Functionalization of TiO2 sol-gel derived films for cell confinement.

The neuroscience field has increased enormously over the last decades, achieving the possible real application of neuronal cultures not only for reproducing neural architectures resembling in vivo tissues, but also for the development of functional devices. In this context, surface patterning for cell confinement is crucial, and new active materials together with new protocols for preparing substrates suitable for confining cells, guiding their processes in the desired configuration are extremely appreciated. Here, TiO2 sol-gel derived films were selected as proof-of-concept materials to grow neurons in suitable confined configurations, taking advantage of the biocompatible properties of modified TiO2 substrates. TiO2 sol-gel derived films were made compatible with the growth of neurons thanks to a stable and controlled poly-lysine coating, obtained by silanization chemistry and streptavidin-biotin interactions. Moreover, a spotting protocol, here described and optimized, allowed the simple preparation of arrays of neurons, where cell adhesion was guided in specific areas and the neurites development driven in the desired arrangement. The resulting arrays were successfully tested for the growth and differentiation of neurons, demonstrating the possible adhesion of cells in specific areas of the film, therefore paving the way to applications such as the direct growth of excitable cells nearby electrodes of devices, with an evident enhancement of cell-electrodes communication.

Prototyping a memristive-based device to analyze neuronal excitability.

Many efforts have been spent in the last decade for the development of nanoscale synaptic devices integrated into neuromorphic circuits, trying to emulate the behavior of natural synapses. The study of brain properties with the standard approaches based on biocompatible electrodes coupled to conventional electronics, however, presents strong limitations, which in turn could be overcame by the in-situ growth of neuronal networks coupled to memristive devices. To meet this challenging task, here two different chips were designed and fabricated for culturing neuronal cells and sensing their electrophysiological activity. The first chip was designed to be connected to an external memristor, while the second chip was coated with TiO2 films owning memristive properties. The biocompatibility of chips was preliminary analyzed by culturing the hybrid motor-neuron cell line NSC-34 and by measuring the electrical activity of cells interfacing the chip with a standard patch-clamp setup. Next, neurons were seeded on chips and their activity measured with the same setup. For both cell types total current and voltage responses were evoked and recorded with optimal results with no breakdowns. In addition, an external stimulation was applied to cells through chip electrodes, being effective and causing no damage or pitfalls to the cells. Finally, the whole bio-hybrid system, i.e. the chip interconnected with a commercial memristor, was tested with promising results. Spontaneous electrical activity of neurons grown on the chip was indeed present and this signal was collected and sent to the memristor, changing its state. Taken together, we demonstrated the ability of memristor to work with a synaptic/plastic response together with natural systems, opening the way for the further implementation of basic computing elements able to perform both storage and processing of data, as in natural neurons.

Azulene revisited: solid-state structure, invariom modeling and lattice-energy minimization of a classical example of disorder.

The molecular and solid-state structure of azulene both raise fundamental questions. Therefore, the disordered crystal structure of azulene was re-refined with invariom non-spherical atomic scattering factors from new single-crystal X-ray diffraction data with a resolution of d = 0.45 Å. An unconstrained refinement results in a molecular geometry with Cs symmetry. Refinements constrained to fulfill C2v symmetry, as observed in the gas phase and in high-level ab initio calculations, lead to similar figures of merit and residual densities as unconstrained ones. Such models are consistent with the structures from microwave spectroscopy and electron diffraction, albeit they are not the same. It is shown that for the disorder present in azulene, the invariom model describes valence electron density as successfully as it does for non-disordered structures, although the disorder still leads to high correlations mainly between positional parameters. Lattice-energy minimizations on a variety of ordered model structures using dispersion-corrected DFT calculations reveal that the local deviations from the average structure are small. Despite the molecular dipole moment there is no significant molecular ordering in any spatial direction. A superposition of all ordered model structures leads to a calculated average structure, which explains not only the experimental determined atomic coordinates, but also the apparently unusual experimental anisotropic displacement parameters.

Giant Pressure Dependence and Dimensionality Switching in a Metal-Organic Quantum Antiferromagnet.

We report an extraordinary pressure dependence of the magnetic interactions in the metal-organic system [CuF_{2}(H_{2}O)_{2}]_{2}pyrazine. At zero pressure, this material realizes a quasi-two-dimensional spin-1/2 square-lattice Heisenberg antiferromagnet. By high-pressure, high-field susceptibility measurements we show that the dominant exchange parameter is reduced continuously by a factor of 2 on compression. Above 18 kbar, a phase transition occurs, inducing an orbital re-ordering that switches the dimensionality, transforming the quasi-two-dimensional lattice into weakly coupled chains. We explain the microscopic mechanisms for both phenomena by combining detailed x-ray and neutron diffraction studies with quantitative modeling using spin-polarized density functional theory.

