Levansucrase from Halomonas smyrnensis AAD6T: first halophilic GH-J clan enzyme recombinantly expressed, purified, and characterized.

Appl Microbiol Biotechnol. 2018 Aug 17;:

Authors: Kirtel O, Menéndez C, Versluys M, Van den Ende W, Hernández L, Toksoy Öner E

Abstract
Fructans, homopolymers of fructose produced by fructosyltransferases (FTs), are emerging as intriguing components in halophiles since they are thought to be associated with osmotic stress tolerance and overall fitness of microorganisms and plants under high-salinity conditions. Here, we report on the full characterization of the first halophilic FT, a levansucrase from Halomonas smyrnensis AAD6T (HsLsc; EC 2.4.1.10). The encoding gene (lsc) was cloned into a vector with a 6xHis Tag at its C-terminus, then expressed in Escherichia coli. The purified recombinant enzyme (47.3 kDa) produces levan and a wide variety of fructooligosaccharides from sucrose, but only in the presence of high salt concentrations (> 1.5 M NaCl). HsLsc showed Hill kinetics and pH and temperature optima of 5.9 and 37 °C, respectively. Interestingly, HsLsc was still very active at salt concentrations close to saturation (4.5 M NaCl) and was selectively inhibited by divalent cations. The enzyme showed high potential in producing novel saccharides derived from raffinose as both fructosyl donor and acceptor and cellobiose, lactose, galactose, and ʟ-arabinose as fructosyl acceptors. With its unique biochemical characteristics, HsLsc is an important enzyme for future research and potential industrial applications in a world faced with drought and diminishing freshwater supplies.

PMID: 30120521 [PubMed - as supplied by publisher]

Hsa-miR-10a-5p downregulation in mutant UQCRB-expressing cells promotes the cholesterol biosynthesis pathway.

Sci Rep. 2018 Aug 17;8(1):12407

Authors: Kim JE, Hong JW, Lee HS, Kim W, Lim J, Cho YS, Kwon HJ

Abstract
Ubiquinol cytochrome c reductase binding protein (UQCRB) is known to play crucial roles in the development of various types of diseases. However, the link between UQCRB and microRNAs remains unknown. In the present study, we performed microRNA sequencing of mutant UQCRB-expressing stable cell lines that exhibited pro-oncogenic activities caused by expression of the mutant UQCRB gene. Results showed that hsa-miR-10a-5p was significantly downregulated in the mutant UQCRB-expressing cell lines. Furthermore, mRNA sequencing and gene ontology analysis of differentially expressed genes (DEGs) revealed that the cholesterol biosynthesis pathway might be activation by mutant UQCRB expression. Moreover, inhibition of cholesterol synthesis in mutant UQCRB-expressing cells via treatment with the specific inhibitors suppressed the cell proliferation. Transfection with a hsa-miR-10a-5p mimic validated that lanosterol synthase (LSS) is a target of hsa-miR-10a-5p. In addition, hsa-miR-10a-5p was found to be downregulated in liver cancer cell lines overexpressing UQCRB. Taken together, our findings highlighted the potential use of hsa-miR-10a-5p as a biomarker for UQCRB related diseases.

PMID: 30120311 [PubMed - in process]

Transcriptional insights into the pyramided resistance to rice bacterial blight.

Sci Rep. 2018 Aug 17;8(1):12358

Authors: Gao L, Fang Z, Zhou J, Li L, Lu L, Li L, Li T, Chen L, Zhang W, Zhai W, Peng H

Abstract
The pyramiding of resistance (R) genes provides broad-spectrum and durable resistance to plant diseases. However, the genetic basis for bacterial blight (BB) resistance remains unclear. The BB R gene pyramided line IRBB54, which expresses xa5 and Xa21, possessed a higher level of resistance than both single R gene lines. Large-scale genotyping of genetic markers in this study revealed similar genetic backgrounds among the near-isogenic lines (NILs), suggesting that resistance in the resistant NILs was mainly conferred by the individual R genes or the interaction between them. Transcriptome analysis demonstrated that more than 50% of the differentially expressed genes (DEGs), and more than 70% of the differentially expressed functions, were shared between IRBB54 and IRBB5 or IRBB21. Most of the DEGs in the resistant NILs were downregulated and are predicted to function in cellular and biological process. The DEGs common among the resistant NILs mainly showed non-additive expression patterns and enrichment in stress-related pathways. The differential expression of agronomic trait-controlled genes in the resistant NILs, especially in IRBB54, indicated the existence of potential side-effects resulting from gene pyramiding. Our findings contribute to the understanding of R gene pyramiding, as well as its effects on targeted and non-targeted trait(s).

