regione, chiamata LOC644096, che codifica per un putativo prodotto genico di 115 aminoacidi a funzione sconosciuta. Allo scopo di valutare se le mutazioni missenso che cosegregavano con la patologia fossero effettivamente responsabili della diminuita attività del complesso II, è stato utilizzato il lievito come sistema modello. Numerosi studi hanno infatti dimostrato che questo organismo è un ottimo modello sia per studiare gli effetti primari delle mutazioni patogene che per determinare i meccanismi molecolari che inducono patologie mitocondriali. Il lievito presentava un putativo ortologo del gene umano, anche esso a funzione sconosciuta che codifica per una piccola proteina di 79 aa: YDR379c-a. L’allineamento delle due sequenze ha evidenziato come l’identità condivisa tra le due sequenze fosse del 21% e come i residui coinvolti nelle due mutazioni, R55P e G57R fossero conservati. Gli studi funzionali condotti nel lievito e in linee cellulari umane hanno indicato che la proteina identificata sia richiesta per l’assemblaggio e il corretto funzionamento del complesso II, dal momento che la sua mancanza determina una drastica riduzione della attività succinato deidrogena sica. Quale sia però il ruolo biochimico di Sdh6 nel processo di assemblaggio della SDH non è ancora stato chiarito. Obiettivo del seguente lavoro di ricerca è stato quello di contribuire alla comprensione della funzione di questo fattore utilizzando approcci genetici e biochimici. Sdh6p è caratterizzata dalla presenza di un motivo LYR che ci ha spinto ad indagare il ruolo di questa proteina nell’inserimento dei centri Fe-S nel backbone del complesso II. Questi motivi proteici difatti sono una signature per proteine coinvolte nel metabolismo dei clusters Fe-S. Sdhp6 inoltre, presenta una significativa similarità di sequenza con Isd11p, proteina nota essere necessaria per la biogenesi dei clusters Fe-S. Sulla base di tali considerazioni si è deciso di valutare se ISD11 in multi copia potesse ripristinare il fenotipo OXPHOS del mutante sdh6 e viceversa. I risultati indicano che la somiglianza strutturale tra i due geni non è accompagnata da una qualche omologia funzionale. E’ stato poi analizzato se SDH5 e TCM62, codificanti gli altri due fattori di assemblaggio del complesso II, espressi in multi copia ripristinassero la crescita ossidativa del mutante sdh6 e viceversa. In nessun caso si è ottenuto il rescue del fenotipo di crescita. In seguito sono state overespresse nel mutante nullo le quattro subunità SDH per valutare se questo potesse incrementare l’attività SDH. A differenza di quanto osservato nel ceppo parentale in cui l’attività SDH aumentava del 50% nel ceppo Δsdh6 che overesprime l’intero complesso non è stato osservato alcun incremento dell’attività SDH. Questo dato ha suggerito come Sdh6p sia un rate limiting factor nel processo di assemblaggio del complesso II. Risultati relativi alla determinazione del valore di Km per il succinato, uguale tra mutante e wild-type, hanno suggerito come l’abbattimento specifico dell’attività SDH nel mutante sdh6 sia dovuto ad un ridotto numero di unità enzimatiche piuttosto che ad un’alterazione qualitativa del complesso. Per aver ulteriore conferma di ciò sono stati dapprima valutati in SDS-PAGE i livelli di Sdh1p e Sdh2p nel ceppo Δsdh6. In secondo luogo è stato valutato lo stato di assemblaggio del complesso in condizioni native attraverso una BN-PAGE. I risultati ottenuti hanno consentito di affermare che la ridotta attività SDH misurata nel mutante è imputabile ad una riduzione del complesso SDH correttamente assemblato. Per valutare se Sdh6p contattasse il complesso o intermedi di questo o altri complessi molecolari sono state costruite delle varianti di Sdh6p taggate sia con l’epitopo HA che con la coda di istidine. I risultati ottenuti suggeriscono che Sdh6p non è associato né al complesso SDH né ad alcun complesso molecolare in quanto migra in forma monomerica. Utilizzando Sdh6 His-tag, sfruttando le interazioni tra Ni-NTA e i residui di 6HIS inseriti nella proteina, è stato dimostrato che Sdh6p non interagisce né con la subunità Sdh1 né con Sdh2. Parallelamente per cercare di ottenere maggiori informazioni circa il ruolo funzionale di SDH6 è stata intrapresa una ricerca di soppressori multi copia nel mutante nullo Δsdh6. Questo screening ha portato all’identificazione di due geni, YAP1 e YAP2 codificanti due fattori di trascrizione coinvolti in diversi processi cellulari. Tuttavia il rescue del difetto OXPHOS non era accompagnato da un ripristino dell’attività SDH. Questo risultato ha suggerito che Sdh6p svolgesse un altro ruolo nella cellula. Peraltro l’osservazione che diversi mutanti sdh, aventi la stessa attività residua SDH crescessero meglio su fonti di carbonio ossidabili rispetto al mutante