The Most Severe Test for Hydrophobicity Scales: Two Proteins with 88% Sequence Identity but Different Structure and Function

Protein-protein interactions (protein functionalities) are mediated by water, which compacts individual proteins and promotes close and temporarily stable large-area protein-protein interfaces. In their classic paper Kyte and Doolittle (KD) concluded that the "simplicity and graphic nature of hydrophobicity scales make them very useful tools for the evaluation of protein structures". In practice, however, attempts to develop hydrophobicity scales (for example, compatible with classical force fields (CFF) in calculating the energetics of protein folding) have encountered many difficulties. Here we suggest an entirely different approach, based on the idea that proteins are self-organized networks, subject to finite-scale criticality (like some network glasses). We test this proposal against two small proteins that are delicately balanced between alpha and alpha/beta structures, with different functions encoded with only 12% of their amino acids. This example explains why protein structure prediction is so challenging, and it provides a severe test for the accuracy and content of hydrophobicity scales. The new method confirms KD's evaluation, and at the same time suggests that protein structure, dynamics and function can be best discussed without using CFF

Hydrophobic gating of mechanosensitive channel of large conductance evidenced by single-subunit resolution

Mechanosensitive (MS) ion channels are membrane proteins that detect and respond to membrane tension in all branches of life. In bacteria, MS channels prevent cells from lysing upon sudden hypoosmotic shock by opening and releasing solutes and water. Despite the importance of MS channels and ongoing efforts to explain their functioning, the molecular mechanism of MS channel gating remains elusive and controversial. Here we report a method that allows single-subunit resolution for manipulating and monitoring “mechanosensitive channel of large conductance” from Escherichia coli. We gradually changed the hydrophobicity of the pore constriction in this homopentameric protein by modifying a critical pore residue one subunit at a time. Our experimental results suggest that both channel opening and closing are initiated by the transmembrane 1 helix of a single subunit and that the participation of each of the five identical subunits in the structural transitions between the closed and open states is asymmetrical. Such a minimal change in the pore environment seems ideal for a fast and energy-efficient response to changes in the membrane tension.

A Small Conductance Calcium-Activated K⁺ Channel in C. elegans, KCNL-2, Plays a Role in the Regulation of the Rate of Egg-Laying

In the nervous system of mice, small conductance calcium-activated potassium (SK) channels function to regulate neuronal excitability through the generation of a component of the medium afterhyperpolarization that follows action potentials. In humans, irregular action potential firing frequency underlies diseases such as ataxia, epilepsy, schizophrenia and Parkinson's disease. Due to the complexity of studying protein function in the mammalian nervous system, we sought to characterize an SK channel homologue, KCNL-2, in C. elegans, a genetically tractable system in which the lineage of individual neurons was mapped from their early developmental stages. Sequence analysis of the KCNL-2 protein reveals that the six transmembrane domains, the potassium-selective pore and the calmodulin binding domain are highly conserved with the mammalian homologues. We used widefield and confocal fluorescent imaging to show that a fusion construct of KCNL-2 with GFP in transgenic lines is expressed in the nervous system of C. elegans. We also show that a KCNL-2 null strain, kcnl-2(tm1885), demonstrates a mild egg-laying defective phenotype, a phenotype that is rescued in a KCNL-2-dependent manner. Conversely, we show that transgenic lines that overexpress KCNL-2 demonstrate a hyperactive egg-laying phenotype. In this study, we show that the vulva of transgenic hermaphrodites is highly innervated by neuronal processes and by the VC4 and VC5 neurons that express GFP-tagged KCNL-2. We propose that KCNL-2 functions in the nervous system of C. elegans to regulate the rate of egg-laying. © 2013 Chotoo et al

Systematic evaluation of patient-reported outcome (PRO) protocol content and reporting in UK cancer clinical trials: the EPiC study protocol.

Emerging evidence suggests that patient-reported outcome (PRO)-specific information may be omitted in trial protocols and that PRO results are poorly reported, limiting the use of PRO data to inform cancer care. This study aims to evaluate the standards of PRO-specific content in UK cancer trial protocols and their arising publications and to highlight examples of best-practice PRO protocol content and reporting where they occur. The objective of this study is to determine if these early findings are generalisable to UK cancer trials, and if so, how best we can bring about future improvements in clinical trials methodology to enhance the way PROs are assessed, managed and reported.Trials in which the primary end point is based on a PRO will have more complete PRO protocol and publication components than trials in which PROs are secondary end points.Completed National Institute for Health Research (NIHR) Portfolio Cancer clinical trials (all cancer specialities/age-groups) will be included if they contain a primary/secondary PRO end point. The NIHR portfolio includes cancer trials, supported by a range of funders, adjudged as high-quality clinical research studies. The sample will be drawn from studies completed between 31 December 2000 and 1 March 2014 (n=1141) to allow sufficient time for completion of the final trial report and publication. Two reviewers will then review the protocols and arising publications of included trials to: (1) determine the completeness of their PRO-specific protocol content; (2) determine the proportion and completeness of PRO reporting in UK Cancer trials and (3) model factors associated with PRO protocol and reporting completeness and with PRO reporting proportion.The study was approved by the ethics committee at University of Birmingham (ERN_15-0311). Trial findings will be disseminated via presentations at local, national and international conferences, peer-reviewed journals and social media including the CPROR twitter account and UOB departmental website (http://www.birmingham.ac.uk/cpro0r)

International perspectives on suboptimal patient-reported outcome trial design and reporting in cancer clinical trials: a qualitative study

