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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.

Abstract: Cenobamate is a novel antiseizure medication (ASM) commercially available in Europe and in the U.S. for the treatment of focal seizures in adults. The mechanisms responsible for its antiseizure activity include enhancement of the inactivated state of voltage-gated sodium channels with blockade of the persistent sodium current and positive allosteric modulation of GABAA receptors at a non-benzodiazepine binding site. Cenobamate has a high oral bioavailability that is not influenced by food intake. The terminal half-life is 50-60 hours, allowing for once-daily dosing. Cenobamate is a CYP2C19 inhibitor and an inducer of CYP3A4 and CYP2B6, and consequently, it can cause a number of drug-drug interactions. Efficacy and safety have been evaluated in two randomized, double-blind, placebo-controlled adjunctive therapy trials in adults with focal seizures. In both trials, cenobamate decreased significantly the frequency of focal seizures, with relatively high seizure freedom rates. Adverse events most commonly reported in double-blind trials included dizziness, somnolence, headache, fatigue, and diplopia. The occurrence of three cases of drug reaction with eosinophilia and systemic symptoms (DRESS) during early clinical development led to the conduction of a Phase 3 open-label long-term safety study in a total of 1339 patients. In this study, no serious idiosyncratic adverse reactions were observed using a start-low and go-slow approach. Further studies are required to determine whether the clinical activity profile of cenobamate extends to protection against other seizure types and to evaluate its efficacy and safety profile in special patient groups such as infants, children, the elderly, and patients with comorbid conditions.

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.

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.

Over the last decade, hair analysis has become a routine procedure in most forensic laboratories and, complementary to blood and urine, hair is a unique biological matrix which gives the opportunity to establish a temporal consumption profile. Despite hair is widely used to identify drug use, environmental contamination continues to represent a challenging factor of this procedure, especially for cocaine (COC). In the last few years several strategies have been proposed in order to distinguish between actual use and external contamination, however the commonly detected COC metabolites probably are insufficient for demonstrating cocaine use through hair testing. Thus, the aim of this study is to develop an ultra high performance liquid cromatography - tandem mass spectrometry (UHPLC-MS/MS) method able to detect and quantify hydroxy-COC metabolites, as specific markers of COC abuse, in hair samples from COC consumers, thus enabling unambiguous evidence of COC consumption. At the beginning, since no commercial reference materials were available, COC-positive hair samples were tested using parent ion scan-based analysis to extract hydroxy COC metabolites target ions. Once identified, the reference materials were synthesized by our analytical laboratory allowing the development of the first UHPLC-MS/MS validated method to quantify p- and m-isomers of hydroxy COC, as well as hydroxy benzoylecgonine (BE) and hydroxy norcocaine (NCOC). The method was successfully applied to a large number of COC-positive hair samples and introduced into a routine procedure for testing drug ingestion in order to evaluate for the first-time hydroxy metabolites of COC ranges in hair and their correlation with COC and BE.

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.

The assay of saliva samples provides a valuable alternative to the use of blood samples for therapeutic drug monitoring (TDM), at least for certain categories of patients. To determine the feasibility of using saliva sampling for the TDM of rufinamide, we compared rufinamide concentrations in paired samples of saliva and plasma collected from 26 patients with epilepsy at steady state. Within-patient relationships between plasma rufinamide concentrations and dose, and the influence of comedication were also investigated. Assay results in the two tested fluids showed a good correlation (r2 = .78, P

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 so-called “sialo-chemical-biology” has become an attractive research area, as an increasing number of natural products containing a sialic acid moiety have been shown to play important roles in biological, pathological, and immunological processes. The intramolecular lactones of sialic acids are a subclass from this crucial family that could have central functions in the discrimination of physiological and pathological conditions. In this review, we report an in-depth analysis of the synthetic achievements in the preparation of the intramolecular lactones of sialic acids (1,4-, 1,7- and γ-lactones), in their free and/or protected form. In particular, recent advances in the synthesis of the 1,7-lactones have allowed the preparation of key sialic acid derivatives. These compounds could be used as authentic reference standards for their correct determination in biological samples, thus overcoming some of the limitations of the previous analytical procedures.

The acidic hydrolysis of

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 facile syntheses of previously unreported 2,3- and 3,4-unsaturated derivatives of N-glycolylneuraminic acid are herein described. The procedure involves a ring-opening of the 4,5-oxazoline of N-acetylneuraminic acid, the acylation of the intermediate free amine, its conversion into the 4,5-oxazoline of N-glycolylneuraminic acid and the subsequent Ferrier reaction or C4 nucleophilic substitution.

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.

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.

An internal rearrangement of the 1,7-lactone of N-acetylneuraminic acid into its γ-lactone in high yield is reported, together with the successive conversion into the free parent sialic acid. Overall, these reactions further characterize the chemical behavior of sialic acids and their derivatives, which are increasingly considered to be key players in many biological processes, including ischemic heart disease.

