Investigating social communication in mice: A two-intruders test approach.

Understanding the complex dynamics of social communication behaviors, such as exploration, communication, courtship, mating, and aggression in animal models, is crucial to reveal key neural and hormonal mechanisms underlying these behaviors. The two-intruders test is designed to investigate residents' behavior toward both male and female intruders within the home cage of the test male. During this test imitating natural conditions, several aspects of social interaction were investigated: Exploration, courtship, mating, and aggressive behavior. As mating and aggression involve overlapping neural circuits, the behavioral setup testing both behaviors is best at reflecting their competitive nature. Our findings demonstrate that resident male mice exhibit strong preference to communicate with a female intruder, which correlates with baseline testosterone levels of test males. Relevant female preference in the two-intruders test was also found in BALB/c males. Behavioral breakdown revealed the anogenital sniffing as a key behavioral feature that discriminates resident male behavior toward intruders of different sex. Furthermore, resident male interaction with female intruder was accompanied by neuronal activation in the ventromedial hypothalamus. We demonstrate that odor recognition underlies preference toward females in male residents, as experimental anosmia reduced communication with a female intruder. We conclude the two-intruders test setup to be a useful tool to study the neurological basis of social communication in animal models, which provides detailed analysis of various aspects of the laboratory animals' social behavior in the most natural conditions.

The Effect of Dietary Phospholipids on the Ultrastructure and Function of Intestinal Epithelial Cells.

Dietary composition substantially determines human health and affects complex diseases, including obesity, inflammation and cancer. Thus, food supplements have been widely used to accommodate dietary composition to the needs of individuals. Among the promising supplements are dietary phospholipids (PLs) that are commonly found as natural food ingredients and as emulsifier additives. The aim of the present study was to evaluate the effect of major PLs found as food supplements on the morphology of intestinal epithelial cells upon short-term and long-term high-dose feeding in mice. In the present report, the effect of short-term and long-term high dietary PL content was studied in terms of intestinal health and leaky gut syndrome in male mice. We used transmission electron microscopy to evaluate endothelial morphology at the ultrastructural level. We found mitochondrial damage and lipid droplet accumulation in the intracristal space, which rendered mitochondria more sensitive to respiratory uncoupling as shown by a mitochondrial respiration assessment in the intestinal crypts. However, this mitochondrial damage was insufficient to induce intestinal permeability. We propose that high-dose PL treatment impairs mitochondrial morphology and acts through extensive membrane utilization via the mitochondria. The data suggest that PL supplementation should be used with precaution in individuals with mitochondrial disorders.

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.

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.

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.

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.

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.

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.

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.

Phospholipid supplementation inhibits male and female odor discrimination in mice.

Dietary phospholipids (PLs) are promising supplements that are commonly found as natural food ingredients and emulsifier additives. The present study aimed to evaluate the effect of major PLs found in food supplements on social behavior in mice. In this study, the effect of short-term high dietary PL content was studied in terms of social odor discrimination and social interactions with male and female intruders in male mice. We used odor discrimination and habituation tests to demonstrate that PL-fed male mice tend to lose preference toward female odor and fail to discriminate against socially significant scents. At the same time, test animals recognize non-social odors. We also found that PL affected the social behavior of the test males, who tend to behave indiscriminately toward male and female intruders during direct contact. Brain metabolomic profiling revealed no major changes in the intermediary metabolism or neurotransmitter biosynthesis. At the same time, intranasal PL application resembled the effects of dietary supplementation. These data suggest that certain PL might suppress pheromone perception in the olfactory system and affect the sense of socially important odor cues.

Perspectives in Genome-Editing Techniques for Livestock.

Genome editing of farm animals has undeniable practical applications. It helps to improve production traits, enhances the economic value of livestock, and increases disease resistance. Gene-modified animals are also used for biomedical research and drug production and demonstrate the potential to be used as xenograft donors for humans. The recent discovery of site-specific nucleases that allow precision genome editing of a single-cell embryo (or embryonic stem cells) and the development of new embryological delivery manipulations have revolutionized the transgenesis field. These relatively new approaches have already proven to be efficient and reliable for genome engineering and have wide potential for use in agriculture. A number of advanced methodologies have been tested in laboratory models and might be considered for application in livestock animals. At the same time, these methods must meet the requirements of safety, efficiency and availability of their application for a wide range of farm animals. This review aims at covering a brief history of livestock animal genome engineering and outlines possible future directions to design optimal and cost-effective tools for transgenesis in farm species.

