scholarly journals Organotypic Epithelial Raft Cultures as a Model for Evaluating Compounds against Alphaherpesviruses

2005 ◽  
Vol 49 (11) ◽  
pp. 4671-4680 ◽  
Author(s):  
Graciela Andrei ◽  
Joost van den Oord ◽  
Pierre Fiten ◽  
Ghislain Opdenakker ◽  
Chris De Wolf-Peeters ◽  
...  
Keyword(s):  
Antiviral Agents ◽  
Plaque Assay ◽  
Three Dimensional ◽  
Liquid Interface ◽  
Dependent Manner ◽  
Wild Type ◽  
Organotypic Cultures ◽  
Concentration Dependent Manner ◽  
Raft Culture ◽  
Air Liquid Interface

ABSTRACT The course of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) and varicella-zoster virus (VZV) infections in squamous epithelial cells cultured in a three-dimensional organotypic raft culture was tested. In these raft cultures, normal human keratinocytes isolated from neonatal foreskins grown at the air-liquid interface stratified and differentiated, reproducing a fully differentiated epithelium. Typical cytopathic changes identical to those found in the squamous epithelium in vivo, including ballooning and reticular degeneration with the formation of multinucleate cells, were observed throughout the raft following infection with HSV and VZV at different times after lifting the cultures to the air-liquid interface. For VZV, the aspects of the lesions depended on the stage of differentiation of the organotypic cultures. The activity of reference antiviral agents, acyclovir (ACV), penciclovir (PCV), brivudin (BVDU), foscarnet (PFA), and cidofovir (CDV), was evaluated against wild-type and thymidine kinase (TK) mutants of HSV and VZV in the raft cultures. ACV, PCV, and BVDU protected the epithelium against cytopathic effect induced by wild-type viruses in a concentration-dependent manner, while treatment with CDV and PFA proved protective against the cytodestructive effects induced by both TK+ and TK− strains. The quantification of the antiviral effects in the rafts were accomplished by measuring viral titers by plaque assay for HSV and by measuring viral DNA load by real-time PCR for VZV. A correlation between the degree of protection as determined by histological examination and viral quantification could be demonstrated The three-dimensional epithelial raft culture represents a novel model for the study of antiviral agents active against HSV and VZV. Since no animal model is available for the evaluation of antiviral agents against VZV, the organotypic cultures may be considered a model to evaluate the efficacy of new anti-VZV antivirals before clinical trials.

scholarly journals A robust SARS-CoV-2 replication model in primary human epithelial cells at the air liquid interface to assess antiviral agents

2021 ◽  
pp. 105122
Author(s):  
Thuc Nguyen Dan Do ◽  
Kim Donckers ◽  
Laura Vangeel ◽  
Arnab K. Chatterjee ◽  
Philippe A. Gallay ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Antiviral Agents ◽  
Liquid Interface ◽  
Human Epithelial Cells ◽  
Replication Model ◽  
Air Liquid Interface

scholarly journals Global Expression Profiling and Physiological Characterization of Corynebacterium glutamicum Grown in the Presence of l-Valine

2003 ◽  
Vol 69 (5) ◽  
pp. 2521-2532 ◽  
Author(s):  
C. Lange ◽  
D. Rittmann ◽  
V. F. Wendisch ◽  
M. Bott ◽  
H. Sahm
Keyword(s):  
Corynebacterium Glutamicum ◽  
Expression Profiling ◽  
Large Subunit ◽  
Mrna Level ◽  
Acetohydroxyacid Synthase ◽  
Limiting Factor ◽  
Unexpected Result ◽  
Dependent Manner ◽  
Wild Type ◽  
Concentration Dependent Manner

