Ribosome-associated Trigger Factor (TF) and the DnaK chaperone system assist the folding of newly synthesized proteins in Escherichia coli. Here, we show that DnaK and TF share a common substrate pool in vivo. In TF-deficient cells, deltatig, depleted for DnaK and DnaJ the amount of aggregated proteins increases with increasing temperature, amounting to 10% of total soluble protein (approximately 340 protein species) at 37 degrees C. A similar population of proteins aggregated in DnaK depleted tig+ cells, albeit to a much lower extent. Ninety-four aggregated proteins isolated from DnaK- and DnaJ-depleted deltatig cells were identified by mass spectrometry and found to include essential cytosolic proteins. Four potential in vivo substrates were screened for chaperone binding sites using peptide libraries. Although TF and DnaK recognize different binding motifs, 77% of TF binding peptides also associated with DnaK. In the case of the nascent polypeptides TF and DnaK competed for binding, however, with competitive advantage for TF. In vivo, the loss of TF is compensated by the induction of the heat shock response and thus enhanced levels of DnaK. In summary, our results demonstrate that the co-operation of the two mechanistically distinct chaperones in protein folding is based on their overlap in substrate specificities.

BACKGROUND

Sepsis is associated with systemic inflammatory responses and induction of coagulation system. Neutrophil extracellular traps (NETs) constitute an antimicrobial mechanism, recently implicated in thrombosis via platelet entrapment and aggregation.

RESULTS

In this study, we demonstrate for the first time the localization of thrombogenic tissue factor (TF) in NETs released by neutrophils derived from patients with gram-negative sepsis and normal neutrophils treated with either serum from septic patients or inflammatory mediators involved in the pathogenesis of sepsis. Localization of TF in acidified autophagosomes was observed during this process, as indicated by positive LC3B and LysoTracker staining. Moreover, phosphatidylinositol 3-kinase inhibition with 3-MA or inhibition of endosomal acidification with bafilomycin A1 hindered the release of TF-bearing NETs. TF present in NETs induced thrombin generation in culture supernatants, which further resulted in protease activated receptor-1 signaling.

CONCLUSIONS

This study demonstrates the involvement of autophagic machinery in the extracellular delivery of TF in NETs and the subsequent activation of coagulation cascade, providing evidence for the implication of this process in coagulopathy and inflammatory response in sepsis.

In macaque monkeys, lesions involving the posterior portion of the inferior temporal cortex, cytoarchitectonic area TEO, produce a severe impairment in visual pattern discrimination. Recently, this area has been shown to contain a complete, though coarse, representation of the contralateral visual field (Boussaoud, Desimone, and Ungerleider: J. Comp. Neurol. 306:554-575, '91). Because the inputs and outputs of area TEO have not yet been fully described, we injected a variety of retrograde and anterograde tracers into 11 physiologically identified sites within TEO of seven rhesus monkeys and analyzed the areal and laminar distribution of its cortical connections. Our results show that TEO receives feedforward, topographically organized inputs from prestriate areas V2, V3, and V4. Additional sparser feedforward inputs arise from areas V3A, V4t, and MT. Each of these inputs is reciprocated by a feedback projection from TEO. TEO was also found to have reciprocal intermediate-type connections with the fundus of the superior temporal area (area FST), cortex in the most posteromedial portion of the superior temporal sulcus (the posterior parietal sulcal zone [area PP]), cortex in the intraparietal sulcus (including the lateral intraparietal area [area LIP]), the frontal eye field, and area TF on the parahippocampal gyrus. The connections with V3A, V4t, and PP were found only after injections in the peripheral field representations of TEO. Finally, TEO was found to project in a feedforward pattern to area TE and to areas anterior to FST on the lateral bank and floor of the superior temporal sulcus (areas TEm, TEa, and IPa, Seltzer and Pandya: Brain Res. 149:1-24, '78), all of which send feedback projections to TEO. Feedback projections also arise from parahippocampal area TH, and areas TG, 36, and possibly 35. These are complemented by only sparse feedforward projections to TG from central field representations in TEO and to TH from peripheral field representations. The results thus indicate that TEO forms an important link in the occipitotemporal pathway for object recognition, sending visual information forward from V1 and prestriate relays in V2-V4 to anterior inferior temporal area TE.

