Characterization of RACK1-depleted mammalian cells by a palette of microscopy approaches reveals defects in cell cycle progression and polarity establishment.

The Receptor for Activated C Kinase 1 (RACK1) is an evolutionarily conserved scaffold protein involved in the regulation of numerous cellular processes. Here, we used CRISPR/Cas9 and siRNA to reduce the expression of RACK1 in Madin-Darby Canine Kidney (MDCK) epithelial cells and Rat2 fibroblasts, respectively. RACK1-depleted cells were examined using coherence-controlled holographic microscopy, immunofluorescence, and electron microscopy. RACK1 depletion resulted in decreased cell proliferation, increased cell area and perimeter, and in the appearance of large binucleated cells suggesting a defect in the cell cycle progression. Our results show that the depletion of RACK1 has a pleiotropic effect on both epithelial and mesenchymal cell lines and support its essential role in mammalian cells.

The influence of verapamil on the pharmacokinetics of the pan-HER tyrosine kinase inhibitor neratinib in rats: the role of P-glycoprotein-mediated efflux.

Neratinib, an irreversible pan-HER tyrosine kinase inhibitor, has been approved for the treatment of HER2-positive (HER2+) early-stage and brain metastatic breast cancer. Thus far, the pharmacology effects and pharmacodynamics of neratinib have been well studied. However, the disposition of neratinib and its influencing factors in vivo remain unclear. P-glycoprotein (P-gp), one of the most extensively studied transporters, substantially restricts penetration of drugs into the body or deeper compartments (i.e., blood-brain barrier, BBB), regarding drug resistance and drug-drug interactions. Thereby, the aim of this study was to investigate the influence of verapamil (a P-gp inhibitor) on the pharmacokinetics of neratinib in rats. Here, we have established a high specific, selective and sensitive ultra-performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method to quantify plasma concentrations of neratinib in rats. Pharmacokinetic results showed that verapamil significantly increased the system exposure of neratinib, as Cmax increased by 2.09-fold and AUC0-t increased by 1.64-fold, respectively. Additionally, the in vitro transport of neratinib was evaluated using Madin-Darby canine kidney II (MDCK II) and human MDR1 gene overexpressed MDCK (MDCK-MDR1) cell line models. As a result, the net flux ratio was over than 2 and decreased over 50% by verapamil, suggesting that neratinib was a substrate of P-gp. Hence, our findings have highlighted the important role of P-gp in the system exposure of neratinib in vivo, and drug-drug interaction should be considered when coadministration of P-gp inhibitors with neratinib. These findings may support the further clinical development and application of neratinib.

Deacetyl epoxyazadiradione protects aminoglycoside antibiotic-induced renal cell apoptosis, in vitro.

Aminoglycoside antibiotics such as gentamicin are used frequently to treat bacterial infections in humans. Excessive consumption of these antibiotics lead to renal dysfunction. One of the factors contributing to renal dysfunction is oxidative damage, which causes apoptosis. Hence, this study investigates the effect of the antioxidant compound deacetyl epoxyazadiradione (DEA) in reducing cell death induced by gentamicin treatment in kidney cells (Madin-Darby canine kidney cells). The antioxidant experiments showed that reactive oxygen species level is decreased up to 27.06 ± 0.18% in 150 µM of DEA treatment. At this concentration, the activity of antioxidant enzymes such as superoxide dismutase increased from 0.4 ± 0.04 to 1.46 ± 0.05 µmol/min/L and catalase increased from 7.48 ± 0.39 to 17.6 ± 0.74 U/mg. The relative folds of gene expression of mitochondrial enzymes such as GST, GPx and GR restored from 0.596 ± 0.019, 0.521 ± 0.013 and 0.775 ± 0.014 to 0.866 ± 0.013, 0.669 ± 0.015 and 0.8615 ± 0.028, respectively. Consequently, the percentage of cell viability increases upto 91.8 ± 2.01 from 61.93 ± 1.63 with much less fragmentation in genomic DNA. Additionally, molecular docking results showed that DEA could bind to Bax, Bcl- 2, Caspase- 3 and Caspase- 9 proteins. These results indicate that DEA could reduce cell apoptosis by reducing oxidative stress due to antibiotics and interrupting the apoptotic signal pathway in kidney cells.

Loss of NKCC1 function increases epithelial tight junction permeability by upregulating claudin-2 expression.

