How to use CRISPR/Cas9 in plants - from target site selection to DNA repair.

A tool for precise, target-specific, efficient and affordable genome editing, it is a dream for many researchers, from those who do basic research to those who use it for applied research. Since 2012, we have the tool that almost fulfils such requirements; it is based on CRISPR/Cas systems. However, even CRISPR/Cas has limitations and obstacles that might surprise its users. In this review, we focus on the most frequently used variant, CRISPR/Cas9 from Streptococcus pyogenes, and highlight the key factors affecting its mutagenesis outcomes. Firstly, factors affecting the CRISPR/Cas9 activity, such as the effect of the target sequence, chromatin state or Cas9 variant, and how long it remains in place after cleavage. Secondly, factors affecting the follow-up DNA repair mechanisms include mostly the cell type and cell cycle phase, but also, for example, the type of DNA ends produced by Cas9 cleavage (blunt/staggered). Moreover, we note some differences between using CRISPR/Cas9 in plants, yeasts and animals, as knowledge from individual kingdoms is not fully transferable. Awareness of these factors can increase the likelihood of achieving the expected results of plant genome editing, for which we provide detailed guidelines.

Dendritic Mechanisms for In Vivo Neural Computations and Behavior.

Dendrites receive the vast majority of a single neuron's inputs, and coordinate the transformation of these signals into neuronal output. Ex vivo and theoretical evidence has shown that dendrites possess powerful processing capabilities, yet little is known about how these mechanisms are engaged in the intact brain or how they influence circuit dynamics. New experimental and computational technologies have led to a surge in interest to unravel and harness their computational potential. This review highlights recent and emerging work that combines established and cutting-edge technologies to identify the role of dendrites in brain function. We discuss active dendritic mediation of sensory perception and learning in neocortical and hippocampal pyramidal neurons. Complementing these physiological findings, we present theoretical work that provides new insights into the underlying computations of single neurons and networks by using biologically plausible implementations of dendritic processes. Finally, we present a novel brain-computer interface task, which assays somatodendritic coupling to study the mechanisms of biological credit assignment. Together, these findings present exciting progress in understanding how dendrites are critical for in vivo learning and behavior, and highlight how subcellular processes can contribute to our understanding of both biological and artificial neural computation.

Sulfadiazine and phosphinothricin selection systems optimised for the transformation of tobacco BY-2 cells.

KEY MESSAGE: We extended the applicability of the BY-2 cell line as a model by introducing two new selection systems. Our protocol provides guidelines for optimising Basta selection in other recalcitrant models. Tobacco BY-2 cell line is the most commonly used cytological model in plant research. It is uniform, can be simply treated by chemicals, synchronised and easily transformed. However, only a few selection systems are available that complicate advanced studies using multiple stacked transgenes and extensive gene editing. In our work, we adopted for BY-2 cell line two other selection systems: sulfadiazine and phosphinothricin (PPT, an active ingredient of Basta herbicide). We show that sulfadiazine can be used in a wide range of concentrations. It is suitable for co-transformation and subsequent double selection with kanamycin or hygromycin, which are standardly used for BY-2 transformation. We also have domesticated the sulfadiazine resistance for the user-friendly GoldenBraid cloning system. Compared to sulfadiazine, establishing selection on phosphinothricin was considerably more challenging. It did not work in any concentration of PPT with standardly cultured cells. Since the selection is based on blocking glutamine synthetase and consequent ammonium toxicity and deficiency of assimilated nitrogen, we tried to manipulate nitrogen availability. We found that the PPT selection reliably works only with nitrogen-starved cells with reduced nitrate reserves that are selected on a medium without ammonium nitrate. Both these adjustments prevent the release of large amounts of ammonium, which can toxify the entire culture in the case of standardly cultured cells. Since high nitrogen reserves can be a common feature of in vitro cultures grown on MS media, nitrogen starvation could be a key step in establishing phosphinothricin resistance in other plant models.

DNA methylation can alter CRISPR/Cas9 editing frequency and DNA repair outcome in a target-specific manner.