In the present study we investigated whether endothelial microparticles (EMPs) can bind to monocytic THP-1 cells and modulate their procoagulant properties. Using flow cytometry, we demonstrated that EMPs express adhesive receptors similar to those expressed by activated endothelial cells. Expression of endothelial antigens by THP-1 cells incubated with EMP was shown by immunoperoxidase staining and flow cytometry using antibodies directed against E-selectin, VCAM-1, and endoglin. EMP binding to THP-1 cells was time- and concentration- dependent, reached a plateau at 15 minutes, and had an EMP-to-monocyte ratio of 50:1. EMP binding was not affected by low temperature and was not followed by the restoration of phosphatidylserine asymmetry, suggesting that adhesion was not followed by fusion. A 4-hour incubation of THP-1 cells with EMP led to an increase in procoagulant activity as measured by clotting assay. Concomitantly, THP-1 exhibited increased levels of tissue factor (TF) antigen and TF mRNA compared to control cells. The ability of EMP to induce THP-1 procoagulant activity was significantly reduced when THP-1 cells were incubated with EMP in the presence of blocking antibodies against ICAM-1 and beta2 integrins. These results demonstrate that EMPs interact with THP-1 cells in vitro and stimulate TF-mediated procoagulant activity that is partially dependent on the interaction of ICAM-1 on EMP and its counterreceptor, beta2 integrins, on THP-1 cells. Induction of procoagulant activity was also demonstrated using human monocytes, suggesting a novel mechanism by which EMP may participate in the dissemination and amplification of procoagulant cellular responses.

The expression of eukaryotic genes is regulated by cis-regulatory elements such as promoters and enhancers, which bind sequence-specific DNA-binding proteins. One of the great challenges in the gene regulation field is to characterise these elements. This involves the identification of transcription factor (TF) binding sites within regulatory elements that are occupied in a defined regulatory context. Digestion with DNase and the subsequent analysis of regions protected from cleavage (DNase footprinting) has for many years been used to identify specific binding sites occupied by TFs at individual cis-elements with high resolution. This methodology has recently been adapted for high-throughput sequencing (DNase-seq). In this study, we describe an imbalance in the DNA strand-specific alignment information of DNase-seq data surrounding protein-DNA interactions that allows accurate prediction of occupied TF binding sites. Our study introduces a novel algorithm, Wellington, which considers the imbalance in this strand-specific information to efficiently identify DNA footprints. This algorithm significantly enhances specificity by reducing the proportion of false positives and requires significantly fewer predictions than previously reported methods to recapitulate an equal amount of ChIP-seq data. We also provide an open-source software package, pyDNase, which implements the Wellington algorithm to interface with DNase-seq data and expedite analyses.

Transcription factors (TFs) are a group of proteins that control cellular processes by regulating the expression of downstream target genes. Recent progress has been made in the cloning and characterization of Arabidopsis TFs on the genome scale, especially on the cloning of open reading frames (ORFs), sequence analysis and the expression profiling of different TF families. Huge difference in numbers of subfamily members were found for Arabidopsis MYB, C2H2 (Zn), C3H-type 1 (Zn), C3H-type 2 (Zn) TFs by independent research groups, mainly because of differences in bioinformatic search stringency. However, the Arabidopsis and rice genomes contain very different numbers of TFs in the WRKY, NAC, bZIP, MADS, ALFIN-like, GRAS and C2C2 (Zn)-dof families, indicating a possible divergence of biological functions from dicots to monocots. TFs have also been found to play key roles in the biosynthesis and signaling of plant hormones, in cell growth and differentiation, and in photomorphogenesis.

Autism spectrum disorders (ASD) represent a class of neurodevelopmental disorders characterized by impairments in social interaction, verbal and non-verbal communication, as well as restricted interests and repetitive behavior. This latter class of symptoms often includes features such as compulsive behaviors and resistance to change. The BTBR T+ tf/J mouse strain has been used as an animal model to investigate the social communication and restricted interest features in ASD. Less is known about whether this mouse strain models cognitive flexibility deficits also observed in ASD. The present experiment investigated performance of BTBR T+ tf/J and C57BL/6J on two different spatial reversal learning tests (100% accurate feedback and 80/20 probabilistic feedback), as well as marble burying and grooming behavior. BTBR T+ tf/J and C57BL/6J mice exhibited similar performance on acquisition and reversal learning with 100% accurate feedback. BTBR T+ tf/J mice were impaired in probabilistic reversal learning compared to that of C57BL/6J mice. BTBR T+ tf/J mice also displayed increased stereotyped repetitive behaviors compared to that of C57BL/6J mice as shown by increased marble burying and grooming behavior. The present findings indicate that BTBR T+ tf/J mice exhibit similar features related to "insistence on sameness" in ASD that include not only stereotyped repetitive behaviors, but also alterations in behavioral flexibility. Thus, BTBR T+ tf/J mice can serve as a model to understand the neural mechanisms underlying alterations in behavioral flexibility, as well as to test potential treatments in alleviating these symptoms.

