A Prognostic Model Based on the Log Odds Ratio of Positive Lymph Nodes Predicts Prognosis of Patients with Rectal Cancer.

OBJECTIVE: This study aimed to compare the prognostic value of rectal cancer by comparing different lymph node staging systems, and a nomogram was constructed based on superior lymph node staging. METHODS: Overall, 8700 patients with rectal cancer was obtained from the Surveillance, Epidemiology, and End Results (SEER) database between 2010 and 2015. The area under the curve (AUC), the C index, and the Akaike informativeness criteria (AIC) were used to examine the predict ability of various lymph node staging methods. Prognostic indicators were assessed using univariate and multivariate COX regression, and further correlation nomograms were created after the data were randomly split into training and validation cohorts. To evaluate the effectiveness of the model, the C index, calibration curves, decision curves (DCA), and receiver operating characteristic curve (ROC) were used. We ran Kaplan-Meier survival analyses to look for variations in risk classification. RESULTS: While compared to the N-stage positive lymph node ratio (LNR), the log odds ratio of positive lymph nodes (LODDS) had the highest predictive effectiveness. Multifactorial COX regression analyses were used to create nomograms for overall survival (OS) and cancer-specific survival (CSS). The C indices of OS and CSS for this model were considerably higher than those for TNM staging in the training cohort. The created nomograms demonstrated good efficacy based on ROC, rectification, and decision curves. Kaplan-Meier survival analysis revealed notable variations in patient survival across various patient strata. CONCLUSIONS: Compared to AJCC staging, the LODDS-based nomograms have a more accurate predictive effectiveness in predicting OS and CSS in patients with rectal cancer.

Activated caspase-6 and caspase-6-cleaved fragments of huntingtin specifically colocalize in the nucleus.

Proteolysis of mutant huntingtin is crucial to the development of Huntington disease (HD). Specifically preventing proteolysis at the capase-6 (C6) consensus sequence at amino acid 586 of mutant huntingtin prevents the development of behavioural, motor and neuropathological features in a mouse model of HD. However, the mechanism underlying the selective toxicity of the 586 amino acid cleavage event is currently unknown. We have examined the subcellular localization of different caspase proteolytic fragments of huntingtin using neo-epitope antibodies. Our data suggest that the nucleus is the primary site of htt cleavage at amino acid 586. Endogenously cleaved 586 amino acid fragments are enriched in the nucleus of immortalized striatal cells and primary striatal neurons where they co-localize with active C6. Cell stress induced by staurosporine results in the nuclear translocation and activation of C6 and an increase in 586 amino acid fragments of huntingtin in the nucleus. In comparison, endogenous caspase-2/3-generated huntingtin 552 amino acid fragments localize to the perinuclear region. The different cellular itineraries of endogenously generated caspase products of huntingtin may provide an explanation for the selective toxicity of huntingtin fragments cleaved at amino acid 586.

Caspase cleavage of mutant huntingtin precedes neurodegeneration in Huntington's disease.

Huntington's disease (HD) results from polyglutamine expansion in huntingtin (htt), a protein with several consensus caspase cleavage sites. Despite the identification of htt fragments in the brain, it has not been shown conclusively that htt is cleaved by caspases in vivo. Furthermore, no study has addressed when htt cleavage occurs with respect to the onset of neurodegeneration. Using antibodies that detect only caspase-cleaved htt, we demonstrate that htt is cleaved in vivo specifically at the caspase consensus site at amino acid 552. We detect caspase-cleaved htt in control human brain as well as in HD brains with early grade neuropathology, including one homozygote. Cleaved htt is also seen in wild-type and HD transgenic mouse brains before the onset of neurodegeneration. These results suggest that caspase cleavage of htt may be a normal physiological event. However, in HD, cleavage of mutant htt would release N-terminal fragments with the potential for increased toxicity and accumulation caused by the presence of the expanded polyglutamine tract. Furthermore, htt fragments were detected most abundantly in cortical projection neurons, suggesting that accumulation of expanded htt fragments in these neurons may lead to corticostriatal dysfunction as an early event in the pathogenesis of HD.

Cross-species characterization of the ALS2 gene and analysis of its pattern of expression in development and adulthood.

Mutations in the ALS2 gene, which encodes alsin, cause autosomal recessive juvenile-onset amyotrophic lateral sclerosis (ALS2) and related conditions. Using both a novel monoclonal antibody and LacZ knock-in mice, we demonstrate that alsin is widely expressed in neurons of the CNS, including the cortex, brain stem and motor neurons of the spinal cord. Interestingly, the highest levels of alsin are found in the molecular layer of the cerebellum, a brain region not previously implicated in ALS2. During development, alsin is expressed by day E9.5, but CNS expression does not become predominant until early postnatal life. At the subcellular level, alsin is tightly associated with endosomal membranes and is likely to be part of a large protein complex that may include the actin cytoskeleton. ALS2 is present in primates, rodents, fish and flies, but not in the nematode worm or yeast, and is more highly conserved than expected among mammals. Additionally, the product of a second, widely expressed gene, ALS2 C-terminal like (ALS2CL), may subserve or modulate some of the functions of alsin as an activator of Rab and Rho GTPases.

ABCA1 mRNA and protein distribution patterns predict multiple different roles and levels of regulation.

