RESUMEN
We experimentally demonstrate optoacoustic cooling via stimulated Brillouin-Mandelstam scattering in a 50 cm long tapered photonic crystal fiber. For a 7.38 GHz acoustic mode, a cooling rate of 219 K from room temperature has been achieved. As anti-Stokes and Stokes Brillouin processes naturally break the symmetry of phonon cooling and heating, resolved sideband schemes are not necessary. The experiments pave the way to explore the classical to quantum transition for macroscopic objects and could enable new quantum technologies in terms of storage and repeater schemes.
RESUMEN
Real-time monitoring of gas-liquid pipe flow is highly demanded in industrial processes in the chemical and power engineering sectors. Therefore, the present contribution describes the novel design of a robust wire-mesh sensor with an integrated data processing unit. The developed device features a sensor body for industrial conditions of up to 400 °C and 135 bar as well as real-time processing of measured data, including phase fraction calculation, temperature compensation and flow pattern identification. Furthermore, user interfaces are included via a display and 4 20 mA connectivity for the integration into industrial process control systems. In the second part of the contribution, we describe the experimental verification of the main functionalities of the developed system. Firstly, the calculation of cross-sectionally averaged phase fractions along with temperature compensation was tested. Considering temperature drifts of up to 55 K, an average deviation of 3.9% across the full range of the phase fraction was found by comparison against image references from camera recordings. Secondly, the automatic flow pattern identification was tested in an air-water two-phase flow loop. The results reveal reasonable agreement with well-established flow pattern maps for both horizontal and vertical pipe orientations. The present results indicate that all prerequisites for an application in industrial environments in the near future are fulfilled.
RESUMEN
Background: Transfusion of red cell concentrates (RCCs) is an integral therapy after severe hemorrhage or trauma. Prehospital transfusion offers an immediate intervention in emergency cases. Air ambulance-based prehospital transfusion, already used in different countries, is currently established in Germany. Limited information is available for regulatory-compliant transport logistics of RCCs and their quality after repeated air rescue missions. Thus, the aim of this study was (i) to validate regulatory-compliant logistics and (ii) to assess product quality, analyzing biochemical parameters and RBC morphology. Study Design and Methods: Due to regulatory requirements, we adapted a rotation system of 1 day transport, 1 day quarantine storage and 1 day storage over the entire RCC shelf life. RCCs transported on air rescue missions (flight group) were compared against a control group, treated identically except for helicopter transport. RCCs were visually inspected, and their temperature was documented throughout the entire rotation cycles. RCCs at the end of shelf life (end point samples) were assessed for levels of hemoglobin, hematocrit, free hemoglobin, hemolysis, mean corpuscular volume, potassium and pH. In addition, morphological changes were assessed using flow morphometry. Results: In total 81 RCCs were assessed in the flight group and 50 in the control group. Within the flight group, 30 RCCs were transfused. RCCs were dispatched on average 11 times (7-13 times). The average flight time was 18.3 h (6.6-28.8 h). The rotation system ensured adherence to regulatory guidelines, especially compliance to storage conditions of +2 to +6°C of intermediate storage. Biochemical and morphological quality parameters did not exhibit any changes upon repeated air rescue missions. A correlation with respect to the flight time was not observed either. Discussion: The quality of RCCs after repeated air rescue missions is noninferior to control samples regarding biochemical and morphological parameters. The product quality is within German regulations for up to 42 days of storage. The logistics and maintenance of the thermal conditions are safe and feasible. Thus, a rotation system of RCCs offers a regulatory-compliant option to supply air rescue missions with RCCs to allow life-saving prehospital transfusions at the incident scene.
RESUMEN
N-glycosylated proteins produced in human embryonic kidney 293 (HEK 293) cells often carry terminal N-acetylgalactosamine (GalNAc) and only low levels of sialylation. On therapeutic proteins, such N-glycans often trigger rapid clearance from the patient's bloodstream via efficient binding to asialoglycoprotein receptor (ASGP-R) and mannose receptor (MR). This currently limits the use of HEK 293 cells for therapeutic protein production. To eliminate terminal GalNAc, we knocked-out GalNAc transferases B4GALNT3 and B4GALNT4 by CRISPR/Cas9 in FreeStyle 293-F cells. The resulting cell line produced a coagulation factor VII-albumin fusion protein without GalNAc but with increased sialylation. This glyco-engineered protein bound less efficiently to both the ASGP-R and MR in vitro and it showed improved recovery, terminal half-life and area under the curve in pharmacokinetic rat experiments. By overexpressing sialyltransferases ST6GAL1 and ST3GAL6 in B4GALNT3 and B4GALNT4 knock-out cells, we further increased factor VII-albumin sialylation; for ST6GAL1 even to the level of human plasma-derived factor VII. Simultaneous knock-out of B4GALNT3 and B4GALNT4 and overexpression of ST6GAL1 further lowered factor VII-albumin binding to ASGP-R and MR. This novel glyco-engineered cell line is well-suited for the production of factor VII-albumin and presumably other therapeutic proteins with fully human N-glycosylation and superior pharmacokinetic properties.
