Erratum to "Resveratrol interrupts the pro-invasive communication between Cancer associated Fibroblasts and Cholangiocarcinoma cells" [Cancer Letters 430C (2018) 160-171].
Publication Date: 10/10/2018, on Cancer letters
by Thongchot S, Ferraresi A, Vidoni C, Loilome W, Yongvanit P, Namwat N, Isidoro C
Adiponectin profile at baseline is correlated to progression and severity of multiple sclerosis.
Publication Date: 09/10/2018, on European journal of neurology
by Signoriello E, Lus G, Polito R, Casertano S, Scudiero O, Coletta M, Monaco ML, Rossi F, Nigro E, Daniele A
Adiponectin is a cytokine linking energy metabolism and immune system. After being assembled, adiponectin circulates as oligomers of different molecular weight, i.e. low, medium and high (HMW) molecular weight. These have the most potent biological effects. Multiple sclerosis (MS) is an autoimmune disease of the human central nervous system. The aim of this study was to characterize the expression levels of both total adiponectin and its oligomerization state in the serum from 99 patients with MS at baseline (i.e. not influenced by therapies). We also investigated the potential relationships between adiponectin and disease progression and severity.
FAAH-Catalyzed C-C Bond Cleavage of a New Multitarget Analgesic Drug.
Publication Date: 04/10/2018, on ACS chemical neuroscience
by Ligresti A, Silvestri C, Vitale RM, Martos JL, Piscitelli F, Wang JW, Allarà M, Carling RW, Luongo L, Guida F, Illiano A, Amoresano A, Maione S, Amodeo P, Woodward DF, Di Marzo V, Marino G
The discovery of extended catalytic versatilities is of great importance in both the chemistry and biotechnology fields. Fatty acid amide hydrolase (FAAH) belongs to the amidase signature superfamily and is a major endocannabinoid inactivating enzyme using an atypical catalytic mechanism involving hydrolysis of amide and occasionally ester bonds. FAAH inhibitors are efficacious in experimental models of neuropathic pain, inflammation, and anxiety, among others. We report a new multitarget drug, AGN220653, containing a carboxyamide-4-oxazole moiety and endowed with efficacious analgesic and anti-inflammatory activities, which are partly due to its capability of achieving inhibition of FAAH, and subsequently increasing the tissue concentrations of the endocannabinoid anandamide. This inhibitor behaves as a noncompetitive, slowly reversible inhibitor. Autoradiography of purified FAAH incubated with AGN220653, opportunely radiolabeled, indicated covalent binding followed by fragmentation of the molecule. Molecular docking suggested a possible nucleophilic attack by FAAH-Ser241 on the carbonyl group of the carboxyamide-4-oxazole moiety, resulting in the cleavage of the C-C bond between the oxazole and the carboxyamide moieties, instead of either of the two available amide bonds. MRM-MS analyses only detected the Ser241-assisted formation of the carbamate intermediate, thus confirming the cleavage of the aforementioned C-C bond. Quantum mechanics calculations were fully consistent with this mechanism. The study exemplifies how FAAH structural features and mechanism of action may override the binding and reactivity propensities of substrates. This unpredicted mechanism could pave the way to the future development of a completely new class of amidase inhibitors, of potential use against pain, inflammation, and mood disorders.
Microwave Ablation in Intermediate Hepatocellular Carcinoma in Cirrhosis: An Italian Multicenter Prospective Study.