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.

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.

A protocol for direct access to C-4-functionalized Neu5Ac2en derivatives by allylic substitution of an α-acetoxy group with various nucleophiles is reported. The DANA acetamido group is exchanged for a trifluoroacetylamido group (as in FANA) to avoid the formation of a stable 4,5-oxazoline. With thiols, the reaction involves an initial attack at the anomeric carbon (Ferrier reaction) under kinetic control, followed by an equilibration of the nucleophile to the thermodynamically more stable 4-position.

Pyridinoline (Pyr), deoxypyridinoline (D-Pyr), galactosyl-pyridinoline (Gal-Pyr) and glucosyl-galactosyl pyridinoline (GluGal-Pyr) are enzymatic mature pyridinium crosslinks. Generally, only total Pyr and D-Pyr urinary amounts (free+bound forms) are evaluated by HPLC as indices of bone resorption. This report describes the validation of an HPLC-fluorescence method for the simultaneous evaluation of free Pyr and D-Pyr, together with GluGal-Pyr and Gal-Pyr, in urine of healthy women (n=20, aged 27-41) and girls (n=20, aged 5-10). The use of an unnatural D-Pyr homologue, here proposed for the first time as internal standard, and of pure Pyr, D-Pyr, GluGal-Pyr and Gal-Pyr synthesized to be used as primary calibrators, guarantees method specificity and correct crosslink quantification. Urine, spiked with IS, was solid-phase extracted prior to HPLC analysis. Total Pyr and D-Pyr amounts were also evaluated after urine hydrolysis. The HPLC method was validated for selectivity, sensitivity, linearity, precision, accuracy, recovery and stability for all measured crosslinks. Both free and total Pyr and D-Pyr as well as GluGal-Pyr and Gal-Pyr amounts were significantly higher in girls than in women (p0.0001), indicating an increased collagen turnover rather than only bone turnover. Gal-Pyr, for the first time evaluated in girls, was under its lower quantification limit (LLOQ,21.20 pmol/mL) in women. The measurement of free and glycosylated pyridinium crosslinks might provide more information on the degradation of various types of collagen than only that of total Pyr and D-Pyr. Moreover, this validated method could be a useful non-invasive technique for studying pathological conditions characterized by modified glycosylation enzyme activity and for more clinical investigation on bone fragility.

The synthesis of four deuterated sialic acids and their 1,7-lactones has been performed in two ways, one based on sialic acid classical chemistry, and the other involving a direct exchange of the unlabeled acyl group of N-acetylneuraminic acid with a labeled one mediated by a perfluorinated amide. The final lactonization is promoted by benzyloxycarbonyl chloride.

The simple addition of mild bases (Et3N or DIPEA) allows a high yielding N-transacylation of secondary amides, performed by perfluorinated anhydrides, in molecules that are very sensitive to acids.

Three homologues of the collagen cross-links deoxypyridinoline, differing in the length of the side chain at the aromatic nitrogen, have been efficiently synthesized as possible internal standards in the quantitative analyses of pyridinolines. The first has a one-carbon shorter N-chain, while other two have a one-carbon and a two-carbon longer N-chain. The stereogenic centers are introduced stereoselectively using Williams’ glycine template methodology and oxazinones to generate chirality and to suitably protect the amino acid functionality during assembly of the pyridinoline nucleus.

“Time-to-build” models of investment expenditures play an important role in many traditional and modern theories of the business cycle, especially for explaining the dynamic propagation of shocks. We estimate the structural parameters of a time-to-build model using annual firm-level investment data on equipment and structures. For expenditures on equipment, we find no evidence of time-to-build effects beyond one year. For expenditures on structures, by contrast, there is clear evidence of such effects in the range of two to three years. The contrast between equipment and structures is intuitively reasonable and consistent with previous results. The estimates for structures also indicate that initial-period expenditures are low and increase as projects near completion. These results provide empirical support for including “time-to-plan” effects for investment in structures. More generally, these results suggest a potential source of specification error for Q models of investment and production-based asset pricing models that ignore the time required to plan, build, and install new capital. (JEL: D24, G31, C33, C34)

We analyse the conditions under which an (S,s) rule may be implemented in the case of labour demand. The (S,s) rule implies a specific ordering of choices: downward adjustment, nonadjustment and upward adjustment with the decision of inaction lying crucially in the middle. This requires an exact negative relation between the choice-specific error terms. The particular ordering of choices implied may be estimated by an ordered probit. We test the (S,s) rule nesting the ordered probit within a multinomial model with correlated error terms. The restriction to a univariate error distribution is rejected by the data.