ABSTRACT Addition of l-valine (50 to 200 mM) to glucose minimal medium had no effect on the growth of wild-type Corynebacterium glutamicum ATCC 13032 but inhibited the growth of the derived valine production strain VAL1 [13032 ΔilvA ΔpanBC(pJC1ilvBNCD)] in a concentration-dependent manner. In order to explore this strain-specific valine effect, genomewide expression profiling was performed using DNA microarrays, which showed that valine caused an increased ilvBN mRNA level in VAL1 but not in the wild type. This unexpected result was confirmed by an increased cellular level of the ilvB protein product, i.e., the large subunit of acetohydroxyacid synthase (AHAS), and by an increased AHAS activity of valine-treated VAL1 cells. The conclusion that valine caused the limitation of another branched-chain amino acid was confirmed by showing that high concentrations of l-isoleucine could relieve the valine effect on VAL1 whereas l-leucine had the same effect as valine. The valine-caused isoleucine limitation was supported by the finding that the inhibitory valine effect was linked to the ilvA deletion that results in isoleucine auxotrophy. Taken together, these results implied that the valine effect is caused by competition for uptake of isoleucine by the carrier BrnQ, which transports all branched-chained amino acids. Indeed, valine inhibition could also be relieved by supplementing VAL1 with the dipeptide isoleucyl-isoleucine, which is taken up by a dipeptide transport system rather than by BrnQ. Interestingly, addition of external valine stimulated valine production by VAL1. This effect is most probably due to a reduced carbon usage for biomass production and to the increased expression of ilvBN, indicating that AHAS activity may still be a limiting factor for valine production in the VAL1 strain.

scholarly journals Preliminary Study of In Vitro Three-Dimensional Skin Model Using an Ovine Collagen Type I Sponge Seeded with Co-Culture Skin Cells: Submerged versus Air-Liquid Interface Conditions

Polymers ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 2784
Author(s):  
Mh Busra Fauzi ◽  
Zahra Rashidbenam ◽  
Aminuddin Bin Saim ◽  
Ruszymah Binti Hj Idrus
Keyword(s):  
Collagen Type ◽  
Three Dimensional ◽  
Collagen Type I ◽  
Liquid Interface ◽  
Type I ◽  
Skin Model ◽  
Skin Cells ◽  
Air Liquid Interface ◽  
In Vitro Skin Model

Three-dimensional (3D) in vitro skin models have been widely used for cosmeceutical and pharmaceutical applications aiming to reduce animal use in experiment. This study investigate capability of ovine tendon collagen type I (OTC-I) sponge suitable platform for a 3D in vitro skin model using co-cultured skin cells (CC) containing human epidermal keratinocytes (HEK) and human dermal fibroblasts (HDF) under submerged (SM) and air-liquid interface (ALI) conditions. Briefly, the extracted OTC-I was freeze-dried and crosslinked with genipin (OTC-I_GNP) and carbodiimide (OTC-I_EDC). The gross appearance, physico-chemical characteristics, biocompatibility and growth profile of seeded skin cells were assessed. The light brown and white appearance for the OTC-I_GNP scaffold and other groups were observed, respectively. The OTC-I_GNP scaffold demonstrated the highest swelling ratio (~1885%) and water uptake (94.96 ± 0.14%). The Fourier transformation infrared demonstrated amide A, B and I, II and III which represent collagen type I. The microstructure of all fabricated sponges presented a similar surface roughness with the presence of visible collagen fibers and a heterogenous porous structure. The OTC-I_EDC scaffold was more toxic and showed the lowest cell attachment and proliferation as compared to other groups. The micrographic evaluation revealed that CC potentially formed the epidermal- and dermal-like layers in both SM and ALI that prominently observed with OTC-I_GNP compared to others. In conclusion, these results suggest that OTC_GNP could be used as a 3D in vitro skin model under ALI microenvironment.

scholarly journals Role ofCaulobacterCell Surface Structures in Colonization of the Air-Liquid Interface

2019 ◽  
Vol 201 (18) ◽  
Author(s):  
Aretha Fiebig
Keyword(s):  
Cell Surface ◽  
Caulobacter Crescentus ◽  
Three Dimensional ◽  
Liquid Interface ◽  
Type Iv Pili ◽  
Surface Structures ◽  
Aquatic Environments ◽  
Content Type ◽  
Type Iv ◽  
Air Liquid Interface

