scholarly journals Cyclic-di-GMP is required for corneal infection byPseudomonas aeruginosaand modulates host immunity

2017 ◽  
Author(s):  
Joey Kuok Hoong Yam ◽  
Thet Tun Aung ◽  
Song Lin Chua ◽  
Yingying Cheng ◽  
Gurjeet Singh Kohli ◽  
...  
Keyword(s):  
Extracellular Polymeric Substances ◽  
Antimicrobial Treatment ◽  
Guanosine Monophosphate ◽  
Rna Seq ◽  
Physiological Characterization ◽  
Wide Range

AbstractBiofilms are extremely tolerant toward antimicrobial treatment and host immune clearance due to their distinct physiology and protection by extracellular polymeric substances. Bis-(3´-5´)-cyclic dimeric guanosine monophosphate (c-di-GMP) is an essential messenger that regulates biofilm formation by a wide range of bacteria. However, there is a lack of physiological characterization of biofilmsin vivoas well as the roles of c-di-GMP signaling in mediating host-biofilm interactions. Here, we employed dual RNA-Seq to characterize the host and pathogen transcriptomes duringPseudomonas aeruginosainfection using a mouse keratitis model.In vivo P. aeruginosabiofilms maintained a distinct physiology compared within vitro P. aeruginosabiofilms, with enhanced virulence and iron uptake capacity. C-di-GMP synthesis was enhanced inP. aeruginosacellsin vivo,potentially due to down-regulation of the expression of several phosphodiesterases (e.g., DipA, NbdA). Increased intracellular c-di-GMP levels were required for long-term ocular colonization ofP. aeruginosaand impaired host innate immunity.

scholarly journals MODL-16. ABEMACICLIB, A SELECTIVE CDK4/6 INHIBITOR, RESTRICTS GROWTH OF PEDIATRIC GLIAL-LINEAGE TUMORS IN VITRO AND IN VIVO

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii414-iii414
Author(s):  
Muh-Lii Liang ◽  
Tsung-Han Hsieh ◽  
Tai-Tong Wong
Keyword(s):  
Dna Repair ◽  
Therapeutic Strategy ◽  
Target Genes ◽  
Rna Seq ◽  
Downstream Target ◽  
Rb Phosphorylation ◽  
Glial Lineage

Abstract BACKGROUND Glial-lineage tumors constitute a heterogeneous group of neoplasms, comprising gliomas, oligodendrogliomas, and ependymomas, which account for 40%–50% of all pediatric central nervous system tumors. Advances in modern neuro-oncological therapeutics are aimed at improving neoadjuvant chemotherapy and deferring radiotherapy because radiation exposure may cause long-term side effects on the developing brain in young children. Despite aggressive treatment, more than half the high-grade gliomas (pHGGs) and one-third of ependymomas exhibit recurrence within 2 years of initial treatment. METHODS By using integrated bioinformatics and through experimental validation, we found that at least one gene among CCND1, CDK4, and CDK6 was overexpressed in pHGGs and ependymomas. RESULTS The use of abemaciclib, a highly selective CDK4/6 inhibitor, effectively inhibited cell proliferation and reduced the expression of cell cycle–related and DNA repair–related gene expression, which was determined through RNA-seq analysis. The efficiency of abemaciclib was validated in vitro in pHGGs and ependymoma cells and in vivo by using subcutaneously implanted ependymoma cells from patient-derived xenograft (PDX) in mouse models. Abemaciclib demonstrated the suppression of RB phosphorylation, downstream target genes of E2F, G2M checkpoint, and DNA repair, resulting in tumor suppression. CONCLUSION Abemaciclib showed encouraging results in preclinical pediatric glial-lineage tumors models and represented a potential therapeutic strategy for treating challenging tumors in children.

Correlation between magnesium and potassium contents in muscle: role of Na(+)-K+ pump

1993 ◽  
Vol 264 (2) ◽  
pp. C457-C463 ◽  
Author(s):  
I. Dorup ◽  
T. Clausen
Keyword(s):  
Extensor Digitorum Longus ◽  
Extensor Digitorum ◽  
Deficient Diet ◽  
Young Rats ◽  
Wide Range ◽  
86Rb Influx

In young rats fed a Mg(2+)-deficient diet for 3 wk, Mg2+ and K+ contents in soleus and extensor digitorum longus muscles were significantly reduced and closely correlated. In isolated soleus muscles, Mg2+ depletion induced an even more pronounced loss of K+, and Mg2+ and K+ contents were correlated over a wide range (r = 0.95, P < 0.001). Extracellular Mg2+ (0-1.2 mM) caused no change in total or ouabain-suppressible 86Rb influx. After long-term incubation in Ca(2+)-Mg(2+)-free buffer with EDTA and EGTA, cellular Mg2+ and K+ contents were reduced by 35 and 15%, respectively, without any reduction in ATP and total or ouabain-suppressible 86Rb influx. In Mg(2+)-depleted muscles 42K efflux was increased by up to 42%, and repletion with Mg2+ produced a graded decrease. We conclude that Mg2+ and K+ contents are closely correlated in muscles Mg2+ depleted in vivo or in vitro and that neither extracellular nor moderate intracellular Mg2+ depletion affects total or Na(+)-K+ pump-mediated K+ influx. The reduced K+ content may rather be related to increased K+ efflux from the muscles.