PMID: 30120263 [PubMed - in process]

Galectin-9 binds IgM-BCR to regulate B cell signaling.

Nat Commun. 2018 Aug 17;9(1):3288

Authors: Cao A, Alluqmani N, Buhari FHM, Wasim L, Smith LK, Quaile AT, Shannon M, Hakim Z, Furmli H, Owen DM, Savchenko A, Treanor B

Abstract
The galectin family of secreted lectins have emerged as important regulators of immune cell function; however, their role in B-cell responses is poorly understood. Here we identify IgM-BCR as a ligand for galectin-9. Furthermore, we show enhanced BCR microcluster formation and signaling in galectin-9-deficient B cells. Notably, treatment with exogenous recombinant galectin-9 nearly completely abolishes BCR signaling. We investigated the molecular mechanism for galectin-9-mediated inhibition of BCR signaling using super-resolution imaging and single-particle tracking. We show that galectin-9 merges pre-existing nanoclusters of IgM-BCR, immobilizes IgM-BCR, and relocalizes IgM-BCR together with the inhibitory molecules CD45 and CD22. In resting naive cells, we use dual-color super-resolution imaging to demonstrate that galectin-9 mediates the close association of IgM and CD22, and propose that the loss of this association provides a mechanism for enhanced activation of galectin-9-deficient B cells.

PMID: 30120235 [PubMed - in process]

Material microenvironmental properties couple to induce distinct transcriptional programs in mammalian stem cells.

Proc Natl Acad Sci U S A. 2018 Aug 17;:

Authors: Darnell M, O'Neil A, Mao A, Gu L, Rubin LL, Mooney DJ

Abstract
Variations in a multitude of material microenvironmental properties have been observed across tissues in vivo, and these have profound effects on cell phenotype. Phenomenological experiments have suggested that certain of these features of the physical microenvironment, such as stiffness, could sensitize cells to other features; meanwhile, mechanistic studies have detailed a number of biophysical mechanisms for this sensing. However, the broad molecular consequences of these potentially complex and nonlinear interactions bridging from biophysical sensing to phenotype have not been systematically characterized, limiting the overall understanding and rational deployment of these biophysical cues. Here, we explore these interactions by employing a 3D cell culture system that allows for the independent control of culture substrate stiffness, stress relaxation, and adhesion ligand density to systematically explore the transcriptional programs affected by distinct combinations of biophysical parameters using RNA-seq. In mouse mesenchymal stem cells and human cortical neuron progenitors, we find dramatic coupling among these substrate properties, and that the relative contribution of each property to changes in gene expression varies with cell type. Motivated by the bioinformatic analysis, the stiffness of hydrogels encapsulating mouse mesenchymal stem cells was found to regulate the secretion of a wide range of cytokines, and to accordingly influence hematopoietic stem cell differentiation in a Transwell coculture model. These results give insights into how biophysical features are integrated by cells across distinct tissues and offer strategies to synthetic biologists and bioengineers for designing responses to a cell's biophysical environment.

PMID: 30120125 [PubMed - as supplied by publisher]

Intestinal bitter taste receptor activation alters hormone secretion and imparts metabolic benefits.

