Signalogs: orthology-based identification of novel signaling pathway components in three metazoans

Background

Uncovering novel components of signal transduction pathways and their interactions within species is a central task in current biological research. Orthology alignment and functional genomics approaches allow the effective identification of signaling proteins by cross-species data integration. Recently, functional annotation of orthologs was transferred across organisms to predict novel roles for proteins. Despite the wide use of these methods, annotation of complete signaling pathways has not yet been transferred systematically between species.

Principal Findings

Here we introduce the concept of ‘signalog’ to describe potential novel signaling function of a protein on the basis of the known signaling role(s) of its ortholog(s). To identify signalogs on genomic scale, we systematically transferred signaling pathway annotations among three animal species, the nematode Caenorhabditis elegans, the fruit fly Drosophila melanogaster, and humans. Using orthology data from InParanoid and signaling pathway information from the SignaLink database, we predict 88 worm, 92 fly, and 73 human novel signaling components. Furthermore, we developed an on-line tool and an interactive orthology network viewer to allow users to predict and visualize components of orthologous pathways. We verified the novelty of the predicted signalogs by literature search and comparison to known pathway annotations. In C. elegans, 6 out of the predicted novel Notch pathway members were validated experimentally. Our approach predicts signaling roles for 19 human orthodisease proteins and 5 known drug targets, and suggests 14 novel drug target candidates.

Conclusions

Orthology-based pathway membership prediction between species enables the identification of novel signaling pathway components that we referred to as signalogs. Signalogs can be used to build a comprehensive signaling network in a given species. Such networks may increase the biomedical utilization of C. elegans and D. melanogaster. In humans, signalogs may identify novel drug targets and new signaling mechanisms for approved drugs.     

Muscle fibre conduction velocity and cardiorespiratory response during incremental cycling exercise in young and older individuals with different training status

We investigated the effect of ageing and training on muscle fibre conduction velocity (MFCV) and cardiorespiratory response during incremental cycling exercise. Eight young (YT; 24 ± 5 yrs) and eight older (OT; 64 ± 3 yrs) cyclists, together with eight young (YU; 27 ± 4 yrs) and eight older (OU; 63 ± 2 yrs) untrained individuals underwent to an incremental maximal test on a cycle ergometer. Ventilatory threshold (VT), respiratory compensation point (RCP) and maximal oxygen uptake (VO₂max) were identified and MFCV recorded from the vastus lateralis muscle using surface electromyography with linear arrays electrodes.In YT MFCV increased with the exercise intensity, reaching a peak of 4.99 ± 1.02 [m/s] at VT. Thereafter, and up to VO₂max, MFCV declined. In YU MFCV showed a similar trend although the peak [4.55 ± 0.53 m/s] was observed, at 75% of VO₂max an intensity higher than VT (66% of VO₂max). In both YT and YU MFCV did not decline until RPC, which occurred at 78% VO₂max in YU and at 92% VO₂max (P < 0.01) in YT. Differently from young individuals, MFCV in older subjects did not increase with exercise intensity. Moreover, maximal MFCV in OU was significantly lower [3.53 ± 0.40 m/s;] than that of YT (P < 0.005) and YU (P < 0.05).The present study shows that, especially in young individuals, MFCV reflects cardiorespiratory response during incremental dynamic cyclic exercise and hence can be used to investigate motor unit recruitment strategies.     

1H NMR-visible mobile lipid domains correlate with cytoplasmic lipid bodies in apoptotic T-lymphoblastoid cells

The presence of nuclear magnetic resonance (NMR)-visible mobile lipid (ML) domains in apoptotic lymphoblasts suggests alterations in neutral lipid metabolism and compartmentation during programmed cell death. The detection of similar ML signals in activated lymphocytes raises questions about common mechanisms of ML formation during apoptosis and upon lymphoblast stimulation. Structure and subcellular localization of ML domains were therefore investigated by NMR, fluorescence and electron microscopy in Jurkat T-lymphoblasts either induced to apoptosis (by anthracyclines or dexamethasone or by serum deprivation) or activated by phorbol myristate acetate (PMA) plus ionomycin. ML contents in drug-treated cells correlated linearly with apoptosis, irrespective of the specific inducer and cell cycle arrest phase (r=0.993, P<0.001). Similar ML levels were measured in drug-induced apoptotic cells (A≈30–40%) and in non-apoptotic PMA/ionomycin-treated lymphoblasts (72 h). Lower ML contents were instead formed in serum-deprived apoptotic cells, with respect to controls. Increases in ML signals were associated, in either apoptotic or activated cells, with the accumulation of cytoplasmic, osmophilic lipid bodies (diameter≤1.0 μm), surrounded by own membrane, possessing intramembrane particles. The results support the hypothesis that ML are formed in the cytoplasm of drug-induced apoptotic cells during an early, ‘biochemically active’ phase of programmed cell death.     

Role of hyperhomocysteinemia in aortic disease.