Electron Density Analysis of Metal Clusters with Semi-Interstitial Main Group Atoms. Chemical Bonding in [Co6X(CO)16]- Species.

In this work, we propose a careful and thorough analysis of the chemical bond nature in high nuclearity metal carbonyl clusters having semi-interstitial main group atoms. We investigated the species [Co6X(CO)16]- (X = As, P), known for a rather interesting conformational flexibility of the cluster (leading to open or closed cages) and a corresponding polymorphism in the solid state (observed at least for X = As). The factors that trigger the molecular isomerism and the nature of X-Co and Co-Co interactions emerge from theoretical calculations and high resolution X-ray diffraction. Both energy and charge density atomic partitioning (QTAIM, EDA, IQA) are employed for this analysis, with the aim of revealing the stabilizing/destabilizing factors of the interaction between the cage and the semi-interstitial atoms in the various conformations.

Influence of volcanic activity and anthropic impact in the trace element contents of fishes from the North Patagonia in a global context.

The elemental contents in salmonid muscle and liver tissues from different lakes around the world were investigated. Fish from pristine areas were compared with those fishes from impacted environments, both by volcanic and anthropogenic activities. Within the data, special attention was given to fishes from the Andean Patagonian lakes in two contexts: local and global. The local evaluation includes geological and limnological parameters and diet composition which were obtained through a data search from published works. The volcanic influence in Andean Patagonian lakes was mainly observed by an increase of cesium (Cs) and rubidium (Rb) concentrations in fishes, influenced by calcium (Ca) and potassium (K) water contents. Zinc (Zn), selenium (Se), iron (Fe), silver (Ag), and mercury (Hg) contents in fishes showed the effect of the geological substratum, and some limnological parameters. The diet composition was another factor which affects the elemental concentration in fishes. The analyzed data showed that the fishes from Andean Patagonian lakes had elemental content patterns corresponding to those of pristine regions with volcanic influence. Selenium and Ag contents from Andean Patagonian fishes were the highest reported.

An interacting quantum atoms analysis of the metal-metal bond in [M2(CO)8]n systems.

The metal-metal interaction in policarbonyl metal clusters remains one of the most challenging and controversial issues in metal-organic chemistry, being at heart of a generalized understanding of chemical bonding and of specific applications of these molecules. In this work, the interacting quantum atoms (IQA) approach is used to study the metal-metal interaction in dimetal polycarbonyl dimers, analyzing bridged (Co2(CO)8)), semibridged ([FeCo(CO)8](-)) and unbridged (Co2(CO)8, [Fe2(CO)8](2-)) clusters. In all systems, a delocalized covalent bond is found to occur, involving the metals and the carbonyls, but the global stability of the dimers mainly originates from the Coulombic attraction between the metals and the oxygens.

Restoring orbital thinking from real space descriptions: bonding in classical and non-classical transition metal carbonyls.

A combined strategy that unifies our interacting quantum atoms approach (IQA), a chemically intuitive energetic perspective within the quantum theory of atoms in molecules (QTAIM), the domain natural orbitals obtained by the diagonalization of the charge-weighted domain-averaged Fermi hole (DAFH), and the statistical analyses of chemical bonding provided by the electron number distribution functions (EDF) is presented. As shown, it allows for recovering traditional orbital images from the orbital invariant descriptions of QTAIM. It does also provide bonding indices (like bond orders) and bond energetics, all in a per orbital basis, still invariant manner, using a single unified framework. The procedure is applied to show how the Dewar, Chatt, and Ducanson model of bonding in simple transition metal carbonyls may be recovered in the real space. The balance between the number of σ-donated and π-backdonated electrons is negative in classical compounds and positive in non-classical ones. The energetic strength of backdonation is, however, smaller than that of donation. Our technique surpasses conventional orbital models by providing physically sound, quantitative energetics of chemical bonds (or interactions) together with effective one-electron pictures, all for arbitrary wavefunctions.

Synthesis, platelet adhesion and cytotoxicity studies of new glycerophosphoryl-containing polyurethanes.

