Nfkb2 deficiency and its impact on plasma cells and immunoglobulin expression in murine small intestinal mucosa.

The alternative (noncanonical) nuclear factor-κB (NF-κB) signaling pathway predominantly regulates the function of the p52/RelB heterodimer. Germline Nfkb2 deficiency in mice leads to loss of p100/p52 protein and offers protection against a variety of gastrointestinal conditions, including azoxymethane/dextran sulfate sodium (DSS)-induced colitis-associated cancer and lipopolysaccharide (LPS)-induced small intestinal epithelial apoptosis. However, the common underlying protective mechanisms have not yet been fully elucidated. We applied high-throughput RNA-Seq and proteomic analyses to characterize the transcriptional and protein signatures of the small intestinal mucosa of naïve adult Nfkb2-/- mice. Those data were validated by immunohistochemistry and quantitative ELISA using both small intestinal tissue lysates and serum. We identified a B-lymphocyte defect as a major transcriptional signature in the small intestinal mucosa and immunoglobulin A as the most downregulated protein by proteomic analysis in Nfkb2-/- mice. Small intestinal immunoglobulins were dramatically dysregulated, with undetectable levels of immunoglobulin A and greatly increased amounts of immunoglobulin M being detected. The numbers of IgA-producing, cluster of differentiation (CD)138-positive plasma cells were also reduced in the lamina propria of the small intestinal villi of Nfkb2-/- mice. This phenotype was even more striking in the small intestinal mucosa of RelB-/- mice, although these mice were equally sensitive to LPS-induced intestinal apoptosis as their RelB+/+ wild-type counterparts. NF-κB2/p52 deficiency confers resistance to LPS-induced small intestinal apoptosis and also appears to regulate the plasma cell population and immunoglobulin levels within the gut.NEW & NOTEWORTHY Novel transcriptomic analysis of murine proximal intestinal mucosa revealed an unexpected B cell signature in Nfkb2-/- mice. In-depth analysis revealed a defect in the CD38+ B cell population and a gut-specific dysregulation of immunoglobulin levels.

Tetrahydrobiopterin and alkylglycerol monooxygenase substantially alter the murine macrophage lipidome.

Tetrahydrobiopterin is a cofactor synthesized from GTP with well-known roles in enzymatic nitric oxide synthesis and aromatic amino acid hydroxylation. It is used to treat mild forms of phenylketonuria. Less is known about the role of tetrahydrobiopterin in lipid metabolism, although it is essential for irreversible ether lipid cleavage by alkylglycerol monooxygenase. Here we found intracellular alkylglycerol monooxygenase activity to be an important regulator of alkylglycerol metabolism in intact murine RAW264.7 macrophage-like cells. Alkylglycerol monooxygenase was expressed and active also in primary mouse bone marrow-derived monocytes and "alternatively activated" M2 macrophages obtained by interleukin 4 treatment, but almost missing in M1 macrophages obtained by IFN-γ and lipopolysaccharide treatment. The cellular lipidome of RAW264.7 was markedly changed in a parallel way by modulation of alkylglycerol monooxygenase expression and of tetrahydrobiopterin biosynthesis affecting not only various ether lipid species upstream of alkylglycerol monooxygenase but also other more complex lipids including glycosylated ceramides and cardiolipins, which have no direct connection to ether lipid pathways. Alkylglycerol monooxygenase activity manipulation modulated the IFN-γ/lipopolysaccharide-induced expression of inducible nitric oxide synthase, interleukin-1ß, and interleukin 1 receptor antagonist but not transforming growth factor ß1, suggesting that alkylglycerol monooxygenase activity affects IFN-γ/lipopolysaccharide signaling. Our results demonstrate a central role of tetrahydrobiopterin and alkylglycerol monooxygenase in ether lipid metabolism of murine macrophages and reveal that alteration of alkylglycerol monooxygenase activity has a profound impact on the lipidome also beyond the class of ether lipids.

