Passage Of Cells Research Articles

Photomodulation alleviates cellular senescence of aging adipose-derived stem cells

BackgroundMesenchymal stem cells (MSCs) therapies are emerging as a promising approach to therapeutic regeneration. Therapeutic persistence and reduced functional stem cells following cell delivery remain critical hurdles for clinical investigation due to the senescence of freshly isolated cells and extensive in-vitro passage.MethodsCultured adipose-derived stem cells (ASCs) were derived from subcutaneous white adipose tissue isolated from mice fed a normal diet. We performed senescence-associated-β-galactosidase (SA-β-gal) staining, real-time PCR, and Westernblot to evaluate the levels related to cellular senescence markers.ResultsThe mRNA expression levels of senescence markers were significantly increased in the later passage of ASCs. We show that light activation reduced the expression of senescent genes, and SA-β-Gal in all cells at passages. Moreover, the light-activated ASCs-derived exosomes decrease the expression of senescence, and SA-β-Gal in the later passage cells. We further investigated the photoreceptive effect of Opsin3 (Opn3) in light-activated ASCs. Deletion of Opn3 abolished the differences of light activation in reduced expression of senescent genes, increased Ca 2+ influx, and cAMP levels.ConclusionsASCs can undergo cellular senescence in-vitro passage. Photomodulation might be better preserved over senescence and Opn3-dependent activation in aged ASCs. Light-activated ASCs-derived exosomes could be served as e a new protective paradigm for cellular senescence in-vitro passage.AY8VxFmVMVxVLQ-spERSupVideo

Cytokine-Mediated Degradation of the Transcription Factor ERG Impacts the Pulmonary Vascular Response to Systemic Inflammatory Challenge.

During infectious diseases, proinflammatory cytokines transiently destabilize interactions between adjacent vascular endothelial cells (ECs) to facilitate the passage of immune molecules and cells into tissues. However, in the lung, the resulting vascular hyperpermeability can lead to organ dysfunction. Previous work identified the transcription factor ERG (erythroblast transformation-specific-related gene) as a master regulator of endothelial homeostasis. Here we investigate whether the sensitivity of pulmonary blood vessels to cytokine-induced destabilization is due to organotypic mechanisms affecting the ability of endothelial ERG to protect lung ECs from inflammatory injury. Cytokine-dependent ubiquitination and proteasomal degradation of ERG were analyzed in cultured human umbilical vein ECs. Systemic administration of TNFα (tumor necrosis factor alpha) or the bacterial cell wall component lipopolysaccharide was used to cause a widespread inflammatory challenge in mice; ERG protein levels were assessed by immunoprecipitation, immunoblot, and immunofluorescence. Murine Erg deletion was genetically induced in ECs (Ergfl/fl;Cdh5[PAC]-CreERT2), and multiple organs were analyzed by histology, immunostaining, and electron microscopy. In vitro, TNFα promoted the ubiquitination and degradation of ERG in human umbilical vein ECs, which was blocked by the proteasomal inhibitor MG132. In vivo, systemic administration of TNFα or lipopolysaccharide resulted in a rapid and substantial degradation of ERG within lung ECs but not ECs of the retina, heart, liver, or kidney. Pulmonary ERG was also downregulated in a murine model of influenza infection. Ergfl/fl;Cdh5(PAC)-CreERT2 mice spontaneously recapitulated aspects of inflammatory challenges, including lung-predominant vascular hyperpermeability, immune cell recruitment, and fibrosis. These phenotypes were associated with a lung-specific decrease in the expression of Tek-a gene target of ERG previously implicated in maintaining pulmonary vascular stability during inflammation. Collectively, our data highlight a unique role for ERG in pulmonary vascular function. We propose that cytokine-induced ERG degradation and subsequent transcriptional changes in lung ECs play critical roles in the destabilization of pulmonary blood vessels during infectious diseases.

