How Difficult Is Inference of Mammalian Causal Gene Regulatory Networks?

Gene regulatory networks (GRNs) play a central role in systems biology, especially in the study of mammalian organ development. One key question remains largely unanswered: Is it possible to infer mammalian causal GRNs using observable gene co-expression patterns alone? We assembled two mouse GRN datasets (embryonic tooth and heart) and matching microarray gene expression profiles to systematically investigate the difficulties of mammalian causal GRN inference. The GRNs were assembled based on pieces of experimental genetic perturbation evidence from manually reading primary research articles. Each piece of perturbation evidence records the qualitative change of the expression of one gene following knock-down or over-expression of another gene. Our data have thorough annotation of tissue types and embryonic stages, as well as the type of regulation (activation, inhibition and no effect), which uniquely allows us to estimate both sensitivity and specificity of the inference of tissue specific causal GRN edges. Using these unprecedented datasets, we found that gene co-expression does not reliably distinguish true positive from false positive interactions, making inference of GRN in mammalian development very difficult. Nonetheless, if we have expression profiling data from genetic or molecular perturbation experiments, such as gene knock-out or signalling stimulation, it is possible to use the set of differentially expressed genes to recover causal regulatory relationships with good sensitivity and specificity. Our result supports the importance of using perturbation experimental data in causal network reconstruction. Furthermore, we showed that causal gene regulatory relationship can be highly cell type or developmental stage specific, suggesting the importance of employing expression profiles from homogeneous cell populations. This study provides essential datasets and empirical evidence to guide the development of new GRN inference methods for mammalian organ development.     

PDCD6 additively cooperates with anti-cancer drugs through activation of NF-κB pathways

The expression of programmed cell death 6 (PDCD6) is known to be down-regulated in cancer cell lines and ovarian cancer tissues compared to normal cells and tissues. In the current study, we characterized the specific function of PDCD6 as a novel pro-apoptotic protein. To define the roles of PDCD6 and cisplatin in tumorigenesis, we either over-expressed PDCD6 or treated it with cisplatin in SKOV-3 ovarian cancer cells. Both PDCD6 and cisplatin respectively inhibited cancer cell proliferation in a dose-dependent manner. The combined treatment of PDCD6 and cisplatin was more effective at suppressing cell growth than with either drug treatment alone, but had no effect with the treatment of caspase-3 and caspase-9 inhibitors. Cleavages of caspase-3, -8, -9, and poly (ADP-ribose) polymerase (PARP) in PDCD6-overexpressing cells were significantly increased after cisplatin treatment. Cell cycle analysis highly correlated with down-regulation of cyclin D1 and CDK4, and the induction of p16 and p27 as a cyclin-dependent kinase inhibitor. Additionally, PDCD6 also suppressed the phosphorylation of signaling regulators downstream of PI3K, including PDK1 and Akt. PDCD6 promotes TNFα-dependent apoptosis through the activation of NF-κB signaling pathways, increasing Bax, p53, and p21 expression, while also down-regulating Bcl-2 and Bcl-xL expression. The p21 and p53 promoter luciferase activities were enhanced by PDCD6, while there was no affect in p53^−/− and p21^−/−. At the same time, p53 activity was confirmed by UV irradiation and siPDCD6. Taken together, these results provide evidence that PDCD6 can mediate the pro-apoptotic activity of cisplatin or TNFα through the down-regulation of NF-κB expression.     

Chaperonins GroEL and GroES: views from atomic force microscopy.

The Escherichia coli chaperonins, GroEL and GroES, as well as their complexes in the presence of a nonhydrolyzable nucleotide AMP-PNP, have been imaged with the atomic force microscope (AFM). We demonstrate that both GroEL and GroES that have been adsorbed to a mica surface can be resolved directly by the AFM in aqueous solution at room temperature. However, with glutaraldehyde fixation of already adsorbed molecules, the resolution of both GroEL and GroES was further improved, as all seven subunits were well resolved without any image processing. We also found that chemical fixation was necessary for the contact mode AFM to image GroEL/ES complexes, and in the AFM images. GroEL with GroES bound can be clearly distinguished from those without. The GroEL/ES complex was about 5 nm higher than GroEL alone, indicating a 2 nm upward movement of the apical domains of GroEL. Using a slightly larger probe force, unfixed GroEL could be dissected: the upper heptamer was removed to expose the contact surface of the two heptamers. These results clearly demonstrate the usefulness of cross-linking agents for the determination of molecular structures with the AFM. They also pave the way for using the AFM to study the structural basis for the function of GroE system and other molecular chaperones.     

