Histone H3 lysine 27 acetylation is altered in colon cancer

Background

Histone post-translational modifications (PTMs) play an important role in the regulation of the expression of genes, including those involved in cancer development and progression. However, our knowledge of PTM patterns in human tumours is limited.

Methods

MS-based analyses were used to quantify global alterations of histone PTMs in colorectal cancer (CRC) samples. Histones isolated from 12 CRCs and their corresponding normal mucosa by acidic extraction were separated by SDS-PAGE and analysed by liquid chromatography-mass spectrometry.

Results

Among 96 modified peptides, 41 distinct PTM sites were identified, of which 7, 13, 11, and 10 were located within the H2A, H2B, H3, and H4 sequences, respectively, and distributed among the amino-terminal tails and the globular domain of the four histones. Modification intensities were quantified for 33 sites, of which 4 showed significant (p-value ≤ 0.05) differences between CRC tissues and healthy mucosa samples. We identified histone H3 lysine 27 acetylation (H3K27Ac) as a modification upregulated in CRC, which had not been shown previously.

Conclusions

The present results indicate the usefulness of a bottom-up proteomic approach for the detection of histone modifications at a global scale. The differential abundance of H3K27Ac mark in CRC, a PTM associated with active enhancers, suggests its role in regulating genes whose expression changes in CRC.     

PhosphOrtholog: a web-based tool for cross-species mapping of orthologous protein post-translational modifications

Background

Most biological processes are influenced by protein post-translational modifications (PTMs). Identifying novel PTM sites in different organisms, including humans and model organisms, has expedited our understanding of key signal transduction mechanisms. However, with increasing availability of deep, quantitative datasets in diverse species, there is a growing need for tools to facilitate cross-species comparison of PTM data. This is particularly important because functionally important modification sites are more likely to be evolutionarily conserved; yet cross-species comparison of PTMs is difficult since they often lie in structurally disordered protein domains. Current tools that address this can only map known PTMs between species based on known orthologous phosphosites, and do not enable the cross-species mapping of newly identified modification sites. Here, we addressed this by developing a web-based software tool, PhosphOrtholog (www.phosphortholog.com) that accurately maps protein modification sites between different species. This facilitates the comparison of datasets derived from multiple species, and should be a valuable tool for the proteomics community.

Results

Here we describe PhosphOrtholog, a web-based application for mapping known and novel orthologous PTM sites from experimental data obtained from different species. PhosphOrtholog is the only generic and automated tool that enables cross-species comparison of large-scale PTM datasets without relying on existing PTM databases. This is achieved through pairwise sequence alignment of orthologous protein residues. To demonstrate its utility we apply it to two sets of human and rat muscle phosphoproteomes generated following insulin and exercise stimulation, respectively, and one publicly available mouse phosphoproteome following cellular stress revealing high mapping and coverage efficiency. Although coverage statistics are dataset dependent, PhosphOrtholog increased the number of cross-species mapped sites in all our example data sets by more than double when compared to those recovered using existing resources such as PhosphoSitePlus.

Conclusions

PhosphOrtholog is the first tool that enables mapping of thousands of novel and known protein phosphorylation sites across species, accessible through an easy-to-use web interface. Identification of conserved PTMs across species from large-scale experimental data increases our knowledgebase of functional PTM sites. Moreover, PhosphOrtholog is generic being applicable to other PTM datasets such as acetylation, ubiquitination and methylation.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1820-x) contains supplementary material, which is available to authorized users.     

Computational reconstruction of proteome-wide protein interaction networks between HTLV retroviruses and Homo sapiens

Background

Human T-cell leukemia viruses (HTLV) tend to induce some fatal human diseases like Adult T-cell Leukemia (ATL) by targeting human T lymphocytes. To indentify the protein-protein interactions (PPI) between HTLV viruses and Homo sapiens is one of the significant approaches to reveal the underlying mechanism of HTLV infection and host defence. At present, as biological experiments are labor-intensive and expensive, the identified part of the HTLV-human PPI networks is rather small. Although recent years have witnessed much progress in computational modeling for reconstructing pathogen-host PPI networks, data scarcity and data unavailability are two major challenges to be effectively addressed. To our knowledge, no computational method for proteome-wide HTLV-human PPI networks reconstruction has been reported.