Skeletal muscle mitochondria of NDUFS4-/- mice display normal maximal pyruvate oxidation and ATP production.

Mitochondrial ATP production is mediated by the oxidative phosphorylation (OXPHOS) system, which consists of four multi-subunit complexes (CI-CIV) and the FoF1-ATP synthase (CV). Mitochondrial disorders including Leigh Syndrome often involve CI dysfunction, the pathophysiological consequences of which still remain incompletely understood. Here we combined experimental and computational strategies to gain mechanistic insight into the energy metabolism of isolated skeletal muscle mitochondria from 5-week-old wild-type (WT) and CI-deficient NDUFS4-/- (KO) mice. Enzyme activity measurements in KO mitochondria revealed a reduction of 79% in maximal CI activity (Vmax), which was paralleled by 45-72% increase in Vmax of CII, CIII, CIV and citrate synthase. Mathematical modeling of mitochondrial metabolism predicted that these Vmax changes do not affect the maximal rates of pyruvate (PYR) oxidation and ATP production in KO mitochondria. This prediction was empirically confirmed by flux measurements. In silico analysis further predicted that CI deficiency altered the concentration of intermediate metabolites, modestly increased mitochondrial NADH/NAD+ ratio and stimulated the lower half of the TCA cycle, including CII. Several of the predicted changes were previously observed in experimental models of CI-deficiency. Interestingly, model predictions further suggested that CI deficiency only has major metabolic consequences when its activity decreases below 90% of normal levels, compatible with a biochemical threshold effect. Taken together, our results suggest that mouse skeletal muscle mitochondria possess a substantial CI overcapacity, which minimizes the effects of CI dysfunction on mitochondrial metabolism in this otherwise early fatal mouse model.

Suprabasin as a novel tumor endothelial cell marker.

Recent studies have reported that stromal cells contribute to tumor progression. We previously demonstrated that tumor endothelial cells (TEC) characteristics were different from those of normal endothelial cells (NEC). Furthermore, we performed gene profile analysis in TEC and NEC, revealing that suprabasin (SBSN) was upregulated in TEC compared with NEC. However, its role in TEC is still unknown. Here we showed that SBSN expression was higher in isolated human and mouse TEC than in NEC. SBSN knockdown inhibited the migration and tube formation ability of TEC. We also showed that the AKT pathway was a downstream factor of SBSN. These findings suggest that SBSN is involved in the angiogenic potential of TEC and may be a novel TEC marker.

Gut microbiota signatures in inflammatory bowel disease.

BACKGROUND: Inflammatory bowel diseases (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), affect millions of people worldwide with increasing incidence. OBJECTIVES: Several studies have shown a link between gut microbiota composition and IBD, but results are often limited by small sample sizes. We aimed to re-analyze publicly available fecal microbiota data from IBD patients. METHODS: We extracted original fecal 16S rRNA amplicon sequencing data from 45 cohorts of IBD patients and healthy individuals using the BioProject database at the National Center for Biotechnology Information. Unlike previous meta-analyses, we merged all study cohorts into a single dataset, including sex, age, geography, and disease information, based on which microbiota signatures were analyzed, while accounting for varying technical platforms. RESULTS: Among 2518 individuals in the combined dataset, we discovered a hitherto unseen number of genera associated with IBD. A total of 77 genera associated with CD, of which 38 were novel associations, and a total of 64 genera associated with UC, of which 28 represented novel associations. Signatures were robust across different technical platforms and geographic locations. Reduced alpha diversity in IBD compared to healthy individuals, in CD compared to UC, and altered microbiota composition (beta diversity) in UC and especially in CD as compared to healthy individuals were found. CONCLUSIONS: Combining original microbiota data from 45 cohorts, we identified a hitherto unseen large number of genera associated with IBD. Identification of microbiota features robustly associated with CD and UC may pave the way for the identification of new treatment targets.