Asunto(s)
Glicoproteínas , Sialiltransferasas , Animales , Técnicas de Inactivación de Genes , Glicoproteínas/biosíntesis , Glicoproteínas/genética , Glicoproteínas/farmacocinética , Glicosilación , Células HEK293 , Humanos , Ratas , Sialiltransferasas/genética , Sialiltransferasas/metabolismoRESUMEN
Wire-mesh sensors are well-established scientific instruments for measuring the spatio-temporal phase distribution of two-phase flows based on different electrical conductivities of the phases. Presently, these instruments are also applied in industrial processes and need to cope with dynamic operating conditions increasingly. However, since the quantification of phase fractions is achieved by normalizing signals with respect to a separately recorded reference measurement, the results are sensitive to temperature differences in any application. Therefore, the present study aims at proposing a method to compensate temperature effects in the data processing procedure. Firstly, a general approach is theoretically derived from the underlying measurement principle and compensation procedures for the electrical conductivity from literature models. Additionally, a novel semi-empirical model is developed on the basis of electrochemical fundamentals. Experimental investigations are performed using a single-phase water loop with adjustable fluid temperature in order to verify the theoretical approach for wire-mesh sensor applications and to compare the different compensation models by means of real data. Finally, the preferred model is used to demonstrate the effect of temperature compensation with selected sets of experimental two-phase data from a previous study. The results are discussed in detail and show that temperature effects need to be handled carefully-not merely in industrial applications, but particularly in laboratory experiments.
RESUMEN
We present a novel automated system for morphology analysis of red blood cells (RBC) under flow. RBC concentrates collected by blood banks for transfusions are stored for periods of up to several weeks, during which time a number of changes occur, collectively termed the storage lesion. Typically the extent of hemolysis is the defining criterion to determine the acceptability of the RBCs for transfusions. Morphological changes are related with biochemical alteration during the storage of RBCs. The typical blood smear procedure for determining such changes is a labor-intensive and potentially biased manual process. The advantage of the flow morphometry system presented here is that it provides fully automated morphological classification of RBCs with large sample numbers in a short time. Our system uses a commercially available flow cell and flow conditions that prevent adhesion of RBCs, thus eliminating the need for blocking agents such as albumin that affect the distribution of cell shapes. Our morphometry results are validated by comparison with standard biochemical assays (hemolysis, ATP) for blood from 17 donors stored under blood bank conditions for 13 weeks. We show that the percentage of spherocytes present can be used to estimate the status of RBC concentrates. © 2017 International Society for Advancement of Cytometry.
Asunto(s)
Eritrocitos/citología , Adenosina Trifosfato/metabolismo , Adolescente , Adulto , Almacenamiento de Sangre/métodos , Conservación de la Sangre/métodos , Recuento de Eritrocitos/métodos , Citometría de Flujo/métodos , Humanos , Persona de Mediana Edad , Adulto JovenRESUMEN
Biosensors for continuous glucose monitoring in bioreactors could provide a valuable tool for optimizing culture conditions in biotechnological applications. We have developed an optical biosensor for long-term continuous glucose monitoring and demonstrated a tight glucose level control during cell culture in disposable bioreactors. The in-line sensor is based on a commercially available oxygen sensor that is coated with cross-linked glucose oxidase (GOD). The dynamic range of the sensor was tuned by a hydrophilic perforated diffusion membrane with an optimized permeability for glucose and oxygen. The biosensor was thoroughly characterized by experimental data and numerical simulations, which enabled insights into the internal concentration profile of the deactivating by-product hydrogen peroxide. The simulations were carried out with a one-dimensional biosensor model and revealed that, in addition to the internal hydrogen peroxide concentration, the turnover rate of the enzyme GOD plays a crucial role for biosensor stability. In the light of this finding, the glucose sensor was optimized to reach a long functional stability (>52 days) under continuous glucose monitoring conditions with a dynamic range of 0-20 mM and a response time of t 90 ≤ 10 min. In addition, we demonstrated that the sensor was sterilizable with beta and UV irradiation and only subjected to minor cross sensitivity to oxygen, when an oxygen reference sensor was applied. Graphical abstract Measuring setup of a glucose biosensor in a shake flask for continuous glucose monitoring in mammalian cell culture.
