Harvest year effects on Apulian EVOOs evaluated by (1)H NMR based metabolomics

none5noNine hundred extra virgin olive oils (EVOO) were extracted from individual olive trees of four olive cultivars (Coratina, Cima di Mola, Ogliarola, Peranzana), originating from the provinces of Bari and Foggia (Apulia region, Southern Italy) and collected during two consecutive harvesting seasons (2013/14 and 2014/15). Following genetic identification of individual olive trees, a detailed Apulian EVOO NMR database was built using 900 oils samples obtained from 900 cultivar certified single trees. A study on the olive oil lipid profile was carried out by statistical multivariate analysis (Principal Component Analysis, PCA, Partial Least-Squares Discriminant Analysis, PLS-DA, Orthogonal Partial Least-Squares Discriminant Analysis, OPLS-DA). Influence of cultivar and weather conditions, such as the summer rainfall, on the oil metabolic profile have been evaluated. Mahalanobis distances and J2 criterion have been measured to assess the quality of resulting scores clusters for each cultivar in the two harvesting campaigns. The four studied cultivars showed non homogeneous behavior. Notwithstanding the geographical spread and the wide number of samples, Coratina showed a consistent behavior of its metabolic profile in the two considered harvests. Among the other three Peranzana showed the second more consistent behavior, while Cima di Mola and Ogliarola having the biggest change over the two years.Girelli, Chiara R; Del Coco, Laura; Papadia, Paride; De Pascali, Sandra A; Fanizzi, Francesco PGirelli, CHIARA ROBERTA; DEL COCO, Laura; Papadia, Paride; DE PASCALI, SANDRA ANGELICA; Fanizzi, Francesco Paol

Parentification, distress, and relationship with parents as factors shaping the relationship between adult siblings and their brother/sister with disabilities

According to parentification theory, when the siblings of a brother/sister with disabilities assume parent-like duties, this role reversal is known as sibling-focused parentification. It has a significant impact on these siblings’ distress and the quality of their family relationships; 605 Italian adult siblings (19–26 years) of people with disabilities completed the online survey. Measures of siblings’ parentification, distress, quality of family relationships, social support, and perceived benefits of parentification were used. The hypothesized model aims to test, on the target sample, the distress and the quality of the relationship with parents as mediators on the interplay between the siblings’ parentification and their sibling relationship. Additionally, social support and perceived benefits of parentification as protective factors were considered. Results showed that the distress and the low quality of the relationship with parents negatively affected the interplay between the siblings’ parentification and the relationship with their own brother/sister with disabilities. Social support and the perceived benefits of parentification decreased the siblings’ distress levels; the perceived benefits of parentification served as a protective factor for the quality of the relationship with parents. Current findings extend the knowledge regarding the risk and protective factors of the siblings’ mental health when disability occurs in the family. Additionally, they inform family-based intervention programs, which should involve the whole family system for reducing distress and improving the wellbeing of siblings without disabilities

Widespread occurrence of non-canonical transcription termination by human RNA polymerase III

Human RNA polymerase (Pol) III-transcribed genes are thought to share a simple termination signal constituted by four or more consecutive thymidine residues in the coding DNA strand, just downstream of the RNA 3′-end sequence. We found that a large set of human tRNA genes (tDNAs) do not display any T≥4 stretch within 50 bp of 3′-flanking region. In vitro analysis of tDNAs with a distanced T≥4 revealed the existence of non-canonical terminators resembling degenerate T≥5 elements, which ensure significant termination but at the same time allow for the production of Pol III read-through pre-tRNAs with unusually long 3′ trailers. A panel of such non-canonical signals was found to direct transcription termination of unusual Pol III-synthesized viral pre-miRNA transcripts in gammaherpesvirus 68-infected cells. Genome-wide location analysis revealed that human Pol III tends to trespass into the 3′-flanking regions of tDNAs, as expected from extensive terminator read-through. The widespread occurrence of partial termination suggests that the Pol III primary transcriptome in mammals is unexpectedly enriched in 3′-trailer sequences with the potential to contribute novel functional ncRNA

Parentification, distress, and relationship with parents as factors shaping the relationship between adult siblings and their brother/sister with disabilities

