scholarly journals Triple, Mutually Orthogonal Cycloadditions Through the Design of Electronically Activated SNO-OCTs

2020 ◽  
Author(s):  
Yun Hu ◽  
Jessica M. Roberts ◽  
Henry R. Kilgore ◽  
Amirah Mat Lani ◽  
Ronald Raines ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Experimental Studies ◽  
Order Rate Constant ◽  
Cell Labeling ◽  
Type I ◽  
Electronic Tuning ◽  
Live Cell Labeling ◽  
New Compounds

Interest in mutually exclusive pairs of bioorthogonal labeling reagents continues to drive the design of new compounds capable of fast and predictable reactions. The ability to easily modify heterocyclic strained cyclooctynes containing sulfamate backbones (SNO-OCTs) enables electronic tuning of the relative rates of reactions of SNO-OCTs in cycloadditions with Type I–III dipoles. As opposed to optimizations based on just one specific dipole class, the electrophilicity of the alkynes in SNO-OCTs can be manipulated to achieve divergent reactivities and furnish mutually orthogonal dual ligation systems. Significant rate enhancements for reactions of a difluorinated SNO-OCT derivative compared to the parent scaffold were noted, with the second-order rate constant in cycloadditions with diazoacetamides exceeding 1 M−1 s −1 . Computational and experimental studies were employed to inform the design of triple ligation systems that encompass three orthogonal reactivities. Finally, polar SNO-OCTs are rapidly internalized by mammalian cells and remain functional in the cytosol for live-cell labeling, highlighting their potential for diverse in vitro and in vivo applications.

scholarly journals Triple, Mutually Orthogonal Cycloadditions Through the Design of Electronically Activated SNO-OCTs

2020 ◽  
Author(s):  
Yun Hu ◽  
Jessica M. Roberts ◽  
Henry R. Kilgore ◽  
Amirah Mat Lani ◽  
Ronald Raines ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Experimental Studies ◽  
Order Rate Constant ◽  
Cell Labeling ◽  
Type I ◽  
Electronic Tuning ◽  
Live Cell Labeling ◽  
New Compounds

Interest in mutually exclusive pairs of bioorthogonal labeling reagents continues to drive the design of new compounds capable of fast and predictable reactions. The ability to easily modify heterocyclic strained cyclooctynes containing sulfamate backbones (SNO-OCTs) enables electronic tuning of the relative rates of reactions of SNO-OCTs in cycloadditions with Type I–III dipoles. As opposed to optimizations based on just one specific dipole class, the electrophilicity of the alkynes in SNO-OCTs can be manipulated to achieve divergent reactivities and furnish mutually orthogonal dual ligation systems. Significant rate enhancements for reactions of a difluorinated SNO-OCT derivative compared to the parent scaffold were noted, with the second-order rate constant in cycloadditions with diazoacetamides exceeding 1 M−1 s −1 . Computational and experimental studies were employed to inform the design of triple ligation systems that encompass three orthogonal reactivities. Finally, polar SNO-OCTs are rapidly internalized by mammalian cells and remain functional in the cytosol for live-cell labeling, highlighting their potential for diverse in vitro and in vivo applications.

scholarly journals HIVEP1 Is a Negative Regulator of NF-κB That Inhibits Systemic Inflammation in Sepsis

2021 ◽  
Vol 12 ◽  
Author(s):  
Hisatake Matsumoto ◽  
Brendon P. Scicluna ◽  
Kin Ki Jim ◽  
Fahimeh Falahi ◽  
Wanhai Qin ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Negative Regulator ◽  
Type I ◽  
Promoter Regions ◽  
Monocytic Cells ◽  
Site Analysis ◽  
Enhancer Binding Protein ◽  
Transcriptomic Changes

