Changes in the permeability of blood brain barrier and endothelial cell damage after cerebral ischemia

OBJECTIVE： To investigate the effect of endothelial cells on the permeability of blood brain barrier （BBB） after brain injury and its effect mechanism. DATA SOURCES： We searched for the articles of permeability of BBB and endothelial cell injury after brain is- chemia, which were published between January 1982 and December 2005, with the key words of ＂cerebral ischemia damage,blood brain barrier （ BBB）,permeability,effect of endothelial cell （EC） and its variation mechanism＂in English. STUDY SELECTION： The materials were primarily selected. The articles related to the changes in the permeability of BBB and the effect of endothelial cells as well as the change mechanism after cerebral ischemia damage were chosen. Repetitive studies or review articles were excluded. DATA EXTRACTION： Totally 55 related articles were collected, and 35 were excluded due to repetitive or review articles, finally 20 articles were involved. DATA SYNTHESIS： The content or viewpoints of involved literatures were analyzed. Cerebral ischemia had damage for endothelial cells, such as the inflow of a lot of Ca2^＋, the production of nitrogen monoxide and oxygen free radical, and aggravated destruction of BBB. After acceptors of inflammatory mediators on cerebrovascular endothelial cell membrane, such as histamine, bradykinin , 5-hydroxytryptamine and so on are activated, endothelial cells shrink and the permeability of BBB increases. Its mechanism involves in the inflow of extracellular Ca^＋2and the release of intracellular Ca^2＋ in the cells. Glycocalyx molecule on the surface of endothelial cell, having structural polytropy, is the determinative factor of the permeability of BBB. VEGF, intensively increasing the vasopermeability and mainly effecting on postcapillary vein and veinlet, is the strongest known blood vessel permeation reagent. Its chronic overexpression in the brain can lead the destruction of BBB. CONCLUSION： The injury of endothelial cell participants in the pathological mechanism of BBB destruction after cerebral ischemla.

TNF-a induce the F-actin arrangement and permeability increase in endothelial cells by RhoA-ERK1/2 pathway

Background This study aimed to determine the effects of tumor necrosis factor(TNF-a) on endothelial cytoskeleton morphology and permeability,and to detect the underlying signaling mechanisms involved in these responses. Methods Cultured endothelial cells(ECs) were exposed to TNF-a,and EC cytoskeletal changes were evaluated by observing fluorescence of F-actin following ligation with labeled antibodies.Endothelial permeability was detected by measuring the flux of HRP-albumin across the EC monolayers.To explore the signaling pathways behind TNF-a-induced EC alteration, ECs were treated with either the RhoGTPase inhibitor Y27632 or the MAPK inhibitors PD98059 and SB203580 before TNF-a administration.To further elucidate possible involvement of the RhoA and ERK pathways in TNF-induced EC changes,retrovirus-carried recombinant dominant-negative forms and constitutive-activative forms of RhoA,namely T19NRhoA and Q63LRhoA,were pre-infect-ed into ECs prior to TNF-a exposure.Results TNF-a induced F-actin cytoskeleton rearrangement,as well as EC hyperpermeability in a dose and time-dependent manner.The effects were attenuated in cells pretreated with Y27632 or PD98059,respectively.EC pre-infection with T19NRhoA also alleviated the effects of TNF-a.Furthermore,retrovirus-mediated administration of activated forms of Q63LRhoA alone induced rearrangement of F-actin and hyperpermeability as well as induced the activation of pERK.Conclusions These results indicate that RhoA-ERK/MAPK signal pathway play important roles in the mediation of TNF-a induced EC barrier dysfunction associated with morphological changes of the Factin.

Effects of exogenous Ca^(2+) on the membrane permeability, MDA and SH group content of Alexandrium sp. LC3 under surfactant stress

The effect of Ca 2+ on the removal of Alexandrium sp. LC3 under HDTMAB stress was investigated. The results showed that the toxic effect of HDTMAB on Alexandrium sp. LC3 was significantly reduced in the presence of Ca 2+, especially under 4 mmol/L of Ca 2+. To understand the underlying mechanism, the SH group and MDA content of the cell membrane and membrane permeability were measured. It was found that the SH content of cell member increased, the MDA content and membrane permeability decreased when Alexandrium sp. was treated with Ca 2+ and HDTMAB complex, compared with using HDTMAB only. The data suggested that Ca 2+ might promote HDTMAB stress resistance of Alexandrium sp. LC3 by reducing the permeability and increasing the stability of cell membrane.