Mol Metab. 2018 Aug 04;:

Authors: Kok BP, Galmozzi A, Littlejohn NK, Albert V, Godio C, Kim W, Kim SM, Bland JS, Grayson N, Fang M, Meyerhof W, Siuzdak G, Srinivasan S, Behrens M, Saez E

Abstract
OBJECTIVES: Extracts of the hops plant have been shown to reduce weight and insulin resistance in rodents and humans, but elucidation of the mechanisms responsible for these benefits has been hindered by the use of heterogeneous hops-derived mixtures. Because hop extracts are used as flavoring agents for their bitter properties, we hypothesized that bitter taste receptors (Tas2rs) could be mediating their beneficial effects in metabolic disease. Studies have shown that exposure of cultured enteroendocrine cells to bitter tastants can stimulate release of hormones, including glucagon-like peptide 1 (GLP-1). These findings have led to the suggestion that activation of Tas2rs may be of benefit in diabetes, but this tenet has not been tested. Here, we have assessed the ability of a pure derivative of a hops isohumulone with anti-diabetic properties, KDT501, to signal through Tas2rs. We have further used this compound as a tool to systematically assess the impact of bitter taste receptor activation in obesity-diabetes.
METHODS: KDT501 was tested in a panel of bitter taste receptor signaling assays. Diet-induced obese mice (DIO) were dosed orally with KDT501 and acute effects on glucose homeostasis determined. A wide range of metabolic parameters were evaluated in DIO mice chronically treated with KDT501 to establish the full impact of activating gut bitter taste signaling.
RESULTS: We show that KDT501 signals through Tas2r108, one of 35 mouse Tas2rs. In DIO mice, acute treatment stimulated GLP-1 secretion and enhanced glucose tolerance. Chronic treatment caused weight and fat mass loss, increased energy expenditure, enhanced glucose tolerance and insulin sensitivity, normalized plasma lipids, and induced broad suppression of inflammatory markers. Chronic KDT501 treatment altered enteroendocrine hormone levels and bile acid homeostasis and stimulated sustained GLP-1 release. Combined treatment with a dipeptidyl peptidase IV inhibitor amplified the incretin-based benefits of this pure isohumulone.
CONCLUSIONS: Activation of Tas2r108 in the gut results in a remodeling of enteroendocrine hormone release and bile acid metabolism that ameliorates multiple features of metabolic syndrome. Targeting extraoral bitter taste receptors may be useful in metabolic disease.

PMID: 30120064 [PubMed - as supplied by publisher]

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"systems biology"

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Influence of inter-domain dynamics and surrounding environment flexibility on the direct electrochemistry and electrocatalysis of self-sufficient cytochrome P450 3A4-BMR chimeras.

J Inorg Biochem. 2018 Aug 04;188:9-17

Authors: Castrignanò S, Di Nardo G, Sadeghi SJ, Gilardi G

Abstract
The linker region of multi-domain enzymes has a very important role for the interconnection of different enzyme modules and for the efficiency of catalytic activity. This is particularly evident for artificial chimeric systems. We characterised an artificial self-sufficient enzyme developed by genetic fusion of the catalytic domain of cytochrome P450 3A4 and reductase domain of Bacillus megaterium BM3 (BMR). Here we report the direct electrochemistry of 3A4-BMR chimeras immobilised on glassy carbon electrodes and we investigated the effect of inter-domain loop length and immobilising environment flexibility on both redox properties and electrocatalysis. We observe that redox potential can be modulated by the linker length and the immobilising layer flexibility. In addition, enzyme inter-domain dynamics and environment flexibility also modulate 3A4-BMR turnover efficiency on electrode system. Vmax values are increased up to about 100% in the presence of testosterone and up to about 50% in presence of tamoxifen by decreasing immobilising film rigidity. The effect on 3A4-BMR Vmax values is dependent on inter-domain loop length with 3A4-5GLY-BMR chimera being the more affected. The underlying reason for these observations is the potential motion of the FMN domain that is the key to shuttle electrons from FAD to haem.

PMID: 30098472 [PubMed - as supplied by publisher]

Early behavioral and metabolomic change after mild to moderate traumatic brain injury in the developing brain.

