b Dysbiosis in the intestinal microbiota of persons with inflammatory bowel disease (IBD) has been described, but there are still varied reports on changes in the abundance of Bifidobacterium and Lactobacillus organisms in patients with IBD. The aim of this investigation was to compare the compositions of mucosa-associated and fecal bacteria in patients with IBD and in healthy controls (HCs). Fecal and biopsy samples from 21 HCs, 21 and 15 Crohn's disease (CD) patients, and 34 and 29 ulcerative colitis (UC) patients, respectively, were analyzed by quantitative real-time PCR targeting the 16S rRNA gene. The bacterial numbers were transformed into relative percentages for statistical analysis. The proportions of bacteria were uniformly distributed along the colon regardless of the disease state. Bifidobacterium was significantly increased in the biopsy specimens of active UC patients compared to those in the HCs (4.6% versus 2.1%, P ‫؍‬ 0.001), and the proportion of Bifidobacterium was significantly higher in the biopsy specimens than in the fecal samples in active CD patients (2.7% versus 2.0%, P ‫؍‬ 0.012). The Lactobacillus group was significantly increased in the biopsy specimens of active CD patients compared to those in the HCs (3.4% versus 2.3%, P ‫؍‬ 0.036). Compared to the HCs, Faecalibacterium prausnitzii was sharply decreased in both the fecal and biopsy specimens of the active CD patients (0.3% versus 14.0%, P < 0.0001 for fecal samples; 0.8% versus 11.4%, P < 0.0001 for biopsy specimens) and the active UC patients (4.3% versus 14.0%, P ‫؍‬ 0.001 for fecal samples; 2.8% versus 11.4%, P < 0.0001 for biopsy specimens). In conclusion, Bifidobacterium and the Lactobacillus group were increased in active IBD patients and should be used more cautiously as probiotics during the active phase of IBD. Butyrate-producing bacteria might be important to gut homeostasis.

We carried out a test sample study to try to identify errors leading to irreproducibility, including incompleteness of peptide sampling, in LC-MS-based proteomics. We distributed a test sample consisting of an equimolar mix of 20 highly purified recombinant human proteins, to 27 laboratories for identification. Each protein contained one or more unique tryptic peptides of 1250 Da to also test for ion selection and sampling in the mass spectrometer. Of the 27 labs, initially only 7 labs reported all 20 proteins correctly, and only 1 lab reported all the tryptic peptides of 1250 Da. Nevertheless, a subsequent centralized analysis of the raw data revealed that all 20 proteins and most of the 1250 Da peptides had in fact been detected by all 27 labs. The centralized analysis allowed us to determine sources of problems encountered in the study, which include missed identifications (false negatives), environmental contamination, database matching, and curation of protein identifications. Improved search engines and databases are likely to increase the fidelity of mass spectrometry-based proteomics.

Gastrulation of the mouse embryo entails progressive restriction of lineage potency and the organization of the lineage progenitors into a body plan. Here we performed a high-resolution RNA sequencing analysis on single mid-gastrulation mouse embryos to collate a spatial transcriptome that correlated with the regionalization of cell fates in the embryo. 3D rendition of the quantitative data enabled the visualization of the spatial pattern of all expressing genes in the epiblast in a digital whole-mount in situ format. The dataset also identified genes that (1) are co-expressed in a specific cell population, (2) display similar global pattern of expression, (3) have lineage markers, (4) mark domains of transcriptional and signaling activity associated with cell fates, and (5) can be used as zip codes for mapping the position of single cells isolated from the mid-gastrula stage embryo and the embryo-derived stem cells to the equivalent epiblast cells for delineating their prospective cell fates.

Receptor−ligand interactions (RLIs) that play pivotal roles in living organisms are often depicted with the classic keys-and-locks model. Nevertheless, RLIs on the cell surface are generally highly complex and nonlinear, partially due to the noncontinuous and dynamic distribution of receptors on extracellular membranes. Here, we develop a tetrahedral DNA framework (TDF)-programmed approach to topologically engineer RLIs on the cell membrane, which enables active recruitment-binding of clustered receptors for high-affinity capture of circulating tumor cells (CTCs). The four vertices of a TDF afford orthogonal anchoring of ligands with spatial organization, based on which we synthesized n-simplexes harboring 1−3 aptamers targeting epithelial cell adhesion molecule (EpCAM) that are overexpressed on the membrane of tumor cells. The 2-simplex with three aptamers not only shows increased binding affinity (∼19-fold) but prevents endocytosis by cells. By using 2-simplex as the capture probe, we demonstrate the high-efficiency CTC capture, which is challenged in real clinical breast cancer patient samples. This TDFprogrammed platform thus provides a powerful means for studying RLIs in physiological settings and for cancer diagnosis.

