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Ensuring the safety of parenteral drugs before injection into patients is of utmost importance. New regulations around the globe and the need to refrain from using animals however, have highlighted the need for new cell sources to be used in next-generation bioassays to detect the entire spectrum of possible contaminating pyrogens. Given the current drawbacks of the Monocyte-Activation-Test (MAT) with respect to the use of primary peripheral blood mono-nuclear cells or the use of monocytic cell lines, we here demonstrate the manufacturing of sensor monocytes/macrophages from human induced pluripotent stem cells (iMonoMac), which are fully defined and superior to current cell products. Using a modern and scalable manufacturing platform, iMonoMac showed typical macrophage-like morphology and stained positive for several Toll like receptor (TLRs) such as TLR-2, TLR-5, TLR-4. Furthermore, iMonoMac derived from the same donor were sensitive to endotoxins, non-endotoxins, and process related pyrogens at a high dynamic range and across different cellular densities. Of note, iMonoMac showed increased sensitivity and reactivity to a broad range of pyrogens, demonstrated by the detection of interleukin-6 at low concentrations of LPS and MALP-2 which could not be reached using the current MAT cell sources. To further advance the system, iMonoMac or genetically engineered iMonoMac with NF-κB-luciferase reporter cassette could reveal a specific activation response while correlating to the classical detection method employing enzyme-linked immunosorbent assay to measure cytokine secretion. Thus, we present a valuable cellular tool to assess parenteral drugs safety, facilitating the future acceptance and design of regulatory-approved bioassays.
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Células-Tronco Pluripotentes Induzidas , Macrófagos , Pirogênios , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Humanos , Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/citologia , Contaminação de Medicamentos , Receptores Toll-Like/metabolismo , Endotoxinas , Interleucina-6/metabolismo , Monócitos/citologia , Monócitos/metabolismo , Monócitos/efeitos dos fármacos , Infusões ParenteraisRESUMO
Inflammatory bowel diseases are characterized by the chronic relapsing inflammation of the gastrointestinal tract. While the molecular causality between endoplasmic reticulum (ER) stress and intestinal inflammation is widely accepted, the metabolic consequences of chronic ER stress on the pathophysiology of IBD remain unclear. By using in vitro, in vivo models, and patient datasets, we identified a distinct polarization of the mitochondrial one-carbon metabolism and a fine-tuning of the amino acid uptake in intestinal epithelial cells tailored to support GSH and NADPH metabolism upon ER stress. This metabolic phenotype strongly correlates with IBD severity and therapy response. Mechanistically, we uncover that both chronic ER stress and serine limitation disrupt cGAS-STING signaling, impairing the epithelial response against viral and bacterial infection and fueling experimental enteritis. Consequently, the antioxidant treatment restores STING function and virus control. Collectively, our data highlight the importance of serine metabolism to allow proper cGAS-STING signaling and innate immune responses upon gut inflammation.
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Modeling tumor metabolism in vitro remains challenging. Here, we used galactose as an in vitro tool compound to mimic glycolytic limitation. In contrast to the established idea that high glycolytic flux reduces pyruvate kinase isozyme M2 (PKM2) activity to support anabolic processes, we have discovered that glycolytic limitation also affects PKM2 activity. Surprisingly, despite limited carbon availability and energetic stress, cells induce a near-complete block of PKM2 to divert carbons toward serine metabolism. Simultaneously, TCA cycle flux is sustained, and oxygen consumption is increased, supported by glutamine. Glutamine not only supports TCA cycle flux but also serine synthesis via distinct mechanisms that are directed through PKM2 inhibition. Finally, deleting mitochondrial one-carbon (1C) cycle reversed the PKM2 block, suggesting a potential formate-dependent crosstalk that coordinates mitochondrial 1C flux and cytosolic glycolysis to support cell survival and proliferation during nutrient-scarce conditions.