Effective Penetration of Cell-permeable Peptide Mimic of Tyrosine Residue 654 Domain of β-catenin into Human Renal Tubular Epithelial Cells

Phosphorylation of β-catenin tyrosine residue 654 plays an important role in the epithelial to myofibroblast transition （EMT）. Introducing mimic peptide of tyrosine residue 654 domain of β-catenin into cells may influence phosphorylation of β-catenin tyrosine residue 654. To deliver this mimic peptide into renal epithelial cells, we used penetratin as a vector, which is a novel cell permeable peptide, to deliver hydrophilic molecules into cells. A tyrosine 654 residue domain mimic peptide of β-catenin （PM） with fused penetratin was constructed, purified and then detected for the penetration of the mimic peptide into human renal tubular epithelial cells （HK-2）. The results showed that purified fusion mimic peptide could efficiently and rapidly translocate into human renal tubular epithelial cells. It is concluded that a cell-permeable peptides mimic of tyrosine residue 654 domain of β-catenin was successfully obtained, which may provide a useful reagent for interfering the human renal tubular epithelial-mesenchymal transition.

Non-Thermal Biological Effect of Graphene Far-Infrared Ray on Saccharomyces cerevisiae Cells

Graphene materials can emit far-infrared ray, but the biological effects of graphene far-infrared ray have not been studied. Furthermore, the non-thermal biological effect of far-infrared ray on organism has not been systematically studied independently of the thermal effect. The purpose of this study was to investigate the non-thermal biological effect of graphene far-infrared ray (gFIR) on Saccharomyces cerevisiae cells. In this work, stringent control of the cultivation conditions was carried out to ensure the stability and constancy of the culture and its temperature. Flow cytometry was used to detect the non-thermal effect of gFIR irradiation on cell membrane permeability, mitochondrial membrane potential (MMP) and intracellular reactive oxygen species (ROS) content. Compared with the control group, cell membrane permeability of the gFIR exposure cells decreased by 4.7%, MMP increased by 16% and intracellular ROS reduced by 10.7%. The results revealed the valuable features of the non-thermal biological effect of gFIR on Saccharomyces cerevisiae cells, and the further analysis demonstrated that graphene far-infrared materials should have great application value in disease prevention and health promotion.

Involvement of CRF2 signaling in enterocyte differentiation

To determine the role of corticotropin releasing factor receptor (CRF2) in epithelial permeability and enterocyte cell differentiation.METHODSFor this purpose, we used rat Sprague Dawley and various colon carcinoma cell lines (SW620, HCT8R, HT-29 and Caco-2 cell lines). Expression of CRF2 protein was analyzed by fluorescent immunolabeling in normal rat colon and then by western blot in dissociated colonic epithelial cells and in the lysates of colon carcinoma cell lines or during the early differentiation of HT-29 cells (ten first days). To assess the impact of CRF2 signaling on colonic cell differentiation, HT-29 and Caco-2 cells were exposed to Urocortin 3 recombinant proteins (Ucn3, 100 nmol/L). In some experiments, cells were pre-exposed to the astressin 2b (A2b) a CRF2 antagonist in order to inhibit the action of Ucn3. Intestinal cell differentiation was first analyzed by functional assays: the trans-cellular permeability and the para-cellular permeability were determined by Dextran-FITC intake and measure of the transepithelial electrical resistance respectively. Morphological modifications associated to epithelial dysfunction were analyzed by confocal microscopy after fluorescent labeling of actin (phaloidin-TRITC) and intercellular adhesion proteins such as E-cadherin, p120ctn, occludin and ZO-1. The establishment of mature adherens junctions (AJ) was monitored by following the distribution of AJ proteins in lipid raft fractions, after separation of cell lysates on sucrose gradients. Finally, the mRNA and the protein expression levels of characteristic markers of intestinal epithelial cell (IEC) differentiation such as the transcriptional factor krüppel-like factor 4 (KLF4) or the dipeptidyl peptidase IV (DPPIV) were performed by RT-PCR and western blot respectively. The specific activities of DPPIV and alkaline phosphatase (AP) enzymes were determined by a colorimetric method.RESULTSCRF2 protein is preferentially expressed in undifferentiated epithelial cells from the crypts of colon and in human colon carcinoma cell lines. Furthermore, CRF2 expression is down regulated according to the kinetic of HT-29 cell differentiation. By performing functional assays, we found that Ucn3-induced CRF2 signaling alters both para- and trans-cellular permeability of differentiated HT-29 and Caco-2 cells. These effects are partly mediated by Ucn3-induced morphological changes associated with the disruption of mature AJ in HT-29 cells and tight junctions (TJ) in Caco-2 cells. Ucn3-mediated activation of CRF2 decreases mRNA and protein expression levels of KLF4 a transcription factor involved in IEC differentiation. This signaling is correlated to a down-regulation of key IEC markers such as DPPIV and AP, at both transcriptional and post-transcriptional levels.CONCLUSIONOur findings suggest that CRF2 signaling could modulate IEC differentiation. These mechanisms could be relevant to the stress induced epithelial alterations found in inflammatory bowel diseases.