According to parentification theory, when the siblings of a brother/sister with disabilities assume parent-like duties, this role reversal is known as sibling-focused parentification. It has a significant impact on these siblings’ distress and the quality of their family relationships; 605 Italian adult siblings (19–26 years) of people with disabilities completed the online survey. Measures of siblings’ parentification, distress, quality of family relationships, social support, and perceived benefits of parentification were used. The hypothesized model aims to test, on the target sample, the distress and the quality of the relationship with parents as mediators on the interplay between the siblings’ parentification and their sibling relationship. Additionally, social support and perceived benefits of parentification as protective factors were considered. Results showed that the distress and the low quality of the relationship with parents negatively affected the interplay between the siblings’ parentification and the relationship with their own brother/sister with disabilities. Social support and the perceived benefits of parentification decreased the siblings’ distress levels; the perceived benefits of parentification served as a protective factor for the quality of the relationship with parents. Current findings extend the knowledge regarding the risk and protective factors of the siblings’ mental health when disability occurs in the family. Additionally, they inform family-based intervention programs, which should involve the whole family system for reducing distress and improving the wellbeing of siblings without disabilities

Widespread occurrence of non-canonical transcription termination by human RNA polymerase III

Human RNA polymerase (Pol) III-transcribed genes are thought to share a simple termination signal constituted by four or more consecutive thymidine residues in the coding DNA strand, just downstream of the RNA 3′-end sequence. We found that a large set of human tRNA genes (tDNAs) do not display any T≥4 stretch within 50 bp of 3′-flanking region. In vitro analysis of tDNAs with a distanced T≥4 revealed the existence of non-canonical terminators resembling degenerate T≥5 elements, which ensure significant termination but at the same time allow for the production of Pol III read-through pre-tRNAs with unusually long 3′ trailers. A panel of such non-canonical signals was found to direct transcription termination of unusual Pol III-synthesized viral pre-miRNA transcripts in gammaherpesvirus 68-infected cells. Genome-wide location analysis revealed that human Pol III tends to trespass into the 3′-flanking regions of tDNAs, as expected from extensive terminator read-through. The widespread occurrence of partial termination suggests that the Pol III primary transcriptome in mammals is unexpectedly enriched in 3′-trailer sequences with the potential to contribute novel functional ncRNAs

High Risk of Secondary Infections Following Thrombotic Complications in Patients With COVID-19

Background. This study’s primary aim was to evaluate the impact of thrombotic complications on the development of secondary infections. The secondary aim was to compare the etiology of secondary infections in patients with and without thrombotic complications. Methods. This was a cohort study (NCT04318366) of coronavirus disease 2019 (COVID-19) patients hospitalized at IRCCS San Raffaele Hospital between February 25 and June 30, 2020. Incidence rates (IRs) were calculated by univariable Poisson regression as the number of cases per 1000 person-days of follow-up (PDFU) with 95% confidence intervals. The cumulative incidence functions of secondary infections according to thrombotic complications were compared with Gray’s method accounting for competing risk of death. A multivariable Fine-Gray model was applied to assess factors associated with risk of secondary infections. Results. Overall, 109/904 patients had 176 secondary infections (IR, 10.0; 95% CI, 8.8–11.5; per 1000-PDFU). The IRs of secondary infections among patients with or without thrombotic complications were 15.0 (95% CI, 10.7–21.0) and 9.3 (95% CI, 7.9–11.0) per 1000-PDFU, respectively (P = .017). At multivariable analysis, thrombotic complications were associated with the development of secondary infections (subdistribution hazard ratio, 1.788; 95% CI, 1.018–3.140; P = .043). The etiology of secondary infections was similar in patients with and without thrombotic complications. Conclusions. In patients with COVID-19, thrombotic complications were associated with a high risk of secondary infections

Study of the osteoblastic differentiation of SCAP before and after their laser-assisted bio-printing

