The general mode of translation inhibition by macrolide antibiotics.

Macrolides are clinically important antibiotics thought to inhibit bacterial growth by impeding the passage of newly synthesized polypeptides through the nascent peptide exit tunnel of the bacterial ribosome. Recent data challenged this view by showing that macrolide antibiotics can differentially affect synthesis of individual proteins. To understand the general mechanism of macrolide action, we used genome-wide ribosome profiling and analyzed the redistribution of ribosomes translating highly expressed genes in bacterial cells treated with high concentrations of macrolide antibiotics. The metagene analysis indicated that inhibition of early rounds of translation, which would be characteristic of the conventional view of macrolide action, occurs only at a limited number of genes. Translation of most genes proceeds past the 5'-proximal codons and can be arrested at more distal codons when the ribosome encounters specific short sequence motifs. The problematic sequence motifs are confined to the nascent peptide residues in the peptidyl transferase center but not to the peptide segment that contacts the antibiotic molecule in the exit tunnel. Therefore, it appears that the general mode of macrolide action involves selective inhibition of peptide bond formation between specific combinations of donor and acceptor substrates. Additional factors operating in the living cell but not functioning during in vitro protein synthesis may modulate site-specific action of macrolide antibiotics

The general mode of translation inhibition by macrolide antibiotics.

Macrolides are clinically important antibiotics thought to inhibit bacterial growth by impeding the passage of newly synthesized polypeptides through the nascent peptide exit tunnel of the bacterial ribosome. Recent data challenged this view by showing that macrolide antibiotics can differentially affect synthesis of individual proteins. To understand the general mechanism of macrolide action, we used genome-wide ribosome profiling and analyzed the redistribution of ribosomes translating highly expressed genes in bacterial cells treated with high concentrations of macrolide antibiotics. The metagene analysis indicated that inhibition of early rounds of translation, which would be characteristic of the conventional view of macrolide action, occurs only at a limited number of genes. Translation of most genes proceeds past the 5'-proximal codons and can be arrested at more distal codons when the ribosome encounters specific short sequence motifs. The problematic sequence motifs are confined to the nascent peptide residues in the peptidyl transferase center but not to the peptide segment that contacts the antibiotic molecule in the exit tunnel. Therefore, it appears that the general mode of macrolide action involves selective inhibition of peptide bond formation between specific combinations of donor and acceptor substrates. Additional factors operating in the living cell but not functioning during in vitro protein synthesis may modulate site-specific action of macrolide antibiotics

The IRF4 Gene Regulatory Module Functions as a Read-Write Integrator to Dynamically Coordinate T Helper Cell Fate

Transcriptional regulation during CD4+ T cell fate decisions enables their differentiation into distinct states, guiding immune responses toward antibody production via Tfh cells or inflammation by Teff cells. Tfh-Teff cell fate commitment is regulated by mutual antagonism between the transcription factors Bcl6 and Blimp-1. Here we examined how T cell receptor (TCR) signals establish and arbitrate Bcl6-Blimp-1 counter-antagonism. We found that the TCR-signal-induced transcription factor Irf4 is essential for the differentiation of Bcl6-expressing Tfh and Blimp-1-expressing Teff cells. Increased TCR signaling raised Irf4 amounts and promoted Teff cell fates at the expense of Tfh ones. Importantly, orthogonal induction of Irf4 expression redirected Tfh cell fate trajectories toward those of Teff. Mechanistically, we linked greater Irf4 abundance with its recruitment toward low-affinity binding sites within Teff cell cis-regulatory elements, including those of Prdm1. We propose that the Irf4 locus functions as the "reader" of TCR signal strength, and in turn, concentration-dependent activity of Irf4 "writes" T helper fate choice

Improved Statistical Methods Enable Greater Sensitivity in Rhythm Detection for Genome-Wide Data