PGF2α induces a pro-labour phenotypical switch in human myometrial cells that can be inhibited with PGF2α receptor antagonists.

Preterm birth is the leading cause of infant morbidity and mortality. There has been an interest in developing prostaglandin F2α (PGF2α) antagonists as a new treatment for preterm birth, although much of the rationale for their use is based on studies in rodents where PGF2α initiates labour by regressing the corpus luteum and reducing systemic progesterone concentrations. How PGF2α antagonism would act in humans who do not have a fall in systemic progesterone remains unclear. One possibility, in addition to an acute stimulation of contractions, is a direct alteration of the myometrial smooth muscle cell state towards a pro-labour phenotype. In this study, we developed an immortalised myometrial cell line, MYLA, derived from myometrial tissue obtained from a pregnant, non-labouring patient, as well as a novel class of PGF2α receptor (FP) antagonist. We verified the functionality of the cell line by stimulation with PGF2α, resulting in Gαq-specific coupling and Ca2+ release, which were inhibited by FP antagonism. Compared to four published FP receptor antagonists, the novel FP antagonist N582707 was the most potent compound [Fmax 7.67 ± 0.63 (IC50 21.26 nM), AUC 7.30 ± 0.32 (IC50 50.43 nM), and frequency of Ca2+ oscillations 7.66 ± 0.41 (IC50 22.15 nM)]. RNA-sequencing of the MYLA cell line at 1, 3, 6, 12, 24, and 48 h post PGF2α treatment revealed a transforming phenotype from a fibroblastic to smooth muscle mRNA profile. PGF2α treatment increased the expression of MYLK, CALD1, and CNN1 as well as the pro-labour genes OXTR, IL6, and IL11, which were inhibited by FP antagonism. Concomitant with the inhibition of a smooth muscle, pro-labour transition, FP antagonism increased the expression of the fibroblast marker genes DCN, FBLN1, and PDGFRA. Our findings suggest that in addition to the well-described acute contractile effect, PGF2α transforms myometrial smooth muscle cells from a myofibroblast to a smooth muscle, pro-labour-like state and that the novel compound N582707 has the potential for prophylactic use in preterm labour management beyond its use as an acute tocolytic drug.

Metabolic modeling and analysis of the metabolic switch in Streptomyces coelicolor.

BACKGROUND: The transition from exponential to stationary phase in Streptomyces coelicolor is accompanied by a major metabolic switch and results in a strong activation of secondary metabolism. Here we have explored the underlying reorganization of the metabolome by combining computational predictions based on constraint-based modeling and detailed transcriptomics time course observations. RESULTS: We reconstructed the stoichiometric matrix of S. coelicolor, including the major antibiotic biosynthesis pathways, and performed flux balance analysis to predict flux changes that occur when the cell switches from biomass to antibiotic production. We defined the model input based on observed fermenter culture data and used a dynamically varying objective function to represent the metabolic switch. The predicted fluxes of many genes show highly significant correlation to the time series of the corresponding gene expression data. Individual mispredictions identify novel links between antibiotic production and primary metabolism. CONCLUSION: Our results show the usefulness of constraint-based modeling for providing a detailed interpretation of time course gene expression data.

The dynamic architecture of the metabolic switch in Streptomyces coelicolor.

BACKGROUND: During the lifetime of a fermenter culture, the soil bacterium S. coelicolor undergoes a major metabolic switch from exponential growth to antibiotic production. We have studied gene expression patterns during this switch, using a specifically designed Affymetrix genechip and a high-resolution time-series of fermenter-grown samples. RESULTS: Surprisingly, we find that the metabolic switch actually consists of multiple finely orchestrated switching events. Strongly coherent clusters of genes show drastic changes in gene expression already many hours before the classically defined transition phase where the switch from primary to secondary metabolism was expected. The main switch in gene expression takes only 2 hours, and changes in antibiotic biosynthesis genes are delayed relative to the metabolic rearrangements. Furthermore, global variation in morphogenesis genes indicates an involvement of cell differentiation pathways in the decision phase leading up to the commitment to antibiotic biosynthesis. CONCLUSIONS: Our study provides the first detailed insights into the complex sequence of early regulatory events during and preceding the major metabolic switch in S. coelicolor, which will form the starting point for future attempts at engineering antibiotic production in a biotechnological setting.

