Applied Microbiology and Biotechnology / The bud tip is the cellular hot spot of protein secretion in yeasts

Abstract in deutscher Sprache nicht verf\ufcgbarYeasts are valuable hosts for recombinant protein production. Among them, Pichia pastoris is frequently used for production of secreted proteins, and much effort was made to improve the secretion efficiency of this expression platform. However, the knowledge on the secretion machinery is mainly based on studies in Saccharomyces cerevisiae. Therefore, it is of great interest for targeted improvement of the system to learn more about the secretion process in P. pastoris. Using human serum albumin, a protein which is produced in high quantities in P. pastoris, we show here the secretion pathway of this protein. During passage of the secretory route, the recombinant protein is mainly localized in the endoplasmic reticulum (ER) and in COPII vesicles, and is inherited to the daughter cell via the perinuclear ER. The final release to the cell exterior occurs at the bud, initiating at the bud tip and later spreading over the entire bud surface. The same polarized secretion pattern was observed for a recombinant antibody light chain and the native secretory protein Epx1 of P. pastoris. Clarifying the point of release of secretory proteins will have major impact on engineering the secretory pathway of P. pastoris and other budding yeasts

An overview of atmospheric deposition chemistry over the Alps: present status and long-term trends

Several research programs monitoring atmospheric deposition have been launched in the Alpine countries in the last few decades. This paper uses data from previous and ongoing projects to: (i) investigate geographical variability in wet deposition chemistry over the Alps; (ii) assess temporal trends of the major chemical variables in response to changes in the atmospheric emission of pollutants; (iii) discuss the potential relationship between the status of atmospheric deposition and its effects on forest ecosystems in the alpine and subalpine area, focusing particularly on nitrogen input. We also present results of studies performed at a local level on specific topics such as long-term changes in lead deposition and the role of occult deposition in total nitrogen input. The analysis performed here highlights the marked geographical variability of atmospheric deposition in the Alpine region. Apart from some evidence of geographically limited effects, due to local sources, no obvious gradients were identified in the major ion deposition. The highest ionic loads were recorded in areas in the foothills of the Alps, such as the pre-alpine area in North-Western Italy and the area of Canton Ticino, Switzerland. Trend analysis shows a widespread decrease in the acidity of precipitation in the last 15–20 years as a consequence of the reduced emission of S compounds. On the other hand, nitrate concentrations in rain have not changed so much, and ammonium has decreased significantly only at the Austrian sampling sites. The deposition of N is still well above the estimated critical loads of nutrient N at some forest sites in the alpine and subalpine areas, thus confirming the critical situation of both terrestrial and aquatic ecosystems regarding N inputs. Existing data highlights the importance of continuously monitoring atmospheric deposition chemistry in the Alpine area, taking account of acidifying elements, nutrients and other pollutants such as heavy metals and organic compounds. There is also a need for unifying sampling and analytical methods in order to obtain comparable data from the different regions of the Alps

On the effects of organic matter and sulphur-containing compounds on the CCN activation of combustion particles

The European PartEmis project (Measurement and prediction of emissions of aerosols and gaseous precursors from gas turbine engines) was focussed on the characterisation and quantification of exhaust emissions from a gas turbine engine. The combustion aerosol characterisation included on-line measurements of mass and number concentration, size distribution, mixing state, thermal stability of internally mixed particles, hygroscopicity, cloud condensation nuclei (CCN) activation potential, and off-line analysis of chemical composition. Based on this extensive data set, the role of sulphuric acid coating and of the organic fraction of the combustion particles for the CCN activation was investigated. Modelling of CCN activation was conducted using microphysical and chemical properties obtained from the measurements as input data. Coating the combustion particles with water-soluble sulphuric acid, increases the potential CCN activation, or lowers the activation diameter, respectively. The adaptation of a Köhler model to the experimental data yielded coatings from 0.1 to 3 vol-% of water-soluble matter, which corresponds to an increase in the fraction of CCN-activated combustion particles from ≤10^−4 to ≌10^−2 at a water vapour saturation ratio S_w=1.006. Additional particle coating by coagulation of combustion particles and aqueous sulphuric acid particles formed by nucleation further reduces the CCN activation diameter. In contrast, particles containing a large fraction of non-volatile organic compounds grow significantly less at high relative humidity than particles with a lower content of non-volatile OC. The resulting reduction in the potential CCN activation with an increasing fraction of non-volatile OC becomes visible as a trend in the experimental data. While a coating of water-soluble sulphuric acid increases the potential CCN activation, or lowers the activation diameter, respectively, the non-volatile organic compounds, mainly found at lower combustion temperatures, can partially compensate this sulphuric acid-related enhancement of CCN activation of carbonaceous combustion aerosol particles