Conditions that cause the loss of epithelial barrier integrity are often accompanied by dysregulation of tight junction protein expression and/or localization. Recently, we have reported that patients with mutations in SLC12A2, the gene encoding the basolateral Na+-K+-2Cl- cotransporter (NKCC1), suffer from severe gastrointestinal deficits, including chronic gastrointestinal inflammation, gastrointestinal hemorrhage, intestinal obstruction, and constipation. Although the intestinal inflammation observed in patients with loss of NKCC1 function may or may not be due to tight junction dysfunction, we investigated whether the loss of NKCC1 function affects paracellular ion transport and epithelial barrier function. Wild-type HT29-MTX-E12 and CRISPR/Cas9-mediated NKCC1 knockout (KO) HT29 clones were tested for tight junction protein expression and localization. Tightness of epithelial cell monolayer was assessed by measurement of transepithelial electrical resistance and permeability of molecular tracers in transwell filters. Tight junction protein localization was assessed by immunofluorescence. Loss of NKCC1 expression strongly increases the expression of claudin-2 and occludin in epithelial cell monolayers. Loss of NKCC1 significantly reduces the transepithelial electrical resistance (TER) indicating an increase in paracellular ions flux, consistent with upregulation of the cation-selective and channel-forming claudin-2. In addition, NKCC1-KO monolayers showed a significant increase in the paracellular flux of small molecules like fluorescein (0.33 kDa), whereas the permeability of higher molecular weight TRITC-Dextran (4 kDa and 70 kDa) remained unchanged. Thus, NKCC1 regulates tight junction protein expression and loss of NKCC1 function affects epithelial barrier integrity.

Anti-inflammatory role demonstrated both in vitro and in vivo models using nonsteroidal tetranortriterpenoid, Nimbin (N1) and its analogs (N2 and N3) that alleviate the domestication of alternative medicine.

Human health may benefit from the study of natural compounds and phytoconstituents that can protect from inflammation. We investigated Nimbin (N1), a member of the ring C Seco-tetranortriterpenoids family, and its semi-natural analog deacetyl Nimbin namely N2 and N3 for their anti-inflammatory properties. As key findings, N1, N2, and N3 were able to improve wound healing by cell proliferation in a period of 24 h and were able to reduce the reactive oxygen species (ROS) production in Madin-Darby Canine Kidney cells which were screened using dichloro-dihydro fluorescein diacetate (DCF-DA) staining. When the zebrafish larvae were subjected to DCF-DA assay N1, N2, and N3 were able to substantially reduce the ROS levels in a dose-dependent manner. In zebrafish larvae, the cell death indicates the fluorescent intensity due to acridine orange staining that was found to be dramatically decreasing upon the treatment of N1, N2, and N3. The cell membrane lipid peroxidation levels were also reduced in a dose-dependent manner upon the treatment of Nimbin and its analogs indicating lesser blue fluorescent levels. Among the Nimbin and its analogs, N2 was subjected to have better activity. To confirm the activity of N1, N2, and N3, in silico characterization was performed using Density functional theory and molecular docking. As a result, N2 exhibited the lowest electronegative value and highest binding energy when docked with anti-inflammatory and antioxidant proteins CAT, COX, GP, IL-1, and MPO. Furthermore, the therapeutic potential of N2 must be explored at the molecular level as well as in clinical studies for the treatment of inflammation-associated diseases.

Suitability of NIID-MDCK cells as a substrate for cell-based influenza vaccine development from the perspective of adventitious virus susceptibility.

The practical use of cell-based seasonal influenza vaccines is currently being considered in Japan. From the perspective of adventitious virus contamination, we assessed the suitability of NIID-MDCK cells (NIID-MDCK-Cs) as a safe substrate for the isolation of influenza viruses from clinical specimens. We first established a sensitive multiplex real-time PCR system to screen for 27 respiratory viruses and used it on 34 virus samples that were isolated by passaging influenza-positive clinical specimens in NIID-MDCK-Cs. Incidentally, the limit of detection (LOD) of the system was 100 or fewer genome copies per reaction. In addition to influenza viruses, human enterovirus 68 (HEV-D68) genomes were detected in two samples after two or three passages in NIID-MDCK-Cs. To further investigate the susceptibility of NIID-MDCK-Cs to adventitious viruses, eight common respiratory viruses were subjected to passages in NIID-MDCK-Cs. The genome copy numbers of seven viruses other than parainfluenza 3 decreased below the LOD by passage 4. By passaging in NIID-MDCK-Cs, the genome numbers of the input HEV-D68, 1 × 108 copies, declined to 102 at passage 3 and to under the LOD at passage 4, whereas those of the other six viruses were under the LOD by passage 3. These results implied that during the process of isolating influenza viruses with NIID-MDCK-Cs, contaminating viruses other than parainfluenza 3 can be efficiently removed by passages in NIID-MDCK-Cs. NIID-MDCK-Cs could be a safe substrate for isolating influenza viruses that can be used to develop cell-based influenza vaccine candidate viruses.

