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A subclass of the sialic acid family consists of intramolecular lactones that may function as key indicators of physiological and pathological states. However, the existence of these compounds in free form is highly improbable, since they are unlikely to exist in an aqueous solution due to their lability. Current analytical method used to detect them in biological fluids has not recognized their reactivity in solution and is prone to misidentification. However, recent advances in synthetic methods for 1,7-lactones have allowed the preparation of these sialic acid derivatives as authentic reference standards. We report here the development of a new HPLC–MS method for the simultaneous detection of the 1,7-lactone of N-acetylneuraminic acid, its γ-lactone derivative, and N-acetylneuraminic acid that overcomes the limitations of the previous analytical procedure for their identification.

Brugada Syndrome (BrS) is an inherited arrhythmogenic disorder with an increased risk of sudden cardiac death. Recent evidence suggests that BrS should be considered as an oligogenic or polygenic condition. Mutations in genes associated with BrS are found in about one-third of patients and they mainly disrupt the cardiac sodium channel NaV1.5, which is considered the main cause of the disease. However, voltage-gated channel’s activity could be impacted by post-translational modifications such as sialylation, but their role in BrS remains unknown. Thus, we analyzed high risk BrS patients (n = 42) and healthy controls (n = 42) to assess an involvement of sialylation in BrS. Significant alterations in gene expression and protein sialylation were detected in Peripheral Blood Mononuclear Cells (PBMCs) from BrS patients. These changes were significantly associated with the phenotypic expression of the disease, as the size of the arrhythmogenic substrate and the duration of epicardial electrical abnormalities. Moreover, protein desialylation caused a reduction in the sodium current in an in vitro NaV1.5-overexpressing model. Dysregulation of the sialylation machinery provides definitive evidence that BrS affects extracardiac tissues, suggesting an underlying cause of the disease. Moreover, detection of these changes at the systemic level and their correlation with the clinical phenotype hint at the existence of a biomarker signature for BrS.

The salvage of myocardium after acute myocardial infarction (AMI) can only be achieved by timely reperfusion. This is life-saving, however it may also lead to additional damage in the form of ischemia and reperfusion injury (IRI). Protecting cardiac tissue from IRI remains one of the greatest unmet clinical needs in cardiology. Therefore, a better understanding of IRI pathophysiology is necessary. There has been increasing recognition that the activation of specific molecular mechanisms, including the Reperfusion Injury Salvage Kinase (RISK) pathway and HIF-1α contributes to cardioprotection against IRI. Along this line, we previously reported that sialidase Neu3, which removes sialic acid from glycosphingolipids, controls HIF-1α activation via a prolyl hydroxylase-independent pathway. In fact, we found that Neu3 was up-regulated under chronic hypoxia in cyanotic congenital cardiac patients and its overexpression increased muscle cells’ resistance to hypoxic stress by activating the RISK pathway. In light of these premises, the goal of this study was to determine Neu3's involvement in the cardiac response to IRI and establish whether its inducible activation could promote cardioprotection. An IRI model in mice, obtained by temporarily blocking the LAD coronary artery, showed that Neu3 is modulated during this process. Notably, Neu3 showed progressive downregulation during the ischemic phase followed by its reactivation during reperfusion. These results were similar to the Neu3 modulation we observed in human cardiac cells (AC16) exposed to an IRI model in vitro . The overexpression of Neu3 during ischemia had beneficial effects both in terms of cell proliferation and reduction of oxidative stress after reperfusion. We found that Neu3 promotes the activation of glycolytic enzymes during the ischemic phase as well as the maintenance of the mitochondrial membrane potential, suggesting that the Neu3-overexpressing human cardiomyocyte undergoes more aerobic metabolism even in oxygen-deficient environments. In conclusion, these results support a role for Neu3 in cardioprotection against IRI, suggesting that it may be a useful therapeutic target for improving the recovery of patients who have undergone acute myocardial infarction.

