Entwicklung eines neuen Expressionssystemes für die Produktion rekombinanter Proteine in Chinese hamster ovary Zellen basierend auf der Selektion mit einem metabolischen Enzym

The Chinese hamster ovary (CHO) cell line is currently the most important mammalian production cell line for therapeutic proteins. The aim of this study was to use the proline auxotrophy of the CHO cell line as a selection system for the production of recombinant proteins. The gene of the enzyme pyrroline-5-carboxylate synthetase (P5CS), one key enzyme of proline synthesis, was used as a selection marker. Using the CHO-S cells, selection times and expression levels comparable to those of antibiotic selection with neomycin or hygromycin B could be reached. Expressing the green fluorescent protein (GFP), three different monoclonal antibodies (anti-CD303, anti-CD14, anti-Biotin) in a two-vector strategy in combination with a zeocin selection or the cytokine hTGF-?1, stable cell lines with producer rates of over 90% were generated in less than 20 days. Stable cell lines with titers up to 8 mg/L (static, adherent, in minimal medium) and clones expressing up to 140 mg/L (limiting dilution, orbital shake reactor, suspension, fed-batch process) were generated for the production of anti-CD303. To further improve the proline selection system, a tricistronic vector allowing coexpression of fluorescent reporter proteins for the use of fluorescence as well as magnetic assisted cell sorting (FACS and MACS) was developed. Using a stable cell line expressing anti-CD303, FACS improved clone isolation when compared to a limiting dilution. MACS enrichment of stable cell lines revealed an 2.5- to 10-fold increase of titer. A combination of MACS enrichment of stable producer cell lines with clone isolation by FACS was proven to dramatically improve timelines and the manual amount of work. Clones producing up to 130 mg/L anti-CD303 antibody (orbital shake reactor, suspension, batch process) could be isolated by this combination. The developed P5CS expression systems offers a selection process without the need for any cytotoxic substances.Die Chinese hamster ovary (CHO Zelllinie) die am häufigsten verwendete Mammaliazelllinie zur Produktion therapeutischer Proteine. Das Ziel dieser Arbeit war die Prolin-Auxotrophie als Selektionssystem für die Produktion rekombinanter Proteine zu nutzen. Das Gen des Enzyms Pyrrolin-5-Carboxylat-Synthetase (P5CS), welches die Reaktion von Glutamat zu Pyrrolin-5-Carboxylat katalysiert, wurde als Selektionsmarker in einen dizistronischen Expressionsvektor kloniert. Mit der CHO-S-Zelllinie können sowohl Selektionszeiten als auch Expressionslevels vergleichbar zu Antibiotikaselektionssystemen mit Neomycin oder Hygromycin B erreicht werden. Stabile Zelllinien, die das grün-fluoreszierende Protein GFP, drei verschiedene Antikörper (anti-CD303, anti-CD14, anti-Biotin) oder das Zytokin hTGF-?1 exprimieren, konnten in weniger als 20 Tagen mit Produzentenraten von über 90% generiert werden. Für die anti-CD303-Produktion konnten so stabile Zelllinien mit Titern von 8 mg/L (statisch, adhärent, in Minimalmedium) und Klone mit Titern von bis zu 140 mg/L (limiting dilution, Schüttelinkubator, Suspension, fed-batch) isoliert werden. Um das neu etablierte Prolinselektionssystem weiter zu optimieren, wurde ein tricistronischer Expressionsvektor entwickelt, der die Koexpression von fluoreszierenden Reporter-proteinen erlaubt und sowohl für magnetische als auch durchflusszytometrische Zell-sortierung (MACS und FACS) genutzt werden kann. Im Vergleich zu einer traditionellen limiting dilution konnten mittels FACS Klone mit höheren durchschnittlichen Produktivitäten isoliert werden. Membrangebundene Fluoreszenzproteine können außerdem für eine schnelle Anreicherung mittels MACS genutzt werden. Eine Kombination aus einer MACS-Anreicherung von stabilen Zelllinien mit einer anschließenden Klonisolation mittels FACS ist geeignet sowohl Zeit- als auch Arbeitsaufwand zu reduzieren. Das entwickelte P5CS-Expressionssystem erlaubt eine Selektion ohne zytotoxische Substanzen

Context-based Normalization of Histological Stains using Deep Convolutional Features