ABSTRACTIn aquatic environments,Caulobacterspp. can be found at the boundary between liquid and air known as the neuston. I report an approach to study temporal features ofCaulobacter crescentuscolonization and pellicle biofilm development at the air-liquid interface and have defined the role of cell surface structures in this process. At this interface,C. crescentusinitially forms a monolayer of cells bearing a surface adhesin known as the holdfast. When excised from the liquid surface, this monolayer strongly adheres to glass. The monolayer subsequently develops into a three-dimensional structure that is highly enriched in clusters of stalked cells known as rosettes. As this pellicle film matures, it becomes more cohesive and less adherent to a glass surface. A mutant strain lacking a flagellum does not efficiently reach the surface, and strains lacking type IV pili exhibit defects in organization of the three-dimensional pellicle. Strains unable to synthesize the holdfast fail to accumulate at the boundary between air and liquid and do not form a pellicle. Phase-contrast images support a model whereby the holdfast functions to trapC. crescentuscells at the air-liquid boundary. Unlike the holdfast, neither the flagellum nor type IV pili are required forC. crescentusto partition to the air-liquid interface. While it is well established that the holdfast enables adherence to solid surfaces, this study provides evidence that the holdfast has physicochemical properties that allow partitioning of nonmotile mother cells to the air-liquid interface and facilitate colonization of this microenvironment.IMPORTANCEIn aquatic environments, the boundary at the air interface is often highly enriched with nutrients and oxygen. Colonization of this niche likely confers a significant fitness advantage in many cases. This study provides evidence that the cell surface adhesin known as a holdfast enablesCaulobacter crescentusto partition to and colonize the air-liquid interface. Additional surface structures, including the flagellum and type IV pili, are important determinants of colonization and biofilm formation at this boundary. Considering that holdfast-like adhesins are broadly conserved inCaulobacterspp. and other members of the diverse classAlphaproteobacteria, these surface structures may function broadly to facilitate colonization of air-liquid boundaries in a range of ecological contexts, including freshwater, marine, and soil ecosystems.

Increased susceptibility to exacerbated liver injury in hypercholesterolemic ApoE-deficient mice: potential involvement of oxysterols

2009 ◽  
Vol 296 (3) ◽  
pp. G553-G562 ◽  
Author(s):  
Natàlia Ferré ◽  
Marcos Martínez-Clemente ◽  
Marta López-Parra ◽  
Ana González-Périz ◽  
Raquel Horrillo ◽  
...  
Keyword(s):  
Liver Injury ◽  
Smooth Muscle Actin ◽  
Cell Types ◽  
Dependent Manner ◽  
Wild Type ◽  
Sod Activity ◽  
Concentration Dependent Manner ◽  
Oil Red O Staining ◽  
Cytochrome P 450 ◽  
Oxidized Products

The contribution of metabolic factors to the severity of liver disease is not completely understood. In this study, apolipoprotein E-deficient (ApoE−/−) mice were evaluated to define potential effects of hypercholesterolemia on the severity of carbon tetrachloride (CCl4)-induced liver injury. Under baseline conditions, hypercholesterolemic ApoE−/− mice showed increased hepatic oxidative stress (SOD activity/4-hydroxy-2-nonenal immunostaining) and higher hepatic TGF-β1, MCP-1, and TIMP-1 expression than wild-type control mice. After CCl4 challenge, ApoE−/− mice exhibited exacerbated steatosis (Oil Red O staining), necroinflammation (hematoxylin-eosin staining), macrophage infiltration (F4/80 immunohistochemistry), and fibrosis (Sirius red staining and α-smooth muscle actin immunohistochemistry) and more severe liver injury [alanine aminotransferase (ALT) and aspartate aminotransferase] than wild-type controls. Direct correlations were identified between serum cholesterol and hepatic steatosis, fibrosis, and ALT levels. These changes did not reflect the usual progression of the disease in ApoE−/− mice, since exacerbated liver injury was not present in untreated age-paired ApoE−/− mice. Moreover, hepatic cytochrome P-450 expression was unchanged in ApoE−/− mice. To explore potential mechanisms, cell types relevant to liver pathophysiology were exposed to selected cholesterol-oxidized products. Incubation of hepatocytes with a mixture of oxysterols representative of those detected by GC-MS in livers from ApoE−/− mice resulted in a concentration-dependent increase in total lipoperoxides and SOD activity. In hepatic stellate cells, oxysterols increased IL-8 secretion through a NF-κB-independent mechanism and upregulated TIMP-1 expression. In macrophages, oxysterols increased TGF-β1 secretion and MCP-1 expression in a concentration-dependent manner. Oxysterols did not compromise cell viability. Taken together, these findings demonstrate that hypercholesterolemic mice are sensitized to liver injury and that cholesterol-derived products (i.e., oxysterols) are able to induce proinflammatory and profibrogenic mechanisms in liver cells.

