Quantitative model for inferring dynamic regulation of the tumour suppressor gene p53

Background: The availability of various "omics" datasets creates a prospect of performing the study of genome-wide genetic regulatory networks. However, one of the major challenges of using mathematical models to infer genetic regulation from microarray datasets is the lack of information for protein concentrations and activities. Most of the previous researches were based on an assumption that the mRNA levels of a gene are consistent with its protein activities, though it is not always the case. Therefore, a more sophisticated modelling framework together with the corresponding inference methods is needed to accurately estimate genetic regulation from "omics" datasets. Results: This work developed a novel approach, which is based on a nonlinear mathematical model, to infer genetic regulation from microarray gene expression data. By using the p53 network as a test system, we used the nonlinear model to estimate the activities of transcription factor (TF) p53 from the expression levels of its target genes, and to identify the activation/inhibition status of p53 to its target genes. The predicted top 317 putative p53 target genes were supported by DNA sequence analysis. A comparison between our prediction and the other published predictions of p53 targets suggests that most of putative p53 targets may share a common depleted or enriched sequence signal on their upstream non-coding region. Conclusions: The proposed quantitative model can not only be used to infer the regulatory relationship between TF and its down-stream genes, but also be applied to estimate the protein activities of TF from the expression levels of its target genes

Definition of the Gene Content of the Human Genome: The Need for Deep Experimental Verification

Based on the analysis of the drafts of the human genome sequence, it is being speculated that our species may possess an unexpectedly low number of genes. The quality of the drafts, the impossibility of accurate gene prediction and the lack of sufficient transcript sequence data, however, render such speculations very premature. The complexity of human gene structure requires additional and extensive experimental verification of transcripts that may result in major revisions of these early estimates of the number of human genes

Use of tobacco and alcohol by Swiss primary care physicians: a cross-sectional survey

BACKGROUND: Health behaviours among doctors has been suggested to be an important marker of how harmful lifestyle behaviours are perceived. In several countries, decrease in smoking among physicians was spectacular, indicating that the hazard was well known. Historical data have shown that because of their higher socio-economical status physicians take up smoking earlier. When the dangers of smoking become better known, physicians began to give up smoking at a higher rate than the general population. For alcohol consumption, the situation is quite different: prevalence is still very high among physicians and the dangers are not so well perceived. To study the situation in Switzerland, data of a national survey were analysed to determine the prevalence of smoking and alcohol drinking among primary care physicians. METHODS: 2'756 randomly selected practitioners were surveyed to assess subjective mental and physical health and their determinants, including smoking and drinking behaviours. Physicians were categorised as never smokers, current smokers and former smokers, as well as non drinkers, drinkers (AUDIT-C < 4 for women and < 5 for men) and at risk drinkers (higher scores). RESULTS: 1'784 physicians (65%) responded (men 84%, mean age 51 years). Twelve percent were current smokers and 22% former smokers. Sixty six percent were drinkers and 30% at risk drinkers. Only 4% were never smokers and non drinkers. Forty eight percent of current smokers were also at risk drinkers and 16% of at risk drinkers were also current smokers. Smoking and at risk drinking were more frequent among men, middle aged physicians and physicians living alone. When compared to a random sample of the Swiss population, primary care physicians were two to three times less likely to be active smokers (12% vs. 30%), but were more likely to be drinkers (96% vs. 78%), and twice more likely to be at risk drinkers (30% vs. 15%). CONCLUSION: The prevalence of current smokers among Swiss primary care physicians was much lower than in the general population in Switzerland, reflecting that the hazards of smoking are well known to doctors. However, the opposite was found for alcohol use, underlining the importance of making efforts in this area to increase awareness among physicians of the dangers of alcohol consumption

