ResearchPad - cultured-tumor-cells https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Patient-derived oral mucosa organoids as an <i>in vitro</i> model for methotrexate induced toxicity in pediatric acute lymphoblastic leukemia]]> https://www.researchpad.co/article/elastic_article_15720 We have recently established a protocol to grow wildtype human oral mucosa organoids. These three-dimensional structures can be maintained in culture long-term, do not require immortalization, and recapitulate the multilayered composition of the epithelial lining of the oral mucosa. Here, we validate the use of this model to study the effect of Leucovorin (LV) on Methotrexate (MTX)-induced toxicity. MTX is a chemotherapeutic agent used in the treatment of pediatric acute lymphoblastic leukemia. Although effective, the use of MTX often results in severe side-effects, including oral mucositis, which is characterized by epithelial cell death. Here, we show that organoids are sensitive to MTX, and that the addition of LV reduces MTX toxicity, in both a concentration- and timing-dependent manner. Additionally, we show that a 24 hour ‘pretreatment’ with LV reduces MTX-induced cell death, suggesting that such a pretreatment could decrease mucositis in patients. Taken together, we provide the first in vitro model to study the effect of MTX on wildtype oral mucosa cells. Our findings underscore the relevance of the clinically applied LV regimen and highlight the potential of this model to further optimize modifications in dosing and timing of Leucovorin on oral mucosa cells.

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<![CDATA[<i>Salmonella</i> Typhimurium discreet-invasion of the murine gut absorptive epithelium]]> https://www.researchpad.co/article/elastic_article_14650 Bacterial pathogens can use secreted effector molecules to drive entry into host cells. Studies of the intestinal pathogen S.Tm have been central to uncover the mechanistic basis for the entry process. More than two decades of research have resulted in a detailed model for how S.Tm invades gut epithelial cells through effector triggering of large Rho-GTPase-dependent actin ruffles. However, the evidence for this model comes predominantly from studies in cultured cell lines. These experimental systems lack many of the architectural and signaling features of the intact gut epithelium. Our study surprisingly reveals that in the intact mouse gut, S.Tm invades absorptive epithelial cells through a process that does not require the Rho-GTPase-activating effectors and can proceed in the absence of the prototypical ruffling response. Instead, S.Tm exploits another effector, SipA, to sneak in through discreet entry structures close to cell–cell junctions. Our results challenge the current model for S.Tm epithelial cell entry and emphasizes the need of taking a physiological host cell context into account when studying bacterium–host cell interactions.

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<![CDATA[A mathematical model coupling polarity signaling to cell adhesion explains diverse cell migration patterns]]> https://www.researchpad.co/article/5989db5cab0ee8fa60be0154

Protrusion and retraction of lamellipodia are common features of eukaryotic cell motility. As a cell migrates through its extracellular matrix (ECM), lamellipod growth increases cell-ECM contact area and enhances engagement of integrin receptors, locally amplifying ECM input to internal signaling cascades. In contrast, contraction of lamellipodia results in reduced integrin engagement that dampens the level of ECM-induced signaling. These changes in cell shape are both influenced by, and feed back onto ECM signaling. Motivated by experimental observations on melanoma cells lines (1205Lu and SBcl2) migrating on fibronectin (FN) coated topographic substrates (anisotropic post-density arrays), we probe this interplay between intracellular and ECM signaling. Experimentally, cells exhibited one of three lamellipodial dynamics: persistently polarized, random, or oscillatory, with competing lamellipodia oscillating out of phase (Park et al., 2017). Pharmacological treatments, changes in FN density, and substrate topography all affected the fraction of cells exhibiting these behaviours. We use these observations as constraints to test a sequence of hypotheses for how intracellular (GTPase) and ECM signaling jointly regulate lamellipodial dynamics. The models encoding these hypotheses are predicated on mutually antagonistic Rac-Rho signaling, Rac-mediated protrusion (via activation of Arp2/3 actin nucleation) and Rho-mediated contraction (via ROCK phosphorylation of myosin light chain), which are coupled to ECM signaling that is modulated by protrusion/contraction. By testing each model against experimental observations, we identify how the signaling layers interact to generate the diverse range of cell behaviors, and how various molecular perturbations and changes in ECM signaling modulate the fraction of cells exhibiting each. We identify several factors that play distinct but critical roles in generating the observed dynamic: (1) competition between lamellipodia for shared pools of Rac and Rho, (2) activation of RhoA by ECM signaling, and (3) feedback from lamellipodial growth or contraction to cell-ECM contact area and therefore to the ECM signaling level.