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Glutamina , Piruvato Quinase , Piruvato Quinase/metabolismo , Glutamina/metabolismo , Glicólise , Carbono , Serina/metabolismoRESUMO
BACKGROUND AND AIMS: The Swiss Autoimmune Hepatitis Cohort Study is a nationwide registry, initiated in 2017, that collects retrospective and prospective clinical data and biological samples from patients of all ages with autoimmune hepatitis treated at Swiss hepatology centres. Here, we report the analysis of the first 5 years of registry data. RESULTS: A total of 291 patients with autoimmune hepatitis have been enrolled, 30 of whom were diagnosed before 18 years of age and composed the paediatric cohort. Paediatric cohort: median age at diagnosis 12.5 years (range 1-17, interquartile range (IQR) 8-15), 16 (53%) girls, 6 (32%) with type 2 autoimmune hepatitis, 8 (27%) with autoimmune sclerosing cholangitis, 1 with primary biliary cholangitis variant syndrome, 4 (15%) with inflammatory bowel disease and 10 (41%) with advanced liver fibrosis at diagnosis. Adult cohort: median age at diagnosis 54 years (range 42-64, IQR 18-81), 185 (71%) women, 51 (20%) with primary biliary cholangitis variant syndrome, 22 (8%) with primary sclerosing cholangitis variant syndrome, 9 (4%) with inflammatory bowel disease and 66 (32%) with advanced liver fibrosis at diagnosis. The median follow-up time for the entire cohort was 5.2 years (IQR 3-9.3 years). Treatment in children: 29 (97%) children were initially treated with corticosteroids, 28 of whom received combination treatment with azathioprine. Budesonide was used in four children, all in combination with azathioprine. Mycophenolate mofetil was used in five children, all of whom had previously received corticosteroids and thiopurine. Treatment in adults (data available for 228 patients): 219 (96%) were treated with corticosteroids, mostly in combination with azathioprine. Predniso(lo)ne was the corticosteroid used in three-quarters of patients; the other patients received budesonide. A total of 78 (33%) patients received mycophenolate mofetil, 62 of whom had previously been treated with azathioprine. Complete biochemical response was achieved in 13 of 19 (68%) children and 137 of 182 (75%) adults with available follow-up data. All children were alive at the last follow-up, and none had undergone liver transplantation. Five (2%) adults underwent liver transplantation, two of whom had a fulminant presentation. Four (2%) adults with autoimmune hepatitis died (two from liver-associated causes). CONCLUSION: Patients with autoimmune hepatitis in Switzerland had clinical features similar to those in other cohorts. The proportion of patients diagnosed with primary biliary cholangitis variant syndrome was higher than expected. Autoimmune hepatitis was managed according to guidelines, except for the use of budesonide in a small proportion of paediatric patients. The outcomes were excellent, but the findings must be confirmed over a longer follow-up period.
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Hepatite Autoimune , Doenças Inflamatórias Intestinais , Cirrose Hepática Biliar , Adulto , Humanos , Criança , Feminino , Lactente , Pré-Escolar , Adolescente , Pessoa de Meia-Idade , Masculino , Azatioprina/uso terapêutico , Estudos Retrospectivos , Hepatite Autoimune/complicações , Hepatite Autoimune/diagnóstico , Hepatite Autoimune/tratamento farmacológico , Estudos Prospectivos , Suíça/epidemiologia , Estudos de Coortes , Cirrose Hepática Biliar/complicações , Cirrose Hepática Biliar/tratamento farmacológico , Ácido Micofenólico/uso terapêutico , Cirrose Hepática , Doenças Inflamatórias Intestinais/tratamento farmacológico , Budesonida/uso terapêuticoRESUMO
Introduction: Information on the use of telemedical approaches in the context of veterinary medicine is evolving. As in human medicine, veterinary medicine is subject to an increasing digitalization effort. The aim of the current study was to investigate the perspective of German veterinarians regarding their awareness and usage of telemedical approaches. Furthermore, the degree of implementation of different digital approaches in the context of German veterinary medicine was elaborated. Methods: A literature review, that also aimed to address the necessary framework or standardization of these digitalization efforts and potential barriers such as legal or infrastructural aspects, provided information for the empirical research. Using a quantitative research approach, the perspective of German veterinarians was surveyed. Results: In total, responses from 169 veterinarians were analyzed. The results show that digital approaches were used by veterinarians and the usage was enhanced by the COVID-19 crisis. Discussion: However, the lack of a clear legal framework may be a significant barrier for further implementation. This survey provides a basis for a critical discussion on the use of veterinary telemedicine in Germany. The results may contribute to future strategies for the implementation and development of necessary policies, training, and service applications within Germany, which may be transferable for the profession in other countries.
