Istituto Neurologico Mediterraneo

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is thatthere is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the completeprocess including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increasedautophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in manycases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as forreviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multipleassays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagyrelated protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.

The DJ-1 gene encodes a ubiquitous, highly conserved protein. Here, we show that DJ-1 mutations are associated with PARK7, a monogenic form of human parkinsonism. The function of the DJ-1 protein remains unknown, but evidence suggests its involvement in the oxidative stress response. Our findings indicate that loss of DJ-1 function leads to neurodegeneration. Elucidating the physiological role of DJ-1 protein may promote understanding of the mechanisms of brain neuronal maintenance and pathogenesis of Parkinson's disease.

Friedreich's ataxia (FRDA) is an autosomal recessive, degenerative disease that involves the central and peripheral nervous systems and the heart. A gene, X25, was identified in the critical region for the FRDA locus on chromosome 9q13. This gene encodes a 210-amino acid protein, frataxin, that has homologs in distant species such as Caenorhabditis elegans and yeast. A few FRDA patients were found to have point mutations in X25, but the majority were homozygous for an unstable GAA trinucleotide expansion in the first X25 intron.

autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

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Cardiac myocytes have been traditionally regarded as terminally differentiated cells that adapt to increased work and compensate for disease exclusively through hypertrophy. However, in the past few years, compelling evidence has accumulated suggesting that the heart has regenerative potential. Recent studies have even surmised the existence of resident cardiac stem cells, endothelial cells generating cardiomyocytes by cell contact or extracardiac progenitors for cardiomyocytes, but these findings are still controversial. We describe the isolation of undifferentiated cells that grow as self-adherent clusters (that we have termed "cardiospheres") from subcultures of postnatal atrial or ventricular human biopsy specimens and from murine hearts. These cells are clonogenic, express stem and endothelial progenitor cell antigens/markers, and appear to have the properties of adult cardiac stem cells. They are capable of long-term self-renewal and can differentiate in vitro and after ectopic (dorsal subcutaneous connective tissue) or orthotopic (myocardial infarction) transplantation in SCID beige mouse to yield the major specialized cell types of the heart: myocytes (ie, cells demonstrating contractile activity and/or showing cardiomyocyte markers) and vascular cells (ie, cells with endothelial or smooth muscle markers).

Hyperglycemia and insulin resistance are key players in the development of atherosclerosis and its complications. A large body of evidence suggest that metabolic abnormalities cause overproduction of reactive oxygen species (ROS). In turn, ROS, via endothelial dysfunction and inflammation, play a major role in precipitating diabetic vascular disease. A better understanding of ROS-generating pathways may provide the basis to develop novel therapeutic strategies against vascular complications in this setting. Part I of this review will focus on the most current advances in the pathophysiological mechanisms of vascular disease: (i) emerging role of endothelium in obesity-induced insulin resistance; (ii) hyperglycemia-dependent microRNAs deregulation and impairment of vascular repair capacities; (iii) alterations of coagulation, platelet reactivity, and microparticle release; (iv) epigenetic-driven transcription of ROS-generating and proinflammatory genes. Taken together these novel insights point to the development of mechanism-based therapeutic strategies as a promising option to prevent cardiovascular complications in diabetes.

Nonmotor symptoms (NMS) of Parkinson's disease (PD) are not well recognized in clinical practice, either in primary or in secondary care, and are frequently missed during routine consultations. There is no single instrument (questionnaire or scale) that enables a comprehensive assessment of the range of NMS in PD both for the identification of problems and for the measurement of outcome. Against this background, a multidisciplinary group of experts, including patient group representatives, has developed an NMS screening questionnaire comprising 30 items. This instrument does not provide an overall score of disability and is not a graded or rating instrument. Instead, it is a screening tool designed to draw attention to the presence of NMS and initiate further investigation. In this article, we present the results from an international pilot study assessing feasibility, validity, and acceptability of a nonmotor questionnaire (NMSQuest). Data from 123 PD patients and 96 controls were analyzed. NMS were highly significantly more prevalent in PD compared to controls (PD NMS, median = 9.0, mean = 9.5 vs. control NMS, median = 5.5, mean = 4.0; Mann-Whitney, Kruskal-Wallis, and t test, P < 0.0001), with PD patients reporting at least 10 different NMS on average per patient. In PD, NMS were highly significantly more prevalent across all disease stages and the number of symptoms correlated significantly with advancing disease and duration of disease. Furthermore, frequently, problems such as diplopia, dribbling, apathy, blues, taste and smell problems were never previously disclosed to the health professionals.