Protective effect of morin by targeting mitochondrial reactive oxygen species induced by hydrogen peroxide demonstrated at a molecular level in MDCK epithelial cells.

BACKGROUND: The development of diabetic nephropathy is aided by the presence of oxidative stress. Morin, a natural flavonoid molecule, has been shown to have antioxidant and anti-diabetic properties. However, little is known about the mechanism of its protective effect in diabetic nephropathy pathogenesis caused by oxidative stress. METHODS: Using Madin-Darby canine kidney (MDCK) cells as a working model, the current study investigates the detailed mechanism of morin's beneficial action. In hydrogen peroxide-induced oxidative stressed MDCK cells, there was a considerable rise in intracellular ROS and decreased antioxidant enzyme levels. RESULTS: Morin has a higher binding affinity for the antioxidant receptor; according to in silico study using molecular docking and ADMET, it is predicted to be an orally active molecule. While morin administration increased SOD and CAT activity in oxidative stress-induced MDCK cells, it also reduced mitochondrial oxidative stress and apoptosis. Furthermore, the present study discovered the molecular mechanism through which morin reduced oxidative stress in MDCK cells by upregulating antioxidant enzyme molecules including GST, GPx, and GCS. CONCLUSION: These findings suggest that morin reduces H2O2-induced oxidative stress, reduces DNA oxidative damage, and prevents the depletion of antioxidant genes in MDCK cells.

Selenomethionine alleviated Ochratoxin A induced pyroptosis and renal fibrotic factors expressions in MDCK cells.

Ochratoxin A (OTA) is universally known to induce nephrotoxicity via inducing oxidative stress and apoptosis, inhibiting protein synthesis and activating autophagy. Our previous studies have proved that OTA induces nephrotoxicity in vitro and in vivo by adjusting the NOD-like receptor protein 3 (NLRP3) inflammasome activation and caspase-1-dependent pyroptosis. Based on these findings, we further investigated the protective role of selenomethionine (SeMet) on OTA-caused nephrotoxicity using the Madin-Darby canine kidney (MDCK) epithelial cells as an in vitro model, proposing to offer a new way for remedying OTA-induced nephrotoxicity by nutritional manipulation. We measured the cell vitality, lactate dehydrogenase (LDH) activity and the expression of renal fibrotic genes, NLRP3 inflammasome and pyroptosis related genes. MTT and LDH results indicated that SeMet supplementation significantly mitigated 2.0 µg/ml OTA-induced cytotoxicity in MDCK cells (p < 0.05). Meanwhile, SeMet alleviated OTA induced increase of reactive oxygen species in MDCK cells. Then, the expressions of α-SMA, Vimentin, and TGF-ß were detected both in mRNA and protein levels. The results indicated 8 µM SeMet supplementation could significantly downregulate the expression of OTA-induced renal fibrosis-related genes (p < 0.05). In addition, the upregulation of OTA-induced NLRP3 inflammasome and pyroptosis downstream genes was also significantly inhibited by 8 µM of SeMet (p < 0.05). In summary, SeMet could alleviate OTA-induced renal fibrotic genes expression and reduce NLRP3-caspase-1-dependent pyroptosis. Therefore, SeMet supplementation may become an effective approach for preserving animals from renal injury exposed to OTA.

Metabolic reprogramming and alteration of the redox state in hyper-productive MDCK cells for influenza a virus production.