The impact of epigenetic modifications on the efficacy of CRISPR/Cas9-mediated double-stranded DNA breaks and subsequent DNA repair is poorly understood, especially in plants. In this study, we investigated the effect of the level of cytosine methylation on the outcome of CRISPR/Cas9-induced mutations at multiple Cas9 target sites in Nicotiana benthamiana leaf cells using next-generation sequencing. We found that high levels of promoter methylation, but not gene-body methylation, decreased the frequency of Cas9-mediated mutations. DNA methylation also influenced the ratio of insertions and deletions and potentially the type of Cas9 cleavage in a target-specific manner. In addition, we detected an over-representation of deletion events governed by a single 5'-terminal nucleotide at Cas9-induced DNA breaks. Our findings suggest that DNA methylation can indirectly impair Cas9 activity and subsequent DNA repair, probably through changes in the local chromatin structure. In addition to the well described Cas9-induced blunt-end double-stranded DNA breaks, we provide evidence for Cas9-mediated staggered DNA cuts in plant cells. Both types of cut may direct microhomology-mediated DNA repair by a novel, as yet undescribed, mechanism.

Is There an Association Between Bone Microarchitecture and Fracture in Patients who were Treated for High-grade Osteosarcoma? A Controlled Study at Long-term Follow-up Using High-resolution Peripheral Quantitative CT.

BACKGROUND: Neoadjuvant chemotherapy in patients with primary osteosarcoma improves survival rates, but it also causes side effects in various organs including bone. Low bone mineral density (BMD) can occur owing partly to chemotherapy or limited mobility. This can cause a higher risk of fractures compared with those who do not receive such treatment. Changes in BMD alone cannot explain the propensity of fractures. Studying microarchitectural changes of bone might help to understand the effect. QUESTIONS/PURPOSES: (1) Do patients who were treated for osteosarcoma (more than 20 years previously) have low BMD? (2) Do these patients experience more fractures than controls who do not have osteosarcoma? (3) What differences in bone microarchitecture are present between patients treated for high-grade osteosarcoma and individuals who have never had osteosarcoma? METHODS: We contacted 48 patients who were treated for osteosarcoma and who participated in an earlier study. These patients underwent multimodal treatment including chemotherapy more than 20 years ago. Of the original patient group, 60% (29 of 48) were missing, leaving 40% (19 of 48) available for inclusion in this study; all 19 agreed to participate. There were nine men and 10 women with a mean age of 46 ± 4 years and a mean time from surgery to examination of 28 ± 3 years. BMD was measured by dual-energy x-ray absorptiometry, and any fracture history was assessed using a questionnaire. Additionally, high-resolution peripheral quantitative CT was performed to compare the groups in terms of microarchitectural changes, such as cortical and trabecular area, cortical and trabecular thickness, cortical porosity, and endocortical perimeter. Participants in the control group were selected from a cohort consisting of a population-based random sample of 499 healthy adult women and men. Osteoporosis or low BMD was not an exclusion criterion for entering this study; however, the patients in the control group were selected based on a normal BMD (that is, T score > -1.0 at both the spine and hip). Also, the participants were matched based on age and sex. Differences between patients and controls were assessed using the Wilcoxon rank sum test for continuous variables and a chi-square test for categorical variables. A multiple regression analysis was performed. Model assumptions were checked using histograms and quantile-quantile plots of residuals. RESULTS: Twelve of 19 patients who were treated for osteosarcoma had either osteopenia (eight patients) or osteoporosis (four patients). More patients with osteosarcoma reported sustaining fractures (11 of 19 patients) than did control patients (2 of 19 controls; p < 0.001). Among all microarchitectural parameters, only the endocortical perimeter was increased in patients compared with the control group (75 ± 15 mm versus 62 ± 18 mm; p = 0.04); we found no differences between the groups in terms of cortical and trabecular area, cortical and trabecular thickness, or cortical porosity. CONCLUSION: Although patients who were treated for osteosarcoma had osteopenic or osteoporotic BMD and a higher proportion of patients experienced fractures than did patients in the control group, we could not confirm differences in microarchitectural parameters using high-resolution peripheral quantitative CT. Therefore, it seems that bone geometry and microstructural parameters are not likely the cause of the increased proportion of fractures observed in our patients who were treated for osteosarcoma. Until we learn more about the bone changes associated with chemotherapy in patients with osteosarcoma, we recommend that patients undergo regular BMD testing, and we recommend that physicians consider osteoporosis treatment in patients with low BMD. These data might provide the impetus for future multicenter prospective studies examining the association between chemotherapy and bone microarchitecture. LEVEL OF EVIDENCE: Level III, therapeutic study.