L’Ingénierie Tissulaire osseuse est une stratégie thérapeutique innovante, visant à créer du tissu osseux pour régénérer des éléments anatomiques lésés. Elle fait appel à l'utilisation de cellules souches, qui peuvent être disposées dans des matrices 3D mimant le tissu à obtenir. Les SCAPS (Stem Cells from Apical Papilla, cellules souches issues des papilles apicales des dents permanentes immatures) représentent des candidates idéales pour cette approche, car elles ont un potentiel ostéogénique et elles peuvent être organisées dans des motifs 3D par bio-impression assistée par laser (Laser Assisted Bioprinting, LAB). Ce travail de thèse s’est proposé d’évaluer si la LAB peut affecter la capacité ostéogénique des SCAPS. Dans ce but, l’expression de marqueurs ostéoblastiques a été étudiée au long de l’ostéoinduction de SCAPS bio-imprimées par LAB et, en parallèle, de SCAPS non bio-imprimées. Les résultats obtenus démontrent que les SCAPS imprimées sont capables de répondre à l’induction ostéogénique, car l’expression des marqueurs osseux est activée pendant leur différenciation ostéoblastique. Ceci indique que la bio-impression par LAB n’inhibe pas leur potentiel ostéogénique. Toutefois, les SCAPS non imprimées ont manifesté une réponse ostéogénique plus accentuée. Ce phénomène semblerait lié aux faibles propriétés mécaniques des constructions bio-imprimées, plutôt qu’à l’effet direct de la LAB. Des modifications du support, aptes à augmenter sa rigidité, pourraient améliorer la réponse ostéogénique des SCAPS imprimées. L’ostéopontine s’est démarquée parmi les gènes activés pendant l’ostéogenèse de SCAPS. Son rôle dans la différenciation ostéoblastique n’est pas encore élucidé et mériterait d’être approfondi.Bone Tissue Engineering is an innovative therapeutic strategy, aimed at creating bone tissue in order to regenerate damaged anatomical elements. Tissue engineering uses a combination of cells, biomaterials and engineering technologies to fabricate biological constructs that mimic the functions of their counterparts in human body. SCAPs (Stem Cells from Apical Papilla) represent ideal candidates for this approach, as they have an osteogenic potential et they can be organized in 3D patterns by Laser Assisted Bioprinting (LAB). The aim of this study was to evaluate whether LAB could affect the osteogenic capacity of SCAPs. For this purpose, we followed the expression of bone markers during osteoinduction of LAB-bio-printed SCAPs versus unprinted SCAPs. Our results show that bio-printed SCAPs respond to osteogenic induction, since their expression of bone markers is activated after osteoinduction. Then, LAB doesn’t inhibit their osteoblastic differentiation. However, the unprinted SCAPs showed a higher response to osteogenic induction. That seems to be related to the reduced stiffness and cell density in printed structures, rather than to LAB influence. Some modifications of scaffolds, aimed to increase their rigidity, could improve the osteogenic response of printed SCAPs. The expression of osteopontin seemed to be enhanced during osteogenesis of SCAPs. Its role is still unclear and it should be investigated

Genome wide identification of genes transcribed by two isoforms of human RNA

En 2010, Haurie et al. ont identifié deux isoformes différentes de la Pol III humaine : Pol IIIα, quin’est présente que dans les cellules souches embryonnaires et dans celles tumorales, et Pol IIIβ,exprimée constitutivement.L’expression ectopique de Pol IIIα affecte profondément l’équilibre de cellules différentiées, jusqu’àen induire la transformation oncogénique. Ceci n’est pas observé dans le cas de l’expression ectopiquede Pol IIIβ.A fin de définir les causes moléculaires de la transformation cellulaire induite par Pol IIIα, nousavons décidé d’étudier son transcriptome en comparaison avec celui de Pol IIIβ, à la recherche desgènes qui pourraient soutenir l’oncogenèse observée. Dans notre étude, nous avons adopté latechnique du ChIP-Seq, une approche innovatrice et puissante qui permet de cartographier tous lessites de liaison d’une protéine sur le génome. Elle comporte la purification de la protéine d’intérêtcomplexe avec ces cibles génomiques et le séquençage de ces dernières de manière massive.Le premier but de cette thèse a été représenté par la mise a point d’un protocole de ChIP-Seqoptimisé spécifiquement pour Pol IIIα et Pol IIIβ. Une fois obtenue une préparation de chromatine dequalité adéquate aux standards du ChIP-Seq, nous avons effectué les séquençages massifs etcartographié sur le génome tous les loci contactés par les deux isoformes de polymérase.De cette manière, nous avons identifié 1287 loci occupés par Pol IIIα et 1281 par Pol IIIβ. Les deuxpolymérases se co-localisent sur la plupart de ces sites, mais montrent aussi une occupationpréférentielle et spécifique sur une fraction de loci Pol III. Cette disproportion pourrait contribuer auphénomène cellulaire observé par Haurie et al.Le manuscrit de thèse détaille les résultats de cette analyse et les conclusions qui en dérivent.Nous y avons ajouté une synthèse des travaux réalisés antérieurement et concertants la caractérisationdes promoteurs de gène qui codent pour les snoARN chez la levure S.cerevisiae, positionnée en toutefin de document.In eukaryotes, transcription is carried out by DNA-dependent RNA polymerases I, II and III (or I-V inplants). These RNA polymerases are specialized in the transcription of specific groups of genes.Human RNA polymerase III (Pol III) transcribes small noncoding RNAs involved in the regulation oftranscription (7SK RNA), RNA processing (U6 RNA, RNAse P, RNAse MRP), translation (tRNAs,5S RNA) or other cellular processes (vault RNAs [multidrug resistance], adenoviral RNAs [VA-I,VA-II], Epstein-Barr virus RNAs [EBER1, EBER2]). It has furthermore been reported that somemicroRNAs of viral or cellular origin may also be transcribed by Pol III.Interestingly, increased Pol III transcription levels accompany or cause cell transformation. Themechanisms underlying this phenomenon are still largely unknown. Recently, two distinct isoforms ofhuman Pol III have been discovered (Haurie et al., 2010). RPC32β-containing Pol IIIβ is ubiquitouslyexpressed and essential for growth and survival of human cells. In contrast, RPC32α-containing PolIIIα is dispensable for cell survival and its expression is restricted to undifferentiated embryonic stem(ES) cells and to tumor cells.The distinct effects of Pol IIIα and Pol III β on cell growth and transformation may be explained bythe transcription of isoform-specific target genes. To identify such isoform-specific target genes, wespecifically targeted RPC32α and of RPC32β subunits in chromatin immunoprecipitation (ChIP)experiments and analyzed the co-precipitated DNA sequences by high throughput sequencing (ChIPseq.).Genome wide localization of RPC32α and RPC32β subunits of Pol III revealed the presence of bothsubunits on many of the known Pol III-transcribed genes, suggesting redundant activities of bothisoforms of Pol III in transcription of these genes. We also found that some of the genes known to betranscribed by Pol III are only occupied by either RPC32α or by RPC32β, suggesting that these genesare exclusively transcribed by Pol IIIα or by Pol IIIβ, respectively.RPC32α and RPC32β ChIP-seq. results furthermore led to the identification of novel Pol III candidategenes in HeLa cells. Moreover, we found high levels of Pol IIIα or Pol III β at some of the annotatedtRNA pseudogenes, implicating that these genes may be transcribed. The functions of RNAstranscribed from novel putative Pol III genes or from tRNA pseudogenes remain to be determined

