Reporting Recommendations for Tumor Marker Prognostic Studies (REMARK): An Abridged Explanation and Elaboration.

The Reporting Recommendations for Tumor Marker Prognostic Studies (REMARK) were developed to address widespread deficiencies in the reporting of such studies. The REMARK checklist consists of 20 items to report for published tumor marker prognostic studies. A detailed paper was published explaining the rationale behind checklist items, providing positive examples and giving empirical evidence of the quality of reporting. REMARK provides a comprehensive overview to educate on good reporting and provide a valuable reference for the many issues to consider when designing, conducting, and analyzing tumor marker studies and prognostic studies in medicine in general. Despite support for REMARK from major cancer journals, prognostic factor research studies remain poorly reported. To encourage dissemination and uptake of REMARK, we have produced this considerably abridged version of the detailed explanatory manuscript, which may also serve as a brief guide to key issues for investigators planning tumor marker prognostic studies. To summarize the current situation, more recent papers investigating the quality of reporting and related reporting guidelines are cited, but otherwise the literature is not updated. Another important impetus for this paper is that it serves as a basis for literal translations into other languages. Translations will help to bring key information to a larger audience world-wide. Many more details can be found in the original paper.

Criteria for the use of omics-based predictors in clinical trials: explanation and elaboration.

High-throughput 'omics' technologies that generate molecular profiles for biospecimens have been extensively used in preclinical studies to reveal molecular subtypes and elucidate the biological mechanisms of disease, and in retrospective studies on clinical specimens to develop mathematical models to predict clinical endpoints. Nevertheless, the translation of these technologies into clinical tests that are useful for guiding management decisions for patients has been relatively slow. It can be difficult to determine when the body of evidence for an omics-based test is sufficiently comprehensive and reliable to support claims that it is ready for clinical use, or even that it is ready for definitive evaluation in a clinical trial in which it may be used to direct patient therapy. Reasons for this difficulty include the exploratory and retrospective nature of many of these studies, the complexity of these assays and their application to clinical specimens, and the many potential pitfalls inherent in the development of mathematical predictor models from the very high-dimensional data generated by these omics technologies. Here we present a checklist of criteria to consider when evaluating the body of evidence supporting the clinical use of a predictor to guide patient therapy. Included are issues pertaining to specimen and assay requirements, the soundness of the process for developing predictor models, expectations regarding clinical study design and conduct, and attention to regulatory, ethical, and legal issues. The proposed checklist should serve as a useful guide to investigators preparing proposals for studies involving the use of omics-based tests. The US National Cancer Institute plans to refer to these guidelines for review of proposals for studies involving omics tests, and it is hoped that other sponsors will adopt the checklist as well.

Criteria for the use of omics-based predictors in clinical trials.

The US National Cancer Institute (NCI), in collaboration with scientists representing multiple areas of expertise relevant to 'omics'-based test development, has developed a checklist of criteria that can be used to determine the readiness of omics-based tests for guiding patient care in clinical trials. The checklist criteria cover issues relating to specimens, assays, mathematical modelling, clinical trial design, and ethical, legal and regulatory aspects. Funding bodies and journals are encouraged to consider the checklist, which they may find useful for assessing study quality and evidence strength. The checklist will be used to evaluate proposals for NCI-sponsored clinical trials in which omics tests will be used to guide therapy.

Modifications to the C-terminus of Arf1 alter cell functions and protein interactions.

Arf family proteins are approximately 21-kDa GTP-binding proteins that are critical regulators of membrane traffic and the actin cytoskeleton. Studies examining the complex signaling pathways underlying Arf action have relied on recombinant proteins comprised of Arf fused to epitope tags or proteins, such as glutathione S-transferase or green fluorescent protein, for both cell-based mammalian cell studies and bacterially expressed recombinant proteins for biochemical assays. However, the effects of such protein fusions on the biochemical properties relevant to the cellular function have been only incompletely studied at best. Here, we have characterized the effect of C-terminal tagging of Arf1 on (i) function in Saccharomyces cerevisiae, (ii) in vitro nucleotide exchange and (iii) interaction with guanine nucleotide exchange factors and GTPase-activating proteins. We found that the tagged Arfs were substantially impaired or altered in each assay, compared with the wild-type protein, and these changes are certain to alter actions in cells. We discuss the results related to the interpretation of experiments using these reagents and we propose that authors and editors consistently adopt a few simple rules for describing and discussing results obtained with Arf family members that can be readily applied to other proteins.

The G-protein coupling properties of the human sweet and amino acid taste receptors.