On the effects of hydrocarbon and sulphur-containing compounds on the CCN activation of combustion particles

International audienceThe European PartEmis project (''Measurement and prediction of emissions of aerosols and gaseous precursors from gas turbine engines'') was focussed on the characterisation and quantification of exhaust emissions from a gas turbine engine. A comprehensive suite of aerosol, gas and chemi-ion measurements were conducted under different combustor operating conditions and fuel sulphur concentrations. Combustion aerosol characterisation included on-line measurements of mass and number concentration, size distribution, mixing state, thermal stability of internally mixed particles, hygroscopicity, cloud condensation nuclei (CCN) activation potential, and off-line analysis of chemical composition. Modelling of CCN activation of combustion particles was conducted using microphysical and chemical properties obtained from the measurements as input data. Based on this unique data set, the role of sulphuric acid coatings on the combustion particles, formed in the cooling exhaust plume through either direct condensation of gaseous sulphuric acid or coagulation with volatile condensation particles nucleating from gaseous sulphuric acid, and the role of the organic fraction for the CCN activation of combustion particles was investigated. It was found that particles containing a large fraction of non-volatile organic compounds grow significantly less at high relative humidity than particles with a lower content of non-volatile OC. Also the effect of the non-volatile OC fraction on the potential CCN activation is significant. While a coating of water-soluble sulphuric acid increases the potential CCN activation, or lowers the activation diameter, respectively, the non-volatile organic compounds, mainly found at lower combustion temperatures, can partially compensate this sulphuric acid-related enhancement of CCN activation of carbonaceous combustion aerosol particles

An intercomparison of measurement systems for vapor and particulate phase concentrations of formic and acetic acids

During June 1986, eight systems for measuring vapor phase and four for measuring particulate phase concentrations of formic acid (HCOOH) and acetic acid (CH_3COOH) were intercompared in central Virginia. HCOOH and CH_3COOH vapors were sampled by condensate, mist, Chromosorb 103 GC resin, NaOH-coated annular denuders, NaOH impregnated quartz filters, K_2CO_3 and Na_2CO_3 impregnated cellulose filters, and Nylasorb membranes. Atmospheric aerosol was collected on Teflon and Nuclepore filters using both hi-vol and lo-vol systems to measure particulate phase concentrations. Samples were collected during 31 discrete day and night intervals of 0.5–2 hour duration over a 4-day period. Performance of the mist chamber and K_2CO_3 impregnated filter techniques were also evaluated using zero air and ambient air spiked with HCOOH_g, CH_3COOH_g, and formaldehyde (CH_2O_g) from permeation sources. Results of this intercomparison show significant systematic and episodic artifacts among many currently deployed measurement systems for HCOOH_g and CH_3COOH_g. The spiking experiments revealed no significant interferences for the mist chamber technique and results generated by the mist chamber and denuder techniques were statistically indistinguishable. The condensate technique showed general agreement with the mist chamber and denuder methods, but episodic bias between these systems was inferred from large and significant differences observed during the first day of sampling. Nylasorb membranes are unacceptable for collecting carboxylic acid vapors as they did not retain HCOOH_g and CH_3COOH_g quantitatively. Strong base impregnated filter and GC resin sampling techniques are prone to large positive interferences apparently resulting, in part, from reactions involving CH_2O_g to generate HCOOH and CH_3COOH subsequent to collection. Significant bias presumably associated with differences in postcollection handling was observed for particulate phase measurements by participating groups. Analytical bias did not contribute significantly to differences in vapor and particulate phase measurements

Organic compounds in PM2.5 emitted from fireplace and woodstove combustion of typical Portuguese wood species