Phospholipid Acyl Chain Diversity Controls the Tissue-Specific Assembly of Mitochondrial Cardiolipins.

Cardiolipin (CL) is a phospholipid specific for mitochondrial membranes and crucial for many core tasks of this organelle. Its acyl chain configurations are tissue specific, functionally important, and generated via post-biosynthetic remodeling. However, this process lacks the necessary specificity to explain CL diversity, which is especially evident for highly specific CL compositions in mammalian tissues. To investigate the so far elusive regulatory origin of CL homeostasis in mice, we combine lipidomics, integrative transcriptomics, and data-driven machine learning. We demonstrate that not transcriptional regulation, but cellular phospholipid compositions are closely linked to the tissue specificity of CL patterns allowing artificial neural networks to precisely predict cross-tissue CL compositions in a consistent mechanistic specificity rationale. This is especially relevant for the interpretation of disease-related perturbations of CL homeostasis, by allowing differentiation between specific aberrations in CL metabolism and changes caused by global alterations in cellular (phospho-)lipid metabolism.

The pathophysiology of bile acid diarrhoea: differences in the colonic microbiome, metabolome and bile acids.

Bile acid diarrhoea (BAD) is a common disorder resulting from increased loss of bile acids (BAs), overlapping irritable bowel syndrome with diarrhoea (IBS-D). The gut microbiota metabolises primary BAs to secondary BAs, with differing impacts on metabolism and homeostasis. The aim of this study was to profile the microbiome, metabolic products and bile acids in BAD. Patients with BAD diagnosed by SeHCAT testing, were compared with other IBS-D patients, and healthy controls. Faecal 16S ribosomal RNA gene analysis was undertaken. Faecal short chain fatty acid (SCFA) and urinary volatile organic compounds (VOCs) were measured. BAs were quantified in serum and faeces. Faecal bacterial diversity was significantly reduced in patients with BAD. Several taxa were enriched compared to IBS-D. SCFA amounts differed in BAD, controls and IBS-D, with significantly more propionate in BAD. Separation of VOC profiles was evident, but the greatest discrimination was between IBS-D and controls. Unconjugated and primary BA in serum and faeces were significantly higher in BAD. The faecal percentage primary BA was inversely related to SeHCAT. BAD produces dysbiosis, with metabolite differences, including VOC, SCFA and primary BAs when compared to IBS-D. These findings provide new mechanistic insights into the pathophysiology of BAD.

The Glycosyltransferase EOGT Regulates Adropin Expression in Decidualizing Human Endometrium.

In pregnancy, resistance of endometrial decidual cells to stress signals is critical for the integrity of the fetomaternal interface and, by extension, survival of the conceptus. O-GlcNAcylation is an essential posttranslational modification that links glucose sensing to cellular stress resistance. Unexpectedly, decidualization of primary endometrial stromal cells (EnSCs) was associated with a 60% reduction in O-linked ß-N-acetylglucosamine (O-GlcNAc)‒modified proteins, reflecting downregulation of the enzyme that adds O-GlcNAc to substrates (O-GlcNAc transferase; OGT) but not the enzyme that removes the modification (O-GlcNAcase). Notably, epidermal growth factor domain-specific O-linked GlcNAc transferase (EOGT), an endoplasmic reticulum-specific OGT that modifies a limited number of secreted and membrane proteins, was markedly induced in differentiating EnSCs. Knockdown of EOGT perturbed a network of decidual genes involved in multiple cellular functions. The most downregulated gene upon EOGT knockdown in decidualizing cells was the energy homeostasis-associated gene (ENHO), which encodes adropin, a metabolic hormone involved in energy homeostasis and glucose and fatty acid metabolism. Analysis of midluteal endometrial biopsies revealed an inverse correlation between endometrial EOGT and ENHO expression and body mass index. Taken together, our findings revealed that obesity impairs the EOGT-adropin axis in decidual cells, which in turn points toward a mechanistic link between metabolic disorders and adverse pregnancy outcome.