The remarkable effect of amino hydrogen on membrane permeability and organelle staining of 1,8-naphthalimide dyes

Membrane permeability and intracellular diffusion of fluorescent probes determine staining selectivity of intracellular substructures.However,the relationship between the molecular structure of fluorescent probes and their membrane permeability and intracellular distribution is poorly understood.In this paper,we reported a series of 1,8-naphthalimide dyes and carried out cell imaging experiments,and found that the presence of amino hydrogen in these dyes played a crucial role in their cell membrane permeability and intracellular distribution.The secondary amino group containing compounds 1-4 show excellent membrane permeability and strong fluorescence in living cells.While the tertiary amine containing dyes 5 and 6 can hardly permeate the cell membrane though they show extremely similar structure with compounds 2-4.Compound 1 can selectively image lipid droplets by selecting the wavelength of excitation light.With the specificity for lysosomes,2 and 4 have been used in long-term time-lapses imaging of lysosomal dynamics and tracking the process of lysosome-lysosome interaction,fusion and movement.The effect of hydrogen-containing amino substituent on the cell membrane permeability of fluorescent molecules is promising for the development of better biocompatible probes.

Temporal changes in inflammatory mitochondria-enriched microRNAs following traumatic brain injury and effects of miR-146a nanoparticle delivery

MicroRNAs(miRNAs)are small non-coding RNA molecules that regulate post-transcriptional gene expression and contribute to all aspects of cellular function.We previously reported that the activities of several mitochondria-enriched miRNAs regulating inflammation(i.e.,miR-142-3p,miR-142-5p,and miR-146a)are altered in the hippocampus at 3–12 hours following a severe traumatic brain injury.In the present study,we investigated the temporal expression profile of these inflammatory miRNAs in mitochondria and cytosol fractions at more chronic post-injury times following severe controlled cortical impact injury in rats.In addition,several inflammatory genes were analyzed in the cytosol fractions.The analysis showed that while elevated levels were observed in cytoplasm,the mitochondria-enriched miRNAs,miR-142-3p and miR-142-5p continued to be significantly reduced in mitochondria from injured hippocampi for at least 3 days and returned to near normal levels at 7 days post-injury.Although not statistically significant,miR-146a also remained at reduced levels for up to 3 days following controlled cortical impact injury,and recovered by 7 days.In contrast,miRNAs that are not enriched in mitochondria,including miR-124a,miR-150,miR-19b,miR-155,and miR-223 were either increased or demonstrated no change in their levels in mitochondrial fractions for 7 days.The one exception was that miR-223 levels were reduced in mitochondria at 1 day following injury.No major alterations were observed in sham operated animals.This temporal pattern was unique to mitochondria-enriched miRNAs and correlated with injury-induced changes in mitochondrial bioenergetics as well as expression levels of several inflammatory markers.These observations suggested a potential compartmental re-distribution of the mitochondria-enriched inflammatory miRNAs and may reflect an intracellular mechanism by which specific miRNAs regulate injury-induced inflammatory signaling.To test this,we utilized a novel peptide-based nanoparticle strategy for in vitro and in vivo delivery of a miR-146a mimic as a potential therapeutic strategy for targeting nuclear factor-kappa B inflammatory modulators in the injured brain.Nanoparticle delivery of miR-146a to BV-2 or SH-SY5Y cells significantly reduced expression of TNF receptor-associated factor 6(TRAF6)and interleukin-1 receptor-associated kinase 1(IRAK1),two important modulators of the nuclear factor-kappa B(NF-κB)pro-inflammatory pathway.Moreover,injections of miR-146a containing nanoparticles into the brain immediately following controlled cortical impact injury significantly reduced hippocampal TNF receptor-associated factor 6 and interleukin-1 receptor-associated kinase 1 levels.Taken together,our studies demonstrate the subcellular alteration of inflammatory miRNAs after traumatic brain injury and establish proof of principle that nanoparticle delivery of miR-146a has therapeutic potential for modulating pro-inflammatory effectors in the injured brain.All of the studies performed were approved by the University of Kentucky Institutional Animal Care and Usage Committee(IACUC protocol#2014-1300)on August 17,2017.

