Cell fusion reprogramming leads to a specific hepatic expression pattern during mouse bone marrow derived hepatocyte formation In Vivo

The fusion of bone marrow (BM) hematopoietic cells with hepatocytes to generate BM derived hepatocytes (BMDH) is a natural process, which is enhanced in damaged tissues. However, the reprogramming needed to generate BMDH and the identity of the resultant cells is essentially unknown. In a mouse model of chronic liver damage, here we identify a modification in the chromatin structure of the hematopoietic nucleus during BMDH formation, accompanied by the loss of the key hematopoietic transcription factor PU.1/Sfpi1 (SFFV proviral integration 1) and gain of the key hepatic transcriptional regulator HNF-1A homeobox A (HNF-1A/Hnf1a). Through genome-wide expression analysis of laser captured BMDH, a differential gene expression pattern was detected and the chromatin changes observed were confirmed at the level of chromatin regulator genes. Similarly, Tranforming Growth Factor-β1 (TGF-β1) and neurotransmitter (e.g. Prostaglandin E Receptor 4 [Ptger4]) pathway genes were over-expressed. In summary, in vivo BMDH generation is a process in which the hematopoietic cell nucleus changes its identity and acquires hepatic features. These BMDHs have their own cell identity characterized by an expression pattern different from hematopoietic cells or hepatocytes. The role of these BMDHs in the liver requires further investigation.

Classifier combination for in vivo magnetic resonance spectra of brain tumours

Peer-reviewed

Classifier combination for in vivo magnetic resonance spectra of brain tumours

In this paper we present a multi-stage classifier for magnetic resonance spectra of human brain tumours which is being developed as part of a decision support system for radiologists. The basic idea is to decompose a complex classification scheme into a sequence of classifiers, each specialising in different classes of tumours and trying to reproducepart of the WHO classification hierarchy. Each stage uses a particular set of classification features, which are selected using a combination of classical statistical analysis, splitting performance and previous knowledge.Classifiers with different behaviour are combined using a simple voting scheme in order to extract different error patterns: LDA, decision trees and the k-NN classifier. A special label named "unknown¿ is used when the outcomes of the different classifiers disagree. Cascading is alsoused to incorporate class distances computed using LDA into decision trees. Both cascading and voting are effective tools to improve classification accuracy. Experiments also show that it is possible to extract useful information from the classification process itself in order to helpusers (clinicians and radiologists) to make more accurate predictions and reduce the number of possible classification mistakes.

Overexpression of guanylate cyclase activating protein 2 in rod photoreceptors in vivo leads to morphological changes at the synaptic ribbon.

Guanylate cyclase activating proteins are EF-hand containing proteins that confer calcium sensitivity to retinal guanylate cyclase at the outer segment discs of photoreceptor cells. By making the rate of cGMP synthesis dependent on the free intracellular calcium levels set by illumination, GCAPs play a fundamental role in the recovery of the light response and light adaptation. The main isoforms GCAP1 and GCAP2 also localize to the synaptic terminal, where their function is not known. Based on the reported interaction of GCAP2 with Ribeye, the major component of synaptic ribbons, it was proposed that GCAP2 could mediate the synaptic ribbon dynamic changes that happen in response to light. We here present a thorough ultrastructural analysis of rod synaptic terminals in loss-of-function (GCAP1/GCAP2 double knockout) and gain-of-function (transgenic overexpression) mouse models of GCAP2. Rod synaptic ribbons in GCAPs−/− mice did not differ from wildtype ribbons when mice were raised in constant darkness, indicating that GCAPs are not required for ribbon early assembly or maturation. Transgenic overexpression of GCAP2 in rods led to a shortening of synaptic ribbons, and to a higher than normal percentage of club-shaped and spherical ribbon morphologies. Restoration of GCAP2 expression in the GCAPs−/− background (GCAP2 expression in the absence of endogenous GCAP1) had the striking result of shortening ribbon length to a much higher degree than overexpression of GCAP2 in the wildtype background, as well as reducing the thickness of the outer plexiform layer without affecting the number of rod photoreceptor cells. These results indicate that preservation of the GCAP1 to GCAP2 relative levels is relevant for maintaining the integrity of the synaptic terminal. Our demonstration of GCAP2 immunolocalization at synaptic ribbons at the ultrastructural level would support a role of GCAPs at mediating the effect of light on morphological remodeling changes of synaptic ribbons.