Asunto(s)
Técnicas Biosensibles/métodos , Técnicas de Cultivo de Célula/métodos , Glucosa/análisis , Animales , Aspergillus niger/enzimología , Reactores Biológicos , Técnicas Biosensibles/instrumentación , Células CHO , Técnicas de Cultivo de Célula/instrumentación , Cricetulus , Enzimas Inmovilizadas/química , Diseño de Equipo , Glucosa Oxidasa/químicaRESUMEN
BACKGROUND: In the activated sludge process, problems of filamentous bulking and foaming can occur due to overgrowth of certain filamentous bacteria. Nowadays, these microorganisms are typically monitored by means of light microscopy, commonly combined with staining techniques. As drawbacks, these methods are susceptible to human errors, subjectivity and limited by the use of discontinuous microscopy. The in situ microscope appears as a suitable tool for continuous monitoring of filamentous bacteria, providing real-time examination, automated analysis and eliminating sampling, preparation and transport of samples. In this context, a proper image processing algorithm is proposed for automated recognition and measurement of filamentous objects. METHODS: This work introduces a method for real-time evaluation of images without any staining, phase-contrast or dilution techniques, differently from studies present in the literature. Moreover, we introduce an algorithm which estimates the total extended filament length based on geodesic distance calculation. For a period of twelve months, samples from an industrial activated sludge plant were weekly collected and imaged without any prior conditioning, replicating real environment conditions. RESULTS: Trends of filament growth rate-the most important parameter for decision making-are correctly identified. For reference images whose filaments were marked by specialists, the algorithm correctly recognized 72 % of the filaments pixels, with a false positive rate of at most 14 %. An average execution time of 0.7 s per image was achieved. CONCLUSIONS: Experiments have shown that the designed algorithm provided a suitable quantification of filaments when compared with human perception and standard methods. The algorithm's average execution time proved its suitability for being optimally mapped into a computational architecture to provide real-time monitoring.
Asunto(s)
Bacterias/citología , Bacterias/aislamiento & purificación , Procesamiento de Imagen Asistido por Computador/métodos , Algoritmos , Curva ROC , Aguas del Alcantarillado/microbiologíaRESUMEN
The present study demonstrates the application of in situ microscopy for monitoring the growth of filamentous bacteria which can induce disturbances in an industrial activated sludge process. An in situ microscope (ISM) is immersed directly into samples of activated sludge with Microthrix parvicella as dominating species. Without needing further preparatory steps, the automatic evaluation of the ISM-images generates two signals: the number of individual filaments per image (ISM-filament counting) and the total extended filament length (TEFL) per image (ISM-online TEFL). In this first version of the image-processing algorithm, closely spaced crossing filament-segments or filaments within bulk material are not detected. The signals show highly linear correlation both with the standard filament index and the TEFL. Correlations were further substantiated by comparison with real-time polymerase chain reaction (real-time PCR) measurements of M. parvicella and of the diluted sludge volume index. In this case study, in situ microscopy proved to be a suitable tool for straightforward online-monitoring of filamentous bacteria in activated sludge systems. With future adaptation of the system to different filament morphologies, including cross-linking filaments, bundles, and attached growth, the system will be applicable to other wastewater treatment plants.
Asunto(s)
Actinobacteria/citología , Microscopía , Aguas Residuales/microbiología , Actinobacteria/fisiología , Reacción en Cadena en Tiempo Real de la Polimerasa , Aguas del Alcantarillado/microbiología , Instalaciones de Eliminación de Residuos , Eliminación de Residuos Líquidos , Microbiología del AguaRESUMEN
Current state of the art to determine the viability of animal cell suspension cultures is based on sampling and subsequent counting using specific staining assays. We demonstrate for the first time a noninvasive in situ imaging cytometry capable of determining the statistics of a morphologic transition during cell death in suspension cultures. To this end, we measure morphometric inhomogeneity--defined as information entropy--in cell in situ micrographs. We found that the cells are partitioned into two discrete entropy states broadened by phenotypical variability. During the normal course of a culture or by inducing cell death, we observe the transition of cells between these states. As shown by comparison with ex situ diagnostics, the entropy transition happens before or while the cytoplasmatic membrane is loosing its ability to exclude charged dyes. Therefore, measurement of morphometric inhomogeneity constitutes a noninvasive assessment of viability in real time.
