Discovery of imidazo[1,2-a]-, [1,2,4]triazolo[4,3-a]-, and [1,2,4]triazolo[1,5-a]pyridine-8-carboxamide negative allosteric modulators of metabotropic glutamate receptor subtype 5

Based on a hypothesis that an intramolecular hydrogen bond was present in our lead series of picolinamide mGlu₅ NAMs, we reasoned that an inactive nicotinamide series could be modified through introduction of a fused heterocyclic core to generate potent mGlu₅ NAMs. In this Letter, we describe the synthesis and evaluation of compounds that demonstrate the viability of that approach. Selected analogs were profiled in a variety of in vitro assays, and two compounds were evaluated in rat pharmacokinetic studies and a mouse model of obsessive-compulsive disorder. Ancillary pharmacology screening revealed that members of this series exhibited moderate inhibition of the dopamine transporter (DAT), and SAR was developed that expanded the selectivity for mGlu₅ versus DAT.     

Discovery and optimization of 3-(4-aryl/heteroarylsulfonyl)piperazin-1-yl)-6-(piperidin-1-yl)pyridazines as novel,CNS penetrant pan-muscarinic antagonists

This letter describes the synthesis and structure activity relationship (SAR) studies of structurally novel M₄ antagonists, based on a 3-(4-aryl/heteroarylsulfonyl)piperazin-1-yl)-6-(piperidin-1-yl)pyridazine core, identified from a high-throughput screening campaign. A multi-dimensional optimization effort enhanced potency at human M₄ (hM₄ IC₅₀s < 200 nM), with only moderate species differences noted, and with enantioselective inhibition. Moreover, CNS penetration proved attractive for this series (rat brain:plasma K_p = 2.1, K_p,uu = 1.1). Despite the absence of the prototypical mAChR antagonist basic or quaternary amine moiety, this series displayed pan-muscarinic antagonist activity across M_1-5 (with 9- to 16-fold functional selectivity at best). This series further expands the chemical diversity of mAChR antagonists.     

Latency associated peptide has in vitro and in vivo immune effects independent of TGF-beta1

Latency Associated Peptide (LAP) binds TGF-beta1, forming a latent complex. Currently, LAP is presumed to function only as a sequestering agent for active TGF-beta1. Previous work shows that LAP can induce epithelial cell migration, but effects on leukocytes have not been reported. Because of the multiplicity of immunologic processes in which TGF-beta1 plays a role, we hypothesized that LAP could function independently to modulate immune responses. In separate experiments we found that LAP promoted chemotaxis of human monocytes and blocked inflammation in vivo in a murine model of the delayed-type hypersensitivity response (DTHR). These effects did not involve TGF-beta1 activity. Further studies revealed that disruption of specific LAP-thrombospondin-1 (TSP-1) interactions prevented LAP-induced responses. The effect of LAP on DTH inhibition depended on IL-10. These data support a novel role for LAP in regulating monocyte trafficking and immune modulation.     

Differential toxicity of TAR DNA‐binding protein 43 isoforms depends on their submitochondrial localization in neuronal cells

TAR DNA ‐binding protein 43 (TDP ‐43) is an RNA ‐binding protein and a major component of protein aggregates found in amyotrophic lateral sclerosis and several other neurodegenerative diseases. TDP ‐43 exists as a full‐length protein and as two shorter forms of 25 and 35 kD a. Full‐length mutant TDP ‐43s found in amyotrophic lateral sclerosis patients re‐localize from the nucleus to the cytoplasm and in part to mitochondria, where they exert a toxic role associated with neurodegeneration. However, induction of mitochondrial damage by TDP ‐43 fragments is yet to be clarified. In this work, we show that the mitochondrial 35 kD a truncated form of TDP ‐43 is restricted to the intermembrane space, while the full‐length forms also localize in the mitochondrial matrix in cultured neuronal NSC ‐34 cells. Interestingly, the full‐length forms clearly affect mitochondrial metabolism and morphology, possibly via their ability to inhibit the expression of Complex I subunits encoded by the mitochondrial‐transcribed mRNA s, while the 35 kD a form does not. In the light of the known differential contribution of the full‐length and short isoforms to generate toxic aggregates, we propose that the presence of full‐length TDP ‐43s in the matrix is a primary cause of mitochondrial damage. This in turn may cause oxidative stress inducing toxic oligomers formation, in which short TDP ‐43 forms play a major role.