Infectious bronchitis virus nucleocapsid protein suppressed type I interferon production by interfering with the binding of MDA5-dsRNA and interacting with LGP2

The type I interferon (IFN-I) is a critical component of the innate immune responses, and Coronaviruses (CoVs) from both the Alphacoronavirus and Betacoronavirus genera interfere with the IFN-I signaling pathway in various ways. Of the gammacoronaviruses that mainly infect birds, little is known about how infectious bronchitis virus (IBV), evades or interferes with the innate immune responses in avian hosts since few IBV strains have been adapted to grow in avian passage cells. Previously, we reported that a highly pathogenic IBV strain GD17/04 has adaptability in an avian cell line, providing a material basis for further study on the interaction mechanism. In the present work, we describe the suppression of IBV to IFN-I and the potential role of IBV-encoded nucleocapsid (N) protein. We show that IBV significantly inhibits the poly I: C-induced IFN-I production, accordingly the nuclear translocation of STAT1, and the expression of IFN-stimulated genes (ISGs). A detailed analysis revealed that N protein, acting as an IFN-I antagonist, significantly impedes the activation of the IFN-β promoter stimulated by MDA5 and LGP2 but does not counteract its activation by MAVS, TBK1, and IRF7. Further results showed that IBV N protein, verified to be an RNA-binding protein, interferes with MDA5 recognizing double-stranded RNA (dsRNA). Moreover, we found that the N protein targets LGP2, which is required in the chicken IFN-I signaling pathway. Taken together, this study provides a comprehensive analysis of the mechanism by which IBV evades avian innate immune responses.

Surface zwitterionization of polypropylene nonwoven fabric by dielectric barrier discharge plasma for efficient leukodepletion

This work reports the development of highly effective leukoreduction filters using dielectric barrier discharge (DBD) plasma applied to polypropylene (PP) nonwoven coated with zwitterionic sulfobetaine methacrylate (SBMA) monomer. The surface modification had no effect on the structural characteristics (pore size, porosity) of the material, but significantly affected its surface chemistry. Mapping IR showed that a homogeneous layer of polySBMA was grown on the surface, resulting in the formation of a superhydrophilic interface that could be immediately wetted by water (the water contact angle reached zero in 0.14 s). This enabled gravity-driven filtration of whole blood. The use of 1, 3, or 5 layers of the optimized grafted membrane obtained after 160 scans in the DBD reactor resulted in 78%, 96.1%, and 99.8% leukocytes removal, respectively, outperforming the commercial alternatives tested. At the same time, more than 90% of erythrocytes were recovered. The large pore size and the wetting behavior of the membrane allow the passage of small cells while limiting cell interactions with the membrane material as the modification reduced leukocyte adhesion by approximately 91%. After 3 washings with PBS, 43% of the attached leukocytes could be detached from the grafted membrane, which is significantly higher than the recovery measured with commercial membranes. Therefore, the membranes are not only efficient as leukoreduction filters, but could also be a viable solution for cell recovery.

Evaluation of chorionic girdle organoids for ultrastructure and equine Chorionic Gonadotropin production

The equine chorionic girdle (CG) is comprised of specialized invasive trophoblast cells that begin formation approximately 25 days after ovulation (Day 0). The CG cells differentiate into binucleate endometrial cup cells that secrete the glycoprotein hormone equine chorionic gonadotropin (eCG), which has LH-like activity in the horse but variable LH- and FSH-like activity in other species. eCG has been utilized for these properties both in vivo for estrous cycle manipulation and in vitro. To collect this hormone, large volumes of whole blood must be collected from pregnant mares, which negatively impacts equine welfare. Attempts to produce eCG in vitro using long-term culture of CG explants have not been successful beyond 180 days, with peak eCG production at 30 days of culture. Organoids are three-dimensional cell clusters that self-organize and remain genetically and phenotypically stable throughout long-term (i.e. months) culture. Human trophoblast organoids have been reported to successfully produce human chorionic gonadotropin long-term. The objective of this study was to evaluate whether organoids derived from equine chorionic girdle maintain physiological functionality. CG was collected from Day 33 equine conceptuses (n=3). CG was isolated manually using a dissection microscope and then enzymaticallydigested and cultured as organoids using human trophoblast organoid medium. A monolayer culture was performed as a control 2D culture model for comparison. CG organoids were assessed morphologically using brightfield microscopy, immunohistochemistry (IHC) with an anti-horse trophoblast antibody, and transmission electron microscopy (TEM), and all cultures were assessed physiologically by quantifying equine LH (as a proxy for eCG) via radioimmunoassay of conditioned media. The dense, trophoblast organoid phenotype, as described for human trophoblast organoids, was maintained for 5 passages, a period of 6 weeks. TEM of organoids revealed characteristic binucleate cells in both passages 1 and 4 and exhibited the presence of girdle cell processes, which have been described previously in endometrial cups in vivo. Furthermore, eCG was secreted at high concentrations (up to 67,000 ng/mL) up to passage 4 in organoids but only for passage 0 in monolayer cultures (up to 1,100 ng/mL). Therefore, equine chorionic girdle organoids provide a physiologically representative 3D in vitro model for early equine pregnancy. Funding was provided by Foundation for the Horse.