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<![CDATA[Analysis of the nucleocytoplasmic shuttling RNA-binding protein HNRNPU using optimized HITS-CLIP method]]> https://www.researchpad.co/article/Nb5a6160c-8969-498c-b6ff-671487ce7810

RNA-binding proteins (RBPs) control many types of post-transcriptional regulation, including mRNA splicing, mRNA stability, and translational efficiency, by directly binding to their target RNAs and their mutation and dysfunction are often associated with several human neurological diseases and tumorigenesis. Crosslinking immunoprecipitation (CLIP), coupled with high-throughput sequencing (HITS-CLIP), is a powerful technique for investigating the molecular mechanisms underlying disease pathogenesis by comprehensive identification of RBP target sequences at the transcriptome level. However, HITS-CLIP protocol is still required for some optimization due to experimental complication, low efficiency and time-consuming, whose library has to be generated from very small amounts of RNAs. Here we improved a more efficient, rapid, and reproducible CLIP method by optimizing BrdU-CLIP. Our protocol produced a 10-fold greater yield of pre-amplified CLIP library, which resulted in a low duplicate rate of CLIP-tag reads because the number of PCR cycles required for library amplification was reduced. Variance of the yields was also reduced, and the experimental period was shortened by 2 days. Using this, we validated IL-6 expression by a nuclear RBP, HNRNPU, which directly binds the 3’-UTR of IL-6 mRNA in HeLa cells. Importantly, this interaction was only observed in the cytoplasmic fraction, suggesting a role of cytoplasmic HNRNPU in mRNA stability control. This optimized method enables us to accurately identify target genes and provides a snapshot of the protein-RNA interactions of nucleocytoplasmic shuttling RBPs.

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<![CDATA[Aqueous extract of Hibiscus sabdariffa inhibits pedestal induction by enteropathogenic E. coli and promotes bacterial filamentation in vitro]]> https://www.researchpad.co/article/5c8c197bd5eed0c484b4d750

Diarrheic diseases account for the annual death of approximately 1.9 million children under the age of 5 years, and it is a major cause of work absenteeism in developed countries. As diarrheagenic bacteria, enteropathogenic Escherichia coli (EPEC) attach to cells in the small intestine, causing local disappearance of microvilli and inducing the formation of actin-rich pedestals that disrupt the intestinal barrier and help EPEC adhere to and infect intestinal cells. Antibiotics and other bioactive compounds can often be found by analyzing traditional medicines. Here a crude aqueous extract of Hibiscus sabdariffa, which typically grows in subtropical and tropical areas and is a popular medicinal tisane in many countries, was analyzed for antibacterial activity against EPEC. In standard microdilution assays, the extract showed a minimum inhibitory concentration of 6.5 mg/ml against EPEC growth. Time-kill kinetics assays demonstrated significant 24 h bactericidal activity at 25 mg/ml. The extract is able to impede pedestal induction. Not only did the extract inhibit preformed pedestals but it prevented pedestal induction as well. Remarkably, it also promoted the formation of EPEC filaments, as observed with other antibiotics. Our results in vitro support the potential of Hibiscus sabdariffa as an antimicrobial agent against EPEC.

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<![CDATA[Adolescent idiopathic scoliosis associated POC5 mutation impairs cell cycle, cilia length and centrosome protein interactions]]> https://www.researchpad.co/article/5c8accc4d5eed0c48498ff55

Adolescent Idiopathic Scoliosis (AIS) is a spinal deformity that affects approximately 3 percent of human adolescents. Although the etiology and molecular basis of AIS is unclear, several genes such as POC5 have been identified as possible causes of the condition. In order to understand the role of POC5 in the pathogenesis of AIS, we investigated the subcellular localization of POC5 in cilia of cells over-expressing either the wild type (wt) or an AIS-related POC5 variant POC5A429V. Mutation of POC5 was found to alter its subcellular localization and to induce ciliary retraction. Furthermore, we observed an impaired cell-cycle progression with the accumulation of cells in the S-phase in cells expressing POC5A429V. Using immunoprecipitation coupled to mass spectrometry, we identified specific protein interaction partners of POC5, most of which were components of cilia and cytoskeleton. Several of these interactions were altered upon mutation of POC5. Altogether, our results demonstrate major cellular alterations, disturbances in centrosome protein interactions and cilia retraction in cells expressing an AIS-related POC5 mutation. Our study suggests that defects in centrosomes and cilia may underlie AIS pathogenesis.

