Engineering cofactor metabolism for improved protein and glucoamylase production in Aspergillus niger

Background: Nicotinamide adenine dinucleotide phosphate (NADPH) is an important cofactor ensuring intracellular redox balance, anabolism and cell growth in all living systems. Our recent multi-omics analyses of glucoamylase (GlaA) biosynthesis in the filamentous fungal cell factory Aspergillus niger indicated that low availability of NADPH might be a limiting factor for GlaA overproduction. Results: We thus employed the Design-Build-Test-Learn cycle for metabolic engineering to identify and prioritize effective cofactor engineering strategies for GlaA overproduction. Based on available metabolomics and 13C metabolic flux analysis data, we individually overexpressed seven predicted genes encoding NADPH generation enzymes under the control of the\ua0Tet-on gene switch in two A. niger recipient strains, one carrying a single and one carrying seven glaA gene copies, respectively, to test their individual effects on GlaA and total protein overproduction. Both strains were selected to understand if a strong pull towards glaA biosynthesis (seven gene copies) mandates a higher NADPH supply compared to the native condition (one gene copy). Detailed analysis of all 14 strains cultivated in shake flask cultures uncovered that overexpression of the gsdA gene (glucose 6-phosphate dehydrogenase), gndA gene (6-phosphogluconate dehydrogenase) and maeA gene (NADP-dependent malic enzyme) supported GlaA production on a subtle (10%) but significant level in the background strain carrying seven glaA gene copies. We thus performed maltose-limited chemostat cultures combining metabolome analysis for these three isolates to characterize metabolic-level fluctuations caused by cofactor engineering. In these cultures, overexpression of either the gndA or maeA gene increased the intracellular NADPH pool by 45% and 66%, and the yield of GlaA by 65% and 30%, respectively. In contrast, overexpression of the gsdA gene had a negative effect on both total protein and glucoamylase production. Conclusions: This data suggests for the first time that increased NADPH availability can indeed underpin protein and especially GlaA production in strains where a strong pull towards GlaA biosynthesis exists. This data also indicates that the highest impact on GlaA production can be engineered on a genetic level by increasing the flux through the pentose phosphate pathway (gndA gene) followed by engineering the flux through the reverse TCA cycle (maeA gene). We thus propose that NADPH cofactor engineering is indeed a valid strategy for metabolic engineering of A. niger to improve GlaA production, a strategy which is certainly also applicable to the rational design of other microbial cell factories.[Figure not available: see fulltext.]

Measurement of event shape distributions and moments in e+e- -> hadrons at 91-209 GeV and a determination of alpha_s

We have studied hadronic events from e+e- annihilation data at centre-of-mass energies from 91 to 209 GeV. We present distributions of event shape observables and their moments at each energy and compare with QCD Monte Carlo models. From the event shape distributions we extract the strong coupling alpha_s and test its evolution with energy scale. The results are consistent with the running of alpha_s expected from QCD. Combining all data, the value of alpha_s(M_Z) is determined to be alpha_s(M_Z) = 0.1191 +- 0.0005 (stat.) +- 0.0010 (expt.) +- 0.0011 (hadr.) +- 0.0044 (theo.). The energy evolution of the moments is also used to determine a value of alpha_s with slightly larger errors: alpha_s(M_Z) = 0.1223 +- 0.0005 (stat.) +- 0.0014 (expt.) +- 0.0016 (hadr.) +0.0054 -0.0036 (theo.).Comment: 63 pages 26 fi

Flavour Independent hA Search and Two Higgs Doublet Model Interpretation of Neutral Higgs Boson Searches at LEP

Upper limits on the cross-section of the pair-production process e+e- -> h0A0 assuming 100% decays into hadrons, are derived from a new search for the h0A0 -> hadrons topology, independent of the hadronic flavour of the decay products. Searches for the neutral Higgs bosons h0 and A0, are used to obtain constraints on the Type II Two Higgs Doublet Model (2HDM(11)) with no CP violation in the Higgs sector and no additional non Standard Model particles besides the five Higgs bosons. The analysis combines LEP1 and LEP2 data collected with the OPAL detctor up to the highest available centre-of-mass energies. The searches are sensitive to the h0, A0 -> qq, gg,tau+tau- and h0 -> A0A0 decay modes of the Higgs bosons. The 2HDM(II) parameter space is explored in a detailed scan. Large regions of the 2HDM(II) parameter space are excluded at the 95% CL in the (mh, mA), (mh, tanb) and (mA, tanb) planes, using both direct neutral Higgs boson searches and indirect limits derived from Standard Model high precision measurements. The region 1 lesssim mh lesssim 55 GeV and 3 lesssim mA lesssim 63 GeV is excluded at 95% CL independently of the choice of the 2HDM(II) parameters.Comment: 37 pages, 11 figures, Submitted to Eur. Phys. J.