In this work we synthesized new MDI -based poly(ether)urethanes (PEUs) with phospholipid-like residue as chain extender. Polymers were prepared by a conventional two-step solution polymerization procedure using 4,4' diphenylmethanediisocyanate (MDI) and poly(1,4- butanediol) with 1000 as molecular weight to form prepolymers which were successively polymerized with 1 glycerophosphorylcholine (1-GPC), 2-glycerophosphorylcholine (2-GPC) or glycerophosphorylserine (GPS) as chain extenders. Two reference polymers bearing 1,4-butandiol (BD) have been also synthesized. The polymers obtained were characterized by Fourier transform infrared spectroscopy (FT-IR), nuclear magnetic resonance (NMR), differential scanning calorimetry (DSC) and modulated scanning calorimetry (MDSC). The biocompatibility of synthesized segmented polyurethanes was then investigated by platelet-rich plasma contact studies and related scanning electron microscopy (SEM) photographs for blood compatibility and cytotoxicity assay (MTT test) on material elution to assess the effect of any toxic leachables on cellular viability. Three polymers among all have given very satisfactory results suggesting to investigate more deeply their possible use in biomedical devices.

Rb-Cs ratio as an indicator of fish diet in lakes of the Patagonia, Argentina.

The ratios of Rb to Cs contents were studied in five fish species from seven lakes located in the Patagonia Andean Range, Argentina in order to trace fish diet. The species studied were native velvet catfish (Diplomistes viedmensis) and creole perch (Percichthys trucha), and exotic brown trout (Salmo trutta), rainbow trout (Oncorhynchus mykiss), and brook trout (Salvenilus fontinalis). Rainbow trout specimens from two farms were also studied, as well as fish food items and native mussels (Diplodon chilensis). Rb and Cs concentrations were determined by instrumental neutron activation analysis. A positive correlation of Cs concentration in the muscle of velvet catfish with fish length was observed, probably associated with the long biological half-life of this element in this species, whereas the Rb concentration remained constant, hence inhibiting the use of Rb-Cs ratios as a tracer in this case. Seasonal variations observed for rainbow trout and Cs concentration background bias in one of the lakes studied are also a limiting factor to the use of Rb-Cs ratios as a diet tracer. Rb-Cs ratios allowed clear differentiation of rainbow trout raised in farms from the natural specimens that lived in the same environment, in agreement with Rb-Cs ratios determined in both diets. Rb-Cs ratios in rainbow trout showed significant differences between Rivadavia and Futalaufquen lakes compared to Moreno and Nahuel Huapi lakes, which could be associated with a higher participation of plankton in the diet in the first case. No relevant variations in Rb-Cs ratios of brown trout were observed, probably because of the similarity in the diet.

The non-psychoactive cannabidiol triggers caspase activation and oxidative stress in human glioma cells.

Recently, we have shown that the non-psychoactive cannabinoid compound cannabidiol (CBD) induces apoptosis of glioma cells in vitro and tumor regression in vivo. The present study investigated a possible involvement of caspase activation and reactive oxygen species (ROS) induction in the apoptotic effect of CBD. CBD produced a gradual, time-dependent activation of caspase-3, which preceded the appearance of apoptotic death. In addiction, release of cytochrome c and caspase-9 and caspase-8 activation were detected. The exposure to CBD caused in glioma cells an early production of ROS, depletion of intracellular glutathione and increase activity of glutathione reductase and glutathione peroxidase enzymes. Under the same experimental condition, CBD did not impair primary glia. Thus, we found a different sensitivity to the anti-proliferative effect of CBD in human glioma cells and non-transformed cells that appears closely related to a selective ability of CBD in inducing ROS production and caspase activation in tumor cells.

Relativistic analytical wave functions and scattering factors for neutral atoms beyond Kr and for all chemically important ions up to I-.

Relativistic wave functions for elements with Z = 37-54 [Su & Coppens (1998). Acta Cryst. A54, 646-652] have been fitted with a linear combination of Slater-type functions as defined by Bunge, Barrientos & Bunge [At. Data Nucl. Data Tables (1993), 53, 113-162], for use in charge-density analysis and other applications. In addition, numerical relativistic wave functions have been calculated for all chemically relevant ions up to Z = 54, and corresponding analytical expressions have been derived. X-ray scattering factors calculated from the numerical wave functions are parameterized [in the sin(straight theta)/lambda ranges 0.0-2.0, 2.0-4.0 and 4.0-6.0 A(-1)] with six Gaussian functions, using the same method applied previously by Su & Coppens [Acta Cryst. (1997), A53, 749-762].

An evaluation of the brachial plexus block at the humeral canal using a neurostimulator (1417 patients): the efficacy, safety, and predictive criteria of failure.