Influenza is a global public health issue leading to widespread morbidity and mortality with devastating economic loss annually. Madin-Darby Canine Kidney (MDCK) cell line has been a major cell line for influenza vaccine applications. Though many details of the host metabolic responses upon influenza A virus (IAV) infection have been documented, little is known about the metabolic reprogramming features of a hyper-productive host for IAV vaccine production. In this study, a MDCK cell clone H1 was shown to have a particular high productivity of 30 × 103 virions/cell. The glucose and amino acid metabolism of H1 were evaluated, indicating that the high producer had a particular metabolic reprogramming phenotype compared to its parental cell line (P): elevated glucose uptake, superior tricarboxylic acid cycle flux, moderate amino acid consumption, and better regulation of reactive oxygen species. Combined with the stronger mitochondrial function and mild antiviral and inflammatory responses characterized previously, our results indicated that the high producer had a sufficient intracellular energy supply, and balanced substrate distribution for IAV and host protein synthesis as well as the intracellular redox status. Understanding of these metabolic alterations paves the way for the rational cell line development and reasonable process optimization for high-yield influenza vaccine production.

Cell culture-based production and in vivo characterization of purely clonal defective interfering influenza virus particles.

BACKGROUND: Infections with influenza A virus (IAV) cause high morbidity and mortality in humans. Additional to vaccination, antiviral drugs are a treatment option. Besides FDA-approved drugs such as oseltamivir or zanamivir, virus-derived defective interfering (DI) particles (DIPs) are considered promising new agents. IAV DIPs typically contain a large internal deletion in one of their eight genomic viral RNA (vRNA) segments. Consequently, DIPs miss the genetic information necessary for replication and can usually only propagate by co-infection with infectious standard virus (STV), compensating for their defect. In such a co-infection scenario, DIPs interfere with and suppress STV replication, which constitutes their antiviral potential. RESULTS: In the present study, we generated a genetically engineered MDCK suspension cell line for production of a purely clonal DIP preparation that has a large deletion in its segment 1 (DI244) and is not contaminated with infectious STV as egg-derived material. First, the impact of the multiplicity of DIP (MODIP) per cell on DI244 yield was investigated in batch cultivations in shake flasks. Here, the highest interfering efficacy was observed for material produced at a MODIP of 1E-2 using an in vitro interference assay. Results of RT-PCR suggested that DI244 material produced was hardly contaminated with other defective particles. Next, the process was successfully transferred to a stirred tank bioreactor (500 mL working volume) with a yield of 6.0E+8 PFU/mL determined in genetically modified adherent MDCK cells. The produced material was purified and concentrated about 40-fold by membrane-based steric exclusion chromatography (SXC). The DI244 yield was 92.3% with a host cell DNA clearance of 97.1% (99.95% with nuclease digestion prior to SXC) and a total protein reduction of 97.2%. Finally, the DIP material was tested in animal experiments in D2(B6).A2G-Mx1r/r mice. Mice infected with a lethal dose of IAV and treated with DIP material showed a reduced body weight loss and all animals survived. CONCLUSION: In summary, experiments not only demonstrated that purely clonal influenza virus DIP preparations can be obtained with high titers from animal cell cultures but confirmed the potential of cell culture-derived DIPs as an antiviral agent.

Exploring In Vitro Biological Cellular Responses of Pegylated ß-Cyclodextrins.

ßCDPEG5 and ßCDPEG2 are two derivatives comprising seven PEG linear chains of 5 and 2 kDa, respectively, conjugated to ßCD. As ßCDPEGs display different physicochemical properties than their precursors, they could also trigger distinct cellular responses. To investigate the biological behavior of ßCDPEGs in comparison to their parent compounds, we performed broad toxicological assays on RAW 264.7 macrophages, MC3T3-E1 osteoblasts, and MDCK cells. By analyzing ROS and NO2- overproduction in macrophages, we found that ßCDPEGs induced a moderate stress response without affecting cell viability. Although MC3T3-E1 osteoblasts were more sensitive than MDCK cells to ßCDPEGs and the parent compounds, a similar pattern was observed: the effect of ßCDPEG5 on cell viability and cell cycle progression was larger than that of ßCDPEG2; PEG2 affected cell viability and cell cycle more than ßCDPEG2; cell post-treatment recovery was favorable in all cases, and the compounds had similar behaviors regarding ROS generation. The effect on MDCK cell migration followed a similar pattern. In contrast, for osteoblasts, the interference of ßCDPEG5 with cell migration was smaller than that of ßCDPEG2; likewise, the effect of PEG2 was shorter than its conjugate. Overall, the covalent conjugation of ßCD and PEGs, particularly to yield ßCDPEG2, improved the biocompatibility profile, evidencing that a favorable biological response can be tuned through a thoughtful combination of materials. Moreover, this is the first time that an in vitro evaluation of ßCD and PEG has been presented for MC3T3-E1 and MDCK cells, thus providing valuable knowledge for designing biocompatible nanomaterials constructed from ßCD and PEGs.