Sequence diversity and natural selection at domain I of the apical membrane antigen 1 among Indian Plasmodium falciparum populations.

BACKGROUND: The Plasmodium falciparum apical membrane antigen 1 (AMA1) is a leading malaria vaccine candidate antigen. The complete AMA1 protein is comprised of three domains where domain I exhibits high sequence polymorphism and is thus named as the hyper-variable region (HVR). The present study describes the extent of genetic polymorphism and natural selection at domain I of the ama1 gene among Indian P. falciparum isolates. METHODS: The part of the ama1 gene covering domain I was PCR amplified and sequenced from 157 P. falciparum isolates collected from five different geographical regions of India. Statistical and phylogenetic analyses of the sequences were done using DnaSP ver. 4. 10. 9 and MEGA version 3.0 packages. RESULTS: A total of 57 AMA1 haplotypes were observed among 157 isolates sequenced. Forty-six of these 57 haplotypes are being reported here for the first time. The parasites collected from the high malaria transmission areas (Assam, Orissa, and Andaman and Nicobar Islands) showed more haplotypes (H) and nucleotide diversity pi as compared to low malaria transmission areas (Uttar Pradesh and Goa). The comparison of all five Indian P. falciparum subpopulations indicated moderate level of genetic differentiation and limited gene flow (Fixation index ranging from 0.048 to 0.13) between populations. The difference between rates of non-synonymous and synonymous mutations, Tajima's D and McDonald-Kreitman test statistics suggested that the diversity at domain I of the AMA1 antigen is due to positive natural selection. The minimum recombination events were also high indicating the possible role of recombination in generating AMA1 allelic diversity. CONCLUSION: The level of genetic diversity and diversifying selection were higher in Assam, Orissa, and Andaman and Nicobar Islands populations as compared to Uttar Pradesh and Goa. The amounts of gene flow among these populations were moderate. The data reported here will be valuable for the development of AMA1-based malaria vaccine.

Pretransition and progressive softening of bovine carbonic anhydrase II as probed by single molecule atomic force microscopy.

To develop a simple method for probing the physical state of surface adsorbed proteins, we adopted the force curve mode of an atomic force microscope (AFM) to extract information on the mechanical properties of surface immobilized bovine carbonic anhydrase II under native conditions and in the course of guanidinium chloride-induced denaturation. A progressive increase in the population of individually softened molecules was probed under mildly to fully denaturing conditions. The use of the approach regime of force curves gave information regarding the height and rigidity of the molecule under compressive stress, whereas use of the retracting regime of the curves gave information about the tensile characteristics of the protein. The results showed that protein molecules at the beginning of the transition region possessed slightly more flattened and significantly more softened conformations compared with that of native molecules, but were still not fully denatured, in agreement with results based on solution studies. Thus the force curve mode of an AFM was shown to be sensitive enough to provide information concerning the different physical states of single molecules of globular proteins.

Self-establishing communities enable cooperative metabolite exchange in a eukaryote.

Metabolite exchange among co-growing cells is frequent by nature, however, is not necessarily occurring at growth-relevant quantities indicative of non-cell-autonomous metabolic function. Complementary auxotrophs of Saccharomyces cerevisiae amino acid and nucleotide metabolism regularly fail to compensate for each other's deficiencies upon co-culturing, a situation which implied the absence of growth-relevant metabolite exchange interactions. Contrastingly, we find that yeast colonies maintain a rich exometabolome and that cells prefer the uptake of extracellular metabolites over self-synthesis, indicators of ongoing metabolite exchange. We conceived a system that circumvents co-culturing and begins with a self-supporting cell that grows autonomously into a heterogeneous community, only able to survive by exchanging histidine, leucine, uracil, and methionine. Compensating for the progressive loss of prototrophy, self-establishing communities successfully obtained an auxotrophic composition in a nutrition-dependent manner, maintaining a wild-type like exometabolome, growth parameters, and cell viability. Yeast, as a eukaryotic model, thus possesses extensive capacity for growth-relevant metabolite exchange and readily cooperates in metabolism within progressively establishing communities.