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<![CDATA[Ex vivo-expanded highly purified natural killer cells in combination with temozolomide induce antitumor effects in human glioblastoma cells in vitro]]> https://www.researchpad.co/article/5c89771fd5eed0c4847d24e7

Glioblastoma is the leading malignant glioma with a poor prognosis. This study aimed to investigate the antitumor effects of natural killer cells in combination with temozolomide as the standard chemotherapeutic agent for glioblastoma. Using a simple, feeder-less, and chemically defined culture method, we expanded human peripheral blood mononuclear cells and assessed the receptor expression, natural killer cell activity, and regulatory T cell frequency in expanded cells. Next, using the standard human glioblastoma cell lines (temozolomide-sensitive U87MG, temozolomide-resistant T98G, and LN-18), we assessed the ligand expressions of receptors on natural killer cells. Furthermore, the antitumor effects of the combination of the expanded natural killer cells and temozolomide were assessed using growth inhibition assays, apoptosis detection assays, and senescence-associated β-galactosidase activity assays in the glioblastoma cell lines. Novel culture systems were sufficient to attain highly purified (>98%), expanded (>440-fold) CD3/CD56+ peripheral blood-derived natural killer cells. We designated the expanded population as genuine induced natural killer cells. Genuine induced natural killer cells exhibited a high natural killer activity and low regulatory T cell frequency compared with lymphokine-activated killer cells. Growth inhibition assays revealed that genuine induced natural killer cells inhibited the glioblastoma cell line growth but enhanced temozolomide-induced inhibition effects in U87MG. Apoptosis detection assays revealed that genuine induced natural killer cells induced apoptosis in the glioblastoma cell lines. Furthermore, senescence-associated β-galactosidase activity assays revealed that temozolomide induced senescence in U87MG. Genuine induced natural killer cells induce apoptosis in temozolomide-sensitive and temozolomide-resistant glioblastoma cells and enhances temozolomide-induced antitumor effects in different mechanisms. Hence, the combination of genuine induced natural killer cells and temozolomide may prove to be a promising immunochemotherapeutic approach in patients with glioblastoma if the antitumor effects in vivo can be demonstrated.

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<![CDATA[Rescue of collapsed replication forks is dependent on NSMCE2 to prevent mitotic DNA damage]]> https://www.researchpad.co/article/5c6730a3d5eed0c484f37e1c

NSMCE2 is an E3 SUMO ligase and a subunit of the SMC5/6 complex that associates with the replication fork and protects against genomic instability. Here, we study the fate of collapsed replication forks generated by prolonged hydroxyurea treatment in human NSMCE2-deficient cells. Double strand breaks accumulate during rescue by converging forks in normal cells but not in NSMCE2-deficient cells. Un-rescued forks persist into mitosis, leading to increased mitotic DNA damage. Excess RAD51 accumulates and persists at collapsed forks in NSMCE2-deficient cells, possibly due to lack of BLM recruitment to stalled forks. Despite failure of BLM to accumulate at stalled forks, NSMCE2-deficient cells exhibit lower levels of hydroxyurea-induced sister chromatid exchange. In cells deficient in both NSMCE2 and BLM, hydroxyurea-induced double strand breaks and sister chromatid exchange resembled levels found in NSCME2-deficient cells. We conclude that the rescue of collapsed forks by converging forks is dependent on NSMCE2.

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<![CDATA[Targeted fluorescence lifetime probes reveal responsive organelle viscosity and membrane fluidity]]> https://www.researchpad.co/article/5c6f151fd5eed0c48467ae1c

The only way to visually observe cellular viscosity, which can greatly influence biological reactions and has been linked to several human diseases, is through viscosity imaging. Imaging cellular viscosity has allowed the mapping of viscosity in cells, and the next frontier is targeted viscosity imaging of organelles and their microenvironments. Here we present a fluorescent molecular rotor/FLIM framework to image both organellar viscosity and membrane fluidity, using a combination of chemical targeting and organelle extraction. For demonstration, we image matrix viscosity and membrane fluidity of mitochondria, which have been linked to human diseases, including Alzheimer’s Disease and Leigh’s syndrome. We find that both are highly dynamic and responsive to small environmental and physiological changes, even under non-pathological conditions. This shows that neither viscosity nor fluidity can be assumed to be fixed and underlines the need for single-cell, and now even single-organelle, imaging.