Integrating cell‐free biosyntheses of heme prosthetic group and apoenzyme for the synthesis of functional P450 monooxygenase

Harnessing the isolated protein synthesis machinery, cell‐free protein synthesis reproduces the cellular process of decoding genetic information in artificially controlled environments. More often than not, however, generation of functional proteins requires more than simple translation of genetic sequences. For instance, many of the industrially important enzymes require non‐protein prosthetic groups for biological activity. Herein, we report the complete cell‐free biogenesis of a heme prosthetic group and its integration with concurrent apoenzyme synthesis for the production of functional P450 monooxygenase. Step reactions required for the syntheses of apoenzyme and the prosthetic group have been designed so that these two separate pathways take place in the same reaction mixture, being insulated from each other. Combined pathways for the synthesis of functional P450 monooxygenase were then further integrated with in situ assay reactions to enable real‐time measurement of enzymatic activity during its synthesis. Biotechnol. Bioeng. 2013; 110: 1193–1200. © 2012 Wiley Periodicals, Inc.     

A novel human anti‐VCAM‐1 monoclonal antibody ameliorates airway inflammation and remodelling

Asthma is a chronic inflammatory disease induced by Type 2 helper T cells and eosinophils. Vascular cell adhesion molecule‐1 (VCAM‐1) has been implicated in recruiting eosinophils and lymphocytes to pathological sites in asthma as a regulatory receptor. Accordingly, monoclonal antibody (mAb) against VCAM‐1 may attenuate allergic inflammation and pathophysiological features of asthma. We attempted to evaluate whether a recently developed human anti‐VCAM‐1 mAb can inhibit the pathophysiological features of asthma in a murine asthma model induced by ovalbumin (OVA). Leucocyte adhesion inhibition assay was performed to evaluate the in vitro blocking activity of human anti‐VCAM‐1 mAb. OVA‐sensitized BALB/c mice were treated with human anti‐VCAM‐1 mAb or isotype control Ab before intranasal OVA challenge. We evaluated airway hyperresponsiveness (AHR) and bronchoalveolar lavage fluid analysis, measured inflammatory cytokines and examined histopathological features. The human anti‐VCAM‐1 mAb bound to human and mouse VCAM‐1 molecules and inhibited adhesion of human leucocytes in vitro. AHR and inflammatory cell counts in bronchoalveolar lavage fluid were reduced in mice treated with human anti‐VCAM‐1 mAb as compared with a control Ab. The levels of interleukin (IL)‐5 and IL‐13, as well as transforming growth factor‐β, in lung tissue were decreased in treated mice. Human anti‐VCAM‐1 mAb reduced goblet cell hyperplasia and peribronchial fibrosis. In vivo VCAM‐1 expression decreased in the treated group. In conclusion, human anti‐VCAM‐1 mAb attenuated allergic inflammation and the pathophysiological features of asthma in OVA‐induced murine asthma model. The results suggested that human anti‐VCAM‐1 mAb could potentially be used as an additional anti‐asthma therapeutic medicine.     