Brugada syndrome (BrS) is a cardiac arrhythmia associated with a higher risk of SCD. BrS is considered a genetic disorder, and the most commonly mutated gene is SCN5A, encoding the alpha subunit of the voltage-gated cardiac sodium channel (NaV1.5). Mutations of SCN5A usually lead to impairment of NaV1.5 functionality, which is considered the main mechanism of the disease. However, SCN5A mutations are responsible for only 30% of BrS cases. Therefore, it is conceivable that other mechanisms such as post-translational modifications (PTMs) affect NaV1.5 activity. Among others, sialylation may alter ion channel activity by carrying a sugar with a negative charge. Alterations in sialylation have been described in several cardiovascular diseases, as myocardial infarction, Chagas disease, and congenital disorders of glycosylation. For these reasons, the aim was to investigate changes in sialylation in BrS patients to obtain new information on the pathogenesis of BrS. Peripheral blood mononuclear cells (PBMCs) were collected from BrS patients and healthy controls. SNA lectin, a sialic acid-binding protein, was used to characterize the protein sialylation status of PBMCs by Western blot and flow cytometry. Gene expression of enzymes involved in the sialic acid pathway was also examined. The results showed a significant decrease in intracellular and extracellular protein sialylation levels in BrS PBMCs compared with controls. In addition, changes in gene expression of enzymes involved in the sialic acid pathway were detected. Moreover, these changes correlated with clinical parameters associated with phenotypic expression of the disease, such as arrhythmogenic BrS substrate area and potential duration. These findings support that BrS should be considered a systemic disease and are consistent with the presence of overlapping non-cardiac pathologies, as epilepsy, cancer, diabetes, skeletal muscle channelopathies, and laminopathies in BrS patients. Moreover, the discovery that molecular alterations can be found in the peripheral blood of BrS patients supports the existence of a biomarker of the disease. It is a challenge to develop an effective diagnostic test to screen broadly for BrS. In this direction, a multiomic study is ongoing in our center.

Sarcopenia, an age-related decline in muscle mass and strength, is associated with metabolic disease and increased risk of cardiovascular morbidity and mortality. It is associated with decreased tissue vascularization and muscle atrophy. In this work, we investigated the role of the hypoxia inducible factor HIF-1α in sarcopenia. To this end, we obtained skeletal muscle biopsies from elderly sarcopenic patients and compared them with those from young individuals. We found a decrease in the expression of HIF-1α and its target genes in sarcopenia, as well as of PAX7, the major stem cell marker of satellite cells, whereas the atrophy marker MURF1 was increased. We also isolated satellite cells from muscle biopsies and cultured them in vitro. We found that a pharmacological activation of HIF-1α and its target genes caused a reduction in skeletal muscle atrophy and activation of PAX7 gene expression. In conclusion, in this work we found that HIF-1α plays a role in sarcopenia and is involved in satellite cell homeostasis. These results support further studies to test whether pharmacological reactivation of HIF-1α could prevent and counteract sarcopenia.

Aim To systemically review the available evidence on the clinical performance of osseous resective surgery (ORS) in the treatment of residual periodontal defects in terms of pocket elimination and biological costs in patients with chronic periodontitis. Materials and methods Three databases (PubMed, EMBASE and Cochrane) were searched up to January 2019. Clinical trials with a follow-up duration of at least 12 months after ORS with or without fibre retention technique were included. Quantitative synthesis was conducted with random-effect meta-analysis. Results Overall, 1,765 studies were retrieved, of which 53 full-text articles were screened by two reviewers. Finally, a total of three RCTs were included in the meta-analysis. Random-effect meta-analysis showed a weighted mean percentage of pocket elimination (final PD ≤ 4 mm) at 12 months of 98.3% (95% CI: 96.8; 99.7) with I2 of 26%. The weighted mean amount of resected bone was 0.87 mm (95% CI: 0.49; 1.25), and the weighted mean increase in gingival recession was 2.13 mm (95% CI: 1.49; 2.78) at 12 months. Conclusions ORS represents an effective surgical approach for the elimination of residual periodontal pockets in the short to medium term. Additional randomized controlled clinical trials with data on pocket elimination are warranted.

The acidic hydrolysis of N-acetylneuraminic 4,5-oxazoline affords the corresponding 2,3-unsaturated amino ester, which was not previously detected (nor isolated) due to the unexpected rearrangement into its corresponding alcohol. In this work, we unveiled the mechanism of these reactions and optimized the conditions to obtain either synthetic intermediate.

AIM To analyse the efficacy of non-surgical therapy (NST) in terms of pocket closure (PC) and changes in percentage and number of pockets. MATERIALS AND METHODS Three databases (PubMed, EMBASE, and Scopus) were searched up to January 2020. Prospective studies with a minimum follow-up of 12 months and presenting data in terms of PC or number or percentage of pocket depths (PDs) before and after NST on systemically healthy patients were included. Random-effect meta-analyses were performed. RESULTS After screening 4610 titles and abstracts, 27 studies were included. Of these, 63.9% of PC was reported by one study. The percentage of PDs ≤3 mm changed from 39.06% to 64.11% with a weighted mean difference (WMD) of 26.14% (p

Cardiovascular diseases (CVDs) are the leading cause of death globally and the number of cardiovascular patients, which is estimated to be over 30 million in 2018, represent a challenging issue for the healthcare systems worldwide. Therefore, the identification of novel molecular targets to develop new treatments is an ongoing challenge for the scientific community. In this context, sphingolipids (SLs) have been progressively recognized as potent bioactive compounds that play crucial roles in the modulation of several key biological processes, such as proliferation, differentiation, and apoptosis. Furthermore, SLs involvement in cardiac physiology and pathophysiology attracted much attention, since these molecules could be crucial in the development of CVDs. Among SLs, ceramide and sphingosine-1-phosphate (S1P) represent the most studied bioactive lipid mediators, which are characterized by opposing activities in the regulation of the fate of cardiac cells. In particular, maintaining the balance of the so-called ceramide/S1P rheostat emerged as an important novel therapeutical target to counteract CVDs. Thus, this review aims at critically summarizing the current knowledge about the antithetic roles of ceramide and S1P in cardiomyocytes dysfunctions, highlighting how the modulation of their metabolism through specific molecules, such as myriocin and FTY720, could represent a novel and interesting therapeutic approach to improve the management of CVDs.