Rapid growth of organic aerosol nanoparticles over a wide tropospheric temperature range.

Nucleation and growth of aerosol particles from atmospheric vapors constitutes a major source of global cloud condensation nuclei (CCN). The fraction of newly formed particles that reaches CCN sizes is highly sensitive to particle growth rates, especially for particle sizes <10 nm, where coagulation losses to larger aerosol particles are greatest. Recent results show that some oxidation products from biogenic volatile organic compounds are major contributors to particle formation and initial growth. However, whether oxidized organics contribute to particle growth over the broad span of tropospheric temperatures remains an open question, and quantitative mass balance for organic growth has yet to be demonstrated at any temperature. Here, in experiments performed under atmospheric conditions in the Cosmics Leaving Outdoor Droplets (CLOUD) chamber at the European Organization for Nuclear Research (CERN), we show that rapid growth of organic particles occurs over the range from [Formula: see text]C to [Formula: see text]C. The lower extent of autoxidation at reduced temperatures is compensated by the decreased volatility of all oxidized molecules. This is confirmed by particle-phase composition measurements, showing enhanced uptake of relatively less oxygenated products at cold temperatures. We can reproduce the measured growth rates using an aerosol growth model based entirely on the experimentally measured gas-phase spectra of oxidized organic molecules obtained from two complementary mass spectrometers. We show that the growth rates are sensitive to particle curvature, explaining widespread atmospheric observations that particle growth rates increase in the single-digit-nanometer size range. Our results demonstrate that organic vapors can contribute to particle growth over a wide range of tropospheric temperatures from molecular cluster sizes onward.

Risk stratification and prognostic factors in patients with pulmonary arterial hypertension and comorbidities a cross-sectional cohort study with survival follow-up.

BACKGROUND: The objective of this study was to analyze prognostic factors and risk stratification in patients with pulmonary arterial hypertension (PAH) and comorbidities. METHODS: Patients with invasively diagnosed PAH were included in the analysis. Comorbidities were clinically diagnosed as proposed in the 6th World Symposium of pulmonary hypertension. Uni- and multivariate analysis were employed for identification of factors predicting survival and time to first clinical worsening (TTCW). Risk stratification was based on parameters from ESC/ERS-guidelines 2015. RESULTS: In total 142 patients were enrolled in the study, 90 of them were diagnosed as PAH without and 52 with comorbidities. All patients received targeted PAH therapy and were followed for 3.3 ± 2.4 years. In PAH patients without comorbidities survival and TTCW were significantly associated with reduced 6-min walking distance (6MWD), elevated N-terminal pro brain natriuretic peptide (NT-proBNP), WHO-functional class (WHO-FC) and right atrial (RA) area. In the multivariate analysis, 6MWD was an independent predictor for survival (p = 0.002) and WHO-FC for TTCW (p = 0.001). In patients with PAH and comorbidities these parameters had no significant association with survival and TTCW. Average risk score was significantly associated with survival (p = 0.001) and TTCW (p = 0.013) in PAH but not in PAH with comorbidities (both p > 0.05; figure 1). CONCLUSION: Risk stratification based on ESC/ERS-guidelines could only be confirmed in patients without comorbidities, but not in patients with PAH and comorbidities. The data of this study suggest, that a different risk stratification needs to be applied to PAH patients with comorbidities. Further studies are needed to confirm these results. TRIAL REGISTRATION: Not applicable, retrospective registry.