Results

In this work we develop Multi-instance Adaboost method to conduct homolog knowledge transfer for computationally reconstructing proteome-wide HTLV-human PPI networks. In this method, the homolog knowledge in the form of gene ontology (GO) is treated as auxiliary homolog instance to address the problems of data scarcity and data unavailability, while the potential negative knowledge transfer is automatically attenuated by AdaBoost instance reweighting. The cross validation experiments show that the homolog knowledge transfer in the form of independent homolog instances can effectively enrich the feature information and substitute for the missing GO information. Moreover, the independent tests show that the method can validate 70.3% of the recently curated interactions, significantly exceeding the 2.1% recognition rate by the HT-Y2H experiment. We have used the method to reconstruct the proteome-wide HTLV-human PPI networks and further conducted gene ontology based clustering of the predicted networks for further biomedical research. The gene ontology based clustering analysis of the predictions provides much biological insight into the pathogenesis of HTLV retroviruses.

Conclusions

The Multi-instance AdaBoost method can effectively address the problems of data scarcity and data unavailability for the proteome-wide HTLV-human PPI interaction networks reconstruction. The gene ontology based clustering analysis of the predictions reveals some important signaling pathways and biological modules that HTLV retroviruses are likely to target.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2105-15-245) contains supplementary material, which is available to authorized users.     

SNaPAfu: A Novel Single Nucleotide Polymorphism Multiplex Assay for Aspergillus fumigatus Direct Detection,Identification and Genotyping in Clinical Specimens

Objective

Early diagnosis of invasive aspergillosis is essential for positive patient outcome. Likewise genotyping of fungal isolates is desirable for outbreak control in clinical setting. We designed a molecular assay that combines detection, identification, and genotyping of Aspergillus fumigatus in a single reaction.

Methods

To this aim we combined 20 markers in a multiplex reaction and the results were seen following mini-sequencing readings. Pure culture extracts were firstly tested. Thereafter, Aspergillus-DNA samples obtained from clinical specimens of patients with possible, probable, or proven aspergillosis according to European Organization for the Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG) criteria.

Results

A new set of designed primers allowed multilocus sequence typing (MLST) gene amplification in a single multiplex reaction. The newly proposed SNaPAfu assay had a specificity of 100%, a sensitivity of 89% and detection limit of 1 ITS copy/mL (∼0.5 fg genomic Aspergillus-DNA/mL). The marker A49_F was detected in 89% of clinical samples. The SNaPAfu assay was accurately performed on clinical specimens using only 1% of DNA extract (total volume 50 µL) from 1 mL of used bronchoalveolar lavage.

Conclusions

The first highly sensitive and specific, time- and cost-economic multiplex assay was implemented that allows detection, identification, and genotyping of A. fumigatus strains in a single amplification followed by mini-sequencing reaction. The new test is suitable to clinical routine and will improve patient management.     

Regulation of BMAL1 Protein Stability and Circadian Function by GSK3β-Mediated Phosphorylation

Background

Circadian rhythms govern a large array of physiological and metabolic functions. To achieve plasticity in circadian regulation, proteins constituting the molecular clock machinery undergo various post-translational modifications (PTMs), which influence their activity and intracellular localization. The core clock protein BMAL1 undergoes several PTMs. Here we report that the Akt-GSK3β signaling pathway regulates BMAL1 protein stability and activity.

Principal Findings

GSK3β phosphorylates BMAL1 specifically on Ser 17 and Thr 21 and primes it for ubiquitylation. In the absence of GSK3β-mediated phosphorylation, BMAL1 becomes stabilized and BMAL1 dependent circadian gene expression is dampened. Dopamine D2 receptor mediated signaling, known to control the Akt-GSK3β pathway, influences BMAL1 stability and in vivo circadian gene expression in striatal neurons.

Conclusions

These findings uncover a previously unknown mechanism of circadian clock control. The GSK3β kinase phosphorylates BMAL1, an event that controls the stability of the protein and the amplitude of circadian oscillation. BMAL1 phosphorylation appears to be an important regulatory step in maintaining the robustness of the circadian clock.     