sdh6, rafforzava l’ipotesi relativa ad un duplice ruolo di Sdh6p. Per capire in quale modo potesse agire il meccanismo di soppressione esercitato da Yap1p e Yap2p e quindi avere qualche indicazione circa l’altro ruolo di Sdh6 sono state eseguite alcune sperimentazioni sulla base di due funzioni svolte da Yap1 eYap2: risposta allo stress ossidativo e metabolismo del ferro. L’insieme dei risultati ottenuti favoriscono l’ipotesi che il meccanismo di soppressione esercitato da YAP1 e YAP2 sia da attribuire al loro ruolo legato al metabolismo/omeostasi del ferro. Infatti l’aggiunta di ferro al terreno di crescita consente il rescue del fenotipo OXPHOS nel mutante Δsdh6 non accompagnato da un incremento dell’attività SDH. Pertanto i risultati ottenuti suggeriscono che il secondo ruolo di Sdh6p possa essere correlato al metabolismo del ferro.Yeast is a well recognised model for the study of human mitochondrial pathologies thanks its exceptionally ability to survive without a functional mitochondrial respiratory metabolism, provided that a fermentable carbon source is made available. Succinate dehydrogenase (SDH, complex II) is a conserved mitochondrial enzyme of the inner membrane that catalyzes the oxidation of succinate to fumarate during TCA cycle, using at the same time the reducing equivalents in the electron transport chain (ETC). Despite the extensive knowledge of the structural and catalytic properties of the complex, only two assembly factors specific for the SDH complex has been recently found: SDHAF1 and SDHAF2. The SDHAF1 gene was identified in humans as linked to infantile leukoencephalopathy. A yeast strain deleted in SDH6, the SDHAF1 ortholog, was OXPHOS incompetent, due to a severe and specific reduction of SDH activity. However, the Km value for succinate was similar in wild-type and in the null mutant, suggesting that defective SDH activity was caused by reduced number of enzyme units rather than by qualitative alterations of complex II. We have given more insights into the knowledge of SDH6 through genetic and biochemical studies performed in S. cerevisiae. Affinity purification analyses have shown that Sdh6p is not a physical interactor of subunit 1 and 2 of SDH complex. Furthermore Sdh6p is not part of complex II or other molecular complexes, as shown in BN-PAGE analyses. Moreover, Sdh6p is a rate limiting factor of SDH assembly: indeed despite the overexpression of all four SDH subunits in the null mutant the SDH activity is not restored unlike what occurred in the wild type strain suggesting the pivotal role of this protein in the complex II assembly. The results obtained in this work suggested that SDH6 plays a different role from that played by SDH5, the yeast ortholog of SDHAF2. Indeed the overexpression of SDH6 did not rescue the OXPHOS defect of Δsdh5 and viceversa. Therefore the two SDH specific assembly factors are not interchangeable. The presence of a LYR motif in the Sdh6 protein prompted us to investigate a putative role in the insertion of Fe-S clusters in complex II. Isd11p, a protein involved in the iron-sulfur biogenesis, shares a significant sequence similarity with Sdh6p and contains a LYR motif. We tried to rescue the OXPHOS deficient phenotype of sdh6 mutant by overexpressing ISD11 but the results obtained indicated that the two proteins do not share similar functions. To further investigate the functional role of SDH6 a search for multicopy suppressors was performed. This analysis identified YAP1 and YAP2, two transcription factors involved in several cellular processes. Although YAP1 and YAP2 are able to rescue the OXPHOS defect of sdh6 mutant, their action is not exerted by increasing the SDH activity. Their suppression mechanism seems to be independent of complex II. Thus Sdh6p might have an additional role besides the SDH assembly: if this is the case the OXPHOS negative phenotype of sdh6 null mutant would be due to both a reduced SDH activity and to a lack of some other putative function. The observation that several sdh mutants with a 30-40% of residual SDH activity are able to grow on oxidative carbon sources much better than Δsdh6 strengthens this hypothesis. In order to understand the mechanism of suppression exerted by YAP1 and YAP2 we focused on two main functions linked to these genes: the oxidative stress and the iron metabolism. All together these analyses favor the hypothesis that YAP1/YAP2 suppression mechanism could be mainly ascribed to their role in iron metabolism/homeostasis. Moreover when the effects of iron supplementation on the oxidative growth of the Δsdh6 strain were tested a rescue of the OXPHOS phenotype was observed without any increase of SDH activity. The results obtained support the view that the second role of Sdh6p could be related to iron metabolism/homeostasis

