Characterization of the LysR-type Transcriptional Regulator HsdR Gene and Its Adjacent Short-chain Dehydrogenase/Reductase SDRx Gene in Comamonas testosteroni ATCC 11996

3a-hydroxysteroid dehydrogenase/carbonyl reductase (3a-HSD/CR) from Comamonas testosteroni (C. testosteroni) is a key enzyme in the degradation of steroid compounds in soil and water. Interestingly, 3a-HSD/CR gene (hsdA) expression can be induced by steroids like testosterone and progesterone. Thus, the regulatory mechanism of 3a-HSD/CR induction has attracted considerable attention of our group. Previously, it has been shown that induction of hsdA expression by steroids is a derepression where steroid inducers bind to two repressors, RepA and RepB, thereby preventing blocking of hsdA transcription and translation, respectively. In the present study, a new LysR-type transcriptional factor HsdR for 3a-HSD/CR expression in C. testosteroni was identified. The hsdR gene locates 2.58 kb downstream of the hsdA gene on the C. testosteroni ATCC 11996 chromosome with an orientation opposite to hsdA. The hsdR gene was cloned and the recombinant HsdR protein was overproduced, and an anti-HsdR polyclonal antibody was subsequently prepared. While heterologous transformation systems revealed that HsdR activates the expression of the hsdA gene, electrophoretic mobility shift assays (EMSA) showed that HsdR specifically binds to the hsdA promoter region. Furthermore, the activity of HsdR is dependent on the decreased repression by RepA. in vitro binding assays clearly indicated that HsdR can contact with RNA polymerase. Interestingly, an hsdR disrupted mutant expressed low levels of 3a-HSD/CR compared to wild type C. testosteroni after testosterone induction. In addition, HsdR itself cannot be induced by testosterone. As a member of LysR-type regulators, HsdR may also repress it own expression. Here, electrophoretic mobility shift assays indicated that HsdR specifically binds to its own promoter. As expected, mutated HsdR expression in an hsdR-gfp fusion mutant and an hsdR gene disrupted mutant of C. testosteroni increased compared to that in the wild type strain, largely because autorepression of HsdR in these mutants is prevented. This result revealed that HsdR negatively regulates its own expression. Phylogenetic analyses indicated that HsdR is related to the contact-regulated gene A (CrgA) from Neisseria meningitidis, which exists as an octamer. To detect the active form of HsdR, three truncated proteins, HsdRΔN (residues 1-86 deleted), HsdRΔC (residues 221-303 deleted), and HsdRΔNC (residues 1-86 and 221-303 deleted), were constructed and purified. These deleted domains are important for the positive control of HsdR on 3a-HSD/CR expression. Western blotting indicated that HsdR may also exist as an octamer, where the central domain is crucial for the multimerization of HsdR. Unexpectedly, gel filtration chromatography showed that there are two dominant oligomers (octamer and hexamer) present for HsdR and its truncated proteins. Taken together, HsdR is a positive transcription factor for 3a-HSD/CR expression in C. testosteroni, and it may also negatively regulate its own expression. In addition, a novel gene SDRx, which is divergently transcribed from the hsdR gene, was found to be a member of the short-chain dehydrogenase/reductase (SDR) superfamily. The open reading frame of this SDRx consists of 768 bp and translates into a protein of 255 amino acids. Two consensus sequences of the SDR superfamily were found, an N-terminal Gly-X-X-X-Gly-X-Gly cofactor-binding motif and a Tyr-X-X-X-Lys segment (residues 160-164 in the SDRx sequence) essential for catalytic activity of SDR proteins. Phylogenetic analyses indicated that the novel SDRx gene from C. testosteroni, which is active in steroid metabolism, is related to 7α-hydroxysteroid dehydrogenase (7α-HSD). Degradation of the steroids testosterone and estradiol decreased in the SDRx knock-out mutant strain. Furthermore, growth on the steroids cholic acid, estradiol and testosterone was impaired in the SDRx knock-out strain. Combined, the novel SDRx in C. testosteroni was identified as 7α-HSD that