Improvement in oral bioavailability of 2,4-diaminopyrimidine c-Met inhibitors by incorporation of a 3-amidobenzazepin-2-one group

The hepatocyte growth factor (HGF)-c-Met signaling axis is involved in the mediation of many biological activities, including angiogenesis, proliferation, cell survival, cell motility, and morphogenesis. Dysregulation of c-Met signaling (e.g., overexpression or increased activation) is associated with the proliferation and metastasis of a wide range of tumor types, including breast, liver, lung, colorectal, gastric, bladder, and prostate, among others. Inhibiting the HGF-c-Met pathway is predicted to lead to anti-tumor effects in many cancers. Elaboration of the SAR around a series of 2,4-diaminopyrimidines led to a number of c-Met inhibitors in which pharmaceutical properties were modulated by substituents appended on the C2-benzazepinone ring. In particular, certain-3-amidobenzazepin-2-one analogs had improved oral bioavailability and were evaluated in PK/PD and efficacy models. Lead compounds demonstrated tumor stasis with partial regressions when evaluated in a GTL-16 tumor xenograft mouse model.     

Reducing bias in bacterial community analysis of lower respiratory infections

High-throughput pyrosequencing and quantitative PCR (Q-PCR) analysis offer greatly improved accuracy and depth of characterisation of lower respiratory infections. However, such approaches suffer from an inability to distinguish between DNA derived from viable and non-viable bacteria. This discrimination represents an important step in characterising microbial communities, particularly in contexts with poor clearance of material or high antimicrobial stress, as non-viable bacteria and extracellular DNA can contribute significantly to analyses. Pre-treatment of samples with propidium monoazide (PMA) is an effective approach to non-viable cell exclusion (NVCE). However, the impact of NVCE on microbial community characteristics (abundance, diversity, composition and structure) is not known. Here, adult cystic fibrosis (CF) sputum samples were used as a paradigm. The effects of PMA treatment on CF sputum bacterial community characteristics, as analysed by pyrosequencing and enumeration by species-specific (Pseudomonas aeruginosa) and total bacterial Q-PCR, were assessed. At the local community level, abundances of both total bacteria and of P. aeruginosa were significantly lower in PMA-treated sample portions. Meta-analysis indicated no overall significant differences in diversity; however, PMA treatment resulted in a significant alteration in local community membership in all cases. In contrast, at the metacommunity level, PMA treatment resulted in an increase in community evenness, driven by an increase in diversity, predominately representing rare community members. Importantly, PMA treatment facilitated the detection of both recognised and emerging CF pathogens, significantly influencing ‘core'' and ‘satellite'' taxa group membership. Our findings suggest failure to implement NVCE may result in skewed bacterial community analyses.     

Genome-wide analysis of core promoter elements from conserved human and mouse orthologous pairs

Background  

The canonical core promoter elements consist of the TATA box, initiator (Inr), downstream core promoter element (DPE), TFIIB recognition element (BRE) and the newly-discovered motif 10 element (MTE). The motifs for these core promoter elements are highly degenerate, which tends to lead to a high false discovery rate when attempting to detect them in promoter sequences.     

Different properties of ACPA and IgM-RF derived from a large dataset: further evidence of two distinct autoantibody systems

Introduction  

The aim of this study was to examine seroconversion and the relationship with age and inflammation of autoantibodies in a large group of patients attending an outpatient rheumatology clinic.     

Molecular adaptation of a leaf-eating bird: stomach lysozyme of the hoatzin

This report describes a lysozyme expressed at high levels in the stomach of the hoatzin, the only known foregut-fermenting bird. Evolutionary comparison places it among the calcium-binding lysozymes rather than among the conventional types. Conventional lysozymes were recruited as digestive enzymes twice in the evolution of mammalian foregut fermenters, and these independently recruited lysozymes share convergent structural changes attributed to selective pressures in the stomach. Biochemical convergence and parallel amino acid replacements are observed in the hoatzin stomach lysozyme even though it has a different genetic origin from the mammalian examples and has undergone more than 300 million years of independent evolution.      