Asunto(s)
Técnicas Citológicas/métodos , Entropía , Procesamiento de Imagen Asistido por Computador/métodos , Microscopía/métodos , Animales , Supervivencia CelularRESUMEN
Yeast morphology and counting are highly important in fermentation as they are often associated with productivity and can be influenced by process conditions. At present, time-consuming and offline methods are utilized for routine analysis of yeast morphology and cell counting using a haemocytometer. In this study, we demonstrate the application of an in situ microscope to obtain a fast stream of pseudohyphae images from agitated sample suspensions of a Saccharomyces cerevisiae strain, whose morphology in cell clusters is frequently found in the bioethanol fermentation industry. The large statistics of microscopic images allow for online determination of the principal morphological characteristics of the pseudohyphae, including the number of constituent cells, cell-size, number of branches, and length of branches. The distributions of these feature values are calculated online, constituting morphometric monitoring of the pseudohyphae population. By providing representative data, the proposed system can improve the effectiveness of morphological characterization, which in turn can help to improve the understanding and control of bioprocesses in which pseudohyphal-like morphologies are found.
Asunto(s)
Recuento de Colonia Microbiana/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Microscopía/métodos , Saccharomyces cerevisiae/citología , Algoritmos , Fermentación , Microbiología Industrial , Microscopía/instrumentación , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/aislamiento & purificaciónRESUMEN
Great efforts are directed towards improving productivity, consistency and quality of biopharmaceutical processes and products. One particular area is the development of new sensors for continuous monitoring of critical bioprocess parameters by using online or in-line monitoring systems. Recently, we developed a glucose biosensor applicable in single-use, in-line and long-term glucose monitoring in mammalian cell bioreactors. Now, we integrated this sensor in an automated glucose monitoring and feeding system capable of maintaining stable glucose levels, even at very low concentrations. We compared this fed-batch feedback system at both low (< 1 mM) and high (40 mM) glucose levels with traditional batch culture methods, focusing on glycosylation and glycation of the recombinant protein darbepoetin alfa (DPO) produced by a CHO cell line. We evaluated cell growth, metabolite and product concentration under different glucose feeding strategies and show that continuous feeding, even at low glucose levels, has no harmful effects on DPO quantity and quality. We conclude that our system is capable of tight glucose level control throughout extended bioprocesses and has the potential to improve performance where constant maintenance of glucose levels is critical.
Asunto(s)
Automonitorización de la Glucosa Sanguínea , Glucemia , Animales , Técnicas de Cultivo Celular por Lotes , Reactores Biológicos , Células CHO , Cricetinae , Cricetulus , Darbepoetina alfa , GlucosaRESUMEN
In industrial yeast fermentation processes, single-cell yeast suspensions are usually preferable to cells in aggregates, as single cells exhibit a larger contact area with the nutrient medium, which in many cases helps optimize the process. In addition to affecting fermentation time and efficiency, cell aggregates (e.g., pseudohyphal yeast morphology) may also impair centrifugation systems, one of the most expensive and complex steps of the production process that involves the recovery of yeast cells for subsequent fermentation cycles. To date, no standard technique allows for a systematic diagnosis of yeast morphology in real time during sugarcane biofuel fermentation. Accordingly, we investigate an in situ microscope (ISM) for online monitoring of the density and morphology of an industrial Saccharomyces cerevisiae strain widely used in Brazilian distilleries (PE-2). During batch and repeated batch sugarcane molasses fermentation, the instrument revealed single cells, budding yeast cells, and pseudohyphae, all in a variety of sizes and shapes. The ISM image analysis indicated that the volume of single yeast cells increased by roughly 40% over the lag phase before budding and remained approximately constant thereafter. Pseudohyphae with three and more cells appeared mostly during the stationary phase. Cooling problems were simulated by raising the temperature from 33 to 45 °C. During this thermal stress, single cells as well as budding cells and pseudohyphae forming cells became smaller and exhibited intracellular inhomogeneities. From these results, we conclude that an ISM is a useful tool for monitoring yeast morphology during sugarcane fermentation. Atypical morphologies can be detected early and be used as an automatic warning system.