Transcription factor (TF) binding site (TFBS) models are crucial for computational reconstruction of transcription regulatory networks. In existing repositories, a TF often has several models (also called binding profiles or motifs), obtained from different experimental data. Having a single TFBS model for a TF is more pragmatic for practical applications. We show that integration of TFBS data from various types of experiments into a single model typically results in the improved model quality probably due to partial correction of source specific technique bias. We present the Homo sapiens comprehensive model collection (HOCOMOCO, http://autosome.ru/HOCOMOCO/, http://cbrc.kaust.edu.sa/hocomoco/) containing carefully hand-curated TFBS models constructed by integration of binding sequences obtained by both low- and high-throughput methods. To construct position weight matrices to represent these TFBS models, we used ChIPMunk software in four computational modes, including newly developed periodic positional prior mode associated with DNA helix pitch. We selected only one TFBS model per TF, unless there was a clear experimental evidence for two rather distinct TFBS models. We assigned a quality rating to each model. HOCOMOCO contains 426 systematically curated TFBS models for 401 human TFs, where 172 models are based on more than one data source.

Transcription factors (TFs), which are central to the regulation of gene expression, are usually members of multigene families. In plants, they are involved in diverse processes such as developmental control and elicitation of defense and stress responses. To investigate if differences exist in the expansion patterns of TF gene families between plants and other eukaryotes, we first used Arabidopsis (Arabidopsis thaliana) TFs to identify TF DNA-binding domains. These DNA-binding domains were then used to identify related sequences in 25 other eukaryotic genomes. Interestingly, among 19 families that are shared between animals and plants, more than 14 are larger in plants than in animals. After examining the lineage-specific expansion of TF families in two plants, eight animals, and two fungi, we found that TF families shared among these organisms have undergone much more dramatic expansion in plants than in other eukaryotes. Moreover, this elevated expansion rate of plant TF is not simply due to higher duplication rates of plant genomes but also to a higher degree of expansion compared to other plant genes. Further, in many Arabidopsis-rice (Oryza sativa) TF orthologous groups, the degree of lineage-specific expansion in Arabidopsis is correlated with that in rice. This pattern of parallel expansion is much more pronounced than the whole-genome trend in rice and Arabidopsis. The high rate of expansion among plant TF genes and their propensity for parallel expansion suggest frequent adaptive responses to selection pressure common among higher plants.

The microRNAs (miRNAs) are a class of small, 20-22 nucleotides in length, endogenously expressed noncoding RNAs that regulate multiple targets posttranscriptionally. Interestingly, miRNAs have emerged as regulators of most physiological and pathological processes, including metastatic tumor progression, in part by controlling a reversible process called epithelial-to-mesenchymal transition (EMT). The activation of EMT increases the migratory and invasive properties fundamental for tumor cell spread while activation of the reverse mesenchymal-to-epithelial transition is required for metastasis outgrowth. The EMT triggering leads to the activation of a core of transcription factors (EMT-TFs) - SNAIL1/SNAIL2, bHLH (E47, E2-2, and TWIST1/TWIST2), and ZEB1/ZEB2 - that act as E-cadherin repressors and, ultimately, coordinate EMT. Recent evidence indicates that several miRNAs regulate the expression of EMT-TFs or EMT-activating signaling pathways. Interestingly, some miRNAs and EMT-TFs form tightly interconnected negative feedback loops that control epithelial cell plasticity, providing self-reinforcing signals and robustness to maintain the epithelial or mesenchymal cell status. Among the most significant feedback loops, we focus on the ZEB/miR-200 and the SNAIL1/miR-34 networks that hold a clear impact in the regulation of the epithelial-mesenchymal state. Recent insights into the p53 modulation of the EMT-TF/miRNA loops and epigenetic regulatory mechanisms in the context of metastasis dissemination will also be discussed. Understanding the regulation of EMT by miRNAs opens new avenues for the diagnosis and prognosis of tumors and identifies potential therapeutic targets that might help to negatively impact on metastasis dissemination and increasing patient survival.

