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Muography is an imaging technique based on the measurement of absorption profiles for muons as they pass through rocks and earth. Muons are produced in the interactions of high-energy cosmic rays in the Earth’s atmosphere. The technique is conceptually similar to usual X-ray radiography, but with extended capabilities of investigating over much larger thicknesses of matter thanks to the penetrating power of high-energy muons. Over the centuries a complex system of cavities has been excavated in the yellow tuff of Mt. Echia, the site of the earliest settlement of the city of Naples in the 8th century BC. A new generation muon detector designed by us, was installed under a total rock overburden of about 40 metres. A 26 days pilot run provided about 14 millions of muon events. A comparison of the measured and expected muon fluxes improved the knowledge of the average rock density. The observation of known cavities proved the validity of the muographic technique. Hints on the existence of a so far unknown cavity was obtained. The success of the investigation reported here demonstrates the substantial progress of muography in underground imaging and is likely to open new avenues for its widespread utilisation.

Background Microbial lipids are produced by many oleaginous organisms including the well-characterized yeast Yarrowia lipolytica, which can be engineered for increased lipid yield by up-regulation of the lipid biosynthetic pathway and down-regulation or deletion of competing pathways. Results We describe a strain engineering strategy centered on diacylglycerol acyltransferase (DGA) gene overexpression that applied combinatorial screening of overexpression and deletion genetic targets to construct a high lipid producing yeast biocatalyst. The resulting strain, NS432, combines overexpression of a heterologous DGA1 enzyme from Rhodosporidium toruloides, a heterlogous DGA2 enzyme from Claviceps purpurea, and deletion of the native TGL3 lipase regulator. These three genetic modifications, selected for their effect on lipid production, enabled a 77 % lipid content and 0.21 g lipid per g glucose yield in batch fermentation. In fed-batch glucose fermentation NS432 produced 85 g/L lipid at a productivity of 0.73 g/L/h. Conclusions The yields, productivities, and titers reported in this study may further support the applied goal of cost-effective, large -scale microbial lipid production for use as biofuels and biochemicals.

Background Lipids are important precursors in the biofuel and oleochemical industries. Yarrowia lipolytica is among the most extensively studied oleaginous microorganisms and has been a focus of metabolic engineering to improve lipid production. Yield improvement, through rewiring of the central carbon metabolism of Y. lipolytica from glucose to the lipid precursor acetyl-CoA, is a key strategy for achieving commercial success in this organism. Results Building on YB-392, a Y. lipolytica isolate known for stable non-hyphal growth and low citrate production with demonstrated potential for high lipid accumulation, we assembled a heterologous pathway that redirects carbon flux from glucose through the pentose phosphate pathway (PPP) to acetyl-CoA. We used phosphofructokinase (Pfk) deletion to block glycolysis and expressed two non-native enzymes, phosphoketolase (Xpk) and phosphotransacetylase (Pta), to convert PPP-produced xylulose-5-P to acetyl-CoA. Introduction of the pathway in a pfk deletion strain that is unable to grow and accumulate lipid from glucose in defined media ensured maximal redirection of carbon flux through Xpk/Pta. Expression of Xpk and Pta restored growth and lipid production from glucose. In 1-L bioreactors, the engineered strains recorded improved lipid yield and cell-specific productivity by up to 19 and 78%, respectively. Conclusions Yields and cell-specific productivities are important bioprocess parameters for large-scale lipid fermentations. Improving these parameters by engineering the Xpk/Pta pathway is an important step towards developing Y. lipolytica as an industrially preferred microbial biocatalyst for lipid production.

A large number of particle detectors employ liquid argon as their target material owing to its high scintillation yield and its ability to drift ionization charge over large distances. Scintillation light from argon is peaked at 128 nm and a wavelength shifter is required for its efficient detection. In this work, we directly compare the light yield achieved in two identical liquid argon chambers, one of which is equipped with polyethylene naphthalate (PEN) and the other with tetraphenyl butadiene (TPB) wavelength shifter. Both chambers are lined with enhanced specular reflectors and instrumented with SiPMs with a coverage fraction of approximately 1%, which represents a geometry comparable to the future large scale detectors. We measured the light yield of the PEN chamber to be 39.4±0.4(stat)±1.9(syst)% of the yield of the TPB chamber. Using a Monte Carlo simulation this result is used to extract the wavelength shifting efficiency of PEN relative to TPB equal to 47.2±5.7%. This result paves the way for the use of easily available PEN foils as a wavelength shifter, which can substantially simplify the construction of future liquid argon detectors.

BackgroundOleate-enriched triacylglycerides are well-suited for lubricant applications that require high oxidative stability. Fatty acid carbon chain length and degree of desaturation are key determinants of triacylglyceride properties and the ability to manipulate fatty acid composition in living organisms is critical to developing a source of bio-based oil tailored to meet specific application requirements.ResultsWe sought to engineer the oleaginous yeast Yarrowia lipolytica for production of high-oleate triacylglyceride oil. We studied the effect of deletions and overexpressions in the fatty acid and triacylglyceride synthesis pathways to identify modifications that increase oleate levels. Oleic acid accumulation in triacylglycerides was promoted by exchanging the native ∆9 fatty acid desaturase and glycerol-3-phosphate acyltransferase with heterologous enzymes, as well as deletion of the Δ12 fatty acid desaturase and expression of a fatty acid elongase. By combining these engineering steps, we eliminated polyunsaturated fatty acids and created a Y. lipolytica strain that accumulates triglycerides with > 90% oleate content.ConclusionsHigh-oleate content and lack of polyunsaturates distinguish this triacylglyceride oil from plant and algal derived oils. Its composition renders the oil suitable for applications that require high oxidative stability and further demonstrates the potential of Y. lipolytica as a producer of tailored lipid profiles.