TRPV4 activity regulates nuclear Ca2+ and transcriptional functions of ß-catenin in a renal epithelial cell model.

TRPV4 is a nonselective cationic channel responsive to several physical and chemical stimuli. Defects in TRPV4 channel function result in human diseases, such as skeletal dysplasias, arthropathies, and peripheral neuropathies. Nonetheless, little is known about the role of TRPV4 in other cellular functions, such as nuclear Ca2+ homeostasis or Ca2+ -regulated transcription. Here, we confirmed the presence of the full-length TRPV4 channel in the nuclei of nonpolarized Madin-Darby canine kidney cells. Confocal Ca2+ imaging showed that activation of the channel increases cytoplasmic and nuclear Ca2+ leading to translocation of TRPV4 out of the nucleus together with ß-catenin, a transcriptional regulator in the Wnt signaling pathway fundamental in embryogenesis, organogenesis, and cellular homeostasis. TRPV4 inhibits ß-catenin transcriptional activity through a direct interaction dependent upon channel activity. This interaction also occurs in undifferentiated osteoblastoma and neuroblastoma cell models. Our results suggest a mechanism in which TRPV4 may regulate differentiation in several cellular contexts.

Shear stress-induced nuclear shrinkage through activation of Piezo1 channels in epithelial cells.

The cell nucleus responds to mechanical cues with changes in size, morphology and motility. Previous work has shown that external forces couple to nuclei through the cytoskeleton network, but we show here that changes in nuclear shape can be driven solely by calcium levels. Fluid shear stress applied to MDCK cells caused the nuclei to shrink through a Ca2+-dependent signaling pathway. Inhibiting mechanosensitive Piezo1 channels through treatment with GsMTx4 prevented nuclear shrinkage. Piezo1 knockdown also significantly reduced the nuclear shrinkage. Activation of Piezo1 with the agonist Yoda1 caused similar nucleus shrinkage in cells not exposed to shear stress. These results demonstrate that the Piezo1 channel is a key element for transmitting shear force input to nuclei. To ascertain the relative contribution of Ca2+ to cytoskeleton perturbation, we examined F-actin reorganization under shear stress and static conditions, and showed that reorganization of the cytoskeleton is not necessary for nuclear shrinkage. These results emphasize the role of the mechanosensitive channels as primary transducers in force transmission to the nucleus.

Comparison of N-linked glycosylation on hemagglutinins derived from chicken embryos and MDCK cells: a case of the production of a trivalent seasonal influenza vaccine.

N-linked glycosylation plays critical roles in folding, receptor binding, and immunomodulating of hemagglutinin (HA), the main antigen in influenza vaccines. Chicken embryos are the predominant production host for influenza vaccines, but Madin-Darby canine kidney (MDCK) cells have emerged as an important alternative host. In this study, we compared glycosylation patterns, including the occupancy of potential glycosylation sites and the distribution of different glycans, on the HAs of three strains of influenza viruses for the production a trivalent seasonal flu vaccine for the 2015-2016 Northern Hemisphere season (i.e., A/California/7/2009 (H1N1) X179A, A/Switzerland/9715293/2013 (H3N2) NIB-88, and B/Brisbane/60/2008 NYMC BX-35###). Of the 8, 12, and 11 potential glycosylation sites on the HAs of H1N1, H3N2, and B strains, respectively, most were highly occupied. For the H3N2 and B strains, MDCK-derived HAs contained more sites being partially occupied (<95%) than embryo-derived HAs. A highly sensitive glycan assay was developed where 50 different glycans were identified, which was more than what has been reported previously, and their relative abundance was quantified. In general, MDCK-derived HAs contain more glycans of higher molecular weight. High-mannose species account for the most abundant group of glycans, but at a lower level as compared to those reported in previous studies, presumably due to that lower abundance, complex structure glycans were accounted for in this study. The different glycosylation patterns between MDCK- and chicken embryo-derived HAs may help elucidate the role of glycosylation on the function of influenza vaccines. KEY POINTS: • For the H3N2 and B strains, MDCK-derived HAs contained more partially (<95%) occupied glycosylation sites. • MDCK-derived HAs contained more glycans of higher molecular weight. • A systematic comparison of glycosylation on HAs used for trivalent seasonal flu vaccines was conducted.