Analysis of in Vitro and in Vivo Characteristics of Human Embryonic Stem Cell-Derived Neural Precursors

2010 ◽  
Vol 19 (4) ◽  
pp. 471-486 ◽  
Author(s):  
Nataliya Kozubenko ◽  
Karolina Turnovcova ◽  
Miroslava Kapcalova ◽  
Olena Butenko ◽  
Miroslava Anderova ◽  
...  
Keyword(s):  
Embryonic Stem ◽  
Cellular Heterogeneity ◽  
Neural Cells ◽  
Lineage Specification ◽  
Neural Precursors ◽  
Selection Strategies

During the last decade, much progress has been made in developing protocols for the differentiation of human embryonic stem cells (hESCs) into a neural phenotype. The appropriate agent for cell therapy is neural precursors (NPs). Here, we demonstrate the derivation of highly enriched and expandable populations of proliferating NPs from the CCTL14 line of hESCs. These NPs could differentiate in vitro into functionally active neurons, as confirmed by immunohistochemical staining and electrophysiological analysis. Neural cells differentiated in vitro from hESCs exhibit broad cellular heterogeneity with respect to developmental stage and lineage specification. To analyze the population of the derived NPs, we used fluorescence-activated cell sorting (FACS) and characterized the expression of several pluripotent and neural markers, such as Nanog, SSEA-4, SSEA-1, TRA-1-60, CD24, CD133, CD56 (NCAM), β-III-tubulin, NF70, nestin, CD271 (NGFR), CD29, CD73, and CD105 during long-term propagation. The analyzed cells were used for transplantation into the injured rodent brain; the tumorigenicity of the transplanted cells was apparently eliminated following long-term culture. These results complete the characterization of the CCTL14 line of hESCs and provide a framework for developing cell selection strategies for neural cell-based therapies.

scholarly journals Functional characterization of a stable, noncytolytic stage of macrophage activation in tumors.

1977 ◽  
Vol 146 (6) ◽  
pp. 1511-1520 ◽  
Author(s):  
S W Russell ◽  
W F Doe ◽  
A T McIntosh
Keyword(s):  
Macrophage Activation ◽  
Functional Characterization ◽  
Cytolytic Activity ◽  
Target Cells ◽  
Tumor Target ◽  
Total Absence

The state in which macrophages (Mphi) from regressing Moloney sarcomas could kill tumor target cells was a highly labile one which decayed rapidly in vitro. Thereafter, regressor Mphi were noncytolytic. Mphi from several different progressing sarcomas failed to kill, even when challenged with target cells immediately after explantation. Similarly, thioglycollate-induced peritoneal Mphi (TG-Mphi) did not kill. Noncytolygic Mphi derived either from progressing sarcomas or from long-term (up to 96 h) cultures of regressor Mphi were exquisitely sensitive to stimulation by bacterial lipopolysaccharide (LPS); picogram/milliliter amounts induced killing. Similar concentrations of LPS had no demonstrable effect on TG-Mphi. Thus, tumor Mphi generally appeared to have been primed in vivo, with those in regressing sarcomas having additionally acquired cytolytic activity. Inability of progressor Mphi to kill apparently stemmed from lack of, or failure to respond to, the signal needed in vivo to trigger cytolytic activity, rather than the total absence of activation.

scholarly journals Characterization of Andean Blueberry in Bioactive Compounds, Evaluation of Biological Properties and in vitro Bioaccessibility

2020 ◽  
Author(s):  
Nieves Baenas ◽  
Jenny Ruales ◽  
Diego A. Moreno ◽  
Daniel Alejandro Barrio ◽  
Carla M. Stinco ◽  
...  
Keyword(s):  
Antioxidant Capacity ◽  
Bioactive Compounds ◽  
Biological Activities ◽  
In Vitro Digestion ◽  
Biological Properties ◽  
Wide Range ◽  
Zebrafish Embryogenesis