Reperfusion strategies, together with thrombolytic therapies, represent life-saving approaches to restore the blood flow in the cardiac tissue after acute myocardial infarction (AMI). However, they inevitably induce the so-called ischemia/reperfusion injury (IRI), resulting in increased cardiomyocytes damage and heart failure. In this context, many efforts have been made to clarify the molecular mechanisms involved in IRI, and the activation of pro-survival kinases, such as Akt and Erk, as well as of the hypoxia-inducible factor (HIF) has been recognized to be critical. Along this line, we discovered a novel mechanism of HIF-1α activation mediated by sialidase Neu3, which is PHDs independent, and that it increased muscle cells resistance to hypoxic stress, through the activation of Akt and Erk pathways. Moreover, an upregulation of Neu3 expression was observed under chronic hypoxia in cyanotic congenital cardiac patients.On these premises, this study aims at investigating the role of Neu3 in protecting cardiac cells against IRI. In particular, H9C2 rat cardiomyoblasts were exposed to an IRI model in vitro revealing a marked reduction in cell proliferation. This was accompanied by the modulation of Neu3 that was characterized by its progressive down-regulation during the ischemic phase, followed by its reactivation during reperfusion. These experiments resembled Neu3 modulation we observed in an IRI mouse model, obtained by the temporary occlusion of the LAD coronary vessel. Interestingly, overexpression of Neu3 significantly increased cardiomyoblasts resistance to IRI, both in terms of cell proliferation and resistance to apoptosis, as well as promoted HIF-1α and Akt/Erk activation. Remarkably, the treatment with Akt and Erk inhibitors completely reverted the beneficial effects mediated by Neu3 upregulation. Likewise, sialidase Neu3 inhibition reduced Akt/Erk activation, resulting in the complete loss of Neu3-mediated cardioprotection. In conclusion, our results demonstrate the role of sialidase Neu3 in counteracting the detrimental effects of IRI, calling for further studies to unveil its full potential as a therapeutic target to support current strategies to manage cardiac damage and to improve patients recover after AMI.

Cardiac fibrosis is a key physiological response to cardiac tissue injury to protect the heart from wall rupture. However, its progression increases heart stiffness, eventually causing a decrease in heart contractility. Unfortunately, to date, no efficient antifibrotic therapies are available to the clinic. This is primarily due to the complexity of the process, which involves several cell types and signaling pathways. For instance, the transforming growth factor beta (TGF-β) signaling pathway has been recognized to be vital for myofibroblasts activation and fibrosis progression. In this context, complex sphingolipids, such as ganglioside GM3, have been shown to be directly involved in TGF-β receptor 1 (TGF-R1) activation. In this work, we report that an induced up-regulation of sialidase Neu3, a glycohydrolytic enzyme involved in ganglioside cell homeostasis, can significantly reduce cardiac fibrosis in primary cultures of human cardiac fibroblasts by inhibiting the TGF-β signaling pathway, ultimately decreasing collagen I deposition. These results support the notion that modulating ganglioside GM3 cell content could represent a novel therapeutic approach for cardiac fibrosis, warranting for further investigations.

The development of new therapeutic applications for adult and embryonic stem cells has dominated regenerative medicine and tissue engineering for several decades. However, since 2006, induced Pluripotent Stem Cells (iPSCs) have taken center stage in the field, as they promised to overcome several limitations of the other stem cell types. Nonetheless, other promising approaches for adult cell reprogramming have been attempted over the years, even before the generation of iPSCs. In particular, two years before the discovery of iPSCs, the possibility of synthesizing libraries of large organic compounds, as well as the development of high-throughput screenings to quickly test their biological activity, enabled the identification of a 2,6-disubstituted purine, named reversine, which was shown to be able to reprogram adult cells to a progenitor-like state. Since its discovery, the effect of reversine has been confirmed on different cell types, and several studies on its mechanism of action have revealed its central role in inhibitory activity on several kinases implicated in cell cycle regulation and cytokinesis. These key features, together with its chemical nature, suggested a possible use of the molecule as an anti-cancer drug. Remarkably, reversine exhibited potent cytotoxic activity against several tumor cell lines in vitro and a significant effect in decreasing tumor progression and metastatization in vivo. Thus, 15 years since its discovery, this review aims at critically summarizing the current knowledge to clarify the dual role of reversine as a dedifferentiating agent and anti-cancer drug.