Background Yarrowia lipolytica is an oleaginous yeast that can be genetically engineered to produce lipid and non-lipid biochemicals from a variety of feedstocks. Metabolic engineering of this organism usually requires genetic markers in order to select for modified cells. The potential to combine multiple genetic manipulations depends on the availability of multiple or recyclable selectable markers. Results We found that Y. lipolytica has the ability to utilize acetamide as the sole nitrogen source suggesting that the genome contains an acetamidase gene. Two potential Y. lipolytica acetamidase gene candidates were identified by homology to the A. nidulans acetamidase amdS. These genes were deleted in the wild-type Y. lipolytica strain YB-392, and deletion strains were evaluated for acetamide utilization. One deletion strain was unable to grow on acetamide and a putative acetamidase gene YlAMD1 was identified. Transformation of YlAMD1 followed by selection on acetamide media and counterselection on fluoroacetamide media showed that YlAMD1 can be used as a recyclable genetic marker in Saccharomyces cerevisiae and Ylamd1Δ Y. lipolytica . Conclusions These findings add to our understanding of Y. lipolytica nitrogen utilization and expand the set of genetic tools available for engineering this organism, as well as S. cerevisiae .

Background Despite the environmental value of biobased lubricants, they account for less than 2% of global lubricant use due to poor thermo-oxidative stability arising from the presence of unsaturated double bonds. Methyl branched fatty acids (BFAs), particularly those with branching near the acyl-chain mid-point, are a high-performance alternative to existing vegetable oils because of their low melting temperature and full saturation. Results We cloned and characterized two pathways to produce 10-methyl BFAs isolated from actinomycetes and γ-proteobacteria. In the two-step bfa pathway of actinomycetes, BfaB methylates Δ9 unsaturated fatty acids to form 10-methylene BFAs, and subsequently, BfaA reduces the double bond to produce a fully saturated 10-methyl branched fatty acid. A BfaA-B fusion enzyme increased the conversion efficiency of 10-methyl BFAs. The ten-methyl palmitate production (tmp) pathway of γ-proteobacteria produces a 10-methylene intermediate, but the TmpA putative reductase was not active in E. coli or yeast. Comparison of BfaB and TmpB activities revealed a range of substrate specificities from C14-C20 fatty acids unsaturated at the Δ9, Δ10 or Δ11 position. We demonstrated efficient production of 10-methylene and 10-methyl BFAs in S. cerevisiae by secretion of free fatty acids and in Y. lipolytica as triacylglycerides, which accumulated to levels more than 35% of total cellular fatty acids. Conclusions We report here the characterization of a set of enzymes that can produce position-specific methylene and methyl branched fatty acids. Yeast expression of bfa enzymes can provide a platform for the large-scale production of branched fatty acids suitable for industrial and consumer applications.

In the recent years, argon-based experiments looking for Dark Matter in the Universe have explored the non-standard scenario in which Dark Matter is made by low-mass Weakly Interacting Massive Particles, of mass in the range of 1–10 GeV instead of the canonical hundreds of GeV. Detecting such particles is challenging, as their expected signatures are nuclear recoils with energies below 10 keV, observable solely via ionization. This necessitates a precise understanding of the detector response in this energy regime, which remains incomplete for argon. To address this, the ReD experiment was developed within the framework of the DarkSide-20k Collaboration to produce and characterize few-keV nuclear recoils. A compact dual-phase argon Time Projection Chamber (TPC) was irradiated with neutrons from a$$^{252}$$252 Cf source, to produce Ar recoils in the energy range of interest via (n,n’) elastic scattering. A downstream spectrometer composed of 18 plastic scintillators detected the neutrons scattered off Ar nuclei, enabling recoil energy reconstruction via two-body kinematics. The ionization yield$$Q_{y}$$Q y of argon, defined as the number of electrons produced per unit energy deposit, was measured in a model-independent way between 2 and 10 keV. These measurements extend direct experimental coverage well below the previous limit of approximately 7 keV. The results are consistent with existing data above 7 keV, while they indicate a higher$$Q_{y}$$Q y at lower energies.

Silicon Photomultipliers (SiPMs) are excellent devices to detect the faint and short Cherenkov light emitted in high energy atmospheric showers, and therefore suitable for use in imaging air Cherenkov Telescopes. The high density Near Ultraviolet Violet SiPMs (NUV-HD3) produced by Fondazione Bruno Kessler (FBK) in collaboration with INFN were used to equip optical modules for a possible upgrade of the Schwarzschild-Couder Telescope camera prototype, in the framework of the Cherenkov Telescope Array project. SiPMs are 6×6 mm 2 devices based on 40×40 μm 2 microcells optimized for photo-detection at the NUV wavelengths. More than 40 optical modules, each composed by a 4×4 array of SiPMs, were assembled. In this contribution we report on the development and on the assembly of the optical modules, their validation and integration in the camera.

Protons and helium nuclei are the most abundant components of the cosmic radiation. Precise measurements of their fluxes are needed to understand the acceleration and subsequent propagation of cosmic rays in our Galaxy. We report precision measurements of the proton and helium spectra in the rigidity range 1 gigavolt to 1.2 teravolts performed by the satellite-borne experiment PAMELA (payload for antimatter matter exploration and light-nuclei astrophysics). We find that the spectral shapes of these two species are different and cannot be described well by a single power law. These data challenge the current paradigm of cosmic-ray acceleration in supernova remnants followed by diffusive propagation in the Galaxy. More complex processes of acceleration and propagation of cosmic rays are required to explain the spectral structures observed in our data.