The current analytical protocol used for the GC–MS determination of free or 1,7-lactonized natural sialic acids (Sias), as heptafluorobutyrates, overlooks several transformations. Using authentic reference standards and by combining GC–MS and NMR analyses, flaws in the analytical protocol were pinpointed and elucidated, thus establishing the scope and limitations of the method. It was demonstrated that (a) Sias 1,7-lactones, even if present in biological samples, decompose under the acidic hydrolysis conditions used for their release; (b) Sias 1,7-lactones are unpredicted artifacts, accidentally generated from their parent acids; (c) the N-acetyl group is quantitatively exchanged with that of the derivatizing perfluorinated anhydride; (d) the partial or complete failure of the Sias esterification-step with diazomethane leads to the incorrect quantification and structure attribution of all free Sias. While these findings prompt an urgent correction and improvement of the current analytical protocol, they could be instrumental for a critical revision of many incorrect claims reported in the literature.

We consider a dynamic model in which firms decide whether or not to vary labor in the presence of fixed costs. By exploiting the first-order condition for optimality, we derive a semireduced form in which firms' intertemporal employment is defined by a standard marginal productivity condition augmented by a forward-looking term. We obtain a marginal productivity equilibrium relation that takes into account the future alternatives of adjustment or nonadjustment that firms face. We use the structural parameter from this condition to estimate the fixed cost within a discrete decision process. Fixed costs are about 15 months' labor cost.

An efficient protocol for the 4β-acylamidation of the N-acylated glycals of Neu5Ac by using the corresponding N-perfluoroacylated congeners as key tools has been developed and applied to the synthesis of glycals combining the features of the molecules with antiviral properties.

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.

We analyse the Italian labour market, which is typically considered to be highly regulated. We focus on the costs imposed on firms by the institutional environment in which they operate, and on the adjustment strategies that they pursue as a result. We have followed two complementary approaches: the analysis of the development of the laws relating to hiring and firing, together with a set of interviews of 61 manufacturing firms. This also allows us to quantify the extent of turnover costs on the total labour costs. Hiring costs, which include recruiting and training, range between 2.0 and 2.6 months of labour costs; firing costs range from less than half a monthly labour cost to 20 months of labour costs in cases of conflict.

This review focuses on the chemical structure, biosynthesis and synthesis of free and glycosylated pyridinolines (Pyds), fluorescent collagen cross-links, with a pyridinium salt structure. Pyds derive from the degradation of bone collagen and have attracted attention for their use as biochemical markers of bone resorption and to assess fracture risk prediction in persons suffering from osteoporosis, bone cancer and other bone or collagen diseases. We consider and critically discuss all reported syntheses of free and glycosylated Pyds evidencing an unrevised chemistry, original and of general utility, analysis of which allows us to also support a previously suggested non-enzymatic formation of Pyds in collagen better rationalizing and justifying the chemical events.

Two simple and rapid procedures affording 4β-acylamido- and 4β-acetoxyneuraminic acid glycals acylated or perfluoroacylated at the 5-amino group are reported. The first protocol avoids the formation of oxazolines to synthesize the 4β-acetamido glycals through the Ritter reaction. The second passes through the oxazoline derivative to prepare 4β-acetoxyneuraminic acid glycals. Both protocols start from peracetylated methyl neuraminate, acylated at the amino group with a normal, a glycolyl, or a perfluoroacyl group, or with the corresponding peracetylated glycal methyl esters (of the DANA or FANA series).

The chemoselective synthesis of the 1,7-lactones of N-acetylneuraminic acid, N-glycolylneuraminic acid, and 3-deoxy-d-glycero-d-galacto-nononic acid is accomplished in two steps: a simple treatment of the corresponding free sialic acid with benzyloxycarbonyl chloride and a successive hydrogenolysis of the formed 2-benzyloxycarbonyl 1,7-lactone. The instability of the 1,7-lactones to protic solvents has been also evidenced together with the rationalization of the mechanism of their formation under acylation conditions. The results permit to dispose of authentic 1,7-sialolactones to be used as reference standards and of a procedure useful for the preparation of their isotopologues to be used as inner standards in improved analytical procedures for the gas liquid chromatography−mass spectrometry (GLC−MS) analysis of 1,7-sialolactones in biological media.

N-Acetylneuraminic acid is transformed into its until now unavailable and rather unwieldy 1,7-lactone, via the manageable 2-benzyloxycarbonyl N-acetylneuraminic acid 1,7-lactone which generates the free lactone in quantitative yield by hydrogenolysis.

N-Acetylneuraminic acid is transformed into its until now unavailable and rather unwieldy 1,7-lactone, via the manageable 2-benzyloxycarbonyl N-acetylneuraminic acid 1,7-lactone which generates the free lactone in quantitative yield by hydrogenolysis.