Mammals normally maintain their core body temperature (CBT) despite changes in environmental temperature. Exceptions to this norm include suspended animation-like states such as hibernation, torpor, and estivation. These states are all characterized by marked decreases in metabolic rate, followed by a loss of homeothermic control in which the animal's CBT approaches that of the environment. We report that hydrogen sulfide can induce a suspended animation-like state in a nonhibernating species, the house mouse (Mus musculus). This state is readily reversible and does not appear to harm the animal. This suggests the possibility of inducing suspended animation-like states for medical applications.

To learn more about holocentric chromosome structure and function, we generated a monoclonal antibody (mAb), 6C4, that recognizes the poleward face of mitotic chromosomes in Caenorhabditis elegans. Early in mitosis, mAb 6C4 stains dots throughout the nucleoplasm. Later in prophase, mAb 6C4 stains structures on opposing faces of chromosomes which orient towards the centrosomes at metaphase. Colocalization with an antibody against a centromeric histone H3–like protein and the MPM-2 antibody, which identifies a kinetochore-associated phosphoepitope present in a variety of organisms, shows that the mAb 6C4 staining is present adjacent to the centromere.Expression screening using mAb 6C4 identified a protein in C. elegans that we named HCP-1 (for holocentric protein 1). We also identified a second protein from the C. elegans genome sequence database, HCP-2, that is 54% similar to HCP-1. When expression of HCP-1 is reduced by RNA interference (RNAi), staining with mAb 6C4 is eliminated, indicating that hcp-1 encodes the major mAb 6C4 antigen. RNAi with hcp-1 and hcp-2 together results in aberrant anaphases and embryonic arrest at ∼100 cells with different amounts of DNA in individual nuclei. These results suggest that HCP-1 is a centromere-associated protein that is involved in the fidelity of chromosome segregation.

The expression of alternatively spliced mRNAs from genes is an ubiquitous phenomenon in metazoa. A screen for trans-acting factors that alter the expression of alternatively spliced mRNAs reveals that the smg genes of Caenorhabditis elegans participate in this process. smg genes have been proposed to function in degradation of nonsense mutant mRNAs. Here we show that smg genes affect normal gene expression by modulating the levels of alternatively spliced SRp20 and SRp30b mRNAs. These SR genes contain alternatively spliced exons that introduce upstream stop codons. The effect of smg genes on SR transcripts is specific, because the gene encoding the catalytic subunit of the cAMPdependent protein kinase, which also contains an alternatively spliced exon that introduces upstream stop codon, is not effected in a smg background. These results suggest that the levels of alternatively spliced mRNAs may, in part, be regulated by alternative mRNA stability.In multicellular organisms many genes express developmentally regulated or tissue-specific alternative messenger RNAs. Through the use of different splice sites, alternative premRNA splicing can dramatically alter the coding capacity of a gene (1). Many proteins have been identified as alternative splicing factors, and cis-acting elements are important for splice site selection (2-4). Though modulation of splicing is required for the synthesis of alternatively spliced mRNAs, differential stability of the resulting mRNAs could determine the level of expression of any particular alternative message.Regulation of mRNA stability is thought to play a critical role in a variety of processes, including development (5), growth and differentiation (6), and cytoskeleton dynamics (7). Untranslated sequences as well as sequences within coding regions are necessary and͞or sufficient for proper mRNA turnover (8-12). Trans-acting factors have been identified that positively or negatively regulate mRNA degradation (8, 13). However, genetic description of trans-acting factors involved in metazoan mRNA turnover is limited to the smg genes of Caenorhabditis elegans. The smg mutants (suppressor with morphogenetic effects on genitalia) exhibit allele-specific suppression of certain nonsense mutations (14). Molecular analysis suggested that some unc-54 alleles containing premature stop codons exhibit reduced mRNA expression. This reduced expression could be restored by mutations in any of the six smg genes (15). For this reason it has been proposed that the smg genes participate in the degradation of mutant mRNAs.We present results of a genetic screen designed to identify factors involved in alternative pre-mRNA processing. Using a method analagous to yeast screens for temperature-sensitive pre-mRNA splicing mutants (16, 17), we screened mRNA from temperature-sensitive embryo and early larval lethal worm mutants by Northern blotting with alternative exon probes. Through this analysis we found that smg mutants affect the expression of alternatively spliced mRNAs. These results indica...