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Electromethanogenesis is an interesting next-generation technology to produce methane from CO2 and electricity by using methanogens. Iron-corroding methanogens might be of special interest for that application due to their natural ability for electron uptake. Methanococcus maripaludis Mic1c10 and KA1 were tested in bioelectrochemical systems. Strain Mic1c10 showed a 120% higher current density and an 84% higher methane production rate (16.2 mmol m-2 d-2) than the non-corrosive strain Methanococcus maripaludis S2, which was identified earlier as the best methane producer under the same experimental conditions. Interestingly, strain KA1 also showed a 265% higher current density than strain S2. Deposits at the cathodes were detected and analyzed, which were not described earlier. A comparative genome analysis between the corrosive methanogen and the S2 strain enables new insights into proteins that are involved in enhanced electron transfer.
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The electrochemical process of microbial electrosynthesis (MES) is used to drive the metabolism of electroactive microorganisms for the production of valuable chemicals and fuels. MES combines the advantages of electrochemistry, engineering, and microbiology and offers alternative production processes based on renewable raw materials and regenerative energies. In addition to the reactor concept and electrode design, the biocatalysts used have a significant influence on the performance of MES. Thus, pure and mixed cultures can be used as biocatalysts. By using mixed cultures, interactions between organisms, such as the direct interspecies electron transfer (DIET) or syntrophic interactions, influence the performance in terms of productivity and the product range of MES. This review focuses on the comparison of pure and mixed cultures in microbial electrosynthesis. The performance indicators, such as productivities and coulombic efficiencies (CEs), for both procedural methods are discussed. Typical products in MES are methane and acetate, therefore these processes are the focus of this review. In general, most studies used mixed cultures as biocatalyst, as more advanced performance of mixed cultures has been seen for both products. When comparing pure and mixed cultures in equivalent experimental setups a 3-fold higher methane and a nearly 2-fold higher acetate production rate can be achieved in mixed cultures. However, studies of pure culture MES for methane production have shown some improvement through reactor optimization and operational mode reaching similar performance indicators as mixed culture MES. Overall, the review gives an overview of the advantages and disadvantages of using pure or mixed cultures in MES. KEY POINTS: ⢠Undefined mixed cultures dominate as inoculums for the MES of methane and acetate, which comprise a high potential of improvement ⢠Under similar conditions, mixed cultures outperform pure cultures in MES ⢠Understanding the role of single species in mixed culture MES is essential for future industrial applications.
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Dióxido de Carbono , Metano , Acetatos/metabolismo , Dióxido de Carbono/metabolismo , Eletroquímica , Eletrodos , Metano/metabolismoRESUMO
BACKGROUND: Carcinoid heart disease is the cardiac manifestation of carcinoid syndrome. There is limited research on rhythm management in patients with carcinoid heart disease. The association of typical atrial flutter and carcinoid heart disease in particular is poorly described. CASE PRESENTATION: Here we present a case of a 77-year-old German woman with carcinoid heart disease and recurrent typical atrial flutter complicating the postoperative course after tricuspid valve replacement and its successful long-term rhythm control by ablation therapy. CONCLUSION: There is limited evidence on rhythm management in patients with the rare diagnosis of carcinoid heart disease. Typical atrial flutter repeatedly complicated the postoperative course of our patient with carcinoid heart disease and could finally be treated curatively by ablation. Radiofrequency ablation should be considered as a valuable therapeutic component in the challenging therapy of this disease.