Post-translational Modification of LipL32 during Leptospira interrogans Infection

Background

Leptospirosis, a re-emerging disease of global importance caused by pathogenic Leptospira spp., is considered the world''s most widespread zoonotic disease. Rats serve as asymptomatic carriers of pathogenic Leptospira and are critical for disease spread. In such reservoir hosts, leptospires colonize the kidney, are shed in the urine, persist in fresh water and gain access to a new mammalian host through breaches in the skin.

Methodology/Principal Findings

Previous studies have provided evidence for post-translational modification (PTM) of leptospiral proteins. In the current study, we used proteomic analyses to determine the presence of PTMs on the highly abundant leptospiral protein, LipL32, from rat urine-isolated L. interrogans serovar Copenhageni compared to in vitro-grown organisms. We observed either acetylation or tri-methylation of lysine residues within multiple LipL32 peptides, including peptides corresponding to regions of LipL32 previously identified as epitopes. Intriguingly, the PTMs were unique to the LipL32 peptides originating from in vivo relative to in vitro grown leptospires. The identity of each modified lysine residue was confirmed by fragmentation pattern analysis of the peptide mass spectra. A synthetic peptide containing an identified tri-methylated lysine, which corresponds to a previously identified LipL32 epitope, demonstrated significantly reduced immunoreactivity with serum collected from leptospirosis patients compared to the peptide version lacking the tri-methylation. Further, a subset of the identified PTMs are in close proximity to the established calcium-binding and putative collagen-binding sites that have been identified within LipL32.

Conclusions/Significance

The exclusive detection of PTMs on lysine residues within LipL32 from in vivo-isolated L. interrogans implies that infection-generated modification of leptospiral proteins may have a biologically relevant function during the course of infection. Although definitive determination of the role of these PTMs must await further investigations, the reduced immune recognition of a modified LipL32 epitope suggests the intriguing possibility that LipL32 modification represents a novel mechanism of immune evasion within Leptospira.     

Evaluation of Rapid Blood Sample Collection in the Detection of Circulating Filarial Antigens for Epidemiological Survey by rWbSXP-1 Capture Assay

Background

Lymphatic filariasis is a neglected tropical disease leading to profound disfiguring causing socio economic burden in the tropics. Current diagnosis strategies available during field surveys and epidemics are based on traditional microscopic detections and a few antigen/antibody assays. We have compared different sampling methodologies and standardized the highly sensitive and reliable rWbSXP-1 antigen detection assay to our new sampling methodology.

Methodology

Samples collected as serum, whole blood, whole blood on filter paper and whole blood on microscopic slides from patients belonging to various clinical groups of filariasis [endemic normal(EN), chronic pathology(CP), microfilaraemic(MF) and non-endemic normal(NEN)] were collected and standardized the rWbSXP-1 antigen detection assay using monoclonal antibody raised against rWbSXP-1 protein. The whole blood collected on microscopic slide based sampling method was employed in the field and the presence of circulating filarial antigen (CFA) was assessed using the rWbSXP-1 assay.

Principal Findings

The sampling methods were compared and no significant difference was observed for the detection of CFA (MF, P = 0.304, EN, P = 0.675, CP, P = 0.5698, NEN, P = 0.4494). Further the optimized sampling method was utilized to collect the 1106 samples from Polur, Tiruvannamalai. The rWbSXP-1 assay gave 98 antigen positive results whereas the microscopic method gave only 17.

Conclusions

Four sampling methodologies were analyzed and the new sampling methodology of whole blood collected on microscopic slide was found to be convenient for the detection of CFA using rWbSXP-1 antigen detection assay. The 1106 samples from Polur were collected using the new method. The rWbSXP-1 antigen assay perceived a 7.32% increased result which was read as false negatives on the conventional microscopic staining method. This new sampling methodology coupled with the rWbSXP-1 antigen assay can be used in epidemiological surveys for lymphatic filariasis and the same sampling methodology can be expanded to other antigen based high affinity assays.     