is involved in steroid degradation.Die 3α-Hydroxysteroid-Dehydrogenase/Carbonyl-Reduktase (3α-HSD/CR) von C. testosteroni ist ein Schlüsselenzym im Abbau von Steroid-Verbindungen in Boden und Wasser. Bemerkenswerterweise kann die Expression des 3α-HSD/CR Gens (hsdA) durch Steroide wie Testosteron und Progesteron induziert werden. Die Aufklärung dieses Regulationsmechanismus der 3α-HSD/CR Induktion ist Gegenstand intensiver Forschung. Bisher hat sich gezeigt, dass die Induktion der hsdA Expression durch Steroide eigentlich eine Derepression darstellt, bei der Steroid-Induktoren an zwei Repressoren (RepA und RepB) binden, diese inaktivieren und so die Expression einleiten. In der vorliegenden Studie wurde ein neuer LysR-artiger Transkriptionsfaktor (hsdR) der 3α-HSD/CR Expression in C. testosteroni identifiziert. Das hsdR-Gen ist 2,58 kb stromabwärts des hsdA-Gens im Genom von C. testosteroni ATCC 11996 in entgegengesetzter Leserichtung lokalisiert. Das hsdR-Gen wurde in E. coli-Zellen kloniert, überexprimiert und gereinigt. Mit dem rekombinanten Protein wurde ein polyklonaler Antikörper gewonnen. Während mit heterologen Transformations-Systemen gezeigt wurde, dass hsdR die Expression des hsdA-Gens induziert, konnte durch „band-shift“-Versuche nachgewiesen werden, dass hsdR spezifisch an die hsdA Promotor-Region bindet. Dabei ist die HsdR-Aktivität eng mit der hsdA-Repression durch RepA verbunden, insofern als HsdR erst nach der Freigabe des Promotors durch RepA aktiv wird. Weiterhin zeigten in-vitro Bindungsstudien, dass hsdR an RNA-Polymerase bindet. Wie erwartet, ließen sich in hsdR knock-out Mutanten nach Testosteron-Induktion nur erniedrigte 3α-HSD/CR Level im Vergleich zum Wildtyp finden. Die hsdR-Expression wiederum ist nicht durch Testosteron induzierbar. Von LysR-Typ Regulatoren ist bekannt, dass sie im Sinne einer feed-back Regulation ihre eigene Expression unterdrücken können. Es konnte durch weitere „band-shift“-Versuche gezeigt werden, dass HsdR an seinen eigenen Promotor bindet. Wie erwartet war die Expression eines hsdR-gfp Fusionsproteins und einer hsdR-Disruptionsmutante erhöht im Vergleich zum Wildtyp, da in diesen Mutanten die Autorepression nicht stattfinden konnte. Folglich wird hsdR ebenfalls durch einen feed-back Mechanismus reguliert. hsdR ähnelt dem Kontakt-regulierten Gen A (CrgA) von Neisseria meningitidis, einem Octamer, wie sich durch phylogenetische Untersuchungen nachweisen ließ. Für weitere Analysen wurden drei Deletionsmutanten von hsdR hergestellt, denen folgende Aminosäuren fehlten: HsdRΔN (1-86); HsdRΔC (221-303); HsdRΔNC (1-86 und 221-303). Diese deletierten Domänen sind wichtig für die positive HsdR-regulierte 3α-HSD/CR Expression. Western-Blot-Ergebnisse mit diesen Mutanten zeigten, dass HsdR ebenfalls als Octamer vorliegen könnte und die zentrale Domäne entscheidend für die Multimerisierung ist. Ergebnisse aus Gelfiltrations Versuchen mit dem Wildtyp-Protein sowie den Deletionsmutanten deuten das Vorliegen von zwei Oligomeren (Octamer und Hexamer) an. Zusammenfassend ist hsdR als positiver Transkriptionsfaktor der 3α-HSD/CR Expression in C. testosteroni identifiziert worden, der die eigene Expression negativ reguliert. Darüber hinaus wurde ein neues SDR-Gen (SDRx) gefunden, das abweichend von hsdR transkribiert wird. Der open-reading-frame von SDRx umfasst 768 bp, die für ein Protein aus 255 Aminosäuren kodieren. Zwei Konsensus-Sequenzen der SDR-Superfamilie wurden gefunden, das N-terminale Gly-XXX-Gly-X-Gly-Cofaktor-Bindungsmotiv und ein Tyr-XXX-Lys-Segment (Aminosäuren 160-164 in der SDRx Sequenz), das essentiell für die katalytische Aktivität der SDR-Proteine ist. Phylogenetische Analysen lassen vermuten, dass das neu gefundene SDRx-Gen in C. testosteroni homolog zu einer 7α-Hydroxysteroid-Dehydrogenase (7α-HSD) ist, die am Steroidstoffwechsel beteiligt ist. Dies steht im Einklang mit der Beobachtung, dass der Abbau der Steroide Testosteron und Östradiol in SDRx knock-out Mutanten verringert ist. Außerdem war das Wachstum des SDRx knock-out-Stamms mit den Steroiden Cholsäure, Estradiol oder Testosteron als Kohlenstoffquelle geringer als das des Wildtyps

