Associations of clock genes polymorphisms with soft tissue sarcoma susceptibility and prognosis

BACKGROUND: Dysfunction of the circadian clock and polymorphisms of some circadian genes have been linked to cancer development and progression. We investigated the relationship between circadian genes germline variation and susceptibility or prognosis of patients with soft tissue sarcoma. PATIENTS AND METHODS: We considered the 14 single nucleotide polymorphisms (SNPs) of 6 core circadian genes that have a minor allele frequency >\u20095% and that are known to be associated with cancer risk or prognosis. Genotyping was performed by q-PCR. Peripheral blood and clinic-pathological data were available for 162 patients with liposarcoma or leiomyosarcoma and 610 healthy donors. Associations between the selected clock genes polymorphisms and sarcoma susceptibility or prognosis were tested assuming 3 models of inheritance: additive, recessive and dominant. Subgroup analysis based on sarcoma histotype was performed under the additive genetic model. Multivariate logistic regression and multivariate Cox proportional hazard regression analyses were utilized to assess the association between SNPs with patient susceptibility and survival, respectively. Pathway variation analysis was conducted employing the Adaptive Rank Truncated Product method. RESULTS: Six out of the 14 analyzed SNPs were statistically significantly associated with susceptibility or prognosis of soft tissue sarcoma (P <\u20090.05). The present analysis suggested that carriers of the minor allele of the CLOCK polymorphism rs1801260 (C) or of PER2 rs934945 (T) had a reduced predisposition to sarcoma (26% and 35% respectively with the additive model) and liposarcoma (33% and 41% respectively). The minor allele (A) of NPAS2 rs895520 was associated with an increased predisposition to sarcoma of 33% and leiomyosarcoma of 44%. RORA rs339972 C allele was associated with a decreased predisposition to develop sarcoma assuming an additive model (29%) and leiomyosarcoma (36%). PER1 rs3027178 was associated with a reduced predisposition only in liposarcoma subgroup (32%). rs7602358 located upstream PER2 was significantly associated with liposarcoma survival (HR: 1.98; 95% CI 1.02-3.85; P\u2009=\u20090.04). Germline genetic variation in the circadian pathway was associated with the risk of developing soft tissue sarcoma (P\u2009=\u20090.035). CONCLUSIONS: Genetic variation of circadian genes appears to play a role in the determinism of patient susceptibility and prognosis. These findings prompt further studies to fully dissect the molecular mechanisms

First steps to define murine amniotic fluid stem cell microenvironment

Stem cell niche refers to the microenvironment where stem cells reside in living organisms. Several elements define the niche and regulate stem cell characteristics, such as stromal support cells, gap junctions, soluble factors, extracellular matrix proteins, blood vessels and neural inputs. In the last years, different studies demonstrated the presence of cKit+ cells in human and murine amniotic fluid, which have been defined as amniotic fluid stem (AFS) cells. Firstly, we characterized the murine cKit+ cells present both in the amniotic fluid and in the amnion. Secondly, to analyze the AFS cell microenvironment, we injected murine YFP+ embryonic stem cells (ESC) into the amniotic fluid of E13.5 wild type embryos. Four days after transplantation we found that YFP+ sorted cells maintained the expression of pluripotency markers and that ESC adherent to the amnion were more similar to original ESC in respect to those isolated from the amniotic fluid. Moreover, cytokines evaluation and oxygen concentration analysis revealed in this microenvironment the presence of factors that are considered key regulators in stem cell niches. This is the first indication that AFS cells reside in a microenvironment that possess specific characteristics able to maintain stemness of resident and exogenous stem cells

Endothelial properties of third-trimester amniotic fluid stem cells cultured in hypoxia

open12siopenSchiavo, Andrea Alex; Franzin, Chiara; Albiero, Mattia; Piccoli, Martina; Spiro, Giovanna; Bertin, Enrica; Urbani, Luca; Visentin, Silvia; Cosmi, Erich; Fadini, Gian Paolo; De Coppi, Paolo; Pozzobon, MichelaSchiavo, ANDREA ALEX; Franzin, Chiara; Albiero, Mattia; Piccoli, Martina; Spiro, Giovanna; Bertin, Enrica; Urbani, Luca; Visentin, Silvia; Cosmi, Erich; Fadini, GIAN PAOLO; DE COPPI, Paolo; Pozzobon, Michel

