Bedaquiline (BDQ), an ATP synthase inhibitor, is the first drug to be approved for treatment of multidrug-resistant tuberculosis in decades. Though BDQ has shown excellent efficacy in clinical trials, its early bactericidal activity during the first week of chemotherapy is minimal. Here, using microfluidic devices and time-lapse microscopy of Mycobacterium tuberculosis, we confirm the absence of significant bacteriolytic activity during the first 3–4 days of exposure to BDQ. BDQ-induced inhibition of ATP synthesis leads to bacteriostasis within hours after drug addition. Transcriptional and proteomic analyses reveal that M. tuberculosis responds to BDQ by induction of the dormancy regulon and activation of ATP-generating pathways, thereby maintaining bacterial viability during initial drug exposure. BDQ-induced bacterial killing is significantly enhanced when the mycobacteria are grown on non-fermentable energy sources such as lipids (impeding ATP synthesis via glycolysis). Our results show that BDQ exposure triggers a metabolic remodelling in mycobacteria, thereby enabling transient bacterial survival.

We describe a method that combines an optimized titanium dioxide protocol and hydrophilic interaction liquid chromatography to simultaneously enrich, identify and quantify phosphopeptides and formerly N-linked sialylated glycopeptides to monitor changes associated with cell signaling during mouse brain development. We initially applied the method to enriched membrane fractions from HeLa cells, which allowed the identification of 4468 unique phosphopeptides and 1809 formerly N-linked sialylated glycopeptides. We subsequently combined the method with isobaric tagging for relative quantification to compare changes in phosphopeptide and formerly N- The development of novel methods to simultaneously monitor multiple protein post-translational modifications (PTMs) 1 is an attractive tool for researchers. There is increasing evidence that both phosphorylation and glycosylation play important roles in cellular signaling networks during development and transformation of cells. Development of the mammalian brain is initiated during the embryonic stage and continues until adulthood. The brain originates through the proliferation of the telencephalon, the anterior part of the neural tube. Following differentiation, cells begin to migrate and associate into different brain structures. The brain structures are reorganized with the extension of axons and dendrites to communicate via synaptic terminal interactions (1, 2). These molecular interactions are governed by cell surface receptors that are often post-translationally modified with both N-linked glycans and phosphate groups, and studies have suggested that extracellular glycans play vital roles in the regulation of signal transduction pathways (3). For example, the myelin-associated glycoprotein (MAG) binds to cell surface glyco-conjugates GD1a, GT1b and Nogo receptors to form signaling complexes that inhibit axon outgrowth, whereas inhibition of Rho kinase reverses this process in a number of nerve cell types (4). There is growing evidence that both the differentiation and migration of neurons and the guidance of axons are regulated by sialic acid-containing glycoconjugates (5-7). Dietary supplementation of sialic acid leads to increases in sialic acid-containing glycoproteins in the frontal cortex and is associated with faster learning and memory in piglets (8). The nervous system contains an abundant array of sialylated molecules and it is therefore not sur-

Human embryonic stem cells (hESCs) can differentiate into neural stem cells (NSCs), which can further be differentiated into neurons and glia cells. Therefore, these cells have huge potential as source for treatment of neurological diseases. Membrane-associated proteins are very important in cellular signaling and recognition, and their function and activity are frequently regulated by posttranslational modifications such as phosphorylation and glycosylation. To obtain information about membraneassociated proteins and their modified amino acids potentially involved in changes of hESCs and NSCs as well as to investigate potential new markers for these two cell stages, we performed large-scale quantitative membrane-proteomic of hESCs and NSCs. This approach employed membrane purification followed by peptide dimethyl labeling and peptide enrichment to study the membrane subproteome as well as changes in phosphorylation and sialylation between hESCs and NSCs. Combining proteomics and modification specific proteomics we identified a total of 5105 proteins whereof 57% contained transmembrane domains or signal peptides. The enrichment strategy yielded a total of 10,087 phosphorylated peptides in which 78% of phosphopeptides were identified with >99% confidence in site assignment and 1810 unique formerly sialylated N-linked glycopeptides. Several proteins were identified as significantly regulated in hESCs and NSC, including proteins involved in the early embryonic and neural development. In the latter group of proteins, we could identify potential NSC markers as Pluripotent embryonic stem cell (ESC)1 -derived neural stem cells (NSCs) can differentiate into neurons and glia cells of the central nervous system (1), including specialized neuron types like dopaminergic, representing a potential source for treatment of neurological diseases, such as ParkinsonЈs disease. Therefore, a better understanding of the cellular processes behind the changes of hESCs into NSCs, including solid markers for each cell type, is fundamental to move forward with a successful regenerative cell therapy and to investigate the early human neurogenesis processes.Many markers have been reported for the two types of stem cells (2, 3), however several of these markers are also identified in other stem or progenitor cells such as CD133 (Prominin-1) (4). Discovery of cell surface specific markers for differentiated stem cells is highly relevant for future clinical applications. In particular being able to distinguish the developmental stages of the differentiation from parental stem cells to fully mature cells would allow a correct manipulation and isolation of the cell type of interest. Moreover, such study 1 The abbreviations used are: CID, collision-induced dissociation; FDR, false discovery rate; FLR, false localization rate; HCD, highenergy collision induced dissociation; hESC, human embryonic stem cell; LC-MS/MS, liquid chromatography-tandem mass spectrometry; MSA, multistage activation; NSC, neural stem cell; PTM, post-translational modificat...

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A comprehensive quantitative analysis of changes in protein phosphorylation preceding or accompanying transcriptional activation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in 5L rat hepatoma cells was performed using the SILAC approach. Following exposure of the cells to DMSO or 1 nM TCDD for 0.5 to 2 h, 5648 phosphorylated peptides corresponding to 2156 phosphoproteins were identified. Eight peptides exhibited a statistically significantly altered phosphorylation because of TCDD exposure and 22 showed a regulation factor of ≥ 1.5 in one of the experiments per time point. The vast majority of the TCCD-induced phosphorylation changes had not been reported before. The transcription factor ARNT, the obligate partner for gene activation by the TCDD-bound Ah receptor, exhibited an up-regulation of its Ser77 phosphorylation, a modification known to control the differential binding of ARNT homodimers and heterodimers to different enhancers suggesting that this phosphorylation represents a novel mechanism contributing to the alteration of gene expression by TCDD. Other proteins with altered phosphorylation included, among others, various transcriptional coregulators previously unknown to participate in TCDD-induced gene activation, regulators of small GTPases of the Ras superfamily, UBX domain-containing proteins and the oncogenic protein LYRIC. The results open up new directions for research on the molecular mechanisms of dioxin action and toxicity.

Cells must continuously adapt to changing environments and, thus, have evolved mechanisms allowing them to respond to repeated stimuli. For example, faster gene induction upon a repeated stimulus aids adaptation - a process known as reinduction memory. However, whether such a memory exists for gene repression is unclear. Here, we studied gene repression across repeated carbon source shifts in over 2,500 single Saccharomyces cerevisiae cells. By monitoring the expression of a carbon source-responsive gene, galactokinase 1 (Gal1), and mathematical modeling, we discovered repression memory at the population and single-cell level. Using a repressor model to estimate single-cell repression parameters, we show that repression memory is due to a shortened repression delay, the estimated time gap between carbon source shift and Gal1 expression termination, upon the repeated carbon source shift. Additionally, we show that cells lacking Elp6 display a gain-of-repression-memory phenotype characterized by a stronger decrease in repression delay between two consecutive carbon source shifts. Collectively, our study provides the first quantitative description of repression memory in single cells.

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