Photo-oxidation of Aromatic Hydrocarbons Produces Low-Volatility Organic Compounds.

To better understand the role of aromatic hydrocarbons in new-particle formation, we measured the particle-phase abundance and volatility of oxidation products following the reaction of aromatic hydrocarbons with OH radicals. For this we used thermal desorption in an iodide-adduct Time-of-Flight Chemical-Ionization Mass Spectrometer equipped with a Filter Inlet for Gases and AEROsols (FIGAERO-ToF-CIMS). The particle-phase volatility measurements confirm that oxidation products of toluene and naphthalene can contribute to the initial growth of newly formed particles. Toluene-derived (C7) oxidation products have a similar volatility distribution to that of α-pinene-derived (C10) oxidation products, while naphthalene-derived (C10) oxidation products are much less volatile than those from toluene or α-pinene; they are thus stronger contributors to growth. Rapid progression through multiple generations of oxidation is more pronounced in toluene and naphthalene than in α-pinene, resulting in more oxidation but also favoring functional groups with much lower volatility per added oxygen atom, such as hydroxyl and carboxylic groups instead of hydroperoxide groups. Under conditions typical of polluted urban settings, naphthalene may well contribute to nucleation and the growth of the smallest particles, whereas the more abundant alkyl benzenes may overtake naphthalene once the particles have grown beyond the point where the Kelvin effect strongly influences the condensation driving force.

Recovery, Purification, and Reusability of Building Blocks for Solid Phase Synthesis.

Solid phase synthesis (SPS) is well established for the synthesis of biomacromolecules such as peptides, oligonucelotides, and oligosaccharides, and today is also used for the synthesis of synthetic macromolecules and polymers. The key feature of this approach is the stepwise assembly of building blocks on solid support, enabling monodispersity and monomer sequence control. However, in order to achieve such control, a high excess of building blocks is required during the reaction. Herein, the recovery, purification, and reusability of building blocks used in SPS, including representative examples of tailor-made building blocks, Fmoc-protected amino acids, and functionalized carbohydrate ligands, are reported for the first time. Results demonstrate the general applicability with recovery in high yields and high purity. Furthermore, the described recovery process can be applied in both manual and automated synthesis using a standard peptide synthesizer. Overall, this process is envisioned to be applicable for a large variety of building blocks used in the SPS of different types of molecules, and to contribute to more resourceful SPS syntheses.

Oxygen-Bridged Ga2 (Et)3 (OTeF5 )3 and the Weakly Coordinating Anions [Ga(Et)(OTeF5 )3 ]- and [Ga(OTeF5 )4 ].

Salts of the weakly coordinating anions [Ga(OTeF5 )4 ]- as well as [Ga(Et)(OTeF5 )3 ]- and the neutral Ga2 (Et)3 (OTeF5 )3 were synthesized and characterized by spectroscopic methods and single-crystal X-ray diffraction. Ga2 (Et)3 (OTeF5 )3 was formed by treating GaEt3 with pentafluoroorthotelluric acid (HOTeF5 ) and reacted with PPh4 Cl and CPh3 Cl to [PPh4 ][Ga(Et)(OTeF5 )3 ] and [CPh3 ][Ga(Et)(OTeF5 )3 ]. In contrast, Ag[Ga(OTeF5 )4 ] was prepared from AgOTeF5 and GaCl3 and was used as a versatile starting material for further reactions. Starting with Ag[Ga(OTeF5 )4 ] the substrates [PPh4 ][Ga(OTeF5 )4 ] and [CPh3 ][Ga(OTeF5 )4 ] were formed from PPh4 Cl and CPh3 Cl.

Molecular Composition and Volatility of Nucleated Particles from α-Pinene Oxidation between -50 °C and +25 °C.