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Accurate morphological classification of endometrial hyperplasia is crucial as treatments vary widely between the different categories of hyperplasia and are dependent, in part, on the histological diagnosis. However, previous studies have shown considerable inter-observer variation in the classification of endometrial hyperplasias. The aim of this study was to develop a decision support system (DSS) for the classification of endometrial hyperplasias. The system used a Bayesian belief network to distinguish proliferative endometrium, simple hyperplasia, complex hyperplasia, atypical hyperplasia and grade 1 endometrioid adenocarcinoma. These diagnostic outcomes were held in the decision node. Four morphological features were selected as diagnostic clues used routinely in the discrimination of endometrial hyperplasias. These represented the evidence nodes and were linked to the decision node by conditional probability matrices. The system was designed with a computer user interface (CytoInform) where reference images for a given clue were displayed to assist the pathologist in entering evidence into the network. Reproducibility of diagnostic classification was tested on 50 cases chosen by a gynaecological pathologist. These comprised ten cases each of proliferative endometrium, simple hyperplasia, complex hyperplasia, atypical hyperplasia and grade 1 endometrioid adenocarcinoma. The DSS was tested by two consultant pathologists, two junior pathologists and two medical students. Intra- and inter-observer agreement was calculated following conventional histological examination of the slides on two occasions by the consultants and junior pathologists without the use of the DSS. All six participants then assessed the slides using the expert system on two occasions, enabling inter- and intra-observer agreement to be calculated. Using unaided conventional diagnosis, weighted kappa values for intra-observer agreement ranged from 0.645 to 0.901. Using the DSS, the results for the four pathologists ranged from 0.650 to 0.845. Both consultant pathologists had slightly worse weighted kappa values using the DSS, while both junior pathologists achieved slightly better values using the system. The grading of morphological features and the cumulative probability curve provided a quantitative record of the decision route for each case. This allowed a more precise comparison of individuals and identified why discordant diagnoses were made. Taking the original diagnoses of the consultant gynaecological pathologist as the 'gold standard', there was excellent or moderate to good inter-observer agreement between the 'gold standard' and the results obtained by the four pathologists using the expert system, with weighted kappa values of 0.586-0.872. The two medical students using the expert system achieved weighted kappa values of 0.771 (excellent) and 0.560 (moderate to good) compared to the 'gold standard'. This study illustrates the potential of expert systems in the classification of endometrial hyperplasias.

A large body of evidence has implicated mitochondria in control of cell death, where key apoptotic mechanisms involve change in mitochondrial membrane permeability and depolarisation of mitochondrial membrane potential (Δψm). Assessment of Δψm is traditionally conducted using the lipophilic cation JC-1 on the flow cytometer or by fluorescent microscopy. Here we assess JC-1 aggregation using the novel tool of digital texture analysis to establish mitochondrial phenotypic changes induced by the K+ ionophore, valinomycin in a unique model comprising SW480 and SW620 cell lines. This provides an opportunity to study these phenomena in the context of colorectal cancer. Valinomycin-induced apoptosis was detected using morphology and analysis of DNA content. Cells were treated with valinomycin, images digitally recorded on a calibrated video photometer and subjected to high resolution digital texture analysis. This demonstrated that the HARAM texture features (Mean of the Haralick texture features) were highly valuable in describing the transition of Δψm as the cell undergoes apoptosis. In Conclusion this study illustrates the potential of texture analysis as a novel and additional technique for quantifying JC-1 aggregation and revealing the spectrum of collapse of Δψm during apoptosis.

Background: Graves disease is caused by autoantibodies that target the thyroid-stimulating hormone receptor (TSHR). Anti-TSHR autoantibody measurement is routinely performed to differentiate between Graves disease and other causes of hyperthyroidism. We evaluated the clinical performance of a reference laboratory bioassay [the Thyretain thyroid-stimulating immunoglobulin (TSI) Bioassay by Diagnostic Hybrids] and 2 commercially available immunoassays: the TSI Bridge immunoassay by Siemens and the thyroid-stimulating hormone receptor antibody (TRAb) immunoassay by Roche. We further evaluated the analytical performance of the Siemens TSI and Roche TRAb assays. Methods: We performed method comparisons using 125 patient specimens submitted for TSI testing for clinical purposes. Concordance of patient results was assessed between the 3 methods, and chart review was performed to further evaluate samples that generated discordant results. All 3 methods were also evaluated for potential interference caused by human chorionic gonadotropin (hCG). Results: The Roche and Siemens assays demonstrated acceptable day-today precision, within-run precision, and precision at the clinical decision cutoffs. Despite manufacturer-defined analytical measuring ranges up to 40 IU/L, the Roche and Siemens assays were linear to 20 IU/L and 15 IU/L, respectively. hCG concentrations up to 150 000 IU/L did not interfere with any of the methods evaluated. Moderate agreement between methods was observed when testing patient specimens that generated negative (≤1.3) or weakly positive (1.4-3.8) results by the Thyretain assay. One hundred percent agreement was observed when the Thyretain assay was strongly positive (≥3.9). Conclusions: The 3 commercially available anti-TSHR autoantibody measurement methods demonstrated equivalent performance in patients with untreated Graves disease. Discordant results were observed when testing specimens collected from patients undergoing treatment for Graves disease. In these patients, the Siemens TSI assay more frequently generated results consistent with clinical history, results of other laboratory tests, and imaging studies than the Thyretain Bioassay and Roche TRAb assay. IMPACT STATEMENT Laboratories considering in-house measurement of anti-thyroid-stimulating hormone receptor antibodies will benefit from the information summarized here, as we discuss the performance and practical considerations of 3 commercially available methods.