Hydrodeoxygenation of 2,5-dimethyltetrahydrofuran over bifunctional Pt-Cs2.5H0.5PW12O40 catalyst in the gas phase: enhancing effect of gold.

2,5-Dimethyltetrahydrofuran (DMTHF) is deoxygenated to n-hexane with >99% selectivity at mild conditions (90 °C, 1 bar H2 pressure, fixed-bed reactor) in the presence of the bifunctional metal-acid catalyst Pt-CsPW comprising Pt and Cs2.5H0.5PW12O40 (CsPW), an acidic Cs salt of Keggin-type heteropoly acid H3PW12O40. Addition of gold to the Pt-CsPW catalyst increases the turnover rate at Pt sites more than twofold, whereas the Au alone without Pt is not active. The enhancement of catalyst activity is attributed to PtAu alloying, which is supported by STEM-EDX and XRD analysis.

A Clinical Case of Nosocomial Pneumonia as a Complication of COVID-19: How to Balance Benefits and Risks of Immunosuppressive Therapy?

We report a Russian case of a 61-year-old male patient with confirmed COVID-19 infection who developed nosocomial pneumonia complicated by lung abscess associated with multi-drug-resistant isolates of Klebsiella pneumoniae and Acinetobacter baumannii, which could have been provoked due to the immunosuppressive therapy. We discuss the existing literature highlighting the issue of the prudent balance between benefits and risks when prescribing immunomodulators to hospitalized patients with COVID-19 due to the risk of difficult-to-treat nosocomial infections caused by MDR Gram-negative bacterial pathogens. Currently, there is evidence of a substantial positive effect of dexamethasone on the course of COVID-19 in patients requiring supplemental oxygen or anti-interleukin-6 drugs in individuals with prominent systemic inflammation. However, it seems that in real clinical practice, the proposed criteria for initiating treatment with immunomodulators are interpreted arbitrarily, and the doses of dexamethasone can significantly exceed those recommended.

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.

Facile gas-phase hydrodeoxygenation of 2,5-dimethylfuran over bifunctional metal-acid catalyst Pt-Cs2.5H0.5PW12O40.

2,5-Dimethylfuran is deoxygenated to n-hexane with 100% yield on a bifunctional Pt/C-Cs2.5H0.5PW12O40 catalyst under very mild conditions (90 °C, 1 bar H2) in a one-step gas-phase process. A proposed mechanism includes a sequence of hydrogenolysis, hydrogenation and dehydration steps occurring on Pt and proton sites of the bifunctional catalyst.

Diethyl Ether Conversion to Ethene and Ethanol Catalyzed by Heteropoly Acids.

The conversion of diethyl ether (DEE) to ethene and ethanol was studied at a gas-solid interface over bulk and supported Brønsted solid acid catalysts based on tungsten Keggin heteropoly acids (HPAs) at 130-250 °C and ambient pressure. The yield of ethene increased with increasing reaction temperature and reached 98% at 220-250 °C (WHSV = 2.2 h-1). The most active HPA catalysts were silica-supported H3PW12O40 and H4SiW12O40 and the bulk heteropoly salt Cs2.5H0.5PW12O40. The HPA catalysts outperformed zeolites HZSM-5 and USY reported elsewhere. A correlation between catalyst activity and catalyst acid strength was established, which indicates that Brønsted acid sites play an important role in DEE elimination over HPA catalysts. The results point to the reaction occurring through the consecutive reaction pathway: DEE → C2H4 + EtOH followed by EtOH → C2H4 + H2O, where ethene is both a primary product of DEE elimination and a secondary product via dehydration of the primary product EtOH. Evidence is provided that DEE elimination over bulk HPA and high-loaded HPA/SiO2 catalysts proceeds via the surface-type mechanism.

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.