Mesenchymal stromal cells and CAR-T cells in regenerative medicine: The homing procedure and their effective parameters.

Mesenchymal stromal cells (MSCs) and chimeric antigen receptor (CAR)-T cells are two core elements in cell therapy procedures. MSCs have significant immunomodulatory effects that alleviate inflammation in the tissue regeneration process, while administration of specific chemokines and adhesive molecules would primarily facilitate CAR-T cell trafficking into solid tumors. Multiple parameters affect cell homing, including the recipient's age, the number of cell passages, proper cell culture, and the delivery method. In addition, several chemokines are involved in the tumor microenvironment, affecting the homing procedure. This review discusses parameters that improve the efficiency of cell homing and significant cell therapy challenges. Emerging comprehensive mechanistic strategies such as non-systemic and systemic homing that revealed a significant role in cell therapy remodeling were also reviewed. Finally, the primary implications for the development of combination therapies that incorporate both MSCs and CAR-T cells for cancer treatment were discussed.

Membrane Order Effect on the Photoresponse of an Organic Transducer.

Non-genetic photostimulation, which allows for control over cellular activity via the use of cell-targeting phototransducers, is widely used nowadays to study and modulate/restore biological functions. This approach relies on non-covalent interactions between the phototransducer and the cell membrane, thus implying that cell conditions and membrane status can dictate the effectiveness of the method. For instance, although immortalized cell lines are traditionally used in photostimulation experiments, it has been demonstrated that the number of passages they undergo is correlated to the worsening of cell conditions. In principle, this could impact cell responsivity against exogenous stressors, including photostimulation. However, these aspects have usually been neglected in previous experiments. In this work, we investigated whether cell passages could affect membrane properties (such as polarity and fluidity). We applied optical spectroscopy and electrophysiological measurements in two different biological models: (i) an epithelial immortalized cell line (HEK-293T cells) and (ii) liposomes. Different numbers of cell passages were compared to a different morphology in the liposome membrane. We demonstrated that cell membranes show a significant decrease in ordered domains upon increasing the passage number. Furthermore, we observed that cell responsivity against external stressors is markedly different between aged and non-aged cells. Firstly, we noted that the thermal-disordering effect that is usually observed in membranes is more evident in aged cells than in non-aged ones. We then set up a photostimulation experiment by using a membrane-targeted azobenzene as a phototransducer (Ziapin2). As an example of a functional consequence of such a condition, we showed that the rate of isomerization of an intramembrane molecular transducer is significantly impaired in aged cells. The reduction in the photoisomerization rate translates in cells with a sustained reduction of the Ziapin2-related hyperpolarization of the membrane potential and an overall increase in the molecule fluorescence. Overall, our results suggest that membrane stimulation strongly depends on membrane order, highlighting the importance of cell passage during the characterization of the stimulation tools. This study can shine light on the correlation between aging and the development of diseases driven by membrane degradation as well as on the different cell responsivities against external stressors, such as temperature and photostimulation.

Identification of Host Proteins Interacting with IBV S1 Based on Tracheal Organ Culture.

