Effects of microgravity simulation on zebrafish transcriptomes and bone physiology-exposure starting at 5 days post fertilization.

Physiological modifications in near weightlessness, as experienced by astronauts during space flight, have been the subject of numerous studies. Various animal models have been used on space missions or in microgravity simulation on ground to understand the effects of gravity on living animals. Here, we used the zebrafish larvae as a model to study the effect of microgravity simulation on bone formation and whole genome gene expression. To simulate microgravity (sim-µg), we used two-dimensional (2D) clinorotation starting at 5 days post fertilization to assess skeletal formation after 5 days of treatment. To assess early, regulatory effects on gene expression, a single day clinorotation was performed. Clinorotation for 5 days caused a significant decrease of bone formation, as shown by staining for cartilage and bone structures. This effect was not due to stress, as assessed by measuring cortisol levels in treated larvae. Gene expression results indicate that 1-day simulated microgravity affected musculoskeletal, cardiovascular, and nuclear receptor systems. With free-swimming model organisms such as zebrafish larvae, the 2D clinorotation setup appears to be a very appropriate approach to sim-µg. We provide evidence for alterations in bone formation and other important biological functions; in addition several affected genes and pathways involved in bone, muscle or cardiovascular development are identified.

Stem Cells toward the Future: The Space Challenge.

Astronauts experience weightlessness-induced bone loss due to an unbalanced process of bone remodeling that involves bone mesenchymal stem cells (bMSCs), as well as osteoblasts, osteocytes, and osteoclasts. The effects of microgravity on osteo-cells have been extensively studied, but it is only recently that consideration has been given to the role of bone MSCs. These live in adult bone marrow niches, are characterized by their self-renewal and multipotent differentiation capacities, and the published data indicate that they may lead to interesting returns in the biomedical/bioengineering fields. This review describes the published findings concerning bMSCs exposed to simulated/real microgravity, mainly concentrating on how mechanosignaling, mechanotransduction and oxygen influence their proliferation, senescence and differentiation. A comprehensive understanding of bMSC behavior in microgravity and their role in preventing bone loss will be essential for entering the future age of long-lasting, manned space exploration.

The challenging environment on board the International Space Station affects endothelial cell function by triggering oxidative stress through thioredoxin interacting protein overexpression: the ESA-SPHINX experiment.

Exposure to microgravity generates alterations that are similar to those involved in age-related diseases, such as cardiovascular deconditioning, bone loss, muscle atrophy, and immune response impairment. Endothelial dysfunction is the common denominator. To shed light on the underlying mechanism, we participated in the Progress 40P mission with Spaceflight of Human Umbilical Vein Endothelial Cells (HUVECs): an Integrated Experiment (SPHINX), which consisted of 12 in-flight and 12 ground-based control modules and lasted 10 d. Postflight microarray analysis revealed 1023 significantly modulated genes, the majority of which are involved in cell adhesion, oxidative phosphorylation, stress responses, cell cycle, and apoptosis. Thioredoxin-interacting protein was the most up-regulated (33-fold), heat-shock proteins 70 and 90 the most down-regulated (5.6-fold). Ion channels (TPCN1, KCNG2, KCNJ14, KCNG1, KCNT1, TRPM1, CLCN4, CLCA2), mitochondrial oxidative phosphorylation, and focal adhesion were widely affected. Cytokine detection in the culture media indicated significant increased secretion of interleukin-1α and interleukin-1ß. Nitric oxide was found not modulated. Our data suggest that in cultured HUVECs, microgravity affects the same molecular machinery responsible for sensing alterations of flow and generates a prooxidative environment that activates inflammatory responses, alters endothelial behavior, and promotes senescence.

Oxidative stress and alterations in actin cytoskeleton trigger glutathione efflux in Saccharomyces cerevisiae.

