High-throughput isolation of ultra-pure plasmid DNA by a robotic system

BACKGROUND: With the availability of complete genomes, a systematic inventory of cellular processes becomes achievable. This requires assessing the function of all individual genes. Transfection of plasmid DNA into cell culture cells is an essential technique for this aim as it allows functional overexpression or downregulation of genes. While many robotic systems isolate plasmids for sequencing purposes, for more demanding applications such as transfections there is a shortage of robots for the high-throughput isolation of plasmid DNA. RESULTS: Here we describe a custom-made, automated device, which uses a special protocol to isolate plasmid DNAs with a purity sufficient for efficient transfections into mammalian cells. Approximately 1,600 ultra pure plasmids can be isolated in a 96-well plate format within 12 hours. As a unique feature the robot comprises the integration of a centrifuge instead of expensive columns, the use of a custom-made pipetting head with a movable gripper, especially designed shaking platforms and an acetone wash facility. CONCLUSION: Using this robot we demonstrate how centrifugation steps with multiple precipitations, most notably through a precipitation step of SDS in isopropanol, lead to high purity plasmid DNA and make possible high-throughput transfections into mammalian cells for functional gene annotations

Rapid Decline of Serum Proprotein Convertase Subtilisin/Kexin 9 (PCSK9) in Non-Cirrhotic Patients with Chronic Hepatitis C Infection Receiving Direct-Acting Antiviral Therapy.

Direct-acting antivirals (DAAs) efficiently eradicate the hepatitis C virus (HCV). Low-density lipoprotein (LDL) levels increase rapidly upon DAA treatment. Proprotein convertase subtilisin/kexin 9 (PCSK9) induces degradation of the hepatic LDL receptor and thereby elevates serum LDL. The aim of this study was to determine serum PCSK9 concentrations during and after DAA therapy to identify associations with LDL levels. Serum PCSK9 was increased in 82 chronic HCV-infected patients compared to 55 patients not infected with HCV. Serum PCSK9 was low in HCV patients with liver cirrhosis, but patients with HCV-induced liver cirrhosis still exhibited higher serum PCSK9 than patients with non-viral liver cirrhosis. Serum PCSK9 correlated with measures of liver injury and inflammation in cirrhotic HCV patients. In patients without liver cirrhosis, a positive association of serum PCSK9 with viral load existed. Serum PCSK9 was not different between viral genotypes. Serum PCSK9 did not correlate with LDL levels in HCV patients irrespective of cirrhotic status. Serum PCSK9 was reduced, and LDL was increased at four weeks after DAA therapy start in non-cirrhotic HCV patients. Serum PCSK9 and LDL did not change upon DAA treatment in the cirrhotic group. The rapid decline of PCSK9 after the start of DAA therapy in conjunction with raised LDL levels in non-cirrhotic HCV patients shows that these changes are not functionally related

Origen de la casiterita detrítica en los aluviones recientes de Tirados de la Vega-Golpejas (Salamanca)

9 páginas, 6 figuras, 5 tablas.[ES] En el sector Tirados de la Vega-Golpejas han sido detectadas dos anomalías de casiterita en los aluviones del Regato de los Lentiscos y en otros existentes al sur de Tirados de la Vega. El estudio granométrico y morfométrico de éstas casiteritas. contrastado con el de la casiterita procedente del granito de Golpejas. permite asegurar que la anomalía del Regato de los Lentiscos se debe a la dispersión de la casiterita de aquel granito. mientras que la de Tirados de la Vega procede de filones estanníferos. La aplicación del análisis factoral corrobora este hecho y permite diferenciar las casiteritas que proceden de rocas graníticas de aquellas que vienen de filones estanníferos. Según esto se puede indicar que la capacidad de migración de la casiterita desde el yacimiento estannífero de Golpejas es de unos 3 Kms.[EN] Two cassiterite anomalies have been detected in the Regato de los Lentisco and Tirados de la Vega alluviums, in the Golpejas area (Salamanca). The granometric and morphometric parameters of these cassiterites have been compared with those shown by the cassiterite crystal ocurrying in the Golpejas granite. The comparison between the two groups of data allow us to conclude that the anomaly observed in the Regato de los Lentistos has been originated by the disperssion of the cassiterite from the primary deposit of Golpejas. On the contrary, the anomaly ocurring in the alloviums to the south of Tirados de la Vega shows a different origin, and is coming from stanniferous veins. The factor anylisis corroborates the differentiation mentionated above, and indicates a migration capacite of about 3 kms. from the stannifeous deposits of Golpejas for the alluvial cassiterite.Peer reviewe

Numerical Simulation of a Model Spray Flame under MILD conditions using Stochastic Upstream Flow and Temperature Forcing

Numerical simulations of the Delft Spray in Hot Co-flow (DSHC) flame are presented, in order to aid the understanding of reacting multiphase flows under moderate or low-oxygen dilution (MILD) conditions. The test case consists of a single swirled pressure atomizer installed in the center of a cylindrical hot co-flow, operated with ethanol fuel. A large variety of experimental data is available for the burner’s MILD combustion configuration. Here, the particular H-II case is studied. Three different modelling approaches are employed, an unsteady RANS simulation and two scale-resolving methods, namely LES (Large Eddy Simulation) and SAS-SST (Scale Adaptive Simulation) in combination with a Shear Stress Transport turbulence model. Here, for the scale resolving SAS and LES, transient inflow boundary conditions are necessary in order to propagate turbulent flow and temperature structures into the computational domain, supporting the evolution of a full turbulent energy cascade. However, preliminary simulations have shown that due to the low Reynolds number of the co-flow, artificially imposed spatial and temporal turbulent fluctuations of temperature and velocity field are subject to strong artificial decay, prior to reaching the actual combustion zone. Therefore, a simplified stochastic forcing approach based on a first order Langevin model is adopted, reducing the boundary condition to a time dependent function which generates time-coherent structures featuring turbulent decay in time. The accurate implementation of the methodology is verified by means of analytical solutions and validated with the Delft Spray flame test case