Infectious bronchitis virus (IBV) belongs to the gamma-coronavirus genus of Coronaviridae and causes serious infectious diseases in the poultry industry. However, only a few IBV strains can infect avian passage cell lines, seriously hindering the progress of basic research on IBV pathogenesis. Whereas IBV field strains can replicate in tracheal ring organ culture (TOC) without any previous adaptation in chicken embryos or primary cells. In this study, to investigate the potential use of TOC as an in vitro infection model for the study of IBV-host interaction, we first established a chicken embryo TOC culture system and carried out an investigation on the IBV replication kinetics in the system. We found that the selected strains of the IBV GI-1, GI-7, GI-13, GI-19, and GI-22 genotypes could successfully replicate in TOC and bring about damage to the infected trachea. Next, we identified host proteins of the chicken embryo trachea that interact with the IBV S1 protein by immunoprecipitation and protein mass spectrometry. A total of 127 candidate proteins were initially identified with major involvement in cell adhesion pathways and apoptosis- and autophagy-related pathways. The heat shock protein 70 (HSP70) was selected for further investigation in the interaction with IBV viral proteins. Our results showed that HSP70 interacted with IBV S1 in both TOC and CEK cells, whereas HSP70 overexpression inhibited viral replication. This study indicates that TOC is a good system for the elucidation of IBV-host interactions and HSP70 is a potential host antiviral factor.

Effects and mechanism of annexin A1-overexpressing human adipose-derived mesenchymal stem cells in the treatment of mice with acute respiratory distress syndrome

Effects and mechanism of annexin A1-overexpressing human adipose-derived mesenchymal stem cells in the treatment of mice with acute respiratory distress syndrome

Spike-Independent Infection of Human Coronavirus 229E in Bat Cells.

Bats are the reservoir for numerous human pathogens, including coronaviruses. Despite many coronaviruses having descended from bat ancestors, little is known about virus-host interactions and broader evolutionary history involving bats. Studies have largely focused on the zoonotic potential of coronaviruses with few infection experiments conducted in bat cells. To determine genetic changes derived from replication in bat cells and possibly identify potential novel evolutionary pathways for zoonotic virus emergence, we serially passaged six human 229E isolates in a newly established Rhinolophus lepidus (horseshoe bat) kidney cell line. Here, we observed extensive deletions within the spike and open reading frame 4 (ORF4) genes of five 229E viruses after passaging in bat cells. As a result, spike protein expression and infectivity of human cells was lost in 5 of 6 viruses, but the capability to infect bat cells was maintained. Only viruses that expressed the spike protein could be neutralized by 229E spike-specific antibodies in human cells, whereas there was no neutralizing effect on viruses that did not express the spike protein inoculated on bat cells. However, one isolate acquired an early stop codon, abrogating spike expression but maintaining infection in bat cells. After passaging this isolate in human cells, spike expression was restored due to acquisition of nucleotide insertions among virus subpopulations. Spike-independent infection of human coronavirus 229E may provide an alternative mechanism for viral maintenance in bats that does not rely on the compatibility of viral surface proteins and known cellular entry receptors. IMPORTANCE Many viruses, including coronaviruses, originated from bats. Yet, we know little about how these viruses switch between hosts and enter human populations. Coronaviruses have succeeded in establishing in humans at least five times, including endemic coronaviruses and the recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In an approach to identify requirements for host switches, we established a bat cell line and adapted human coronavirus 229E viruses by serial passage. The resulting viruses lost their spike protein but maintained the ability to infect bat cells, but not human cells. Maintenance of 229E viruses in bat cells appears to be independent of a canonical spike receptor match, which in turn might facilitate cross-species transmission in bats.

