Metatranscriptomic analyses of microbial assemblages (< 5 μm) from surface water at the Hawaiian Ocean Time-Series (HOT) revealed community-wide metabolic activities and day/night patterns of ...differential gene expression. Pyrosequencing produced 75 558 putative mRNA reads from a day transcriptome and 75 946 from a night transcriptome. Taxonomic binning of annotated mRNAs indicated that Cyanobacteria contributed a greater percentage of the transcripts (54% of annotated sequences) than expected based on abundance (35% of cell counts and 21% 16S rRNA of libraries), and may represent the most actively transcribing cells in this surface ocean community in both the day and night. Major heterotrophic taxa contributing to the community transcriptome included α-Proteobacteria (19% of annotated sequences, most of which were SAR11-related) and γ-Proteobacteria (4%). The composition of transcript pools was consistent with models of prokaryotic gene expression, including operon-based transcription patterns and an abundance of genes predicted to be highly expressed. Metabolic activities that are shared by many microbial taxa (e.g. glycolysis, citric acid cycle, amino acid biosynthesis and transcription and translation machinery) were well represented among the community transcripts. There was an overabundance of transcripts for photosynthesis, C1 metabolism and oxidative phosphorylation in the day compared with night, and evidence that energy acquisition is coordinated with solar radiation levels for both autotrophic and heterotrophic microbes. In contrast, housekeeping activities such as amino acid biosynthesis, membrane synthesis and repair, and vitamin biosynthesis were overrepresented in the night transcriptome. Direct sequencing of these environmental transcripts has provided detailed information on metabolic and biogeochemical responses of a microbial community to solar forcing.
Here we describe the generation of an antibody–drug conjugate (ADC) consisting of a humanized anti-CD79b antibody that is conjugated to monomethylauristatin E (MMAE) through engineered cysteines ...(THIOMABs) by a protease cleavable linker. By using flow cytometry, we detected the surface expression of CD79b in almost all non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia patients, suggesting that anti–CD79b-vcMMAE could be widely used in these malignancies. By using NHL cell lines to simulate a patient population we discovered that a minimal cell-surface expression level of CD79b was required for in vitro activity. Within the subpopulation of cell lines above this minimal threshold, we found that sensitivity to free MMAE, mutation of cancer genes, and cell doubling time were poorly correlated with in vitro activity; however, the expression level of BCL-XL was correlated with reduced sensitivity to anti–CD79b-vcMMAE. This observation was supported by in vivo data showing that a Bcl-2 family inhibitor, ABT-263, strikingly enhanced the activity of anti–CD79b-vcMMAE. Furthermore, anti–CD79b-vcMMAE was significantly more effective than a standard-of-care regimen, R-CHOP (ie, rituximab with a single intravenous injection of 30 mg/kg cyclophosphamide, 2.475 mg/kg doxorubicin, 0.375 mg/kg vincristine, and oral dosing of 0.15 mg/kg prednisone once a day for 5 days), in 3 xenograft models of NHL. Together, these data suggest that anti–CD79b-vcMMAE could be broadly efficacious for the treatment of NHL.
The p53 transcription factor is a potent suppressor of tumor growth. We report here an analysis of its direct transcriptional program using Global Run-On sequencing (GRO-seq). Shortly after MDM2 ...inhibition by Nutlin-3, low levels of p53 rapidly activate ∼200 genes, most of them not previously established as direct targets. This immediate response involves all canonical p53 effector pathways, including apoptosis. Comparative global analysis of RNA synthesis vs steady state levels revealed that microarray profiling fails to identify low abundance transcripts directly activated by p53. Interestingly, p53 represses a subset of its activation targets before MDM2 inhibition. GRO-seq uncovered a plethora of gene-specific regulatory features affecting key survival and apoptotic genes within the p53 network. p53 regulates hundreds of enhancer-derived RNAs. Strikingly, direct p53 targets harbor pre-activated enhancers highly transcribed in p53 null cells. Altogether, these results enable the study of many uncharacterized p53 target genes and unexpected regulatory mechanisms.DOI: http://dx.doi.org/10.7554/eLife.02200.001.
Focused ion beam milling of ∼200 nm polymer thin films is investigated using a multibeam ion microscope equipped with a gallium liquid metal ion source and a helium/neon gas field-ionization source. ...The quality of gallium, neon, and helium ion milled edges in terms of ion implantation artifacts is analyzed using a combination of helium ion microscopy, transmission electron microscopy and light microscopy. Results for a synthetic polymer thin film, in the form of cryo-ultramicrotomed sections from a co-extruded polymer multilayer, and a biological polymer thin film, in the form of the base layer of a butterfly wing scale, are presented. While gallium and neon ion milling result in the implantation of ions up to tens of nanometers from the milled edge and local thinning near the edge, helium ion milling produces much sharper edges with dramatically reduced implantation. These effects can be understood in terms of the minimal lateral scatter and larger stopping distance of helium compared with the heavier ions, whereby due to the thin film geometry, most of the incident helium ions will pass straight through the material. The basic result demonstrated here for polymer thin films is also expected for thin films of hard materials such as metals and ceramics.
Increased activity of matrix metalloproteinases (MMPs) is associated with worse prognosis in different cancer types. The wild-type protective antigen (PA-WT) of the binary anthrax lethal toxin was ...modified to form a pore in cell membranes only when cleaved by MMPs (to form PA-L1). Anthrax lethal factor (LF) is then able to translocate through these pores. Here, we used a
In-radiolabeled form of LF with the PA/LF system for noninvasive in vivo imaging of MMP activity in tumor tissue by SPECT.