Effect of Drought Stress on Growth and Water Physiological Characteris­tics of Poa sibirica

In order to develop the resources of native turfgrass,the morphological traits and drought resistance of native Siberian bluegrass(Poa sibirica,abbreviated as PS)was evaluated using the introduced Kentucky bluegrass'Midnight'(Poa pratensis,abbreviated as PP)as a control.Two water schemes were imposed to plants in this pot culture study in greenhouse.One was with drought stress persistent limiting water supply for 20 days,the other was re-hydrated until 14 days after drought.The leaf shape,turf color,water status and cell plasma membrane permeability were evaluated.Similar changing trends with these parameters were shown for both species,and there were not significant differences with most evaluations during drought and re-water periods.The values leaf width and length of PS were higher while leaf color intensity was slightly lower than that of PP,but the greenness of PS leaf was still visually acceptable.There were not significant differences with cell membrane stability between the two species.In comparison,the native wild species PS possessed the potential for to be domesticated into a new cultivar for turf industry.

Promotion of hexadecyltrimethyleamine bromide to the damage of Alexandrium sp.LC3 by cupric glutamate

The effect of hexadecyltrimethyleamine bromide （HDTMAB） on the removal of A lexandrium sp. LC3 under cupric glutamate stress was investigated. Toxic effect of cupric glutamate on A lexandrium sp. LC3 was significantly promoted in the presence of HDTMAB, especially at 3.0 cmc of HDTMAB. It was found that the sulfhydryl group content of the cell decreased, while the malonaldehyde content and membrane permeability increased when A lexandrium sp. LC3 was treated with HDTMAB and cupric glutamate complex, compared with cupric glutamate alone. The data suggest that HDTMAB might stimulate the damage of A lexandrium sp. LC3 by enhancing the membrane permeability.

Realgar induces differentiation through ROS-dependent mitochondrial pathway in HL-60 cells

Realgar （As 4 S 4 ）, as a mineral drug in traditional Chinese medicine, is currently used as the remedy for acute promyelocytic leukemia and has been proven to have relatively milder side effects as compared to the arsenolite （As 2 O 3 ）-based drugs. We have previously demonstrated that realgar induces differentiation in HL-60 cells, and the differentiation is associated with serine/threonine protein phosphatases, MAPK signaling pathways, and mitochondrial transmembrane potential decrease. In this study, we further explore the roles of mitochondrial permeability transition pore and reactive oxygen species （ROS） in realgar-induced differentiation in HL-60 cells. The differentiation was preceded by marked changes in the cellular level of ROS, and could be enhanced by SB202190, a p38 MAPK inhibitor. In addition, the efficacy of realgar was suppressed by closing the MPTP with an inhibitor. Taken together, these findings indicate that the opening of MPTP and the alteration of ROS generation were involved in realgar-induced differentiation.

Improving the biotransformation efficiency of soybean phytosterols in Mycolicibacterium neoaurum by the combined deletion of fbpC3 and embC in cell envelope synthesis

Biotransformation of soybean phytosterols into 9α-hydroxy-4-androstene-3,17-dione(9-OHAD)by mycobacteria is the core step in the synthesis of adrenocortical hormone.However,the low permeability of the dense cell envelope largely inhibits the overall conversion efficiency of phytosterols.The antigen 85(Ag85)complex encoded by fbpA,fbpB,and fbpC was proposed as the key factor in the combined catalysis of mycoloyl for producing mycolyl-arabinogalactan(m-AG)and trehalose dimycolate(TDM)in mycobacterial cell envelope.Herein,we confirmed that fbpC3 was essential for the biotransformation of trehalose monomycolate(TMM)to TDM in Mycolicibacterium neoaurum.The deficiency of this gene raised the cell permeability,thereby enhancing the steroid uptake and utilization.The 9-OHAD yield in the fbpC3-deficient 9-OHAD-producing strain was increased by 21.3%.Moreover,the combined deletion of fbpC3 and embC further increased the 9-OHAD yield compared to the single deletion of fbpC3.Finally,after 96 h of bioconversion in industrial resting cells,the 9-OHAD yield of 11.2 g/L was achieved from 20 g/L phytosterols and the productivity reached 0.116 g/L/h.In summary,this study suggested the critical role of the fbpC3 gene in the synthesis of TDM in M.neoaurum and verified the feasibility of improving the bioconversion efficiency of phytosterols through the cell envelope en-gineering strategy.