Our previous work identified human immunodeficiency virus type I enhancer binding protein 1 (HIVEP1) as a putative driver of LPS-induced NF-κB signaling in humans in vivo. While HIVEP1 is known to interact with NF-ĸB binding DNA motifs, its function in mammalian cells is unknown. We report increased HIVEP1 mRNA expression in monocytes from patients with sepsis and monocytes stimulated by Toll-like receptor agonists and bacteria. In complementary overexpression and gene deletion experiments HIVEP1 was shown to inhibit NF-ĸB activity and induction of NF-ĸB responsive genes. RNA sequencing demonstrated profound transcriptomic changes in HIVEP1 deficient monocytic cells and transcription factor binding site analysis showed enrichment for κB site regions. HIVEP1 bound to the promoter regions of NF-ĸB responsive genes. Inhibition of cytokine production by HIVEP1 was confirmed in LPS-stimulated murine Hivep1-/- macrophages and HIVEP1 knockdown zebrafish exposed to the common sepsis pathogen Streptococcus pneumoniae. These results identify HIVEP1 as a negative regulator of NF-κB in monocytes/macrophages that inhibits proinflammatory reactions in response to bacterial agonists in vitro and in vivo.

p21-Activated Kinase 1 Regulates Hyperproliferation of Nf1 Haploinsufficient Mast Cells In Vitro and In Vivo Via Activation of the MAPK Pathway.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3063-3063
Author(s):  
Andrew S. McDaniel
Keyword(s):  
Mast Cells ◽  
Mammalian Cells ◽  
Rho Gtpase ◽  
Mapk Pathway ◽  
Mapk Signaling ◽  
Type I ◽  
Wild Type ◽  
Kit Ligand

Abstract p21-activated kinases (Paks) are downstream mediators of Rho GTPase proteins and have been implicated in yeast and immortalized cells as positive regulators of MAPK pathway members in modulating cell growth and cytoskeletal functions. However, their role in primary mammalian cells has not been described. NF1 encodes neurofibromin, which negatively regulates p21Ras activity by stimulating its intrinsic GTPase activity, and accelerating hydrolysis of Ras from the GTP to the GDP confirmation. Disruption of the NF1 locus results in neurofibromatosis type I (NF1), an inherited disorder characterized by the development of neurofibromas that contain large numbers of degranulating mast cells that have been implicated in tumor progression. Utilizing a genetic intercross of Pak 1−/− mice with mice haploinsufficient at the Nf1 locus, we studied the role of Pak1 in the context of normal and hyperactivated Ras-MAPK signaling in primary inflammatory mast cells. Pak1 was found to directly contribute to Ras-dependent signaling by modulating both Raf-1, Mek-1 and ERK1/2 activation. Loss of Pak1 fully corrects the hyperphosphorylation of ERK1/2 found in Nf1+/− mast cells to that of wild type controls. Deletion of Pak1 in Nf1+/− mast cells is associated with a correction of Kit ligand mediated proliferation to wild type levels in vitro. Further, after subcutaneous administration of Kit ligand via micro osmotic pumps, which is an established model that stimulates local proliferation of mast cells in vivo (Ingram, JEM 2001), we confirmed that genetic disruption of Pak1 corrects the proliferation of Nf1+/− mast cells in vivo to that of wild type controls. These data provide direct genetic evidence that Pak1 modulates the Ras-Raf-Mek-Erk pathway and identifies a specific molecular target within the inflammatory tumor microenvironment for the treatment or prevention of neurofibromas.

Abstract 654: Loss of SPRR3 In ApoE -/- Mice Leads to Increased Atheroma Vulnerability and Evidence of Plaque Rupture and Cardiac Infarcts

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Amanda K Segedy ◽  
Bin Li ◽  
Caressa D Lietman ◽  
MacRae F Linton ◽  
Pampee P Young
Keyword(s):  
Myocardial Infarction ◽  
Plaque Rupture ◽  
Experimental Studies ◽  
Intraplaque Hemorrhage ◽  
Type I ◽  
Gelatinase Activity ◽  
Plaque Instability ◽  
Atherosclerosis Progression