Genome-wide identification of genes transcribed by two isoforms of human RNA Polymerase III

L’RNA polimerasi III (Pol III) trascrive piccoli RNA non codificanti, tra i quali l’RNA 5S, i tRNA, l’RNA U6, l’RNA 7SK e 7SL. La trascrizione Pol III è finemente regolata in condizioni di normalità,ma il processo perde d’equilibrio e modulazione durante la tumorigenesi. Un considerevole incremento dei trascritti Pol III accompagna, infatti, il processo di trasformazione neoplastica e potrebbe rappresentarne l’origine. Le cause molecolari di tale variazione trascrizionale restano sconosciute, così come il loro nesso con gli altri eventi cellulari associati alla cancerogenesi. Nel laboratorio del Prof. Martin Teichmann è stata identificata una proteina omologa alla subunità RPC32 della Pol III (Haurie et al., 2010). Tale proteina, battezzata RPC32beta, risulta ubiquitaria ed essenziale per la sopravvivenza cellulare. Invece, la presenza e l’attività di RPC32alpha (la variante nota in letteratura) sembrano limitate alle cellule staminali embrionali indifferenziate ed a quelle tumorali. L’overespressione di RPC32alpha, inoltre, favorisce ed accelera il processo neoplastico ed apporta un cambiamento drammatico all’espressione di molti markers tumorali e di un subset di geni Pol III. E’ verosilmile che tale subunità partecipi attivamente alla riprogrammazione trascrizionale durante la carcinogenesi, reindirizzando l’RNA polimerasi III su nuovi geni che poi contribuiscono alla trasformazione neoplastica. Alternativamente, il suo effetto potrebbe essere quello di modificare il tasso trascrizionale di geni Pol III, generando così uno squilibrio molecolare che induce la cellula all’oncogenesi. Il lavoro di tesi in questione ha portato all'identificazione, tramite esperimenti di Immunoprecipitazione cromatinica, ChIP cloning e Genome Wide Sequencing, dei geni trascritti dalle due varianti di Pol III: la Pol IIIalpha (che incorpora RPC32alpha) e la Pol IIIbeta (che invece si distingue per la presenza di RPC32beta). La localizzazione su scala genomica delle due polimerasi rivela la presenza di entrambe su molti geni Pol III, suggerendo un’attività ridondante per le due forme nella trascrizione di questi geni. Appare evidente, al contempo, che alcuni geni sono occupati solamente o preferenzialmente da una delle due varianti (tra questi geni, numerose Alu). La genome wide location delle due polimerasi ha condotto anche all’identificazione di nuovi loci PolIII. Tra questi degli pseudogeni dei tRNAs, alcuni snoRNA, delle MIRs (Mammalian-wide Interspersed Repeats). La funzione di tali RNAs nel processo di trasformazione neoplastica che vede partecipe RPC32alpha rimane ancora da chiarire. La presenza dell’intero macchinario Pol III in questi siti è stato confermata tramite immunoprecipitazione cromatica di RPC53, RPC39, Brf1, Brf2, TFIIIC35

