Establishment of CRISPR/Cas9 genome editing in witloof (Cichorium intybus var. foliosum)

Cichorium intybus var. foliosum (witloof) is an economically important crop with a high nutritional value thanks to many specialized metabolites, such as polyphenols and terpenoids. However, witloof plants are rich in sesquiterpene lactones (SL) which are important for plant defense but also impart a bitter taste, thus limiting industrial applications. Inactivating specific genes in the SL biosynthesis pathway could lead to changes in the SL metabolite content and result in altered bitterness. In this study, a CRISPR/Cas9 genome editing workflow was implemented for witloof, starting with polyethylene glycol (PEG) mediated protoplast transfection for CRISPR/Cas9 vector delivery, followed by whole plant regeneration and mutation analysis. Protoplast transfection efficiencies ranged from 20 to 26 %. A CRISPR/Cas9 vector targeting the first exon of the phytoene desaturase (CiPDS) gene was transfected into witloof protoplasts and resulted in the knockout of CiPDS, giving rise to an albino phenotype in 23% of the regenerated plants. Further implementing our protocol, the SL biosynthesis pathway genes germacrene A synthase (GAS), germacrene A oxidase (GAO), and costunolide synthase (COS) were targeted in independent experiments. Highly multiplex (HiPlex) amplicon sequencing of the genomic target loci revealed plant mutation frequencies of 27.3, 42.7, and 98.3% in regenerated plants transfected with a CRISPR/Cas9 vector targeting CiGAS, CiGAO, and CiCOS, respectively. We observed different mutation spectra across the loci, ranging from consistently the same +1 nucleotide insertion in CiCOS across independent mutated lines, to a complex set of 20 mutation types in CiGAO across independent mutated lines. These results demonstrate a straightforward workflow for genome editing based on transfection and regeneration of witloof protoplasts and subsequent HiPlex amplicon sequencing. Our CRISPR/Cas9 workflow can enable gene functional research and faster incorporation of novel traits in elite witloof lines in the future, thus facilitating the development of novel industrial applications for witloof

Testing for news and noise in non-stationary time series subject to multiple historical revisions

This paper focuses on testing non-stationary real-time data for forecastability, i.e., whether data revisions reduce noise or are news, by putting data releases in vector-error correction forms. To deal with historical revisions which affect the whole vintage of time series due to redefinitions, methodological innovations etc., we employ the recently developed impulse indicator saturation approach, which involves potentially adding an indicator dummy for each observation to the model. We illustrate our procedures with the U.S. real GNP/GDP series of the Federal Reserve Bank of Philadelphia and find that revisions to this series neither reduce noise nor can be considered as news

Excess Spin and the Dynamics of Antiferromagnetic Ferritin

Temperature-dependent magnetization measurements on a series of synthetic ferritin proteins containing from 100 to 3000 Fe(III) ions are used to determine the uncompensated moment of these antiferromagnetic particles. The results are compared with recent theories of macroscopic quantum coherence which explicitly include the effect of this excess moment. The scaling of the excess moment with protein size is consistent with a simple model of finite size effects and sublattice noncompensation.Comment: 4 pages, 3 Postsript figures, 1 table. Submitted to PR

Ainu Prayer Text Asahikawa Ainu\u27s prayer (published in the KAMIKAWA Ainu Kumamatsuri )

欧文抄録:p.256These texts were told by Tuakanno SUNAZAWA,Nankeainu MONNO,Hautomtei MONNO, Atsumiyashikuru ISHIYAMA, and recoded by Hideaki KURAMITSU in city MONBETSU in Hokkaido on October.25, 1953. 30 prayers texts upon Bear ceremony(iomante) in Ainu northern diaIect (Ishikari dialect). Roman transcription.1. Prayer to the fire goddess 2.Prayer to the fire goddess 3.Tapkar dance 4.Prayer to the fire goddess 5.Prayer to the fire goddess 6.Prayer to the guardian of village 7.Prayer to the guardian of village 8.Prayer to the god of forest 9.Prayer to the god of wolf 10.Prayer to the god of fox 11.Prayer to the god of owl 12.Prayer to the god of bear 13.Prayer to the god of cliff 14. Prayer to the god of altar 15.Prayer to the god of altar 16.Prayer to the god of wren 17.Prayer to the god of waesel 18.Prayer to the Siberian black bellied dipper 19.Prayer to the water goddess 20.Prayer to the god of pile 21.Prayer to the god of pile 22.Prayer to the cubs 23.Prayer to the cubs 24.Prayer to the cubs 25.Prayer to the god of heaven 26.Prayer to the god of bear 27.Prayer to the cubs 28.Prayer to the god of bear 29. Prayer to the god of bear 30.Tapkar dance 31. Tapkar dance 32.Prayer to the fire goddess 33.Prayer to the cub