UNLABELLED: To evaluate the efficacy and safety of the multiple peripheral nerve block technique at the humeral canal (humeral block) with the use of a neurostimulator, we prospectively studied 1417 patients undergoing upper limb surgery with a brachial plexus block at the humeral canal (1468 blocks). The success rate (defined as sensory block [in all nerve distributions] and/or the absence of another anesthetic technique required to allow surgery) was 95%. The threshold of minimal stimulation used to locate each nerve before injecting the anesthetic solution was the unique predictive factor for identified failure. For the median nerve, the threshold was 0.8 mA with a relative risk of failure (RRf: relative risk evaluated by series of Taylor with a 95% confidence interval) = 1.49 (P = 0.04), for the radial nerve the threshold was 0.6 mA (RRf 1.3, P = 0.02), and 0.7 mA for the ulnar nerve (RRf 1.36, P = 0.04). For any equal or higher stimulation level, the risk of failure of the humeral block increased. For the musculocutaneous nerve, we did not observe a significant stimulation threshold for the risk of failure; although beyond 0.7 mA, the RRf was always more than 1.3. Adverse events occurred in 7% of all cases and were usually minor (nausea/vomiting, anxiety, local pain). Our study provides supplementary information on the efficacy and safety of this technique. Stimulation thresholds are clinically identified for the first time as the main factor linked to the failure of a technique using a neurostimulator. We conclude that the humeral block is a reliable peripheral block allowing good success rates results with minor complications, which can be used as an alternative to the axillary block. IMPLICATIONS: We prospectively evaluated the feasibility and the factors causing failure of a peripheral nerve block technique (humeral block) using neurostimulation in a large number of patients. The importance of the level of stimulation for the success of the block was evaluated for the first time.

[Pt]

The intermediacy of CO/NO substitution in the condensation of [Pt(19)(CO)(22)](4-) into [Pt(38)(CO)(44)](2-) (structure shown) has been demonstrated. Two high-nuclearity carbonyl metal clusters, including one with an unprecedented nitrosyl ligand, have been synthesized and structurally characterized.

Partial respecification of nasotemporal polarity in double-temporal chick and chimeric chick-quail eyes.

In chick embryos, naso-temporal polarity of the retina becomes established before Hamburger-Hamilton stage 10. To examine the plasticity of the early eye anlage, double-temporal eyes were made using stage 10-11 (E1.5) chick embryos and stage 8-9 quail embryos. In vivo and in vitro experiments revealed that these double-temporal compound eyes were not completely temporal but nasal in a large peripheral part of the graft. Four hours after transplantation, the nasal-specific fork head transcription factor CBF1 was not expressed in double-temporal eyes but was clearly detectable 24 h later. This suggests that in the peripheral part of the graft, temporal positional values were changed into nasal positional values by a respecification process.

Structural and functional basis for JAK3-deficient severe combined immunodeficiency.

Mutations of the Janus family kinase JAK3 have been found to be responsible for autosomal recessive severe combined immunodeficiency (SCID) in humans. We report here the analysis of four new unrelated patients affected by JAK3-deficient SCID. The genetic defects were heterogeneous and included a large intragenic deletion as well as different point mutations, leading to missense substitutions, early stop codons, or splicing defects. We performed a series of studies of the biochemical events induced by cytokines on lymphoblastoid B-cell lines obtained from these patients. Abnormalities in tyrosine phosphorylation of JAK3 in response to interleukin-2 (IL-2) and IL-4 were present in all patients. Accordingly, IL-2-mediated phosphorylation of STAT5 was also absent or barely detectable. On the contrary, in all cases, we could show reduced but clear phosphorylation of STAT6 upon IL-4 stimulation. In one patient carrying a single amino acid change (Glu481Gly) in the JH3 domain of JAK3, we observed partially conserved IL-2 responses resulting in reduced but detectable levels of JAK3 and STAT5 phosphorylation. Interestingly, the patient bearing this mutation developed a substantial number of circulating CD4(+)/CD45RO+ activated T lymphocytes that were functionally impaired. In two cases, patients' cells expressed JAK3 proteins with mutations in the JH2 pseudo-kinase domain. A single cysteine to arginine substitution (Cys759Arg) in this region resulted in high basal levels of constitutive JAK3 tyrosine phosphorylation unresponsive to either downregulation by serum starvation or cytokine-mediated upregulation. The characterization of the genetic defects and biochemical abnormalities in these JAK3-deficient patients will help define the role of JAK3 in the ontogeny of a competent immune system and may lead to a better understanding of the JAK3 functional domains.

Partial regression, yet incomplete eradication of mammary tumors in transgenic mice by retrovirally mediated HSVtk transfer 'in vivo'.