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Flutter Atrial , Doença Cardíaca Carcinoide , Ablação por Cateter , Ablação por Radiofrequência , Idoso , Flutter Atrial/cirurgia , Doença Cardíaca Carcinoide/complicações , Doença Cardíaca Carcinoide/diagnóstico por imagem , Doença Cardíaca Carcinoide/cirurgia , Feminino , Humanos , Recidiva Local de NeoplasiaRESUMO
Human kidney anion exchanger 1 (kAE1) facilitates simultaneous efflux of bicarbonate and absorption of chloride at the basolateral membrane of α-intercalated cells. In these cells, kAE1 contributes to systemic acid-base balance along with the proton pump v-H+ -ATPase and the cytosolic carbonic anhydrase II. Recent electron microscopy analyses in yeast demonstrate that heterologous expression of several kAE1 variants causes a massive accumulation of the anion transporter in intracellular membrane structures. Here, we examined the origin of these kAE1 aggregations in more detail. Using various biochemical techniques and advanced light and electron microscopy, we showed that accumulation of kAE1 mainly occurs in endoplasmic reticulum (ER) membranes which eventually leads to strong unfolded protein response (UPR) activation and severe growth defect in kAE1 expressing yeast cells. Furthermore, our data indicate that UPR activation is dose dependent and uncoupled from the bicarbonate transport activity. By using truncated kAE1 variants, we identified the C-terminal region of kAE1 as crucial factor for the increased ER stress level. Finally, a redistribution of ER-localized kAE1 to the cell periphery was achieved by boosting the ER folding capacity. Our findings not only demonstrate a promising strategy for preventing intracellular kAE1 accumulation and improving kAE1 plasma membrane targeting but also highlight the versatility of yeast as model to investigate kAE1-related research questions including the analysis of structural features, protein degradation and trafficking. Furthermore, our approach might be a promising strategy for future analyses to further optimize the cell surface targeting of other disease-related PM proteins, not only in yeast but also in mammalian cells.
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Proteína 1 de Troca de Ânion do Eritrócito , Saccharomyces cerevisiae , Animais , Proteína 1 de Troca de Ânion do Eritrócito/genética , Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Linhagem Celular , Retículo Endoplasmático/metabolismo , Humanos , Rim/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Resposta a Proteínas não DobradasRESUMO
Biomedicines are complex biochemical formulations with multiple components that require extensive quality control during manufacturing and in subsequent batch testing. A proof-of-concept study has shown that an application of Raman spectroscopy can be beneficial for a classification of vaccines. However, the complexity of biomedicines introduces new challenges to spectroscopic methodology that require advanced experimental protocols. We further show the impact of analytical protocols on vaccine classification using R as an Open Source data analysis platform. In conclusion, we advocate for standardized and transparent experimental and analytical procedures and discuss current findings and open challenges.
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Análise Espectral Raman , Controle de QualidadeRESUMO
OBJECTIVE: Acute experimental variations in glycemia decelerate (hyperglycemia) or accelerate (hypoglycemia) gastric emptying. Whether spontaneous variations in fasting plasma glucose (FPG) have a similar influence on gastric emptying is yet unclear. RESEARCH DESIGN AND METHODS: Gastric emptying of a mixed meal was prospectively studied three times in 20 patients with type 1 diabetes and 10 healthy subjects with normal glucose tolerance using a 13C-CO2 octanoate breath test with Wagner-Nelson analysis. The velocity of gastric emptying was related to FPG measured before the test (grouped as low, intermediate, or high). In addition, gastric emptying data from 255 patients with type 1 diabetes studied for clinical indications were compared by tertiles of baseline FPG. RESULTS: Despite marked variations in FPG (by 4.8 [95% CI 3.4; 6.2] mmol/L), gastric emptying did not differ among the three prospective examinations in patients with type 1 diabetes (Δ T1/2 between highest and lowest FPG: 1 [95% CI -35; 37] min; P = 0.90). The coefficient of variation for T1/2 determined three times was 21.0%. Similar results at much lower variations in FPG were found in healthy subjects. In the cross-sectional analysis, gastric emptying did not differ between the tertiles of FPG (Δ T1/2 between highest and lowest FPG: 7 [95% CI -10; 23] min; P = 0.66), when FPG varied by 7.2 (6.7; 7.8) mmol/L. However, higher HbA1c was significantly related to slower gastric emptying. CONCLUSIONS: Day-to-day variations in FPG not induced by therapeutic measures do not influence gastric emptying significantly. These findings are in contrast with those obtained after rapidly clamping plasma glucose in the hyper- or hypoglycemic concentrations range and challenge the clinical importance of short-term glucose fluctuations for gastric emptying in patients with type 1 diabetes. Rather, chronic hyperglycemia is associated with slowed gastric emptying.