Stability and breakdown of Fermi polarons in a strongly interacting Fermi-Bose mixture

We investigate the properties of a strongly interacting imbalanced mixture of bosonic $^{41}$K impurities immersed in a Fermi sea of ultracold $^6$Li atoms. This enables us to explore the Fermi polaron for large impurity concentrations including the case where they form a Bose-Einstein condensate. The system is characterized by means of radio-frequency injection spectroscopy for tunable interactions using an interspecies Feshbach resonance. We find that the energy of the Fermi polarons formed in the thermal fraction of the impurity cloud remains rather insensitive to the impurity concentration, even as we approach equal densities for both species. The apparent insensitivity to high concentration is consistent with the theoretical prediction, based on Landau's quasiparticle theory, of a weak effective interaction between the polarons. The condensed fraction of the bosonic $^{41}$K gas is much denser than its thermal component, which leads to a break-down of the Fermi polaron description. Instead, we observe a new branch in the radio-frequency spectrum with a small energy shift, which is consistent with the presence of Bose polarons formed by $^{6}$Li fermions inside the $^{41}$K condensate. A closer investigation of the behavior of the condensate by means of Rabi oscillation measurements support this observation, indicating that we have realized Fermi and Bose polarons, two fundamentally different quasiparticles, in one cloud.Comment: 16 pages, 10 figure

Stability and breakdown of Fermi polarons in a strongly interacting Fermi-Bose mixture

We investigate the properties of a strongly interacting imbalanced mixture of bosonic 41K impurities immersed in a Fermi sea of ultracold 6 Li atoms. This enables us to explore the Fermi polaron scenario for large impurity concentrations including the case where they form a Bose-Einstein condensate. The system is characterized by means of radio-frequency injection spectroscopy and interspecies interactions are widely tunable by means of a well-characterized Feshbach resonance. We find that the energy of the Fermi polarons formed in the thermal fraction of the impurity cloud remains rather insensitive to the impurity concentration, even as we approach equal densities for both speciesPostprint (published version

Mammalian WDR12 is a novel member of the Pes1–Bop1 complex and is required for ribosome biogenesis and cell proliferation

Target genes of the protooncogene c-myc are implicated in cell cycle and growth control, yet the linkage of both is still unexplored. Here, we show that the products of the nucleolar target genes Pes1 and Bop1 form a stable complex with a novel member, WDR12 (PeBoW complex). Endogenous WDR12, a WD40 repeat protein, is crucial for processing of the 32S precursor ribosomal RNA (rRNA) and cell proliferation. Further, a conditionally expressed dominant-negative mutant of WDR12 also blocks rRNA processing and induces a reversible cell cycle arrest. Mutant WDR12 triggers accumulation of p53 in a p19ARF-independent manner in proliferating cells but not in quiescent cells. Interestingly, a potential homologous complex of Pes1–Bop1–WDR12 in yeast (Nop7p–Erb1p–Ytm1p) is involved in the control of ribosome biogenesis and S phase entry. In conclusion, the integrity of the PeBoW complex is required for ribosome biogenesis and cell proliferation in mammalian cells

Gender-Specific Differences in Serum Sphingomyelin Species in Patients with Hepatitis C Virus Infection—Sphingomyelin Species Are Related to the Model of End-Stage Liver Disease (MELD) Score in Male Patients

Hepatitis C virus (HCV) replication depends on cellular sphingomyelin (SM), but serum SM composition in chronic HCV infection has been hardly analyzed. In this work, 18 SM species could be quantified in the serum of 178 patients with chronic HCV infection before therapy with direct-acting antivirals (DAAs) and 12 weeks later, when therapy was completed. Six SM species were higher in the serum of females than males before therapy and nine at the end of therapy; thus, sex-specific analysis was performed. Type 2 diabetes was associated with lower serum levels of SM 36:2;O2 and 38:2;O2 in men. Serum SM species did not correlate with the viral load in both sexes. Of note, three SM species were lower in males infected with HCV genotype 3 in comparison to genotype 1 infection. These SM species normalized after viral cure. SM 38:1;O2, 40:1;O2, 41:1;O2, and 42:1;O2 (and, thus, total SM levels) were higher in the serum of both sexes at the end of therapy. In males, SM 39:1;O2 was induced in addition, and higher levels of all of these SM species were already detected at 4 weeks after therapy has been started. Serum lipids are related to liver disease severity, and in females 15 serum SM species were low in patients with liver cirrhosis before initiation of and after treatment with DAAs. The serum SM species did not correlate with the model of end-stage liver disease (MELD) score in the cirrhosis and the non-cirrhosis subgroups in females. In HCV-infected male patients, nine SM species were lower in the serum of patients with cirrhosis before DAA treatment and eleven at the end of the study. Most of the SM species showed strong negative correlations with the MELD score in the male cirrhosis patients before DAA treatment and at the end of therapy. Associations of SM species with the MELD score were not detected in the non-cirrhosis male subgroup. In summary, the current analysis identified sex-specific differences in the serum levels of SM species in HCV infection, in liver cirrhosis, and during DAA therapy. Correlations of SM species with the MELD score in male but not in female patients indicate a much closer association between SM metabolism and liver function in male patients