     

Effects of Anabolic Steroids and High-Intensity Aerobic Exercise on Skeletal Muscle of Transgenic Mice

In an attempt to shorten recovery time and improve performance, strength and endurance athletes occasionally turn to the illicit use of anabolic-androgenic steroids (AAS). This study evaluated the effects of AAS treatment on the muscle mass and phenotypic characteristics of transgenic mice subjected to a high-intensity, aerobic training program (5d/wk for 6 weeks). The transgenic mice (CETP^+/-LDLr^-/+) were engineered to exhibit a lipid profile closer to humans. Animals were divided into groups of sedentary (Sed) and/or training (Ex) mice (each treated orally with AAS or gum arabic/vehicle: Sed-C, Sed-M, ex-C, ex-M). The effects of AAS (mesterolone: M) on specific phenotypic adaptations (muscle wet weight, cross-sectional area, and fiber type composition) in three hindlimb muscles (soleus:SOL, tibialis anterior:TA and gastrocnemius:GAS) were assessed. In order to detect subtle changes in fiber type profile, the entire range of fiber types (I, IC, IIAC, IIA, IIAD, IID, IIDB, IIB) was delineated using mATPase histochemistry. Body weight gain occurred throughout the study for all groups. However, the body weight gain was significantly minimized with exercise. This effect was blunted with mesterolone treatment. Both AAS treatment (Sed-M) and high-intensity, aerobic training (ex-C) increased the wet weights of all three muscles and induced differential hypertrophy of pure and hybrid fibers. Combination of AAS and training (ex-M) resulted in enhanced hypertrophy. In the SOL, mesterolone treatment (Sed-M and ex-M) caused dramatic increases in the percentages of fiber types IC, IIAC, IIAD, IID, with concomitant decrease in IIA, but had minimal impact on fiber type percentages in the predominantly fast muscles. Overall, the AAS-induced differential adaptive changes amounted to significant fiber type transformations in the fast-to-slow direction in SOL. AAS treatment had a significant effect on muscle weights and fiber type composition in SOL, TA and GAS which was even maximized in animals subjected to metabolically high-intensity aerobic exercise.     

Differential Denaturation of Serum Proteome Reveals a Significant Amount of Hidden Information in Complex Mixtures of Proteins

Recently developed proteomic technologies allow to profile thousands of proteins within a high-throughput approach towards biomarker discovery, although results are not as satisfactory as expected. In the present study we demonstrate that serum proteome denaturation is a key underestimated feature; in fact, a new differential denaturation protocol better discriminates serum proteins according to their electrophoretic mobility as compared to single-denaturation protocols. Sixty nine different denaturation treatments were tested and the 3 most discriminating ones were selected (TRIDENT analysis) and applied to human sera, showing a significant improvement of serum protein discrimination as confirmed by MALDI-TOF/MS and LC-MS/MS identification, depending on the type of denaturation applied. Thereafter sera from mice and patients carrying cutaneous melanoma were analyzed through TRIDENT. Nine and 8 protein bands were found differentially expressed in mice and human melanoma sera, compared to healthy controls (p<0.05); three of them were found, for the first time, significantly modulated: α2macroglobulin (down-regulated in melanoma, p<0.001), Apolipoprotein-E and Apolipoprotein-A1 (both up-regulated in melanoma, p<0.04), both in mice and humans. The modulation was confirmed by immunological methods. Other less abundant proteins (e.g. gelsolin) were found significantly modulated (p<0.05).Conclusions: i) serum proteome contains a large amount of information, still neglected, related to proteins folding; ii) a careful serum denaturation may significantly improve analytical procedures involving complex protein mixtures; iii) serum differential denaturation protocol highlights interesting proteomic differences between cancer and healthy sera.     