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Glicemia , Diabetes Mellitus Tipo 1 , Estudos Transversais , Jejum , Esvaziamento Gástrico , Humanos , Estudos ProspectivosRESUMO
Ethyl-Toxic Steatohepatitis, the "Quiet Killer" - from Inflamed Fat to Multi-Organ Failure Abstract. The spectrum of the alcohol-associated liver disease (ALD) includes the potentially reversible simple fatty degeneration of the liver (AFLD), ethyl-toxic steatohepatitis (ASH), and the ethyl-toxic liver cirrhosis. Despite an interdisciplinary andn intensive care approach, alcohol-associated steatohepatitis (ASH), clinically characterized by jaundice and SIRS, may, in case of a fulminant course and due to the limited therapeutic options, have a mortality rate of up to 40 % . In highly selected cases, if the basic medical care of corticosteroids combined with N-acetylcysteine fails, a liver transplantation might be discussed, which occasionally shows a satisfactory long-term course. With the presented case we would like to raise awareness about the underlying disease, emphasize prevention, and summarize the most important facts for the clinical practice.
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Fígado Gorduroso Alcoólico , Fígado Gorduroso , Hepatopatias Alcoólicas , Transplante de Fígado , HumanosRESUMO
In eukaryotic cells, KDEL receptors (KDELRs) facilitate the retrieval of endoplasmic reticulum (ER) luminal proteins from the Golgi compartment back to the ER. Apart from the well-documented retention function, recent findings reveal that the cellular KDELRs have more complex roles, e.g. in cell signalling, protein secretion, cell adhesion and tumorigenesis. Furthermore, several studies suggest that a sub-population of KDELRs is located at the cell surface, where they could form and internalize KDELR/cargo clusters after K/HDEL-ligand binding. However, so far it has been unclear whether there are species- or cell-type-specific differences in KDELR clustering. By comparing ligand-induced KDELR clustering in different mouse and human cell lines via live cell imaging, we show that macrophage cell lines from both species do not develop any clusters. Using RT-qPCR experiments and numerical analysis, we address the role of KDELR expression as well as endocytosis and exocytosis rates on the receptor clustering at the plasma membrane and discuss how the efficiency of directed transport to preferred docking sites on the membrane influences the exponent of the power-law distribution of the cluster size.
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Receptores de Peptídeos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Linhagem Celular , Membrana Celular/metabolismo , Endocitose , Exocitose , Expressão Gênica , Humanos , Macrófagos/metabolismo , Camundongos , Transporte Proteico , RNA Mensageiro/análise , RNA Mensageiro/genética , Receptores de Peptídeos/análise , Receptores de Peptídeos/genética , Proteínas de Transporte Vesicular/análise , Proteínas de Transporte Vesicular/genéticaRESUMO
OBJECTIVES: The authors sought to estimate possible interference of the Medtronic Evolut R/Pro transcatheter heart valve (THV) frame with coronary access using multislice computed tomography (MSCT) data. BACKGROUND: Lower-risk patients undergoing transcatheter aortic valve replacement (TAVR) endure a high cumulative risk of coronary events, but coronary access can be challenging. METHODS: In 101 patients who received an Evolut R/Pro THV, post-TAVR MSCT (performed at a median of 30 days after TAVR) was used to assess possible interference of the elements of the THV frame with coronary access. RESULTS: The closest cell of the THV frame vertically aligned with the coronary ostium was located opposite the ostium in 58% and 63%, below the ostium in 22% and 30%, or above the ostium in 20% and 7% of left and right coronary arteries, respectively. The free sinus of Valsalva space between the THV frame and the coronary ostium was 0.45 ± 0.17 cm and 0.44 ± 0.17 cm for the left and right coronary arteries, respectively, and showed a stepwise decrease with decreasing THV size (p < 0.001). Bioprosthetic valve commissures were antianatomic (i.e., not aligned with native commissures) in 45 patients (47%), and the commissural post was overlapping a coronary ostium in 15 patients (16%). Two patients (2.0%) had a possible interference of the paravalvular sealing skirt with coronary access. CONCLUSIONS: Using post-TAVR MSCT data, the main mechanism of potential interference of Evolut R/Pro frame with coronary access was an antianatomic commissural post overlapping the coronary ostium.