Functional EF-hands in neuronal calcium sensor GCAP2 determine its phosphorylation state and subcellular distribution in vivo, and are essential for photoreceptor cell integrity

The neuronal calcium sensor proteins GCAPs (guanylate cyclase activating proteins) switch between Ca2+-free and Ca2+-bound conformational states and confer calcium sensitivity to guanylate cyclase at retinal photoreceptor cells. They play a fundamental role in light adaptation by coupling the rate of cGMP synthesis to the intracellular concentration of calcium. Mutations in GCAPs lead to blindness. The importance of functional EF-hands in GCAP1 for photoreceptor cell integrity has been well established. Mutations in GCAP1 that diminish its Ca2+ binding affinity lead to cell damage by causing unabated cGMP synthesis and accumulation of toxic levels of free cGMP and Ca2+. We here investigate the relevance of GCAP2 functional EF-hands for photoreceptor cell integrity. By characterizing transgenic mice expressing a mutant form of GCAP2 with all EF-hands inactivated (EF(-)GCAP2), we show that GCAP2 locked in its Ca2+-free conformation leads to a rapid retinal degeneration that is not due to unabated cGMP synthesis. We unveil that when locked in its Ca2+-free conformation in vivo, GCAP2 is phosphorylated at Ser201 and results in phospho-dependent binding to the chaperone 14-3-3 and retention at the inner segment and proximal cell compartments. Accumulation of phosphorylated EF(-)GCAP2 at the inner segment results in severe toxicity. We show that in wildtype mice under physiological conditions, 50% of GCAP2 is phosphorylated correlating with the 50% of the protein being retained at the inner segment. Raising mice under constant light exposure, however, drastically increases the retention of GCAP2 in its Ca2+-free form at the inner segment. This study identifies a new mechanism governing GCAP2 subcellular distribution in vivo, closely related to disease. It also identifies a pathway by which a sustained reduction in intracellular free Ca2+ could result in photoreceptor damage, relevant for light damage and for those genetic disorders resulting in 'equivalent-light'' scenarios.

Correlation between in vitro cytotoxicity and in vivo lethal activity in mice of epsilon toxin mutants from Clostridium perfringens

Epsilon toxin (Etx) from Clostridium perfringens is a pore-forming protein with a lethal effect on livestock, producing severe enterotoxemia characterized by general edema and neurological alterations. Site-specific mutations of the toxin are valuable tools to study the cellular and molecular mechanism of the toxin activity. In particular, mutants with paired cysteine substitutions that affect the membrane insertion domain behaved as dominant-negative inhibitors of toxin activity in MDCK cells. We produced similar mutants, together with a well-known non-toxic mutant (Etx-H106P), as green fluorescent protein (GFP) fusion proteins to perform in vivo studies in an acutely intoxicated mouse model. The mutant (GFP-Etx-I51C/A114C) had a lethal effect with generalized edema, and accumulated in the brain parenchyma due to its ability to cross the blood-brain barrier (BBB). In the renal system, this mutant had a cytotoxic effect on distal tubule epithelial cells. The other mutants studied (GFP-Etx-V56C/F118C and GFP-Etx-H106P) did not have a lethal effect or cross the BBB, and failed to induce a cytotoxic effect on renal epithelial cells. These data suggest a direct correlation between the lethal effect of the toxin, with its cytotoxic effect on the kidney distal tubule cells, and the ability to cross the BBB.

In vivo co-ordinated interactions between inhibitory systems to control glutamate mediated hippocampal excitability.

We present an overview of the long-term adaptation of hippocampal neurotransmission to cholinergic and GABAergic deafferentation caused by excitotoxic lesion of the medial septum. Two months after septal microinjection of 2.7 nmol a -amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA), a 220% increase of GABA A receptor labelling in the hippo- campal CA3 and the hilus was shown, and also changes in hippocampal neurotransmission characterised by in vivo microdialysis and HPLC. Basal amino acid and purine extra- cellular levels were studied in control and lesioned rats. In vivo effects of 100 m M KCl perfusion and adenosine A 1 receptor blockade with 1,3-dipropyl- 8-cyclopentylxanthine (DPCPX) on their release were also investigated. In lesioned animals GABA, glutamate and glutamine basal levels were decreased and taurine, adenosine and uric acid levels increased. A similar response to KCl infusion occurred in both groups except for GABA and glutamate, which release decreased in lesioned rats. Only in lesioned rats, DPCPX increased GABA basal level and KCl-induced glutamate release, and decreased glutamate turnover. Our results evidence that an excitotoxic septal lesion leads to increased hippocampal GABA A receptors and decreased glutamate neurotransmis- sion. In this situation, a co-ordinated response of hippocampal retaliatory systems takes place to control neuron excitability.

Multigram synthesis and in vivo efficacy studies of a novel multitarget anti-Alzheimer's compound

We describe the multigram synthesis and in vivo efficacy studies of a donepezil‒huprine hybrid that has been found to display a promising in vitro multitarget profile of interest for the treatment of Alzheimer's disease (AD). Its synthesis features as the key step a novel multigram preparative chromatographic resolution of intermediate racemic huprine Y by chiral HPLC. Administration of this compound to transgenic CL4176 and CL2006 Caenorhabditis elegans strains expressing human Aβ42, here used as simplified animal models of AD, led to a significant protection from the toxicity induced by Aβ42. However, this protective effect was not accompanied, in CL2006 worms, by a reduction of amyloid deposits. Oral administration for 3 months to transgenic APPSL mice, a well-established animal model of AD, improved short-term memory, but did not alter brain levels of Aβ peptides nor cortical and hippocampal amyloid plaque load. Despite the clear protective and cognitive effects of AVCRI104P4, the lack of Aβ lowering effect in vivo might be related to its lower in vitro potency toward Aβ aggregation and formation as compared with its higher anticholinesterase activities. Further lead optimization in this series should thus focus on improving the anti-amyloid/anticholinesterase activity ratio.

