The circular RNA landscape in multiple sclerosis: Disease-specific associated variants and exon methylation shape circular RNA expression profile

BACKGROUND: Circular RNAs (circRNAs) are a class of non-coding RNAs increasingly emerging as crucial actors in the pathogenesis of human diseases, including autoimmune and neurological disorders as multiple sclerosis (MS). Despite several efforts, the mechanisms regulating circRNAs expression are still largely unknown and the circRNA profile and regulation in MS-relevant cell models has not been completely investigated. In this work, we aimed at exploring the global landscape of circRNA expression in MS patients, also evaluating a possible correlation with their genetic and epigenetic background. METHODS: We performed RNA-seq experiments on circRNA-enriched samples, derived from peripheral blood mononuclear cells (PBMCs) of 10 MS patients and 10 matched controls and performed differential circRNA expression. The genetic background was evaluated using array genotyping, and an expression quantitative trait loci (eQTL) analysis was carried out. RESULTS: Expression analysis revealed 166 differentially expressed circRNAs in MS patients, 125 of which are downregulated. One of the top dysregulated circRNAs, hsa_circ_0007990, derives from the PGAP3 gene, encoding a protein relevant for the control of autoimmune responses. The downregulation of this circRNA was confirmed in two independent replication cohorts, suggesting its implementation as a possible RNA-based biomarker. The eQTL analysis evidenced a significant association between 89 MS-associated loci and the expression of at least one circRNA, suggesting that MS-associated variants could impact on disease pathogenesis by altering circRNA profiles. Finally, we found a significant correlation between exon methylation and circRNA expression levels, supporting the hypothesis that epigenetic features may play an important role in the definition of the cell circRNA pool. CONCLUSION: We described the circRNA expression profile of PBMCs in MS patients, suggesting that MS-associated variants may tune the expression levels of circRNAs acting as circ-QTLs , and proposing a role for exon-based DNA methylation in regulating circRNA expression

Astrocyte contribution to the excessive glutamate release in the spinal cord of the SOD1G93A mouse model of amyotrophic lateral sclerosis

Glutamate (Glu) excitotoxicity plays a major role in amyotrophic lateral sclerosis (ALS) and elevated extracellular Glu levels were found in patients and animal models of the disease. Reduced astrocyte uptake was suggested as the main cause for increased Glu availability. On the basis of our experiments, we postulated that abnormal neurotransmitter release represents another source for elevated Glu. Indeed, we found that neuronal Glu release from spinal cord synaptosomes is abnormally high in the spinal cord of pre-symptomatic and symptomatic SOD1G93A mice, a widely used experimental model of ALS, under resting conditions and upon exposure to different stimuli able to induce Glu exocytosis, including KCl depolarization, ionomycin, hypertonic sucrose or activation of Group I metabotropic Glu receptors. Also GABA and glycine induced Glu release in mouse spinal cord synaptosomes by activation of the respective transportes expressed at Glu-releasing terminals (heterotransporters) and also this effect was more elevated in SOD1G93A mice than in controls. We report here the effect of GABA on Glu release from gliosomes, an astrocytic preparation obtained by tissue homogenization and Percoll\uae gradient purification, that represent viable particles originating from the astrocytes sorrounding the synapses. Interestingly, GABA induced Glu release by a heterotransporter-mediated mechanism also from gliosomes purified from the spinal cord of SOD1G93A mice and the effect of GABA was up regulated, leading to over release of Glu. The excessive release of Glu evoked by GABA was already present in the pre-symptomatic stage of the disease. Our results indicate that abnormal Glu release from astrocytes is present in pre-symptomatic and symptomatic SOD1G93A mice. Thus, astrocytes may contribute to the increased concentration of Glu at glutamatergic synapses, to the activation of presynaptic and post-synaptic Glu receptors, and to excitotoxicity

Modello di innesto a blocco di tipo “over-inlay” per lo studio della rigenerazione ossea nel ratto