Increasing evidence suggests that high consumption of ultra-processed foods (UPF) is associated with an increase in non-communicable diseases, overweight and obesity. The present study systematically reviewed all observational studies that investigated the association between UPF consumption and health status. A comprehensive search of MEDLINE, Embase, Scopus, Web of Science and Google Scholar was conducted, and reference lists of included articles were checked. Only cross-sectional and prospective cohort studies were included. At the end of the selection process, twenty-three studies (ten cross-sectional and thirteen prospective cohort studies) were included in the systematic review. As regards the cross-sectional studies, the highest UPF consumption was associated with a significant increase in the risk of overweight/obesity (+39 %), high waist circumference (+39 %), low HDL-cholesterol levels (+102 %) and the metabolic syndrome (+79 %), while no significant associations with hypertension, hyperglycaemia or hypertriacylglycerolaemia were observed. For prospective cohort studies evaluating a total population of 183 491 participants followed for a period ranging from 3·5 to 19 years, highest UPF consumption was found to be associated with increased risk of all-cause mortality in five studies (risk ratio (RR) 1·25, 95 % CI 1·14, 1·37; P < 0·00001), increased risk of CVD in three studies (RR 1·29, 95 % CI 1·12, 1·48; P = 0·0003), cerebrovascular disease in two studies (RR 1·34, 95 % CI 1·07, 1·68; P = 0·01) and depression in two studies (RR 1·20, 95 % CI 1·03, 1·40; P = 0·02). In conclusion, increased UPF consumption was associated, although in a limited number of studies, with a worse cardiometabolic risk profile and a higher risk of CVD, cerebrovascular disease, depression and all-cause mortality.

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion.

Cyclin-dependent kinases (Cdks) are positive regulators of cell proliferation, whereas Cdk inhibitors (CKIs) inhibit proliferation. We describe a new CKI, p57KIP2, which is related to p21CIP1 and p27KIP1. p57KIP2 is a potent, tight-binding inhibitor of several G1 cyclin/Cdk complexes, and its binding is cyclin dependent. Unlike CIP1, KIP2 is not regulated by p53. Overexpression of p57KIP2 arrests cells in G1. p57KIP2 proteins have a complex structure. Mouse p57KIP2 consists of four structurally distinct domains: an amino-terminal Cdk inhibitory domain, a proline-rich domain, an acidic-repeat region, and a carboxy-terminal domain conserved with p27KIP1. Human p57KIP2 appears to have conserved the amino- and carboxy-terminal domains but has replaced the internal regions with sequences containing proline-alanine repeats. In situ hybridization during mouse embryogenesis revealed that KIP2 mRNA displays a striking pattern of expression during development, showing high level expression in skeletal muscle, brain, heart, lungs, and eye. Most of the KIP2-expressing cells are terminally differentiated, suggesting that p57KIP2 is involved in decisions to exit the cell cycle during development and differentiation. Human KIP2 is located at 11p15.5, a region implicated in both sporadic cancers and Beckwith-Wiedemann syndrome, a familial cancer syndrome, marking it as a candidate tumor suppressor. The discovery of a new member of the p21CIP1 inhibitor family with novel structural features and expression patterns suggests a complex role for these proteins in cell cycle control and development.