Preclinical cardiac MR is challenging and time-consuming. A fast and comprehensive acquisition protocol and standardized image post-processing may improve preclinical research, reducing acquisition time, costs and variability of results. In the present study, we evaluated the feasibility of a contrast-enhanced 3D IntraGate steady-state cine sequence (ce-3D-IG-cine) with short acquisition time (11 min) for a single-shot combined characterization of left ventricle (LV) remodeling and infarct size (IS) in a mouse model of acute ischemia-reperfusion injury. Sixteen male C57BL/6N mice underwent 7T cardiac MR (Bruker, BioSpec 70/30) including optimized ce-3D-IG-cine (total scan time 11 min) at day 1, 5 and 28 after surgery. LV end-diastolic volume (EDVMR) and ejection fraction (EFMR) extracted from MR were compared to ones from short-axis (SA-EDVecho, SA-EFecho) and parasternal long-axis (LA-EDVecho, LA-EFecho) echocardiography. IS was manually and semiautomatically segmented from ce-3D-IG-cine using different standard deviation (SD +2, +3, +4, +5, +6 in respect to a reference tissue). Mice were sacrificed at day 28, immediately after imaging. IS at day 28 was compared to injury burden at histology. MR and echocardiographic morpho-functional parameters were compared, as IS from MR and histology. Bland-Altman plots were used to assess the agreement in ischemic burden segmentation. Volumetric and functional parameters measured on ce-3D-IG-cine correlated to the correspondent echocardiographic parameter (EDVMR vs SA-EDVecho: ρ = 0.813; EDVMR vs LA-EDVecho: ρ = 0.845; EFMR vs SA-EFecho ρ = 0.612; EFMR vs LA-EFecho ρ = 0.791; p < 0.001 in all cases). Manually segmented IS strongly correlated with the scar at histology (ρ = 0.904, p < 0.001). A threshold of +3SD showed the highest performance for semiautomatic assessment of IS compared to manual segmentation (ρ = 0.965, p < 0.001), with an overall reproducibility of 73%, and a peak reproducibility of 80% at day 1. The ce-3D-IG-cine sequence, manually or semiautomatically segmented using 3SD threshold, allows fast and comprehensive LV morpho-functional and structural characterization in myocardial ischemia-reperfusion injury model.

Adult stem cell-based therapeutic approaches for tissue regeneration have been proposed for several years. However, adult stem cells are usually limited in number and difficult to be expanded in vitro, and they usually tend to quickly lose their potency with passages, as they differentiate and become senescent. Culturing stem cells under reduced oxygen tensions (below 21%) has been proposed as a tool to increase cell proliferation, but many studies reported opposite effects. In particular, cell response to hypoxia seems to be very stem cell type specific. Nonetheless, it is clear that a major role in this process is played by the hypoxia inducible factor (HIF), the master regulator of cell response to oxygen deprivation, which affects cell metabolism and differentiation. Herein, we report that a chemical activation of HIF in human tendon stem cells reduces their proliferation and inhibits their differentiation in a reversible and dose-dependent manner. These results support the notion that hypoxia, by activating HIF, plays a crucial role in preserving stem cells in an undifferentiated state in the “hypoxic niches” present in the tissue in which they reside before migrating in more oxygenated areas to heal a damaged tissue.

Newcastle Disease Virus (NDV), belonging to the Paramyxoviridae family, causes a serious infectious disease in birds, resulting in severe losses in the poultry industry every year. Haemagglutinin neuraminidase glycoprotein (HN) has been recognized as a key protein in the viral infection mechanism, and its inhibition represents an attractive target for the development of new drugs based on sialic acid glycals, with the 2-deoxy-2,3-didehydro-D-N-acetylneuraminic acid (Neu5Ac2en) as their backbone. Herein we report the synthesis of several Neu5Ac2en glycals and of their perfluorinated C-5 modified derivatives, including their respective stereoisomers at C-4, together with evaluation of their in vitro antiviral activity. While all synthesized compounds were found to be active HN inhibitors in the micromolar range, we found that their potency was influenced by the chain-length of the C-5 perfluorinated acetamido functionality. Thus, the binding modes of the inhibitors were also investigated by performing a docking study. Moreover, the perfluorinated glycals were found to be more active than the corresponding normal C-5 acylic derivatives. Finally, cell-cell fusion assays on NDV infected cells revealed that the addition of a newly synthesized C-4 alpha heptafluorobutyryl derivative almost completely inhibited NDV-induced syncytium formation.

