Colitis-associated intestinal microbiota regulates brain glycine and host behavior in mice.

Inflammatory bowel diseases (IBD) are chronic and relapsing inflammatory disorders of the gastrointestinal tract with complex etiology and no strategies for complete cure. IBD are often complicated by mental disorders like anxiety and depression, indicating substantial shifts in the microbiota gut-brain axis. However, the mechanisms connecting IBD to mental diseases are still under debate. Here we use Muc2 knockout mouse model of chronic colitis to uncouple the effects of the intestinal microbiota on host behavior from chronic inflammation in the gut. Muc2 knockout male mice exhibit high exploratory activity, reduced anxiety-related behaviors, impaired sensorimotor gating, and altered social preference towards males and females. Microbial transfer to wild-type mice via littermate co-housing shows that colitis-associated microbiota rather than inflammation per se defines behavioral features in Muc2 colitis model. Metagenomic profiling and combination of antibiotic treatments revealed that bacterial species Akkermansia muciniphila is associated with the behavioral phenotype in mutants, and that its intestinal abundance correlates with social preference towards males. Metabolomic analysis together with pharmacological inhibition of Gly and NMDA receptors helped us to determine that brain glycine is responsible for the behavioral phenotype in Muc2 mice. Blood and brain metabolic profiles suggest that microbiota-dependent changes in choline metabolism might be involved in regulation of central glycine neurotransmission. Taken together, our data demonstrates that colitis-associated microbiota controls anxiety, sensorimotor gating and social behavior via metabolic regulation of the brain glycinergic system, providing new venues to combat neurological complications of IBD.

Role of Mucin 2 Glycoprotein and L-Fucose in Interaction of Immunity and Microbiome within the Experimental Model of Inflammatory Bowel Disease.

Many factors underlie the development of inflammatory bowel disease (IBD) in humans. In particular, imbalance of microbiota and thinning of the mucosal layer in the large intestine play a huge role. Pathogenic microorganisms also exacerbate the course of diseases. In this research the role of mucin 2 deficiency in the formation of intestinal microflora in the experimental model using the Muc2 gene knockout mice in the presence of Helicobacter spp. was investigated. Also, restorative and anti-inflammatory effect of the dietary L-fucose in the Muc2-/- mice on microflora and immunity was evaluated. For this purpose, bacterial diversity in feces was studied in the animals before and after antibiotic therapy and role of the dietary L-fucose in their recovery was assessed. To determine the effect of bacterial imbalance and fucose on the immune system, mRNA levels of the genes encoding pro-inflammatory cytokines (Tnf, Il1a, Il1b, Il6) and transcription factors of T cells (Foxp3 - Treg, Rorc - Th17, Tbx21 - Th1) were determined in the colon tissue of the Muc2-/- mice. Significant elimination of bacteria due to antibiotic therapy caused decrease of the fucose levels in the intestine and facilitated reduction of the regulatory T cell transcription factor (Foxp3). When the dietary L-fucose was added to antibiotics, the level of bacterial DNA of Bacteroides spp. in the feces of the Muc2-/- mice was partially restored. T regulatory cells are involved in the regulation of inflammation in the Muc2-/- mice. Antibiotics reduced the number of regulatory T cell but did not decrease the inflammatory response to infection. Fucose, as a component of mucin 2, helped to maintain the level of Bacteroides spp. during antibiotic therapy of the Muc2-/- mice and restored biochemical parameters, but did not affect the inflammatory response.

Optogenetic and Chemical Induction Systems for Regulation of Transgene Expression in Plants: Use in Basic and Applied Research.