Forecasting Seizures Using Intracranial EEG Measures and SVM in Naturally Occurring Canine Epilepsy

Management of drug resistant focal epilepsy would be greatly assisted by a reliable warning system capable of alerting patients prior to seizures to allow the patient to adjust activities or medication. Such a system requires successful identification of a preictal, or seizure-prone state. Identification of preictal states in continuous long- duration intracranial electroencephalographic (iEEG) recordings of dogs with naturally occurring epilepsy was investigated using a support vector machine (SVM) algorithm. The dogs studied were implanted with a 16-channel ambulatory iEEG recording device with average channel reference for a mean (st. dev.) of 380.4 (+87.5) days producing 220.2 (+104.1) days of intracranial EEG recorded at 400 Hz for analysis. The iEEG records had 51.6 (+52.8) seizures identified, of which 35.8 (+30.4) seizures were preceded by more than 4 hours of seizure-free data. Recorded iEEG data were stratified into 11 contiguous, non-overlapping frequency bands and binned into one-minute synchrony features for analysis. Performance of the SVM classifier was assessed using a 5-fold cross validation approach, where preictal training data were taken from 90 minute windows with a 5 minute pre-seizure offset. Analysis of the optimal preictal training time was performed by repeating the cross validation over a range of preictal windows and comparing results. We show that the optimization of feature selection varies for each subject, i.e. algorithms are subject specific, but achieve prediction performance significantly better than a time-matched Poisson random predictor (p<0.05) in 5/5 dogs analyzed.     

HIV-1 Coreceptor Usage Assessment by Ultra-Deep Pyrosequencing and Response to Maraviroc

Background

Maraviroc is an HIV entry inhibitor that alters the conformation of CCR5 and is poorly efficient in patients infected by viruses that use CXCR4 as an entry coreceptor. The goal of this study was to assess the capacity of ultra-deep pyrosequencing (UDPS) and different data analysis approaches to characterize HIV tropism at baseline and predict the therapeutic outcome on maraviroc treatment.

Methods

113 patients with detectable HIV-1 RNA on HAART were treated with maraviroc. The virological response was assessed at months 1, 3 and 6. The sequence of the HIV V3 loop was determined at baseline and prediction of maraviroc response by different software and interpretation algorithms was analyzed.

Results

UDPS followed by analysis with the Pyrotrop software or geno2pheno algorithm provided better prediction of the response to maraviroc than Sanger sequencing. We also found that the H34Y/S substitution in the V3 loop was the strongest individual predictor of maraviroc response, stronger than substitutions at positions 11 or 25 classically used in interpretation algorithms.

Conclusions

UDPS is a powerful tool that can be used with confidence to predict maraviroc response in HIV-1-infected patients. Improvement of the predictive value of interpretation algorithms is possible and our results suggest that adding the H34S/Y substitution would substantially improve the performance of the 11/25/charge rule.     

A rise in NAD precursor nicotinamide mononucleotide (NMN) after injury promotes axon degeneration