Investigation and identification of functional post-translational modification sites associated with drug binding and protein-protein interactions

Background

Protein post-translational modification (PTM) plays an essential role in various cellular processes that modulates the physical and chemical properties, folding, conformation, stability and activity of proteins, thereby modifying the functions of proteins. The improved throughput of mass spectrometry (MS) or MS/MS technology has not only brought about a surge in proteome-scale studies, but also contributed to a fruitful list of identified PTMs. However, with the increase in the number of identified PTMs, perhaps the more crucial question is what kind of biological mechanisms these PTMs are involved in. This is particularly important in light of the fact that most protein-based pharmaceuticals deliver their therapeutic effects through some form of PTM. Yet, our understanding is still limited with respect to the local effects and frequency of PTM sites near pharmaceutical binding sites and the interfaces of protein-protein interaction (PPI). Understanding PTM’s function is critical to our ability to manipulate the biological mechanisms of protein.

Results

In this study, to understand the regulation of protein functions by PTMs, we mapped 25,835 PTM sites to proteins with available three-dimensional (3D) structural information in the Protein Data Bank (PDB), including 1785 modified PTM sites on the 3D structure. Based on the acquired structural PTM sites, we proposed to use five properties for the structural characterization of PTM substrate sites: the spatial composition of amino acids, residues and side-chain orientations surrounding the PTM substrate sites, as well as the secondary structure, division of acidity and alkaline residues, and solvent-accessible surface area. We further mapped the structural PTM sites to the structures of drug binding and PPI sites, identifying a total of 1917 PTM sites that may affect PPI and 3951 PTM sites associated with drug-target binding. An integrated analytical platform (CruxPTM), with a variety of methods and online molecular docking tools for exploring the structural characteristics of PTMs, is presented. In addition, all tertiary structures of PTM sites on proteins can be visualized using the JSmol program.

Conclusion

Resolving the function of PTM sites is important for understanding the role that proteins play in biological mechanisms. Our work attempted to delineate the structural correlation between PTM sites and PPI or drug-target binding. CurxPTM could help scientists narrow the scope of their PTM research and enhance the efficiency of PTM identification in the face of big proteome data. CruxPTM is now available at http://csb.cse.yzu.edu.tw/CruxPTM/.

     

FIH-1, a Novel Interactor of Mindbomb,Functions as an Essential Anti-Angiogenic Factor during Zebrafish Vascular Development

Objective

It has been shown that Mindbomb (Mib), an E3 Ubiquitin ligase, is an essential modulator of Notch signaling during development. However, its effects on vascular development remain largely unknown.

Approaches and Results

We identified a number of novel proteins that physically interact with Mib, including the Factor Inhibiting Hypoxia Inducible Factor 1 (FIH-1, also known as HIF1AN) from a yeast two hybrid screen, as previously reported. In cultured cells, FIH-1 colocalizes with Mib1, corroborating their potential interaction. In zebrafish embryos, FIH-1 appears to modulate VEGF-A signaling activity; depletion of fih-1 induces ectopic expression of vascular endothelial growth factor–a (vegfa) and leads to exuberant ectopic sprouts from intersegmental vessels (ISVs). Conversely, over-expression of fih-1 substantially attenuates the formation of ISVs, which can be rescued by concurrent over-expression of vegfa, indicating that FIH-1/HIF1AN may fine tune VEGF-A signaling.

Conclusions

Taken together, our data suggest that FIH-1 interacts with Mib E3 Ubiquitin ligase and modulates vascular development by attenuating VEGF-A signaling activity.     

Evaluation of phenolic profile,antioxidant and anticancer potential of two main representants of Zingiberaceae family against B164A5 murine melanoma cells

Background

Curcuma longa Linnaeus and Zingiber officinale Roscoe are two main representatives of Zingiberaceae family studied for a wide range of therapeutic properties, including: antioxidant, anti-inflammatory, anti-angiogenic, antibacterial, analgesic, immunomodulatory, proapoptotic, anti-human immunodeficiency virus properties and anticancer effects. This study was aimed to analyse the ethanolic extracts of Curcuma rhizome (Curcuma longa Linnaeus) and Zingiber rhizome (Zingiber officinale Roscoe) in terms of polyphenols, antioxidant activity and anti-melanoma potential employing the B164A5 murine melanoma cell line.

Results

In order to evaluate the total content of polyphenols we used Folin-Ciocâlteu method. The antioxidant activity of the two ethanolic extracts was determined by DPPH assay, and for the control of antiproliferative effect it was used MTT proliferation assay, DAPI staining and Annexin-FITC-7AAD double staining test. Results showed increased polyphenols amount and antioxidant activity for Curcuma rhizome ethanolic extract. Moreover, 100 μg/ml of ethanolic plant extract from both vegetal products presented in a different manner an antiproliferative, respectively a proapoptotic effect on the selected cell line.