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<![CDATA[On the influence of cannabinoids on cell morphology and motility of glioblastoma cells]]> https://www.researchpad.co/article/5c6c75a3d5eed0c4843cff4f

The mechanisms behind the anti-tumoral effects of cannabinoids by impacting the migratory activity of tumor cells are only partially understood. Previous studies demonstrated that cannabinoids altered the organization of the actin cytoskeleton in various cell types. As actin is one of the main contributors to cell motility and is postulated to be linked to tumor invasion, we tested the following hypothesizes: 1) Can cannabinoids alter cell motility in a cannabinoid receptor dependent manner? 2) Are these alterations associated with reorganizations in the actin cytoskeleton? 3) If so, what are the underlying molecular mechanisms? Three different glioblastoma cell lines were treated with specific cannabinoid receptor 1 and 2 agonists and antagonists. Afterwards, we measured changes in cell motility using live cell imaging and alterations of the actin structure in fixed cells. Additionally, the protein amount of phosphorylated p44/42 mitogen-activated protein kinase (MAPK), focal adhesion kinases (FAK) and phosphorylated FAK (pFAK) over time were measured. Cannabinoids induced changes in cell motility, morphology and actin organization in a receptor and cell line dependent manner. No significant changes were observed in the analyzed signaling molecules. Cannabinoids can principally induce changes in the actin cytoskeleton and motility of glioblastoma cell lines. Additionally, single cell motility of glioblastoma is independent of their morphology. Furthermore, the observed effects seem to be independent of p44/42 MAPK and pFAK pathways.

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<![CDATA[Yellow fever virus is susceptible to sofosbuvir both in vitro and in vivo]]> https://www.researchpad.co/article/5c5b52b6d5eed0c4842bcec1

Yellow fever virus (YFV) is a member of the Flaviviridae family. In Brazil, yellow fever (YF) cases have increased dramatically in sylvatic areas neighboring urban zones in the last few years. Because of the high lethality rates associated with infection and absence of any antiviral treatments, it is essential to identify therapeutic options to respond to YFV outbreaks. Repurposing of clinically approved drugs represents the fastest alternative to discover antivirals for public health emergencies. Other Flaviviruses, such as Zika (ZIKV) and dengue (DENV) viruses, are susceptible to sofosbuvir, a clinically approved drug against hepatitis C virus (HCV). Our data showed that sofosbuvir docks onto YFV RNA polymerase using conserved amino acid residues for nucleotide binding. This drug inhibited the replication of both vaccine and wild-type strains of YFV on human hepatoma cells, with EC50 values around 5 μM. Sofosbuvir protected YFV-infected neonatal Swiss mice and adult type I interferon receptor knockout mice (A129-/-) from mortality and weight loss. Because of its safety profile in humans and significant antiviral effects in vitro and in mice, Sofosbuvir may represent a novel therapeutic option for the treatment of YF. Key-words: Yellow fever virus; Yellow fever, antiviral; sofosbuvir

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<![CDATA[Immunomagnetic isolation of circulating melanoma cells and detection of PD-L1 status]]> https://www.researchpad.co/article/5c673061d5eed0c484f37a3b

Personalised medicine targeted to specific biomarkers such as BRAF and c-Kit has radically improved the success of melanoma therapy. More recently, further advances have been made using therapies targeting the immune response. In particular, therapies targeting the PD-1/PD-L1 or CTLA-4 axes alone or in combination have shown more sustained responses in 30–60% of patients. However, these therapies are associated with considerable toxicities and useful biomarkers to predict responders and non-responders are slow to emerge. Here we developed a reliable melanoma circulating tumor cell (CTC) detection method with PD-L1 evaluation on CTCs. A set of melanoma cell surface markers was tested as candidates for targeted melanoma CTC isolation and a melanoma specific immunostaining-based CTC identification protocol combined with PD-L1 detection was established. In vitro testing of the effect of exposure to blood cells on melanoma cell PD-L1 expression was undertaken. Immunomagnetic targeting isolated melanoma CTCs in up to 87.5% of stage IV melanoma patient blood samples and 3 8.6% of these had some PD-L1 expressing CTCs. Our in vitro data demonstrate PD-L1 induction on melanoma cells in the blood.This study established a robust, reliable method to isolate melanoma CTCs and detect expression of PD-L1 on these cells.