Mutations in ABCA1 cause the allelic disorders familial hypolipoproteinemia and Tangier Disease. To identify where ABCA1 was likely to have a functional role, we determined the cellular and tissue-specific patterns of murine ABCA1 expression. RT-PCR and Western blot analysis on dissected murine tissues demonstrated broad expression of ABCA1 mRNA and protein in many tissues with prominent protein expression in liver, testis, and adrenal tissue. In situ hybridization and immunohistochemistry experiments demonstrated specific patterns of ABCA1 expression at the cellular level, with hepatocytes, the epithelial lining of the digestive system and bladder, the proximal convoluted tubule of the kidney, and Purkinje and cortical pyramidal neurons containing abundant ABCA1 protein. Significant discordance between relative mRNA and protein expression patterns suggests the possibility of post-transcriptional regulation of ABCA1 expression in selected cells or tissues. We also show that ABCA1 protein levels are up-regulated specifically in the liver after exposure to an atherogenic diet for 7 days, supporting a major role for the liver in dietary modulation of HDL-C levels. Our observations show that ABCA1 is expressed in a pattern consistent with its role in HDL-C metabolism. Additionally, ABCA1 may have important functional roles in other cell types independent of HDL-C regulation.

Truncation mutations in ABCA1 suppress normal upregulation of full-length ABCA1 by 9-cis-retinoic acid and 22-R-hydroxycholesterol.

Mutations in ABCA1 uniformly decrease plasma HDL-cholesterol (HDL-C) and reduce cholesterol efflux, yet different mutations in ABCA1 result in different phenotypic effects in heterozygotes. For example, truncation mutations result in significantly lower HDL-C and apoliprotein A-I (apoA-I) levels in heterozygotes compared with nontruncation mutations, suggesting that truncation mutations may negatively affect the wild-type allele. To specifically test this hypothesis, we examined ABCA1 protein expression in response to 9-cis-retinoic acid (9-cis-RA) and 22-R-hydroxycholesterol (22-R-OH-Chol) in a collection of human fibroblasts representing eight different mutations and observed that truncation mutations blunted the response to oxysterol stimulation and dominantly suppressed induction of the remaining full-length allele to 5-10% of wild-type levels. mRNA levels between truncation and nontruncation mutations were comparable, suggesting that ABCA1 expression was suppressed at the protein level. Dominant negative activity of truncated ABCA1 was recapitulated in an in vitro model using transfected Cos-7 cells. Our results suggest that the severe reduction of HDL-C in patients with truncation mutations may be at least partly explained by dominant negative suppression of expression and activity of the remaining full-length ABCA1 allele. These data suggest that ABCA1 requires a physical association with itself or other molecules for normal function and has important pharmacogenetic implications for individuals with truncation mutations.

Alterations of plasma lipids in mice via adenoviral-mediated hepatic overexpression of human ABCA1.

ATP binding cassette transporter A1 (ABCA1) is a widely expressed lipid transporter essential for the generation of HDL. ABCA1 is particularly abundant in the liver, suggesting that the liver may play a major role in HDL homeostasis. To determine how hepatic ABCA1 affects plasma HDL cholesterol levels, we treated mice with an adenovirus (Ad)-expressing human ABCA1 under the control of the cytomegalovirus promoter. Treated mice showed a dose-dependent increase in hepatic ABCA1 protein, ranging from 1.2-fold to 8.3-fold using doses from 5 x 108 to 1.5 x 109 pfu, with maximal expression observed on Day 3 posttreatment. A selective increase in HDL cholesterol occurred at Day 3 in mice treated with 5 x 108 pfu Ad-ABCA1, but higher doses did not further elevate HDL cholesterol levels. In contrast, total cholesterol, triglycerides, phospholipids, non-HDL cholesterol, and apolipoprotein B levels all increased in a dose-dependent manner, suggesting that excessive overexpression of hepatic ABCA1 in the absence of its normal regulatory sequences altered total lipid homeostasis. At comparable expression levels, bacterial artificial chromosome transgenic mice, which express ABCA1 under the control of its endogenous regulatory sequences, showed a greater and more specific increase in HDL cholesterol than Ad-ABCA1-treated mice. Our results suggest that appropriate regulation of ABCA1 is critical for a selective increase in HDL cholesterol levels.

Selective striatal neuronal loss in a YAC128 mouse model of Huntington disease.

An expanded CAG repeat is the underlying genetic defect in Huntington disease, a disorder characterized by motor, psychiatric and cognitive deficits and striatal atrophy associated with neuronal loss. An accurate animal model of this disease is crucial for elucidation of the underlying natural history of the illness and also for testing experimental therapeutics. We established a new yeast artificial chromosome (YAC) mouse model of HD with the entire human HD gene containing 128 CAG repeats (YAC128) which develops motor abnormalities and age-dependent brain atrophy including cortical and striatal atrophy associated with striatal neuronal loss. YAC128 mice exhibit initial hyperactivity, followed by the onset of a motor deficit and finally hypokinesis. The motor deficit in the YAC128 mice is highly correlated with striatal neuronal loss, providing a structural correlate for the behavioral changes. The natural history of HD-related changes in the YAC128 mice has been defined, demonstrating the presence of huntingtin inclusions after the onset of behavior and neuropathological changes. The HD-related phenotypes of the YAC128 mice show phenotypic uniformity with low inter-animal variability present, which together with the age-dependent striatal neurodegeneration make it an ideal mouse model for the assessment of neuroprotective and other therapeutic interventions.