Obiettivo: L’obiettivo di questo studio è di descrivere un modello di innesto a blocco per la valutazione della rigenerazione ossea nel ratto. Materiali e Metodi: Difetti critici standardizzati dal diametro di 5 mm sono stati creati nell’osso parietale di 12 ratti Wistar maschi di 4 mesi. Blocchi di osso bovino deproteinizzato sono stati inseriti all’interno dei difetti, in modo che una parte del blocco fosse alloggiata nella cavità chirurgica (parte “inlay”) ed una parte eccedesse in altezza lo spessore della teca cranica esterna (parte “over”). Sei animali sono stati sacrificati a 1 mese e 6 a 3 mesi e all’interno di ogni campione sono state identificate diverse regioni di interesse (ROI): area periostale (PA), aree laterali adiacenti all’osso nativo (BA), aree centrali (CA). In ogni ROI sono stati quantificati osso neoformato (NB) e tessuto fibroso (FT). Densità di capillari, espressione di Osterix (OSX) e collagene1 (COL1) sono state valutate con immunoistochimica. I dati sono stati analizzati tramite test 2way-ANOVA e post-test di Sidak, p ≤ 0.05. I risultati sono espressi come media ± SEM. Risultati: NB è risultato maggiore in BA rispetto a CA, a 1 mese (42183.89±6477.27 μm2, p< 0.05) e a 3 mesi (64131.11±7520.61 μm2, p<0.01). FT è risultato maggiore in PA rispetto alle altre ROI a 1 mese (PA 179192.50±29187.19 μm2, p<0.01) e 3 mesi (243367.80±55447.25 μm2, p<0.001). I dati sulla densità capillare hanno evidenziato una progressiva migrazione del processo di neoangiogenesi da BA verso CA. Positività per OSX e COL1 è stata riscontrata soprattutto in PA. Conclusioni: Il modello “over-inlay” rende possibile un approccio critico per la valutazione del contributo distinto di diverse ROI nel processo di integrazione dell’innesto: la parte inlay consente di valutare BA e CA, la parte “over,” priva dell’apporto dell’osso nativo, la PA. Questo consente di ottenere dati più facilmente interpretabili con conseguente riduzione della numerosità del modello animale

"Over-inlay" block graft and differential morphometry: A novel block graft model to study bone regeneration and host-to-graft interfaces in rats

Purpose: The aim of this study was to present new a model that allows the study of the bone healing process, with an emphasis on the biological behavior of different graft-to-host interfaces. A standardized "over-inlay" surgical technique combined with a differential histomorphometric analysis is presented in order to optimize the use of critical-size calvarial defects in pre-clinical testing. Methods: Critical-size defects were created into the parietal bone of 8 male Wistar rats. Deproteinized bovine bone (DBBM) blocks were inserted into the defects, so that part of the block was included within the calvarial thickness and part exceeded the calvarial height (an "over-inlay" graft). All animals were sacrificed at 1 or 3 months. Histomorphometric and immunohistochemical evaluation was carried out within distinct regions of interest (ROIs): the areas adjacent to the native bone (BA), the periosteal area (PA) and the central area (CA). Results: The animals healed without complications. Differential morphometry allowed the examination of the tissue composition within distinct regions: the BA presented consistent amounts of new bone formation (NB), which increased over time (24.53%±1.26% at 1 month; 37.73%±0.39% at 3 months), thus suggesting that this area makes a substantial contribution toward NB. The PA was mainly composed of fibrous tissue (71.16%±8.06% and 78.30%±2.67%, respectively), while the CA showed high amounts of DBBM at both time points (78.30%±2.67% and 74.68%±1.07%, respectively), demonstrating a slow remodeling process. Blood vessels revealed a progressive migration from the interface with native bone toward the central area of the graft. Osterix-positive cells observed at 1 month within the PA suggested that the periosteum was a source of osteoprogenitor elements. Alkaline phosphatase data on matrix deposition confirmed this observation. Conclusions: The present model allowed for a standardized investigation of distinct graft-to-host interfaces both at vertically augmented and inlay-augmented sites, thus possibly limiting the number of animals required for pre-clinical investigations

Lenalidomide and dexamethasone in patients with POEMS syndrome: results of a prospective, open-label trial

Given its anti-angiogenic activity, lenalidomide may have a role in the treatment of POEMS (Peripheral neuropathy, Organomegaly, Endocrinopathy, Monoclonal plasma cell disorder and Skin changes) syndrome. This prospective, open-label, pilot study evaluated the combination of lenalidomide\uc2\ua0+\uc2\ua0dexamethasone (RD) in 18 POEMS syndrome patients (13 pre-treated, 5 newly-diagnosed but ineligible for high-dose therapy). Treatment consisted of six cycles of lenalidomide (25\uc2\ua0mg/day for 21\uc2\ua0days followed by 7\uc2\ua0days rest) plus dexamethasone (40\uc2\ua0mg/once a week). Patients responding after six cycles continued treatment until progression or unbearable toxicity. The primary endpoint was the proportion of patients with either neurological or clinical improvement. The RD combination was considered as deserving further evaluation if 9 of the first 15 patients responded. Ten responses were observed among the first 15 enrolled patients, meeting the primary endpoint. Fifteen of 18 patients (83%) completed six RD cycles: 13 (72%) patients responded and nine had both clinical and neurological improvement. Among the 15 patients who completed the six RD cycles, four were still on treatment after a 25-month follow-up. At 39\uc2\ua0months of follow-up, all patients were alive with a 3-year progression-free survival of 59%. No patient discontinued RD for toxicity. Overall, the RD regimen showed a high incidence of prolonged symptoms improvement and was well tolerated in most POEMS patients

