Ras effector switching promotes divergent cell fates in C. elegans vulval patterning

The C. elegans vulva is patterned by epidermal growth factor (EGF) activation of Ras to control 1° fate, and 1° fate induces antagonistic Notch-dependent 2° fate. Furthermore, a spatial EGF gradient, in addition to inducing 1° fate, directly contributes to 2° fate via an unknown pathway. We find that in addition to its canonical effector, Raf, vulval Ras utilizes an exchange factor for the Ral small GTPase (RalGEF), such that Ras-RalGEF-Ral antagonizes Ras-Raf pro-1° fate activity. Consistent with its restricted expression pattern, Ral participates in EGF pro-2° activity. Thus, we have delineated a Ras effector-switching mechanism whereby position within the morphogen gradient dictates that Ras effector usage is switched to RalGEF from Raf to promote 2° instead of 1° fate. Our observations define the utility of Ras effector switching during normal development and may provide a possible mechanistic basis for cell and cancer-type differences in effector dependency and activation.     

Prolactin-mediated regulation of lipid biosynthesis genes in vivo in the lactating mammary epithelial cell

Prolactin (PRL) is known to play an essential role in mammary alveolar proliferation in the pregnant mouse, but its role in lactation has been more difficult to define. Genetic manipulations that alter expression of the PRL receptor and its downstream signaling molecules resulted in developmental defects that may directly or indirectly impact secretory activation and lactation. To examine the in vivo role of PRL specifically in lactation, bromocriptine (BrCr) was administered every 8 h to lactating mice on the second day postpartum, resulting in an ~95% decrease in serum PRL levels. Although morphological changes in secretory alveoli were slight, by 8 h of BrCr, pup growth was inhibited significantly. Phosphorylated STAT5 fell to undetectable levels within 4 h. Decreased milk protein gene expression, β-casein, and α-lactalbumin, was observed after 8 h of treatment. To assess mammary-specific effects on lipid synthesis genes, we isolated mammary epithelial cells (MECs) depleted of mammary adipocytes. Expression of genes involved in glucose uptake, glycolysis, pentose phosphate shunt, de novo synthesis of fatty acids, and biosynthesis of triacylglycerides was decreased up to 19-fold in MECs by just 8 h of BrCr treatment. Glands from BrCr-treated mice showed a twofold reduction in intracellular cytoplasmic lipid droplets and a reduction in cytosolic β-casein. These data demonstrate that PRL signaling regulates MEC-specific lipogenic gene expression and that PRL signals coordinate the milk synthesis and mammary epithelial cell survival during lactation in the mouse.     

Discovery of a piperazine urea based compound as a potent, selective, orally bioavailable melanocortin subtype-4 receptor partial agonist

We report the discovery of piperazine urea based compound 1, a potent, selective, orally bioavailable melanocortin subtype-4 receptor partial agonist. Compound 1 shows anti-obesity efficacy without potentiating erectile activity in the rodent models.     

Functional insight into LOAD-associated microglial response genes

Alzheimer''s disease (AD) is characterized by the presence of amyloid beta (Aβ) plaques and neurofibrillary tangles (NFTs), neuronal and synaptic loss and inflammation of the central nervous system (CNS). The majority of AD research has been dedicated to the understanding of two major AD hallmarks (i.e. Aβ and NFTs); however, recent genome-wide association studies (GWAS) data indicate neuroinflammation as having a critical role in late-onset AD (LOAD) development, thus unveiling a novel avenue for AD therapeutics. Recent evidence has provided much support to the innate immune system''s involvement with AD progression; however, much remains to be uncovered regarding the role of glial cells, specifically microglia, in AD. Moreover, numerous variants in immune and/or microglia-related genes have been identified in whole-genome sequencing and GWAS analyses, including such genes as TREM2, CD33, APOE, API1, MS4A, ABCA7, BIN1, CLU, CR1, INPP5D, PICALM and PLCG2. In this review, we aim to provide an insight into the function of the major LOAD-associated microglia response genes.     

Direct measurement of single-stranded DNA intermediates in mammalian cells by quantitative polymerase chain reaction

Single-stranded DNA (ssDNA) intermediates are formed in multiple cellular processes, including DNA replication and recombination. Here, we describe a quantitative polymerase chain reaction (qPCR)-based assay to quantitate ssDNA intermediates, specifically the 3′ ssDNA product of resection at specific DNA double-strand breaks induced by the AsiSI restriction enzyme in human cells. We protect the large mammalian genome from shearing by embedding the cells in low-gelling-point agar during genomic DNA extraction and measure the levels of ssDNA intermediates by qPCR following restriction enzyme digestion. This assay is more quantitative and precise compared with existing immunofluorescence-based methods.     

