Transcriptional effects of CRP* expression in Escherichia coli

<p>Abstract</p> <p>Background</p> <p><it>Escherichia coli </it>exhibits diauxic growth in sugar mixtures due to CRP-mediated catabolite repression and inducer exclusion related to phosphotransferase system enzyme activity. Replacement of the native <it>crp </it>gene with a catabolite repression mutant (referred to as <it>crp</it>*) enables co-utilization of glucose and other sugars in <it>E. coli</it>. While previous studies have examined the effects of expressing CRP* mutants on the expression of specific catabolic genes, little is known about the global transcriptional effects of CRP* expression. In this study, we compare the transcriptome of <it>E. coli </it>W3110 (expressing wild-type CRP) to that of mutant strain PC05 (expressing CRP*) in the presence and absence of glucose.</p> <p>Results</p> <p>The glucose effect is significantly suppressed in strain PC05 relative to strain W3110. The expression levels of glucose-sensitive genes are generally not altered by glucose to the same extent in strain PCO5 as compared to W3110. Only 23 of the 80 genes showing significant differential expression in the presence of glucose for strain PC05 are present among the 418 genes believed to be directly regulated by CRP. Genes involved in central carbon metabolism (including several TCA cycle genes) and amino acid biosynthesis, as well as genes encoding nutrient transport systems are among those whose transcript levels are most significantly affected by CRP* expression.</p> <p>We present a detailed transcription analysis and relate these results to phenotypic differences between strains expressing wild-type CRP and CRP*. Notably, CRP* expression in the presence of glucose results in an elevated intracellular NADPH concentration and reduced NADH concentration relative to wild-type CRP. Meanwhile, a more drastic decrease in the NADPH/NADP⁺ratio is observed for the case of CRP* expression in strains engineered to reduce xylose to xylitol via a heterologously expressed, NADPH-dependent xylose reductase. Altered expression levels of transhydrogenase and TCA cycle genes, among others, are consistent with these observations.</p> <p>Conclusion</p> <p>While the simplest model of CRP*-mediated gene expression assumes insensitivity to glucose (or cAMP), our results show that gene expression in the context of CRP* is very different from that of wild-type in the absence of glucose, and is influenced by the presence of glucose. Most of the transcription changes in response to CRP* expression are difficult to interpret in terms of possible systematic effects on metabolism. Elevated NADPH availability resulting from CRP* expression suggests potential biocatalytic applications of <it>crp* </it>strains that extend beyond relief of catabolite repression.</p

How Deep Is Deep enough for RNA-Seq Profiling of Bacterial Transcriptomes?

Background: High-throughput sequencing of cDNA libraries (RNA-Seq) has proven to be a highly effective approach for studying bacterial transcriptomes. A central challenge in designing RNA-Seq-based experiments is estimating a priori the number of reads per sample needed to detect and quantify thousands of individual transcripts with a large dynamic range of abundance. Results: We have conducted a systematic examination of how changes in the number of RNA-Seq reads per sample influences both profiling of a single bacterial transcriptome and the comparison of gene expression among samples. Our findings suggest that the number of reads typically produced in a single lane of the Illumina HiSeq sequencer far exceeds the number needed to saturate the annotated transcriptomes of diverse bacteria growing in monoculture. Moreover, as sequencing depth increases, so too does the detection of cDNAs that likely correspond to spurious transcripts or genomic DNA contamination. Finally, even when dozens of barcoded individual cDNA libraries are sequenced in a single lane, the vast majority of transcripts in each sample can be detected and numerous genes differentially expressed between samples can be identified. Conclusions: Our analysis provides a guide for the many researchers seeking to determine the appropriate sequencing depth for RNA-Seq-based studies of diverse bacterial species

Preliminary Findings from an Interventional Study using Network Analysis to Support Management in Local Health Departments in Florida

Management is the core service that integrates and coordinates essential public health services. Managers of local health departments (LHDs) are experts in practice but may not have expertise in organizational management. We conducted an evidence-based training intervention in 10 LHDs in Florida to support managers’ decision-making on organizational integration and coordination. We deployed a standard survey to collect organizational network measurements pre and post intervention. We presented results as evidence-based performance feedback and interviewed managers to document how they used the results in the context of each organization. Post intervention we found unexpected, significantly higher network centralization in daily work. We attributed this increase in hierarchical communication to preparations for a statewide accreditation initiative. When QI initiatives are undertaken globally within a state, managers and leaders need to be alert for possible impact on autonomous decision-making of professionals at the point of service which could affect service delivery