Conclusions

The study concludes that Curcuma rhizome may be a promising natural source for active compounds against malignant melanoma.     

Citation Analysis May Severely Underestimate the Impact of Clinical Research as Compared to Basic Research

Background

Citation analysis has become an important tool for research performance assessment in the medical sciences. However, different areas of medical research may have considerably different citation practices, even within the same medical field. Because of this, it is unclear to what extent citation-based bibliometric indicators allow for valid comparisons between research units active in different areas of medical research.

Methodology

A visualization methodology is introduced that reveals differences in citation practices between medical research areas. The methodology extracts terms from the titles and abstracts of a large collection of publications and uses these terms to visualize the structure of a medical field and to indicate how research areas within this field differ from each other in their average citation impact.

Results

Visualizations are provided for 32 medical fields, defined based on journal subject categories in the Web of Science database. The analysis focuses on three fields: Cardiac & cardiovascular systems, Clinical neurology, and Surgery. In each of these fields, there turn out to be large differences in citation practices between research areas. Low-impact research areas tend to focus on clinical intervention research, while high-impact research areas are often more oriented on basic and diagnostic research.

Conclusions

Popular bibliometric indicators, such as the h-index and the impact factor, do not correct for differences in citation practices between medical fields. These indicators therefore cannot be used to make accurate between-field comparisons. More sophisticated bibliometric indicators do correct for field differences but still fail to take into account within-field heterogeneity in citation practices. As a consequence, the citation impact of clinical intervention research may be substantially underestimated in comparison with basic and diagnostic research.     

Antioxidant and Anti-Inflammatory Activities of Extracts from Cassia alata,Eleusine indica,Eremomastax speciosa,Carica papaya and Polyscias fulva Medicinal Plants Collected in Cameroon

Background

The vast majority of the population around the world has always used medicinal plants as first source of health care to fight infectious and non infectious diseases. Most of these medicinal plants may have scientific evidence to be considered in general practice.

Objective

The aim of this work was to investigate the antioxidant capacities and anti-inflammatory activities of ethanol extracts of leaves of Cassia alata, Eleusine indica, Carica papaya, Eremomastax speciosa and the stem bark of Polyscias fulva, collected in Cameroon.

Methods

Chemiluminescence was used to analyze the antioxidant activities of plant extracts against hydrogen peroxide or superoxide anion. Comet assays were used to analyze the protection against antioxidant-induced DNA damage induced in white blood cells after treating with hydrogen peroxide. Flow cytometry was used to measure γδ T cells proliferation and anti-inflammatory activity of γδ T cells and of immature dendritic cells (imDC) in the presence of different concentrations of plant extracts.

Results

Ethanol extracts showed strong antioxidant properties against both hydrogen peroxide and superoxide anion. Cassia alata showed the highest antioxidant activity. The effect of plant extracts on γδ T cells and imDC was evidenced by the dose dependent reduction in TNF-α production in the presence of Cassia alata, Carica papaya, Eremomastax speciosa Eleusine indica, and Polyscias fulva. γδ T cells proliferation was affected to the greatest extent by Polyscias fulva.

Conclusion

These results clearly show the antioxidant capacity and anti-inflammatory activities of plant extracts collected in Cameroon. These properties of leaves and stem bark extracts may contribute to the value for these plants in traditional medicine and in general medical practice.     

Methanol extract from Vietnamese Caesalpinia sappan induces apoptosis in HeLa cells

Background

This study evaluated the cytotoxic activity of extracts from Caesalpinia sappan heartwood against multiple cancer cell lines using an MTT cell viability assay. The cell death though induction of apoptosis was as indicated by DNA fragmentation and caspase-3 enzyme activation.

Results

A methanol extract from C. sappan (MECS) showed cytotoxic activity against several of the cancer cell lines. The most potent activity exhibited by the MECS was against HeLa cells with an IC₅₀ value of 26.5 ± 3.2 μg/mL. Treatment of HeLa cells with various MECS concentrations resulted in growth inhibition and induction of apoptosis, as indicated by DNA fragmentation and caspase-3 enzyme activation.