Continuous and ubiquitous expression of foreign genes sometimes results in harmful effects on the growth, development and metabolic activities of plants. Tissue-specific promoters help to overcome this disadvantage, but do not allow one to precisely control transgene expression over time. Thus, inducible transgene expression systems have obvious benefits. In plants, transcriptional regulation is usually driven by chemical agents under the control of chemically-inducible promoters. These systems are diverse, but usually contain two elements, the chimeric transcription factor and the reporter gene. The commonly used chemically-induced expression systems are tetracycline-, steroid-, insecticide-, copper-, and ethanol-regulated. Unlike chemical-inducible systems, optogenetic tools enable spatiotemporal, quantitative and reversible control over transgene expression with light, overcoming limitations of chemically-inducible systems. This review updates and summarizes optogenetic and chemical induction methods of transgene expression used in basic plant research and discusses their potential in field applications.

Fat of the Gut: Epithelial Phospholipids in Inflammatory Bowel Diseases.

Inflammatory bowel diseases (IBD) comprise a distinct set of clinical symptoms resulting from chronic inflammation within the gastrointestinal (GI) tract. Despite the significant progress in understanding the etiology and development of treatment strategies, IBD remain incurable for thousands of patients. Metabolic deregulation is indicative of IBD, including substantial shifts in lipid metabolism. Recent data showed that changes in some phospholipids are very common in IBD patients. For instance, phosphatidylcholine (PC)/phosphatidylethanolamine (PE) and lysophosphatidylcholine (LPC)/PC ratios are associated with the severity of the inflammatory process. Composition of phospholipids also changes upon IBD towards an increase in arachidonic acid and a decrease in linoleic and a-linolenic acid levels. Moreover, an increase in certain phospholipid metabolites, such as lysophosphatidylcholine, sphingosine-1-phosphate and ceramide, can result in enhanced intestinal inflammation, malignancy, apoptosis or necroptosis. Because some phospholipids are associated with pathogenesis of IBD, they may provide a basis for new strategies to treat IBD. Current attempts are aimed at controlling phospholipid and fatty acid levels through the diet or via pharmacological manipulation of lipid metabolism.

Fucose Ameliorates Tritrichomonas sp.-Associated Illness in Antibiotic-Treated Muc2-/- Mice.

The mucus layer in the intestine plays a critical role in regulation of host-microbe interactions and maintaining homeostasis. Disruptions of the mucus layer due to genetic, environmental, or immune factors may lead to inflammatory bowel diseases (IBD). IBD frequently are accompanied with infections, and therefore are treated with antibiotics. Hence, it is important to evaluate risks of antibiotic treatment in individuals with vulnerable gut barrier and chronic inflammation. Mice with a knockout of the Muc2 gene, encoding the main glycoprotein component of the mucus, demonstrate a close contact of the microbes with the gut epithelium which leads to chronic inflammation resembling IBD. Here we demonstrate that the Muc2-/- mice harboring a gut protozoan infection Tritrichomonas sp. are susceptible to an antibiotic-induced depletion of the bacterial microbiota. Suppression of the protozoan infection with efficient metronidazole dosage or L-fucose administration resulted in amelioration of an illness observed in antibiotic-treated Muc2-/- mice. Fucose is a monosaccharide presented abundantly in gut glycoproteins, including Mucin2, and is known to be involved in host-microbe interactions, in particular in microbe adhesion. We suppose that further investigation of the role of fucose in protozoan adhesion to host cells may be of great value.

Female Scent Activated Expression of Arginase1 and Inducible NO-Synthetase in Lung of BALB/c Male Mice.

Scent signals play an important role in the life of rodents. The scent of the opposite sex can modulate immunity. In mice populations with natural specific pathogens, in males, the scent of a female leads to a redistribution of leukocytes between the lung and the blood, resistance to the influenza virus, and a decrease in antibody production, but not in the development of inflammation induced by bacterial endotoxins. This study demonstrates the effect of the scent of soiled bedding of specific pathogen-free (SPF) status female mice on the percentage of different types of leukocytes in the blood, the expression of Nos2, Arg1, and Foxp3 genes, and the presence of M1/M2 macrophages in the lungs of male BALB/c mice. The scent of the female SPF mice caused a redistribution between T- and B-cells in the blood, the increase in the expression of Nos2, Arg1 genes, and the percentage of M1 type macrophages in the lung, but did not affect the different types of T-cells in the periphery or the lungs. Activation of macrophages in the lung is part of mucosal immunity, which is necessary for males as an adaptive mechanism to prevent potential infection during the search for a sexual partner.