The acidic hydrolysis of

Reperfusion strategies are life-saving approaches to restore the blood flow in the cardiac tissue after acute myocardial infarction (AMI). However, they come with the drawback that they inevitably induce ischemia/reperfusion injury (IRI), resulting in increased cardiomyocytes damage and heart failure. In this context, the physiological activation of several pro-survival kinases, such as Akt and Erk, as well as of the hypoxia inducible factor (HIF) has been recognized to be critical during IRI. Along with this line, we recently discovered a novel, PHDs independent, mechanism of HIF-1; activation mediated by sialidase Neu3. Interestingly, Neu3 is upregulated under chronic hypoxia in cyanotic congenital cardiac patients. Moreover, induced activation of Neu3 increased myoblast resistance to hypoxic stress. Thus, in this study, we further investigated the possible role of Neu3 in protecting cardiac myoblasts during IRI. In particular, we set-up an in-vitro model of IRI on H9C2 rat cardiomyoblasts. Results showed a modulation of Neu3 during IRI, with a progressive down-regulation during the ischemic phase, followed by a reactivation during the reperfusion phase. Remarkably, overexpression of Neu3 significantly increased cardiomyoblasts resistance to IRI, both in terms of cell proliferation and resistance to apoptosis. Treatment with Akt and Erk inhibitors, as well as with a Neu3 specific inhibitor completely reverted the beneficial effects mediated by Neu3 upregulation. In conclusion, our results show that Neu3 activation has a cardioprotective effect during IRI, calling for further studies to unveil its full potential as a therapeutic target to treat cardiac ischemia and reperfusion injury and to improve patients recover after AMI.

Assigning the correct configuration at C2 in sialosides is a standing problem because of the absence of an anomeric hydrogen. All different empirical rules that have been proposed over the years lack general applicability. In particular, the correct configuration of several 3,4-unsaturated derivatives of N-acetylneuraminic acid (Neu5Ac), which have been recently shown to be novel sialidase/neuraminidase inhibitors, could only be tentatively assigned by similarity with the reported 3,4-unsaturated 2O-methyl sialosides. In this work, we overcome this problem as we devised a rapid synthetic method to unequivocally resolve the anomeric configuration of the 3,4-unsaturated Neu5Ac derivatives through the synthesis of the corresponding unreported unsaturated 1,7-lactones. Moreover, we discovered a diagnostic 13 C nuclear magnetic resonance signal that allows the formulation of a new empirical rule for the direct assignment of the C2 stereochemistry of these molecules, even when only one of the two C2 epimers is available.

Dilated cardiomyopathy (DCM) is the leading indication for heart transplantation. TTN gene truncating mutations account for about 25% of familial DCM cases and for 18% of sporadic DCM cases. The clinical relevance of specific variants in TTN has been difficult to determine because of the sheer size of the protein for which TTN encodes, as well as existing extensive genetic variation. Clinicians should communicate novel clinically-relevant variants and genotype–phenotype associations, so that animal studies evaluating the molecular mechanisms are always conducted with a focus on clinical significance. In the present study, we report for the first time the novel truncating heterozygous variant NM_001256850.1:c.72777_72783del (p.Phe24259Leufs*51) in the TTN gene and its association with DCM in a family with sudden death. This variant occurs in the A-band region of the sarcomere, in a known mutational hotspot of the gene. Truncating titin variants that occur in this region are the most common cause of DCM and have been rarely reported in asymptomatic individuals, differently from other pathogenic TTN gene variants. Further studies are warranted to better understand this particular clinically-relevant variant.

The possibility of generating induced pluripotent stem cells from mouse embryonic fibroblasts and human adult fibroblasts has introduced new perspectives for possible therapeutic strategies to repair damaged hearts. However, obtaining large numbers of adult stem cells is still an ongoing challenge, and the safety of genetic reprogramming with lenti- or retro-viruses has several drawbacks not easy to be addressed. Furthermore, the majority of adult stem cell-based clinical trials for heart regeneration have had generally poor and controversial results. Nonetheless, it is now clear that the injected cells activate the growth and differentiation of progenitor cells that are already present in the heart. This is achieved by the release of signalling factors and/or exosomes carrying them. Along this line, chemistry may play a major role in developing new strategies for activating resident stem cells to regenerate the heart. In particular, this review focuses on small molecule approaches for cell reprogramming, cell differentiation, and activation of cell protection.

The optimization of the synthetic protocol to obtain the 3,4-unsaturated sialic acid derivatives, through the fine-tuning of both the Ferrier glycosylation conditions and the subsequent hydrolysis work-up, is herein reported. The accomplishment of the desired β-anomers and some selected α-ones, in pure form, led us to evaluate their specific inhibitory activity towards NDV-HN and human sialidase NEU3. Importantly, the resulting data allowed the identification, for the first time, of three active 3,4-unsaturated sialic acid analogs, showing IC50 values against NDV-HN in the micromolar range.