Inversion Effects in the Expert Classification of Mammograms and Faces

A hallmark of a perceptual expert is the ability to detect and categorize stimuli in their domain of expertise after brief exposure. For example, expert radiologists can differentiate between “abnormal” & “normal” mammograms after a 250 msec exposure. It has been speculated that rapid detection depends on a global analysis referred to as holistic perception. Holistic processing in radiology seems similar to holistic perception in which a stimulus like a face is perceived as an integrated whole, not in terms of its individual features. Holistic processing is typically subject to inversion effects in which the inverted image is harder to process/recognize. Is radiological perception similarly subject to inversion effects? Eleven experienced radiologists (> 5 years of radiological experience) and ten resident radiologists (<5 years of radiological experience) judged upright and inverted bilateral mammograms as “normal” or “abnormal”. For comparison, the same participants judged whether upright and inverted faces were “happy” or “neutral”. We obtained the expected inversion effect for faces. Expression discrimination was superior for upright faces. For mammograms, experienced radiologists exhibited a similar inversion effect, showing higher accuracy for upright than for inverted mammograms. Less experienced radiology residents performed more poorly than experienced radiologists and demonstrated no inversion effect with mammograms. These results suggest that the ability to discriminate normal from abnormal mammograms is a form of learned, holistic processing

The Life and Death of Dense Molecular Clumps in the Large Magellanic Cloud

We report the results of a high spatial (parsec) resolution HCO+ (J = 1-0) and HCN (J = 1-0) emission survey toward the giant molecular clouds of the star formation regions N105, N113, N159, and N44 in the Large Magellanic Cloud. The HCO+ and HCN observations at 89.2 and 88.6 GHz, respectively, were conducted in the compact configuration of the Australia Telescope Compact Array. The emission is imaged into individual clumps with masses between 10^2 and 10^4 solar masses and radii of <1 pc to ~2 pc. Many of the clumps are coincident with indicators of current massive star formation, indicating that many of the clumps are associated with deeply-embedded forming stars and star clusters. We find that massive YSO-bearing clumps tend to be larger (>1 pc), more massive (M > 10^3 solar masses), and have higher surface densities (~1 g cm^-2), while clumps without signs of star formation are smaller (<1 pc), less massive (M < 10^3 solar masses), and have lower surface densities (~0.1 g cm^-2). The dearth of massive (M >10^3 solar masses) clumps not bearing massive YSOs suggests the onset of star formation occurs rapidly once the clump has attained physical properties favorable to massive star formation. Using a large sample of LMC massive YSO mid-IR spectra, we estimate that ~2/3 of the massive YSOs for which there are Spitzer mid-IR spectra are no longer located in molecular clumps; we estimate that these young stars/clusters have destroyed their natal clumps on a time scale of at least 3 x 10^{5}$ yrs.Comment: Accepted to ApJ 3-19-201

Flow-induced HDAC1 phosphorylation and nuclear export in angiogenic sprouting

Angiogenesis requires the coordinated growth and migration of endothelial cells (ECs), with each EC residing in the vessel wall integrating local signals to determine whether to remain quiescent or undergo morphogenesis. These signals include vascular endothelial growth factor (VEGF) and flow-induced mechanical stimuli such as interstitial flow, which are both elevated in the tumor microenvironment. However, it is not clear how VEGF signaling and mechanobiological activation due to interstitial flow cooperate during angiogenesis. Here, we show that endothelial morphogenesis is histone deacetylase-1- (HDAC1) dependent and that interstitial flow increases the phosphorylation of HDAC1, its activity, and its export from the nucleus. Furthermore, we show that HDAC1 inhibition decreases endothelial morphogenesis and matrix metalloproteinase-14 (MMP14) expression. Our results suggest that HDAC1 modulates angiogenesis in response to flow, providing a new target for modulating vascularization in the clinic

The SH3 and cysteine-rich domain 3 (Stac3) gene is important to growth, fiber composition, and calcium release from the sarcoplasmic reticulum in postnatal skeletal muscle