Publication Date: 28/09/2018, on Journal of clinical and translational hepatology
by Giorgio A, Gatti P, Montesarchio L, Merola MG, Amendola F, Calvanese A, Iaquinto G, Fontana M, Ciracì E, Semeraro S, Santoro B, Coppola C, Matteucci P, Giorgio V
To report long-term results in treatment of intermediate hepatocellular carcinoma (HCC) in cirrhotics using new high-powered microwaves (MWS) ablation alone. This multicenter study included 215 cirrhotics (age range: 67-84 years; 137 males; 149 Child A, 66 Child B) who underwent percutaneous ultrasound-guided high-powered MWS ablation instead of transarterial chemoembolization. Among the patient population, 109 had a single nodule (Ø 5.3-8 cm) [group A], 70 had 2 nodules (Ø 3-6 cm) [group B] and 36 had 3-5 nodules (Ø 1.5-6.8 cm) [group C]. MWS ablation efficacy was evaluated using enhanced-computed tomography and/or magnetic resonance imaging. Primary end-point was 5-year cumulative overall survival (OS). On enhanced-computed tomography and/or magnetic resonance imaging, complete ablation rates were 100% for 1.5-3.5 cm nodules. In nodules >3.5-5 cm, it was 89% for the first ablation and 100% for the second. For lesions >5-8 cm, ablation was up to 92%. Overall, 1-, 3- and 5-year survival rates were 89, 60, and 21%, respectively. The cumulative OS rate of group A was 89%, 66% and 34% at 1, 3 and 5 years. The cumulative OS rate of group B was 88%, 60% and 11% at 1, 3 and 5 years. The cumulative OS rate of group C was 86%, 55% and 0%. The 5-year survival rate was significantly different among the groups ( <0.001). One patient died from rupture of HCC. Upon multivariate analysis, preablation total bilirubin >1.5 mg/dL was an independent factor for predicting lower survival. Percutaneous MWS ablation of intermediate HCC is safe and effective in inducing large volume of necrosis in intermediate HCC nodules, providing long-term survival rates similar to transarterial chemoembolization. Preablation total bilirubin >1.5 mg/dL as expression of liver function reserve is the main factor predicting a worse outcome.
Fatigue in Parkinson's disease: A systematic review and meta-analysis.
Publication Date: 28/09/2018, on Movement disorders : official journal of the Movement Disorder Society
by Siciliano M, Trojano L, Santangelo G, De Micco R, Tedeschi G, Tessitore A
We conducted a systematic review and meta-analysis aimed at establishing robust prevalence estimates and identifying clinical correlates of fatigue in PD. From 2,459 titles and abstracts, we selected 44 relevant studies (n = 7427 patients). Overall, the meta-analysis showed a prevalence of fatigue of 50% in PD. This prevalence estimate, however, was significantly moderated by study heterogeneity in measurement scales and cut-off thresholds. In contrast, demographic features, disease severity, cognitive impairment, and depression did not moderate prevalence estimates. Moreover, fatigue prevalence did not differ between de novo and treated PD patients. Compared to nonfatigued patients, fatigued patients had sligthly higher age (1.44 years), disease duration (0.93 years), l-dopa equivalent daily dose (50.89 units), UPDRS-III (4.99 points), and H & Y (0.33 points), as well as risk of comorbid depression (risk ratio = 1.89) and had a little lower MMSE score (-0.66 points). Fatigue was moderately associated with apathy (Hedges' g = 0.55), anxiety (Hedges' g = 0.67), daytime somnolence (Hedges' g = 0.43), sleep disturbances (Hedges' g = 0.66), and poorer quality of life (Hedges' g = 1.23). Our analyses suggest that fatigue is a frequent, independent nonmotor symptom in PD appearing early and persisting throughout the disease course, and that establishing uniform diagnostic criteria for PD-related fatigue is critical. In addition, several nonmotor symptoms appear to be associated with fatigue and negatively impact quality of life. Pharmacological and nonpharmacological interventions targeting fatigue and associated symptoms may improve quality of life in patients with PD. © 2018 International Parkinson and Movement Disorder Society.
Fronto-Temporal Circuits in Musical Hallucinations: A PET-MR Case Study.
