Profiling Microbial Communities in Idiopathic Granulomatous Mastitis.

Idiopathic granulomatous mastitis (IGM) is a rare and benign inflammatory breast disease with ambiguous aetiology. Contrastingly, lactational mastitis (LM) is commonly diagnosed in breastfeeding women. To investigate IGM aetiology, we profiled the microbial flora of pus and skin in patients with IGM and LM. A total of 26 patients with IGM and 6 patients with LM were included in the study. The 16S rRNA sequencing libraries were constructed from 16S rRNA gene amplified from total DNA extracted from pus and skin swabs in patients with IGM and LM controls. Constructed libraries were multiplexed and paired-end sequenced on HiSeq4000. Metagenomic analysis was conducted using modified microbiome abundance analysis suite customised R-resource for paired pus and skin samples. Microbiome multivariable association analyses were performed using linear models. A total of 21 IGM and 3 LM paired pus and skin samples underwent metagenomic analysis. Bray−Curtis ecological dissimilarity distance showed dissimilarity across four sample types (IGM pus, IGM skin, LM pus, and LM skin; PERMANOVA, p < 0.001). No characteristic dominant genus was observed across the IGM samples. The IGM pus samples were more diverse than corresponding IGM skin samples (Shannon and Simpson index; Wilcoxon paired signed-rank tests, p = 0.022 and p = 0.07). Corynebacterium kroppenstedtii, reportedly associated with IGM in the literature, was higher in IGM pus samples than paired skin samples (Wilcoxon, p = 0.022). Three other species and nineteen genera were statistically significant in paired IGM pus−skin comparison after antibiotic treatment adjustment and multiple comparisons correction. Microbial profiles are unique between patients with IGM and LM. Inter-patient variability and polymicrobial IGM pus samples cannot implicate specific genus or species as an infectious cause for IGM.

Narciclasine attenuates diet-induced obesity by promoting oxidative metabolism in skeletal muscle.

Obesity develops when caloric intake exceeds metabolic needs. Promoting energy expenditure represents an attractive approach in the prevention of this fast-spreading epidemic. Here, we report a novel pharmacological strategy in which a natural compound, narciclasine (ncls), attenuates diet-induced obesity (DIO) in mice by promoting energy expenditure. Moreover, ncls promotes fat clearance from peripheral metabolic tissues, improves blood metabolic parameters in DIO mice, and protects these mice from the loss of voluntary physical activity. Further investigation suggested that ncls achieves these beneficial effects by promoting a shift from glycolytic to oxidative muscle fibers in the DIO mice thereby enhancing mitochondrial respiration and fatty acid oxidation (FAO) in the skeletal muscle. Moreover, ncls strongly activates AMPK signaling specifically in the skeletal muscle. The beneficial effects of ncls treatment in fat clearance and AMPK activation were faithfully reproduced in vitro in cultured murine and human primary myotubes. Mechanistically, ncls increases cellular cAMP concentration and ADP/ATP ratio, which further lead to the activation of AMPK signaling. Blocking AMPK signaling through a specific inhibitor significantly reduces FAO in myotubes. Finally, ncls also enhances mitochondrial membrane potential and reduces the formation of reactive oxygen species in cultured myotubes.

Epigenetic regulation of olfactory receptor gene expression by the Myb-MuvB/dREAM complex.

In both mammals and insects, an olfactory neuron will usually select a single olfactory receptor and repress remaining members of large receptor families. Here we show that a conserved multiprotein complex, Myb-MuvB (MMB)/dREAM, plays an important role in mediating neuron-specific expression of the carbon dioxide (CO(2)) receptor genes (Gr63a/Gr21a) in Drosophila. Activity of Myb in the complex is required for expression of Gr63a/Gr21a and acts in opposition to the histone methyltransferase Su(var)3-9. Consistent with this, we observed repressive dimethylated H3K9 modifications at the receptor gene loci, suggesting a mechanism for silencing receptor gene expression. Conversely, other complex members, Mip120 (Myb-interacting protein 120) and E2F2, are required for repression of Gr63a in inappropriate neurons. Misexpression in mutants is accompanied by an increase in the H3K4me3 mark of active chromatin at the receptor gene locus. Nuclei of CO(2) receptor-expressing neurons contain reduced levels of the repressive subunit Mip120 compared with surrounding neurons and increased levels of Myb, suggesting that activity of the complex can be regulated in a cell-specific manner. Our evidence suggests a model in which olfactory receptors are regulated epigenetically and the MMB/dREAM complex plays a critical role in specifying, maintaining, and modulating the receptor-to-neuron map.