A rapid procedure to generate stably transfected HEK293 suspension cells for recombinant protein manufacturing: Yield improvements, bioreactor production and downstream processing

The human cell line HEK293 is one of the preferred choices for manufacturing therapeutic proteins and viral vectors for human applications. Despite its increased use, it is still considered in disadvantage in production aspects compared to cell lines such as the CHO cell line. We provide here a simple workflow for the rapid generation of stably transfected HEK293 cells expressing an engineered variant of the SARS-CoV-2 Receptor Binding Domain (RBD) carrying a coupling domain for linkage to VLPs through a bacterial transpeptidase-sortase (SrtA). To generate stable suspension cells expressing the RBD-SrtA, a single two plasmids transfection was performed, with hygromycin selection. The suspension HEK293 were grown in adherent conditions, with 20% FBS supplementation. These transfection conditions increased cell survival, allowing the selection of stable cell pools, which was otherwise not possible with standard procedures in suspension. Six pools were isolated, expanded and successfully re-adapted to suspension with a gradual increase of serum-free media and agitation. The complete process lasted four weeks. Stable expression with viability over 98% was verified for over two months in culture, with cell passages every 4–5 days. With process intensification, RBD-SrtA yields reached 6.4 μg/mL and 13.4 μg/mL in fed-batch and perfusion-like cultures, respectively. RBD-SrtA was further produced in fed-batch stirred tank 1L-bioreactors, reaching 10-fold higher yields than perfusion flasks. The trimeric antigen displayed the conformational structure and functionality expected. This work provides a series of steps for stable cell pool development using suspension HEK293 cells aimed at the scalable production of recombinant proteins.

Infection tracing and organ tropism of Siniperca chuatsi rhabdovirus expressing enhanced green fluorescent protein

Siniperca chuatsi Rhabdovirus (SCRV) is one of the important pathogens that infect mandarin fish. Recovery of recombinant viruses expressing fluorescent protein is an important method for virology research. In this study, the enhanced green fluorescent protein (EGFP) gene was inserted between the leader and the N gene of the SCRV genome. The recombinant SCRV of rS3-LeNEGFP expressing the EGFP was rescued, and the virus titer was as high as 2 × 106 PFU/mL in EPC cells. The insertion of the EGFP gene in the genome of SCRV was stable. EGFP was expressed efficiently after 10 passages in EPC cells. In vitro growth characterization, pathogenicity and organ tropism in vivo were studied. Compared to the parental rS3, rS3-LeNEGFP was defective in vitro, but not attenuated in vivo. The mortality of red zebrafish caused by rS3-LeNEGFP was the same as the parental SCRV rS3. The earliest expression of EGFP was detectable at 2 h, and clear fluorescence spots appeared at 8 h post-infection in EPC cells. The liver, heart, and brain were the major organ targets of rS3-LeNEGFP in vivo, and the kidney, gill, and spermatocyte were also invaded. The organ infection in vitro was different from that in vivo. This study is of great significance for revealing the pathogenesis of SCRV and developing vaccines and antiviral drugs for SCRV in the future.

Establishing extended pluripotent stem cells from human urine cells

BackgroundExtended pluripotent stem cells (EPSCs) can contribute to both embryonic and trophectoderm-derived extraembryonic tissues. Therefore, EPSCs have great application significance for both research and industry. However, generating EPSCs from human somatic cells remains inefficient and cumbersome.ResultsIn this study, we established a novel and robust EPSCs culture medium OCM175 with defined and optimized ingredients. Our OCM175 medium contains optimized concentration of L-selenium-methylcysteine as a source of selenium and ROCK inhibitors to maintain the single cell passaging ability of pluripotent stem cells. We also used Matrigel or the combination of laminin 511 and laminin 521(1:1) to bypass the requirement of feeder cells. With OCM175 medium, we successfully converted integration-free iPSCs from easily available human Urine-Derived Cells (hUC-iPSCs) into EPSCs (O-IPSCs). We showed that our O-IPSCs have the ability to form both intra- and extra- embryonic chimerism, and could contribute to the trophoblast ectoderm lineage and three germ layer cell lineages.ConclusionsIn conclusion, our novel OCM175 culture medium has defined, optimized ingredients, which enables efficient generation of EPSCs in a feeder free manner. With the robust chimeric and differentiation potential, we believe that this system provides a solid basis to improve the application of EPSCs in regenerative medicine.

Emergence of biased hypermutation in a heterologous additional transcription unit in recombinant lentogenic Newcastle disease virus.