Despite considerable improvements in the treatment of cardiovascular diseases, heart failure (HF) still represents one of the leading causes of death worldwide. Poor prognosis is mostly due to the limited regenerative capacity of the adult human heart, which ultimately leads to left ventricular dysfunction. As a consequence, heart transplantation is virtually the only alternative for many patients. Therefore, novel regenerative approaches are extremely needed, and several attempts have been performed to improve HF patients’ clinical conditions by promoting the replacement of the lost cardiomyocytes and by activating cardiac repair. In particular, cell-based therapies have been shown to possess a great potential for cardiac regeneration. Different cell types have been extensively tested in clinical trials, demonstrating consistent safety results. However, heterogeneous efficacy data have been reported, probably because precise end-points still need to be clearly defined. Moreover, the principal mechanism responsible for these beneficial effects seems to be the paracrine release of antiapoptotic and immunomodulatory molecules from the injected cells. This review covers past and state-of-the-art strategies in cell-based heart regeneration, highlighting the advantages, challenges, and limitations of each approach.

Gangliosides, the sialic acid-conjugated glycosphingolipids present in the lipid rafts, have been recognized as important regulators of cell proliferation, migration, and apoptosis. Due to their peculiar localization in the cell membrane, they modulate the activity of several key cell receptors, and increasing evidence supports their involvement also in stem cell differentiation. In this context, herein we report the role played by the ganglioside GM1 in the osteogenic differentiation of human tendon stem cells (hTSCs). In particular, we found an increase of GM1 levels during osteogenesis that is instrumental for driving the process. In fact, supplementation of the ganglioside in the medium significantly increased the osteogenic differentiation capability of hTSCs. Mechanistically, we found that GM1 supplementation caused a reduction in the phosphorylation of the platelet-derived growth factor receptor-β(PDGFR-β), which is a known inhibitor of osteogenic commitment. These results were further corroborated by the observation that GM1 supplementation was able to revert the inhibitory effects on osteogenesis when the process was inhibited with exogenous PDGF.

Transient lateral patellar dislocation (TLPD) is a common lesion in young adults. Vascular injury as a complication of TLPD has not been previously described. We report a case of descending genicular artery (DGA) injury after TLPD. Immediate angiography demonstrated rupture of DGA. Embolization was performed with sudden interruption of bleeding. DGA injury should be considered as a complication after TLPD and prompt diagnosis and intervention are required. We propose selective embolization as a safe and effective procedure to stop bleeding.

Neuraminidase activity is essential for the infection and propagation of paramyxoviruses, including human parainfluenza viruses (hPIVs) and the Newcastle disease virus (NDV). Thus, many inhibitors have been developed based on the 2-deoxy-2,3-didehydro-d-N-acetylneuraminic acid inhibitor (DANA) backbone. Along this line, herein we report a series of neuraminidase inhibitors, having C4 (p-toluenesulfonamido and azido substituents) and C5 (N-perfluorinated chains) modifications to the DANA backbone, resulting in compounds with 5- to 15-fold greater potency than the currently most active compound, the N-trifluoroacetyl derivative of DANA (FANA), toward the NDV hemagglutinin-neuraminidase (NDV-HN). Remarkably, these inhibitors were found to be essentially inactive against the human sialidase NEU3, which is present on the outer layer of the cell membrane and is highly affected by the current NDV inhibitor FANA.

Background Hypoxia is a common feature of many congenital heart defects (CHDs) and significantly contributes to their pathophysiology. Thus, understanding the mechanism underlying cell response to hypoxia is vital for the development of novel therapeutic strategies. Certainly, the hypoxia inducible factor (HIF) has been extensively investigated and it is now recognized as the master regulator of cell defense machinery counteracting hypoxic stress. Along this line, we recently discovered and reported a novel mechanism of HIF activation, which is mediated by sialidase NEU3. Thus, aim of this study was to test whether NEU3 played any role in the cardiac cell response to chronic hypoxia in congenital cyanotic patients. Methods Right atrial appendage biopsies were obtained from pediatric patients with cyanotic/non-cyanotic CHDs and processed to obtain mRNA and proteins. Real-Time PCR and Western Blot were performed to analyze HIF-1α and its downstream targets expression, NEU3 expression, and the NEU3 mediated effects on the EGFR signaling cascade. Results Cyanotic patients showed increased levels of HIF-1α, NEU3, EGFR and their downstream targets, as compared to acyanotic controls. The same patients were also characterized by increased phosphorylation of the EGFR signaling cascade proteins. Moreover, we found that HIF-1α expression levels positively correlated with those recorded for NEU3 in both cyanotic and control patients. Conclusions Sialidase NEU3 plays a central role in activating cell response to chronic hypoxia inducing the up-regulation of HIF-1α, and this represent a possible novel tool to treat several CHD pathologies.