Long-term effects of prenatal stress: Changes in adult cardiovascular regulation and sensitivity to stress

Prenatal environment exerts profound influences on the development of an organism and stressful events during pregnancy can bring about long-term physiological/behavioral alterations in the offspring. Epidemiological evidence points to a relationship between intrauterine growth restriction (IUGR), body weight at birth, and adult cardiovascular disease. Experimental research employed different models of IUGR, including altered maternal nutrition, exposure to elevated glucocorticoids, and reduced placental perfusion, all of which can program, when acting during sensitive temporal windows of foetal life, alterations in cardiovascular regulation and stress sensitivity. Original data are presented indicating that prenatal psychological stress (intermittent restraint) does not induce in the rat adult offspring changes of plasma corticosterone levels, cardiac autonomic modulation, and circadian rhythmicity of heart rate (HR), body temperature (T) and physical activity (Act) at rest. However, prenatally stressed rats – when further stimulated in adulthood – exhibit prolonged adrenocortical stress responsivity, disturbed circadian rhythmicity of HR, T, and Act, and increased adrenal weight. This evidence supports the idea that prenatal stress per se does not change dramatically a given structure or function, but it affects resilience and renders the animal more susceptible to pathophysiological outcomes when further insults occur during adulthood.

VERDI [Dataset]

File1: VERDI_samples_parameters.xlsx - Physico-chemical variables obtained from CTD profile - Inorganic nutrients concentrations - Chlorophyll-a concentrations - Organic carbon and nitrogen concentrations - Phytoplankton abundances - Detections of chytrids File 2: VERDI_asv_table.tbl: ASV abundances from natural samples and incubations File 3: VERDI_tax_table.tbl: Taxonomic assignments of ASVs File 4: VERDI_asv_seqs.fa: Sequences of ASVs File 5: VERDI_incubations_images.zip - Compilation of images taken during incubations with diatomsThe presence of phytoplankton parasites along the water column was explored at the Long Term Ecological Station MareChiara (LTER-MC) in the Gulf of Naples (Mediterranean Sea) in October 2019. Microscopy analyses showed diatoms dominating the phytoplankton community in the upper layers (0-20 m). Metabarcoding data from the water column showed the presence of Chytridiomycota predominantly in the upper layers coinciding with the vertical distribution of diatoms. Laboratory incubations of natural samples enriched with different diatom cultures confirmed parasitic interactions of some of those chytrids – including members of Kappamyces – with diatom taxa. The temporal dynamics of diatoms and chytrids was also explored in a three-year metabarcoding time-series (2011-2013) from surface waters of the study area and in sediment samples. Chytrids were recurrently present at low relative abundances, and some taxa found to infect diatoms in the incubation experiments were also identified in the ASV time-series. However, co-occurrence analyses did not show any clear or recurrent pairing patterns for chytrid and diatom taxa along the three years. The chytrid community in the sediments showed a clearly different species composition compared to the recorded in the water column samples, with higher diversity and relative abundance. The combination of observations, incubations and metabarcoding confirmed that parasites are a common component of marine protist communities at LTER-MC. Host-parasite interactions must be determined and quantified to understand their role and the impact they have on phytoplankton dynamics- European Union’s Horizon 2020 research and innovation programme under grant agreement No 730984, ASSEMBLE Plus project. - Spanish MICINN Project SMART (PID2020-112978GB-I00) - The research program LTER-MC is funded by the Stazione Zoologica Anton Dohrn- Physico-chemical variables obtained from CTD profile - Inorganic nutrients concentrations - Chlorophyll-a concentrations - Organic carbon and nitrogen concentrations - Phytoplankton abundances - Detections of chytrids - Metabarcoding ASV abundances from natural samples and incubations - Metabarcoding Taxonomic assignments of ASVs - Metabarcoding Sequences of ASVs - Compilation of images taken during incubations with diatomsPeer reviewe