Purpose: Evidence suggests that the patient-reported outcome (PRO) content of cancer trial protocols is frequently inadequate and non-reporting of PRO findings is widespread. This qualitative study examined the factors influencing suboptimal PRO protocol content, implementation, and reporting, and use of PRO data during clinical interactions. Methods: Semi-structured interviews were conducted with four stakeholder groups: (1) trialists and chief investigators; (2) people with lived experience of cancer; (3) international experts in PRO cancer trial design; (4) journal editors, funding panelists, and regulatory agencies. Data were analyzed using directed thematic analysis with an iterative coding frame. Results: Forty-four interviews were undertaken. Several factors were identified that could influenced effective integration of PROs into trials and subsequent findings. Participants described (1) late inclusion of PROs in trial design; (2) PROs being considered a lower priority outcome compared to survival; (3) trialists’ reluctance to collect or report PROs due to participant burden, missing data, and perceived reticence of journals to publish; (4) lack of staff training. Strategies to address these included training research personnel and improved communication with site staff and patients regarding the value of PROs. Examples of good practice were identified. Conclusion: Misconceptions relating to PRO methodology and its use may undermine their planning, collection, and reporting. There is a role for funding, regulatory, methodological, and journalistic institutions to address perceptions around the value of PROs, their position within the trial outcomes hierarchy, that PRO training and guidance is available, signposted, and readily accessible, with accompanying measures to ensure compliance with international best practice guidelines

Immunotherapy with allotumour mRNA-transfected dendritic cells in androgen-resistant prostate cancer patients

Here, we present results from a clinical trial employing a new vaccination method using dendritic cells (DCs) transfected with mRNA from allogeneic prostate cancer cell lines (DU145, LNCaP and PC-3). In all, 20 patients were enrolled and 19 have completed vaccination. Each patient received at least four weekly injections with 2 × 107 transfected DCs either intranodally or intradermally. Safety and feasibility of vaccination were determined. Immune responses were measured as delayed-type hypersensitivity and by in vitro immunoassays including ELISPOT and T-cell proliferation in pre- and postvaccination peripheral blood samples. Serum prostate-specific antigen (PSA) levels and bone scans were monitored. No toxicity or serious adverse events related to vaccinations were observed. A total of 12 patients developed a specific immune response to tumour mRNA-transfected DCs. In total, 13 patients showed a decrease in log slope PSA. This effect was strengthened by booster vaccinations. Clinical outcome was significantly related to immune responses (n=19, P=0.002, r=0.68). Vaccination with mRNA-transfected DCs is safe and results in cellular immune responses specific for antigens encoded by mRNA derived from the prostate cancer cell lines. The observation that in some patients vaccination affected the PSA level suggests that this approach may become useful as a treatment modality for prostate cancer patients

Representative transcript sets for evaluating a translational initiation sites predictor

<p>Abstract</p> <p>Background</p> <p>Translational initiation site (TIS) prediction is a very important and actively studied topic in bioinformatics. In order to complete a comparative analysis, it is desirable to have several benchmark data sets which can be used to test the effectiveness of different algorithms. An ideal benchmark data set should be reliable, representative and readily available. Preferably, proteins encoded by members of the data set should also be representative of the protein population actually expressed in cellular specimens.</p> <p>Results</p> <p>In this paper, we report a general algorithm for constructing a reliable sequence collection that only includes mRNA sequences whose corresponding protein products present an average profile of the general protein population of a given organism, with respect to three major structural parameters. Four representative transcript collections, each derived from a model organism, have been obtained following the algorithm we propose. Evaluation of these data sets shows that they are reasonable representations of the spectrum of proteins obtained from cellular proteomic studies. Six state-of-the-art predictors have been used to test the usefulness of the construction algorithm that we proposed. Comparative study which reports the predictors' performance on our data set as well as three other existing benchmark collections has demonstrated the actual merits of our data sets as benchmark testing collections.</p> <p>Conclusion</p> <p>The proposed data set construction algorithm has demonstrated its property of being a general and widely applicable scheme. Our comparison with published proteomic studies has shown that the expression of our data set of transcripts generates a polypeptide population that is representative of that obtained from evaluation of biological specimens. Our data set thus represents "real world" transcripts that will allow more accurate evaluation of algorithms dedicated to identification of TISs, as well as other translational regulatory motifs within mRNA sequences. The algorithm proposed by us aims at compiling a redundancy-free data set by removing redundant copies of homologous proteins. The existence of such data sets may be useful for conducting statistical analyses of protein sequence-structure relations. At the current stage, our approach's focus is to obtain an "average" protein data set for any particular organism without posing much selection bias. However, with the three major protein structural parameters deeply integrated into the scheme, it would be a trivial task to extend the current method for obtaining a more selective protein data set, which may facilitate the study of some particular protein structure.</p

Interpol: An R package for preprocessing of protein sequences

<p>Abstract</p> <p>Background</p> <p>Most machine learning techniques currently applied in the literature need a fixed dimensionality of input data. However, this requirement is frequently violated by real input data, such as DNA and protein sequences, that often differ in length due to insertions and deletions. It is also notable that performance in classification and regression is often improved by numerical encoding of amino acids, compared to the commonly used sparse encoding.</p> <p>Results</p> <p>The software "Interpol" encodes amino acid sequences as numerical descriptor vectors using a database of currently 532 descriptors (mainly from AAindex), and normalizes sequences to uniform length with one of five linear or non-linear interpolation algorithms. Interpol is distributed with open source as platform independent R-package. It is typically used for preprocessing of amino acid sequences for classification or regression.</p> <p>Conclusions</p> <p>The functionality of Interpol widens the spectrum of machine learning methods that can be applied to biological sequences, and it will in many cases improve their performance in classification and regression.</p