Atheroma rupture is the leading cause of myocardial infarction. While studies have examined inflammatory cell-mediated effects on plaque vulnerability, less is known about the role of vascular smooth muscle cells (VSMCs) or specific molecular players in the maintenance of atheroma stability. We reported that loss of small proline-rich repeat protein 3 (SPRR3), enriched in atheroma VSMCs, leads to increased VSMC death and significantly accelerates atherosclerosis progression in ApoE -/- mice. Here, we show that loss of SPRR3 promotes features in plaques of brachiocephalic arteries common to unstable lesions, such as increased necrotic core size, reduced cap collagen content, and reduced VSMC content. Moreover, ApoE -/- mice lacking SPRR3 develop coronary artery lesions with advanced features, including intraplaque hemorrhage. In addition, Sprr3 -/- ApoE -/- mice fed a high-fat diet for 6 months develop spontaneous myocardial infarction. In vitro , SPRR3 deficient VSMCs show reduced expression of procollagen type I, an event associated with Akt activation. SPRR3-deficient VSMCs also show increased expression of MMP2 transcripts, and aortic root lesions of Sprr3 -/- ApoE -/- mice have increased gelatinase activity consistent with MMP2 activation. Our data demonstrate that SPRR3 loss in ApoE -/- mice decreases VSMC survival and collagen I synthesis while increasing MMP2 synthesis and activity, resulting in atheroma instability with evidence of downstream myocardial infarction. Taken together the results present the Sprr3 -/- ApoE -/- mouse as an experimental model of plaque rupture. This model will be used for additional experimental studies including in vivo genetic modulation of the Akt pathway as well as in vitro studies to determine phenotypic outcome, i.e. coronary arterial lesions, myocardial infarction, VSMC survival and collagen synthesis. We hope to establish a mechanistic link between altered Akt signaling and matrix integrity in the context of atheroma rupture, as well as potentially use SPRR3 as a molecular marker which could lead to detection of plaque instability as well as therapeutic intervention methodologies.

scholarly journals Weighted Gene Co-Expression Network Analysis Combined with Machine Learning Validation to Identify Key Modules and Hub Genes Associated with SARS-CoV-2 Infection

2021 ◽  
Vol 10 (16) ◽  
pp. 3567
Author(s):  
Hassan Karami ◽  
Afshin Derakhshani ◽  
Mohammad Ghasemigol ◽  
Mohammad Fereidouni ◽  
Ebrahim Miri-Moghaddam ◽  
...  
Keyword(s):  
Network Analysis ◽  
Signaling Pathway ◽  
Drug Targets ◽  
Experimental Studies ◽  
A549 Cells ◽  
Enrichment Analysis ◽  
Type I ◽  
Hub Genes

The coronavirus disease-2019 (COVID-19) pandemic has caused an enormous loss of lives. Various clinical trials of vaccines and drugs are being conducted worldwide; nevertheless, as of today, no effective drug exists for COVID-19. The identification of key genes and pathways in this disease may lead to finding potential drug targets and biomarkers. Here, we applied weighted gene co-expression network analysis and LIME as an explainable artificial intelligence algorithm to comprehensively characterize transcriptional changes in bronchial epithelium cells (primary human lung epithelium (NHBE) and transformed lung alveolar (A549) cells) during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Our study detected a network that significantly correlated to the pathogenicity of COVID-19 infection based on identified hub genes in each cell line separately. The novel hub gene signature that was detected in our study, including PGLYRP4 and HEPHEL1, may shed light on the pathogenesis of COVID-19, holding promise for future prognostic and therapeutic approaches. The enrichment analysis of hub genes showed that the most relevant biological process and KEGG pathways were the type I interferon signaling pathway, IL-17 signaling pathway, cytokine-mediated signaling pathway, and defense response to virus categories, all of which play significant roles in restricting viral infection. Moreover, according to the drug–target network, we identified 17 novel FDA-approved candidate drugs, which could potentially be used to treat COVID-19 patients through the regulation of four hub genes of the co-expression network. In conclusion, the aforementioned hub genes might play potential roles in translational medicine and might become promising therapeutic targets. Further in vitro and in vivo experimental studies are needed to evaluate the role of these hub genes in COVID-19.