Cardiovascular diseases are the leading cause of death in the Western world. Unfortunately, current therapies are often only palliative, consequently essentially making heart transplantation necessary for many patients. However, several novel therapeutic approaches in the past two decades have yielded quite encouraging results. The generation of induced pluripotent stem cells, through the forced expression of stem cell-specific transcription factors, has inspired the most promising strategies for heart regeneration by direct reprogramming of cardiac fibroblasts into functional cardiomyocytes. Initial attempts at this reprogramming were conducted using a similar approach to the one used with transcription factors, but during years, novel strategies have been tested, e.g., miRNAs, recombinant proteins and chemical molecules. Although preliminary results on animal models are promising, the low reprogramming efficiency, as well as the incomplete maturation of the cardiomyocytes, still represents important obstacles. This review covers direct transdifferentiation strategies that have been proposed and developed and illustrates the pros and cons of each approach. Indeed, as described in the manuscript, there are still many unanswered questions and drawbacks that require a better understanding of the basic signaling pathways and transcription factor networks before functional cells, suitable for cardiac regeneration and safe for the patients, can be generated and used for human therapies.

Regeneration of skeletal muscle is a complex process that requires the activation of quiescent adult stem cells, called satellite cells, which are resident in hypoxic niches in the tissue. Hypoxia has been recognized as a key factor to maintain stem cells in an undifferentiated state. Herein we report that hypoxia plays a fundamental role also in activating myogenesis. In particular, we found that the activation of the hypoxia-inducible factor (HIF)-1α under hypoxia, in murine skeletal myoblasts, leads to activation of MyoD through the noncanonical Wnt/β-catenin pathway. Moreover, chemical inhibition of HIF-1α activity significantly reduces differentiation, thus confirming its crucial role in the process. Furthermore, hypoxia-preconditioned myoblasts, once induced to differentiate under normoxic conditions, tend to form hypertrophic myotubes. These results support the notion that hypoxia plays a pivotal role in activating the regeneration process by directly inducing myogenesis through HIF-1α. Although preliminary, these findings may suggest new perspective for novel therapeutic targets in the treatment of several muscle diseases.-Cirillo, F., Resmini, G., Ghiroldi, A., Piccoli, M., Bergante, S., Tettamanti, G., Anastasia, L. Activation of the hypoxia-inducible factor 1α promotes myogenesis through the noncanonical Wnt pathway, leading to hypertrophic myotubes.

Thromboembolism represents the most significant complication and cause of non-surgical mortality in major urological surgery. The aim of the present study was to assess the association between the type of pharmacological thromboembolism prophylaxis and the postoperative complication rate in a cohort of patients undergoing major urological surgery. All consecutive patients treated with major urological surgery between December 2011 and March 2013 were evaluated. For each patient, clinical and demographic data, as well as information on the post-surgical complications and the type of pharmacological thromboembolism prophylaxis, were collected. In total, 453 patients (mean age, 63.36±12.05 years) were recruited (43.5% for prostate surgery, 33.1% for renal surgery, 12.1% for bladder surgery and 11.3% for other surgery). Postoperative blood transfusions were required in 50 cases (11.0%). A total of 32 patients (7.1%) underwent re-intervention due to the occurrence of grade ≥3 complications, with a readmission rate of 2.0%. According to the Clavien-Dindo Classification, the complications were grade 1 in 36.0% of the cases, grade 2 in 19.4%, grade 3 in 6.0%, grade 4 in 2.0% and grade 5 (mortality) in 0.7%. Only 1 case of deep venous thrombosis not associated with pulmonary thromboembolism was observed. Univariate analyses showed a significant negative association (higher risk of complications) between the use of >4,000 IU enoxaparin as the thromboembolism prophylaxis and postoperative blood transfusion rate (P=0.045), re-intervention rate (P=0.001) and the occurrence of grade ≥3 complications (P4,000 IU enoxaparin and both re-intervention rate (P=0.013) and occurrence of grade ≥3 complications (P=0.002). High doses of enoxaparin (>4,000 IU) may lead to an increased risk of re-intervention and severe postoperative complications following major urological surgery. Randomised, controlled trials comparing the effect of different types of pharmacological thromboembolism prophylaxis on postoperative complications following major urological surgery are required.

NEU3 sialidase has been shown to be a key player in many physio- and pathological processes, including cell differentiation, cellular response to hypoxic stress, and carcinogenesis. The enzyme, peculiarly localized on the outer leaflet of the plasma membrane, has been shown to be able to remove sialic acid residues from the gangliosides present on adjacent cells, thus creating cell to cell interactions. Nonetheless, herein we report that the enzyme localization is dynamically regulated between the plasma membrane and the endosomes, where a substantial amount of NEU3 is stored with low enzymatic activity. However, under opportune stimuli, NEU3 is shifted from the endosomes to the plasma membrane, where it greatly increases the sialidase activity. Finally, we found that NEU3 possesses also the ability to interact with specific proteins, many of which are different in each cell compartment. They were identified by mass spectrometry, and some selected ones were also confirmed by cross-immunoprecipitation with the enzyme, supporting NEU3 involvement in the cell stress response, protein folding, and intracellular trafficking.