Dietary Fucose Affects Macrophage Polarization and Reproductive Performance in Mice.

Intestinal mucus protects epithelial and immune cells from the gut resident microorganisms, and provides growth-promoting factors as mucus-derived O-glycans for beneficial bacteria. A lack of intestinal protective mucus results in changes in the commensal microflora composition, mucosal immune system reprogramming, and inflammation. Previous work has shown that fucose, the terminal glycan chain component of the intestinal glycoprotein Mucin2, and fucoidan polysaccharides have an anti-inflammatory effect in some mouse models of colitis. This study evaluates the effect of fucose on reproductive performance in heterozygous mutant Muc2 female mice. We found that even though Muc2+/- females are physiologically indistinguishable from C57Bl/6 mice, they have a significantly reduced reproductive performance upon dietary fucose supplementation. Metagenomic analysis reveals that the otherwise healthy wild-type siblings of Muc2-/- animals have reduced numbers of some of the intestinal commensal bacterial species, compared to C57BL/6 mice. We propose that the changes in beneficial microflora affect the immune status in Muc2+/- mice, which causes implantation impairment. In accordance with this hypothesis, we find that macrophage polarization during pregnancy is impaired in Muc2+/- females upon addition of fucose. Metabolic profiling of peritoneal macrophages from Muc2+/- females reveals their predisposition towards anaerobic glycolysis in favor of oxidative phosphorylation, compared to C57BL/6-derived cells. In vitro experiments on phagocytosis activity and mitochondrial respiration suggest that fucose affects oxidative phosphorylation in a genotype-specific manner, which might interfere with implantation depending on the initial status of macrophages. This hypothesis is further confirmed in BALB/c female mice, where fucose caused pregnancy loss and opposed implantation-associated M2 macrophage polarization. Taken together, these data suggest that intestinal microflora affects host immunity and pregnancy outcome. At the same time, dietary fucose might act as a differential regulator of macrophage polarization during implantation, depending on the immune status of the host.

Fine gene expression regulation by minor sequence variations downstream of the polyadenylation signal.

The termination of transcription is a complex process that substantially contributes to gene regulation in eukaryotes. Previously, it was noted that a single cytosine deletion at the position + 32 bp relative to the single polyadenylation signal AAUAAA (hereafter the dC mutation) causes a 2-fold increase in the transcription level of the upstream eGFP reporter in mouse embryonic stem cells. Here, we analyzed the conservation of this phenomenon in immortalized mouse, human and drosophila cell lines and the influence of the dC mutation on the choice of the pre-mRNA cleavage sites. We have constructed dual-reporter plasmids to accurately measure the effect of the dC and other nearby located mutations on eGFP mRNA level by RT-qPCR. In this way, we found that the dC mutation leads to a 2-fold increase in the expression level of the upstream eGFP reporter gene in cultured mouse and human, but not in drosophila cells. In addition, 3' RACE analysis demonstrated that eGFP pre-mRNAs are cut at multiple positions between + 14 to + 31, and that the most proximal cleavage site becomes almost exclusively utilized in the presence of the dC mutation. We also identified new short sequence variations located within positions + 25.. + 40 and + 33.. + 48 that increase eGFP expression up to ~2-4-fold. Altogether, the positive effect of the dC mutation seems to be conserved in mouse embryonic stem cells, mouse embryonic 3T3 fibroblasts and human HEK293T cells. In the latter cells, the dC mutation appears to be involved in regulating pre-mRNA cleavage site selection. Finally, a multiplexed approach is proposed to identify motifs located downstream of cleavage site(s) that are essential for transcription termination.

Mucin-2 knockout is a model of intercellular junction defects, mitochondrial damage and ATP depletion in the intestinal epithelium.