Conclusion

This study is the first report of the anticancer properties of the heartwood of C. sappan native to Vietnam. Our findings demonstrate that C. sappan heartwood may have beneficial applications in the field of anticancer drug discovery.     

Structural Analysis of PTM Hotspots (SAPH-ire) – A Quantitative Informatics Method Enabling the Discovery of Novel Regulatory Elements in Protein Families

Predicting the biological function potential of post-translational modifications (PTMs) is becoming increasingly important in light of the exponential increase in available PTM data from high-throughput proteomics. We developed structural analysis of PTM hotspots (SAPH-ire)—a quantitative PTM ranking method that integrates experimental PTM observations, sequence conservation, protein structure, and interaction data to allow rank order comparisons within or between protein families. Here, we applied SAPH-ire to the study of PTMs in diverse G protein families, a conserved and ubiquitous class of proteins essential for maintenance of intracellular structure (tubulins) and signal transduction (large and small Ras-like G proteins). A total of 1728 experimentally verified PTMs from eight unique G protein families were clustered into 451 unique hotspots, 51 of which have a known and cited biological function or response. Using customized software, the hotspots were analyzed in the context of 598 unique protein structures. By comparing distributions of hotspots with known versus unknown function, we show that SAPH-ire analysis is predictive for PTM biological function. Notably, SAPH-ire revealed high-ranking hotspots for which a functional impact has not yet been determined, including phosphorylation hotspots in the N-terminal tails of G protein gamma subunits—conserved protein structures never before reported as regulators of G protein coupled receptor signaling. To validate this prediction we used the yeast model system for G protein coupled receptor signaling, revealing that gamma subunit–N-terminal tail phosphorylation is activated in response to G protein coupled receptor stimulation and regulates protein stability in vivo. These results demonstrate the utility of integrating protein structural and sequence features into PTM prioritization schemes that can improve the analysis and functional power of modification-specific proteomics data.Post-translational modifications (PTMs)¹ are a rapidly expanding and important class of protein feature that broaden the functional diversity of proteins in a proteome. By definition, PTMs change protein structure and therefore have the potential to affect protein function by altering protein interactions, protein stability or catalytic activity (1, 2). As they have been found to occur on nearly every protein in the eukaryotic proteome, PTMs broadly impact nearly all known cellular processes. Over 300 different types of PTM are known, ranging from single atom modifications (e.g. oxide) to small protein modifiers (e.g. ubiquitin), which can occur on all but five amino acid residues resulting from enzymatic or nonenzymatic processes (3). Over 220,000 distinct PTM sites have been experimentally identified across ∼77,000 different proteins to date (dbPTM; http://dbptm.mbc.nctu.edu.tw/statistics.php) – numbers that continue to grow exponentially because of improved methods for high throughput detection by mass spectrometry (MS). By virtue of how they are detected, most PTM data are sequence-linked and lack structural context.The function of most PTMs is unknown because the rate of PTM detection far surpasses the rate at which any one modification can be studied empirically. Moreover, the functional impact of every PTM is likely not equivalent (4). For example, computational analysis of phosphorylation sites in yeast and human proteomes indicate that well-conserved phosphosites are more likely to have a functional consequence compared with poorly conserved sites, yet only a fraction of phosphosites are well conserved (5, 6). Consequently, the development of tools that provide functional prioritization of PTMs could have a broad impact on our understanding of protein regulation, biological mechanism, and molecular evolution.The emerging need for methods that predict the functional impact of a PTM has not yet been met. Longstanding methods capitalize predominantly on the sequence context of PTMs and have been used to predict sites of modification (expasy.org/proteomics/post-translational_modification) and to compare enzyme/substrate interactions (7–9). More recently, studies aimed at expanding the parameters associated with functional PTMs have emerged. In these cases, a set of common features correlated with functional importance