The predictive and prognostic potential of plasma telomerase reverse transcriptase (TERT) RNA in rectal cancer patients

Background: Preoperative chemoradiotherapy (CRT) followed by surgery is the standard care for locally advanced rectal cancer, but tumour response to CRT and disease outcome are variable. The current study aimed to investigate the effectiveness of plasma telomerase reverse transcriptase (TERT) levels in predicting tumour response and clinical outcome. Methods: 176 rectal cancer patients were included. Plasma samples were collected at baseline (before CRT\ubcT0), 2 weeks after CRT was initiated (T1), post-CRT and before surgery (T2), and 4\u20138 months after surgery (T3) time points. Plasma TERT mRNA levels and total cell-free RNA were determined using real-time PCR. Results: Plasma levels of TERT were significantly lower at T2 (Po0.0001) in responders than in non-responders. Post-CRT TERT levels and the differences between pre- and post-CRT TERT levels independently predicted tumour response, and the prediction model had an area under curve of 0.80 (95% confidence interval (CI) 0.73\u20130.87). Multiple analysis demonstrated that patients with detectable TERT levels at T2 and T3 time points had a risk of disease progression 2.13 (95% CI 1.10\u20134.11)-fold and 4.55 (95% CI 1.48\u201313.95)-fold higher, respectively, than those with undetectable plasma TERT levels. Conclusions: Plasma TERT levels are independent markers of tumour response and are prognostic of disease progression in rectal cancer patients who undergo neoadjuvant therapy

Impact of COVID-19 on cardiovascular testing in the United States versus the rest of the world

Objectives: This study sought to quantify and compare the decline in volumes of cardiovascular procedures between the United States and non-US institutions during the early phase of the coronavirus disease-2019 (COVID-19) pandemic. Background: The COVID-19 pandemic has disrupted the care of many non-COVID-19 illnesses. Reductions in diagnostic cardiovascular testing around the world have led to concerns over the implications of reduced testing for cardiovascular disease (CVD) morbidity and mortality. Methods: Data were submitted to the INCAPS-COVID (International Atomic Energy Agency Non-Invasive Cardiology Protocols Study of COVID-19), a multinational registry comprising 909 institutions in 108 countries (including 155 facilities in 40 U.S. states), assessing the impact of the COVID-19 pandemic on volumes of diagnostic cardiovascular procedures. Data were obtained for April 2020 and compared with volumes of baseline procedures from March 2019. We compared laboratory characteristics, practices, and procedure volumes between U.S. and non-U.S. facilities and between U.S. geographic regions and identified factors associated with volume reduction in the United States. Results: Reductions in the volumes of procedures in the United States were similar to those in non-U.S. facilities (68% vs. 63%, respectively; p = 0.237), although U.S. facilities reported greater reductions in invasive coronary angiography (69% vs. 53%, respectively; p < 0.001). Significantly more U.S. facilities reported increased use of telehealth and patient screening measures than non-U.S. facilities, such as temperature checks, symptom screenings, and COVID-19 testing. Reductions in volumes of procedures differed between U.S. regions, with larger declines observed in the Northeast (76%) and Midwest (74%) than in the South (62%) and West (44%). Prevalence of COVID-19, staff redeployments, outpatient centers, and urban centers were associated with greater reductions in volume in U.S. facilities in a multivariable analysis. Conclusions: We observed marked reductions in U.S. cardiovascular testing in the early phase of the pandemic and significant variability between U.S. regions. The association between reductions of volumes and COVID-19 prevalence in the United States highlighted the need for proactive efforts to maintain access to cardiovascular testing in areas most affected by outbreaks of COVID-19 infection

Study of the contribution of muscle inflammation, IMAT origin and microenvironment to sarcopenic obesity