NAD metabolism regulates diverse biological processes, including ageing, circadian rhythm and axon survival. Axons depend on the activity of the central enzyme in NAD biosynthesis, nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2), for their maintenance and degenerate rapidly when this activity is lost. However, whether axon survival is regulated by the supply of NAD or by another action of this enzyme remains unclear. Here we show that the nucleotide precursor of NAD, nicotinamide mononucleotide (NMN), accumulates after nerve injury and promotes axon degeneration. Inhibitors of NMN-synthesising enzyme NAMPT confer robust morphological and functional protection of injured axons and synapses despite lowering NAD. Exogenous NMN abolishes this protection, suggesting that NMN accumulation within axons after NMNAT2 degradation could promote degeneration. Ectopic expression of NMN deamidase, a bacterial NMN-scavenging enzyme, prolongs survival of injured axons, providing genetic evidence to support such a mechanism. NMN rises prior to degeneration and both the NAMPT inhibitor FK866 and the axon protective protein Wld^S prevent this rise. These data indicate that the mechanism by which NMNAT and the related Wld^S protein promote axon survival is by limiting NMN accumulation. They indicate a novel physiological function for NMN in mammals and reveal an unexpected link between new strategies for cancer chemotherapy and the treatment of axonopathies.Axon degeneration in disease shares features with the progressive breakdown of the distal segment of severed axons as described by Augustus Waller in 1850 and named Wallerian degeneration.¹ The serendipitous discovery of Wallerian degeneration slow (Wld^S) mice, where transected axons survive 10 times longer than in wild types (WTs),² suggested that axon degeneration is a regulated process, akin to apoptosis of the cell bodies but distinct in molecular terms.^3,4 This process appears conserved in rats, flies, zebrafish and humans.^{5, 6, 7, 8} Wld^S blocks axon degeneration in some disease models, indicating a mechanistic similarity.³ Therefore understanding the pathway it influences is an excellent route towards novel therapeutic strategies.Wld^S is a modified nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1) enzyme, whose N-terminal extension partially relocates NMNAT1 from nuclei to axons, conferring gain of function.^9,10 In mammals, three NMNAT isoforms, nuclear NMNAT1, cytoplasmic NMNAT2 and mitochondrial NMNAT3, catalyse nicotinamide adenine dinucleotide (NAD) synthesis from nicotinamide mononucleotide (NMN) and adenosine triphosphate (ATP; Figure 1a).^11,12 Several reports indicate Wld^S protects injured axons by maintaining axonal NMNAT activity.^{13, 14, 15} In WT injured axons, without Wld^S, NMNAT activity falls when the labile, endogenous axonal isoform, NMNAT2, is no longer transported from cell bodies.¹⁶ NMNAT2 is required for axon maintenance¹⁶ and for axon growth in vivo and in vitro,^17,18 and modulation of its stability by palmitoylation¹⁹ or ubiquitin-dependent processes both in mice or when ectopically expressed in Drosophila^{19, 20, 21} has a corresponding effect on axon survival.Open in a separate windowFigure 1FK866 acts within axons to delay degeneration after injury. (a) The salvage pathway of NAD biosynthesis from nicotinamide (Nam) and nicotinic acid (Na). Only NAD biosynthesis from Nam is sensitive to FK866, which potently inhibits NAMPT while having no effect on nicotinic acid phosphoribosyltransferase (NaPRT).²⁹ The reaction catalysed by bacterial NMN deamidase is also shown. (b) SCG explants were treated with 100 nM FK866 for the indicated times, and then the whole explants (top panel) or the cell bodies (bottom left panel) and neurite fractions (bottom right panel) were separately collected. NAD was determined with an HPLC-based method (see Materials and Methods; n=3, mean and S.D. shown). (c) SCG neurites untreated (top panels) or treated with 100 nM FK866 the day before transection (bottom panels) and imaged after transection at the indicated time points. (d) SCG explants were treated with 100 nM FK866 1 day before or at the indicated times after cutting their neurites. Degeneration index was calculated from three fields in 2–4 independent experiments. The effect of treatment is highly significant when the drug is preincubated or added at 0–4 h after cut (mean ±S.E.M., n=6–12, one-way ANOVA followed by Bonferroni''s post-hoc test, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, compared with untreated)Wld^S partially colocalizes with mitochondria^14,22 and was shown to increase mitochondria motility and Ca²⁺-buffering capacity.²³ Inhibiting mitochondrial permeability transition pore protects degenerating axons.²⁴ However, Wld^S is protective in axons devoid of mitochondria,⁸ and targeting a cytosolic variant of NMNAT2 to mitochondria abolished its protective effect,¹⁹ suggesting a late mitochondrial involvement in Wallerian degeneration.Despite the importance of NMNAT activity in axon survival and degeneration, the molecular players remain elusive. Although NMNAT activity is required for protection,¹³ the hypothesis that increased NAD levels are responsible^25,26 does not fit some data.^27,28While further investigating the role of NAD, we found that blocking nicotinamide phosphoribosyltransferase (NAMPT, the enzyme preceding NMNAT, Figure 1a), was surprisingly axon-protective despite lowering NAD. NAMPT catalyses the synthesis of NMNAT-substrate NMN, the rate-limiting step in the NAD salvage pathway from nicotinamide (Nam) (Figure 1a). Here, we show that NMN accumulates after axon injury, and we provide genetic and pharmacological evidence supporting a role for this NMN increase in axon degeneration when NMNAT2 is depleted. We reveal an unexpected new direction for research into the degenerative mechanism, a novel class of protective proteins and new players in an axon-degeneration pathway sensitive to drugs under development for cancer.     