The disruption of the protective intestinal barrier-the 'leaky gut'-is a common complication of the inflammatory bowel disease. There is limited data on the mechanisms of the intestinal barrier disruption upon low-grade inflammation characteristic of patients with inflammatory bowel disease in clinical remission. Thus, animal models that recapitulate the complexity of chronic intestinal inflammation in vivo are of particular interest. In this study, we used Mucin-2 (Muc2) knockout mice predisposed to colitis to study intestinal barrier upon chronic inflammation. We used 4-kDa FITC-Dextran assay and transmission electron microscopy to demonstrate the increased intestinal permeability and morphological defects in intercellular junctions in Muc2 knockout mice. Confocal microscopy revealed the disruption of the apical F-actin cytoskeleton and delocalization of tight junction protein Claudin-3 from the membrane. We further demonstrate mitochondrial damage, impaired oxygen consumption and the reduction of the intestinal ATP content in Muc2 knockout mice. Finally, we show that chemically induced mitochondrial uncoupling in the wild type mice mimics the intestinal barrier disruption in vivo and causes partial loss of F-actin and membrane localization of Claudin-3. We propose that mitochondrial damage and metabolic shifts during chronic inflammation contribute to the leaky gut syndrome in Muc2 knockout animal model of colitis.

Modification of Fecal Bacteria Counts and Blood Immune Cells in the Offspring of BALB/c and C57BL/6 Mice Obtained through Interstrain Mouse Embryo Transfer.

The reproducibility of results obtained with rodent models depends on the genetic purity of the strain and the stability of the environment. However, another potential factor is changes in the gut microbiota due to the transmission of mother's bacteria during embryo transfer. In this study, we demonstrate the transmission of the microbiota and immune cell blood phenotype to the offspring of 2 strains, C57BL/6JNskrc and BALB/cJNskrc, from surrogate dams of different genotypes. Interstrain embryo transfer resulted in a change in the number of Enterococcus spp. organisms, as shown by quantitative PCR analysis. The number of blood leukocytes was also affected, as estimated by flow cytometry. The number of blood leukocytes, including B cells and helper T cells, and the number of Enterococcus spp. organisms in male C57BL/6JNskrc offspring bornto BALB/cJNskrc surrogate dams became similar to those of male BALB/cJNskrc mice born to BALB/cJNskrc dams. Likewise, the same parameters of male BALB/cJNskrc mice born to C57BL/6JNskrc dams became similar to those of male C57BL/6JNskrc offspring. Researchers should be aware of the possible transmission of the dam's microbiota and immune cell phenotypes to the experimental strains when planning embryo transfer experiments, because these factors could affect the experimental outcomes or the reproducibility of experimental results.

Fucose Ameliorates Tryptophan Metabolism and Behavioral Abnormalities in a Mouse Model of Chronic Colitis.

Growing evidence suggests that intestinal mucosa homeostasis impacts immunity, metabolism, the Central Nervous System (CNS), and behavior. Here, we investigated the effect of the monosaccharide fucose on inflammation, metabolism, intestinal microbiota, and social behavior in the Dextran Sulfate Sodium (DSS)-induced chronic colitis mouse model. Our data show that chronic colitis is accompanied by the decrease of the serum tryptophan level and the depletion of the intestinal microbiota, specifically tryptophan-producing E. coli and Bifidobacterium. These changes are associated with defects in the male mouse social behavior such as a lack of preference towards female bedding in an odor preference test. The addition of fucose to the test animals' diet altered the bacterial community, increased the abundance of tryptophan-producing E. coli, normalized blood tryptophan levels, and ameliorated social behavior deficits. At the same time, we observed no ameliorating effect of fucose on colon morphology and colitis. Our results suggest a possible mechanism by which intestinal inflammation affects social behavior in male mice. We propose fucose as a promising prebiotic, since it creates a favorable environment for the beneficial bacteria that promote normalization of serum tryptophan level and amelioration of the behavioral abnormalities in the odor preference test.

A toolset to study functions of Cytosolic non-specific dipeptidase 2 (CNDP2) using Drosophila as a model organism.