Abstract Common single-nucleotide polymorphisms (SNPs) are predicted to collectively explain 40–50% of phenotypic variation in human height, but identifying the specific variants and associated regions requires huge sample sizes 1 . Here, using data from a genome-wide association study of 5.4 million individuals of diverse ancestries, we show that 12,111 independent SNPs that are significantly associated with height account for nearly all of the common SNP-based heritability. These SNPs are clustered within 7,209 non-overlapping genomic segments with a mean size of around 90 kb, covering about 21% of the genome. The density of independent associations varies across the genome and the regions of increased density are enriched for biologically relevant genes. In out-of-sample estimation and prediction, the 12,111 SNPs (or all SNPs in the HapMap 3 panel 2 ) account for 40% (45%) of phenotypic variance in populations of European ancestry but only around 10–20% (14–24%) in populations of other ancestries. Effect sizes, associated regions and gene prioritization are similar across ancestries, indicating that reduced prediction accuracy is likely to be explained by linkage disequilibrium and differences in allele frequency within associated regions. Finally, we show that the relevant biological pathways are detectable with smaller sample sizes than are needed to implicate causal genes and variants. Overall, this study provides a comprehensive map of specific genomic regions that contain the vast majority of common height-associated variants. Although this map is saturated for populations of European ancestry, further research is needed to achieve equivalent saturation in other ancestries.

At the end of 2019 a novel virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing severe acute respiratory syndrome expanded globally from Wuhan, China. In March 2020 the World Health Organization declared the SARS-Cov-2 virus a global pandemic. We performed a narrative review to describe existing literature with regard to Corona Virus Disease 2019 (COVID-19) epidemiology, pathophysiology, diagnosis, management and future perspective. MEDLINE, EMBASE and Scopus databases were searched for relevant articles. Although only when the pandemic ends it will be possible to assess the full health, social and economic impact of this global disaster, this review represents a picture of the current state of the art. In particular, we focus on public health impact, pathophysiology and clinical manifestations, diagnosis, case management, emergency response and preparedness.

Co-contraction and overflow of EMG activity of inappropriate muscles are typical features of all dystonic movements whether voluntary or involuntary. Voluntary movements are slow and more variable than normal, and there is particular difficulty switching between component movements of a complex task. Reduced spinal cord and brainstem inhibition is common to many reflex studies (long-latency reflexes, cranial reflexes and reciprocal inhibition). These reflex abnormalities may contribute to the difficulties in voluntary movements but cannot be causal as they can occur outside the clinically involved territory. Clinical and neurophysiological studies have emphasized the possible role of sensory feedback in the generation of dystonic movements. Abnormalities of cortical and basal ganglia function have been described in functional imaging and neurophysiological studies of patients with dystonia and in animal models of primary dystonia. Studies of cortical function have shown reduced preparatory activity in the EEG before the onset of voluntary movements, whilst magnetic brain stimulation has revealed changes in motor cortical excitability. Functional imaging of the brain in primary dystonia has suggested reduced pallidal inhibition of the thalamus with consequent overactivity of medial and prefrontal cortical areas and underactivity of the primary motor cortex during movements. These findings are supported by preliminary neuronal recordings from the globus pallidus and the thalamus at the time of stereotaxic surgery in patients with dystonia. All this evidence suggests that primary dystonia results from a functional disturbance of the basal ganglia, particularly in the striatal control of the globus pallidus (and substantia nigra pars reticulata). This causes altered thalamic control of cortical motor planning and executive areas, and abnormal regulation of brainstem and spinal cord inhibitory interneuronal mechanisms.