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Estenose da Valva Aórtica/cirurgia , Valva Aórtica/cirurgia , Bioprótese , Angiografia por Tomografia Computadorizada , Angiografia Coronária , Estenose Coronária/diagnóstico por imagem , Vasos Coronários/diagnóstico por imagem , Próteses Valvulares Cardíacas , Tomografia Computadorizada Multidetectores , Substituição da Valva Aórtica Transcateter/instrumentação , Idoso , Idoso de 80 Anos ou mais , Valva Aórtica/diagnóstico por imagem , Valva Aórtica/fisiopatologia , Estenose da Valva Aórtica/diagnóstico por imagem , Estenose da Valva Aórtica/fisiopatologia , Estenose Coronária/etiologia , Estenose Coronária/fisiopatologia , Vasos Coronários/fisiopatologia , Feminino , Humanos , Masculino , Valor Preditivo dos Testes , Desenho de Prótese , Sistema de Registros , Fatores de Risco , Substituição da Valva Aórtica Transcateter/efeitos adversos , Resultado do TratamentoRESUMO
Saccharomyces cerevisiae has been frequently used to study biogenesis, functionality, and intracellular transport of various renal proteins, including ion channels, solute transporters, and aquaporins. Specific mutations in genes encoding most of these renal proteins affect kidney function in such a way that various disease phenotypes ultimately occur. In this context, human kidney anion exchanger 1 (kAE1) represents an important bicarbonate/chloride exchanger which maintains the acid-base homeostasis in the human body. Malfunctions in kAE1 lead to a pathological phenotype known as distal renal tubular acidosis (dRTA). Here, we evaluated the potential of baker's yeast as a model system to investigate different cellular aspects of kAE1 physiology. For the first time, we successfully expressed yeast codon-optimized full-length versions of tagged and untagged wild-type kAE1 and demonstrated their partial localization at the yeast plasma membrane (PM). Finally, pH and chloride measurements further suggest biological activity of full-length kAE1, emphasizing the potential of S. cerevisiae as a model system for studying trafficking, activity, and/or degradation of mammalian ion channels and transporters such as kAE1 in the future.IMPORTANCE Distal renal tubular acidosis (dRTA) is a common kidney dysfunction characterized by impaired acid secretion via urine. Previous studies revealed that α-intercalated cells of dRTA patients express mutated forms of human kidney anion exchanger 1 (kAE1) which result in inefficient plasma membrane targeting or diminished expression levels of kAE1. However, the precise dRTA-causing processes are inadequately understood, and alternative model systems are helpful tools to address kAE1-related questions in a fast and inexpensive way. In contrast to a previous study, we successfully expressed full-length kAE1 in Saccharomyces cerevisiae Using advanced microscopy techniques as well as different biochemical and functionality assays, plasma membrane localization and biological activity were confirmed for the heterologously expressed anion transporter. These findings represent first important steps to use the potential of yeast as a model organism for studying trafficking, activity, and degradation of kAE1 and its mutant variants in the future.