Measurement of Bell-type inequalities and quantum entanglement from Λ\Lambda-hyperon spin correlations at high energy colliders

Spin correlations of $\Lambda$-hyperons embedded in the QCD strings formed in high-energy collider experiments provide unique insight into their locality and entanglement features. We show from general considerations that while the Clauser-Horne-Shimony-Holt inequality is less stringent for such states, they provide a benchmark for quantum-to-classical transitions induced by varying i) the associated hadron multiplicity, ii) the spin of nucleons, iii) the separation in rapidity between pairs, and iv) the kinematic regimes accessed. These studies also enable the extraction of quantitative measures of quantum entanglement. We first explore such questions within a simple model of a QCD string comprised of singlets of two partial distinguishable fermion flavors and compare analytical results to those obtained on quantum hardware. We further discuss a class of spin Hamiltonians that model the dynamics of $\Lambda$ spin correlations. Prospects for extracting quantum features of QCD strings from hyperon measurements at current and future colliders are outlined.Comment: Revised manuscrip

Blood preoperative neutrophil-to-lymphocyte ratio is correlated with TNM stage in patients with papillary thyroid cancer

OBJECTIVE: To predict the American Joint Cancer Committee tumor-node-metastasis stage in patients with papillary thyroid carcinoma by evaluating the relationship between the preoperative neutrophil-to-lymphocyte ratio and the tumor-node-metastasis stage. METHODS: We retrospectively examined 161 patients with a diagnosis of papillary thyroid carcinoma. The Neutrophil-to-Lymphocyte Ratio was calculated according to the absolute neutrophil counts and absolute lymphocyte counts on routine blood tests obtained prior to surgery and patients with a Neutrophil-to-Lymphocyte Ratio of 2.0 or more were classified as the high NLR group, while those with a Neutrophil-to-Lymphocyte Ratio less than 2.0 were classified as the low Neutrophil-to-Lymphocyte Ratio group. Clinicopathological variables, which were stratified by the Neutrophil-to-Lymphocyte Ratio, were analyzed. A multivariate analysis was performed to determine factors that affect the Neutrophil-to-Lymphocyte Ratio. The association between the Neutrophil-to-Lymphocyte Ratio and the TNM stage in patients ≥45 years of age was analyzed using the Spearman rank correlation. RESULTS: Various blood indices, including hemoglobin, platelet and thyroid-stimulating hormone levels in the two groups showed no significant differences. Lymph node metastasis, multifocality and tumor size exhibited significant differences in the two groups (p=0.000, p=0.000 and p=0.035, respectively). Correlation analysis indicated that a higher preoperative Neutrophil-to-Lymphocyte Ratio was observed in patients with lymph node metastasis, larger tumor size and multifocality (r=0.341, p=0.000; r=0.271, p=0.000; and r=0.182, p=0.010, respectively). For patients ≥45 years of age, the number of patients with an advanced TNM stage in the high NLR group was higher than that in the low Neutrophil-to-Lymphocyte Ratio group (p=0.013). A linear regression analysis showed that the preoperative Neutrophil-to-Lymphocyte Ratio was positively correlated with the American Joint Cancer Committee tumor-node-metastasis stage (rho=0.403, p=0.000). CONCLUSION: The preoperative Neutrophil-to-Lymphocyte Ratio was closely related to the stage of papillary thyroid carcinoma. The increase in the preoperative Neutrophil-to-Lymphocyte Ratio contributed to the advanced tumor-node-metastasis stage of papillary thyroid carcinoma patients ≥45 years of age

Serum Metabolomic Profiling of Piglets Infected with Virulent Classical Swine Fever Virus