PURPOSE: to investigate the factors affecting survival and toxicity in patients treated with stereotactic radiosurgery (SRS), with special attention to volumes of brain receiving a specific dose (V10 - V16 Gy) as predictors for brain radionecrosis. PATIENTS AND METHODS: Two hundred six consecutive patients with 310 cerebral metastases less than 3.5 cm were treated with SRS as primary treatment and followed prospectively at University of Rome La Sapienza Sant'Andrea Hospital. Overall survival, brain control, and local control were estimated using the Kaplan-Meier method calculated from the time of SRS. Univariate and multivariate analysis using a Cox proportional hazards regression model were performed to determine the predictive value of prognostic factors for treatment outcome and SRS-related complications. RESULTS: Median overall survival and brain control were 14.1 months and 10 months, respectively. The 1-year and 2-year survival rates were 58% and 24%, and respective brain control were 43% and 22%. Sixteen patients recurred locally after SRS, with 1-year and 2-year local control rates of 92% and 84%, respectively. On multivariate analysis, stable extracranial disease and KPS >70 were associated with the most significant survival benefit. Neurological complications were recorded in 27 (13%) patients. Severe neurological complications (RTOG Grade 3 and 4) occurred in 5.8% of patients. Brain radionecrosis occurred in 24% of treated lesions, being symptomatic in 10% and asymptomatic in 14%. On multivariate analysis, V10 through V16 Gy were independent risk factors for radionecrosis, with V10 Gy and V12 Gy being the most predictive (p = 0.0001). For V10 Gy >12.6 cm3 and V12 Gy >10.9 cm3 the risk of radionecrosis was 47%. CONCLUSIONS: SRS alone represents a feasible option as initial treatment for patients with brain metastases, however a significant subset of patients may develop neurological complications. Lesions with V12 Gy >8.5 cm3 carries a risk of radionecrosis >10% and should be considered for hypofractionated stereotactic radiotherapy especially when located in/near eloquent areas.

Abstract Body-mass index (BMI) has increased steadily in most countries in parallel with a rise in the proportion of the population who live in cities 1,2 . This has led to a widely reported view that urbanization is one of the most important drivers of the global rise in obesity 3–6 . Here we use 2,009 population-based studies, with measurements of height and weight in more than 112 million adults, to report national, regional and global trends in mean BMI segregated by place of residence (a rural or urban area) from 1985 to 2017. We show that, contrary to the dominant paradigm, more than 55% of the global rise in mean BMI from 1985 to 2017—and more than 80% in some low- and middle-income regions—was due to increases in BMI in rural areas. This large contribution stems from the fact that, with the exception of women in sub-Saharan Africa, BMI is increasing at the same rate or faster in rural areas than in cities in low- and middle-income regions. These trends have in turn resulted in a closing—and in some countries reversal—of the gap in BMI between urban and rural areas in low- and middle-income countries, especially for women. In high-income and industrialized countries, we noted a persistently higher rural BMI, especially for women. There is an urgent need for an integrated approach to rural nutrition that enhances financial and physical access to healthy foods, to avoid replacing the rural undernutrition disadvantage in poor countries with a more general malnutrition disadvantage that entails excessive consumption of low-quality calories.

Dendritic cells (DCs) exhibit a specialized antigen-presenting function and play crucial roles in both innate and adaptive immune responses. Due to their ability to cross-present tumor cell-associated antigens to naïve T cells, DCs are instrumental in the generation of specific T-cell-mediated antitumor effector responses in the control of tumor growth and tumor cell dissemination. Within an immunosuppressive tumor microenvironment, DC antitumor functions can, however, be severely impaired. In this review, we focus on the mechanisms of DC capture and activation by tumor cell antigens and the role of the tumor microenvironment in shaping DC functions, taking advantage of recent studies showing the phenotype acquisition, transcriptional state and functional programs revealed by scRNA-seq analysis. The therapeutic potential of DC-mediated tumor antigen sensing in priming antitumor immunity is also discussed.

Using functional MRI (fMRI), we have studied the changes induced by the performance of a complex sequential motor task in the cortical areas of six akinetic patients with Parkinson's disease and six normal subjects. Compared with the normal subjects, the patients with Parkinson's disease exhibited a relatively decreased fMRI signal in the rostral part of the supplementary motor area (SMA) and in the right dorsolateral prefrontal cortex, as previously shown in PET studies. Concomitantly, the same patients exhibited a significant bilateral relative increase in fMRI signal in the primary sensorimotor cortex, lateral premotor cortex, inferior parietal cortex, caudal part of the SMA and anterior cingulate cortex. These fMRI data confirm that the frontal hypoactivation observed in patients with Parkinson's disease is restricted to the rostral part of the SMA and to the dorsolateral prefrontal cortex. These results also show that, apart from the lateral premotor and parietal cortices, increased fMRI signals can be found in other cortical motor areas of these patients, including the posterior SMA, the anterior cingulate cortex and the primary sensorimotor cortices, which are then likely to participate in the same putative attempt by the dopamine-denervated brain to recruit parallel motor circuits in order to overcome the functional deficit of the striatocortical motor loops.