scholarly journals Transcriptional analysis of lung epithelial cells using WGCNA revealed the role of IRF9 and IFI6 genes in SARS-CoV-2 pathogenicity

2020 ◽  
Author(s):  
Hassan Karami ◽  
Afshin Derakhshani ◽  
Mohammad Fereidouni ◽  
Ebrahim Miri-Moghaddam ◽  
Behzad Baradaran ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Experimental Studies ◽  
A549 Cells ◽  
Lung Epithelial Cells ◽  
Transcriptional Analysis ◽  
Type I ◽  
Hub Genes

Abstract The coronavirus disease 2019 (COVID-19) outbreak is an ongoing global health emergence, but the pathogenesis remains unclear. Here, we applied weighted gene co-expression network analysis to comprehensively characterize transcriptional changes in bronchial epithelium cells (NHBE and A549 cells) during SARS-CoV-2 infection. Our analysis identified a network highly correlated to COVID-19 pathogenicity based on MX1, IFIT1, ISG15, IFI6, DDX60, IRF9, PARP9, PGLYRP4, IL36G, SAA2 and IL-8 hub genes. The results also indicated a unique transcriptional signatures of infected cells including IFI6 and IRF9 as novel gene candidates and suggested their prospective mechanism in COVID-19 pathogenesis. The result of hub genes enrichment showed that the most correlation topic in biological process and KEGG were type I interferon signaling pathway, IL-17 signaling pathway, cytokine mediated signaling pathway, and defense response to virus categories which all play significant roles in restricting viral infection. Also according to the drug-target network, we recognized 54 FDA-approved drug candidates for other indications could potentially use for the treatment of COVID-19 patients through regulation of six hub genes of the co-expression network. Our findings also showed that the 19 experimentally validated miRNAs regulated the co-expression network through 5 hub genes (SLC19A3, FAM13A, PLA2G16, and HRASLS5). In conclusion, these hub genes had potential roles in the translational medicine and might become promising therapeutic targets further in vitro and in vivo experimental studies are needed to evaluate the role of above mentioned genes in COVID-19.

Vacuoles Induced in Mammalian Cells by Helicobacter Cytotoxin are Acidic Organelles

1990 ◽  
Vol 48 (3) ◽  
pp. 168-169
Author(s):  
M. H. Chestnut ◽  
C. E. Catrenich
Keyword(s):  
Acridine Orange ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Ulcer Disease ◽  
Gastroduodenal Disease ◽  
H Pylori

Helicobacter pylori is a non-invasive, Gram-negative spiral bacterium first identified in 1983, and subsequently implicated in the pathogenesis of gastroduodenal disease including gastritis and peptic ulcer disease. Cytotoxic activity, manifested by intracytoplasmic vacuolation of mammalian cells in vitro, was identified in 55% of H. pylori strains examined. The vacuoles increase in number and size during extended incubation, resulting in vacuolar and cellular degeneration after 24 h to 48 h. Vacuolation of gastric epithelial cells is also observed in vivo during infection by H. pylori. A high molecular weight, heat labile protein is believed to be responsible for vacuolation and to significantly contribute to the development of gastroduodenal disease in humans. The mechanism by which the cytotoxin exerts its effect is unknown, as is the intracellular origin of the vacuolar membrane and contents. Acridine orange is a membrane-permeant weak base that initially accumulates in low-pH compartments. We have used acridine orange accumulation in conjunction with confocal laser scanning microscopy of toxin-treated cells to begin probing the nature and origin of these vacuoles.