The SH3 and cysteine-rich domain 3 (Stac3) gene is specifically expressed in the skeletal muscle. Stac3 knockout mice die perinatally. In this study, we determined the potential role of Stac3 in postnatal skeletal muscle growth, fiber composition, and contraction by generating conditional Stac3 knockout mice. We disrupted the Stac3 gene in 4-week-old male mice using the Flp-FRT and tamoxifen-inducible Cre-loxP systems. RT-qPCR and western blotting analyses of the limb muscles of target mice indicated that nearly all Stac3 mRNA and more than 70 % of STAC3 protein were deleted 4 weeks after tamoxifen injection. Postnatal Stac3 deletion inhibited body and limb muscle mass gains. Histological staining and gene expression analyses revealed that postnatal Stac3 deletion decreased the size of myofibers and increased the percentage of myofibers containing centralized nuclei, with no effect on the total myofiber number. Grip strength and grip time tests indicated that postnatal Stac3 deletion decreased limb muscle strength in mice. Muscle contractile tests revealed that postnatal Stac3 deletion reduced electrostimulation-induced but not the ryanodine receptor agonist caffeine-induced maximal force output in the limb muscles. Calcium imaging analysis of single flexor digitorum brevis myofibers indicated that postnatal Stac3 deletion reduced electrostimulation- but not caffeine-induced calcium release from the sarcoplasmic reticulum. This study demonstrates that STAC3 is important to myofiber hypertrophy, myofiber-type composition, contraction, and excitation-induced calcium release from the sarcoplasmic reticulum in the postnatal skeletal muscle.https://doi.org/10.1186/s13395-016-0088-

Toward the Elucidation of Cytoplasmic Diversity in North American Grape Breeding Programs

Plants have an intriguing tripartite genetic system: Nuclear genome 9 Mitochondria 9 Plastids and their interactions may impact germplasm breeding. In grapevine, the study of cytoplasmic genomes has been limited, and their role with respect to grapevine germplasm diversity has yet to be elucidated. In the present study, the results of an analysis of the cytoplasmic diversity among 6073 individuals (comprising cultivars, interspecific hybrids and segregating progenies) are presented. Genotyping by sequencing (GBS) was used to elucidate plastid and mitochondrial DNA sequences, and results were analyzed using multivariate techniques. Single nucleotide polymorphism (SNP) effects were annotated in reference to plastid and mitochondrial genome sequences. The cytoplasmic diversity identified was structured according to synthetic domestication groups (wine and raisin/table gr.ape types) and interspecific-hybridization-driven groups with introgression from North American Vitis species, identifying five cytoplasmic groups and four major clusters. Fifty-two SNP markers were used to describe the diversity of the germplasm. Ten organelle genes showed distinct SNP annotations and effect predictions, of which six were chloroplast-derived and three were mitochondrial genes, in addition to one mitochondrial SNP affecting a nonannotated open reading frame. The results suggest that the application of GBS will aid in the study of cytoplasmic genomes in grapevine, which will enable further studies on the role of cytoplasmic genomes in grapevine germplasm, and then allow the exploitation of these sources of diversity in breeding

Nonmuscle myosin heavy chain IIA mediates integrin LFA-1 de-adhesion during T lymphocyte migration

Precise spatial and temporal regulation of cell adhesion and de-adhesion is critical for dynamic lymphocyte migration. Although a great deal of information has been learned about integrin lymphocyte function–associated antigen (LFA)-1 adhesion, the mechanism that regulates efficient LFA-1 de-adhesion from intercellular adhesion molecule (ICAM)-1 during T lymphocyte migration is unknown. Here, we show that nonmuscle myosin heavy chain IIA (MyH9) is recruited to LFA-1 at the uropod of migrating T lymphocytes, and inhibition of the association of MyH9 with LFA-1 results in extreme uropod elongation, defective tail detachment, and decreased lymphocyte migration on ICAM-1, without affecting LFA-1 activation by chemokine CXCL-12. This defect was reversed by a small molecule antagonist that inhibits both LFA-1 affinity and avidity regulation, but not by an antagonist that inhibits only affinity regulation. Total internal reflection fluorescence microscopy of the contact zone between migrating T lymphocytes and ICAM-1 substrate revealed that inactive LFA-1 is selectively localized to the posterior of polarized T lymphocytes, whereas active LFA-1 is localized to their anterior. Thus, during T lymphocyte migration, uropodal adhesion depends on LFA-1 avidity, where MyH9 serves as a key mechanical link between LFA-1 and the cytoskeleton that is critical for LFA-1 de-adhesion