Eating Disorders: Assessment of Knowledge on a Dentist’s Sample

Aim: To analyze the knowledge and experience of dentists with private practices in the Sassari district with regard to eating disorders and clinical stomatology manifestations. Methods: A questionnaire to investigate dentists’ knowledge and experience was created and submitted to a random sample of dentists. Data were collected and analysed using the Stata SE 10 software. Results: In total, 150 dentists were enrolled (a representative sample). After questionnaire evaluation, the following results were obtained: 80.7% (121) of the participants had a degree in dentistry and 19.3% (29) had degrees in medicine with a dentistry master of science diploma, 46% (69) were males and 54% (81) were females, their age range was 25-62 (mean, 36.1) years, and the average work experience was 11.5 (range, 1-36) years. More than 90% of the participants defined eating disorders only as bulimia and anorexia. Of the dentists, 77.3% (116) correctly identified Western populations as the most affected, whereas 52.7% identified only whites as the most affected people. In total, 80% of the dentists recognised dental erosion and abrasion as typical oral manifestations. Only 62% stated that salivary pH decreases in these conditions, and 63% did not recognise parotid gland tumefaction as a clinical sign. Regarding operator experience, 60.7% (91 dentists) had clinical experience of patients with eating disorders: 43.9% of them had made diagnoses from oral manifestations, 51.3% (77) were not able to treat these patients, and 69.3% (104) would refer patients to specialized centres for treatment. Moreover, 119 (79.3%) considered that a dental hygienist was a valid collaborator for the treatment of oral cavity manifestations. Regarding prophylaxis, 16.7% (25) suggested fluoride prophylaxis and 21.3% (32) oral hygiene education. Only 1.3% indicated the use of bicarbonate to change salivary pH values, suggested conservative or prosthetic restorations, suggested the use of saliva substitutes, or the sealing of permanent teeth. Difficulties in clinically treating these patients were related to a lack of knowledge for 72% (108) of participants. Of them, 36.7% (55) had studied the clinical aspects of this pathology while attending dental school and 24.7% (37) after their degree. In total, 94.7% (142) expressed the need for further information and only 2% (3) considered they had sufficient clinical knowledge. Conclusions: It is evident that there is a lack of knowledge regarding the problem and there would seem to be an urgent need to provide more training programs and to establish guidelines for the diagnosis and treatment of eating disorders among dentists. In our opinion, the acquisition of such knowledge will change the approach to related pathology, improving clinical skills, and subsequently diagnosis and treatment

Less air pollution did not explain the decline in admissions for AMI during the first wave of COVID-19 pandemic in Sardinia, Italy

During the COVID-19 pandemic, hospitalizations for acute myocardial infarction (AMI) decreased worldwide. We compared the admissions for AMI in the four regional 24/7 cath lab during the national lockdown, the 8 weeks before the lockdown, the 8 weeks after the e lockdown, and the corresponding time period in 2019 and we analyzed the average level of pollution in the studies areas. A marked decline in AMI admissions was observed during the lockdown period in comparison with the 8 weeks before the lockdown (p < 0.0001) and a significant increase in the 8 weeks after the lockdown (p < 0.00001). No significant change in air pollutants density were highlighted. Since air pollution did not change substantially in our region, the environment factor cannot explain the decline in the number of admissions for AMI we recorded during the lockdown. Fear of contagion is the most plausible reason for the drop of hospitalizations during the lockdown period

Conservazione ex situ e in situ della biodiversità vegetale dell'Area Marina Protetta di Capo Carbonara (Sardegna sud-orientale)

The first results of the project "Plant Biodiversity Conservation in the Protected Marine Area of Capo Carbonara (PMA)" are illustrated. This initiative, promoted by the CCB and the Municipality of Villasimius (CA), was funded for three-year period (2005/07) by the Ministry of the Environment and Territory protection. The project provides the study, monitoring and conservation of the coastal habitat, principally on all the Sites of Communitary Importance (pSIC) of the Villasimius Municipality territory, in order to plan a harmonic development between coastal and sea areas unifying the environmental protection secured by the PMA with that relatives to the Natura 2000 network. During the first year there were started ecological, floristic-sociological and chorological studies, in order to value the actual state of conservation and have a possibility to propose adequate measures for a sustainable management. In order to guarantee the effective plant biodiversity conservation, the following ex situ actions have been made: collection, study and conservation for a long time of the seed of selected species in the structures of the Sardinian Seed Bank (BG-SAR), according to international procedures and protocols. At the end of the first year the seed harvest was completed relatively to all the species with a greater risk of extinction. The project provides also actions directed to sensitize the population, local and non, on the thematics of the conservation and the sustainable development of the territory