Caspase-8-dependent HER-2 cleavage in response to tumor necrosis factor alpha stimulation is counteracted by nuclear factor kappa B through c-FLIP-L expression

The oncoprotein HER-2/neu is a prosurvival factor, and its overexpression has been correlated with poor prognosis in patients with breast cancer. We report that HER-2 is a new substrate for caspase-8 and that tumor necrosis factor alpha (TNF-alpha) stimulation leads to an early caspase-8-dependent HER-2 cleavage in MCF7 A/Z breast adenocarcinoma cells defective for nuclear factor kappaB (NFkappaB) activation. We show that the antiapoptotic transcription factor NFkappaB counteracts this cleavage through induction of the caspase-8 inhibitor c-FLIP. Our results also demonstrate that this HER-2 cleavage contributes to the TNF-alpha-induced apoptosis pathway because ectopic expression of an uncleavable HER-2 protects NFkappaB-defective cells against TNF-alpha-mediated cell death. Therefore, we propose an original model in which NFkappaB exerts a new antiapoptotic function by counteracting TNF-alpha-triggered cleavage of the HER-2 survival factor

M-protein diagnostics in multiple myeloma patients using ultra-sensitive targeted mass spectrometry and an off-the-shelf calibrator

Objectives: Minimal residual disease status in multiple myeloma is an important prognostic biomarker. Recently, personalized blood-based targeted mass spectrometry (MS-MRD) was shown to provide a sensitive and minimally invasive alternative to measure minimal residual disease. However, quantification of MS-MRD requires a unique calibrator for each patient. The use of patient-specific stable isotope labelled (SIL) peptides is relatively costly and time-consuming, thus hindering clinical implementation. Here, we introduce a simplification of MS-MRD by using an off-the-shelf calibrator. SILuMAB-based MS-MRD was performed by spiking a monoclonal stable isotope labeled IgG, Methods: SILuMAB-K1, in the patient serum. The abundance of both M-protein-specific peptides and SILuMAB-specific peptides were monitored by mass spectrometry. The relative ratio between M-protein peptides and SILuMAB peptides allowed for M-protein quantification. We assessed linearity, sensitivity and reproducibility of SILuMAB-based MS-MRD in longitudinally collected sera from the IFM-2009 clinical trial. Results: A linear dynamic range was achieved of over 5 log scales, allowing for M-protein quantification down to 0.001 » g/L. The inter-assay CV of SILuMAB-based MS-MRD was on average 11 » %. Excellent concordance between SIL- and SILuMAB-based MS-MRD was shown (R2&gt;0.985). Additionally, signal intensity of spiked SILuMAB can be used for quality control purpose to assess system performance and incomplete SILuMAB digestion can be used as quality control for sample preparation. Conclusion:Compared to SIL peptides, SILuMAB-based MS-MRD improves the reproducibility, turn-around-times and cost-efficacy of MS-MRD without diminishing its sensitivity and specificity. Furthermore, SILuMAB can be used as a MS-MRD quality control tool to monitor sample preparation efficacy and assay performance.</p

Science in the Swiss public: the state of science communication and public engagement with science in Switzerland