Transcription factors (TFs) are sequence-specific DNA-binding proteins acting as critical regulators of gene expression. The Plant Promoter Analysis Navigator (PlantPAN; http://PlantPAN2.itps.ncku.edu.tw) provides an informative resource for detecting transcription factor binding sites (TFBSs), corresponding TFs, and other important regulatory elements (CpG islands and tandem repeats) in a promoter or a set of plant promoters. Additionally, TFBSs, CpG islands, and tandem repeats in the conserve regions between similar gene promoters are also identified. The current PlantPAN release (version 2.0) contains 16 960 TFs and 1143 TF binding site matrices among 76 plant species. In addition to updating of the annotation information, adding experimentally verified TF matrices, and making improvements in the visualization of transcriptional regulatory networks, several new features and functions are incorporated. These features include: (i) comprehensive curation of TF information (response conditions, target genes, and sequence logos of binding motifs, etc.), (ii) co-expression profiles of TFs and their target genes under various conditions, (iii) protein-protein interactions among TFs and their co-factors, (iv) TF-target networks, and (v) downstream promoter elements. Furthermore, a dynamic transcriptional regulatory network under various conditions is provided in PlantPAN 2.0. The PlantPAN 2.0 is a systematic platform for plant promoter analysis and reconstructing transcriptional regulatory networks.

We present a major update of the HOCOMOCO collection that consists of patterns describing DNA binding specificities for human and mouse transcription factors. In this release, we profited from a nearly doubled volume of published in vivo experiments on transcription factor (TF) binding to expand the repertoire of binding models, replace low-quality models previously based on in vitro data only and cover more than a hundred TFs with previously unknown binding specificities. This was achieved by systematic motif discovery from more than five thousand ChIP-Seq experiments uniformly processed within the BioUML framework with several ChIP-Seq peak calling tools and aggregated in the GTRD database. HOCOMOCO v11 contains binding models for 453 mouse and 680 human transcription factors and includes 1302 mononucleotide and 576 dinucleotide position weight matrices, which describe primary binding preferences of each transcription factor and reliable alternative binding specificities. An interactive interface and bulk downloads are available on the web: http://hocomoco.autosome.ru and http://www.cbrc.kaust.edu.sa/hocomoco11. In this release, we complement HOCOMOCO by MoLoTool (Motif Location Toolbox, http://molotool.autosome.ru) that applies HOCOMOCO models for visualization of binding sites in short DNA sequences.

In this investigation the efferent projections from ventral temporal neocortical and limbic cortical areas to the entorhinal and perirhinal cortices have been investigated in the rhesus monkey using silver impregnation methods. It was observed that virtually all ventral temporal neocortical areas contribute some afferents to the transitional zones of periallocortex (perirhinal and prorhinal cortices) forming the walls of the rhinal sulcus. These areas in turn project medially to the entorhinal cortex and hippocampus. Additional direct sources of afferent input to the entorhinal cortex were found to originate in Brodmann's areas 51, 49 and 27, and Bonin and Bailey's areas TF and TH. These connections have been characterized as final relays in multisynaptic cortico-cortical pathways linking the entorhinal cortex and, ultimately, hippocampus to the association areas of the frontal, parietal, temporal, and occipital lobes.

Two related families of transposons were isolated from schizosaccharomyces pombe, an organism which has been the object of extensive genetic studies which had previously produced no evidence for the existence of such elements. These two classes of repeated DNAs, dubbed <em>Tf</em>1 (transposon of fission yeast 1) and <em>Tf</em>2 have many properties of retrotransposons. <em>Tf</em>1 and <em>Tf</em>2 both possess long terminal repeats and predicted protein sequences that resemble the protease, reverse transcriptase, and integrase domains of retroviruses. The chromosomal locations and total numbers of <em>Tf</em>1 and <em>Tf</em>2 differ greatly in various isolates of S. pombe. The <em>Tf</em> elements are expressed in the form of 4.5-kb mRNAs. The complete sequence of <em>Tf</em>1 was determined and suggests that a novel mechanism for regulating its gene expression may be used.