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<![CDATA[Bclaf1 critically regulates the type I interferon response and is degraded by alphaherpesvirus US3]]> https://www.researchpad.co/article/5c57e68bd5eed0c484ef366f

Type I interferon response plays a prominent role against viral infection, which is frequently disrupted by viruses. Here, we report Bcl-2 associated transcription factor 1 (Bclaf1) is degraded during the alphaherpesvirus Pseudorabies virus (PRV) and Herpes simplex virus type 1 (HSV-1) infections through the viral protein US3. We further reveal that Bclaf1 functions critically in type I interferon signaling. Knockdown or knockout of Bclaf1 in cells significantly impairs interferon-α (IFNα) -mediated gene transcription and viral inhibition against US3 deficient PRV and HSV-1. Mechanistically, Bclaf1 maintains a mechanism allowing STAT1 and STAT2 to be efficiently phosphorylated in response to IFNα, and more importantly, facilitates IFN-stimulated gene factor 3 (ISGF3) binding with IFN-stimulated response elements (ISRE) for efficient gene transcription by directly interacting with ISRE and STAT2. Our studies establish the importance of Bclaf1 in IFNα-induced antiviral immunity and in the control of viral infections.

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<![CDATA[Single-molecule dynamics and genome-wide transcriptomics reveal that NF-kB (p65)-DNA binding times can be decoupled from transcriptional activation]]> https://www.researchpad.co/article/5c4a3091d5eed0c4844c0568

Transcription factors (TFs) regulate gene expression in both prokaryotes and eukaryotes by recognizing and binding to specific DNA promoter sequences. In higher eukaryotes, it remains unclear how the duration of TF binding to DNA relates to downstream transcriptional output. Here, we address this question for the transcriptional activator NF-κB (p65), by live-cell single molecule imaging of TF-DNA binding kinetics and genome-wide quantification of p65-mediated transcription. We used mutants of p65, perturbing either the DNA binding domain (DBD) or the protein-protein transactivation domain (TAD). We found that p65-DNA binding time was predominantly determined by its DBD and directly correlated with its transcriptional output as long as the TAD is intact. Surprisingly, mutation or deletion of the TAD did not modify p65-DNA binding stability, suggesting that the p65 TAD generally contributes neither to the assembly of an “enhanceosome,” nor to the active removal of p65 from putative specific binding sites. However, TAD removal did reduce p65-mediated transcriptional activation, indicating that protein-protein interactions act to translate the long-lived p65-DNA binding into productive transcription.

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<![CDATA[Golgin-160 and GMAP210 play an important role in U251 cells migration and invasion initiated by GDNF]]> https://www.researchpad.co/article/5c59feb7d5eed0c484135327

Gliomas are the most common malignant tumors of the brain and are characteristic of severe migration and invasion. Glial cell line-derived neurotrophic factor (GDNF) promotes glioma development process. However, the regulatory mechanisms of promoting occurrence and development of glioma have not yet been clearly elucidated. In the present study, the mechanism by which GDNF promotes glioma cell migration and invasion through regulating the dispersion and location of the Golgi apparatus (GA) is described. Following GDNF treatment, a change in the volume and position of GA was observed. The stack area of the GA was enlarged and it was more concentrated near the nucleus. Golgin-160 and Golgi microtubule-associated protein 210 (GMAP210) were identified as target molecules regulating GA positioning. In the absence of either golgin-160 or GMAP210 using lentivirus, the migration and invasion of U251 cells were decreased, while it was increased following GDNF. It was also found that the GA was decreased in size and dispersed following golgin-160 or GMAP210 knockdown, as determined by GA green fluorescence assay. Once GDNF was added, the above phenomenon would be twisted, and the concentrated location and volume of the GA was restored. In combination, the present data suggested that the regulation of the position and size of the GA by golgin-160 and GMAP210 play an important role in U251 cell migration and invasion.