High-Throughput miRNA and mRNA Sequencing of Paired Colorectal Normal,Tumor and Metastasis Tissues and Bioinformatic Modeling of miRNA-1 Therapeutic Applications

MiRNAs are discussed as diagnostic and therapeutic molecules. However, effective miRNA drug treatments with miRNAs are, so far, hampered by the complexity of the miRNA networks. To identify potential miRNA drugs in colorectal cancer, we profiled miRNA and mRNA expression in matching normal, tumor and metastasis tissues of eight patients by Illumina sequencing. We validated six miRNAs in a large tissue screen containing 16 additional tumor entities and identified miRNA-1, miRNA-129, miRNA-497 and miRNA-215 as constantly de-regulated within the majority of cancers. Of these, we investigated miRNA-1 as representative in a systems-biology simulation of cellular cancer models implemented in PyBioS and assessed the effects of depletion as well as overexpression in terms of miRNA-1 as a potential treatment option. In this system, miRNA-1 treatment reverted the disease phenotype with different effectiveness among the patients. Scoring the gene expression changes obtained through mRNA-Seq from the same patients we show that the combination of deep sequencing and systems biological modeling can help to identify patient-specific responses to miRNA treatments. We present this data as guideline for future pre-clinical assessments of new and personalized therapeutic options.     

DEVELOPMENTAL CHANGES IN Na+, K+ AND ATP AND IN THE LEVELS AND TRANSPORT OF AMINO ACIDS IN INCUBATED SLICES OF RAT BRAIN

Abstract—

• 1 Upon incubation, slices of brain tissue took up fluid; the degree of swelling increased with increasing age. No sweiling occurred in slices from foetal brain. Since this swelling was associated with increases in the inulin space, the percentage of inulin space in slices at the end of incubation increased during brain development.
• 2 Most of the capacity for ion transport seemed to be absent from foetal brain. In vivo and in slices, Na⁺ was very high and K⁺ was very low in comparison to levels at other ages. There was a rapid change around birth, but no significant change at later ages. Upon incubation, Na⁺ levels increased in other slices, but not in slices of foetal brain.
• 3 Upon incubation of the slices, ATP levels were restored to levels close to those in the living brain; there were no significant alterations in available energy during development to explain changes in amino acid transport.
• 4 The composition of the free pool of cerebral amino acids in vivo changed with development, with some compounds (glutamic acid and related compounds) increasing, others (mostly‘essential’amino acids) decreasing, with age. These changes were not linear with time, and the level of a compound might exhibit several peaks during development.
• 5 The uptake (influx) of taurine, glutamate and glycine into brain slices increased rapidly during the foetal and early neonatal periods, reached a maximum between 2 and 3 weeks of postnatal age and then declined to adult levels. The levels of steady-state uptake with glycine also exhibited a maximal peak at 2-3 weeks of postnatal age. Steady-state uptake of taurine and glutamate reached adult levels by about 3 weeks of age.
• 6 The pattern of inhibition of amino acid transport by two specific amino acid analogues changed during development for some amino acids (GABA, glycine and glutamate), indicating an alteration in substrate specificity.
• 7 The results demonstrate complex changes in cerebral amino acid transport during development, with several maxima or minima and with changes in specificity for at least some compounds.

     

A karyotype analysis of the genus Dicentrarchus by different staining techniques

This study presents a cytogenetic analysis of the genus Dicentrarchus , represented by two species, D. labrax and D. punctatus . The karyotypes are very similar, even after staining with different techniques. Both species show 48 subtelocentric and acrocentric chromosomes, gradually decreasing in size. One pair of small size chromosomes has heteropycnotic and heteromorphtc short arms of longer size. These short arms are C- and Ag-positive, i.e. nucleolar organizer regions, NORs, are located there. Constitutive heterochromatin is also evident as a subcentromeric band on the long arms of a large chromosome pair. CMA₃-staining confirms the location and the heteromorphism of NORs. DAPI and quinacrine produce homogeneous staining of chromosomes. A review of cytogenetic studies on 'serranid' species is also presented.