Mean Fluorescence Intensity Of Cells Research Articles

Effective delivery and selective insecticidal activity of double-stranded RNA via complexation with diblock copolymer varies with polymer block composition.

Chemical insecticides are an important tool to control damaging pest infestations. However, lack of species specificity, the rise of resistance and the demand for biological alternatives with improved ecotoxicity profiles means that chemicals with new modes of action are required. RNA interference (RNAi)-based strategies using double-stranded RNA (dsRNA) as a species-specific bio-insecticide offer an exquisite solution that addresses these issues. Many species, such as the fruit pest Drosophila suzukii, do not exhibit RNAi when dsRNA is orally administered due to degradation by gut nucleases and slow cellular uptake pathways. Thus, delivery vehicles that protect and deliver dsRNA are highly desirable. In this work, we demonstrate the complexation of D. suzukii-specific dsRNA for degradation of vha26 mRNA with bespoke diblock copolymers. We study the ex vivo protection of dsRNA against enzymatic degradation by gut enzymes, which demonstrates the efficiency of this system. Flow cytometry then investigates the cellular uptake of Cy3-labelled dsRNA, showing a 10-fold increase in the mean fluorescence intensity of cells treated with polyplexes. The polymer/dsRNA polyplexes induced a significant 87% decrease in the odds of survival of D. suzukii larvae following oral feeding only when formed with a diblock copolymer containing a long neutral block length (1:2 cationic block/neutral block). However, there was no toxicity when fed to the closely related Drosophila melanogaster. We provide evidence that dsRNA complexation with diblock copolymers is a promising strategy for RNAi-based species-specific pest control, but optimisation of polymer composition is essential for RNAi success. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Roles of CD28, CTLA4, and Inducible Costimulator in Acute Graft-versus-Host Disease in Mice

T cells deficient for CD28 have reduced ability to expand and survive, but still cause graft-versus-host disease (GVHD). Inducible costimulator (ICOS), a member of the CD28 family, is expressed on antigen-activated T cells and plays unique roles in T cell activation and effector function. We hypothesized that ICOS contributes to the development of GVHD in the absence of B7:CD28/CTLA4 costimulation. In this study, we evaluated the roles of CD28, CTLA4, and ICOS in the pathogenesis of acute GVHD after myeloablative allogeneic bone marrow transplantation. Unexpectedly, we found that blocking CD28 and CTLA4 signals using the clinically relevant reagent CTLA4-Ig increases the severity of GVHD mediated by CD4(+) T cells, and that such treatment does not add any benefit to the blockade of ICOS. In contrast, selectively blocking CD28 and ICOS, but not CTLA4, prevents GVHD more effectively than blocking either CD28 or ICOS alone. Taken together, these results indicate that CD28 and ICOS are synergistic in promoting GVHD, whereas the CTLA4 signal is required for T cell tolerance regardless of ICOS signaling. Thus, blocking CD28 and ICOS while sparing CTLA4 represents a promising approach for abrogating pathogenic T cell responses after allogeneic bone marrow transplantation.

Cytomegalovirus Promoter Up-regulation Is the Major Cause of Increased Protein Levels of Unstable Reporter Proteins after Treatment of Living Cells with Proteasome Inhibitors

Fluorescent unstable proteins obtained by the fusion of a fluorescent protein coding sequence with specific amino acid sequences that promote its fast degradation have become popular to gauge the activity of the ubiquitin/proteasome system in living cells. The steady-state levels of expression of these unstable proteins is low in agreement with their short half-lives, and they accumulate in the cell upon treatment with proteasome inhibitors. We show here that this accumulation is mainly due to transcriptional up-regulation of the cytomegalovirus promoter by proteasome inhibitors and mediated, at least in part, by AP1 transactivation. These simple facts put under quarantine conclusions reached about the activity of the ubiquitin/proteasome pathway in animal cells in culture or in transgenic mice, where popular cytomegalovirus-driven constructs are used, as transcriptional regulation of the expression of the reporter protein construct and not degradation of the unstable protein by the ubiquitin/proteasome system may contribute significantly to the interpretation of the results observed.

The antibacterial mechanism of carvacrol and thymol againstEscherichia coli

To investigate the antibacterial mechanism of carvacrol and thymol against Escherichia coli. The time-kill curve results showed that carvacrol and thymol at 200 mg l(-1) could inhibit the growth of E. coli. Flow cytometry and fluorescent dyes were used to explore the effect of two components on membrane permeability and membrane potential. In membrane permeability experiment, the mean fluorescence intensity of cells treated with 200 mg l(-1) carvacrol or thymol were lower than nonexposed cells. The ratio of red to green fluorescence intensity of DiOC2(3) reflected the change of membrane potential. Carvacrol and thymol at 200 mg l(-1) caused the ratio of red/green decreasing from 0.42 of control to 0.08 and 0.07, respectively. Carvacrol and thymol had desired antimicrobial effect on E. coli. The antibacterial effects were attributed to their ability to permeabilize and depolarize the cytoplasmic membrane. This study showed the potential use of flow cytometry as a suitable method to investigate the mode of antibacterial action of essential oil components.