A marked deficiency in glutathione (GSH), the most abundant antioxidant in living systems, plays a major role in aging and the pathogenesis of diseases ranging from neurological disorders to early atherosclerosis and the impairment of various immunological functions. In an attempt to shed light on GSH homeostasis, we carried out the space experiment SCORE (Saccharomyces cerevisiae oxidative stress response evaluation) during the FOTON-M3 mission. Microgravity and hyperoxic conditions induced an enormous extracellular release of GSH from S. cerevisiae cells (≈40% w/dw), changed the distribution of the buds, and activated the high osmolarity glycerol (HOG) and cell integrity/PKC pathways, as well as protein carbonylation. The results from the single spaceflight experiment were validated by a complete set of experiments under conditions of simulated microgravity and indicate that cytoskeletal alterations are mainly responsible for the observed effects. The results of ground experiments in which we induced cytoskeletal modifications by means of treatment with dihydrocytochalasin B (DHCB), a potent inhibitor of actin polymerisation, or (R)-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632), a selective ROCK (Rho-associated coiled-coil forming protein serine/threonine kinase) inhibitor, confirmed the role of actin in GSH efflux. We also found that the GSH release can be inhibited using the potent chloride channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB).

Assessment of water-soluble pi-extended squaraines as one- and two-photon singlet oxygen photosensitizers: design, synthesis, and characterization.

Singlet oxygen sensitization by organic molecules is a topic of major interest in the development of both efficient photodynamic therapy (PDT) and aerobic oxidations under complete green chemistry conditions. We report on the design, synthesis, biology, and complete spectroscopic characterization (vis-NIR linear and two-photon absorption spectroscopy, singlet oxygen generation efficiencies for both one- and two-photon excitation, electrochemistry, intrinsic dark toxicity, cellular uptake, and subcellular localization) of three classes of innovative singlet oxygen sensitizers pertaining to the family of symmetric squaraine derivatives originating from pi-excessive heterocycles. The main advantage of pi-extended squaraine photosensitizers over the large number of other known photosensitizers is their exceedingly strong two-photon absorption enabling, together with sizable singlet oxygen sensitization capabilities, for their use at the clinical application relevant wavelength of 806 nm. We finally show encouraging results about the dark toxicity and cellular uptake capabilities of water-soluble squaraine photosensitizers, opening the way for clinical small animal PDT trials.

Alterations of the actin cytoskeleton and increased nitric oxide synthesis are common features in human primary endothelial cell response to changes in gravity.

Because endothelial cells are fundamental to the maintenance of the functional integrity of the vascular wall, endothelial modifications in altered gravity conditions might offer some insights into the mechanisms leading to circulatory impairment in astronauts. We cultured human endothelial cells in a dedicated centrifuge (MidiCAR) to generate hypergravity and in two different devices, namely the Rotating Wall Vessel and the Random Positioning Machine, to generate hypogravity. Hypogravity stimulated endothelial growth, did not affect migration, and enhanced nitric oxide production. It also remodeled the actin cytoskeleton and reduced the total amounts of actin. Hypergravity did not affect endothelial growth, markedly stimulated migration, and enhanced nitric oxide synthesis. In addition, hypergravity altered the distribution of actin fibers without, however, affecting the total amounts of actin. A short exposure to hypergravity (8 min) abolished the hypogravity induced growth advantage. Our results indicate that cytoskeletal alterations and increased nitric oxide production represent common denominators in endothelial responses to both hypogravity and hypergravity.

Novel nontoxic mitochondrial probe for confocal fluorescence microscopy.

We propose a 2,5-Bis[1-(4-N-methylpyridinium)ethen-2-yl)]-N-methylpyrrole ditriflate (PEPEP) as a novel nontoxic, nonpotentiometric mitochondrial probe for confocal fluorescence microscopy. PEPEP is a representative chromophore of a large family of heterocyclic fluorescent dyes that show fluorescence emission in aqueous media and great DNA affinity. We check its cytotoxicity and intracellular localization in mammalian and yeast cell cultures. We demonstrate that PEPEP is a very efficient dye for fluorescence confocal microscopy and a valuable alternative to the most frequently used mitochondrial stains.

New pi-extended water-soluble squaraines as singlet oxygen generators.