Andean blueberries are wild berries grown and consumed in Ecuador which contain high values of bioactive compounds, mainly anthocyanins, with powerful antioxidant activity. The aim of this study was to evaluate the profile and contents of (poly)phenols and carotenoids in Andean blueberry by HPLC-DAD-MSn and determine a wide range of its biological activities. The antioxidant capacity of this fruit was evaluated in vitro by three different methods and in vivo using the zebrafish animal model, also the toxicity effect was determined by the zebrafish embryogenesis test. Besides, the antimicrobial activity and the capacity of Andean blueberry to produce hemagglutination in blood cells were evaluated. Finally, the bioaccessibility of (poly)phenols and related antioxidant capacity were determined in the different phases of an in vitro digestion. The global results indicated no toxicity of Andean blueberry, weakly bacteriostatic activity, and high contents of anthocyanins and antioxidant capacity, which were partially bioaccesible in vitro (~ 50 % at the final intestinal step), contributing to the knowledge of its health benefits for consumers and its potential use in the food and pharmaceutical industry as functional ingredient.

scholarly journals Semi-automated high-throughput substrate screening assay for nucleoside kinases

2021 ◽  
Author(s):  
Katja Hellendahl ◽  
Maryke Fehlau ◽  
Sebastian Hans ◽  
Peter Neubauer ◽  
Anke Kurreck
Keyword(s):  
High Throughput ◽  
Viral Infections ◽  
Screening Assay ◽  
Liquid Handling ◽  
Wide Range ◽  
High Throughput Assay ◽  
Liquid Handling Robot

Nucleoside kinases (NKs) are key enzymes involved in the in vivo phosphorylation of nucleoside analogues used as drugs to treat cancer or viral infections. Having different specificities, the characterization of NKs is essential for drug design and the production of nucleotide analogues in an in vitro enzymatic process. Therefore, a fast and reliable substrate screening assay for NKs is of great importance. Here, we report the validation of a well-known luciferase-based assay for the detection of NK activity in 96-well plate format. The assay was semi-automated using a liquid handling robot. A good linearity was demonstrated (r² >0.98) in the range of 0 to 500 µM ATP, and it was shown that also alternative phosphate donors like dATP or CTP were accepted by the luciferase. The developed high-throughput assay revealed comparable results to HPLC analysis. The assay was exemplary used for the comparison of the substrate spectra of four nucleoside kinases using 20 (8 natural and 12 modified) substrates. The screening results correlated well with literature data and, additionally, previously unknown substrates were identified for three of the NKs studied. Our results demonstrate that the developed semi-automated high-throughput assay is suitable to identify best performing NKs for a wide range of substrates.

F.05 Characterization of NBCA glue polymerization for embolization of brain AVM’s

2016 ◽  
Vol 43 (S2) ◽  
pp. S19-S20
Author(s):  
BH Wang ◽  
D Pelz ◽  
D Lee ◽  
MR Boulton ◽  
SP Lownie
Keyword(s):  
High Speed ◽  
Contact Angles ◽  
Polymerization Rate ◽  
Physical Interaction ◽  
Wide Range ◽  
Glue Injection ◽  
Glue Embolization

Background: Brain arteriovenous malformations (AVM’s) are abnormal connections between arteries and veins. Endovascular glue embolization with N-butyl cyanoacrylate (NBCA) is an accepted form of treatment, with most complications related to timing of polymerization. Current literature reports a wide range of polymerization times with large discrepancies between in-vivo and in-vitro results. Methods: Polymerization time was measured for mixtures of lipiodol/NBCA of 50/50, 60/40, 70/30. The influence of pH, temperature and presence of biological catalysts on polymerization rate was investigated in-vivo using submerged droplet tests. PVA-C, silicone and endothelium surfaces were compared and contact angles were measured to assess physical interaction with NBCA. High-speed video of glue injection through a microcatheter was captured to characterize coaxial flow. Results: Polymerization rate increases with pH and temperature. A hydrophilic substrate such as PVA-C provides surface properties that are most similar to endothelium. Endothelium provides a catalytic surface that increases the rate of polymerization. Blood products further increase the polymerization rate with RBC’s providing almost instantaneous polymerization of NBCA upon contact. Characterization of coaxial flow shows dripping to jetting transition with significant wall effect. Conclusions: We have successfully deconstructed and characterized the dynamic behavior of NBCA embolization. A refined understanding of NBCA behavior could help reduce embolization-related complications.