Recombinant Newcastle disease virus (rNDV) strains engineered to express foreign genes from an additional transcription unit (ATU) are considered as candidate live-attenuated vector vaccines for human and veterinary use. Early during the COVID-19 pandemic we and others generated COVID-19 vaccine candidates based on rNDV expressing a partial or complete SARS-CoV-2 spike (S) protein. In our studies, a number of the rNDV constructs did not show high S expression levels in cell culture or seroconversion in immunized hamsters. Sanger sequencing showed the presence of frequent A-to-G transitions characteristic of adenosine deaminase acting on RNA (ADAR). Subsequent whole genome rNDV sequencing revealed that this biased hypermutation was exclusively localized in the ATU expressing the spike gene, and was related to deamination of adenosines in the negative strand viral genome RNA. The biased hypermutation was found both after virus rescue in chicken cell line DF-1 followed by passaging in embryonated chicken eggs, and after direct virus rescue and subsequent passaging in Vero E6 cells. Levels of biased hypermutation were higher in constructs containing codon-optimized as compared to native S gene sequences, suggesting potential association with increased GC content. These data show that deep sequencing of candidate recombinant vector vaccine constructs in different phases of development is of crucial importance in the development of NDV-based vaccines.

117 The Impact of Cell Passage Number on Elucidating Toxicological Mechanisms in Vitro

Abstract Cell passage number is of high importance in experimental design and can cause biochemical, genetic, and/or morphological changes to a cell. This project aimed to understand the impact of increasing cell passage number on the structure and function of epithelial cells from different organs. Two epithelial cell types were studied, NCI-H441 lung cells (type II alveolar cells), and HepG2 liver cells. Predefined ranges of 11-20, 21-30, 31-40, and 41-50 were studied. Cells were exposed to 1µg/mL tumour necrosis factor α (TNFα) and 15µM hydrogen peroxide (H2O2) via submerged or quasi-air liquid interface, according to GIVIMP guidelines. Endpoints tests included cytotoxicity (lactate dehydrogenase (LDH) release), cell death (BCL2, BAX, FAS), (pro-) inflammatory mediators (IL6 & IL8), oxidative stress (SOD1) and genetic toxicology (DNA damage (p21 & p53)). In addition, confocal microscopy was used to observe any changes to cell morphology. Preliminary data indicates the NCI-H441 epithelial cell passage range has an effect on the secretion of (pro-)inflammatory mediators, with an increase in IL8 and IL6 in higher passage ranges (41-50) compared to lower passage ranges (11-20) when stimulated with TNFα as a positive control. In HepG2 cells there is also an increase in IL8 production across passage range, however, it is considerably less than NCI-H441 cells. In addition, IL6 production in HepG2 cells was negligible across all passage ranges. LDH assay showed minimal variation between cell type and passage number. Data obtained suggests passage range, depending on cell type, has an influence on the effect level of the end point measured.

NSCLC patient-derived xenografts perpetuating tumor-associated, tumor-reactive T cells that respond to immunotherapy but do not cause GVHD in host mice

Abstract Non-small cell lung cancer (NSCLC) is the leading cause of malignancy-related deaths worldwide. We wished to overcome a current major limitation of PDX modeling, by providing autologous human T cells suitable for immunotherapy responses, but notably, without graft versus host disease (GVHD). We have developed PDXs from NSCLC by implanting surgically resected patient tumors subcutaneously in NOD scid gamma (NSG) mice. We used a metastatic PDX model to test potential therapeutic efficacy of a novel T cell molecule, anti-human-CD3ɛ monovalent Fab fragment (Mono-OKT3-Fab) and compared tumor growth results with anti-human PD-L1 therapy. Overall engraftment rate of unique, primary patient tumors was 52%. Over multiple passages in vivo, PDX tumors retained expression of primary tumor-matched NSCLC-diagnostic pathological markers. Moreover, primary-tumor-associated T cells were present and passaged in mice in vivo, without producing any signs of GVHD. Immunotherapy showed significant response from a PDL-1+ tumor, consistent with functional tumor-specific T cell passaging in our system. In contrast, control experiments using patient-matched human PBMCs showed vigorous GVHD in the presence or absence of co-transplanted PDX tumor; this demonstrated that non-tumor-associated lymphocytes from these patients were capable of xenoreaction, even though tumor-associated T cells were not. We observed that PDX-NSG mice treated with either modality showed significantly reduced tumor burden and increased survival over control-Ig-treated mice. Overall, our study shows that PDXs implanted in NSG mice from surgically resected-NSCLC retain T cells sufficient for preclinical evaluation of novel T cell-targeting immunotherapies. NIH, National Cancer Institute, IOTN(U01 CA244314) NIH, NIAID, R01 AI097187 Mizzou Start-up funds