A bioinspired peptide scaffold with high antibiotic activity and low in vivo toxicity

Bacterial resistance to almost all available antibiotics is an important public health issue. A major goal in antimicrobial drug discovery is the generation of new chemicals capable of killing pathogens with high selectivity, particularly multi-drug-resistant ones. Here we report the design, preparation and activity of new compounds based on a tunable, chemically accessible and upscalable lipopeptide scaffold amenable to suitable hit-to-lead development. Such compounds could become therapeutic candidates and future antibiotics available on the market. The compounds are cyclic, contain two D-amino acids for in vivo stability and their structures are reminiscent of other cyclic disulfide-containing peptides available on the market. The optimized compounds prove to be highly active against clinically relevant Gram-negative and Gram-positive bacteria. In vitro and in vivo tests show the low toxicity of the compounds. Their antimicrobial activity against resistant and multidrug-resistant bacteria is at the membrane level, although other targets may also be involved depending on the bacterial strain.

A bioinspired peptide scaffold with high antibiotic activity and low in vivo toxicity

Bacterial resistance to almost all available antibiotics is an important public health issue. A major goal in antimicrobial drug discovery is the generation of new chemicals capable of killing pathogens with high selectivity, particularly multi-drug-resistant ones. Here we report the design, preparation and activity of new compounds based on a tunable, chemically accessible and upscalable lipopeptide scaffold amenable to suitable hit-to-lead development. Such compounds could become therapeutic candidates and future antibiotics available on the market. The compounds are cyclic, contain two D-amino acids for in vivo stability and their structures are reminiscent of other cyclic disulfide-containing peptides available on the market. The optimized compounds prove to be highly active against clinically relevant Gram-negative and Gram-positive bacteria. In vitro and in vivo tests show the low toxicity of the compounds. Their antimicrobial activity against resistant and multidrug-resistant bacteria is at the membrane level, although other targets may also be involved depending on the bacterial strain.

A bioinspired peptide scaffold with high antibiotic activity and low in vivo toxicity

Bacterial resistance to almost all available antibiotics is an important public health issue. A major goal in antimicrobial drug discovery is the generation of new chemicals capable of killing pathogens with high selectivity, particularly multi-drug-resistant ones. Here we report the design, preparation and activity of new compounds based on a tunable, chemically accessible and upscalable lipopeptide scaffold amenable to suitable hit-to-lead development. Such compounds could become therapeutic candidates and future antibiotics available on the market. The compounds are cyclic, contain two D-amino acids for in vivo stability and their structures are reminiscent of other cyclic disulfide-containing peptides available on the market. The optimized compounds prove to be highly active against clinically relevant Gram-negative and Gram-positive bacteria. In vitro and in vivo tests show the low toxicity of the compounds. Their antimicrobial activity against resistant and multidrug-resistant bacteria is at the membrane level, although other targets may also be involved depending on the bacterial strain.

A bioinspired peptide scaffold with high antibiotic activity and low in vivo toxicity

Bacterial resistance to almost all available antibiotics is an important public health issue. A major goal in antimicrobial drug discovery is the generation of new chemicals capable of killing pathogens with high selectivity, particularly multi-drug-resistant ones. Here we report the design, preparation and activity of new compounds based on a tunable, chemically accessible and upscalable lipopeptide scaffold amenable to suitable hit-to-lead development. Such compounds could become therapeutic candidates and future antibiotics available on the market. The compounds are cyclic, contain two D-amino acids for in vivo stability and their structures are reminiscent of other cyclic disulfide-containing peptides available on the market. The optimized compounds prove to be highly active against clinically relevant Gram-negative and Gram-positive bacteria. In vitro and in vivo tests show the low toxicity of the compounds. Their antimicrobial activity against resistant and multidrug-resistant bacteria is at the membrane level, although other targets may also be involved depending on the bacterial strain.

A bioinspired peptide scaffold with high antibiotic activity and low in vivo toxicity

Bacterial resistance to almost all available antibiotics is an important public health issue. A major goal in antimicrobial drug discovery is the generation of new chemicals capable of killing pathogens with high selectivity, particularly multi-drug-resistant ones. Here we report the design, preparation and activity of new compounds based on a tunable, chemically accessible and upscalable lipopeptide scaffold amenable to suitable hit-to-lead development. Such compounds could become therapeutic candidates and future antibiotics available on the market. The compounds are cyclic, contain two D-amino acids for in vivo stability and their structures are reminiscent of other cyclic disulfide-containing peptides available on the market. The optimized compounds prove to be highly active against clinically relevant Gram-negative and Gram-positive bacteria. In vitro and in vivo tests show the low toxicity of the compounds. Their antimicrobial activity against resistant and multidrug-resistant bacteria is at the membrane level, although other targets may also be involved depending on the bacterial strain.