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<![CDATA[AMPA receptor antagonist perampanel affects glioblastoma cell growth and glutamate release in vitro]]> https://www.researchpad.co/article/5c61e8d7d5eed0c48496f231

Epileptic seizures are frequent in patients with glioblastoma, and anticonvulsive treatment is often necessary. While clinical guidelines recommend all approved anticonvulsants, so far it is still unclear which of the available drugs is the best therapeutic option for treating glioma-associated seizures, also in view of possible anti-tumorigenic effects. In our study, we employed four patient-derived low-passage cell lines of glioblastoma and three cell lines of brain metastases, and challenged these cultures with four anticonvulsants with different mechanisms of action: levetiracetam, valproic acid, carbamazepine and perampanel. Cell proliferation was determined by bromodeoxyuridine incorporation. To further analyze the effects of perampanel, apoptosis induction was measured by caspase 3/7 activation. Glutamate release was quantified and glucose uptake was determined using 18F-fluorodeoxyglucose. Real-time polymerase chain reaction was employed to assess the expression of genes associated with glutamate release and uptake in brain tumor cells. Of the four anticonvulsants, only perampanel showed systematic inhibitory effects on cell proliferation, whereas all other anticonvulsants failed to inhibit glioma and metastasis cell growth in vitro. Metastasis cells were much more resistant to perampanel than glioblastoma cell lines. Glucose uptake was attenuated in all glioblastoma cells after perampanel exposure, whereas cell death via apoptosis was not induced. Extracellular glutamate levels were found to be significantly higher in glioblastoma cell lines as compared to metastasis cell lines, but could be reduced by perampanel exposure. Incubation with perampanel up-regulated glutamine synthetase expression in glioblastoma cells, whereas treatment with valproic acid and levetiracetam downregulated excitatory amino acid transporter-2 expression. Overall, our data suggest that perampanel acts as an anticonvulsive drug and additionally mediated anti-tumorigenic effects.

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<![CDATA[In-stem molecular beacon targeted to a 5′-region of tRNA inclusive of the D arm that detects mature tRNA with high sensitivity]]> https://www.researchpad.co/article/5c59febad5eed0c484135341

Cellular functions are regulated by the up- and down-regulation and localization of RNA molecules. Therefore, many RNA detection methods have been developed to analyze RNA levels and localization. Molecular beacon (MB) is one of the major methods for quantitative RNA detection and analysis of RNA localization. Most oligonucleotide-based probes, including MB, are designed to target a long flexible region on the target RNA molecule, e.g., a single-stranded region. Recently, analyses of tRNA localization and levels became important, as it has been shown that environmental stresses and chemical reagents induce nuclear accumulation of tRNA and tRNA degradation in mammalian cells. However, tRNA is highly structured and does not harbor any long flexible regions. Hence, only a few methods are currently available for detecting tRNA. In the present study, we attempted to detect elongator tRNAMet (eMet) and initiator tRNAMet (iMet) by using an in-stem molecular beacon (ISMB), characterized by more effective quenching and significantly higher sensitivity than those of conventional MB. We found that ISMB1 targeted a 5′- region that includes the D arm of tRNA and that it detected eMet and iMet transcripts as well as mature eMet with high sensitivity. Moreover, the analysis revealed that the formation of the ISMB/tRNA transcript complex required more time than the formation of an ISMB/unstructured short RNA complex. These results suggest that ISMB-based tRNA detection can be a useful tool for various biological and medical studies.

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<![CDATA[Interaction of two antitumor peptides with membrane lipids – Influence of phosphatidylserine and cholesterol on specificity for melanoma cells]]> https://www.researchpad.co/article/5c57e69ad5eed0c484ef394a

R-DIM-P-LF11-322 and DIM-LF11-318, derived from the cationic human host defense peptide lactoferricin show antitumor activity against human melanoma. While R-DIM-P-LF11-322 interacts specifically with cancer cells, the non-specific DIM-LF11-318 exhibits as well activity against non-neoplastic cells. Recently we have shown that cancer cells expose the negatively charged lipid phosphatidylserine (PS) in the outer leaflet of the plasma membrane, while non-cancer cells just expose zwitterionic or neutral lipids, such as phosphatidylcholine (PC) or cholesterol. Calorimetric and zeta potential studies with R-DIM-P-LF11-322 and cancer-mimetic liposomes composed of PS, PC and cholesterol indicate that the cancer-specific peptide interacts specifically with PS. Cholesterol, however, reduces the effectiveness of the peptide. The non-specific DIM-LF11-318 interacts with PC and PS. Cholesterol does not affect its interaction. The dependence of activity of R-DIM-P-LF11-322 on the presence of exposed PS was also confirmed in vitro upon PS depletion of the outer leaflet of cancer cells by the enzyme PS-decarboxylase. Further corresponding to model studies, cholesterol depleted melanoma plasma membranes showed increased sensitivity to R-DIM-P-LF11-322, whereas activity of DIM-LF11-318 was unaffected. Microscopic studies using giant unilamellar vesicles and melanoma cells revealed strong changes in lateral distribution and domain formation of lipids upon addition of both peptides. Whereas R-DIM-P-LF11-322 enters the cancer cell specifically via PS and reaches an intracellular organelle, the Golgi, inducing mitochondrial swelling and apoptosis, DIM-LF11-318 kills rapidly and non-specifically by lysis of the plasma membrane. In conclusion, the specific interaction of R-DIM-P-LF11-322 with PS and sensitivity to cholesterol seem to modulate its specificity for cancer membranes.