Development of a Cell Line Containing the Chimeric ETV6-NTRK3 Gene. The Search for Mutations of the Tyrosine Kinase Chimeric Domain That Cause Resistance to Larotrectinib.

The development, registration, and further use of entrectinib and larotrectinib for the treatment of tumors resulting from oncogenic stimulation of chimeric neurotrophin receptors (TRK) attracted much interest to the mechanisms of tumor cells resistance to TRK inhibitors during treatment. In the presented study, a cell line carrying the chimeric gene ETV6-NTRK3 (HFF-EN) was created on the basis of human fibroblasts. The transcription level of the chimeric ETV6-NTRK3 gene in HFF-EN was comparable to the transcription level of the household ACTB gene, the expression of the ETV6-NTRKA protein was confirmed by immunoblotting. A comparison of the dose-effect curves of fibroblasts and HFF-EN cells showed a ~38-fold increase in the sensitivity of HFF-EN to larotrectinib. To obtain a cell model of the resistance to larotrectinib in NTRK-dependent cancer, we used cell passages with a gradually increasing concentration of larotrectinib and obtained six resistant clones. p.G623E c.1868G>A mutation was found in five clones, and p.R582W c.1744C>T mutation, previously not described as a resistance mutation, was found in one clone showing significantly less resistance. These results can be further used for more complete understanding of the mechanisms of the resistance to TRK inhibitors and for the development of new drugs.

Comparison of Cell Dynamics and Characterization of Bone Marrow-Derived Stem Cells in Different Passages in Young and Aged Rats

Comparison of Cell Dynamics and Characterization of Bone Marrow-Derived Stem Cells in Different Passages in Young and Aged Rats

Development of recombinant West Nile virus expressing mCherry reporter protein

West Nile virus (WNV) is transmitted to humans and animals by a mosquito and enters the central nervous system, leading to lethal encephalitis. Reporter viruses expressing fluorescent proteins enable detection of infected cells in vitro and in vivo, facilitating evaluation of the dynamics of viral infection, and the development of diagnostic or therapeutic methods. In this study, we developed a method for production of a recombinant replication-competent WNV expressing mCherry fluorescent protein. The expression of mCherry was observed in viral antigen-positive cells in vitro and in vivo, but the growth of the reporter WNV was reduced as compared to the parental WNV. The expression of mCherry was stable during 5 passages in reporter WNV-infected culture cells. Neurological symptoms were observed in mice inoculated intracranially with the reporter WNV. The reporter WNV expressing mCherry will facilitate research into WNV replication in mouse brains.

Phototoxicity and cell passage affect intracellular reactive oxygen species levels and sensitivity towards non-thermal plasma treatment in fluorescently-labeled cancer cells

Live-cell imaging with fluorescence microscopy is a powerful tool, especially in cancer research, widely-used for capturing dynamic cellular processes over time. However, light-induced toxicity (phototoxicity) can be incurred from this method, via disruption of intracellular redox balance and an overload of reactive oxygen species (ROS). This can introduce confounding effects in an experiment, especially in the context of evaluating and screening novel therapies. Here, we aimed to unravel whether phototoxicity can impact cellular homeostasis and response to non-thermal plasma (NTP), a therapeutic strategy which specifically targets the intracellular redox balance. We demonstrate that cells incorporated with a fluorescent reporter for live-cell imaging have increased sensitivity to NTP, when exposed to ambient light or fluorescence excitation, likely through altered proliferation rates and baseline intracellular ROS levels. These changes became even more pronounced the longer the cells stayed in culture. Therefore, our results have important implications for research implementing this analysis technique and are particularly important for designing experiments and evaluating redox-based therapies like NTP.