The future of industrial antibiotic production: from random mutagenesis to synthetic biology.

Natural products derived from the secondary metabolism of microbes constitute a cornerstone of modern medicine. Engineering bugs to produce these products in high quantities is a major challenge for biotechnology, which has usually been tackled by either one of two strategies: iterative random mutagenesis or rational design. Recently, we analyzed the transcriptome of a Streptomyces clavuligerus strain optimized for production of the ß-lactamase inhibitor clavulanic acid by multiple rounds of mutagenesis and selection, and discovered that the observed changes matched surprisingly well with simple changes that have been introduced into these strains by rational engineering. Here, we discuss how in the new field of synthetic biology, random mutagenesis and rational engineering can be implemented complementarily in ways which may enable one to go beyond the status quo that has now been reached by each method independently.

Genome-wide gene expression changes in an industrial clavulanic acid overproduction strain of Streptomyces clavuligerus.

To increase production of the important pharmaceutical compound clavulanic acid, a ß-lactamase inhibitor, both random mutagenesis approaches and rational engineering of Streptomyces clavuligerus strains have been extensively applied. Here, for the first time, we compared genome-wide gene expression of an industrial S. clavuligerus strain, obtained through iterative mutagenesis, with that of the wild-type strain. Intriguingly, we found that the majority of the changes contributed not to a complex rewiring of primary metabolism but consisted of a simple upregulation of various antibiotic biosynthesis gene clusters. A few additional transcriptional changes in primary metabolism at key points seem to divert metabolic fluxes to the biosynthetic precursors for clavulanic acid. In general, the observed changes largely coincide with genes that have been targeted by rational engineering in recent years, yet the presence of a number of previously unexplored genes clearly demonstrates that functional genomic analysis can provide new leads for strain improvement in biotechnology.

The sequence of a 1.8-mb bacterial linear plasmid reveals a rich evolutionary reservoir of secondary metabolic pathways.

Plasmids are mobile genetic elements that play a key role in the evolution of bacteria by mediating genome plasticity and lateral transfer of useful genetic information. Although originally considered to be exclusively circular, linear plasmids have also been identified in certain bacterial phyla, notably the actinomycetes. In some cases, linear plasmids engage with chromosomes in an intricate evolutionary interplay, facilitating the emergence of new genome configurations by transfer and recombination or plasmid integration. Genome sequencing of Streptomyces clavuligerus ATCC 27064, a Gram-positive soil bacterium known for its production of a diverse array of biotechnologically important secondary metabolites, revealed a giant linear plasmid of 1.8 Mb in length. This megaplasmid (pSCL4) is one of the largest plasmids ever identified and the largest linear plasmid to be sequenced. It contains more than 20% of the putative protein-coding genes of the species, but none of these is predicted to be essential for primary metabolism. Instead, the plasmid is densely packed with an exceptionally large number of gene clusters for the potential production of secondary metabolites, including a large number of putative antibiotics, such as staurosporine, moenomycin, beta-lactams, and enediynes. Interestingly, cross-regulation occurs between chromosomal and plasmid-encoded genes. Several factors suggest that the megaplasmid came into existence through recombination of a smaller plasmid with the arms of the main chromosome. Phylogenetic analysis indicates that heavy traffic of genetic information between Streptomyces plasmids and chromosomes may facilitate the rapid evolution of secondary metabolite repertoires in these bacteria.