A growing body of evidence has shown a strong association between elevated plasma homocysteine (Hcy) levels with vascular disease and thrombotic complications. Data available in literature also suggest a role of hyperhomocysteinemia in abdominal and thoracic aortic diseases. In particular, Hcy was investigated in patients with Marfan syndrome and it was demonstrated that Hcy levels were associated with the risk of severe cardiovascular manifestations or dissection. Hcy was significantly higher also in patients with abdominal aortic aneurysms and was associated with the size of aneurysms. It remains to be elucidated if this association is causal or simply an effect of the disease. A number of mechanisms may be evoked to explain these findings. Studies in animal models demonstrated that hyperhomocysteinemia could induce marked remodelling of the extracellular matrix of the arterial wall by inducing elastolysis through the activation of metalloproteinases. In addition, Hcy may directly affect fibrillin-1 or collagen by interfering with intra- and/or inter-molecular disulfide bonds through disulfide exchange, or binding to free sulphydryl groups. Further studies are needed to confirm the role of Hcy in aortic disease and the usefulness of including Hcy determination in the clinical evaluation of these patients.     

Innervation of the urinary bladder of Ovis aries L

The authors have extended a preliminary study about the innervation of urinary bladder, confirming the previous results pointing out the presence of metasympathetic ganglions in the wall of urinary bladder. Therefore nine urinary bladders of Ovis aries of different age and both sexes have been studied by Ruffini, Bodian and Bielschowsky's staining methods. It's possible summarize the data on the innervation of urinary bladder in the following way: in the tunica adventitia there are motor and sensitive bundles of myelinated nervous fiber. The formers, after many divisions, penetrate into the tunica muscularis contacting bundles of smooth muscle fibers, while the latters after several divisions after giving rise to thinner bundles, produce Pacini-like and Ruffini-like sense corpuscles and free nervous terminations. Furthermore, some metasympathetic ganglions of different size have been detected throughout the running of the bundles. In the tunica submucosa a diffuse and peculiar non myelinated network is observed, arising from the vegetative nervous fibers.     

Non-invasive assessment of muscle fiber conduction velocity during an incremental maximal cycling test

Muscle fiber conduction velocity (MFCV) gives critical information on neuromuscular control and can be considered a size principle parameter, being suggestive of motor unit recruitment strategies. MFCV has been recently measured during constant-load sub-maximal cycling exercise and was found to correlate positively with percentage of type I myosin heavy chain.The aim of this study was to test the hypothesis that MFCV measured during an incremental cycling test using surface electromyography (sEMG), can be sensitive to the different metabolic requests elicited by the exercise. In particular, the relationship between ventilatory threshold (T-vent), V^′O_2max and MFCV was explored.Eleven male physically active subjects (age 30 ± 9 years) undertook a 1-min incremental cycling test to exhaustion. T-vent and V^′O_2max were measured using an open circuit breath by breath gas analyzer. The sEMG was recorded from the vastus lateralis muscle with an adhesive 4-electrodes array, and the MFCV was computed on each sEMG burst over the last 30-s of each step.The mean V^′O_2max obtained during the maximal test was 53.32 ± 2.33 ml kg^?1 min^?1, and the T-vent was reached at 80.77 ± 3.49% of V^′O_2max. In all subjects reliable measures of MFCV were obtained at every exercise intensity (cross correlation values >0.8). MFCV increased linearly with the mechanical load, reaching a maximum value of 4.28 ± 0.67 m s^?1 at an intensity corresponding to the T-vent. Thereafter, MFCV declined until maximal work intensities. This study demonstrates that MFCV can be used as non-invasive tool to infer MUs recruitment/derecruitment strategies even during dynamic exercise from low to maximal intensities.     

Congenital asplenia in mice and humans with mutations in a Pbx/Nkx2-5/p15 module

The molecular determinants of spleen organogenesis and the etiology of isolated congenital asplenia (ICA), a life-threatening human condition, are unknown. We previously reported that Pbx1 deficiency causes organ growth defects including asplenia. Here, we show that mice with splenic mesenchyme-specific Pbx1 inactivation exhibit hyposplenia. Moreover, the loss of Pbx causes downregulation of Nkx2-5 and derepression of p15Ink4b in spleen mesenchymal progenitors, perturbing the cell cycle. Removal of p15Ink4b in Pbx1 spleen-specific mutants partially rescues spleen growth. By whole-exome sequencing of a multiplex kindred with ICA, we identify a heterozygous missense mutation (P236H) in NKX2-5 showing reduced transactivation in vitro. This study establishes that a Pbx/Nkx2-5/p15 regulatory module is essential for spleen development.     

Efficiency of expression of transfected genes depends on the cell cycle

Lipofection, a lipid-mediated DNA transfection procedure, was used to transfect synchronized L929 mouse fibroblast cells with a reporter plasmid containing the bacterial chloramphenicol acetyltransferase gene. The efficiency of gene expression was investigated on transfection of cells at different stages of the cell cycle. Our data show that expression of the reporter gene was minimal when transfection was performed in G0-phase and parallel experimental data disproved the possibility that the reduced expression observed was due to differential uptake at different times in the cell cycle. Investigation into the condensation state of the plasmid has shown that the low chloramphenicol acetyltransferase gene expression could be a direct consequence of the packaging of the plasmid into condensed chromatin when transfection occurs in G0-phase. The inactivation of the reporter gene is not reversed by growth of the cells in high serum or by treatment with Trichostatin A, a specific inhibitor of histone deacetylase, suggesting that the inactive chromatin formed in G0-phase cells lacks associated histone acetylase activity. In contrast, the high activity seen when cells in S-phase are transfected is enhanced even further by treatment with Trichostatin A.