While human observers are able to cope with variations in color and appearance of histological stains, digital pathology algorithms commonly require a well-normalized setting to achieve peak performance, especially when a limited amount of labeled data is available. This work provides a fully automated, end-to-end learning-based setup for normalizing histological stains, which considers the texture context of the tissue. We introduce Feature Aware Normalization, which extends the framework of batch normalization in combination with gating elements from Long Short-Term Memory units for normalization among different spatial regions of interest. By incorporating a pretrained deep neural network as a feature extractor steering a pixelwise processing pipeline, we achieve excellent normalization results and ensure a consistent representation of color and texture. The evaluation comprises a comparison of color histogram deviations, structural similarity and measures the color volume obtained by the different methods.Comment: In: 3rd Workshop on Deep Learning in Medical Image Analysis (DLMIA 2017

Effects of Task Experience and Layout on Learning from Text and Pictures with or without Unnecessary Picture Descriptions

The presentation of extraneous (i.e., irrelevant or unnecessary) information may hamper learning with multimedia. The present study examined whether people can learn to ignore unnecessary information with increasing experience with the task and whether this depends on the layout of that information. In two experiments, participants learned about the process of mitosis from a multimedia slideshow, with each slide presenting a combination of expository text and a picture on one of the stages in the process. Slides either contained no unnecessary text (control condition) or unnecessary text (i.e., merely describing the picture) either integrated in the picture (integrated condition) or presented underneath the picture (separated condition). Knowledge about the studied mitosis phase was tested immediately after each slide using a cloze test. Across Experiments 1 and 2, we did not find a reliable negative effect of the unnecessary text on cloze test performance. As a result, the question of whether task experience would reduce or eliminate that negative effect could not be answered. The eye movement data did confirm, however, that participants attended less to the unnecessary information with increasing task experience, suggesting that students can adapt their study strategy and learn to ignore unnecessary information

Replicative senescence of mesenchymal stem cells causes DNA-methylation changes which correlate with repressive histone marks

Cells in culture undergo replicative senescence. In this study, we analyzed functional, genetic and epigenetic sequels of long-term culture in human mesenchymal stem cells (MSC). Already within early passages the fibroblastoid colonyforming unit (CFU-f) frequency and the differentiation potential of MSC declined significantly. Relevant chromosomal aberrations were not detected by karyotyping and SNP-microarrays. Subsequently, we have compared DNA-methylation profiles with the Infinium HumanMethylation27 Bead Array and the profiles differed markedly in MSC derived from adipose tissue and bone marrow. Notably, all MSC revealed highly consistent senescence-associated modifications at specific CpG sites. These DNA-methylation changes correlated with histone marks of previously published data sets, such as trimethylation of H3K9, H3K27 and EZH2 targets. Taken together, culture expansion of MSC has profound functional implications - these are hardly reflected by genomic instability but they are associated with highly reproducible DNA-methylation changes which correlate with repressive histone marks. Therefore replicative senescence seems to be epigenetically controlled

Liver tests, cardiovascular outcomes and effects of empagliflozin in patients with heart failure and preserved ejection fraction: The EMPEROR-Preserved trial

Aim The prognostic implication of elevated liver tests in heart failure with preserved ejection fraction (HFpEF) is uncertain. This analysis investigates the association of liver markers with hospitalization for heart failure (HHF) and cardiovascular death (CVD), and the treatment effect of empagliflozin across the range of liver marker levels. Methods and results The double-blind, placebo-controlled EMPEROR-Preserved (EMPagliflozin outcomE tRial in Patients With chrOnic heaRt Failure with Preserved Ejection Fraction) enrolled 5988 patients with HFpEF (ejection fraction >40%). Patients in New York Heart Association class II–IV and elevated N-terminal pro-B-type natriuretic peptide were randomized to receive empagliflozin 10 mg daily or placebo in addition to usual therapy. Patients with significant liver disease were excluded. The primary endpoint was time to first adjudicated HHF or CVD. We explored the association of liver function abnormalities with heart failure outcomes in patients on placebo, the effects of empagliflozin on liver tests and the treatment effects of empagliflozin on heart failure outcomes across categories of liver laboratory values. High alkaline phosphatase (p trend < 0.0001), low albumin (p trend < 0.0001) and high bilirubin (p = 0.02) were associated with poorer outcomes for HHF or CVD, while high aspartate aminotransferase was not, and high alanine aminotransferase was associated with better outcomes. Empagliflozin had no significant effects on liver tests compared to placebo except for albumin which was significantly increased. The treatment effect of empagliflozin on outcomes was not modified by liver tests. Conclusion Abnormalities of liver function tests are associated differently with heart failure outcomes. Salutary effects of empagliflozin on liver tests were not observed although albumin increased. The treatment benefits of empagliflozin were not affected by baseline values of liver parameters