Preparation of a folate-mediated tumor targeting ultraparamagnetic polymeric micelles and its in vitro experimental study

Objective To evaluate the tumor targeting characteristic of the Folate-SPIO-DOX-Micelles by in vitro studies,and to test the feasibility of monitor tumor targeting using it and clinical MRI.Methods The polymeric micelles,Folate-SPIO-DOXO-Micelles were prepared.The in vitro tumor cell targeting efficacy of these folate modified and DOX or SPIO-loaded micelles (Folate-SPIO-DOX-Micelles)was evaluated by observing the cellular uptake of micelles by human hepatic carcinoma cells(Bel 7402 cells) which overexpressed folate surface receptors. Cell suspensions were incubated with Folate-SPIO-DOXO-Micelles for 1 h.Prussian blue staining was performed to show intracellular irons.Flow cytometry was used to further quantify the cellular uptake of the nanoparticles into Bel 7402 cells.MRl was performed to show the signal intensity changes by using T2 WI sequences at a clinical 1.5 T MR system.Results Prussian blue staining showed much more intracellular iron in cells incubated with Folate-SPIO-DOX-Micelles than the cells incubated with the non-targeting SPIO-DOX-Micelles.As revealed by flow cytometry,the mean fluorescence intensity of cells in the folate group and the non-folate group were 117.88 and 46.33,respectively.The T2 signal intensity in MRI of cells treated with the folate targeting micelles decreased significantly (when the concentration of SPIO in cell culture medium was 5,10,20,40,and 80 μg/ml,respectively,T2 signal intensity decreased by -5.02%,-23.58%,-45.89%,-70.34%,and -92.41%,respectively).In contrast,T2 signal intensity did not show obvious decrease for cells treated with the folate-free micelles (when the concentration of SPIO in cell culture medium was at 5,10,20,40,and 80 μg/ml,respectively,T2 signal intensity decreased by -3.77%,-2.16%,-2.18%,-2.74% and -19.77%,respectively).Conclusion The polymeric micelles,Folate-SPIO-DOX-Micelles has good targeting ability to the hepatic carcinoma cells in vitro,and the cell targeting events of the micelles can be monitored by using a clinical MR scanner. Key words: Folic acid; Magnetic resonance imaging; Carcinoma,hepatocellular; Animals,laboratory

Assessment of antiepileptic drugs as substrates for canine P-glycoprotein

To determine whether antiepileptic drugs (AEDs) are substrates for canine P-glycoprotein (P-gp). Sample Population-OS2.4/Doxo cells (canine osteosarcoma cells induced via exposure to doxorubicin to highly express P-gp). Competitive inhibition of rhodamine 123 efflux from OS2.4/Doxo cells was used to determine whether AEDs were substrates for canine P-gp. Flow cytometry was used to quantify mean fluorescence intensity of cells treated with rhodamine alone and in combination with each experimental drug. Known P-gp substrate drugs ivermectin and cyclosporin A altered rhodamine efflux by 90% and 95%, respectively. Experimental drugs altered rhodamine efflux weakly (diazepam, gabapentin, lamotrigine, levetiracetam, and phenobarbital) or not at all (carbamazepine, felbamate, phenytoin, topirimate, and zonisamide). At clinically relevant doses, it appeared that AEDs were weak substrates (diazepam, gabapentin, lamotrigine, levetiracetam, and phenobarbital) or were not substrates (carbamazepine, felbamate, phenytoin, topirimate, and zonisamide) for canine P-gp. Therefore, it seems unlikely that efficacy of these AEDs is affected by P-gp expression at the blood-brain barrier in dogs.

Engineered T-cell receptor tetramers bind MHC-peptide complexes with high affinity

In this study we extend tetramerization technology to T-cell receptors (TCRs). We identified TCR alpha beta pairs in the absence of accessory molecules, ensuring isolation of high-affinity TCRs that maintain stable binding characteristics after tetramerization. Subtle changes in cognate peptide levels bound to the class I molecule were accurately reflected by parallel changes in the mean fluorescence intensity of cells that bound TCR tetramers, allowing us to accurately assess the binding affinity of a panel of peptides to major histocompatibility complex (MHC) class I. Using a TCR tetramer specific for the Mamu-A(*)01 allele, we identified animals expressing this restricting class I allele from a large cohort of outbred rhesus macaques. TCR tetramers should facilitate analysis of the MHC-peptide interface and, more generally, the design of immunotherapeutics and vaccines.