Ham's F12 medium supplemented with insulin (Ins), transferrin (Tf), epidermal growth factor (EGF), hydrocortisone (HC), T3, cholera toxin (CT), and bovine hypothalamus extract (BHE) was developed for in vitro growth of human nasal epithelial (HNE) cells. The HNE cells were dissociated from freshly excised nasal polyps or turbinates with protease. Colony-forming efficiency of primary HNE cells was approximately 5%. Growth studies showed Ins, BHE, and CT were essential for growth; HC, EGF, Tf, and T3 were also stimulatory for growth. The growth rate in this serum-free, hormone-supplemented medium was 24 h per population doubling. Up to 20 population doublings and 3 passages of dissociated HNE cells could be achieved. Addition of serum to this culture medium inhibited epithelial cell growth. Vitamin A had no apparent effect on cell growth but induced an alteration in the morphologic characteristics of the cell. The epithelial nature of cultured cells was confirmed by positive staining with antihuman keratin antibody, ultrastructural studies, and by formation of a columnar, ciliated epithelium in denuded tracheal grafts repopulated by these cultured HNE cells. Biochemical analyses of glycoproteins (labeled with 3H-glucosamine and/or 35S-sulfate) secreted by cultured HNE cells were unable to demonstrate the secretion of mucinlike glycoproteins in culture. Instead, major secretory products of cultured cells were hyaluronate and heparan sulfate. These results were in agreement with morphologic observations that showed no mucus-secreting granules in cultured cells. Dome formation was observed in high cell density cultures. We conclude that HNE cells can be cultured in well-defined culture media. As indicated by formation of domes, these cells may be useful for in vitro ion transport studies. Further differentiation, however, may be required for studies of mucin synthesis.

Finding functional DNA binding sites of transcription factors (TFs) throughout the genome is a crucial step in understanding transcriptional regulation. Unfortunately, these binding sites are typically short and degenerate, posing a significant statistical challenge: many more matches to known TF motifs occur in the genome than are actually functional. However, information about chromatin structure may help to identify the functional sites. In particular, it has been shown that active regulatory regions are usually depleted of nucleosomes, thereby enabling TFs to bind DNA in those regions. Here, we describe a novel motif discovery algorithm that employs an informative prior over DNA sequence positions based on a discriminative view of nucleosome occupancy. When a Gibbs sampling algorithm is applied to yeast sequence-sets identified by ChIP-chip, the correct motif is found in 52% more cases with our informative prior than with the commonly used uniform prior. This is the first demonstration that nucleosome occupancy information can be used to improve motif discovery. The improvement is dramatic, even though we are using only a statistical model to predict nucleosome occupancy; we expect our results to improve further as high-resolution genome-wide experimental nucleosome occupancy data becomes increasingly available.

BACKGROUND

Transcatheter aortic valve implantation (TAVI) has emerged as a new therapeutic option in high-risk patients with severe aortic stenosis.

OBJECTIVE

PARTNER EU is the first study to evaluate prospectively the procedural and mid-term outcomes of transfemoral (TF) or transapical (TA) implantation of the Edwards SAPIEN® valve involving a multi-disciplinary approach.

RESULTS

Primary safety endpoints were 30 days and 6 months mortality. Primary efficacy endpoints were haemodynamic and functional improvement at 12 months. One hundred and thirty patients (61 TF, 69 TA), aged 82.1 ± 5.5 years were included. TA patients had higher logistic EuroSCORE (33.8 vs. 25.7%, P = 0.0005) and more peripheral disease (49.3 vs. 16.4%, P< 0.0001). Procedures were aborted in four TA (5.8%) and six TF cases (9.8%). Valve implantation was successful in the remaining patients in 95.4 and 96.4%, respectively. Thirty days and 6 months survival were 81.2 and 58.0% (TA) and 91.8 and 90.2% (TF). In both groups, mean aortic gradient decreased from 46.9 ± 18.1 to 10.9 ± 5.4 mmHg 6 months post-TAVI. In total, 78.1 and 84.8% of patients experienced significant improvement in New York Heart Association (NYHA) class, whereas 73.9 and 72.7% had improved Kansas City Cardiomyopathy Questionnaire (KCCQ) scores in TA and TF cohorts, respectively.

CONCLUSIONS

This first team-based multi-centre European TAVI registry shows promising results in high-risk patients treated by TF or TA delivery. Survival rates differ significantly between TF and TA groups and probably reflect the higher risk profile of the TA cohort. Optimal patient screening, approach selection, and device refinement may improve outcomes.