Asunto(s)
Microbiología Industrial/métodos , Microscopía/métodos , Saccharomyces cerevisiae/fisiología , Medios de Cultivo , Fermentación , Melaza , SaccharumRESUMEN
Biosensors for continuous glucose monitoring in bioreactors could provide a valuable tool for optimizing culture conditions in biotechnological applications. We have developed an optical biosensor for long-term continuous glucose monitoring and demonstrated a tight glucose level control during cell culture in disposable bioreactors. The in-line sensor is based on a commercially available oxygen sensor that is coated with cross-linked glucose oxidase (GOD). The dynamic range of the sensor was tuned by a hydrophilic perforated diffusion membrane with an optimized permeability for glucose and oxygen. The biosensor was thoroughly characterized by experimental data and numerical simulations, which enabled insights into the internal concentration profile of the deactivating by-product hydrogen peroxide. The simulations were carried out with a one-dimensional biosensor model and revealed that, in addition to the internal hydrogen peroxide concentration, the turnover rate of the enzyme GOD plays a crucial role for culture monitoring is an integral part of animal cell cultivation. For several culture parameters, in situ sensors exist; others are predominantly monitored off-line. One important cell culture parameter is glucose concentration. Despite many efforts, there is still a lack of in situ sensors for continuous glucose monitoring. Such biosensors could provide a valuable tool for optimizing culture conditions in biotechnological applications. In this contribution, the manufacture of a long-term stable optical glucose sensor is described which is used to demonstrate glucose level monitoring during cell culture in disposable bioreactors. The in situ sensor is based on a commercially available oxygen sensor that is coated with cross-linked glucose oxidase and a hydrophilic perforated diffusion membrane. Glucose was measured in shake flasks and wave bags with only minor drifts of the sensor sensitivity during batch and fed-batch fermentations.
Asunto(s)
Técnicas Biosensibles/instrumentación , Técnicas Biosensibles/métodos , Biotecnología/métodos , Enzimas Inmovilizadas/química , Diseño de Equipo/métodos , Glucosa/análisis , Animales , Reactores Biológicos , Células CHO , Técnicas de Cultivo de Célula , Células/metabolismo , Células Cultivadas , Cricetulus , Difusión , Glucosa/química , Glucosa/metabolismo , Glucosa Oxidasa/química , Peróxido de Hidrógeno/análisis , Peróxido de Hidrógeno/química , Oxígeno/química , Programas InformáticosRESUMEN
The culture of human induced pluripotent stem cells (hiPSCs) at large scale becomes feasible with the aid of scalable suspension setups in continuously stirred tank reactors (CSTRs). Innovative monitoring options and emerging automated process control strategies allow for the necessary highly defined culture conditions. Next to standard process characteristics such as oxygen consumption, pH, and metabolite turnover, a reproducible and steady formation of hiPSC aggregates is vital for process scalability. In this regard, we developed a hiPSC-specific suspension culture unit consisting of a fully monitored CSTR system integrated into a custom-designed and fully automated incubator. As a step towards cost-effective hiPSC suspension culture and to pave the way for flexibility at a large scale, we constructed and utilized tailored miniature CSTRs that are largely made from three-dimensional (3D) printed polylactic acid (PLA) filament, which is a low-cost material used in fused deposition modelling. Further, the monitoring tool for hiPSC suspension cultures utilizes in situ microscopic imaging to visualize hiPSC aggregation in real-time to a statistically significant degree while omitting the need for time-intensive sampling. Suitability of our culture unit, especially concerning the developed hiPSC-specific CSTR system, was proven by demonstrating pluripotency of CSTR-cultured hiPSCs at RNA (including PluriTest) and protein level.
Asunto(s)
Reactores Biológicos , Sistemas de Computación , Automatización , Agregación Celular , Proliferación Celular , Células Cultivadas , Simulación por Computador , Humanos , Células Madre Pluripotentes Inducidas/citología , SuspensionesRESUMEN
Cerebral malaria is a complex neurological syndrome caused by an infection with Plasmodium falciparum parasites and is exclusively attributed to a series of host-parasite interactions at the pathological blood-stage of infection. In contrast, the preceding intra-hepatic phase of replication is generally considered clinically silent and thereby excluded from playing any role in the development of neurological symptoms. In this study, however, we present an antigen PbmaLS_05 that is presented to the host immune system by both pre-erythrocytic and intra-erythrocytic stages and contributes to the development of cerebral malaria in mice. Although deletion of the endogenous PbmaLS_05 prevented the development of experimental cerebral malaria (ECM) in susceptible mice after both sporozoite and infected red blood cell (iRBC) infections, we observed significant differences in contribution of the host immune response between both modes of inoculation. Moreover, PbmaLS_05-specific CD8+ T cells contributed to the development of ECM after sporozoite but not iRBC-infection, suggesting that pre-erythrocytic antigens like PbmaLS_05 can also contribute to the development of cerebral symptoms. Our data thus highlight the importance of the natural route of infection in the study of ECM, with potential implications for vaccine and therapeutic strategies against malaria.