[graph: see text] Condensation of squaric acid with a number of differently substituted 2-pyrrolyl derivatives afforded three new classes of squaraines. Their sharp and intense absorption bands in the biological window (700-900 nm), inherent singlet oxygen generation capabilities, together with proper functionalization allowing good water solubility make them suitable candidates as new non-porphyrinic singlet oxygen photosensitizers for photodynamic therapy (PDT).

Cardiovascular protective effects of resveratrol.

Resveratrol (3,4',5-trihydroxy-trans-stilbene), a phytoalexin found in grape skins, peanuts, and red wine, has been reported to have a wide range of biological and pharmacological properties. It has been speculated that at low doses (such as consumed in the common diet) resveratrol may have cardioprotective activity. In this article we describe recent in vitro and in vivo studies in animal models. The results of these studies suggest that resveratrol modulates vascular cell function, inhibits LDL oxidation, suppresses platelet aggregation and reduces myocardial damage during ischemia-reperfusion. Although the reported biological data indicate that resveratrol is a highly promising cardiovascular protective agent, more studies are needed to establish its bioavailability and in vivo cardioprotective effects, particularly in humans.

Simulated weightlessness in the design and exploitation of a NMR-compatible bioreactor.

Mammalian cells cultured in simulated weightlessness take advantage of a favorable environment, experiencing low shear stress and reduced turbulence. NMR spectroscopy allows the on-line noninvasive monitoring of cell growth and metabolism. With this in mind, we developed a novel bioreactor that fits into a NMR instrument and in which the simulated weightlessness conditions are obtained by a suitable medium and a flow-lift suspension. In detail, the gravitational vector acting on cells is counterbalanced by the hydrodynamic thrusts created by a bottom-up spiral flow of a fluid having increased density. We validate its efficiency (a) by calculating the main physical parameters as relative velocity, shear stress, and oxygen transport, and (b) by comparing the experimental results of growing a cell culture in the proposed bioreactor with those obtained using an established simulated weightlessness system (rotating wall vessel, NASA). As a test study we focused on the proliferation of human umbilical vein endothelial cells (HUVEC) in terms of cell viability and organization of their cytoskeleton.

Novel coordinating motifs for lanthanide(III) ions based on 5-(2-pyridyl)tetrazole and 5-(2-pyridyl-1-oxide)tetrazole. Potential new contrast agents.

Water-soluble and neutral Ln(III) and Zn (II) complexes of pyridine- and (pyridine-1-oxide)tetrazole have been synthesized and the Gd derivatives have great potential as high-relaxivity low-osmolarity MRI contrast agents.

A distinctive example of the cooperative interplay of structure and environment in tuning of intramolecular charge transfer in second-order nonlinear optical chromophores.

The strongly enhanced cooperative influence of medium polarity and organic structural design on the first hyperpolarizability beta of a novel family of highly polarizable azinium-(CH=CH-thienyl)-dicyanomethanido chromophores 1-3 is described. The dyes can be efficiently synthesized by regioselective protonation/alkylation of the corresponding bidentate anion precursors. Consecutive annelation of the pyridyl ring of 1 (pyridine-->quinoline-->acridine) and medium polarity effects are responsible for an extraordinarily variable range of intramolecular charge transfer (ICT), leading to a large set of pi-electron distribution patterns. Accordingly, systems with remarkably different zwitterionic/quinoid character in the ground and excited states present beta values in a broad range, eventually switching from negative to positive. Our investigation is based on a combination of experimental (UV/Vis spectroscopy, multinuclear NMR spectroscopy, and electrooptical absorption measurements) and computational (ab initio) approaches. It is shown that: 1) beta and mubeta are dramatically influenced, even by orders of magnitude, by a complex, non-monotonic interplay of structure and medium action, which in turn affects molecular ICT and bond length alternation (BLA), 2) the computations, validated by different experimental data, are to be recommended as an extremely useful tool in the search for a greatly improved set of molecular nonlinear optical (NLO) responses (in the case of 1-3 they show that such conditions may be attained only in a narrow and limited range of dielectric constants in which the annelation effect operates most efficiently), and 3) the search for the most favorable molecular NLO response of a highly polarizable chromophore both in solution and in solid matrices should simultaneously take into account not only the molecular design supplemented by annelation effects but also the polarity of the medium.