We use a real-time temperature-programmed desorption chemical-ionization mass spectrometer (FIGAERO-CIMS) to measure particle-phase composition and volatility of nucleated particles, studying pure α-pinene oxidation over a wide temperature range (-50 °C to +25 °C) in the CLOUD chamber at CERN. Highly oxygenated organic molecules are much more abundant in particles formed at higher temperatures, shifting the compounds toward higher O/C and lower intrinsic (300 K) volatility. We find that pure biogenic nucleation and growth depends only weakly on temperature. This is because the positive temperature dependence of degree of oxidation (and polarity) and the negative temperature dependence of volatility counteract each other. Unlike prior work that relied on estimated volatility, we directly measure volatility via calibrated temperature-programmed desorption. Our particle-phase measurements are consistent with gas-phase results and indicate that during new-particle formation from α-pinene oxidation, gas-phase chemistry directly determines the properties of materials in the condensed phase. We now have consistency between measured gas-phase product concentrations, product volatility, measured and modeled growth rates, and the particle composition over most temperatures found in the troposphere.

Magnetostriction in magnetic gels and elastomers as a function of the internal structure and particle distribution.

Magnetic gels and elastomers are promising candidates to construct reversibly excitable soft actuators, triggered from outside by magnetic fields. These magnetic fields induce or alter the magnetic interactions between discrete rigid particles embedded in a soft elastic polymeric matrix, leading to overall deformations. It is a major challenge in theory to correctly predict from the discrete particle configuration the type of deformation resulting for a finite-sized system. Considering an elastic sphere, we here present such an approach. The method is in principle exact, at least within the framework of linear elasticity theory and for large enough interparticle distances. Different particle arrangements are considered. We find, for instance, that regular simple cubic configurations show elongation of the sphere along the magnetization if oriented along a face or space diagonal of the cubic unit cell. Contrariwise, with the magnetization along the edge of the cubic unit cell, they contract. The opposite is true in this geometry for body- and face-centered configurations. Remarkably, for the latter configurations but the magnetization along a face or space diagonal of the unit cell, contraction was observed to revert to expansion with decreasing Poisson ratio of the elastic material. Randomized configurations were considered as well. They show a tendency of elongating the sphere along the magnetization, which is more pronounced for compressible systems. Our results can be tested against actual experiments for spherical samples. Moreover, our approach shall support the search of optimal particle distributions for a maximized effect of actuation.

Reduced anthropogenic aerosol radiative forcing caused by biogenic new particle formation.

The magnitude of aerosol radiative forcing caused by anthropogenic emissions depends on the baseline state of the atmosphere under pristine preindustrial conditions. Measurements show that particle formation in atmospheric conditions can occur solely from biogenic vapors. Here, we evaluate the potential effect of this source of particles on preindustrial cloud condensation nuclei (CCN) concentrations and aerosol-cloud radiative forcing over the industrial period. Model simulations show that the pure biogenic particle formation mechanism has a much larger relative effect on CCN concentrations in the preindustrial atmosphere than in the present atmosphere because of the lower aerosol concentrations. Consequently, preindustrial cloud albedo is increased more than under present day conditions, and therefore the cooling forcing of anthropogenic aerosols is reduced. The mechanism increases CCN concentrations by 20-100% over a large fraction of the preindustrial lower atmosphere, and the magnitude of annual global mean radiative forcing caused by changes of cloud albedo since 1750 is reduced by [Formula: see text] (27%) to [Formula: see text] Model uncertainties, relatively slow formation rates, and limited available ambient measurements make it difficult to establish the significance of a mechanism that has its dominant effect under preindustrial conditions. Our simulations predict more particle formation in the Amazon than is observed. However, the first observation of pure organic nucleation has now been reported for the free troposphere. Given the potentially significant effect on anthropogenic forcing, effort should be made to better understand such naturally driven aerosol processes.

Pervasive read-through transcription of T-DNAs is frequent in tobacco BY-2 cells and can effectively induce silencing.