Meat,dairy, and more: Analysis of material,energy, and greenhouse gas flows of the meat and dairy supply chains in the EU28 for 2016

To decarbonize the European Union, protein consumption must transition to diets low in meat and dairy which will drastically change the material and energy flows in current meat and dairy supply chains. To understand the impacts on current flows, a baseline is required. Although recent studies have improved the scope of reported greenhouse gas (GHG) emissions, no quantitative overview exists including intermediate and final product flows. To address this knowledge gap, we structured the meat and dairy supply chains into a connected set of transformation nodes and distribution nodes. The former are processes transforming inputs into outputs, whereas the latter distribute the outputs to other processes using them as inputs. Currently, livestock play a central role in agriculture and other industries through the consumption of 271 Mt fodder crops, 108 Mt grain, 85 Mt grazed biomass, 49 Mt oil meal, and 16 Mt feed by‐products. This feed is transformed into 64 Mt dairy and 35 Mt meat which ensures that the EU28 is a net exporter of meat and dairy while providing 25 Mt of by‐products. This production also leads to 435 Mt CO₂‐eq. with the main contribution from beef cattle (35%), dairy cattle (32%), and swine (20%). Thus, the lower GHG intensities of dairy products compared to meat do not imply a low contribution to the total emissions. By mapping the material, energy, and GHG emission flows, we have created a baseline suitable for identifying potential supply chain changes and their related GHG increase or decrease resulting from the protein transition.     

Chromogranin a expression in phaeochromocytomas associated with von Hippel-Lindau syndrome and multiple endocrine neoplasia type 2.

Chromogranin A (CGA) is a major secretory protein present in the soluble matrix of chromaffin granules of neuroendocrine cells and tumours, such as phaeochromocytomas. CGA has several functions, some of which may be involved in the distinct phenotypic differences of phaeochromocytomas in patients with von Hippel-Lindau (VHL) syndrome compared to multiple endocrine neoplasia type 2 (MEN 2). In this study, we therefore compared tumour and plasma levels of CGA in patients with phaeochromocytoma associated with the two syndromes. We show that phaeochromocytomas from MEN 2 patients express substantially more CGA than tumours from VHL patients at both the mRNA (3-fold greater) and protein (20-fold) level. We further show that relative to increases in plasma catecholamines, patients with phaeochromocytomas associated with MEN 2 have higher plasma concentrations of CGA than those with tumours in VHL syndrome. These data supplement other observations that phaeochromocytomas in VHL compared to MEN 2 patients express lower amounts of catecholamines and other chromaffin granule cargo, such as chromogranin B and neuropeptide Y. Possibly the differences in tumour CGA expression may contribute to differences in secretory vesicle formation and secretion in the two types of tumours. Alternatively the differences in expression in CGA and other secretory constituents may reflect downregulation of the entire regulated secretory pathway in VHL compared to MEN 2 tumours.