The aim of this study is the further characterisation of PM2.5 emissions from the residential wood combustion of common woods grown in Portugal. This new research extends to eight the number of biomass fuels studied and tries to understand the differences that the burning appliance (fireplace versus woodstove) and the combustion temperature (cold and hot start) have on emissions. Pinus pinaster (Maritime pine), Eucalyptus globulus (eucalypt), Quercus suber (cork oak), Acacia longifolia (Golden wattle), Quercus faginea (Portuguese oak), Olea europea (Olive), Quercus ilex rotundifolia (Holm oak) and briquettes produced from forest biomass waste were used in the combustion tests. Determinations included fine particle emission factors, carbonaceous content (OC and EC) by a thermaleoptical transmission technique and detailed identification and quantification of organic compounds by gas chromatographyemass spectrometry. Fine particle emission factors from the woodstove were lower than those from the fireplace. For both combustion appliances, the OC/EC ratio was higher in “cold start” tests (1.56 0.95 for woodstove and 2.03 1.34 for fireplace). These “cold start” OC/EC values were, respectively, for the woodstove and the fireplace, 51% and 69% higher than those obtained in “hot start” experiments. The chromatographically resolved organics included n-alkanes, n-alkenes, PAHs, n-alkanals, ketones, n-alkanols, terpenoids, triterpenoids, phenolic compounds, phytosterols, alcohols, n-alkanoic acids, n-di-acids, unsaturated acids and alkyl esters of acids. The smoke emission rate and composition varied widely depending on fuel type, burning appliance and combustion temperature

BAF complex vulnerabilities in cancer demonstrated via structure-based PROTAC design

Targeting subunits of BAF/PBAF chromatin remodeling complexes has been proposed as an approach to exploit cancer vulnerabilities. Here, we develop proteolysis targeting chimera (PROTAC) degraders of the BAF ATPase subunits SMARCA2 and SMARCA4 using a bromodomain ligand and recruitment of the E3 ubiquitin ligase VHL. High-resolution ternary complex crystal structures and biophysical investigation guided rational and efficient optimization toward ACBI1, a potent and cooperative degrader of SMARCA2, SMARCA4 and PBRM1. ACBI1 induced anti-proliferative effects and cell death caused by SMARCA2 depletion in SMARCA4 mutant cancer cells, and in acute myeloid leukemia cells dependent on SMARCA4 ATPase activity. These findings exemplify a successful biophysics- and structure-based PROTAC design approach to degrade high profile drug targets, and pave the way toward new therapeutics for the treatment of tumors sensitive to the loss of BAF complex ATPases.</p

Oxalic acid in clear and cloudy atmospheres: Analysis of data from International Consortium for Atmospheric Research on Transport and Transformation 2004

Oxalic acid is often the leading contributor to the total dicarboxylic acid mass in ambient organic aerosol particles. During the 2004 International Consortium for Atmospheric Research on Transport and Transformation (ICARTT) field campaign, nine inorganic ions (including SO_4^(2−)) and five organic acid ions (including oxalate) were measured on board the Center for Interdisciplinary Remotely Piloted Aircraft Studies (CIRPAS) Twin Otter research aircraft by a particle-into-liquid sampler (PILS) during flights over Ohio and surrounding areas. Five local atmospheric conditions were studied: (1) cloud-free air, (2) power plant plume in cloud-free air with precipitation from scattered clouds overhead, (3) power plant plume in cloud-free air, (4) power plant plume in cloud, and (5) clouds uninfluenced by local pollution sources. The aircraft sampled from two inlets: a counterflow virtual impactor (CVI) to isolate droplet residuals in clouds and a second inlet for sampling total aerosol. A strong correlation was observed between oxalate and SO_4^(2−) when sampling through both inlets in clouds. Predictions from a chemical cloud parcel model considering the aqueous-phase production of dicarboxylic acids and SO_4^(2−) show good agreement for the relative magnitude of SO_4^(2−) and oxalate growth for two scenarios: power plant plume in clouds and clouds uninfluenced by local pollution sources. The relative contributions of the two aqueous-phase routes responsible for oxalic acid formation were examined; the oxidation of glyoxylic acid was predicted to dominate over the decay of longer-chain dicarboxylic acids. Clear evidence is presented for aqueous-phase oxalic acid production as the primary mechanism for oxalic acid formation in ambient aerosols