Effects of local anesthetics on Na+ channels containing the equine hyperkalemic periodic paralysis mutation

1998 ◽  
Vol 275 (2) ◽  
pp. C389-C400 ◽  
Author(s):  
Rajan L. Sah ◽  
Robert G. Tsushima ◽  
Peter H. Backx
Keyword(s):  
Local Anesthetics ◽  
Time Course ◽  
Slow Component ◽  
Periodic Paralysis ◽  
Dependent Manner ◽  
Wild Type ◽  
Hyperkalemic Periodic Paralysis ◽  
Concentration Dependent Manner ◽  
Drug Concentrations ◽  
Recovery From Inactivation

We examined the ability of local anesthetics to correct altered inactivation properties of rat skeletal muscle Na+channels containing the equine hyperkalemic periodic paralysis (eqHPP) mutation when expressed in Xenopusoocytes. Increased time constants of current decay in eqHPP channels compared with wild-type channels were restored by 1 mM benzocaine but were not altered by lidocaine or mexiletine. Inactivation curves, which were determined by measuring the dependence of the relative peak current amplitude after depolarization to −10 mV on conditioning prepulse voltages, could be shifted in eqHPP channels back toward that observed for wild-type (WT) channels using selected concentrations of benzocaine, lidocaine, and mexiletine. Recovery from inactivation at −80 mV (50-ms conditioning pulse) in eqHPP channels followed a monoexponential time course and was markedly accelerated compared with wild-type channels (τWT= 10.8 ± 0.9 ms; τeqHPP= 2.9 ± 0.4 ms). Benzocaine slowed the time course of recovery (τeqHPP,ben = 9.6 ± 0.4 ms at 1 mM) in a concentration-dependent manner. In contrast, the recovery from inactivation with lidocaine and mexiletine had a fast component (τfast,lid = 3.2 ± 0.2 ms; τfast,mex = 3.1 ± 0.2 ms), which was identical to the recovery in eqHPP channels without drug, and a slow component (τslow,lid = 1,688 ± 180 ms; τslow,mex = 2,323 ± 328 ms). The time constant of the slow component of the recovery from inactivation was independent of the drug concentration, whereas the fraction of current recovering slowly depended on drug concentrations and conditioning pulse durations. Our results show that local anesthetics are generally incapable of fully restoring normal WT behavior in inactivation-deficient eqHPP channels.

scholarly journals Control of Formation and Cellular Detachment from Shewanella oneidensis MR-1 Biofilms by Cyclic di-GMP

2006 ◽  
Vol 188 (7) ◽  
pp. 2681-2691 ◽  
Author(s):  
Kai M. Thormann ◽  
Stefanie Duttler ◽  
Renee M. Saville ◽  
Mamoru Hyodo ◽  
Soni Shukla ◽  
...  
Keyword(s):  
Ectopic Expression ◽  
Three Dimensional ◽  
Shewanella Oneidensis ◽  
Dependent Manner ◽  
Gene Encoding ◽  
Concentration Dependent Manner ◽  
Environmental Perturbations ◽  
Environmental Trigger ◽  
Exclusive Processes ◽  
Stability And Resilience

ABSTRACT Stability and resilience against environmental perturbations are critical properties of medical and environmental biofilms and pose important targets for their control. Biofilm stability is determined by two mutually exclusive processes: attachment of cells to and detachment from the biofilm matrix. Using Shewanella oneidensis MR-1, an environmentally versatile, Fe(III) and Mn(IV) mineral-reducing microorganism, we identified mxdABCD as a new set of genes essential for formation of a three-dimensional biofilm. Molecular analysis revealed that mxdA encodes a cyclic bis(3′,5′)guanylic acid (cyclic di-GMP)-forming enzyme with an unusual GGDEF motif, i.e., NVDEF, which is essential for its function. mxdB encodes a putative membrane-associated glycosyl transferase. Both genes are essential for matrix attachment. The attachment-deficient phenotype of a ΔmxdA mutant was rescued by ectopic expression of VCA0956, encoding another diguanylate cyclase. Interestingly, a rapid cellular detachment from the biofilm occurred upon induction of yhjH, a gene encoding an enzyme that has been shown to have phosphodiesterase activity. In this way, it was possible to bypass the previously identified sudden depletion of molecular oxygen as an environmental trigger to induce biofilm dissolution. We propose a model for c-di-GMP as a key intracellular regulator for controlling biofilm stability by shifting the state of a biofilm cell between attachment and detachment in a concentration-dependent manner.