MMP-mediated activation of PA-L1 was correlated to anthrax receptor expression and MMP activity in a panel of cancer cells (HT1080, MDA-MB-231, B8484, and MCF7). Uptake of
In-radiolabeled PA-L1,
In-PA-WT
, or
In-LF
(a catalytically inactive LF mutant) in tumor and normal tissues was measured using SPECT/CT imaging in vivo.
Activation of PA-L1 in vitro correlated with anthrax receptor expression and MMP activity (HT1080 > MDA-MB-231 > B8484 > MCF7). PA-L1-mediated delivery of
In-LF
was demonstrated and was corroborated using confocal microscopy with fluorescently labeled LF
Uptake was blocked by the broad-spectrum MMP inhibitor GM6001. In vivo imaging showed selective accumulation of
In-PA-L1 in MDA-MB-231 tumor xenografts (5.7 ± 0.9 percentage injected dose %ID/g) at 3 h after intravenous administration.
In-LF
was selectively delivered to MMP-positive MDA-MB-231 tumor tissue by MMP-activatable PA-L1 (5.98 ± 0.62 %ID/g) but not by furin-cleavable PA-WT (1.05 ± 0.21 %ID/g) or a noncleavable PA variant control, PA-U7 (2.74 ± 0.24 %ID/g).
Taken together, our results indicate that radiolabeled forms of mutated anthrax lethal toxin hold promise for noninvasive imaging of MMP activity in tumor tissue.
Trans-splicing of one of two short leader RNAs, SL1 or SL2, occurs at the 5' ends of pre-mRNAs of many C. elegans genes. We have exploited RNA-sequencing data from the modENCODE project to analyze ...the transcriptome of C. elegans for patterns of trans-splicing. Transcripts of ∼70% of genes are trans-spliced, similar to earlier estimates based on analysis of far fewer genes. The mRNAs of most trans-spliced genes are spliced to either SL1 or SL2, but most genes are not trans-spliced to both, indicating that SL1 and SL2 trans-splicing use different underlying mechanisms. SL2 trans-splicing occurs in order to separate the products of genes in operons genome wide. Shorter intercistronic distance is associated with greater use of SL2. Finally, increased use of SL1 trans-splicing to downstream operon genes can indicate the presence of an extra promoter in the intercistronic region, creating what has been termed a "hybrid" operon. Within hybrid operons the presence of the two promoters results in the use of the two SL classes: Transcription that originates at the promoter upstream of another gene creates a polycistronic pre-mRNA that receives SL2, whereas transcription that originates at the internal promoter creates transcripts that receive SL1. Overall, our data demonstrate that >17% of all C. elegans genes are in operons.
Abstract
Background
RUNX1 is a transcription factor and a master regulator for the specification of the hematopoietic lineage during embryogenesis and postnatal megakaryopoiesis. Mutations and ...rearrangements on
RUNX1
are key drivers of hematological malignancies. In humans, this gene is localized to the ‘Down syndrome critical region’ of chromosome 21, triplication of which is necessary and sufficient for most phenotypes that characterize Trisomy 21.
Main body
Individuals with Down syndrome show a higher predisposition to leukemias. Hence, RUNX1 overexpression was initially proposed as a critical player on Down syndrome-associated leukemogenesis. Less is known about the functions of RUNX1 in other tissues and organs, although growing reports show important implications in development or homeostasis of neural tissues, muscle, heart, bone, ovary, or the endothelium, among others. Even less is understood about the consequences on these tissues of RUNX1 gene dosage alterations in the context of Down syndrome. In this review, we summarize the current knowledge on RUNX1 activities outside blood/leukemia, while suggesting for the first time their potential relation to specific Trisomy 21 co-occurring conditions.
Conclusion
Our concise review on the emerging RUNX1 roles in different tissues outside the hematopoietic context provides a number of well-funded hypotheses that will open new research avenues toward a better understanding of RUNX1-mediated transcription in health and disease, contributing to novel potential diagnostic and therapeutic strategies for Down syndrome-associated conditions.
Background
In‐hospital cardiac arrest (IHCA) is a major public health problem with significant mortality. A better understanding of where IHCA occurs in hospitals (intensive care unit ICU versus ...monitored ward telemetry versus unmonitored ward) could inform strategies for reducing preventable deaths.
Methods and Results
This is a retrospective study of adult IHCA events in the Get with the Guidelines—Resuscitation database from January 2003 to September 2010. Unadjusted analyses were used to characterize patient, arrest, and hospital‐level characteristics by hospital location of arrest (ICU versus inpatient ward). IHCA event rates and outcomes were plotted over time by arrest location. Among 85 201 IHCA events at 445 hospitals, 59% (50 514) occurred in the ICU compared to 41% (34 687) on the inpatient wards. Compared to ward patients, ICU patients were younger (64±16 years versus 69±14; P<0.001) and more likely to have a presenting rhythm of ventricular tachycardia/ventricular fibrillation (21% versus 17%; P<0.001). In the ICU, mean event rate/1000 bed‐days was 0.337 (±0.215) compared with 0.109 (±0.079) for telemetry wards and 0.134 (±0.098) for unmonitored wards. Of patients with an arrest in the ICU, the adjusted mean survival to discharge was 0.140 (0.037) compared with the unmonitored wards 0.106 (0.037) and telemetry wards 0.193 (0.074). More IHCA events occurred in the ICU compared to the inpatient wards and there was a slight increase in events/1000 patient bed‐days in both locations.
Conclusions
Survival rates vary based on location of IHCA. Optimizing patient assignment to unmonitored wards versus telemetry wards may contribute to improved survival after IHCA.
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