Core fucosylation (CF) patterns of some glycoproteins are more sensitive and specific than evaluation of their total respective protein levels for diagnosis of many diseases, such as cancers. Global profiling and quantitative characterization of CF glycoproteins may reveal potent biomarkers for clinical applications. However, current techniques are unable to reveal CF glycoproteins precisely on a large scale. Here we developed a robust strategy that integrates molecular weight cutoff, neutral loss-dependent MS 3 , database-independent candidate spectrum filtering, and optimization to effectively identify CF glycoproteins. The rationale for spectrum treatment was innovatively based on computation of the mass distribution in spectra of CF glycopeptides. The efficacy of this strategy was demonstrated by implementation for plasma from healthy subjects and subjects with hepatocellular carcinoma. Over 100 CF glycoproteins and CF sites were identified, and over 10,000 mass spectra of CF glycopeptide were found. The scale of identification results indicates great progress for finding biomarkers with a particular and attractive prospect, and the candidate spectra will be a useful resource for the improvement of database searching methods for glycopeptides. Molecular & Cellular Proteomics 8:913-923, 2009.

Depression is a neuropsychiatric disorder accompanied by a decrease in the brain-derived neurotrophic factor (BDNF) signalling cascade in the hippocampus. Fenofibrate is a selective agonist of PPAR-α. In this study, we investigated the antidepressant-like effects of fenofibrate in C57BL/6J mice. EXPERIMENTAL APPROACHThe antidepressant-like effects of fenofibrate were first identified in the forced swim test (FST) and tail suspension test (TST), and then assessed in the chronic social defeat stress (CSDS) model. The changes in the hippocampal BDNF signalling pathway and adult hippocampal neurogenesis after CSDS and fenofibrate treatment were further investigated. A PPAR-α inhibitor, cannabinoid system inhibitors and BDNF signalling inhibitors were also used to determine the antidepressant mechanisms of fenofibrate. KEY RESULTSFenofibrate administration exhibited antidepressant-like effects in the FST and TST without affecting the locomotor activity of mice. Chronic fenofibrate treatment also prevented the depressive-like symptoms induced by CSDS. Moreover, fenofibrate restored the CSDS-induced decrease in the hippocampal BDNF signalling cascade and adult hippocampal neurogenesis. The antidepressant-like effects of fenofibrate could be blocked by a PPAR-α inhibitor and BDNF signalling inhibitors. CONCLUSIONS AND IMPLICATIONSTaken together, these results suggest that fenofibrate has antidepressant-like effects mediated through the promotion of the hippocampal BDNF signalling cascade. AbbreviationsBDNF, brain-derived neurotrophic factor; BrdU, 5-bromo-2-deoxyuridine; CREB, cAMP response element-binding protein;

The neural fate commitment of pluripotent stem cells requires the repression of extrinsic inhibitory signals and the activation of intrinsic positive transcription factors. However, how these two events are integrated to ensure appropriate neural conversion remains unclear. In this study, we showed that Pou3f1 is essential for the neural differentiation of mouse embryonic stem cells (ESCs), specifically during the transition from epiblast stem cells (EpiSCs) to neural progenitor cells (NPCs). Chimeric analysis showed that Pou3f1 knockdown leads to a markedly decreased incorporation of ESCs in the neuroectoderm. By contrast, Pou3f1-overexpressing ESC derivatives preferentially contribute to the neuroectoderm. Genome-wide ChIP-seq and RNA-seq analyses indicated that Pou3f1 is an upstream activator of neural lineage genes, and also is a repressor of BMP and Wnt signaling. Our results established that Pou3f1 promotes the neural fate commitment of pluripotent stem cells through a dual role, activating internal neural induction programs and antagonizing extrinsic neural inhibitory signals.DOI: http://dx.doi.org/10.7554/eLife.02224.001

Nucleic acids, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), are natural biopolymers composed of nucleotides that store, transmit, and express genetic information. Overexpressed or underexpressed as well as mutated nucleic acids have been implicated in many diseases. Therefore, nucleic acid tests (NATs) are extremely important. Inspired by intracellular DNA replication and RNA transcription, in vitro NATs have been extensively developed to improve the detection specificity, sensitivity, and simplicity. The principles of NATs can be in general classified into three categories: nucleic acid hybridization, thermal-cycle or isothermal amplification, and signal amplification. Driven by pressing needs in clinical diagnosis and prevention of infectious diseases, NATs have evolved to be a rapidly advancing field. During the past ten years, an explosive increase of research interest in both basic research and clinical translation has been witnessed. In this review, we aim to provide comprehensive coverage of the progress to analyze nucleic acids, use nucleic acids as recognition probes, construct detection devices based on nucleic acids, and utilize nucleic acids in clinical diagnosis and other important fields. We also discuss the new frontiers in the field and the challenges to be addressed.