Chicken ovalbumin upstream promoter transcription factor (COUP-TF)-interacting proteins 1 and 2 [CTIP1/Evi9/B cell leukaemia (Bcl) l1a and CTIP2/Bcl11b respectively] are highly related C(2)H(2) zinc finger proteins that are abundantly expressed in brain and the immune system, and are associated with immune system malignancies. A selection procedure was employed to isolate high-affinity DNA binding sites for CTIP1. The core binding site on DNA identified in these studies, 5'-GGCCGG-3' (upper strand), is highly related to the canonical GC box and was bound by a CTIP1 oligomeric complex(es) in vitro. Furthermore, both CTIP1 and CTIP2 repressed transcription of a reporter gene harbouring a multimerized CTIP binding site, and this repression was neither reversed by trichostatin A (an inhibitor of known class I and II histone deacetylases) nor stimulated by co-transfection of a COUP-TF family member. These results demonstrate that CTIP1 is a sequence-specific DNA binding protein and a bona fide transcriptional repressor that is capable of functioning independently of COUP-TF family members. These findings may be relevant to the physiological and/or pathological action(s) of CTIPs in cells that do not express COUP-TF family members, such as cells of the haematopoietic and immune systems.

Abnormal tissue factor (TF) expression has been demonstrated on blood monocytes and circulating endothelial cells in humans with sickle cell anemia. We have now studied sickle transgenic mice to help define the biology of endothelial TF expression in sickle disease. Using immunostaining of tissue sections, we find that this is confined almost exclusively to the pulmonary veins. About 15% and 13% of these exhibit TF-positive endothelium in the wild-type normal mouse and the normal human hemoglobin (HbA)-expressing control transgenic mouse, respectively. The mild sickle mouse is indistinguishable from normal (approximately 14% positive), but TF expression is significantly elevated in the moderate and severe mouse models of sickle disease (approximately 29% and approximately 41% positive, respectively). Exposure of the mild sickle mouse to hypoxia for 3 hours, followed by reoxygenation, converted its TF expression phenotype to that of the severe sickle mouse (approximately 36% positive). Pretreatment with lovastatin eliminated excessive expression of TF in the posthypoxic mild sickle mouse (approximately 16% positive) and in the more severe mouse at ambient air (approximately 21% positive). In addition to identifying tissue expression of endothelial TF in the sickle lung, these studies implicate reperfusion injury physiology in its expression and suggest a rationale for use of statins in sickle disease.

1. In total 1999 consecutive admissions to six medical wards were subjected to a prospective high-intensity drug event monitoring scheme to assess the extent and pattern of admissions caused by adverse drug reactions (ADRs) or dose related therapeutic failures (TF), in a population-based design. The wards were sub-specialised in general medicine, geriatrics, endocrinology, cardiology, respiratory medicine and gastroenterology. 2. Considering definite, probable and possible drug events, the prevalence of drug related hospital admissions was 11.4% of which 8.4% were caused by ADRs and 3.0% by TFs. There were large inter-department differences. 3. The six classes of drugs most frequently involved in admissions caused by ADRs were anti-rheumatics and analgesics (27%), cardiovascular drugs (23%), psychotropic drugs (14%), anti-diabetics (12%), antibiotics (7%), and corticosteroids (5%). Noncompliance accounted for 66% of the TFs with diuretics and anti-asthmatics most frequently involved. 4. The pattern of drugs involved in ADRs was compared with the regional drug sales statistics. Drugs with a particularly high rate of ADR related admissions per unit dispensed were nitrofurantoin and insulin (617 and 182 admissions per 1,000,000 defined daily doses), while low rates were seen for diuretics and benzodiazepines (10 and 7 admissions per 1,000,000 defined daily doses). Confidence intervals were wide. 5. Patients who had their therapy prescribed by a hospital doctor had a slightly higher prevalence of drug events than those who were treated by a general practitioner (12.6% vs 11.8%). The reverse applied for drug events assessed as avoidable (3.3% vs 4.6%). Although these differences were not statistically significant, it may suggest general practitioners as the appropriate target for interventive measures.(ABSTRACT TRUNCATED AT 250 WORDS)

Sequence-specific DNA-binding proteins including transcription factors (TFs) are key determinants of gene regulation and chromatin architecture. TF profiling is commonly carried out by formaldehyde cross-linking and sonication followed by chromatin immunoprecipitation (X-ChIP). We describe a method to profile TF binding at high resolution without cross-linking. We begin with micrococcal nuclease-digested non-cross-linked chromatin and then perform affinity purification of TFs and paired-end sequencing. The resulting occupied regions of genomes from affinity-purified naturally isolated chromatin (ORGANIC) profiles of Saccharomyces cerevisiae Abf1 and Reb1 provide high-resolution maps that are accurate, as defined by the presence of known TF consensus motifs in identified binding sites, that are not biased toward accessible chromatin and that do not require input normalization. We profiled Drosophila melanogaster GAGA factor and Pipsqueak to test ORGANIC performance on larger genomes. Our results suggest that ORGANIC profiling is a widely applicable high-resolution method for sensitive and specific profiling of direct protein-DNA interactions.