Cytological changes induced by platinum-thymine in sarcoma-180 cells: Electron microscopy study

1990 ◽  
Vol 48 (3) ◽  
pp. 290-291
Author(s):  
Gustav Ofosu
Keyword(s):  
Mammalian Cells ◽  
Antitumor Agent ◽  
Microscopy Study ◽  
Electron Microscopy Study ◽  
Limiting Factor ◽  
Sarcoma 180 ◽  
Electron Microscopic ◽  
Cytological Changes

Platinum-thymine has been found to be a potent antitumor agent, which is quite soluble in water, and lack nephrotoxicity as the dose-limiting factor. The drug has been shown to interact with DNA and inhibits DNA, RNA and protein synthesis in mammalian cells in vitro. This investigation was undertaken to elucidate the cytotoxic effects of piatinum-thymine on sarcoma-180 cells in vitro ultrastructurally, Sarcoma-180 tumor bearing mice were treated with intraperitoneal injection of platinum-thymine 40mg/kg. A concentration of 60μg/ml dose of platinum-thymine was used in in vitro experiments. Treatments were at varying time intervals of 3, 7 and 21 days for in vivo experiments, and 30, 60 and 120 min., 6, 12, and 24th in vitro. Controls were not treated with platinum-thymine.Electron microscopic analyses of the treated cells in vivo and in vitro showed drastic cytotoxic effect.

Programmable in vitro Co-Encapsidation of Guest Proteins Inside Protein Nanocages

2018 ◽  
Author(s):  
Noor H. Dashti ◽  
Rufika S. Abidin ◽  
Frank Sainsbury
Keyword(s):  
Self Assembly ◽  
Mammalian Cells ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Super Resolution ◽  
Cell Engineering ◽  
Particle Analysis ◽  
Protein Cages

Bioinspired self-sorting and self-assembling systems using engineered versions of natural protein cages have been developed for biocatalysis and therapeutic delivery. The packaging and intracellular delivery of guest proteins is of particular interest for both <i>in vitro</i> and <i>in vivo</i> cell engineering. However, there is a lack of platforms in bionanotechnology that combine programmable guest protein encapsidation with efficient intracellular uptake. We report a minimal peptide anchor for <i>in vivo</i> self-sorting of cargo-linked capsomeres of the Murine polyomavirus (MPyV) major coat protein that enables controlled encapsidation of guest proteins by <i>in vitro</i> self-assembly. Using Förster resonance energy transfer (FRET) we demonstrate the flexibility in this system to support co-encapsidation of multiple proteins. Complementing these ensemble measurements with single particle analysis by super-resolution microscopy shows that the stochastic nature of co-encapsidation is an overriding principle. This has implications for the design and deployment of both native and engineered self-sorting encapsulation systems and for the assembly of infectious virions. Taking advantage of the encoded affinity for sialic acids ubiquitously displayed on the surface of mammalian cells, we demonstrate the ability of self-assembled MPyV virus-like particles to mediate efficient delivery of guest proteins to the cytosol of primary human cells. This platform for programmable co-encapsidation and efficient cytosolic delivery of complementary biomolecules therefore has enormous potential in cell engineering.

Peculiarities of the course of type I allergic reactions in patients with blood diseases

2020 ◽  
pp. 40-50
Author(s):  
A. Nikitina
Keyword(s):  
Search Engines ◽  
Anaphylactic Shock ◽  
Type I ◽  
Allergic Reactions ◽  
Common Cause ◽  
Blood Diseases ◽  
Treatment Regimens ◽  
Modern Methods

Analysis of literature data presented in search engines — Elibrary, PubMed, Cochrane — concerning the risk of developing type I allergic reactions in patients with blood diseases is presented. It is shown that the most common cause of type I allergic reactions is drugs included in the treatment regimens of this category of patients. The article presents statistics on the increase in the number of drug allergies leading to cases of anaphylactic shock in patients with blood diseases. Modern methods for the diagnosis of type I allergic reactions in vivo and in vitro are considered.

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