OmpD but not OmpC is involved in adherence ofSalmonella entericaserovar Typhimurium to human cells

Conflicting reports exist regarding the role of porins OmpC and OmpD in infections due to Salmonella enterica serovar Typhimurium. This study investigated the role of these porins in bacterial adherence to human macrophages and intestinal epithelial cells. ompC and ompD mutant strains were created by transposon mutagenesis using P22-mediated transduction of Tn10 and Tn5 insertions, respectively, into wild-type strain 14028. Fluorescein-labeled wild-type and mutant bacteria were incubated with host cells at various bacteria to cell ratios for 1 h at 37 degrees C and analyzed by flow cytometry. The mean fluorescence intensity of cells with associated wild-type and mutant bacteria was used to estimate the number of bacteria bound per host cell. Adherence was also measured by fluorescence microscopy. Neither assay showed a significant difference in binding of the ompC mutant and wild-type strains to the human cells. In contrast, the ompD mutant exhibited lowered binding to both cell types. Our findings suggest that OmpD but not OmpC is involved in the recognition of Salmonella serovar Typhimurium by human macrophages and intestinal epithelial cells.

Effects of Exogenous Nitric Oxide and Hyperoxia on Lung Fibroblast Viability and DNA Fragmentation

Effective lung repair after acute injury requires elimination of proliferating mesenchymal and inflammatory cells without inducing an acute inflammatory response or disturbing concomitant repair of lung microvasculature. Previous studies have shown that endogenous NO regulates programmed cell death in fibroblasts and can modulate wound fibroblast synthetic function. We hypothesized that exposure of human lung fibroblasts to NO gas would decrease viability and induce apoptotic cell death. Primary cultures of normal human lung fibroblasts were exposed for 4 h to room air (RA), 80% oxygen, NO (at either 20 or 50 ppm) blended with RA, or NO blended with 80% O2, then incubated for 24 to 72 h. Cell viability was determined by fluorescence viability/cytotoxicity assay and DNA fragmentation by TUNEL assay. Peroxynitrite formation was assessed using immunoblotting for S-nitrosotyrosine. NO plus O2 induced significant cell death at 20 and 50 ppm NO when compared to either RA or O2 alone at both 24 and 72 h (p < 0.05). Incubation with superoxide dismutase (SOD), catalase (CAT) or SOD + CAT significantly decreased cell death in fibroblasts treated with NO20/O2 and NO50/O2 compared with controls (p < 0.05). NO20/O2 and NO50/O2 exposure significantly increased TUNEL mean fluorescence intensity (MFI), consistent with increased DNA fragmentation, compared to RA at 24 and 72 h (p < 0.05). Antioxidants decreased MFI in cells exposed to NO20/O2 (CAT and SOD + CAT) compared to controls at 24 h (p < 0.05). Western blot analysis for S-nitrosotyrosine showed increased signal intensity in fibroblasts exposed to NO at 20 and 50 ppm plus O2 compared to RA or O2 alone. Incubation with SOD + CAT reduced signal intensity for peroxynitrite in cells exposed to NO20/O2. We conclude that NO in hyperoxic conditions induces fibroblast cell death and DNA fragmentation, which could be partially mediated by peroxynitrite synthesis.

A differentiation antigen of human large luteal cells in corpora lutea of the menstrual cycle and early pregnancy.

To investigate the mechanism(s) of luteal cell differentiation, we raised a monoclonal antibody, HCL-1, against human large luteal cells. The antigen was undetectable in growing and preovulatory follicles by immunohistochemistry. The antigen was initially detected on centrally located luteinizing granulosa cells during CL formation. During the midluteal phase, the antigen was expressed at high levels on large luteal cells. Large luteal cells in the CL during late luteal phase and early pregnancy also expressed high levels of HCL-1 antigen, whereas small luteal cells at any stage of the CL did not. Granulosa cells in some atretic follicles weakly expressed HCL-1 antigen. Immunofluorescence staining of enzymatically dispersed luteal cells from human mature CL revealed that HCL-1 antigen was present on the cell surface of large luteal cells. Human granulosa cells isolated from patients who had undergone in vitro fertilization treatment were cultured for 7 days. Indirect immunofluorescence detected HCL-1 antigen on only a few granulosa cells after culture for 1 day and on almost all granulosa cells after culture for 7 days. Flow cytometry of 7-day-cultured cells showed that the percentages of positivity for HCL-1 antigen as well as the mean fluorescence intensities of granulosa cells cultured with hCG (1 IU/ml) were significantly lower than those of the controls (without treatment) (44.3 +/- 3.2% vs. 62.9 +/- 4.0%. p < 0.01; 60.9 +/- 6.7 vs. 82.1 +/- 7.6, p < 0.05). By contrast, the mean fluorescence intensities of cells cultured with interleukin- 1 alpha (10 ng/ml, 105 +/- 6.3, p < 0.05) and tumor necrosis factor alpha (10 ng/ml, 112 +/- 11.2, p < 0.05) were significantly higher than those of controls. These findings showed that the cell surface antigen detected on human large luteal cells by HCL-1 was differentiation-related, and that large luteal cells in the CL of pregnancy are derived from granulosa cells via large luteal cells in the CL of the menstrual cycle.