Human homologues of Delta-1 and Delta-4 function as mitogenic regulators of primitive human hematopoietic cells

Blood ◽  
2001 ◽  
Vol 97 (7) ◽  
pp. 1960-1967 ◽  
Author(s):  
Francis N. Karanu ◽  
Barbara Murdoch ◽  
Tomoyuki Miyabayashi ◽  
Mitsuhara Ohno ◽  
Masahide Koremoto ◽  
...  
Keyword(s):  
Cell Fate ◽  
Hematopoietic Cells ◽  
Functional Roles ◽  
Cell Fate Decisions ◽  
Wide Range ◽  
Deficient Mice

Delta-mediated Notch signaling controls cell fate decisions during invertebrate and murine development. However, in the human, functional roles for Delta have yet to be described. This study reports the characterization of Delta-1 and Delta-4 in the human. Human Delta-4 was found to be expressed in a wide range of adult and fetal tissues, including sites of hematopoiesis. Subsets of immature hematopoietic cells, along with stromal and endothelial cells that support hematopoiesis, were shown to express Notch and both Delta-1 and Delta-4. Soluble forms of human Delta-1 (hDelta-1) and hDelta-4 proteins were able to augment the proliferation of primitive human hematopoietic progenitors in vitro. Intravenous transplantation of treated cultures into immune-deficient mice revealed that hDelta-1 is capable of expanding pluripotent human hematopoietic repopulating cells detected in vivo. This study provides the first evidence for a role of Delta ligands as a mitogenic regulator of primitive hematopoietic cells in the human.

scholarly journals Characterization of a progenitor cell population in childhood T-cell acute lymphoblastic leukemia

Blood ◽  
2006 ◽  
Vol 109 (2) ◽  
pp. 674-682 ◽  
Author(s):  
Charlotte V. Cox ◽  
Hannah M. Martin ◽  
Pamela R. Kearns ◽  
Paul Virgo ◽  
Roger S. Evely ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Lymphoblastic Leukemia ◽  
Significant Proportion ◽  
Serial Passage ◽  
Cell Acute Lymphoblastic Leukemia

Abstract A significant proportion of children with T-cell acute lymphoblastic leukemia (T-ALL) continue to fail therapy. Consequently, characterization of the cells that proliferate to maintain the disease should provide valuable information on the most relevant therapeutic targets. We have used in vitro suspension culture (SC) and nonobese diabetic–severe combined immune deficient (NOD/SCID) mouse assays to phenotypically characterize and purify T-ALL progenitor cells. Cells from 13 pediatric cases were maintained in vitro for at least 4 weeks and expanded in 8 cases. To characterize the progenitors, cells were sorted for expression of CD34 and CD4 or CD7 and the subfractions were evaluated in vitro and in vivo. The majority of cells capable of long-term proliferation in vitro were derived from the CD34+/CD4− and CD34+/CD7− subfractions. Moreover, the CD34+/CD4− or CD7− cells were the only subfractions capable of NOD/SCID engraftment. These T-ALL cells successfully repopulated secondary and tertiary recipients with equivalent levels of engraftment, demonstrating self-renewal ability. The immunophenotype and genotype of the original leukemia cells were preserved with serial passage in the NOD/SCID mice. These data demonstrate the long-term repopulating ability of the CD34+/CD4− and CD34+/CD7− subfractions in T-ALL and suggest that a cell with a more primitive phenotype was the target for leukemic transformation in these cases.

scholarly journals Sweet Basil Has Distinct Synthases for Eugenol Biosynthesis in Glandular Trichomes and Roots with Different Regulatory Mechanisms

2021 ◽  
Vol 22 (2) ◽  
pp. 681
Author(s):  
Vaishnavi Amarr Reddy ◽  
Chunhong Li ◽  
Kumar Nadimuthu ◽  
Jessica Gambino Tjhang ◽  
In-Cheol Jang ◽  
...  
Keyword(s):  
Glandular Trichomes ◽  
Functional Characterization ◽  
Rna Seq ◽  
Post Translational Modifications ◽  
E Coli ◽  
Sweet Basil ◽  
Specific Regulation

Production of a volatile phenylpropene; eugenol in sweet basil is mostly associated with peltate glandular trichomes (PGTs) found aerially. Currently only one eugenol synthase (EGS), ObEGS1 which belongs to PIP family is identified from sweet basil PGTs. Reports of the presence of eugenol in roots led us to analyse other EGSs in roots. We screened for all the PIP family reductase transcripts from the RNA-Seq data. In vivo functional characterization of all the genes in E. coli showed their ability to produce eugenol and were termed as ObEGS2-8. Among all, ObEGS1 displayed highest expression in PGTs and ObEGS4 in roots. Further, eugenol was produced only in the roots of soil-grown plants, but not in roots of aseptically-grown plants. Interestingly, eugenol production could be induced in roots of aseptically-grown plants under elicitation suggesting that eugenol production might occur as a result of environmental cues in roots. The presence of ObEGS4 transcript and protein in aseptically-grown plants indicated towards post-translational modifications (PTMs) of ObEGS4. Bioinformatics analysis showed possibility of phosphorylation in ObEGS4 which was further confirmed by in vitro experiment. Our study reveals the presence of multiple eugenol synthases in sweet basil and provides new insights into their diversity and tissue specific regulation.

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