Science communication and public engagement with science have repeatedly been called for in recent years, particularly during the COVID-19 pandemic. Therefore, die Swiss Academies of the Arts and Sciences have set up an expert group to assess the state of science communication in Switzerland, and to provide recommendations for how to improve it. The expert group report is based on a comprehensive review of the available interdisciplinary scholarship analyzing science communication and public engagement with science in Switzerland. Selectively, it also incorporates original data, international findings, and secondary analyses where little or no published scholarly work was available. The report covers a wide range of facets of science communication and public engagement in Switzerland, from public attitudes towards science over individuals and organizations engaging in science communication and engagement formats to news and social media representations of science. On this basis, it formulates 20 recommendations for improving science communication in Switzerland

N-linked glycosylation of the M-protein variable region:glycoproteogenomics reveals a new layer of personalized complexity in multiple myeloma

Objectives: Multiple myeloma (MM) is a plasma cell malignancy characterized by a monoclonal expansion of plasma cells that secrete a characteristic M-protein. This M-protein is crucial for diagnosis and monitoring of MM in the blood of patients. Recent evidence has emerged suggesting that N-glycosylation of the M-protein variable (Fab) region contributes to M-protein pathogenicity, and that it is a risk factor for disease progression of plasma cell disorders. Current methodologies lack the specificity to provide a site-specific glycoprofile of the Fab regions of M-proteins. Here, we introduce a novel glycoproteogenomics method that allows detailed M-protein glycoprofiling by integrating patient specific Fab region sequences (genomics) with glycoprofiling by glycoproteomics. Methods: Glycoproteogenomics was used for the detailed analysis of de novo N-glycosylation sites of M-proteins. First, Genomic analysis of the M-protein variable region was used to identify de novo N-glycosylation sites. Subsequently glycopeptide analysis with LC-MS/MS was used for detailed analysis of the M-protein glycan sites. Results: Genomic analysis uncovered a more than two-fold increase in the Fab Light Chain N-glycosylation of M-proteins of patients with Multiple Myeloma compared to Fab Light Chain N-glycosylation of polyclonal antibodies from healthy individuals. Subsequent glycoproteogenomics analysis of 41 patients enrolled in the IFM 2009 clinical trial revealed that the majority of the Fab N-glycosylation sites were fully occupied with complex type glycans, distinguishable from Fc region glycans due to high levels of sialylation, fucosylation and bisecting structures. Conclusions: Together, glycoproteogenomics is a powerful tool to study de novo Fab N-glycosylation in plasma cell dyscrasias.</p

N-linked glycosylation of the M-protein variable region:glycoproteogenomics reveals a new layer of personalized complexity in multiple myeloma

Objectives: Multiple myeloma (MM) is a plasma cell malignancy characterized by a monoclonal expansion of plasma cells that secrete a characteristic M-protein. This M-protein is crucial for diagnosis and monitoring of MM in the blood of patients. Recent evidence has emerged suggesting that N-glycosylation of the M-protein variable (Fab) region contributes to M-protein pathogenicity, and that it is a risk factor for disease progression of plasma cell disorders. Current methodologies lack the specificity to provide a site-specific glycoprofile of the Fab regions of M-proteins. Here, we introduce a novel glycoproteogenomics method that allows detailed M-protein glycoprofiling by integrating patient specific Fab region sequences (genomics) with glycoprofiling by glycoproteomics. Methods: Glycoproteogenomics was used for the detailed analysis of de novo N-glycosylation sites of M-proteins. First, Genomic analysis of the M-protein variable region was used to identify de novo N-glycosylation sites. Subsequently glycopeptide analysis with LC-MS/MS was used for detailed analysis of the M-protein glycan sites. Results: Genomic analysis uncovered a more than two-fold increase in the Fab Light Chain N-glycosylation of M-proteins of patients with Multiple Myeloma compared to Fab Light Chain N-glycosylation of polyclonal antibodies from healthy individuals. Subsequent glycoproteogenomics analysis of 41 patients enrolled in the IFM 2009 clinical trial revealed that the majority of the Fab N-glycosylation sites were fully occupied with complex type glycans, distinguishable from Fc region glycans due to high levels of sialylation, fucosylation and bisecting structures. Conclusions: Together, glycoproteogenomics is a powerful tool to study de novo Fab N-glycosylation in plasma cell dyscrasias.</p