Acetylome study in mouse adipocytes identifies targets of SIRT1 deacetylation in chromatin organization and RNA processing.

SIRT1 is a key protein deacetylase that regulates cellular metabolism through lysine deacetylation on both histones and non-histone proteins. Lysine acetylation is a wide-spread post-translational modification found on many regulatory proteins and it plays an essential role in cell signaling, transcription and metabolism. In mice, SIRT1 has known protective functions during high-fat diet but the acetylome regulated by SIRT1 in adipocytes is not completely understood. Here we conducted acetylome analyses in murine adipocytes treated with small-molecule modulators that inhibit or activate the deacetylase activity of SIRT1. We identified a total of 302 acetylated peptides from 78 proteins in this study. From the list of potential SIRT1 targets, we selected seven candidates and further verified that six of them can be deacetylated by SIRT1 in-vitro. Among them, half of the SIRT1 targets are involved in regulating chromatin structure and the other half is involved in RNA processing. Our results provide a resource for further SIRT1 target validation in fat cells and suggest a potential role of SIRT1 in the regulation of chromatin structure and RNA processing, which may possibly extend to other cell types as well.

Delayed colonization of Bifidobacterium spp. and low prevalence of B. infantis among infants of Asian ancestry born in Singapore: insights from the GUSTO cohort study.

Background: The loss of ancestral microbes, or the "disappearing microbiota hypothesis" has been proposed to play a critical role in the rise of inflammatory and immune diseases in developed nations. The effect of this loss is most consequential during early-life, as initial colonizers of the newborn gut contribute significantly to the development of the immune system. Methods: In this longitudinal study (day 3, week 3, and month 3 post-birth) of infants of Asian ancestry born in Singapore, we studied how generational immigration status and common perinatal factors affect bifidobacteria and Bifidobacterium longum subsp. infantis (B. infantis) colonization. Cohort registry identifier: NCT01174875. Results: Our findings show that first-generation migratory status, perinatal antibiotics usage, and cesarean section birth, significantly influenced the abundance and acquisition of bifidobacteria in the infant gut. Most importantly, 95.6% of the infants surveyed in this study had undetectable B. infantis, an early and beneficial colonizer of infant gut due to its ability to metabolize the wide variety of human milk oligosaccharides present in breastmilk and its ability to shape the development of a healthy immune system. A comparative analysis of B. infantis in 12 countries by their GDP per capita showed a remarkably low prevalence of this microbe in advanced economies, especially Singapore. Conclusion: This study provides new insights into infant gut microbiota colonization, showing the impact of generational immigration on early-life gut microbiota acquisition. It also warrants the need to closely monitor the declining prevalence of beneficial microbes such as B. infantis in developed nations and its potential link to increasing autoimmune and allergic diseases.

Modeling the electrophysiology of suprachiasmatic nucleus neurons.

Neurons in the SCN act as the central circadian (approximately 24-h) pacemaker in mammals. Using measurements of the ionic currents in SCN neurons, the authors fit a Hodgkin-Huxley-type model that accurately reproduces slow (approximately 28 Hz) neural firing as well as the contributions of ionic currents during an action potential. When inputs of other SCN neurons are considered, the model accurately predicts the fractal nature of firing rates and the appearance of random bursting. In agreement with experimental data, the molecular clock within these neurons modulates the firing rate through small changes in the concentration of internal calcium, calcium channels, or potassium channels. Predictions are made on how signals from other neurons can start, stop, speed up, or slow down firing. Only a slow sodium inactivation variable and voltage do not reach equilibrium during the interval between action potentials, and based on this finding, a reduced model is formulated.

Regulation of white and brown adipocyte differentiation by RhoGAP DLC1.