Introduction The optimal condition for skeletal muscle function is represented by an efficient activation of processes that regulate muscle development, growth, regeneration and metabolism. When the balance among these events is missing, a pathological condition arise. In particular, loss or decrease of skeletal muscle function and mass is linked with severe health diseases such as muscle dystrophies, cancer, obesity and aging process. In this context, sarcopenic obesity, a metabolic disorder followed by atrophy of muscle fibres and reduction of stem cell reservoir (satellite cells in muscle) is associated with increasing ectopic adipose tissue, impairing glucose tolerance and decreasing strength and mobility in old adults. This excess of fatty acids accumulated in several other organs, including skeletal muscle, induces metabolic dysfunctions, β-oxidative alterations, increase of ROS production, lipotoxicity and insulin resistance. Moreover this intermuscular adipose tissue (IMAT) acts as chemoattractant for inflammatory cells, chiefly macrophages, which produce high amount of pro-inflammatory cytokines (TNF-α, IL-6, IL-1, INFγ) and low of anti-inflammatory cytokines (IL-4, IL-10) resulting in chronic low-grade systemic inflammation and muscle loss. Aims The aim of this work is to investigate the origin of intermuscular fat and the complex mechanisms regulating the crosstalk with the surrounding muscle. In the first part of the study we try to understand the differences in muscle inflammation in obese subjects both normoglycemic and diabetic versus healthy patients using muscle biopsies. Moreover, in the same groups of patients, we attempt to identify serum microRNAs as possible useful markers of muscle alterations in presence of obesity. Because it is well known that in chronic diseases, as obesity and diabetes, multifactorial components contribute to the pathology, we decided to investigate the microenvironment of skeletal muscle, represented by extracellular matrix (ECM). We aim to understand if it could have had a role in the deposition of IMAT and on associated inflammatory processes using an obese mouse model. Material and methods From healthy and obese patients either normoglycemic or diabetic, both muscle biopsies and serum samples were collected. By Real-time PCR the first were analysed for selected inflammation markers, whereas the second for the principal muscle circulating miRNAs. In the second part of the study, a detergent-enzymatic decellularization protocol for wild type (C57BL6J) and ob/ob (B6.Cg-Lepob/J) murine quadriceps were set and samples were characterized for DNA content and ECM structure preservation. After that, a volumetric muscle loss model on wild-type mice was created and ECMs from wt or ob/ob quadriceps mice were implanted. The ability of the matrices to be remodelled and reabsorbed was evaluated by immunofluorescence and molecular analysis after 7, 15 and 30 days. Results and discussion mRNA from three human rectus abdomins biopsies from obese patients both normoglycemic and diabetic and three peroneal muscle biopsies from healthy subjects indicate an increase in pro-inflammatory gene expression both in normoglycemic and diabetic patients in comparison with the controls. However, in contrast to what we expected, diabetic subjects, that were under Metformin treatment from at least 1 year, showed a reduction of inflammation. The serum miR133a, miR133b and miR1 levels of healthy and obese patients both normoglycemic and diabetic, display the same trend previously described for muscle biopsies. This suggests a possible direct correlation between muscle inflammation and the release of these circulating miRNAs, which could be used in the future as indicative markers of patient muscle alterations. On the other hand, the decellularization of wild type (wt) and ob/ob quadriceps did not show significant structural differences between the two samples. In vivo transplantation of the two decellularized muscle matrix (ECMs) in wt injured mice induced after 7 days a strong cells migration in the interface between the native tissue and the matrix that gradually diminished after 15 and 30 days. . Pan macrophages cells (CD68+ cells) were found in both ECM implantation, mainly in the area around the stitches and in the interface between the matrix and the native tissue, but in the inner part of the recipient absence of inflammatory infiltrate was evident at all time points. Alcian blue and Masson’s Trichrome stains highlighted similar GAG deposition and collagen components between wt and ob/ob donor ECM. In particular we observed that the scaffold derived from donor affected by a metabolic disease did no induce alteration in structural matrix deposition but promoted a premature M2 inflammation that was reduced or stabilised after 30 days. On the contrary healthy ECM in wt mice start later the tissue rebuilding leading to a more complete regeneration. No differences were also observed in activation or modulation of myogenic and adipogenic pathways after both implantations.Introduzione La condizione ottimale attraverso cui il muscolo scheletrico può espletare la propria funzione è rappresentata da un’ efficiente attivazione dei processi che regolano lo sviluppo, la crescita, la rigenerazione e il metabolismo del muscolo stesso. Quando viene a mancare l’equilibrio tra questi eventi è il momento in cui insorge una condizione patologica. Nello specifico, una perdita o riduzione di massa