Control of Mitochondrial Membrane Permeabilization by Adenine Nucleotide Translocator Interacting with HIV-1 Viral Protein R and Bcl-2

Viral protein R (Vpr), an apoptogenic accessory protein encoded by HIV-1, induces mitochondrial membrane permeabilization (MMP) via a specific interaction with the permeability transition pore complex, which comprises the voltage-dependent anion channel (VDAC) in the outer membrane (OM) and the adenine nucleotide translocator (ANT) in the inner membrane. Here, we demonstrate that a synthetic Vpr-derived peptide (Vpr52-96) specifically binds to the intermembrane face of the ANT with an affinity in the nanomolar range. Taking advantage of this specific interaction, we determined the role of ANT in the control of MMP. In planar lipid bilayers, Vpr52-96 and purified ANT cooperatively form large conductance channels. This cooperative channel formation relies on a direct protein–protein interaction since it is abolished by the addition of a peptide corresponding to the Vpr binding site of ANT. When added to isolated mitochondria, Vpr52-96 uncouples the respiratory chain and induces a rapid inner MMP to protons and NADH. This inner MMP precedes outer MMP to cytochrome c. Vpr52-96–induced matrix swelling and inner MMP both are prevented by preincubation of purified mitochondria with recombinant Bcl-2 protein. In contrast to König's polyanion (PA10), a specific inhibitor of the VDAC, Bcl-2 fails to prevent Vpr52-96 from crossing the mitochondrial OM. Rather, Bcl-2 reduces the ANT–Vpr interaction, as determined by affinity purification and plasmon resonance studies. Concomitantly, Bcl-2 suppresses channel formation by the ANT–Vpr complex in synthetic membranes. In conclusion, both Vpr and Bcl-2 modulate MMP through a direct interaction with ANT

Empagliflozin Improves Cardiovascular and Renal Outcomes in Heart Failure Irrespective of Systolic Blood Pressure.

BACKGROUND: Empagliflozin reduces the risk of cardiovascular death or heart failure (HF) hospitalization in patients with reduced ejection fraction. Its interplay with systolic blood pressure (SBP) is not known. OBJECTIVES: The goal of this study was to evaluate the interplay of SBP and the effects of empagliflozin in EMPEROR-Reduced (Empagliflozin Outcome Trial in Patients With Chronic Heart Failure With Reduced Ejection Fraction). METHODS: Study patients (N = 3,730) were randomly assigned to groups according to SBP at baseline (130 mm Hg, n = 1,047). This study explored the influence of SBP on the effects of empagliflozin on cardiovascular death or HF hospitalization (primary outcome), as well as on total HF hospitalizations, rate of decline in estimated glomerular filtration rate, renal outcomes, and empagliflozin's effects and significance on SBP. RESULTS: Over a median of 16 months considering only patients receiving placebo, baseline SBP and the risk of cardiovascular death or hospitalization for HF (P trend = 0.0015) were inversely related. Corrected for placebo, a slight early increase was observed in SBP at 130 mm Hg. These between-group differences were of borderline significance (P for interaction trend = 0.05-0.10) after 4 and 12 weeks but were not significant later. SBP at baseline did not influence the effect of empagliflozin to reduce the risk of HF events or renal endpoints. When treated with empagliflozin, patients with SBP <110 mm Hg did not have an increased rate of symptomatic hypotension. CONCLUSIONS: Empagliflozin was effective and safe, with no meaningful interaction between SBP and the effects of empagliflozin in the EMPEROR-Reduced trial. (Empagliflozin Outcome Trial in Patients With Chronic Heart Failure With Reduced Ejection Fraction [EMPEROR-Reduced]; NCT03057977)

Pimasertib Versus Dacarbazine in Patients With UnresectableNRAS-Mutated Cutaneous Melanoma: Phase II, Randomized, Controlled Trial with Crossover