SARNP,a participant in mRNA splicing and export,negatively regulates E-cadherin expression via interaction with pinin

Triple-negative breast cancer (TNBC) is associated with a high mortality rate, which is related to the insufficient number of appropriate biomarkers and targets. Therefore, there is an urgent need to discover appropriate biomarkers and targets for TNBC. SARNP (Hcc-1 and CIP29) is highly expressed in several cancers. It binds to UAP56, an RNA helicase component of the TREX complex in messenger RNA (mRNA) splicing and export. However, the role of SARNP in mRNA splicing and export and in the progression of breast cancer, especially of TNBC, remains unknown. Therefore, we examined the role of SARNP in mRNA splicing and export and progression of TNBC. We confirmed that SARNP binds to UAP56 and Aly and that SARNP overexpression enhances mRNA splicing, whereas its knockdown suppressed mRNA export. The SARNP overexpression induced the proliferation of MCF7 cells, whereas its knockdown induced E-cadherin expression and downregulated vimentin and N-cadherin expressions in SK-BR-3 and MDA-MB-231 cells. SARNP downregulates E-cadherin expression by interaction with pinin. Mice injected with MDA-MB-231_shSARNP cells exhibited a significant reduction in tumor growth and lung metastasis compared with those injected with MDA-MB-231_shCon cells in vivo. These findings suggested that SARNP is involved in mRNA splicing and export. SARNP maintains mesenchymal phenotype by escaping from inhibitory interaction with pinin leading to the downregulation of E-cadherin expression.     

GeSUT4 mediates sucrose import at the symbiotic interface for carbon allocation of heterotrophic Gastrodia elata (Orchidaceae)

Gastrodia elata, a fully mycoheterotrophic orchid without photosynthetic ability, only grows symbiotically with the fungus Armillaria. The mechanism of carbon distribution in this mycoheterotrophy is unknown. We detected high sucrose concentrations in all stages of Gastrodia tubers, suggesting sucrose may be the major sugar transported between fungus and orchid. Thick symplasm‐isolated wall interfaces in colonized and adjacent large cells implied involvement of sucrose importers. Two sucrose transporter (SUT)‐like genes, GeSUT4 and GeSUT3, were identified that were highly expressed in young Armillaria‐colonized tubers. Yeast complementation and isotope tracer experiments confirmed that GeSUT4 functioned as a high‐affinity sucrose‐specific proton‐dependent importer. Plasma‐membrane/tonoplast localization of GeSUT4‐GFP fusions and high RNA expression of GeSUT4 in symbiotic and large cells indicated that GeSUT4 likely functions in active sucrose transport for intercellular allocation and intracellular homeostasis. Transgenic Arabidopsis overexpressing GeSUT4 had larger leaves but were sensitive to excess sucrose and roots were colonized with fewer mutualistic Bacillus, supporting the role of GeSUT4 in regulating sugar allocation. This is not only the first documented carbon import system in a mycoheterotrophic interaction but also highlights the evolutionary importance of sucrose transporters for regulation of carbon flow in all types of plant‐microbe interactions.     

The ABC’s of Suicide Risk Assessment: Applying a Tripartite Approach to Individual Evaluations

There is considerable need for accurate suicide risk assessment for clinical, screening, and research purposes. This study applied the tripartite affect-behavior-cognition theory, the suicidal barometer model, classical test theory, and item response theory (IRT), to develop a brief self-report measure of suicide risk that is theoretically-grounded, reliable and valid. An initial survey (n = 359) employed an iterative process to an item pool, resulting in the six-item Suicidal Affect-Behavior-Cognition Scale (SABCS). Three additional studies tested the SABCS and a highly endorsed comparison measure. Studies included two online surveys (Ns = 1007, and 713), and one prospective clinical survey (n = 72; Time 2, n = 54). Factor analyses demonstrated SABCS construct validity through unidimensionality. Internal reliability was high (α = .86-.93, split-half = .90-.94)). The scale was predictive of future suicidal behaviors and suicidality (r = .68, .73, respectively), showed convergent validity, and the SABCS-4 demonstrated clinically relevant sensitivity to change. IRT analyses revealed the SABCS captured more information than the comparison measure, and better defined participants at low, moderate, and high risk. The SABCS is the first suicide risk measure to demonstrate no differential item functioning by sex, age, or ethnicity. In all comparisons, the SABCS showed incremental improvements over a highly endorsed scale through stronger predictive ability, reliability, and other properties. The SABCS is in the public domain, with this publication, and is suitable for clinical evaluations, public screening, and research.