Mice transgenic for the activated rat neu oncogene under the control of the mouse mammary tumor virus long terminal repeat (MMTV-LTR) (neu+ mice), develop breast tumors in 100% of cases. We have previously reported that double transgenic mice obtained from crossing neu+ mice with mice transgenic for the herpes simplex virus thymidine kinase (HSVtk) gene can be used as a suitable model to test the 'suicide gene' strategy for mammary tumor gene therapy in vivo. In the present study, we evaluated the efficacy of the HSVtk/ganciclovir (GCV) system in the neu+ mice by inoculating cells producing a retroviral vector bearing the HSVtk gene in the mammary tumors on one side of the animals, and comparing their weight with that of the contralateral tumors, after systemic GCV administration. A statistically significant effect of this therapy was clearly seen (P < 0.001) but complete eradication of the tumors could not be achieved. This was not due to the inefficient delivery of GCV, as no HSVtk expression was detected in the residual tumors, but could be related to the low transduction efficiency (< 10%) and to inability of the 'bystander effect' (probably due to the absence of functional gap-junctions among mammary tumor cells) to kill nontransduced neoplastic cells. These data suggest that results obtained by in vivo models using transplanted tumor cell lines as targets for gene therapy might not be immediately transferable to spontaneously arising tumors in animals or humans.

Monocyte function in a severe combined immunodeficient patient with a donor splice site mutation in the Jak3 gene.

Janus kinase-3 (Jak3) is a nonreceptor tyrosine kinase functionally coupled to cytokine receptors which share a "common" gamma chain (gamma c). Mutations in gamma c and Jak3 genes have been identified in X-linked and autosomal severe combined immuno deficiency (SCID), respectively. Jak3 is expressed and activated in myelomonocytic cells. The present study was designed to define the structural alteration responsible for lack of Jak3 in a patient with autosomal SCID and to characterize monocyte function in the absence of this signal transduction element, as well as to establish the whole exon-intron structure. Polymerase chain reaction analysis, performed with primers designed on exon sequences, identified 20 exons spanning approximately 15 kb. These primers, or others designed on the flanking sequences provided in the present report, can be used to amplify the whole gene, allowing the definition of the molecular defects in all cases, including prenatal diagnosis, in which transcript analysis is not possible. On this basis, the deletion transcript found at the homozygous state in patient CM, with both his consanguineous parents being heterozygous for the deletion, was associated with mutation (T to C) of a splice donor site of intron 16 that was also detected in his mother's DNA. Monocytes from Jak3-SCID showed normal cytokine production in response to interleukin-4 (IL-4) (release of IL-1 receptor antagonist) and IL-2 (release of tumor necrosis factor-alpha and IL-8). Lipopolysaccharide-induced cytokine production was also normal and was blocked by IL-4 in Jak3- SCID monocytes. Interferon-gamma induced augmented expression of major histocompatibility class II in Jak3-SCID monocytes. These data indicate that Jak3, expressed and activated in myelomonocytic cells, is dispensable for monocyte differentiation and responsiveness to cytokines that interact with gamma c receptors as well as to other regulatory signals.

The ZNF75 zinc finger gene subfamily: isolation and mapping of the four members in humans and great apes.

We have previously reported (Villa et al. (1993), Genomics 18: 223) the characterization of the human ZNF75 gene located on Xq26, which has only limited homology (less than 65%) to other ZF genes in the databases. Here, we describe three human zinc finger genes with 86 to 95% homology to ZNF75 at the nucleotide level, which represent all the members of the human ZNF75 subfamily. One of these, ZNF75B, is a pseudogene mapped to chromosome 12q13. The other two, ZNF75A and ZNF75C, maintain an ORF in the sequenced region, and at least the latter is expressed in the U937 cell line. They were mapped to chromosomes 16 and 11, respectively. All these genes are conserved in chimpanzees, gorillas, and orangutans. The ZNF75B homologue is a pseudogene in all three great apes, and in chimpanzee it is located on chromosome 10 (phylogenetic XII), at p13 (corresponding to the human 12q13). The chimpanzee homologue of ZNF75 is also located on the Xq26 chromosome, in the same region, as detected by in situ hybridization. As expected, nucleotide changes were clearly more abundant between human and orangutan than between human and chimpanzee or gorilla homologues. Members of the same class were more similar to each other than to the other homologues within the same species. This suggests that the duplication and/or retrotranscription events occurred in a common ancestor long before great ape speciation. This, together with the existence of at least two genes in cows and horses, suggests a relatively high conservation of this gene family.