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Proteína 1 de Troca de Ânion do Eritrócito/fisiologia , Membrana Celular/fisiologia , Saccharomyces cerevisiae , Proteína 1 de Troca de Ânion do Eritrócito/genética , Transporte Biológico , Vetores Genéticos , Microrganismos Geneticamente Modificados , Plasmídeos , Saccharomyces cerevisiae/genética , Transformação GenéticaRESUMO
BACKGROUND: Virus infected killer strains of the baker's yeast Saccharomyces cerevisiae secrete protein toxins such as K28, K1, K2 and Klus which are lethal to sensitive yeast strains of the same or related species. K28 is somewhat unique as it represents an α/ß heterodimeric protein of the A/B toxin family which, after having bound to the surface of sensitive target cells, is taken up by receptor-mediated endocytosis and transported through the secretory pathway in a retrograde manner. While the current knowledge on yeast killer toxins is largely based on genetic screens for yeast mutants with altered toxin sensitivity, in vivo imaging of cell surface binding and intracellular toxin transport is still largely hampered by a lack of fluorescently labelled and biologically active killer toxin variants. RESULTS: In this study, we succeeded for the first time in the heterologous K28 preprotoxin expression and production of fluorescent K28 variants in Pichia pastoris. Recombinant P. pastoris GS115 cells were shown to successfully process and secrete K28 variants fused to mCherry or mTFP by high cell density fermentation. The fluorescent K28 derivatives were obtained in high yield and possessed in vivo toxicity and specificity against sensitive yeast cells. In cell binding studies the resulting K28 variants caused strong fluorescence signals at the cell periphery due to toxin binding to primary K28 receptors within the yeast cell wall. Thereby, the ß-subunit of K28 was confirmed to be the sole component required and sufficient for K28 cell wall binding. CONCLUSION: Successful production of fluorescent killer toxin variants of S. cerevisiae by high cell density fermentation of recombinant, K28 expressing strains of P. pastoris now opens the possibility to study and monitor killer toxin cell surface binding, in particular in toxin resistant yeast mutants in which toxin resistance is caused by defects in toxin binding due to alterations in cell wall structure and composition. This novel approach might be easily transferable to other killer toxins from different yeast species and genera. Furthermore, the fluorescent toxin variants described here might likewise represent a powerful tool in future studies to visualize intracellular A/B toxin trafficking with the help of high resolution single molecule imaging techniques.
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Fatores Matadores de Levedura/metabolismo , Pichia/genética , Pichia/metabolismo , Parede Celular/genética , Parede Celular/metabolismo , Fermentação , Fluorescência , Fatores Matadores de Levedura/química , Fatores Matadores de Levedura/genética , Pichia/química , Pichia/crescimento & desenvolvimento , Saccharomyces cerevisiae/genéticaRESUMO
Bacterial and plant A/B toxins exploit the natural trafficking pathways in eukaryotic cells to reach their intracellular target(s) in the cytosol and to ultimately kill. Such A/B toxins generally consist of an enzymatically active Asubunit (e.g., ricin toxin A (RTA)) and one or more cell binding Bsubunit(s), which are responsible for toxin binding to specific cell surface receptors. Our current knowledge of how A/B toxins are capable of efficiently intoxicating cells helped scientists to understand fundamental cellular mechanisms, like endocytosis and intracellular protein sorting in higher eukaryotic cells. From a medical point of view, it is likewise important to identify the major toxin trafficking routes to find adequate treatment solutions for patients or to eventually develop therapeutic toxin-based applications for cancer therapy. Since genome-wide analyses of A/B toxin trafficking in mammalian cells is complex, time-consuming, and expensive, several studies on A/B toxin transport have been performed in the yeast model organism Saccharomyces cerevisiae. Despite being less complex, fundamental cellular processes in yeast and higher eukaryotic cells are similar and very often results obtained in yeast can be transferred to the mammalian situation. Here, we describe a fast and easy to use reporter assay to analyze the intracellular trafficking of RTA in yeast. An essential advantage of the new assay is the opportunity to investigate not only RTA retro-translocation from the endoplasmic reticulum (ER) into the cytosol, but rather endocytosis and retrograde toxin transport from the plasma membrane into the ER. The assay makes use of a reporter plasmid that allows indirect measurement of RTA toxicity through fluorescence emission of the green fluorescent protein (GFP) after in vivo translation. Since RTA efficiently prevents the initiation of protein biosynthesis by 28S rRNA depurination, this assay allows the identification of host cell proteins involved in intracellular RTA transport through the detection of changes in fluorescence emission.