Herbicide promotes the conjugative transfer of multi-resistance genes by facilitating cellular contact and plasmid transfer

The global dissemination of antibiotic resistance genes(ARGs),especially via plasmid-mediated horizontal transfer,is becoming a pervasive health threat.While our previous study found that herbicides can accelerate the horizontal gene transfer(HGT)of ARGs in soil bacteria,the underlying mechanisms by which herbicides promote the HGT of ARGs across and within bacterial genera are still unclear.Here,the underlying mechanism associ-ated with herbicide-promoted HGT was analyzed by detecting intracellular reactive oxygen species(ROS)production,extracellular polymeric substance composition,cell membrane integrity and proton motive force combined with genome-wide RNA sequencing.Exposure to herbicides induced a series of the above bacterial responses to promote HGT except for the ROS response,including compact cell-to-cell contact by enhancing pilus-encoded gene expression and decreasing cell surface charge,increasing cell membrane permeability,and enhancing the proton motive force,providing additional power for DNA uptake.This study provides a mechanistic understanding of the risk of bacterial resistance spread promoted by herbicides,which elucidates a new perspective on nonantibiotic agrochemical acceleration of the HGT of ARGs.

Current strategies for improving limitations of proteolysis targeting chimeras

Proteolysis targeting chimeras(PROTACs)are bifunctional degrader molecules via hijacking the ubiquitinproteasome system(UPS)to specifically eliminate targeted proteins.PROTACs have gained momentum as a new modality of attractive technologies in the drug discovery landscape,since it allows to degrade disease-related proteins effectively.Although some PROTACs drugs reached the clinical research,they are still facing some bottlenecks and challenges that should not be neglected,such as poor oral bioavailability and potential toxic side effects.To overcome these limitations,herein,we provide an overview of recent strategies for improving the durability of PROTACs by enhancing cell permeability and reducing toxic side effects.Meanwhile,the impact of these strategies on improving oral bioavailability as well as their advantages and drawbacks will also be discussed.This review will give a useful reference toolbox for PROTACs design and further promote its clinical application.

Immunopathogenesis of idiopathic nephrotic syndrome in children:two sides of the coin

Background Idiopathic nephrotic syndrome is a common form of glomerular nephropathy in children,with an incidence rate of 1.15-16.9/100,000 depending on different nationalities and ethnicities.The etiological factors and mechanisms of childhood idiopathic nephrotic syndrome have not yet been fully elucidated.This review summarizes the progress of the immunopathogenesis of idiopathic nephrotic syndrome in children.Data sources We review the literature on the immunopathogenesis of idiopathic nephrotic syndrome in children.Databases including Medline,Scopus,and Web of Science were searched for studies published in any language with the terms"chil-dren","idiopathic nephrotic syndrome","immunopathogenesis","T cells","circulating permeability factors",and"B cells".Results Dysfunction in T lymphocytes and pathogenic circulatory factors were indicated to play key roles in the pathogenesis of idiopathic nephrotic syndrome.Recently,some studies have shown that cellular immune dysfunction may also be involved in the pathogenesis of idiopathic nephrotic syndrome.Conclusions Both T-and B-cell dysfunction may play significant roles in the pathogenesis of idiopathic nephrotic syndrome,like two sides of one coin,but the role of B cell seems more important than T cells.

Testing of a new high voltage electrical discharge generator prototype at high frequencies to assist anthocyanin extraction from blueberries

Traditional extraction methods are based on high-temperature maceration with organic solvents,which are dangerous for human health.A viable alternative to overcome the issues associated with conventional extraction is to increase cell tissue permeability by applying high voltage electrical discharge(HVED)treatments.The objective of this work was to validate the electroporation of blueberry plant cells using a new HVED generator prototype at a high frequency,investigate the effect,intensity,and duration of the applied voltage,and recover anthocyanins from its electroporated cells.The electroporation level of the HVED-treated blueberries was measured qualitatively by transmission electron microscopy(TEM)analysis.Meanwhile,it was quantitatively measured by the cell permeabilization index(Zp)and anthocyanin extraction level.Results of the micrographs(TEM)showed electroporation in all treatments in which Zp was 0.24 when applying a 2 kV treatment for 2 s,whereas a 3-fold increase in tissue damage was revealed with the most powerful treatment(10 kV voltage,30 s).In addition,anthocyanin values ranged from 83.09±1.20(control)to 136.82±0.84(HVED),which was 64.66%higher.The HVED treatment can increase mass transfer rates during conventional extraction processes.It should be noted that the validated prototype required a low specific energy requirement(31–204 kJ/kg)for proper tissue electropermeabilization.In conclusion,we demonstrated the capability of the developed HVED prototype to boost mass transfer phenomena and thus potentially increase its adaptability to assist dissimilar industrial processes or waste(e.g.,peels and seeds)such as freeze-drying operations.