High cell density perfusion process for high yield of influenza A virus production using MDCK suspension cells.

Similar to the recent COVID-19 pandemic, influenza A virus poses a constant threat to the global community. For the treatment of flu disease, both antivirals and vaccines are available with vaccines the most effective and safest approach. In order to overcome limitations in egg-based vaccine manufacturing, cell culture-based processes have been established. While this production method avoids egg-associated risks in face of pandemics, process intensification using animal suspension cells in high cell density perfusion cultures should allow to further increase manufacturing capacities worldwide. In this work, we demonstrate the development of a perfusion process using Madin-Darby canine kidney (MDCK) suspension cells for influenza A (H1N1) virus production from scale-down shake flask cultivations to laboratory scale stirred tank bioreactors. Shake flask cultivations using semi-perfusion mode enabled high-yield virus harvests (4.25 log10(HAU/100 µL)) from MDCK cells grown up to 41 × 106 cells/mL. Scale-up to bioreactors with an alternating tangential flow (ATF) perfusion system required optimization of pH control and implementation of a temperature shift during the infection phase. Use of a capacitance probe for on-line perfusion control allowed to minimize medium consumption. This contributed to a better process control and a more economical performance while maintaining a maximum virus titer of 4.37 log10(HAU/100 µL) and an infectious virus titer of 1.83 × 1010 virions/mL. Overall, this study clearly demonstrates recent advances in cell culture-based perfusion processes for next-generation high-yield influenza vaccine manufacturing for pandemic preparedness. KEY POINTS: • First MDCK suspension cell-based perfusion process for IAV produciton was established. • "Cell density effect" was overcome and process was intensified by reduction of medium use and automated process control. • The process achieved cell density over 40 × 106 cells/mL and virus yield over 4.37 log10(HAU/100 µL).

Ochratoxin A induces nephrotoxicity in vitro and in vivo via pyroptosis.

Ochratoxin A (OTA), a prevalent nephrotoxic mycotoxin contaminant in food and feedstuff, has been reported to induce renal injury. To disclose the nephrotoxicity of continuous administration of OTA and to investigate potential mechanisms related to pyroptosis, male C57BL/6 mice were intraperitoneally injected with 1.0 and 2.0 mg/kg B.W. OTA every other day for 14 days. At 2.0 mg/kg B.W. OTA administration significantly increased histological injury and renal fibrosis molecules (α-SMA, Vimentin, TGF-ß) and activated the NOD-like receptor protein 3 (NLRP3) inflammasome and induced pyroptosis compared with control. In the in vitro tests, Madin-Darby canine kidney (MDCK) epithelial cells were exposed to 0-4.0 µg/ml OTA for 24 h in serum-free medium. Data showed that OTA dose-dependently affected cell viability and significantly up-regulated renal fibrosis genes (α-SMA, Vimentin, TGF-ß). 2.0 µg/ml OTA significantly induced NLRP3 inflammasome activation and caspase-1-dependent pyroptosis, increasing the expression and secretion of pro-inflammatory cytokines (IL-6, TNF-α) and pyroptosis-related genes (GSDMD, IL-1ß, IL-18) in MDCK cells. These outcomes were significantly abrogated after inhibiting NLRP3 activation with inhibitor MCC950 and silencing NLRP3 with small interfering RNA (siRNA). Furthermore, knockdown of caspase-1 also ameliorated OTA-induced renal fibrosis via the inhibition of pyroptosis. Collectively, the chosen doses of OTA-triggered nephrotoxicity through NLRP3 inflammasome activation and caspase-1-dependent pyroptosis both in vitro and in vivo.

Genome-wide identification and characterization of long non-coding RNAs in MDCK cell lines with high and low tumorigenicities.