Publication Date: 27/09/2018, on Frontiers in human neuroscience
by Cavaliere C, Longarzo M, Orsini M, Aiello M, Grossi D
The aim of the study is to investigate morphofunctional circuits underlying musical hallucinations (MH) in a 72-years old female that underwent a simultaneous 18fluoredeoxyglucose positron emission tomography (PET) and advanced magnetic resonance (MR) exam. This represents a particular case of MH occurred in an healthy subject, not displaying neurological or psychopathological disorders, and studied simultaneously with a multimodal approach. For the resting-state fMRI analysis a seed to seed approach was chosen. For the task-based fMRI, 4 different auditory stimuli were presented. Imaging findings were compared with data obtained by ten healthy controls matched for age and sex. Neuropsychological evaluation and questionnaires investigating depression and anxiety were also administered. PET findings showed hypermetabolism of: superior temporal gyri, anterior cingulate, left orbital frontal, and medial temporal cortices. Structural MRI did not show macroscopical lesions except for gliotic spots along the uncinate fascicle pathways with an increased cortical thickness for the right orbitofrontal cortex ( = 0.003). DTI showed increased fractional anisotropy values in the left uncinate fascicle, when compared to controls ( = 0.04). Resting-state fMRI showed increased functional connectivity between the left inferior frontal gyrus and the left temporal fusiform cortex ( = 0.01). Task-based fMRI confirmed PET findings showing an increased activation of the superior temporal gyrus in all the auditory tasks except for the monotone stimulus, with a significant activation of the left orbital frontal cortex only during the song in foreign language, object of MH. Results on cognitive test did not show cognitive impairment, excepting for the performance on Frontal Assessment Battery where the patient fails in the cognitive domains of conceptualization, sensitive to interference, and inhibitory control. The subject did not show depressive or anxiety symptoms. Summarizing, multimodal imaging analyses in the MH case showed a microstructural alteration of the left uncinate fascicle paralleled by an increased metabolism and functional connectivity of cortical regions that receive left uncinate projections (orbital frontal cortex, and medial temporal cortex). This alteration of fronto-hyppocampal circuits could be responsible of retrieval of known songs even in the absence of real stimuli.
Late-Onset Pompe Disease with Nemaline Bodies.
Publication Date: 27/09/2018, on Case reports in neurological medicine
by Frezza E, Terracciano C, Giacanelli M, Rastelli E, Greco G, Massa R
Pompe disease is an autosomal recessive disorder characterized by deficiency of alpha-glucosidase, a lysosomal enzyme, which can lead to glycogen accumulation in skeletal muscle, heart, and nervous system. Clinical presentation is highly variable, with infantile and late-onset (LOPED) forms. Although muscle biopsy findings are rather stereotyped, atypical features have been described. A 52-year-old man without a family history of muscle disorders presented with slowly progressing upper and lower limb girdle weakness and hyperCKemia. At needle EMG, a diffuse neurogenic pattern was detected. Muscle biopsy showed a selective type 1 fiber atrophy with vacuoles of various sizes, filled with PAS and acid phosphatase positive material, confirmed to be glycogen by electron microscopy (EM). Many atrophic fibers contained foci of myofibrillar material recognized as nemaline bodies (NBs) at EM. Low level of alpha-glucosidase activity in blood and molecular genetic testing confirmed the diagnosis of late-onset Pompe disease (LOPED). Major causes of hereditary and acquired NB myopathy were ruled out. In conclusion, NBs represent a novel histological finding in LOPED and characterize the atypical presentation of our case.
A hypothesis of sudden body fluid vaporization in the 79 AD victims of Vesuvius.
Publication Date: 26/09/2018, on PloS one
by Petrone P, Pucci P, Vergara A, Amoresano A, Birolo L, Pane F, Sirano F, Niola M, Buccelli C, Graziano V
In AD 79 the town of Herculaneum was suddenly hit and overwhelmed by volcanic ash-avalanches that killed all its remaining residents, as also occurred in Pompeii and other settlements as far as 20 kilometers from Vesuvius. New investigations on the victims' skeletons unearthed from the ash deposit filling 12 waterfront chambers have now revealed widespread preservation of atypical red and black mineral residues encrusting the bones, which also impregnate the ash filling the intracranial cavity and the ash-bed encasing the skeletons. Here we show the unique detection of large amounts of iron and iron oxides from such residues, as revealed by inductively coupled plasma mass spectrometry and Raman microspectroscopy, thought to be the final products of heme iron upon thermal decomposition. The extraordinarily rare preservation of significant putative evidence of hemoprotein thermal degradation from the eruption victims strongly suggests the rapid vaporization of body fluids and soft tissues of people at death due to exposure to extreme heat.
Neural correlates of apathy in patients with neurodegenerative disorders: an activation likelihood estimation (ALE) meta-analysis.