OBJECTIVE: To comprehensively assess published peer-reviewed studies related to extracorporeal membrane oxygenation (ECMO), focusing on outcomes and complications of ECMO in adult patients. DESIGN: Systematic review and meta-analysis. DATA SOURCES: MEDLINE/PubMed was searched for articles on complications and mortality occurring during or after ECMO. DATA EXTRACTION: Included studies had more than 100 patients receiving ECMO and reported in detail fatal or nonfatal complications occurring during or after ECMO. Primary outcome was mortality at the longest follow-up available; secondary outcomes were fatal and non-fatal complications. DATA SYNTHESIS: Twelve studies were included (1763 patients), mostly reporting on venoarterial ECMO. Criteria for applying ECMO were variable, but usually comprised acute respiratory failure, cardiogenic shock or both. After a median follow-up of 30 days (1st-3rd quartile, 30-68 days), overall mortality was 54% (95% CI, 47%-61%), with 45% (95% CI, 42%-48%) of fatal events occurring during ECMO and 13% (95% CI, 11%-15%) after it. The most common complications associated with ECMO were: renal failure requiring continuous venovenous haemofiltration (occurring in 52%), bacterial pneumonia (33%), any bleeding (33%), oxygenator dysfunction requiring replacement (29%), sepsis (26%), haemolysis (18%), liver dysfunction (16%), leg ischaemia (10%), venous thrombosis (10%), central nervous system complications (8%), gastrointestinal bleeding (7%), aspiration pneumonia (5%), and disseminated intravascular coagulation (5%). CONCLUSIONS: Even with conditions usually associated with a high chance of death, almost 50% of patients receiving ECMO survive up to discharge. Complications are frequent and most often comprise renal failure, pneumonia or sepsis, and bleeding.

BACKGROUND: The use of radial-artery grafts for coronary-artery bypass grafting (CABG) may result in better postoperative outcomes than the use of saphenous-vein grafts. However, randomized, controlled trials comparing radial-artery grafts and saphenous-vein grafts have been individually underpowered to detect differences in clinical outcomes. We performed a patient-level combined analysis of randomized, controlled trials to compare radial-artery grafts and saphenous-vein grafts for CABG. METHODS: Six trials were identified. The primary outcome was a composite of death, myocardial infarction, or repeat revascularization. The secondary outcome was graft patency on follow-up angiography. Mixed-effects Cox regression models were used to estimate the treatment effect on the outcomes. RESULTS: A total of 1036 patients were included in the analysis (534 patients with radial-artery grafts and 502 patients with saphenous-vein grafts). After a mean (±SD) follow-up time of 60±30 months, the incidence of adverse cardiac events was significantly lower in association with radial-artery grafts than with saphenous-vein grafts (hazard ratio, 0.67; 95% confidence interval [CI], 0.49 to 0.90; P=0.01). At follow-up angiography (mean follow-up, 50±30 months), the use of radial-artery grafts was also associated with a significantly lower risk of occlusion (hazard ratio, 0.44; 95% CI, 0.28 to 0.70; P<0.001). As compared with the use of saphenous-vein grafts, the use of radial-artery grafts was associated with a nominally lower incidence of myocardial infarction (hazard ratio, 0.72; 95% CI, 0.53 to 0.99; P=0.04) and a lower incidence of repeat revascularization (hazard ratio, 0.50; 95% CI, 0.40 to 0.63; P<0.001) but not a lower incidence of death from any cause (hazard ratio, 0.90; 95% CI, 0.59 to 1.41; P=0.68). CONCLUSIONS: As compared with the use of saphenous-vein grafts, the use of radial-artery grafts for CABG resulted in a lower rate of adverse cardiac events and a higher rate of patency at 5 years of follow-up. (Funded by Weill Cornell Medicine and others.).