The human T1R taste receptors are family C G-protein-coupled receptors (GPCRs) that act as heterodimers to mediate sweet (hT1R2 + hT1R3) and umami (hT1R1 + hT1R3) taste modalities. Each T1R has a large extracellular ligand-binding domain linked to a seven transmembrane-spanning core domain (7TMD). We demonstrate that the 7TMDs of hT1R1 and hT1R2 display robust ligand-independent constitutive activity, efficiently catalyzing the exchange of GDP for GTP on Galpha subunits. In contrast, relative to the 7TMDs of hT1R1 and hT1R2, the 7TMD of hT1R3 couples poorly to G-proteins, suggesting that in vivo signaling may proceed primarily through hT1R1 and hT1R2. In addition, we provide direct evidence that the hT1Rs selectively signal through Galpha(i/o) pathways, coupling to multiple Galpha(i/o) subunits as well as the taste cell specific Gbeta(1)gamma(13) dimer.

Functional characterization of human bitter taste receptors.

The T2Rs belong to a multi-gene family of G-protein-coupled receptors responsible for the detection of ingested bitter-tasting compounds. The T2Rs are conserved among mammals with the human and mouse gene families consisting of about 25 members. In the present study we address the signalling properties of human and mouse T2Rs using an in vitro reconstitution system in which both the ligands and G-proteins being assayed can be manipulated independently and quantitatively assessed. We confirm that the mT2R5, hT2R43 and hT2R47 receptors respond selectively to micromolar concentrations of cycloheximide, aristolochic acid and denatonium respectively. We also demonstrate that hT2R14 is a receptor for aristolochic acid and report the first characterization of the ligand specificities of hT2R7, which is a broadly tuned receptor responding to strychnine, quinacrine, chloroquine and papaverine. Using these defined ligand-receptor interactions, we assayed the ability of the ligand-activated T2Rs to catalyse GTP binding on divergent members of the G(alpha) family including three members of the G(alphai) subfamily (transducin, G(alphai1) and G(alphao)) as well as G(alphas) and G(alphaq). The T2Rs coupled with each of the three G(alphai) members tested. However, none of the T2Rs coupled to either G(alphas) or G(alphaq), suggesting the T2Rs signal primarily through G(alphai)-mediated signal transduction pathways. Furthermore, we observed different G-protein selectivities among the T2Rs with respect to both G(alphai) subunits and G(betagamma) dimers, suggesting that bitter taste is transduced by multiple G-proteins that may differ among the T2Rs.

Two members of the TRPP family of ion channels, Pkd1l3 and Pkd2l1, are co-expressed in a subset of taste receptor cells.

Taste receptors cells are responsible for detecting a wide variety of chemical stimuli. Several molecules including both G protein coupled receptors and ion channels have been shown to be involved in the detection and transduction of tastants. We report on the expression of two members of the transient receptor potential (TRP) family of ion channels, PKD1L3 and PKD2L1, in taste receptor cells. Both of these channels belong to the larger polycystic kidney disease (PKD or TRPP) subfamily of TRP channels, members of which have been demonstrated to be non-selective cation channels and permeable to both Na(+) and Ca(2+). Pkd1l3 and Pkd2l1 are co-expressed in a select subset of taste receptor cells and therefore may, like other PKD channels, function as a heteromer. We found the taste receptor cells expressing Pkd1l3 and Pkd2l1 to be distinct from those that express components of sweet, bitter and umami signal transduction pathways. These results provide the first evidence for a role of TRPP channels in taste receptor cell function.

Two novel genes, Gpr113, which encodes a family 2 G-protein-coupled receptor, and Trcg1, are selectively expressed in taste receptor cells.

To identify genes important for taste receptor cell function, we analyzed the sequences and expression patterns of clones isolated from a mouse taste receptor cell-enriched cDNA library. Here, we report the analyses of two novel genes, Gpr113 and Trcg1. Gpr113 encodes a G-protein-coupled receptor belonging to family 2B, members of which are characterized by having long N-terminal, extracellular domains. The predicted N-terminal extracellular domain of GPR113 contains 696 amino acids with two functional domains, a peptide hormone-binding domain and a G-protein-coupled receptor proteolytic site. Expression analyses indicate that Gpr113 expression is highly restricted to a subset of taste receptor cells. TRCG1 is also selectively expressed in a subset of taste receptor cells. Trcg1 is alternatively spliced and encodes Trcg1 isoforms of 209 and 825 amino acids. BLAST searches of genomic sequences indicate that a putative homolog of Trcg1 resides on human chromosome 15q22.