Publication Date: 20/09/2018, on Brain imaging and behavior
by Raimo S, Santangelo G, D'Iorio A, Trojano L, Grossi D
Apathy is commonly reported in Alzheimer's Disease (AD), Fronto-Temporal Dementia (FTD) and Parkinson's Disease (PD). In our meta-analysis we analysed a total of 41 studies to identify brain patterns associated with apathy. For these purposes we used activation likelihood estimation meta-analyses. Our main overall analysis showed that apathy is associated to hypometabolism and a decreased gray matter volume in the left inferior frontal gyrus (BA 45, 46). Disorder-specific analyses, not performed by means of meta-analysis, because of the small number of studies, but by means a label-based review, revealed an altered brain perfusion and decreased gray matter volume in anterior cingulate cortex (BA 24, 32) in AD patients and a decreased gray matter volume in inferior frontal gyrus (BA 44, 45) and parietal cortex (BA 40) in FTD patients. These findings suggest that apathy is mainly associated with a cortical dysfunction of areas involved in executive-cognitive processing (i.e. action planning) and emotional regulation (auto-activation and reward processing). Knowledge about the neural underpinnings of apathy is crucial for understanding its clinical characteristics in neurodegenerative diseases and for developing novel strategies of treatment in clinical practice.
Ruminant meat and milk contain δ-valerobetaine, another precursor of trimethylamine N-oxide (TMAO) like γ-butyrobetaine.
Publication Date: 15/09/2018, on Food chemistry
by Servillo L, D'Onofrio N, Giovane A, Casale R, Cautela D, Castaldo D, Iannaccone F, Neglia G, Campanile G, Balestrieri ML
Quaternary ammonium compounds containing N-trimethylamino moiety, such as choline derivatives and carnitine, abundant in meat and dairy products, are metabolic precursors of trimethylamine (TMA). A similar fate is reported for N-trimethyllysine and γ-butyrobetaine. With the aim at investigating the metabolic profile of such metabolites in most employed animal dietary sources, HPLC-ESI-MS/MS analyses on ruminant and non-ruminant milk and meat were performed. Results demonstrate, for the first time, the presence of δ-valerobetaine, occurring at levels higher than γ-butyrobetaine in all ruminant samples compared to non-ruminants. Demonstration of δ-valerobetaine metabolic origin, surprisingly, showed that it originates from rumen through the transformation of dietary N-trimethyllysine. These results highlight our previous findings showing the ubiquity of free N-trimethyllysine in vegetable kingdom. Furthermore, δ-valerobetaine, similarly to γ-butyrobetaine, can be degraded by host gut microbiota producing TMA, precursor of the proatherogenic trimethylamine N-oxide (TMAO), unveiling its possible role in the biosynthetic route of TMAO.
Cardioprotective Effects of Nanoemulsions Loaded with Anti-Inflammatory Nutraceuticals against Doxorubicin-Induced Cardiotoxicity.
Publication Date: 14/09/2018, on Nutrients
by Quagliariello V, Vecchione R, Coppola C, Di Cicco C, De Capua A, Piscopo G, Paciello R, Narciso V, Formisano C, Taglialatela-Scafati O, Iaffaioli RV, Botti G, Netti PA, Maurea N
Doxorubicin is a highly active antineoplastic agent, but its clinical use is limited because of its cardiotoxicity. Although nutraceuticals endowed with anti-inflammatory properties exert cardioprotective activity, their bioavailability and stability are inconsistent. In an attempt to address this issue, we evaluated whether bioavailable nanoemulsions loaded with nutraceuticals (curcumin and fresh and dry tomato extracts rich in lycopene) protect cardiomyoblasts (H9C2 cells) from doxorubicin-induced toxicity. Nanoemulsions were produced with a high-pressure homogenizer. H9C2 cells were incubated with nanoemulsions loaded with different nutraceuticals alone or in combination with doxorubicin. Cell viability was evaluated with a modified MTT method. The levels of the lipid peroxidation products malondialdehyde (MDA) and 4-hydroxy-2-butanone (4-HNA), and of the cardiotoxic-related interleukins IL-6, IL-8, IL-1β and IL-10, tumor necrosis factor-alpha (TNF-α), and nitric oxide were analyzed in cardiomyoblasts. The hydrodynamic size of nanoemulsions was around 100 nm. Cell viability enhancement was 35⁻40% higher in cardiomyoblasts treated with nanoemulsion + doxorubicin than in cardiomyoblasts treated with doxorubicin alone. Nanoemulsions also protected against oxidative stress as witnessed by a reduction of MDA and 4-HNA. Notably, nanoemulsions inhibited the release of IL-6, IL-8, IL-1β, TNF-α and nitric oxide by around 35⁻40% and increased IL-10 production by 25⁻27% versus cells not treated with emulsions. Of the nutraceuticals evaluated, lycopene-rich nanoemulsions had the best cardioprotective profile. In conclusion, nanoemulsions loaded with the nutraceuticals described herein protect against cardiotoxicity, by reducing inflammation and lipid oxidative stress. These results set the stage for studies in preclinical models.