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<![CDATA[Preparation and characterization of size-controlled glioma spheroids using agarose hydrogel microwells]]> https://www.researchpad.co/article/5c536990d5eed0c484a45f62

Treatment of glioblastoma, the most common and aggressive type of primary brain tumors, is a major medical challenge and the development of new alternatives requires simple yet realistic models for these tumors. In vitro spheroid models offer attractive platforms to mimic the tumor behavior in vivo and have thus, been increasingly applied for assessment of drug efficacy in various tumors. The aim of this study was to produce and characterize size-controlled U251 glioma spheroids towards application in glioma drug evaluation studies. To this end, we fabricated agarose hydrogel microwells with cylindrical shape and diameters of 70–700 μm and applied these wells without any surface modification for glioma spheroid formation. The resultant spheroids were homogeneous in size and shape, exhibited high cell viability (> 90%), and had a similar growth rate to that of natural brain tumors. The final size of spheroids depended on cell seeding density and microwell size. The spheroids’ volume increased linearly with the cell seeding density and the rate of this change increased with the well size. Lastly, we tested the therapeutic effect of an anti-cancer drug, Di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT) on the resultant glioma spheroids and demonstrated the applicability of this spheroid model for drug efficacy studies.

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<![CDATA[The role of microtubules and the dynein/dynactin motor complex of host cells in the biogenesis of the Coxiella burnetii-containing vacuole]]> https://www.researchpad.co/article/5c466526d5eed0c484517b0f

Microtubules (Mts) are dynamic cytoskeleton structures that play a key role in vesicular transport. The Mts-mediated transport depends on motor proteins named kinesins and the dynein/dynactin motor complex. The Rab7 adapter protein FYCO1 controls the anterograde transport of the endocytic compartments through the interaction with the kinesin KIF5. Rab7 and its partner RILP induce the recruitment of dynein/dynactin to late endosomes regulating its retrograde transport to the perinuclear area to fuse with lysosomes. The late endosomal-lysosomal fusion is regulated by the HOPS complex through its interaction with RILP and the GTPase Arl8. Coxiella burnetii (Cb), the causative agent of Q fever, is an obligate intracellular pathogen, which generates a large compartment with autophagolysosomal characteristics named Cb-containing vacuole (CCV). The CCV forms through homotypic fusion between small non-replicative CCVs (nrCCV) and through heterotypic fusion with other compartments, such as endosomes and lysosomes. In this work, we characterise the role of Mts, motor proteins, RILP/Rab7 and Arl8 on the CCV biogenesis. The formation of the CCV was affected when either the dynamics and/or the acetylation state of Mts were modified. Similarly, the overexpression of the dynactin subunit non-functional mutants p150Glued and RILP led to the formation of small nrCCVs. This phenomenon is not observed in cells overexpressing WT proteins, the motor KIF5 or its interacting protein FYCO1. The formation of the CCV was normal in infected cells that overexpressed Arl8 alone or together with hVps41 (a HOPS subunit) or in cells co-overexpressing hVps41 and RILP. The dominant negative mutant of Arl8 and the non-functional hVps41 inhibited the formation of the CCV. When the formation of CCV was affected, the bacterial multiplication diminished. Our results suggest that nrCCVs recruit the molecular machinery that regulate the Mts-dependent retrograde transport, Rab7/RILP and the dynein/dynactin system, as well as the tethering processes such as HOPS complex and Arl8 to finally originate the CCV where C. burnetii multiplies.

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