e funzionalità muscolare è collegata a gravi malattie, quali distrofie, cancro, obesità e processi di invecchiamento. In questo contesto, l’obesità sarcopenica, una malattia metabolica che porta ad atrofia delle fibre muscolari e riduzione delle cellule staminali (chiamate nel muscolo cellule satelliti), è associata ad un aumento in zone ectopiche di tessuto adiposo, a riduzione della tolleranza al glucosio e a perdita di forza e motilità soprattutto in adulti e anziani. Questo accumulo di acidi grassi in altri distretti corporei, compreso il muscolo scheletrico, induce disfunzioni metaboliche, alterazioni nella β-ossidazione, aumento di produzione di ROS, lipotossicità e insulino-resistenza. Inoltre questo tessuto adiposo intermuscolare (IMAT) agisce come chemoattrattivo per le cellule infiammatorie, soprattutto macrofagi, che, una volta in sede, producono elevate quantità di citochine pro-infiammatorie (TNF-α, IL-6, IL-1, INFγ) e basse quantità di citochine anti-infiammatorie (IL-4, IL-10). Tutto ciò porta ad un costante basso grado di infiammazione sistemica e a conseguente perdita di massa muscolare. Scopo Scopo di questo lavoro è quello di investigare l’origine del tessuto adiposo intermuscolare e i meccanismi che regolano il dialogo tra questo e il tessuto muscolare circostante. La prima parte dello studio ha lo scopo di capire, utilizzando biopsie muscolari, le differenze che ci sono a livello di infiammazione del muscolo in soggetti obesi normoglicemici e diabetici rispetto ai pazienti sani. Inoltre, analizzando i sieri di pazienti che appartengono alle stesse categorie sopraelencate, saranno individuati possibili miRNA da utilizzare come marcatori identificativi delle alterazioni muscolari in presenza di obesità. Poiché in malattie croniche, quali obesità e diabete di tipo2, diversi fattori contribuiscono alla patologia, abbiamo deciso di investigare anche il microambiente del muscolo scheletrico, inteso come matrice extracellulare (ECM). Utilizzando un modello murino di obesità cercheremo di capire se questo può avere un ruolo nella deposizione dell’IMAT e dei processi infiammatori ad esso associati. Materiali e metodi Sia biopsie muscolari sia campioni di siero sono stati prelevati da pazienti sani, obesi normoglicemici e obesi diabetici. Mediante l’utilizzo di analisi di Real-time PCR le prime sono state analizzate per i principali marcatori infiammatori, mentre i secondi per i principali miRNA muscolari circolanti. Nella seconda parte dello studio, invece, è stato settato un protocollo di decellularizzazione del tipo detergente enzimatico su quadricipiti murini di topi wild type (C57BL6J) e di topi ob/ob (B6.Cg-Lepob/J) e i campioni ottenuti sono stati caratterizzati per il contenuto di DNA e le componenti strutturali della matrice. In seguito è stato creato un modello di perdita consistente di massa muscolare in topi wild type (wt) ed è stata impiantata nelle sede di danno una porzione di matrice proveniente da topi wt o ob/ob. La capacità delle diverse matrici di essere rimodellate e riassorbite è stata analizzata dopo 7, 15 e 30 giorni mediante analisi di immunofluorescenza e molecolari. Risultati e discussione L’mRNA estratto da 3 biopsie di retto addominale di pazienti obesi normoglicemici e obesi diabetici, insieme a 3 biopsie di muscolo peroniere di soggetti sani indica la presenza di una maggiore espressione dei geni pro-infiammatori sia nei soggetti obesi normoglicemici sia diabetici in confronto a quelli sani. Tuttavia, in contrasto con quanto ci aspettavamo, i pazienti diabetici che erano in trattamento con Metformina da almeno 1 anno mostravano un grado infiammatorio inferiore. Osservando poi i livelli di miRNA 133a, miR133b e miR1 presenti nel siero di una coorte di pazienti sani, obesi normoglicemici e obesi diabetici abbiano notato lo stesso andamento descritto in precedenza per le biopsie muscolari. Questo ci suggerisce una possibile correlazione diretta tra infiammazione muscolare e rilascio di questi miRNA circolanti, che in futuro potranno essere utilizzati come marcatori della condizione muscolare del paziente. La decellularizzazione dei quadricipiti wt e ob/ob non ha evidenziato differenze strutturali tra le due matrici. Inoltre il trapianto in vivo di entrambe ha indotto a 7 giorni una forte migrazione cellulare all’interfaccia tra tessuto nativo e matrice, la quale diminuisce a 15 e 30 giorni dopo impianto. Cellule macrofagiche (CD68+) sono state trovate in entrambe le matrici, soprattutto nelle aree attorno ai punti di sutura e all’interfaccia con il tessuto nativo, tuttavia a nessun time point è evidente una condizione infiammatoria nella parte più interna del muscolo nativo. La colorazione Alcian Blue e quella della Tricromica di Masson hanno mostrato un’ induzione nella deposizione di glicosamminoglicani e di collagene simile tra le due matrici. Nello specifico, abbiamo osservato, quindi, che una matrice derivante da un donatore affetto da malattia metabolica non è in grado di indurre un’alterazione nella deposizione di componenti strutturali ma promuove una prematura risposta infiammatoria di tipo M2 che si riduce o stabilizza dopo 30 giorni dall’impianto. Al contrario, la matrice proveniente da topi sani induce una ricostruzione del tessuto più ritardata ma che porta a una miglior e completa rigenerazione. Nessuna differenza, infine, è stata osservata in termini di attivazione e modulazione dei segnali miogenici e adipogenici per entrambi gli impianti.