BACKGROUND: Expression of the CNDP2 gene is frequently up- or down-regulated in different types of human cancers. However, how the product of this gene is involved in cell growth and proliferation is poorly understood. Moreover, our knowledge of the functions of the CNDP2 orthologs in well-established model organisms is scarce. In particular, the function of the D. melanogaster ortholog of CNDP2, encoded by the CG17337 gene (hereafter referred to as dCNDP2), is still unknown. RESULTS: This study was aimed at developing a set of genetic and molecular tools to study the roles of dCNDP2. We generated a dCNDP2 null mutation (hereafter ∆dCNDP2) using CRISPR/Cas9-mediated homologous recombination (HR) and found that the ∆dCNDP2 mutants are homozygous viable, morphologically normal and fertile. We also generated transgenic fly lines expressing eGFP-tagged and non-tagged dCNDP2 protein, all under the control of the UAS promoter, as well as polyclonal antibodies specific to dCNDP2. Using these tools, we demonstrate that only one of the two predicted dCNDP2 isoforms is expressed throughout the different tissues tested. dCNDP2 was detected in both the cytoplasm and the nucleus, and was found to be associated with multiple sites in the salivary gland polytene chromosomes. CONCLUSIONS: The dCNDP2 gene is not essential for fly viability under standard laboratory conditions. The subcellular localization pattern of dCNDP2 suggests that this protein might have roles in both the cytoplasm and the nucleus. The genetic and molecular tools developed in this study will allow further functional characterization of the conserved CNDP2 protein using D. melanogaster as a model system.

Eradication of Helicobacter spp. in mucin2-deficient mice.

Infections with Helicobacter spp. are known to have serious effects on rodent health, especially in immunocompromised animals. In this study three approaches were used to eradicate Helicobacter spp. infection in mice with a deficiency in intestinal proteoglycan (mucin2), namely triple oral antibiotic therapy (amoxicillin, clarithromycin and metronidazole), cross-fostering of neonatal pups by surrogate mothers negative for Helicobacter spp., and in vitro fertilization (IVF) with embryo transfer into Helicobacter-free mothers. However, triple antibiotic therapy in mice with mucin2 deficiency was not effective and had negative effects on reproductive performance, and high susceptibility of mucin2-deficient mice to Helicobacter spp. during the first 12 h after birth rendered cross-fostering impossible. Only IVF with embryo transfer was effective in eradicating Helicobacter infection from transgenic mice with mucin2 deficiency.

Inducible DamID systems for genomic mapping of chromatin proteins in Drosophila.

Dam identification (DamID) is a powerful technique to generate genome-wide maps of chromatin protein binding. Due to its high sensitivity, it is particularly suited to study the genome interactions of chromatin proteins in small tissue samples in model organisms such as Drosophila Here, we report an intein-based approach to tune the expression level of Dam and Dam-fusion proteins in Drosophila by addition of a ligand to fly food. This helps to suppress possible toxic effects of Dam. In addition, we describe a strategy for genetically controlled expression of Dam in a specific cell type in complex tissues. We demonstrate the utility of the latter by generating a glia-specific map of Polycomb in small samples of brain tissue. These new DamID tools will be valuable for the mapping of binding patterns of chromatin proteins in Drosophila tissues and especially in cell lineages.

Tethering of SUUR and HP1 proteins results in delayed replication of euchromatic regions in Drosophila melanogaster polytene chromosomes.