Resveratrol provides late-phase cardioprotection by means of a nitric oxide- and adenosine-mediated mechanism.

We used two experimental models to prove that resveratrol (trans-3,4',5-trihydroxystilbene) reduces cardiac ischemic-reperfusion injury by means of a nitric oxide- and adenosine-dependent mechanism. (1). ACUTE EX VIVO: resveratrol (10 microM, 10 min) infusion in Langendorff-perfused normoxic rat hearts significantly increased adenosine release and coronary flow compared with baseline. After 30-min low-flow ischemia, vasodilation, still present at reperfusion, was completely abolished by resveratrol plus adenosine antagonist 8-(p-sulfophenyl)theophylline (SPT, 50 microM) administration. (2). CHRONIC IN VIVO: rats received tap water containing 25 mg/l resveratrol for 15 days or normal water. Twenty-four hours after, their hearts were Langendorff-perfused and submitted to 60-min low-flow ischemia and reperfusion. The resveratrol-treated hearts showed better functional recovery at reperfusion and significant vasodilation, but no variation in high-energy phosphates (31P Nuclear Magnetic Resonance). N(G)-nitro-L-arginine methyl ester (L-NAME, 30 microM), a nonselective nitric oxide synthase inhibitor, or SPT (50 microM) administered for 10 min prior to the low-flow ischemia cancelled the effects. This suggests that long-term moderate resveratrol consumption could play an important role in late cardioprotective effects.

Carnosine is a quencher of 4-hydroxy-nonenal: through what mechanism of reaction?

The aim of this study was to understand the mechanism of action through which carnosine (beta-alanyl-L-histidine) acts as a quencher of cytotoxic alpha,beta-unsaturated aldehydes, using 4-hydroxy-trans-2,3-nonenal (HNE) as a model aldehyde. In phosphate buffer solution (pH 7.4), carnosine was 10 times more active as an HNE quencher than L-histidine and N-acetyl-carnosine while beta-alanine was totally inactive; this indicates that the two constitutive amino acids act synergistically when incorporated as a dipeptide and that the beta-alanyl residue catalyzes the addition reaction of the histidine moiety to HNE. Two reaction products of carnosine were identified, in a pH-dependent equilibrium: (a) the Michael adduct, stabilized as a 5-member cyclic hemi-acetal and (b) an imine macrocyclic derivative. The adduction chemistry of carnosine to HNE thus appears to start with the formation of a reversible alpha,beta-unsaturated imine, followed by ring closure through an intra-molecular Michael addition. The biological role of carnosine as a quencher of alpha,beta-unsaturated aldehydes was verified by detecting carnosine-HNE reaction adducts in oxidized rat skeletal muscle homogenate.

Production of lovastatin examined by an integrated approach based on chemometrics and DOSY-NMR.

Microbial secondary metabolites are one of the sources of therapeutic molecules in the pharmaceutical industry. Product quality and high yields of secondary metabolites are the main goals for the commercial success of a fermentation process. Our novel approach was based on the decision-tree algorithm to determine the key variables correlated with the process outcome and on DOSY-NMR to identify both co-metabolites and impurities, and it improves fermentation systems and speeds up bioprocess development. The approach has been validated in the case of lovastatin production from Aspergillus terreus.

Metal chelation aptitudes of bis(o-azaheteroaryl)methanes as tuned by heterocycle charge demands.