INTRODUCTION Chronic Lymphocytic Leukemia (CLL) is the most common leukemia in the western world and is characterized by the accumulation of monoclonal B cells, due to both increased proliferation and apoptosis resistance. Although in the last years with the introduction of new kinase inhibitors blocking the pathways mediated by B-cell receptor (BCR) signaling we got an astonishing progress in the comprehension and treatment of this disease, CLL is still an incurable disease and many characteristics of its pathogenesis still remain unclear. Signaling events downstream the BCR engagement are central for the progression of CLL. Focal Adhesion Kinase (FAK), one of the primary enzyme involved in the engagement of integrins and assembly of focal adhesions, plays a major role in cellular adhesion and metastasis of various cancers, being regulated by Calcium (Ca2+) flux and by Src-kinases (e.g. Lyn) through a Calpain-dependent manner following BCR triggering. FAK has been demonstrated to be over-expressed in many human cancers but a down-modulation of its expression has also been reported. Studies concerning FAK expression in CLL are lacking in the literature. However, since an interaction of FAK with molecules implicated in BCR signal transduction, such as the Src-kinase Lyn, has been demonstrated we hypothesize that this kinase could have a key role in CLL pathogenesis. METHODS FAK expression was analyzed in B-lymphocytes from 107 CLL patients and 10 healthy subjects by Western blotting (WB) and the obtained expression data were correlated with the clinical features of the patients. In 25 out of 107 patients studied, surface IgM and IgD expression has been evaluated by flow cytometry (FC). For Ca2+ mobilization assessment, 1x107 cells were incubated with 4μM Fluo-4-AM at 37°C for 30min and then analyzed by FC; after 30s of baseline acquisition, α-IgM F(ab')2 and α-IgD F(ab')2 (10μg/ml) were added and fluorescence intensity was recorded for 5min. Ionomycin was added as positive control. Phosphorylation at Tyr397 was assessed with a specific antibody. Leukemic B cells from patients were treated in vitro with 5μM Defactinib (FAK inhibitor) and the apoptosis induction was evaluated by Annexin V/Propidium Iodide flow cytometry test and by the presence of cleaved PARP by WB. RESULTS By WB analyses we demonstrated a slightly significant difference in FAK expression between patients and controls (p CONCLUSIONS We herein propose that full length-FAK down-modulation could be considered as a new marker of unfavorable prognosis. In our model, poor prognosis CLL patients (particularly IGHV unmutated ones) presenting Ca2+ mobilization, are more prone to activate Calpain, which in turn activates FAK. Together with data from the literature, our results suggest that CLL cells missing the full length-FAK, not only are unaffected by the lack of it, but they rather present a cleaved/activated form of FAK that could favor cell migration and metastatic invasion. Moreover, since Defactinib can induce apoptosis in CLL cells, should these data be confirmed by in vivo studies, this FAK inhibitor could represent a new therapeutic approach for CLL. Disclosures Trentin: Gilead: Research Funding; Abbvie: Honoraria; Roche: Membership on an entity's Board of Directors or advisory committees; Janssen: Research Funding.

Previous studies demonstrated that reducing the GM3 content in myoblasts increased the cell resistance to hypoxic stress, suggesting that a pharmacological inhibition of the GM3 synthesis could be instrumental for the development of new treatments for ischemic diseases. Herein, the synthesis of several dephosphonated CMP-Neu5Ac congeners and their anti-GM3-synthase activity is reported. Biological activity testes revealed that some inhibitors almost completely blocked the GM3-synthase activity in vitro and reduced the GM3 content in living embryonic kidney 293A cells, eventually activating the epidermal growth factor receptor (EGFR) signaling cascade.

Owing to their exposure on the cell surface and the possibility of being directly recognized with specific antibodies, glycosphingolipids have aroused great interest in the field of stem cell biology. In the search for specific markers of the differentiation of human bone marrow mesenchymal stem cells (hBMSCs) toward osteoblasts, we studied their glycosphingolipid pattern, with particular attention to gangliosides. After lipid extraction and fractionation, gangliosides, metabolically (3)H-labeled in the sphingosine moiety, were separated by high-performance TLC and chemically characterized by MALDI MS. Upon induction of osteogenic differentiation, a 3-fold increase of ganglioside GD1a was observed. Therefore, the hypothesis of GD1a involvement in hBMSCs commitment toward the osteogenic phenotype was tested by comparison of the osteogenic propensity of GD1a-highly expressing versus GD1a-low expressing hBMSCs and direct addition of GD1a in the differentiation medium. It was found that either the high expression of GD1a in hBMSCs or the addition of GD1a in the differentiation medium favored osteogenesis, providing a remarkable increase of alkaline phosphatase. It was also observed that ganglioside GD2, although detectable in hBMSCs by immunohistochemistry with an anti-GD2 antibody, could not be recognized by chemical analysis, likely reflecting a case, not uncommon, of molecular mimicry.