Madin-Darby canine kidney(MDCK) cells can be used to prepare cell-based influenza vaccines; however, little is known regarding the effect of lncRNA regulatorson tumorigenicity. In the present study, two cell lines with low tumorigenicity were screened from highly tumorigenic MDCK cell lines using monoclonal cell technology. Accordingly, three groups of lncRNAs were extracted from three cell lines and investigated using strand-specific Ribo-Zero RNA sequencing, detecting 1092 known and 619 novel lncRNAs. Moreover, in pairwise comparisons between the libraries of the nominally tumorigenic clones and the highly tumorigenic parent cell line, a total of 344 transcripts were expressed differentially, which were validated by qPCR using six randomly selected lncRNA genes. Furthermore, 63 target genes were identified in the upstream and downstream 100 kb of lncRNAs and their relative functions were analyzed. It was found that ten GO terms and twelve KEGG terms related to tumor by target genes and functional items. Five lncRNA transcripts and the corresponding differentially expressed target genes were used for co-expression network analysis. In addition, certain classical tumor pathways were also activated by target genes, among which, lncRNA MSTRG.1056.2 directly regulates ERBB3 to activate the PI3K-Akt pathway, contributing to tumorigenesis. Consequently, direct evidence was obtained that lncRNA regulates tumorigenesis, and a variety of target genes regulated by lncRNA were elucidated, which may be significant for non-tumorigenic MDCK cells lines acquisition.

Clathrin and AP1 are required for apical sorting of glycosyl phosphatidyl inositol-anchored proteins in biosynthetic and recycling routes in Madin-Darby canine kidney cells.

Recently, studies in animal models demonstrate potential roles for clathrin and AP1 in apical protein sorting in epithelial tissue. However, the precise functions of these proteins in apical protein transport remain unclear. Here, we reveal mistargeting of endogenous glycosyl phosphatidyl inositol-anchored proteins (GPI-APs) and soluble secretory proteins in Madin-Darby canine kidney (MDCK) cells upon clathrin heavy chain or AP1 subunit knockdown (KD). Using a novel directional endocytosis and recycling assay, we found that these KD cells are not only affected for apical sorting of GPI-APs in biosynthetic pathway but also for their apical recycling and basal-to-apical transcytosis routes. The apical distribution of the t-SNARE syntaxin 3, which is known to be responsible for selective targeting of various apical-destined cargo proteins in both biosynthetic and endocytic routes, is compromised suggesting a molecular explanation for the phenotype in KD cells. Our results demonstrate the importance of biosynthetic and endocytic routes for establishment and maintenance of apical localization of GPI-APs in polarized MDCK cells.

Metabolic machinery encrypted in the Raman spectrum of influenza A virus-inoculated mammalian cells.

Raman spectroscopy was applied with a high spectral resolution to a structural study of Influenza (type A) virus before and after its inoculation into Madin-Darby canine kidney cells. This study exploits the fact that the major virus and cell constituents, namely DNA/RNA, lipid, and protein molecules, exhibit peculiar fingerprints in the Raman spectrum, which clearly differed between cells and viruses, as well as before and after virus inoculation into cells. These vibrational features, which allowed us to discuss viral assembly, membrane lipid evolution, and nucleoprotein interactions of the virus with the host cells, reflected the ability of the virus to alter host cells' pathways to enhance its replication efficiency. Upon comparing Raman signals from the host cells before and after virus inoculation, we were also able to discuss in detail cell metabolic reactions against the presence of the virus in terms of compositional variations of lipid species, the formation of fatty acids, dephosphorylation of high-energy adenosine triphosphate molecules, and enzymatic hydrolysis of the hemagglutinin glycoprotein.

Water-soluble fluorescent probe for simultaneous detection of cyanide, hypochlorite and bisulfite at different emission wavelengths.

A fluorescent probe that responds at distinct wavelengths upon exposure to cyanide, hypochlorite, and bisulfite was synthesized. As a result, an easy to apply analytical methodology was developed for the detection of these ions. The feasibility of this method was evaluated by theoretical calculations. The probe exhibited excellent solubility in the test solution (H2O: DMF = 99: 1, v: v) with low detection limits for cyanide, hypochlorite and bisulfite (4.5 × 10 -8 M, 4.9 × 10 -7 M and 4.3 × 10 -8 M respectively) showing distinct emission wavelengths for each ion without interference in practical application. Furthermore, the probe had low toxicity and was applied for the imaging experiments of cyanide, hypochlorite and bisulfite in living HeLa and MDCK cells.