We describe the synthesis of a number of 1,3-azol-2-yl-, 1,3-benzazol-2-yl-, and azinyl-based bis(o-azaheteroaryl)methanes (LH, L(-) = Het(2)CH(-)) and their coordinating properties toward divalent transition metals (Zn, Cu, Co, Ni, Hg, Pd). This extended investigation includes both symmetrical and unsymmetrical ligands based on several substituted and/or unsubstituted thiazole, benzothiazole, benzoxazole, benzimidazole, pyridine, and quinoline derivatives. Depending on the structure and electron properties of the ligand, a vast set of neutral chelates ML(2) were obtained, where the ligand is present in its carbanionic form L(-). Additionally, we have prepared salt complexes [M(LH)(n)]X(m), where the ligand is present as a neutral system. Neutral chelates were typically obtained by the reaction of the ligand with metal acetates in alcoholic solution; salt complexes were formed by reaction with other metal salts such as chlorides. By exploring the coordinating properties of several bisheteroarylmethane ligands based on heteroaromatics of variable pi-electron structure and substitution pattern, we demonstrate that the formation of neutral chelates is strictly dependent on the electron-withdrawing capacity (charge demand) of the heteroaromatic moiety. The latter primarily dictates the efficiency by which the negative charge of the anionic ligand L(-) is stabilized by delocalization in ML(2) and, therefore, the stability of the chelate itself. On the basis of the large number and the variable nature of the nitrogen ligands used, we confirm the general validity of the charge-demand-dependent formation of chelates. This key factor can therefore be used for the efficient design of new pi-deficient heteroaromatic nitrogen ligands in chelates of great potential in many synthetic, catalytic, and technological fields.

Modulation of human endothelial cell behaviour in simulated microgravity.

Eukaryotic organisms are influenced by gravitational forces in their environment. The low gravitational forces endured by organisms in space alter cellular processes in cultured mammalian cells. Endothelial cells represent an interesting model to study because of their crucial role in the pathogenesis of various diseases, from atherosclerosis to inflammation to any situation characterized by dysregulated angiogenesis. We therefore cultured human endothelial cells derived from the umbilical vein in Rotating Wall Vessels (RWV) that simulate microgravity on earth. Under these experimental conditions, cells are viable and no increase in apoptotic rate was observed. They grow reproducibly faster than controls up to 8 days from seeding. Because endothelial proliferation is crucial in angiogenesis, we evaluated other steps required for new blood vessels to form. We found no variations in the levels of metalloproteases and an increased synthesis of their inhibitors (TIMP), suggesting that hypogravity does not induce a pro-angiogenic phenotype. Since i) alterations of blood pressure have been observed in astronauts and ii) endothelial cell synthesize vasoactive molecules, we evaluated the synthesis of nitric oxide and prostacyclin, both potent vasodilators and inhibitors of platelet aggregation. We observed that human endothelial cells grown in hypogavity synthesize higher amounts of prostacyclin and nitric oxide than controls. More studies are ongoing to understand the molecular basis of these events and their role in altering the physiology of the vascular tree.

Diheteroarylmethanes. 8.(1) Mapping Charge and Electron-Withdrawing Power of the 1,2,4-Triazol-5-yl Substituent.

Three new triazolyl derivatives, bis(1H-1-phenyl-1,2,4-triazol-5-yl)methane (11), 1H-1-phenyl-5-(beta-styryl)-1,2,4-triazole (12), and 1H-5-benzyl-1-phenyl-1,2,4-triazole (13) have been synthesized and the carbanions 11(-)() and 13(-)() investigated in DMSO by multinuclear NMR spectroscopy. By applying previously proposed and widely used pi-charge/(13)C shift relationships on the spectra of the anions, it is possible to rank the pi electron-withdrawing power of the 1,2,4-triazol-5-yl group in terms of charge demand c(X), a quantity that represents the fraction of pi negative charge delocalized by the heterocyclic ring. Our results indicate that the charge demand c(X) of this heterocycle is considerably greater than that of other 1,3-azoles (2-imidazolyl, 2-oxazolyl, 2-benzoimidazolyl), being close to that of some mono- and diazinyl substituents. A single set of resonances is presented by both carbanions 11(-)() and 13(-)(), thus showing that they exist either as a single geometric isomer species or as a mixture of isomers in a rapid (on the NMR time scale) equilibrium. (13)C and (15)N shift/pi-charge relationships allow accurate pi-charge mapping of carbanionic systems. Our results clearly show that, in the case of benzyl carbanion 13(-)(), all of the three nitrogen atoms are almost equally involved in delocalizing the negative charge. Also, the N-phenyl group contributes to charge delocalization. Anion 11(-)()( )()is the first of the bis(heteroaryl)methyl carbanions that we have studied, in which all of the negative charge originated by deprotonation of the carbon acid 11 is hosted on the nitrogen atoms without any appreciable involvement of the heteroaromatic carbon frame.