Study of the contribution of muscle inflammation, IMAT origin and microenvironment to sarcopenic obesity

Introduction The optimal condition for skeletal muscle function is represented by an efficient activation of processes that regulate muscle development, growth, regeneration and metabolism. When the balance among these events is missing, a pathological condition arise. In particular, loss or decrease of skeletal muscle function and mass is linked with severe health diseases such as muscle dystrophies, cancer, obesity and aging process. In this context, sarcopenic obesity, a metabolic disorder followed by atrophy of muscle fibres and reduction of stem cell reservoir (satellite cells in muscle) is associated with increasing ectopic adipose tissue, impairing glucose tolerance and decreasing strength and mobility in old adults. This excess of fatty acids accumulated in several other organs, including skeletal muscle, induces metabolic dysfunctions, β-oxidative alterations, increase of ROS production, lipotoxicity and insulin resistance. Moreover this intermuscular adipose tissue (IMAT) acts as chemoattractant for inflammatory cells, chiefly macrophages, which produce high amount of pro-inflammatory cytokines (TNF-α, IL-6, IL-1, INFγ) and low of anti-inflammatory cytokines (IL-4, IL-10) resulting in chronic low-grade systemic inflammation and muscle loss. Aims The aim of this work is to investigate the origin of intermuscular fat and the complex mechanisms regulating the crosstalk with the surrounding muscle. In the first part of the study we try to understand the differences in muscle inflammation in obese subjects both normoglycemic and diabetic versus healthy patients using muscle biopsies. Moreover, in the same groups of patients, we attempt to identify serum microRNAs as possible useful markers of muscle alterations in presence of obesity. Because it is well known that in chronic diseases, as obesity and diabetes, multifactorial components contribute to the pathology, we decided to investigate the microenvironment of skeletal muscle, represented by extracellular matrix (ECM). We aim to understand if it could have had a role in the deposition of IMAT and on associated inflammatory processes using an obese mouse model. Material and methods From healthy and obese patients either normoglycemic or diabetic, both muscle biopsies and serum samples were collected. By Real-time PCR the first were analysed for selected inflammation markers, whereas the second for the principal muscle circulating miRNAs. In the second part of the study, a detergent-enzymatic decellularization protocol for wild type (C57BL6J) and ob/ob (B6.Cg-Lepob/J) murine quadriceps were set and samples were characterized for DNA content and ECM structure preservation. After that, a volumetric muscle loss model on wild-type mice was created and ECMs from wt or ob/ob quadriceps mice were implanted. The ability of the matrices to be remodelled and reabsorbed was evaluated by immunofluorescence and molecular analysis after 7, 15 and 30 days. Results and discussion mRNA from three human rectus abdomins biopsies from obese patients both normoglycemic and diabetic and three peroneal muscle biopsies from healthy subjects indicate an increase in pro-inflammatory gene expression both in normoglycemic and diabetic patients in comparison with the controls. However, in contrast to what we expected, diabetic subjects, that were under Metformin treatment from at least 1 year, showed a reduction of inflammation. The serum miR133a, miR133b and miR1 levels of healthy and obese patients both normoglycemic and diabetic, display the same trend previously described for muscle biopsies. This suggests a possible direct correlation between muscle inflammation and the release of these circulating miRNAs, which could be used in the future as indicative markers of patient muscle alterations. On the other hand, the decellularization of wild type (wt) and ob/ob quadriceps did not show significant structural differences between the two samples. In vivo transplantation of the two decellularized muscle matrix (ECMs) in wt injured mice induced after 7 days a strong cells migration in the interface between the native tissue and the matrix that gradually diminished after 15 and 30 days. . Pan macrophages cells (CD68+ cells) were found in both ECM implantation, mainly in the area around the stitches and in the interface between the matrix and the native tissue, but in the inner part of the recipient absence of inflammatory infiltrate was evident at all time points. Alcian blue and Masson’s Trichrome stains highlighted similar GAG deposition and collagen components between wt and ob/ob donor ECM. In particular we observed that the scaffold derived from donor affected by a metabolic disease did no induce alteration in structural matrix deposition but promoted a premature M2 inflammation that was reduced or stabilised after 30 days. On the contrary healthy ECM in wt mice start later the tissue rebuilding leading to a more complete regeneration. No differences were also observed in activation or modulation of myogenic and adipogenic pathways after both implantations