Asunto(s)
Antígenos de Protozoos/inmunología , Susceptibilidad a Enfermedades , Malaria Cerebral/inmunología , Malaria Cerebral/parasitología , Plasmodium berghei/inmunología , Animales , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Reactividad Cruzada/inmunología , Modelos Animales de Enfermedad , Expresión Génica , Genes Protozoarios , Genes Reporteros , Estadios del Ciclo de Vida , Imagen por Resonancia Magnética , Malaria Cerebral/diagnóstico , Malaria Cerebral/patología , Ratones , Plasmodium berghei/genética , Plasmodium berghei/crecimiento & desarrolloRESUMEN
This work reviews the state-of-the-art in image-based in situ methods with regard to their potential use for fermentation of Saccharomyces cerevisiae in sugarcane wine. The integration of real time information from fermentation tanks in the control strategies has high potential to promote better fermentative performance. While several image-based techniques for the measurement of cell concentration have been established, a reliable and consistent viability measurement still remains a challenging task. Reagent-free methods that estimate viability from information contained in micrograph images are reviewed. Nevertheless, the inherent complexity of the sugarcane syrup medium imposes extra challenges regarding its representation in microscopic images and their evaluation by real time image analysis.
Asunto(s)
Biotecnología/métodos , Etanol/metabolismo , Microscopía/métodos , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Saccharum/metabolismo , Etanol/toxicidad , Fermentación , Procesamiento de Imagen Asistido por Computador/métodos , Viabilidad Microbiana/efectos de los fármacos , Saccharomyces cerevisiae/fisiologíaRESUMEN
Mammalian cells, especially CHO (Chinese Hamster Ovary), are an important host for the production of biopharmaceuticals. Early detection of cellular stress and the onset of apoptosis, ultimately leading to a reduced viability of the culture, are important with respect to process development and monitoring. In this work, intact cell MALDI mass spectrometry (ICM MS) biotyping was used to rapidly and sensitively detect cell stress and the onset of apoptosis at line in CHO cell cultures. We describe the identification of specific and highly reproducible stress and apoptosis related changes in m/z signal intensities that allowed prediction of upcoming cell viability changes approximately 24h earlier than standard culture monitoring. Furthermore, early identification of apoptosis onset was comparable to that using a sensitive, albeit offline, detection method. By comparison with ICM MS analysis of apoptosis induced cultures, many of the m/z values were identified as apoptosis-specific. A classification model for discrimination of unknown samples regarding their cellular viability/apoptosis status was developed based on a condensed set of 51 m/z values. The fast, robust and automated acquisition of cell state specific MS signatures could become a promising tool for CHO culture monitoring.
Asunto(s)
Apoptosis/genética , Células CHO/citología , Técnicas de Cultivo de Célula , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Animales , Células CHO/fisiología , Supervivencia Celular , Cricetinae , CricetulusRESUMEN
Despite the critical contributions of cilia to embryonic development and human health, key regulators of cilia formation await identification. In this paper, a functional RNA interference-based screen linked 30 novel protein kinases with ciliogenesis. Of them, we have studied the role of the microtubule (MT)-associated protein/MT affinity regulating kinase 4 (MARK4) in depth. MARK4 associated with the basal body and ciliary axoneme in human and murine cell lines. Ultrastructural and functional analyses established that MARK4 kinase activity was required for initiation of axoneme extension. We identified the mother centriolar protein ODF2 as an interaction partner of MARK4 and showed that ODF2 localization to the centriole partially depended on MARK4. Our data indicated that, upon MARK4 or ODF2 knockdown, the ciliary program arrested before the complete removal of the CP110-Cep97 inhibitory complex from the mother centriole, suggesting that these proteins act at this level of axonemal extension. We propose that MARK4 is a critical positive regulator of early steps in ciliogenesis.