Neurochem Int. 2018 Aug 09;120:75-86

Authors: Chitturi J, Li Y, Santhakumar V, Kannurpatti SS

Abstract
Pathophysiology of developmental traumatic brain injury (TBI) is unique due to intrinsic differences in the developing brain. Energy metabolic studies of the brain during early development (P13 to P30) have indicated acute oxidative energy metabolic decreases below 24 h after TBI, which generally recovered by 48 h. However, marked neurodegeneration and altered neural functional connectivity have been observed at later stages into adolescence. As secondary neurodegeneration is most prominent during the first week after TBI in the rat model, we hypothesized that the subacute TBI-metabolome may contain predictive markers of neurodegeneration. Sham and TBI metabolomes were examined at 72 h after a mild to moderate intensity TBI in male Sprague-Dawley rats aged P31. Sensorimotor behavior was assessed at 24, 48 and 72 h after injury, followed by 72-hour postmortem brain removal for metabolomics using Liquid Chromatography/Mass Spectrometry (LC-MS) measurement. Broad TBI-induced metabolomic shifts occurred with relatively higher intensity in the injury-lateralized (ipsilateral) hemisphere. Intensity of metabolomic perturbation correlated with the extent of sensorimotor behavioral deficit. N-acetyl-aspartate (NAA) levels at 72 h after TBI, predicted the extent of neurodegeneration assessed histochemically 7-days post TBI. Results from the multivariate untargeted approach clearly distinguished metabolomic shifts induced by TBI. Several pathways including amino acid, fatty acid and energy metabolism continued to be affected at 72 h after TBI, whose collective effects may determine the overall pathological response after TBI in early development including neurodegeneration.

PMID: 30098378 [PubMed - as supplied by publisher]

Novel vaccine candidates against Mycobacterium tuberculosis.

Int J Biol Macromol. 2018 Aug 08;:

Authors: Khoshnood S, Heidary M, Haeili M, Drancourt M, Darban-Sarokhalil D, Nasiri MJ, Lohrasbi V

Abstract
Tuberculosis (TB) is now among the top ten causes of mortality worldwide being resulted in 1.7 million deaths in 2016. The Bacille Calmette-Guerin (BCG) is the only available TB vaccine which fails to provide consistent protection against pulmonary TB in adults and adolescents despite being efficacious at protecting infants and young children from the most severe, often deadly forms of TB disease. To achieve the goal of global TB elimination by 2050 we will need new interventions including more improved vaccines that are effective in adult individuals who have not been infected with Mycobacterium tuberculosis as well as latently infected or immunocompromised subjects. In recent decades, multiple new vaccine candidates including whole cell vaccines, adjuvanted proteins, and vectored subunit vaccines have entered into the clinical trials. These new TB vaccines are hoped to provide encouraging safety and immunogenicity under various conditions including prevention of TB disease in adolescents and adults, as BCG replacement/boosters, or as therapeutic vaccines to reduce the duration of TB therapy. In this review, we will discuss the status of novel TB vaccine candidates currently under development in preclinical or clinical phases.

PMID: 30098365 [PubMed - as supplied by publisher]

Candidate susceptibility variants in angioimmunoblastic T-cell lymphoma.

Fam Cancer. 2018 Aug 10;:

Authors: Donner I, Katainen R, Kaasinen E, Aavikko M, Sipilä LJ, Pukkala E, Aaltonen LA

Abstract
Angioimmunoblastic T-cell lymphoma (AITL) is a subtype of peripheral T-cell lymphoma with a poor prognosis: the 5-year survival rate is approximately 30%. Somatic driver mutations have been found in TET2, IDH2, DNMT3A, RHOA, FYN, PLCG1, and CD28, whereas germline susceptibility to AITL has to our knowledge not been studied. The homogenous Finnish population is well suited for studies on genetic predisposition. Here, we performed an exome-wide rare variant analysis in 23 AITL patients. No germline mutations were found in the driver genes, implying that they are not frequently involved in genetic AITL predisposition. Potentially pathogenic variants present in at least two patients and showing significant (p < 0.01) enrichment in our sample set were found in ten genes: POLK, PRKCB, ZNF676, PRRC2B, PCDHGB6, GNL3L, TTC36, OTOG, OSGEPL1, and RASSF9. The most significantly enriched variants, causing p.Lys469Ter in a splice variant of POLK and p.Pro588His in PRKCB, are intriguing candidates as Polk deficient mice display a spontaneous mutator phenotype, whereas PRKCB was recently shown to be somatically mutated in 33% of another peripheral T-cell lymphoma, adult T-cell lymphoma. If validated, our findings would provide new insight into the pathogenesis of AITL, as well as tools for early detection in susceptible individuals.