Integrated Cognitive and Neuromotor Rehabilitation in Multiple Sclerosis: A Pragmatic Study.
Publication Date: 05/09/2018, on Frontiers in behavioral neuroscience
by Barbarulo AM, Lus G, Signoriello E, Trojano L, Grossi D, Esposito M, Costabile T, Lanzillo R, Saccà F, Morra VB, Conchiglia G
Few studies examined the effects of combined motor and cognitive rehabilitation in patients with multiple sclerosis (MS). The present prospective, multicenter, observational study aimed to determine the efficacy of an integrated cognitive and neuromotor rehabilitation program versus a traditional neuromotor training on walking, balance, cognition and emotional functioning in MS patients. Sixty three MS patients were selected and assigned either to the Integrated Treatment Group (ITG; = 32), receiving neuropsychological treatment (performed by ERICA software and paper-pencil tasks) complemented by conventional neuromotor rehabilitation, or to the Motor Treatment Group ( = 31) receiving neuromotor rehabilitation only. The intervention included two 60-min sessions per week for 24 weeks. At baseline and at end of the training all patients underwent a wide-range neuropsychological, psychological/emotional, and motor assessment. At baseline the two groups did not differ for demographic, neuropsychological, psychological/emotional, and motor features significantly. After rehabilitation, only ITG group significantly ( for False Discovery Rate) improved on test tapping spatial memory, attention and cognitive flexibility, as well as on scales assessing depression and motor performance (balance and gait). A regression analysis showed that neuropsychological and motor improvement was not related to improvements in fatigue and depression. The present study demonstrated positive effects in emotional, motor, and cognitive aspects in MS patients who received an integrated cognitive and neuromotor training. Overall, results are supportive of interventions combining motor and cognitive training for MS.
Dihydroartemisinin induces apoptosis and autophagy-dependent cell death in cholangiocarcinoma through a DAPK1-BECLIN1 pathway.
Publication Date: 22/08/2018, on Molecular carcinogenesis
by Thongchot S, Vidoni C, Ferraresi A, Loilome W, Yongvanit P, Namwat N, Isidoro C
Cholangiocarcinoma (CCA) is a very aggressive cancer arising from the malignant transformation of cholangiocytes. Intrahepatic CCA is associated with reactive inflammation and intense fibrosis of the hepatobiliary tract. Dihydroartemisinin (DHA), the active compound found in Artemisia annua, has been shown to possess anti-tumor activity in a variety of human cancers, including hepatoma. Here, we tested the ability of DHA to specifically kill CCA cells and have investigated the underlying mechanisms. DHA induced both apoptosis and autophagy-dependent caspase-independent cell death in many CCA cell lines, while being slightly toxic to immortalized cholangiocytes. DHA induced the expression of many apoptosis- and autophagy-related genes in CCA cells. In particular, it greatly induced the expression of DAPK1, and reduced the interaction of BECLIN1 with BCL-2 while promoting its interaction with PI3KC3. Genetic silencing of DAPK1 prevented DHA-induced autophagy. Pharmacologic and genetic inhibition of BECLIN1 function prevented autophagy and cell death induced by DHA in CCA cells. These data unravel a novel pathway of DHA cancer toxicity and open the possibility to introduce DHA in the therapeutic regimen for the treatment of CCA. This article is protected by copyright. All rights reserved.
Inflammatory Cytokines and SIRT1 Levels in Subcutaneous Abdominal Fat: Relationship With Cardiac Performance in Overweight Pre-diabetics Patients.