Genome wide identification of genes transcribed by two isoforms of human RNA

En 2010, Haurie et al. ont identifié deux isoformes différentes de la Pol III humaine : Pol IIIα, quin’est présente que dans les cellules souches embryonnaires et dans celles tumorales, et Pol IIIβ,exprimée constitutivement.L’expression ectopique de Pol IIIα affecte profondément l’équilibre de cellules différentiées, jusqu’àen induire la transformation oncogénique. Ceci n’est pas observé dans le cas de l’expression ectopiquede Pol IIIβ.A fin de définir les causes moléculaires de la transformation cellulaire induite par Pol IIIα, nousavons décidé d’étudier son transcriptome en comparaison avec celui de Pol IIIβ, à la recherche desgènes qui pourraient soutenir l’oncogenèse observée. Dans notre étude, nous avons adopté latechnique du ChIP-Seq, une approche innovatrice et puissante qui permet de cartographier tous lessites de liaison d’une protéine sur le génome. Elle comporte la purification de la protéine d’intérêtcomplexe avec ces cibles génomiques et le séquençage de ces dernières de manière massive.Le premier but de cette thèse a été représenté par la mise a point d’un protocole de ChIP-Seqoptimisé spécifiquement pour Pol IIIα et Pol IIIβ. Une fois obtenue une préparation de chromatine dequalité adéquate aux standards du ChIP-Seq, nous avons effectué les séquençages massifs etcartographié sur le génome tous les loci contactés par les deux isoformes de polymérase.De cette manière, nous avons identifié 1287 loci occupés par Pol IIIα et 1281 par Pol IIIβ. Les deuxpolymérases se co-localisent sur la plupart de ces sites, mais montrent aussi une occupationpréférentielle et spécifique sur une fraction de loci Pol III. Cette disproportion pourrait contribuer auphénomène cellulaire observé par Haurie et al.Le manuscrit de thèse détaille les résultats de cette analyse et les conclusions qui en dérivent.Nous y avons ajouté une synthèse des travaux réalisés antérieurement et concertants la caractérisationdes promoteurs de gène qui codent pour les snoARN chez la levure S.cerevisiae, positionnée en toutefin de document.In eukaryotes, transcription is carried out by DNA-dependent RNA polymerases I, II and III (or I-V inplants). These RNA polymerases are specialized in the transcription of specific groups of genes.Human RNA polymerase III (Pol III) transcribes small noncoding RNAs involved in the regulation oftranscription (7SK RNA), RNA processing (U6 RNA, RNAse P, RNAse MRP), translation (tRNAs,5S RNA) or other cellular processes (vault RNAs [multidrug resistance], adenoviral RNAs [VA-I,VA-II], Epstein-Barr virus RNAs [EBER1, EBER2]). It has furthermore been reported that somemicroRNAs of viral or cellular origin may also be transcribed by Pol III.Interestingly, increased Pol III transcription levels accompany or cause cell transformation. Themechanisms underlying this phenomenon are still largely unknown. Recently, two distinct isoforms ofhuman Pol III have been discovered (Haurie et al., 2010). RPC32β-containing Pol IIIβ is ubiquitouslyexpressed and essential for growth and survival of human cells. In contrast, RPC32α-containing PolIIIα is dispensable for cell survival and its expression is restricted to undifferentiated embryonic stem(ES) cells and to tumor cells.The distinct effects of Pol IIIα and Pol III β on cell growth and transformation may be explained bythe transcription of isoform-specific target genes. To identify such isoform-specific target genes, wespecifically targeted RPC32α and of RPC32β subunits in chromatin immunoprecipitation (ChIP)experiments and analyzed the co-precipitated DNA sequences by high throughput sequencing (ChIPseq.).Genome wide localization of RPC32α and RPC32β subunits of Pol III revealed the presence of bothsubunits on many of the known Pol III-transcribed genes, suggesting redundant activities of bothisoforms of Pol III in transcription of these genes. We also found that some of the genes known to betranscribed by Pol III are only occupied by either RPC32α or by RPC32β, suggesting that these genesare exclusively transcribed by Pol IIIα or by Pol IIIβ, respectively.RPC32α and RPC32β ChIP-seq. results furthermore led to the identification of novel Pol III candidategenes in HeLa cells. Moreover, we found high levels of Pol IIIα or Pol III β at some of the annotatedtRNA pseudogenes, implicating that these genes may be transcribed. The functions of RNAstranscribed from novel putative Pol III genes or from tRNA pseudogenes remain to be determined