Citation: Gong, W. J., Jia, J. J., Zhang, B. K., Mi, S. J., Zhang, L., Xie, X. M., . . . Tu, C. C. (2017). Serum Metabolomic Profiling of Piglets Infected with Virulent Classical Swine Fever Virus. Frontiers in Microbiology, 8, 14. doi:10.3389/fmicb.2017.00731Classical swine fever (CSF) is a highly contagious swine infectious disease and causes significant economic losses for the pig industry worldwide. The objective of this study was to determine whether small molecule metabolites contribute to the pathogenesis of CSF. Birefly, serum metabolomics of CSFV Shimen strain-infected piglets were analyzed by ultraperformance liquid chromatography/electrospray ionization time-of-flight mass spectrometry (UPLC/ESI-Q-TOF/MS) in combination with multivariate statistical analysis. In CSFV-infected piglets at days 3 and 7 post-infection changes were found in metabolites associated with several key metabolic pathways, including tryptophan catabolism and the kynurenine pathway, phenylalanine metabolism, fatty acid and lipid metabolism, the tricarboxylic acid and urea cycles, branched-chain amino acid metabolism, and nucleotide metabolism. Several pathways involved in energy metabolism including fatty acid biosynthesis and beta-oxidation, branched-chain amino acid metabolism, and the tricarboxylic acid cycle were significantly inhibited. Changes were also observed in several metabolites exclusively associated with gut microbiota. The metabolomic profiles indicate that CSFV-host gut microbiome interactions play a role in the development of CSF

Realization of the Based on WAP Push SMS Gateway

WAP PUSH can send message actively and in time,as well as that it can receive message at all time and place, therefore there are favourable application prospect.The paper has simplely introduced the system frame,the push protocol and the push way of WAP PUSH,especially it has introduced the implementation method of the message gateway for WAP PUSH

Growth and characterization of Bi2Se3 thin films by pulsed laser deposition using alloy target

Bi2Se3 thin films were deposited on the (100) oriented Si substrates by pulsed laser deposition technique at different substrate temperatures (room temperature – 400 ºC). The effects of the substrate temperature on the structural and electrical properties of the Bi2Se3 films were studied. The film prepared at room temperature showed a very poor polycrystalline structure with the mainly orthorhombic phase. The crystallinity of the films was improved by heating the substrate during the deposition and the crystal phase of the film changed to the rhombohedral phase as the substrate temperature was higher than 200 ºC. The stoichiometry of the films and the chemical state of Bi and Se elements in the films were studied by fitting the Se 3d and the Bi 4d5/2 peaks of the X-ray photoelectron spectra. The hexagonal structure was seen clearly for the film prepared at the substrate temperature of 400 ºC. The surface roughness of the film increased as the substrate temperature was increased. The electrical resistivity of the film decreased from 1x10-3 to 3 x 10-4 Ω cm as the substrate temperature was increased from room temperature to 400 ºC.Shenyang National Laboratory for Material Science (SYNL), Chin

Bayesian Optimization Enhanced Deep Reinforcement Learning for Trajectory Planning and Network Formation in Multi-UAV Networks

In this paper, we employ multiple UAVs coordinated by a base station (BS) to help the ground users (GUs) to offload their sensing data. Different UAVs can adapt their trajectories and network formation to expedite data transmissions via multi-hop relaying. The trajectory planning aims to collect all GUs' data, while the UAVs' network formation optimizes the multi-hop UAV network topology to minimize the energy consumption and transmission delay. The joint network formation and trajectory optimization is solved by a two-step iterative approach. Firstly, we devise the adaptive network formation scheme by using a heuristic algorithm to balance the UAVs' energy consumption and data queue size. Then, with the fixed network formation, the UAVs' trajectories are further optimized by using multi-agent deep reinforcement learning without knowing the GUs' traffic demands and spatial distribution. To improve the learning efficiency, we further employ Bayesian optimization to estimate the UAVs' flying decisions based on historical trajectory points. This helps avoid inefficient action explorations and improves the convergence rate in the model training. The simulation results reveal close spatial-temporal couplings between the UAVs' trajectory planning and network formation. Compared with several baselines, our solution can better exploit the UAVs' cooperation in data offloading, thus improving energy efficiency and delay performance.Comment: 15 pages, 10 figures, 2 algorithm