Expected Performance of the ATLAS Experiment - Detector, Trigger and Physics

A detailed study is presented of the expected performance of the ATLAS detector. The reconstruction of tracks, leptons, photons, missing energy and jets is investigated, together with the performance of b-tagging and the trigger. The physics potential for a variety of interesting physics processes, within the Standard Model and beyond, is examined. The study comprises a series of notes based on simulations of the detector and physics processes, with particular emphasis given to the data expected from the first years of operation of the LHC at CERN

Search for direct stau production in events with two hadronic tau-leptons in root s=13 TeV pp collisions with the ATLAS detector

A search for the direct production of the supersymmetric partners ofτ-leptons (staus) in final stateswith two hadronically decayingτ-leptons is presented. The analysis uses a dataset of pp collisions corresponding to an integrated luminosity of139fb−1, recorded with the ATLAS detector at the LargeHadron Collider at a center-of-mass energy of 13 TeV. No significant deviation from the expected StandardModel background is observed. Limits are derived in scenarios of direct production of stau pairs with eachstau decaying into the stable lightest neutralino and oneτ-lepton in simplified models where the two staumass eigenstates are degenerate. Stau masses from 120 GeV to 390 GeV are excluded at 95% confidencelevel for a massless lightest neutralino

Crystal structure of 2-(2-naphthyl)-4,6-dimethylpyrimidine, C(16)H(14)N(2)

C(16)H(14)N(2), monoclinic, P12(1)/c1 (no. 14), a = 8.387(1) angstrom, b = 17.453(3) angstrom, c = 8.694(1) angstrom, beta = 93.092(2)degrees, V = 1270.8 angstrom(3), Z = 4, R(gt)(F) = 0.046, wR(ref)(F(2)) = 0.129, T = 294 K.program for innovation of science and technology talents of Henan Province[104200510022]; program for science and technology leaders of Zhengzhou City[10LJRC174]; Doctor Foundation[D09004]; program for innovative research team of Henan Institute of Engineering[2009IRTHNIE05

Design and baseline characteristics of the finerenone in reducing cardiovascular mortality and morbidity in diabetic kidney disease trial

Background: Among people with diabetes, those with kidney disease have exceptionally high rates of cardiovascular (CV) morbidity and mortality and progression of their underlying kidney disease. Finerenone is a novel, nonsteroidal, selective mineralocorticoid receptor antagonist that has shown to reduce albuminuria in type 2 diabetes (T2D) patients with chronic kidney disease (CKD) while revealing only a low risk of hyperkalemia. However, the effect of finerenone on CV and renal outcomes has not yet been investigated in long-term trials. Patients and Methods: The Finerenone in Reducing CV Mortality and Morbidity in Diabetic Kidney Disease (FIGARO-DKD) trial aims to assess the efficacy and safety of finerenone compared to placebo at reducing clinically important CV and renal outcomes in T2D patients with CKD. FIGARO-DKD is a randomized, double-blind, placebo-controlled, parallel-group, event-driven trial running in 47 countries with an expected duration of approximately 6 years. FIGARO-DKD randomized 7,437 patients with an estimated glomerular filtration rate >= 25 mL/min/1.73 m(2) and albuminuria (urinary albumin-to-creatinine ratio >= 30 to <= 5,000 mg/g). The study has at least 90% power to detect a 20% reduction in the risk of the primary outcome (overall two-sided significance level alpha = 0.05), the composite of time to first occurrence of CV death, nonfatal myocardial infarction, nonfatal stroke, or hospitalization for heart failure. Conclusions: FIGARO-DKD will determine whether an optimally treated cohort of T2D patients with CKD at high risk of CV and renal events will experience cardiorenal benefits with the addition of finerenone to their treatment regimen. Trial Registration: EudraCT number: 2015-000950-39; ClinicalTrials.gov identifier: NCT02545049

A Search for Photons with Energies Above 2X10(17) eV Using Hybrid Data from the Low-Energy Extensions of the Pierre Auger Observatory

Ultra-high-energy photons with energies exceeding 10(17) eV offer a wealth of connections to different aspects of cosmic-ray astrophysics as well as to gamma-ray and neutrino astronomy. The recent observations of photons with energies in the 10(15) eV range further motivate searches for even higher-energy photons. In this paper, we present a search for photons with energies exceeding 2 x 10(17) eV using about 5.5 yr of hybrid data from the low-energy extensions of the Pierre Auger Observatory. The upper limits on the integral photon flux derived here are the most stringent ones to date in the energy region between 10(17) and 10(18) eV

Correlated long-range mixed-harmonic fluctuations measured in pp, p+Pb and low-multiplicity Pb+Pb collisions with the ATLAS detector

For abstract see published article