Adipose tissues constitute an important component of metabolism, the dysfunction of which can cause obesity and type II diabetes. Here we show that differentiation of white and brown adipocytes requires Deleted in Liver Cancer 1 (DLC1), a Rho GTPase Activating Protein (RhoGAP) previously studied for its function in liver cancer. We identified Dlc1 as a super-enhancer associated gene in both white and brown adipocytes through analyzing the genome-wide binding profiles of PPARγ, the master regulator of adipogenesis. We further observed that Dlc1 expression increases during differentiation, and knockdown of Dlc1 by siRNA in white adipocytes reduces the formation of lipid droplets and the expression of fat marker genes. Moreover, knockdown of Dlc1 in brown adipocytes reduces expression of brown fat-specific genes and diminishes mitochondrial respiration. Dlc1-/- knockout mouse embryonic fibroblasts show a complete inability to differentiate into adipocytes, but this phenotype can be rescued by inhibitors of Rho-associated kinase (ROCK) and filamentous actin (F-actin), suggesting the involvement of Rho pathway in DLC1-regulated adipocyte differentiation. Furthermore, PPARγ binds to the promoter of Dlc1 gene to regulate its expression during both white and brown adipocyte differentiation. These results identify DLC1 as an activator of white and brown adipocyte differentiation, and provide a molecular link between PPARγ and Rho pathways.

An Alternative Strategy for Pan-acetyl-lysine Antibody Generation.

Lysine acetylation is an important post-translational modification in cell signaling. In acetylome studies, a high-quality pan-acetyl-lysine antibody is key to successful enrichment of acetylated peptides for subsequent mass spectrometry analysis. Here we show an alternative method to generate polyclonal pan-acetyl-lysine antibodies using a synthesized random library of acetylated peptides as the antigen. Our antibodies are tested to be specific for acetyl-lysine peptides/proteins via ELISA and dot blot. When pooled, five of our antibodies show broad reactivity to acetyl-lysine peptides, complementing a commercial antibody in terms of peptide coverage. The consensus sequence of peptides bound by our antibody cocktail differs slightly from that of the commercial antibody. Lastly, our antibodies are tested in a proof-of-concept to analyze the acetylome of HEK293 cells. In total we identified 1557 acetylated peptides from 416 proteins. We thus demonstrated that our antibodies are well-qualified for acetylome studies and can complement existing commercial antibodies.

Acetylome Analysis Identifies SIRT1 Targets in mRNA-Processing and Chromatin-Remodeling in Mouse Liver.

Lysine acetylation is a post-translational modification found on numerous proteins, a strategy used in cell signaling to change protein activity in response to internal or external cues. Sirtuin 1 (SIRT1) is a central lysine deacetylase involved in a variety of cellular processes including metabolism, apoptosis, and DNA repair. Here we characterize the lysine acetylome in mouse liver, and by using a model of Sirt1-/-knockout mouse, show that SIRT1 regulates the deacetylation of 70 proteins in the liver in-vivo. Amongst these SIRT1-regulated proteins, we find that four RNA-processing proteins and a chromatin-remodeling protein can be deacetylated by SIRT1 directly in-vitro. The discovery that SIRT1 has a potential role in RNA-processing suggests a new layer of regulation in the variety of functions performed by SIRT1.

Histone H3 K27 acetylation marks a potent enhancer element on the adipogenic master regulator gene Pparg2.

PPARγ2 is expressed almost exclusively in adipose tissue and plays a central role in adipogenesis. Despite intensive studies over the last 2 decades, the mechanism regulating the expression of the Pparg2 gene, especially the role of cis-regulatory elements, is still not completely understood. Here, we report a comprehensive investigation of the enhancer elements within the murine Pparg2 gene. Utilizing the combined techniques of sequence conservation analysis and chromatin marker examination, we identified a potent enhancer element that augmented the expression of a reporter gene under the control of the Pparg2 promoter by 20-fold. This enhancer element was first identified as highly conserved non-coding sequence 10 (CNS10) and was later shown to be enriched with the enhancer marker H3 K27 acetylation. Further studies identified a binding site for p300 as the essential enhancer element in CNS10. Moreover, p300 physically binds to CNS10 and is required for the enhancer activity of CNS10. The depletion of p300 by siRNA resulted in significantly impaired activation of Pparg2 at the early stages of 3T3-L1 adipogenesis. In summary, our study identified a novel enhancer element on the murine Pparg2 gene and suggested a novel mechanism for the regulation of Pparg2 expression by p300 in 3T3-L1 adipogenesis.

A fluorescent light-up probe with AIE characteristics for specific mitochondrial imaging to identify differentiating brown adipose cells.

We report the design and synthesis of a specific mitochondrial fluorescent probe AIE-MitoGreen-1 with AIE characteristics to monitor the mitochondrial morphology changes and identify the differentiation process of living brown adipose cells. The probe AIE-MitoGreen-1 has significant advantages such as high cellpermeability, good mitochondrial retention, low background fluorescence, large Stokes shift, and low toxicity.