This study investigated the efficacy and safety of pimasertib (MEK1/MEK2 inhibitor) versus dacarbazine (DTIC) in patients with untreated NRAS-mutated melanoma. Phase II, multicenter, open-label trial. Patients with unresectable, stage IIIc/IVM1 NRAS-mutated cutaneous melanoma were randomized 2:1 to pimasertib (60 mg; oral twice-daily) or DTIC (1000 mg/m2 ; intravenously) on Day 1 of each 21-day cycle. Patients progressing on DTIC could crossover to pimasertib. Primary endpoint: investigator-assessed progression-free survival (PFS); secondary endpoints: overall survival (OS), objective response rate (ORR), quality of life (QoL), and safety. Overall, 194 patients were randomized (pimasertib n = 130, DTIC n = 64), and 191 received treatment (pimasertib n = 130, DTIC n = 61). PFS was significantly improved with pimasertib versus DTIC (median 13 versus 7 weeks, respectively; hazard ratio (HR) 0.59, 95% confidence interval (CI) 0.42–0.83; p = 0.0022). ORR was improved with pimasertib (odds ratio 2.24, 95% CI 1.00–4.98; p = 0.0453). OS was similar between treatments (median 9 versus 11 months, respectively; HR 0.89, 95% CI 0.61–1.30); 64% of patients receiving DTIC crossed over to pimasertib. Serious adverse events (AEs) were more frequent for pimasertib (57%) than DTIC (20%). The most common treatment-emergent AEs were diarrhea (82%) and blood creatine phosphokinase (CPK) increase (68%) for pimasertib, and nausea (41%) and fatigue (38%) for DTIC. Most frequent grade ≥3 AEs were CPK increase (34%) for pimasertib and neutropenia (15%) for DTIC. Mean QoL scores (baseline and last assessment) were similar between treatments. Pimasertib has activity in NRAS-mutated cutaneous melanoma and a safety profile consistent with known toxicities of MEK inhibitors. Trial registration: ClinicalTrials.gov, NCT01693068

Palaeogenomics of Upper Palaeolithic to Neolithic European hunter-gatherers

: Modern humans have populated Europe for more than 45,000 years1,2. Our knowledge of the genetic relatedness and structure of ancient hunter-gatherers is however limited, owing to the scarceness and poor molecular preservation of human remains from that period3. Here we analyse 356 ancient hunter-gatherer genomes, including new genomic data for 116 individuals from 14 countries in western and central Eurasia, spanning between 35,000 and 5,000 years ago. We identify a genetic ancestry profile in individuals associated with Upper Palaeolithic Gravettian assemblages from western Europe that is distinct from contemporaneous groups related to this archaeological culture in central and southern Europe4, but resembles that of preceding individuals associated with the Aurignacian culture. This&nbsp;ancestry profile survived during the Last Glacial Maximum (25,000 to 19,000 years ago) in human populations from southwestern Europe associated with the Solutrean&nbsp;culture, and with&nbsp;the following Magdalenian culture&nbsp;that re-expanded northeastward after the Last Glacial Maximum. Conversely, we reveal a genetic turnover in southern Europe suggesting a local replacement of human groups around the time of the Last Glacial Maximum, accompanied by a north-to-south dispersal of populations associated with the Epigravettian culture. From at least 14,000 years ago, an ancestry related to this culture spread from the south across the rest of Europe, largely replacing the Magdalenian-associated gene pool. After a period of limited admixture that spanned the beginning of the Mesolithic, we find genetic interactions between western and eastern European hunter-gatherers,&nbsp;who were also characterized by marked differences in phenotypically relevant variants

Palaeogenomics of Upper Palaeolithic to Neolithic European hunter-gatherers

Publisher Copyright: © 2023, The Author(s).Modern humans have populated Europe for more than 45,000 years1,2. Our knowledge of the genetic relatedness and structure of ancient hunter-gatherers is however limited, owing to the scarceness and poor molecular preservation of human remains from that period3. Here we analyse 356 ancient hunter-gatherer genomes, including new genomic data for 116 individuals from 14 countries in western and central Eurasia, spanning between 35,000 and 5,000 years ago. We identify a genetic ancestry profile in individuals associated with Upper Palaeolithic Gravettian assemblages from western Europe that is distinct from contemporaneous groups related to this archaeological culture in central and southern Europe4, but resembles that of preceding individuals associated with the Aurignacian culture. This ancestry profile survived during the Last Glacial Maximum (25,000 to 19,000 years ago) in human populations from southwestern Europe associated with the Solutrean culture, and with the following Magdalenian culture that re-expanded northeastward after the Last Glacial Maximum. Conversely, we reveal a genetic turnover in southern Europe suggesting a local replacement of human groups around the time of the Last Glacial Maximum, accompanied by a north-to-south dispersal of populations associated with the Epigravettian culture. From at least 14,000 years ago, an ancestry related to this culture spread from the south across the rest of Europe, largely replacing the Magdalenian-associated gene pool. After a period of limited admixture that spanned the beginning of the Mesolithic, we find genetic interactions between western and eastern European hunter-gatherers, who were also characterized by marked differences in phenotypically relevant variants.Peer reviewe