Deep,Quantitative Coverage of the Lysine Acetylome Using Novel Anti-acetyl-lysine Antibodies and an Optimized Proteomic Workflow

Introduction of antibodies specific for acetylated lysine has significantly improved the detection of endogenous acetylation sites by mass spectrometry. Here, we describe a new, commercially available mixture of anti-lysine acetylation (Kac) antibodies and show its utility for in-depth profiling of the acetylome. Specifically, seven complementary monoclones with high specificity for Kac were combined into a final anti-Kac reagent which results in at least a twofold increase in identification of Kac peptides over a commonly used Kac antibody. We outline optimal antibody usage conditions, effective offline basic reversed phase separation, and use of state-of-the-art LC-MS technology for achieving unprecedented coverage of the acetylome. The methods were applied to quantify acetylation sites in suberoylanilide hydroxamic acid-treated Jurkat cells. Over 10,000 Kac peptides from over 3000 Kac proteins were quantified from a single stable isotope labeling by amino acids in cell culture labeled sample using 7.5 mg of peptide input per state. This constitutes the deepest coverage of acetylation sites in quantitative experiments obtained to-date. The approach was also applied to breast tumor xenograft samples using isobaric mass tag labeling of peptides (iTRAQ4, TMT6 and TMT10-plex reagents) for quantification. Greater than 6700 Kac peptides from over 2300 Kac proteins were quantified using 1 mg of tumor protein per iTRAQ 4-plex channel. The novel reagents and methods we describe here enable quantitative, global acetylome analyses with depth and sensitivity approaching that obtained for other well-studied post-translational modifications such as phosphorylation and ubiquitylation, and should have widespread application in biological and clinical studies employing mass spectrometry-based proteomics.Lysine acetylation (Kac)¹ is a well conserved, reversible post-translational modification (PTM) involved in multiple cellular processes (1). Acetylation is regulated by two classes of enzymes: lysine acetyltransferases (KATs) and histone deacetylases (HDACs) (2–4). This modification was originally identified as a nuclear event on histone proteins and has been long appreciated for its role in epigenetic and DNA-dependent processes. With the help of a growing number of large-scale acetylation studies, it has become evident that lysine acetylation is ubiquitous, also occurring on cytoplasmic and mitochondrial proteins and has a role in signaling, metabolism, and immunity (1, 4–6). Therefore, the examination of lysine acetylation on nonhistone proteins has gained a prominent role in PTM analysis.To date, the identification of large numbers of acetylation sites has been challenging because of the substoichiometric nature of this modification (7, 8). Additionally, global acetylation is generally less abundant than phosphorylation and ubiquitylation (1). The introduction of antibodies specific for lysine acetylation has significantly improved the ability to enrich and identify thousands of sites (9–14). A landmark study by Choudhary et al. used anti-Kac antibodies to globally map 3600 lysine acetylation sites on 1750 proteins, thereby demonstrating the feasibility of profiling the acetylome (10). A more recent study by Lundby et al. investigated the function and distribution of acetylation sites in 16 different rat tissues, and identified, in aggregate, 15,474 acetylation sites from 4541 proteins (12).Although anti-acetyl lysine antibodies have been a breakthrough for globally mapping acetylation sites (9–12), it remains a challenge to identify large numbers of lysine acetylation sites from a single sample, as is now routinely possible for phosphorylation and ubiquitylation (13, 15–18). To improve the depth-of-coverage in acetylation profiling experiments there is a clear need for (1) alternative anti-acetyl lysine antibodies with higher specificity, (2) optimized antibody usage parameters, and (3) robust proteomic workflows that permit low to moderate protein input. In this study, we describe a newly commercialized mixture of anti-Kac antibodies and detail a complete proteomic workflow for achieving unprecedented coverage of the acetylome from a single stable isotope labeling by amino acids in cell culture (SILAC) labeled sample as well as isobaric tags for relative and absolute quantitation (iTRAQ)- and tandem mass tag (TMT)-labeled samples.     

Similar but Different: Dynamic Social Network Analysis Highlights Fundamental Differences between the Fission-Fusion Societies of Two Equid Species,the Onager and Grevy’s Zebra

Understanding why animal societies take on the form that they do has benefited from insights gained by applying social network analysis to patterns of individual associations. Such analyses typically aggregate data over long time periods even though most selective forces that shape sociality have strong temporal elements. By explicitly incorporating the temporal signal in social interaction data we re-examine the network dynamics of the social systems of the evolutionarily closely-related Grevy’s zebras and wild asses that show broadly similar social organizations. By identifying dynamic communities, previously hidden differences emerge: Grevy’s zebras show more modularity than wild asses and in wild asses most communities consist of solitary individuals; and in Grevy’s zebras, lactating females show a greater propensity to switch communities than non-lactating females and males. Both patterns were missed by static network analyses and in general, adding a temporal dimension provides insights into differences associated with the size and persistence of communities as well as the frequency and synchrony of their formation. Dynamic network analysis provides insights into the functional significance of these social differences and highlights the way dynamic community analysis can be applied to other species.