are derived from the analysis of PTMs within and between organisms including: number of PTM observations at a multiple sequence alignment position (i.e. hotspots), measures of co-occurrence between different PTMs (e.g. distance between phosphorylation and ubiquitination sites), biological dynamics (up or down-regulation), and protein–protein interaction influence (7, 10–12). Recent efforts to provide structural context by linking individual PTMs to three-dimensional structures in the protein data bank (PDB) have also been described (13, 14). However, these resources are extensions of existing PTM databases that allow visualization of single instances of modification onto individual proteins, but do not provide quantitative or analytical value.In principle, combining PTM hotspot and structural analysis would offer multiple advantages over any one approach used in isolation. Sequence homology provides protein family membership—thereby clustering PTMs into hotspots for groups of proteins to provide information about: (1) the evolutionary conservation and (2) observation frequencies of PTMs within the family. A primary consequence of their sequence homology is that members of a protein family will exhibit similar structures and protein interactions—features that dictate the function of protein systems. A secondary consequence is that PTM hotspots generated by alignment can be projected onto family-representative protein structures, which places each PTM hotspot into a three-dimensional context that can be visualized for each family. The structural context enabled by this projection can also provide spatial information about the PTM site that can supplement the sequence characteristics of the hotspot, namely: (3) solvent accessibility, which provides an estimate of whether a modification could occur on the folded protein; and (4) protein interface residence, which indicates the potential of the PTM to disrupt protein–protein interactions. Despite the theoretical advantages, no single tool has been developed that exploits the quantitative output from both sequence and structural data to evaluate the function potential of PTMs.Here we describe a new analytical method – Structural Analysis of PTM Hotspots (SAPH-ire), which ranks PTM hotspots by their potential to impact biological function for distinct protein families (Fig. 1). We demonstrate the application of SAPH-ire to the complete set of PTMs for eight distinct protein families including large heterotrimeric G proteins—revealing high-ranking hotspots for which a biological function has not yet been determined. In particular, SAPH-ire revealed the N-terminal tail (Nt) of G protein gamma (Gγ) subunits as one of the highest ranking PTM hotspots for heterotrimeric G proteins (Gα, Gβ, and Gγ). We tested this prediction by monitoring the phosphorylation state and mutation effects of phosphorylation sites in the N terminus of the yeast Gγ subunit (Ste18). Consistent with SAPH-ire predictions, we found that phosphorylation of Ste18-Nt is biologically responsive to a GPCR stimulus and that phospho-null or phospho-mimic mutation of these sites controls protein abundance in an opposite manner in vivo. Thus, SAPH-ire is a powerful new method for predicting the function potential of PTM hotspots, which can guide empirical research toward the discovery of new protein regulatory elements based on high-throughput proteomics.Open in a separate windowFig. 1.Schematic diagram of the SAPH-ire method. A, SAPH-ire integrates InterPro, the Protein Data bank (PDB) and a customized database of experimentally validated PTMs. Uniprot entries with PTMs that belong to specific InterPro-classified protein families undergo multiple-sequence alignment (MSA) and PTM hotspot analysis (HSA), which layers all PTMs for a given alignment position in the MSA. The total PTMs observed in each hotspot and the conservation of a modifiable residue (e.g. conservation lysine at a ubiquitination hotspot) at the hotspot are quantified. B, PTM hotspots within the protein family are then projected onto all known crystal structures for the family using the Structural Projection of PTMs (SPoP) tool. From the structural topology of PTM hotspots generated by SPoP, the solvent accessible surface area (SASA) and protein interface residence is quantified for each hotspot. C, PTM Function Potential Calculator (FPC) integrates the output from HSA and SPoP, resulting in PTM function potential scores for each hotspot. The function potential score can be used to rank PTM hotspots within or between protein families – prioritizing hotspots with the greatest potential to be biologically regulated and/or effect a biological function for the protein family of interest.     