We analyze how artificial targeting of Suppressor of Under-Replication (SUUR) and HP1 proteins affects DNA replication in the "open," euchromatic regions. Normally these regions replicate early in the S phase and display no binding of either SUUR or HP1. These proteins were expressed as fusions with DNA-binding domain of GAL4 and recruited to multimerized UAS integrated in three euchromatic sites of the polytene X chromosome: 3B, 8D, and 18B. Using PCNA staining as a marker of ongoing replication, we showed that targeting of SUUR(GAL4DBD) and HP1(GAL4DBD) results in delayed replication of appropriate euchromatic regions. Specifically, replication at these regions starts early, much like in the absence of the fusion proteins; however, replication completion is significantly delayed. Notably, delayed replication was insufficient to induce underreplication. Recruitment of SUUR(GAL4DBD) and HP1(GAL4DBD) had distinct effects on expression of a mini-white reporter, found near UAS. Whereas SUUR(GAL4DBD) had no measurable influence on mini-white expression, HP1(GAL4DBD) targeting silenced mini-white, even in the absence of functional SU(VAR)3-9. Furthermore, recruitment of SUUR(GAL4DBD) and HP1(GAL4DBD) had distinct effects on the protein composition of target regions. HP1(GAL4DBD) but not SUUR(GAL4DBD) could displace an open chromatin marker, CHRIZ, from the tethering sites.

DNA copy-number control through inhibition of replication fork progression.

Proper control of DNA replication is essential to ensure faithful transmission of genetic material and prevent chromosomal aberrations that can drive cancer progression and developmental disorders. DNA replication is regulated primarily at the level of initiation and is under strict cell-cycle regulation. Importantly, DNA replication is highly influenced by developmental cues. In Drosophila, specific regions of the genome are repressed for DNA replication during differentiation by the SNF2 domain-containing protein SUUR through an unknown mechanism. We demonstrate that SUUR is recruited to active replication forks and mediates the repression of DNA replication by directly inhibiting replication fork progression instead of functioning as a replication fork barrier. Mass spectrometry identification of SUUR-associated proteins identified the replicative helicase member CDC45 as a SUUR-associated protein, supporting a role for SUUR directly at replication forks. Our results reveal that control of eukaryotic DNA copy number can occur through the inhibition of replication fork progression.

Metabolic enzyme IMPDH is also a transcription factor regulated by cellular state.

Cells need to coordinate gene expression and metabolic state. Inosine monophosphate dehydrogenase (IMPDH) controls the guanine nucleotide pool and, thereby, cell proliferation. We found that Drosophila IMPDH is also a DNA-binding transcriptional repressor. IMPDH attenuates expression of histone genes and E2f, a key driver of cell proliferation. Nuclear IMPDH accumulates during the G2 phase of the cell cycle or following replicative or oxidative stress. Thus, IMPDH can couple the expression of histones and E2F to cellular state. Genome-wide profiling and in vitro binding assays established that IMPDH binds sequence specifically to single-stranded, CT-rich DNA elements. Surprisingly, this DNA-binding function is conserved in E. coli IMPDH. The catalytic function of IMPDH is not required for DNA binding. Yet substitutions that correspond to human retinitis pigmentosa mutations disrupt IMPDH binding to CT-rich, single-stranded DNA elements. By doubling as nucleotide biosynthetic enzyme or transcription factor, IMPDH can either enable or restrict cell proliferation.

Interaction between the Drosophila heterochromatin proteins SUUR and HP1.

SUUR (Suppressor of Under-Replication) protein is responsible for late replication and, as a consequence, for DNA underreplication of intercalary and pericentric heterochromatin in Drosophila melanogaster polytene chromosomes. However, the mechanism by which SUUR slows down the replication process is not clear. To identify possible partners for SUUR we performed a yeast two-hybrid screen using full-length SUUR as bait. This identified HP1, the well-studied heterochromatin protein, as a strong SUUR interactor. Furthermore, we have determined that the central region of SUUR is necessary and sufficient for interaction with the C-terminal part of HP1, which contains the hinge and chromoshadow domains. In addition, recruitment of SUUR to ectopic HP1 sites on chromosomes provides evidence for their association in vivo. Indeed, we found that the distributions of SUUR and HP1 on polytene chromosomes are interdependent: both absence and overexpression of HP1 prevent SUUR from chromosomal binding, whereas SUUR overexpression causes redistribution of HP1 to numerous sites occupied by SUUR. Finally, HP1 binds to intercalary heterochromatin when histone methyltransferase activity of SU(VAR)3-9 is increased. We propose that interaction with HP1 is crucial for the association of SUUR with chromatin.