BACKGROUND: Plant transformation via Agrobacterium tumefaciens is characterized by integration of commonly low number of T-DNAs at random positions in the genome. When integrated into an active gene region, promoterless reporter genes placed near the T-DNA border sequence are frequently transcribed and even translated to reporter proteins, which is the principle of promoter- and gene-trap lines. RESULTS: Here we show that even internal promotorless regions of T-DNAs are often transcribed. Such spontaneous transcription was observed in the majority of independently transformed tobacco BY-2 lines (over 65%) and it could effectively induce silencing if an inverted repeat was present within the T-DNA. We documented that the transcription often occurred in both directions. It was not directly connected with any regulatory elements present within the T-DNAs and at least some of the transcripts were initiated outside of the T-DNA. The likeliness of this read-through transcription seemed to increase in lines with higher T-DNA copy number. Splicing and presence of a polyA tail in the transcripts indicated involvement of Pol II, but surprisingly, the transcription was able to run across two transcription terminators present within the T-DNA. Such pervasive transcription was observed with three different T-DNAs in BY-2 cells and with lower frequency was also detected in Arabidopsis thaliana. CONCLUSIONS: Our results demonstrate unexpected pervasive read-through transcription of T-DNAs. We hypothesize that it was connected with a specific chromatin state of newly integrated DNA, possibly affected by the adjacent genomic region. Although this phenomenon can be easily overlooked, it can have significant consequences when working with highly sensitive systems like RNAi induction using an inverted repeat construct, so it should be generally considered when interpreting results obtained with the transgenic technology.

Right heart size and function significantly correlate in patients with pulmonary arterial hypertension - a cross-sectional study.

BACKGROUND: The objective of this study was to assess, whether right atrial (RA) and ventricular (RV) size is related to RV pump function at rest and during exercise in patients with pulmonary arterial hypertension (PAH). METHODS: We included 54 patients with invasively diagnosed PAH that had been stable on targeted medication. All patients underwent clinical assessments including right heart catheterization and echocardiography at rest and during exercise. RV output reserve was defined as increase of cardiac index (CI) from rest to peak exercise (∆CIexercise). Patients were classified according to the median of RA and RV-area. RV pump function and further clinical parameters were compared between groups by student's t-test. Uni- and multivariate Pearson correlation analyses were performed. RESULTS: Patients with larger RA and/or RV-areas (above a median of 16 and 20cm2, respectively) showed significantly lower ∆CIexercise, higher mean pulmonary arterial pressure, pulmonary vascular resistance at rest and NT-proBNP levels. Furthermore, patients with higher RV-areas presented with a significantly lower RV stroke volume and pulmonary arterial compliance at peak exercise than patients with smaller RV-size. RV area was identified as the only independent predictor of RV output reserve. CONCLUSION: RV and RA areas represent valuable and easily accessible indicators of RV pump function at rest and during exercise. Cardiac output reserve should be considered as an important clinical parameter. Prospective studies are needed for further evaluation.

Accretion Product Formation from Ozonolysis and OH Radical Reaction of α-Pinene: Mechanistic Insight and the Influence of Isoprene and Ethylene.

α-Pinene (C10H16) represents one of the most important biogenic emissions in the atmosphere. Its oxidation products can significantly contribute to the secondary organic aerosol (SOA) formation. Here, we report on the formation mechanism of C19 and C20 accretion products from α-pinene oxidation, which are believed to be efficient SOA precursors. Measurements have been performed in a free-jet flow system. Detection of RO2 radicals and accretion products was carried out by recent mass spectrometric techniques using different ionization schemes. Observed C10-RO2 radicals from α-pinene ozonolysis were O,O-C10H15(O2) xO2 with x = 0, 1, 2, 3 and from the OH radical reaction HO-C10H16(O2)αO2 with α = 0, 1, 2. All detected C20 accretion products can be explained via the accretion reaction RO2 + R'O2 → ROOR' + O2 starting from the measured C10-RO2 radicals. We speculate that C19 accretion products are formed in an analogous way assuming CH2O elimination. Addition of isoprene (C5H8), producing C5-RO2 radicals, leads to C15 accretion products formed via cross-reactions with C10-RO2 radicals. This process is competing with the formation of C19/C20 products from the pure α-pinene oxidation. A similar behavior has been observed for ethylene additives that form C12 accretion products. In the atmosphere, a complex accretion product spectrum from self- and cross-reactions of available RO2 radicals can be expected. Modeling atmospheric conditions revealed that C19/C20 product formation is only reduced by a factor of 1.2 or 3.6 in isoprene-dominated environments assuming a 2- or 15-fold isoprene concentration over α-pinene, respectively, as present in different forested areas.