Publication Date: 21/08/2018, on Frontiers in physiology
by Sardu C, Pieretti G, D'Onofrio N, Ciccarelli F, Paolisso P, Passavanti MB, Marfella R, Cioffi M, Mone P, Dalise AM, Ferraraccio F, Panarese I, Gambardella A, Passariello N, Rizzo MR, Balestrieri ML, Nicoletti G, Barbieri M
In obese patients the superficial adipose tissue expresses cytokines, and sirtuins, that may affect myocardial function. In this study, we investigated the effect of metformin therapy added to a hypocaloric diet on the inflammatory pattern and cardiac performance (MPI) in obese patients with pre-diabetic condition. Fifty-eight obese patients that were enrolled for abdominoplastic surgery were divided into patients with pre-diabetic condition (n 40) and normo-glycemic patients (n18). Patients with pre-diabetic condition were randomly assigned to metformin therapy added to a hypocaloric diet (group 1, n 20) or to a hypocaloric diet therapy alone (group 2, n20). Patients with normo-glycemic condition were assigned to a hypocaloric diet therapy. During enrollment, obese patients with a pre-diabetic condition (group 1 and 2) presented higher glucose values, lower values of insulin, and higher values of the homeostasis model for the assessment of insulin resistance (HOMA-IR) than obese patients with normo-glycemic condition(group 3). In addition, they had higher values of C Reactive protein (CRP), interleukin 6 (IL6), and lower values of sirtuin 1(SIRT1). In the 12th month of the follow-up, metformin therapy induced in patients with pre-diabetic condition (group 1) a significant reduction of glucose values, HOMA-IR, and inflammatory markers such as CRP (1.04 ± 0.48 vs. 0.49 ± 0.02 mmol/L, < 0.05), IL6 (4.22 ± 0.45 vs. 3.33 ± 0.34 pg/ml, < 0.05), TNFα (6.95 ± 0.59 vs. 5.15 ± 0.44 pg/ml, < 0.05), and Nitrotyrosine (5,214 ± 0,702 vs. 2,151 ± 0,351 nmol/l, < 0.05). This was associated with a significant reduction of Intima-media thickness (1.01 ± 0.15 vs. 0.86 ± 0.15 mm, < 0.05), Septum (14 ± 2.5 vs. 10.5 ± 2 mm, < 0.05), Posterior wall (11 ± 1.5 vs. 8 ± 1 mm, < 0.05), LV mass (192.5 ± 49.5 vs. 133.2 ± 37.6 g, < 0.05) and of MPI (0.58 ± 0.03 vs. 0.38 ± 0.02, < 0.05). At 12 months of follow-up, group 2 experienced only a reduction of cholesterol (4.15 ± 0.94 vs. 4.51 ± 0.88 mmol/L, < 0.05) and triglycerides (1.71 ± 1.18 vs. 1.83 ± 0.54 mmol/L, < 0.05). At 12 months of follow-up, group 3 experienced a significant reduction of inflammatory markers, and also of echographic parameters, associated with amelioration of myocardial performance. To date, IL6 expression was related to higher values of left ventricle mass (-value 0.272, -value 0.039), and to higher IMT (-value 0.272, -value 0.039), such as those observed for CRP (-value 0.308, -value 0.021), for glucose blood values (-value 0.449, -value 0.001), and for HOMA-IR (-value 0.366, -value 0.005). An inverse correlation was found between subcutaneous fat expression of SIRT1 and myocardial performance index (-value-0.236, -value 0.002). In obese patients with pre-diabetic condition a metformin therapy may reduce inflammation and oxidative stress, and this may be associated with the amelioration of the cardiac performance. Clinical research trial number: NCT03439592.
Isolation of Smenopyrone, a Bis-γ-Pyrone Polypropionate from the Caribbean Sponge <i>Smenospongia aurea</i>.
Publication Date: 17/08/2018, on Marine drugs
by Esposito G, Teta R, Della Sala G, Pawlik JR, Mangoni A, Costantino V
The organic extract of the Caribbean sponge has been shown to contain an array of novel chlorinated secondary metabolites derived from a mixed PKS-NRPS biogenetic route such as the smenamides. In this paper, we report the presence of a biogenetically different compound known as smenopyrone, which is a polypropionate containing two γ-pyrone rings. The structure of smenopyrone including its relative and absolute stereochemistry was determined by spectroscopic analysis (NMR, MS, ECD) and supported by a comparison with model compounds from research studies. Pyrone polypropionates are unprecedented in marine sponges but are commonly found in marine mollusks where their biosynthesis by symbiotic bacteria has been hypothesized and at least in one case demonstrated. Since pyrones have recently been recognized as bacterial signaling molecules, we speculate that smenopyrone could mediate inter-kingdom chemical communication between and its symbiotic bacteria.