Learning to control a BMI-driven wheelchair for people with severe tetraplegia

Mind-controlled wheelchairs are an intriguing assistive mobility solution applicable in complete paralysis. Despite technical progress in brain-machine interface (BMI) technology, its translation remains elusive. The primary objective of this study is to probe the hypothesis that BMI skill acquisition by end-users is fundamental to control a non-invasive brain-actuated intelligent wheelchair in real-world settings. Here, we show that three tetraplegic spinal cord injury users could be trained to operate a non-invasive, self-paced thought-controlled wheelchair and execute complex navigation tasks. However, only the two users exhibiting increasing decoding performance and feature discriminancy, as well as significant neuroplasticity changes and improved BMI command latency, achieved high navigation performance. Additionally, we show that dexterous, continuous control of robots is possible through low-degree of freedom, discrete and uncertain control channels like a motor imagery BMI, by blending human and artificial intelligence through shared-control methodologies. We posit that subject learning and shared-control are the key-components paving the way for translational non-invasive BMI

CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering

Prokaryotic type II CRISPR-Cas systems can be adapted to enable targeted genome modifications across a range of eukaryotes.1–7. Here we engineer this system to enable RNA-guided genome regulation in human cells by tethering transcriptional activation domains either directly to a nuclease-null Cas9 protein or to an aptamer-modified single guide RNA (sgRNA). Using this functionality we developed a novel transcriptional activation–based assay to determine the landscape of off-target binding of sgRNA:Cas9 complexes and compared it with the off-target activity of transcription activator–like (TAL) effector proteins8, 9. Our results reveal that specificity profiles are sgRNA dependent, and that sgRNA:Cas9 complexes and 18-mer TAL effector proteins can potentially tolerate 1–3 and 1–2 target mismatches, respectively. By engineering a requirement for cooperativity through offset nicking for genome editing or through multiple synergistic sgRNAs for robust transcriptional activation, we suggest methods to mitigate off-target phenomena. Our results expand the versatility of the sgRNA:Cas9 tool and highlight the critical need to engineer improved specificity

Fluorescent in situ sequencing (FISSEQ) of RNA for gene expression profiling in intact cells and tissues

RNA sequencing measures the quantitative change in gene expression over the whole transcriptome, but it lacks spatial context. On the other hand, in situ hybridization provides the location of gene expression, but only for a small number of genes. Here we detail a protocol for genome-wide profiling of gene expression in situ in fixed cells and tissues, in which RNA is converted into cross-linked cDNA amplicons and sequenced manually on a confocal microscope. Unlike traditional RNA-seq our method enriches for context-specific transcripts over house-keeping and/or structural RNA, and it preserves the tissue architecture for RNA localization studies. Our protocol is written for researchers experienced in cell microscopy with minimal computing skills. Library construction and sequencing can be completed within 14 d, with image analysis requiring an additional 2 d

Novel Evolved Immunoglobulin (Ig)-Binding Molecules Enhance the Detection of IgM against Hepatitis C Virus

Detection of specific antibodies against hepatitis C virus (HCV) is the most widely available test for viral diagnosis and monitoring of HCV infections. However, narrowing the serologic window of anti-HCV detection by enhancing anti-HCV IgM detection has remained to be a problem. Herein, we used LD5, a novel evolved immunoglobulin-binding molecule (NEIBM) with a high affinity for IgM, to develop a new anti-HCV enzyme-linked immunosorbent assay (ELISA) using horseradish peroxidase-labeled LD5 (HRP-LD5) as the conjugated enzyme complex. The HRP-LD5 assay showed detection efficacy that is comparable with two kinds of domestic diagnostic kits and the Abbott 3.0 kit when tested against the national reference panel. Moreover, the HRP-LD5 assay showed a higher detection rate (55.9%, 95% confidence intervals (95% CI) 0.489, 0.629) than that of a domestic diagnostic ELISA kit (Chang Zheng) (53.3%, 95% CI 0.463, 0.603) in 195 hemodialysis patient serum samples. Five serum samples that were positive using the HRP-LD5 assay and negative with the conventional anti-HCV diagnostic ELISA kits were all positive for HCV RNA, and 4 of them had detectable antibodies when tested with the established anti-HCV IgM assay. An IgM confirmation study revealed the IgM reaction nature of these five serum samples. These results demonstrate that HRP-LD5 improved anti-HCV detection by enhancing the detection of anti-HCV IgM, which may have potential value for the early diagnosis and screening of hepatitis C and other infectious diseases