Accretion Product Formation from Self- and Cross-Reactions of RO2 Radicals in the Atmosphere.

Hydrocarbons are emitted into the Earth's atmosphere in very large quantities by human and biogenic activities. Their atmospheric oxidation processes almost exclusively yield RO2 radicals as reactive intermediates whose atmospheric fate is not yet fully unraveled. Herein, we show that gas-phase reactions of two RO2 radicals produce accretion products composed of the carbon backbone of both reactants. The rates for accretion product formation are very high for RO2 radicals bearing functional groups, competing with those of the corresponding reactions with NO and HO2 . This pathway, which has not yet been considered in the modelling of atmospheric processes, can be important, or even dominant, for the fate of RO2 radicals in all areas of the atmosphere. Moreover, the vapor pressure of the formed accretion products can be remarkably low, characterizing them as an effective source for the secondary organic aerosol.

PTR3: An Instrument for Studying the Lifecycle of Reactive Organic Carbon in the Atmosphere.

We have developed and characterized the novel PTR3, a proton transfer reaction-time-of-flight mass spectrometer (PTR-TOF) using a new gas inlet and an innovative reaction chamber design. The reaction chamber consists of a tripole operated with rf voltages generating an electric field only in the radial direction. An elevated electrical field is necessary to reduce clustering of primary hydronium (H3O+) and product ions with water molecules present in the sample gas. The axial movement of the ions is achieved by the sample gas flow only. Therefore, the new design allows a 30-fold longer reaction time and a 40-fold increase in pressure compared to standard PTR-TOF-MS. First calibration tests show sensitivities of up to 18000 counts per second/parts per billion and volume (cps/ppbv) at a mass resolution of >8000 m/Δm (fwhm). The new inlet using center-sampling through a critical orifice reduces wall losses of low volatility compounds. Therefore, the new PTR3 instrument is sensitive to VOC typically present in the ppbv range as well as to semivolatile organic compounds (SVOC) and even highly oxidized organic molecules (HOMs) present in the parts per quadrillion per volume (ppqv) range in the atmosphere.

5-Azacytidine mediated reactivation of silenced transgenes in potato (Solanum tuberosum) at the whole plant level.

KEY MESSAGE: Transient 5-azacytidine treatment of leaf explants from potato plants with transcriptionally silenced transgenes allows de novo regeneration of plants with restored transgene expression at the whole plant level. Transgenes introduced into plant genomes frequently become silenced either at the transcriptional or the posttranscriptional level. Transcriptional silencing is usually associated with DNA methylation in the promoter region. Treatments with inhibitors of maintenance DNA methylation were previously shown to allow reactivation of transcriptionally silenced transgenes in single cells or tissues, but not at the whole plant level. Here we analyzed the effect of DNA methylation inhibitor 5-azacytidine (AzaC) on the expression of two silenced reporter genes encoding green fluorescent protein (GFP) and neomycin phosphotransferase (NPTII) in potato plants. Whereas no obvious reactivation was observed in AzaC-treated stem cuttings, transient treatment of leaf segments with 10 µM AzaC and subsequent de novo regeneration of shoots on the selective medium with kanamycin resulted in the production of whole plants with clearly reactivated expression of previously silenced transgenes. Reactivation of nptII expression was accompanied by a decrease in cytosine methylation in the promoter region of the gene. Using the plants with reactivated GFP expression, we found that re-silencing of this transgene can be accidentally triggered by de novo regeneration. Thus, testing the incidence of transgene silencing during de novo regeneration could be a suitable procedure for negative selection of transgenic lines (insertion events) which have an inclination to be silenced. Based on our analysis of non-specific inhibitory effects of AzaC on growth of potato shoots in vitro, we estimated that AzaC half-life in the culture media is approximately 2 days.