Citizens of Nowhere? Paradoxes of State Parental Responsibility for Unaccompanied Migrant Children in the United Kingdom

Social workers are confronted with a contradictory task: that of acting as state parents for unaccompanied asylum seeking children, in an era of hostile migration policies and austerity. Mobilizing Young’s (2006) concept of ‘responsibility’ we ask: how is state parental responsibility towards unaccompanied minors given meaning, and with what consequences, for both frontline workers and unaccompanied minors alike? Drawing on interviews with frontline workers and unaccompanied minors in the UK (n = 107), we delineate three modes through which responsibility operates: namely outcomes, capacity and morality. We argue that the underlying logic of responsibility shifts the blame from sociopolitical structures to migrant children themselves, with crucial consequences for questions of social justice

Modelling and Automated Implementation of Optimal Power Saving Strategies in Coarse-Grained Reconfigurable Architectures

This paper focuses on how to efficiently reduce power consumption in coarse-grained reconfigurable designs, to allow their effective adoption in heterogeneous architectures supporting and accelerating complex and highly variable multifunctional applications. We propose a design flow for this kind of architectures that, besides their automatic customization, is also capable of determining their optimal power management support. Power and clock gating implementation costs are estimated in advance, before their physical implementation, on the basis of the functional, technological, and architectural parameters of the baseline design. Experimental results, on 90 and 45 nm CMOS technologies, demonstrate that the proposed approach guides the designer towards optimal implementation

Validation of the Treadmill Six-Minute Walk Test in People Following Cardiac Surgery

n/

Health workers' perceptions of access to care for children and pregnant women with precarious immigration status: Health as a right or a privilege?

The Canadian government's recent cuts to healthcare coverage for refugee claimants has rekindled the debate in Canada about what medical services should be provided to individuals with precarious immigration status, and who should pay for these services. This article further explores this debate, focussing on the perceptions of healthcare workers in Montreal, a large multiethnic Canadian city. In April–June 2010, an online survey was conducted to assess how clinicians, administrators, and support staff in Montreal contend with the ethical and professional dilemmas raised by the issue of access to healthcare services for pregnant women and children who are partially or completely uninsured. Drawing on qualitative analysis of answers (n = 237) to three open-ended survey questions, we identify the discursive frameworks that our respondents mobilized when arguing for, or against, universal access to healthcare for uninsured patients. In doing so, we highlight how their positions relate to their self-evaluations of Canada's socioeconomic situation, as well as their ideological representations of, and sense of social connection to, precarious status immigrants. Interestingly, while abstract values lead some healthcare workers to perceive uninsured immigrants as “deserving” of universal access to healthcare, negative perceptions of these migrants, coupled with pragmatic considerations, pushed most workers to view the uninsured as “underserving” of free care. For a majority of our respondents, the right to healthcare of precarious status immigrants has become a “privilege”, that as taxpayers, they are increasingly less willing to contribute to. We conclude by arguing for a reconsideration of access to healthcare as a right, and offer recommendations to move in this direction

Sistema di diagnosi collaborativa per cardiologia pediatrica

In questo rapporto viene dimostrato come è possibile costruire, partendo da tecnologie COTS, un sistema di telemedicina in grado di permettere ad un gruppo di esperti localizzati centralmente di fornire a personale clinico remoto supporto e guida in tempo reale per procedure ecografiche complesse. La prima applicazione del sistema è nella ecocardiologia neonatale, in particolare per la valutazione della pericolosità di potenziali patologie cardiache in neonati immediatamente dopo il parto

An FPGA platform for real-time simulation of spiking neuronal networks

In the last years, the idea to dynamically interface biological neurons with artificial ones has become more and more urgent. The reason is essentially due to the design of innovative neuroprostheses where biological cell assemblies of the brain can be substituted by artificial ones. For closed-loop experiments with biological neuronal networks interfaced with in silico modeled networks, several technological challenges need to be faced, from the low-level interfacing between the living tissue and the computational model to the implementation of the latter in a suitable form for real-time processing. Field programmable gate arrays (FPGAs) can improve flexibility when simple neuronal models are required, obtaining good accuracy, real-time performance, and the possibility to create a hybrid system without any custom hardware, just programming the hardware to achieve the required functionality. In this paper, this possibility is explored presenting a modular and efficient FPGA design of an in silico spiking neural network exploiting the Izhikevich model. The proposed system, prototypically implemented on a Xilinx Virtex 6 device, is able to simulate a fully connected network counting up to 1,440 neurons, in real-time, at a sampling rate of 10 kHz, which is reasonable for small to medium scale extra-cellular closed-loop experiments