Impulsive Action but Not Impulsive Choice Determines Problem Gambling Severity

Background

Impulsivity is a hallmark of problem gambling. However, impulsivity is not a unitary construct and this study investigated the relationship between problem gambling severity and two facets of impulsivity: impulsive action (impaired ability to withhold a motor response) and impulsive choice (abnormal aversion for the delay of reward).

Methods

The recruitment includes 65 problem gamblers and 35 normal control participants. On the basis of DSM-IV-TR criteria, two groups of gamblers were distinguished: problem gamblers (n = 38) and pathological gamblers (n = 27) with similar durations of gambling practice. Impulsive action was assessed using a response inhibition task (the stop-signal task). Impulsive choice was estimated with the delay-discounting task. Possible confounds (e.g., IQ, mood, ADHD symptoms) were recorded.

Results

Both problem and pathological gamblers discounted reward at a higher rate than their controls, but only pathological gamblers showed abnormally low performance on the most demanding condition of the stop-signal task. None of the potential confounds covaried with these results.

Conclusions

These results suggest that, whereas abnormal impulsive choice characterizes all problem gamblers, pathological gamblers'' impairments in impulsive action may represent an important developmental pathway of pathological gambling.     

Triterpenoids Amplify Anti-Tumoral Effects of Mistletoe Extracts on Murine B16.F10 Melanoma In Vivo

Purpose

Mistletoe extracts are often used in complementary cancer therapy although the efficacy of that therapy is controversially discussed. Approved mistletoe extracts contain mainly water soluble compounds of the mistletoe plant, i.e. mistletoe lectins. However, mistletoe also contains water-insoluble triterpenoids (mainly oleanolic acid) that have anti-tumorigenic effects. To overcome their loss in watery extracts we have solubilized mistletoe triterpenoids with cyclodextrins, thus making them available for in vivo cancer experiments.

Experimental design

B16.F10 subcutaneous melanoma bearing C57BL/6 mice were treated with new mistletoe extracts containing both water soluble compounds and solubilized triterpenoids. Tumor growth and survival was monitored. In addition, histological examinations of the tumor material and tumor surrounding tissue were performed.

Results

Addition of solubilized triterpenoids increased the anti-tumor effects of the mistletoe extracts, resulting in reduced tumor growth and prolonged survival of the mice. Histological examination of the treated tumors showed mainly tumor necrosis and some apoptotic cells with active caspase-3 and TUNEL staining. A significant decrease of CD31-positive tumor blood vessels was observed after treatment with solubilized triterpenoids and different mistletoe extracts.

Conclusion

We conclude that the addition of solubilized mistletoe triterpenoids to conventional mistletoe extracts improves the efficacy of mistletoe treatment and may represent a novel treatment option for malignant melanoma.     

A rapid culture independent methodology to quantitatively detect and identify common human bacterial pathogens associated with contaminated high purity water

Background

Water and High Purity Water (HPW) distribution systems can be contaminated with human pathogenic microorganisms. This biocontamination may pose a risk to human health as HPW is commonly used in the industrial, pharmaceutical and clinical sectors. Currently, routine microbiological testing of HPW is performed using slow and labour intensive traditional microbiological based techniques. There is a need to develop a rapid culture independent methodology to quantitatively detect and identify biocontamination associated with HPW.

Results

A novel internally controlled 5-plex real-time PCR Nucleic Acid Diagnostics assay (NAD), was designed and optimised in accordance with Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines, to rapidly detect, identify and quantify the human pathogenic bacteria Stenotrophomonas maltophilia, Burkholderia species, Pseudomonas aeruginosa and Serratia marcescens which are commonly associated with the biocontamination of water and water distribution systems. The specificity of the 5-plex assay was tested against genomic DNA isolated from a panel of 95 microorganisms with no cross reactivity observed. The analytical sensitivities of the S. maltophilia, B. cepacia, P. aeruginosa and the S. marcescens assays are 8.5, 5.7, 3.2 and 7.4 genome equivalents respectively.Subsequently, an analysis of HPW supplied by a Millipore Elix 35 water purification unit performed using standard microbiological methods revealed high levels of naturally occurring microbiological contamination. Five litre water samples from this HPW delivery system were also filtered and genomic DNA was purified directly from these filters. These DNA samples were then tested using the developed multiplex real-time PCR NAD assay and despite the high background microbiological contamination observed, both S. maltophilia and Burkholderia species were quantitatively detected and identified. At both sampling points the levels of both S. maltophilia and Burkholderia species present was above the threshold of 10 cfu/100 ml recommended by both EU and US guidelines.

Conclusions

The novel culture independent methodology described in this study allows for rapid (<5 h), quantitative detection and identification of these four human pathogens from biocontaminated water and HPW distribution systems. We propose that the described NAD assay and associated methodology could be applied to routine testing of water and HPW distribution systems to assure microbiological safety and high water quality standards.

Electronic supplementary material

The online version of this article (doi:10.1186/s12896-015-0124-1) contains supplementary material, which is available to authorized users.