PMID: 30097855 [PubMed - as supplied by publisher]

Physiological dynamic compression regulates central energy metabolism in primary human chondrocytes.

Biomech Model Mechanobiol. 2018 Aug 10;:

Authors: Salinas D, Mumey BM, June RK

Abstract
Chondrocytes use the pathways of central metabolism to synthesize molecular building blocks and energy for cartilage homeostasis. An interesting feature of the in vivo chondrocyte environment is the cyclical loading generated in various activities (e.g., walking). However, it is unknown whether central metabolism is altered by mechanical loading. We hypothesized that physiological dynamic compression alters central metabolism in chondrocytes to promote production of amino acid precursors for matrix synthesis. We measured the expression of central metabolites (e.g., glucose, its derivatives, and relevant co-factors) for primary human osteoarthritic chondrocytes in response to 0-30 minutes of compression. To analyze the data, we used principal components analysis and ANOVA-simultaneous components analysis, as well as metabolic flux analysis. Compression-induced metabolic responses consistent with our hypothesis. Additionally, these data show that chondrocyte samples from different patient donors exhibit different sensitivity to compression. Most importantly, we find that grade IV osteoarthritic chondrocytes are capable of synthesizing non-essential amino acids and precursors in response to mechanical loading. These results suggest that further advances in metabolic engineering of chondrocyte mechanotransduction may yield novel translational strategies for cartilage repair.

PMID: 30097814 [PubMed - as supplied by publisher]

Comprehensive proteomics identification of IFN-λ3-regulated anti-viral proteins in HBV-transfected cells.

Mol Cell Proteomics. 2018 Aug 10;:

Authors: Makjaroen J, Somparn P, Hodge K, Poomipak W, Hirankarn N, Pisitkun T

Abstract
Interferon lambda (IFN-λ) is a relatively unexplored, yet promising anti-viral agent. IFN-λ has recently been tested in clinical trials of chronic hepatitis B virus infection (CHB), with the advantage that side effects may be limited compared with IFN-α, as IFN-λ receptors are found only in epithelial cells. To date, IFN-λ's downstream signaling pathway remains largely unelucidated, particularly via proteomics methods. Here, we report that IFN-λ3 inhibits HBV replication in HepG2.2.15 cells, reducing levels of both HBV transcripts and intracellular HBV DNA. Quantitative proteomic analysis of HBV-transfected cells was performed following 24-hour IFN-λ3 treatment, with parallel IFN-α2a and PBS treatments for comparison using a dimethyl labeling method. The depth of the study allowed us to map the induction of anti-viral proteins to multiple points of the viral life cycle, as well as facilitating the identification of anti-viral proteins not previously known to be elicited upon HBV infection (e.g. IFITM3, XRN2, and NT5C3A). This study also shows up-regulation of many effectors involved in antigen processing/presentation indicating that this cytokine exerted immunomodulatory effects through a number of essential molecules for these processes. Interestingly, the 2 subunits of the immunoproteasome cap (PSME1 and PSME2) were up-regulated while cap components of the constitutive proteasome were down-regulated upon both IFN treatments, suggesting coordinated modulation towards the antigen processing/presentation mode. Furthermore, in addition to confirming canonical activation of interferon-stimulated gene (ISG) transcription through the JAK-STAT pathway, we reveal that IFN-λ3 restored levels of RIG-I and RIG-G, proteins known to be suppressed by HBV. Enrichment analysis demonstrated that several biological processes including RNA metabolism, translation, and ER-targeting were differentially regulated upon treatment with IFN-λ3 vs. IFN-α2a. Our proteomic data suggests that IFN-λ3 regulates an array of cellular processes to control HBV replication.

PMID: 30097535 [PubMed - as supplied by publisher]