Several studies have established a link between blood coagulation and cancer, and more specifically between tissue factor (TF), a transmembrane protein involved in initiating blood coagulation, and tumor metastasis. In the study reported here, a murine model of human melanoma metastasis was used for two experiments. (i) The first experiment was designed to test the effect of varying the level of TF expression in human melanoma cells on their metastatic potential. Two matched sets of cloned human melanoma lines, one expressing a high level and the other a low level of the normal human TF molecule, were generated by retroviral-mediated transfections of a nonmetastatic parental line. The metastatic potential of the two sets of transfected lines was compared by injecting the tumor cells into the tail vein of severe combined immunodeficiency (SCID) mice and later examining the lungs and other tissues for tumor development. Metastatic tumors were detected in 86% of the mice injected with the high-TF lines and in 5% of the mice injected with the low-TF lines, indicating that a high TF level promotes metastasis of human melanoma in the SCID mouse model. This TF effect on metastasis occurs with i.v.-injected melanoma cells but does not occur with primary tumors formed from s.c.-injected melanoma cells, suggesting that TF acts at a late stage of metastasis, after tumor cells have escaped from the primary site and entered the blood. (ii) The second experiment was designed to analyze the mechanism by which TF promotes melanoma metastasis. The procedure involved testing the effect on metastasis of mutations in either the extracellular or cytoplasmic domains of the transmembrane TF molecule. The extracellular mutations introduced two amino acid substitutions that inhibited initiation by TF of the blood-coagulation cascade; the cytoplasmic mutation deleted most of the cytoplasmic domain without impairing the coagulation function of TF. Several human melanoma lines expressing high levels of either of the two mutant TF molecules were generated by retroviral-mediated transfection of the corresponding TF cDNA into the nonmetastatic parental melanoma line, and the metastatic potential of each transfected line was tested in the SCID mouse model. Metastases occurred in most mice injected with the melanoma lines expressing the extracellular TF mutant but were not detected in most mice injected with the melanoma lines expressing the cytoplasmic TF mutant. Results with the extracellular TF mutant indicate that the metastatic effect of TF in the SCID mouse model does not involve products of the coagulation cascade. Results with the cytoplasmic TF mutant indicate that the cytoplasmic domain of TF is important for the metastatic effect, suggesting that the TF could transduce a melanoma cell signal that promotes metastasis.

Investigation of transcription factors (TFs) and their downstream regulated genes (targets) is a significant issue in post-genome era, which can provide a brand new vision for some vital biological process. However, information of TFs and their targets in mammalian is far from sufficient. Here, we developed an integrated TF platform (ITFP), which included abundant TFs and their targets of mammalian. In current release, ITFP includes 4105 putative TFs and 69 496 potential TF-target pairs for human, 3134 putative TFs and 37 040 potential TF-target pairs for mouse, and 1114 putative TFs and 18 055 potential TF-target pairs for rat. In short, ITFP will serve as an important resource for the research community of transcription and provide strong support for regulatory network study.

The WRKY gene family is among the largest families of transcription factors (TFs) in higher plants. By regulating the plant hormone signal transduction pathway, these TFs play critical roles in some plant processes in response to biotic and abiotic stress. Various bodies of research have demonstrated the important biological functions of WRKY TFs in plant response to different kinds of biotic and abiotic stresses and working mechanisms. However, very little summarization has been done to review their research progress. Not just important TFs function in plant response to biotic and abiotic stresses, WRKY also participates in carbohydrate synthesis, senescence, development, and secondary metabolites synthesis. WRKY proteins can bind to W-box (TGACC (A/T)) in the promoter of its target genes and activate or repress the expression of downstream genes to regulate their stress response. Moreover, WRKY proteins can interact with other TFs to regulate plant defensive responses. In the present review, we focus on the structural characteristics of WRKY TFs and the research progress on their functions in plant responses to a variety of stresses.