Robust methods for identifying patterns of expression in genome-wide data are important for generating hypotheses regarding gene function. To this end, several analytic methods have been developed for detecting periodic patterns. We improve one such method, JTK_CYCLE, by explicitly calculating the null distribution such that it accounts for multiple hypothesis testing and by including non-sinusoidal reference waveforms. We term this method empirical JTK_CYCLE with asymmetry search, and we compare its performance to JTK_CYCLE with Bonferroni and Benjamini-Hochberg multiple hypothesis testing correction, as well as to five other methods: cyclohedron test, address reduction, stable persistence, ANOVA, and F24. We find that ANOVA, F24, and JTK_CYCLE consistently outperform the other three methods when data are limited and noisy; empirical JTK_CYCLE with asymmetry search gives the greatest sensitivity while controlling for the false discovery rate. Our analysis also provides insight into experimental design and we find that, for a fixed number of samples, better sensitivity and specificity are achieved with higher numbers of replicates than with higher sampling density. Application of the methods to detecting circadian rhythms in a metadataset of microarrays that quantify time-dependent gene expression in whole heads of Drosophila melanogaster reveals annotations that are enriched among genes with highly asymmetric waveforms. These include a wide range of oxidation reduction and metabolic genes, as well as genes with transcripts that have multiple splice forms.</p

Heat-Map of Gene Expression Analysis of Lacrimal Gland and Other Ectodermal Tissues.

<p>Heat-Map showing genes with significant differential expression (FDR<0.01 for both t-test and U test) for all genes studied. The heat-map shows all the genes that were significant in at least one of the comparisons, which was 7787 total. The number significant in the LG vs all other comparisons (LG-specific genes) was 1855. The tissue types are color coded: bars along the top indicate which tissue each sample belongs to; bars down the left-hand side indicate which tissues were significantly differentially expressed with respect to LG for each gene.</p

Human Lacrimal Gland Gene Expression

<div><p>Background</p><p>The study of human lacrimal gland biology and development is limited. Lacrimal gland tissue is damaged or poorly functional in a number of disease states including dry eye disease. Development of cell based therapies for lacrimal gland diseases requires a better understanding of the gene expression and signaling pathways in lacrimal gland. Differential gene expression analysis between lacrimal gland and other embryologically similar tissues may be helpful in furthering our understanding of lacrimal gland development.</p><p>Methods</p><p>We performed global gene expression analysis of human lacrimal gland tissue using Affymetrix ^® gene expression arrays. Primary data from our laboratory was compared with datasets available in the NLM GEO database for other surface ectodermal tissues including salivary gland, skin, conjunctiva and corneal epithelium.</p><p>Results</p><p>The analysis revealed statistically significant difference in the gene expression of lacrimal gland tissue compared to other ectodermal tissues. The lacrimal gland specific, cell surface secretory protein encoding genes and critical signaling pathways which distinguish lacrimal gland from other ectodermal tissues are described.</p><p>Conclusions</p><p>Differential gene expression in human lacrimal gland compared with other ectodermal tissue types revealed interesting patterns which may serve as the basis for future studies in directed differentiation among other areas.</p></div

Differentially Regulated Signaling Pathways in Lacrimal Gland.

<p>Bar plot showing the top 10 differentially regulated developmental signaling pathways in lacrimal gland in addition to 9 (depicted in a lighter shade of red) pathways of potential interest in lacrimal gland development.</p

List of Drugs and Their Action Targeting Differentially Expressed Genes in Lacrimal Gland.

<p>Drugs categorized by differentially expressed targeted genes in lacrimal gland and their action (inhibition blue, activation red).</p

Heat Map of Top-50 Genes Expressed in Lacrimal Gland Versus Other Ectodermal Tissues.

<p>Heat-map of the most differentially expressed genes in lacrimal gland versus other tissue types (top 50). Only genes that have FDR <0.01 in all pairwise comparisons and prioritized by average fold change either up or down regulated between lacrimal gland and each other tissue. The inset pie chart shows the total number of differentially expressed genes which are up, down or ambiguously differentially expressed.</p