Genetic susceptibility to bone and soft tissue sarcomas: a field synopsis and meta-analysis

Background: The genetic architecture of bone and soft tissue sarcomas susceptibility is yet to be elucidated. We aimed to comprehensively collect and meta-analyze the current knowledge on genetic susceptibility in these rare tumors. Methods: We conducted a systematic review and meta-analysis of the evidence on the association between DNA variation and risk of developing sarcomas through searching PubMed, The Cochrane Library, Scopus and Web of Science databases. To evaluate result credibility, summary evidence was graded according to the Venice criteria and false positive report probability (FPRP) was calculated to further validate result noteworthiness. Integrative analysis of genetic and eQTL (expression quantitative trait locus) data was coupled with network and pathway analysis to explore the hypothesis that specific cell functions are involved in sarcoma predisposition. Results: We retrieved 90 eligible studies comprising 47,796 subjects (cases: 14,358, 30%) and investigating 1,126 polymorphisms involving 320 distinct genes. Meta-analysis identified 55 single nucleotide polymorphisms (SNPs) significantly associated with disease risk with a high (N=9), moderate (N=38) and low (N=8) level of evidence, findings being classified as noteworthy basically only when the level of evidence was high. The estimated joint population attributable risk for three independent SNPs (rs11599754 of ZNF365/EGR2, rs231775 of CTLA4, and rs454006 of PRKCG) was 37.2%. We also identified 53 SNPs significantly associated with sarcoma risk based on single studies. Pathway analysis enabled us to propose that sarcoma predisposition might be linked especially to germline variation of genes whose products are involved in the function of the DNA repair machinery. Conclusions: We built the first knowledgebase on the evidence linking DNA variation to sarcomas susceptibility, which can be used to generate mechanistic hypotheses and inform future studies in this field of oncology

Irisin prevents and restores bone loss and muscle atrophy in hind-limb suspended mice

We previously showed that Irisin, a myokine released from skeletal muscle after physical exercise, plays a central role in the control of bone mass. Here we report that treatment with recombinant Irisin prevented bone loss in hind-limb suspended mice when administered during suspension (preventive protocol) and induced recovery of bone mass when mice were injected after bone loss due to a suspension period of 4 weeks (curative protocol). MicroCT analysis of femurs showed that r-Irisin preserved both cortical and trabecular bone mineral density, and prevented a dramatic decrease of the trabecular bone volume fraction. Moreover, r-Irisin protected against muscle mass decline in the hind-limb suspended mice, and maintained the fiber cross-sectional area. Notably, the decrease of myosin type II expression in unloaded mice was completely prevented by r-Irisin administration. Our data reveal for the first time that Irisin retrieves disuse-induced bone loss and muscle atrophy. These findings may lead to development of an Irisin-based therapy for elderly immobile osteoporotic and physically disable patients, and might represent a countermeasure for astronauts subjected to microgravity-induced bone and muscle losses