8-Purinyl versus 2-Benzimidazolyl Carbanions: Charge Demands of the Heterocycles and Ligand Properties of the Bis(heteroaryl)methanes(1).

Two new purinyl derivatives, 8-benzyl-7-methylpurine (8) and bis(7-methylpurin-8-yl)methane (9), have been synthesized and their corresponding carbanions investigated in DMSO. The application of our previously proposed pi-charge/shift relationships to the (13)C and (15)N shifts of the carbanions has made it possible to map pi-charges and obtain the values of the charge demand of the heterocyclic rings, a resonance index of the pi-electron-withdrawing power of the substituent. The charge demand of the 7-methylpurin-8-yl substituent is found to be the highest among the previously investigated azinyl and azolyl substituents and is comparable with that of the strongest classical electron-withdrawing functions. The replacement of the fused benzene ring in the benzimidazolyl substituent by a pyrimidine ring containing the nitrogen atoms in appropriate positions causes a considerable increase in the electron-withdrawing capacity of the heterocycle. Spectroscopic and reactivity data confirm the strong electron-withdrawing nature of the purinyl ring. The (13)C NMR spectrum shows the existence of the carbanions of both purinyl derivatives as a mixture of geometric isomers, a consequence of the high double-bond character along the bond linking the carbanionic carbon to C(8) of the purinyl ring. Bis(7-methylpurin-8-yl)methane can be easily converted to its stable NH tautomer by means of basic catalysis. It behaves as an "active methylene" compound giving high-yield condensations with electrophiles. Finally, unlike the corresponding benzimidazol-2-yl derivative, bis(7-methylpurin-8-yl)methane reacts with metal acetates to give neutral methanates [ML(2)] (M = Zn, Cu, Co; LH = bis(7-methylpurin-8-yl)methane), where the ligand is present as an anionic system. On the basis of this and previous data, it is concluded that charge demand plays a strategic role in obtaining stable chelates of this sort.

Diheteroarylmethanes. 5.(1) E-Z Isomerism of Carbanions Substituted by 1,3-Azoles: (13)C and (15)N pi-Charge/Shift Relationships as Source for Mapping Charge and Ranking the Electron-Withdrawing Power of Heterocycles.

Previously proposed pi-charge/shift relationships have been applied to (13)C and (15)N shifts of the carbanions of 2-benzylazoles (thiazole, oxazole, and imidazole), their corresponding benzo-fused analogs, and bis(2-azolyl)methanes (azolyl groups as above). In this way it is possible to rank the pi electron-withdrawing power of these heterocycles in terms of charge demands c(X), a quantity representing the fraction of pi negative charge withdrawn (delocalized) by the ring. The results indicate that c(thiaz) > c(oxaz) > c(imidaz); furthermore, benzoazoles are more efficient than monocyclic systems in delocalizing the negative charge. The charge demand c(X) of imidazole is the smallest among the heteroaromatics so far considered, being even smaller than that of the phenyl ring. As a consequence, the negative charge in the anion of 2-benzyl-N-methylimidazole is predominantly transferred from the carbanionic carbon to the phenyl group rather than to the imidazolyl residue. The high double bond character of the bond linking the carbanionic and ipso phenyl ring carbons leads to room temperature (13)C shift anisochrony of the meta and meta' and ortho and ortho' positions of the phenyl ring. In all of the other cases, hindered rotation is observed at room temperature between the carbanionic carbon and position 2 of the heterocycle. A single set of resonances is presented by the bis(heteroaryl)methyl carbanions. pi-Charge/shift relationships allow for the accurate pi-charge mapping in these carbanionic systems, and the results point to considerable delocalization of the electron pair(s) of the oxygen and pyrrolic nitrogen atoms at position 1 in oxazole and imidazole toward the pyridic nitrogen at position 3 of the rings (in both the neutrals and the carbanionic species). On the contrary, not only does the sulfur atom in thiazole derivatives not delocalize any negative charge in the anions but it is barely involved in any pi-donation to the pyridic nitrogen atom at position 3 also in the neutrals.