Chloroplast division protein ARC3 acts on FtsZ2 by preventing filament bundling and enhancing GTPase activity

2018 ◽  
Vol 475 (1) ◽  
pp. 99-115 ◽  
Author(s):  
Rahamthulla S. Shaik ◽  
Min Woo Sung ◽  
Stanislav Vitha ◽  
Andreas Holzenburg
Keyword(s):  
Sequence Similarity ◽  
Three Dimensional ◽  
Chloroplast Division ◽  
Particle Analysis ◽  
Temperature Sensitive ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Z Ring

Chloroplasts evolved from cyanobacterial endosymbiotic ancestors and their division is a complex process initiated by the assembly of cytoskeletal FtsZ (Filamentous temperature sensitive Z) proteins into a ring structure at the division site (Z-ring). The cyanobacterial Z-ring positioning system (MinCDE proteins) is also conserved in chloroplasts, except that MinC was lost and replaced by the eukaryotic ARC3 (accumulation and replication of chloroplasts). Both MinC and ARC3 act as negative regulators of FtsZ assembly, but ARC3 bears little sequence similarity with MinC. Here, light scattering assays, co-sedimentation, GTPase assay and transmission electron microscopy in conjunction with single-particle analysis have been used to elucidate the structure of ARC3 and its effect on its main target in chloroplast division, FtsZ2. Analysis of FtsZ2 in vitro assembly reactions in the presence and absence of GMPCPP showed that ARC3 promotes FtsZ2 debundling and disassembly of existing filaments in a concentration-dependent manner and requires GTP hydrolysis. Three-dimensional reconstruction of ARC3 revealed an almost circular molecule in which the FtsZ-binding N-terminus and the C-terminal PARC6 (paralog of ARC6)-binding MORN (Membrane Occupation and Recognition Nexus) domain are in close proximity and suggest a model for PARC6-enabled binding of ARC3 to FtsZ2. The latter is corroborated by in vivo data.

Factor XIII cross-links fibrin(ogen) independent of fibrin polymerization in experimental acute liver injury

Blood ◽  
2021 ◽  
Author(s):  
Lauren G. Poole ◽  
Anna K Kopec ◽  
Dafna Groeneveld ◽  
Asmita Pant ◽  
Kevin Baker ◽  
...  
Keyword(s):  
Liver Injury ◽  
Acute Liver Injury ◽  
Factor Xiiia ◽  
Cross Linking ◽  
Dependent Manner ◽  
Wild Type ◽  
Concentration Dependent Manner ◽  
Fibrin Polymerization ◽  
Apap Overdose ◽  
Injured Liver

Intravascular fibrin clot formation follows a well-ordered series of reactions catalyzed by thrombin cleavage of fibrinogen leading to fibrin polymerization and cross-linking by factor XIIIa (FXIIIa). Extravascular fibrin(ogen) deposits are observed in injured tissues; however, the mechanisms regulating fibrin(ogen) polymerization and cross-linking in this setting are unclear. The objective of this study was to determine the mechanisms of fibrin polymerization and cross-linking in acute liver injury induced by acetaminophen (APAP) overdose. Hepatic fibrin(ogen) deposition and cross-linking were measured following APAP overdose in wild-type mice, mice lacking the catalytic subunit of FXIII (FXIII-/-), and in FibAEK mice, which express mutant fibrinogen insensitive to thrombin-mediated fibrin polymer formation. Hepatic fibrin(ogen) deposition was similar in APAP-challenged wild-type and FXIII-/- mice yet cross-linking of hepatic fibrin(ogen) was dramatically reduced (>90%) by FXIII deficiency. Surprisingly, hepatic fibrin(ogen) deposition and cross-linking were only modestly reduced in APAP-challenged FibAEK mice, suggesting that in the APAP-injured liver fibrin polymerization is not strictly required for the extravascular deposition of cross-linked fibrin(ogen). We hypothesized that the oxidative environment in the injured liver, containing high levels of reactive mediators (e.g., peroxynitrite), modifies fibrin(ogen) such that fibrin polymerization is impaired without impacting FXIII-mediated cross-linking. Notably, fibrin(ogen) modified with 3-nitrotyrosine adducts was identified in the APAP-injured liver. In biochemical assays, peroxynitrite inhibited thrombin-mediated fibrin polymerization in a concentration-dependent manner without affecting fibrin(ogen) cross-linking over time. These studies depict a unique pathology wherein thrombin-catalyzed fibrin polymerization is circumvented to allow tissue deposition and FXIII-dependent fibrin(ogen) cross-linking.

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