Repetitive Passages Research Articles

The rutting performance of cold bitumen emulsion mix using ground granulated blast furnace slag and lime as fillers

Abstract Rutting is a worldwide recognized distress in bituminous pavement caused by repetitive passage of traffic loads at high temperature. High air voids and poor early strength of cold bitumen emulsion mix (CBEM) can cause rutting deformation even at lower temperatures, making it undesirable to practitioners. The CBEM can be effectively modified by using hydraulic materials as filler. The present study examines the effect of ground granulated blast furnace slag (GGBFS) and lime as fillers on rutting characteristics of CBEM at varying temperatures (i.e., 40, 50, 60oC). The conventional stone dust (SD) filler was partially replaced by GGBFS. For further modification, lime was added in CBEM containing GGBFS. Initially mix were selected for rutting test based on the results obtained by Marshall stability, indirect tensile strength, retained Marshall stability, and tensile strength ratio tests. Based on these results, 80% of GGBFS was optimized for CBEM production. Incorporating 20% lime with 80% GGBFS in CBEM further improved the properties mentioned above. Both mixes were selected for the rutting test, along with CBEM containing 100% SD and CBEM containing 20% lime, which were also selected for comparison purposes. Wheel tracking device was used for rutting test on CBEM at varying temperatures. Based on results, it was found that increasing testing temperature increases the rutting deformation. However, the CBEM containing GGBFS and lime by 80% and 20% of total weight of filler in the mix, respectively, gave better rutting resistance at each testing temperature and provide less temperature sensitivity. So, this paper introduces the blended GGBFS and lime as a new composition in CBEM that provides improved laboratory strength, moisture susceptibility resistance, and rutting resistance.

A novel study of brain microvascular endothelial cells induced by astrocyte conditioned medium for constructing blood brain barrier model in vitro: A promising tool for meningitis of teleost

The blood-brain barrier (BBB) is mainly composed of specialized endothelial cells, which can resist harmful substances, transport nutrients, and maintain the stability of the brain environment. In this study, an endothelial cell line from tilapia (Oreochromis niloticus) named TVEC-01 was successfully established. During the earlier establishment phase of the cell line, the TVEC-01 cells were persistently exposed to an astrocyte-conditioned medium (ACM). TVEC-01 cells were identified as an endothelial cell line. TVEC-01 cells retained the multiple functions of endothelial cells and were capable of performing various experiments in vitro. Furthermore, TVEC-01 cells efficiently expressed BBB-related tight junctions and key efflux transporters. From the results of the qRT-PCR, we found that the TVEC-01 cell line did not gradually lose BBB characteristics after persistent and repetitive passages, which was different from the vast majority of immortalized endothelial cells. The results showed that ACM induced up-regulation of the expression levels of multiple BBB-related genes in TVEC-01 cells. We confirmed that Streptococcus agalactiae was capable of invading the TVEC-01 cells and initiating a series of immune responses, which provided a theoretical basis for S. agalactiae to break through the BBB of teleost through the transcellular traversal pathway. In summary, we have successfully constructed an endothelial cell line of teleost, named TVEC-01, which can be used in many experiments in vitro and even for constructing BBB in vitro. Moreover, it was confirmed that S. agalactiae broke through the BBB of teleost through the transcellular traversal pathway and caused meningitis.

Effects of Loading and Compaction Conditions on Permanent Strains of Unbound Lateritic Soils from Senegal and Burkina Faso

Permanent strains of granular materials result from the repetitive passage of heavy trucks on the pavement. Pavement design relies mainly on permanent strains to predict rut depths. In order to contribute to the improvement of technical documents used for the design and modelling of pavements in tropical African countries, the researchers have focused on the determination of the permanent deformation parameters of laterites (axial (εp,1) and radial (εp,3) permanent strains). The Repeated Triaxial cyclic Loading (RTL) apparatus was used according to the European standard (EN 13286-7: 2004, Multistage method). Four sites of gravelly lateritic soils chosen, two from Burkina Faso (Badnogo2 and Dédougou) and two from Senegal (Sindia and Lam-Lam) were tested in this research. They are compacted to 95% and more of the optimum with a variation in water content of ±2% of the optimum. The permanents deformations are influenced by the water content, the compaction rate and the stress level. The axial permanent deformation ε1,p (max) decreases by 83.4% when the water content decreases from 12.10% to 7.59%. Moreover with a compactness rate that varies from 95.58% to 100.06%, respectively from a water content that varies from 10.04% to 9.36%, we observe a decrease in deformation ε_(1,p) from 224.340*10-4 to 148.150*10-4 from the 50,000 cycles, which shows the effect of the compactness rate and the stress level on the lateritic gravels materials soils. however, for a good control of permanent deformations on flexible pavements using gravel materials, it is necessary to compact at water contents lower than the optimum added to average compaction rates. Keywords: permanent strains (p), lateritic soils, Repeated Triaxial cyclic Loading (RTL), Sindia, Badnogo2, Dedougou, Lam-lam.

Effectiveness of Picture Concept Activity on Improving the Cognitive Proficiency Skill and Reading Performance of Nigerian Pupils with Dyslexia

This study determines the effectiveness of a non-digital picture concept programme to treat cognitive deficits in pupils with dyslexia. Using a sample of 38 primary two pupils (aged 7-9 years) in Ebonyi State, Nigeria, a Picture Concept Activity and the traditional method of repetitive passage drill were administered for eight weeks and results were compared. Standard cognitive and reading measures were used and data were analysed with descriptive and inferential statistics. Results show that the experimental participants performed higher than the control group on the overall reading and cognitive proficiency performances. Improved cognitive proficiency was associated with improved reading tasks.

The role of Spirulina platensis on the proliferation of rat bone marrow-derived mesenchymal stem cells.

Spirulina platensis micro-algae have some effects on cellular procedures. The proliferative potential of mesenchymal stem cells (MSCs) will be decreased after repetitive passage. The stromal cells were isolated, and then proven by differentiating to adipogenesis and osteoblastic lineage. The cell markers such as CD90 and CD105 were detected by flowcytometry. MSCs were treated with extract of S. platensis in logarithmic concentrations. MTT and ATP assays were done to determine cell proliferation capacity. The antioxidant and antimicrobial properties of the extract were evaluated. The results obtained from differentiation confirm cells' potential for osteoblastic and adipoblastic differentiation. Detection of CD90 and CD105 markers over 70% proved that the majority of cells are MSCs. Statistical analyzes revealed a significant increase in MSCs proliferation in the concentration of 0.9 μl/ml S. platensis. DPPH assay demonstrated that the extract could scavenge free radicals up to 57%. Additionally, the extract showed the inhibition zone up to 11 mm against a different strain of bacteria by agar well diffusion assay. Secreting nutritional elements, S. platensis extract can be used as an antioxidant, antimicrobial, and growth agent for enhancing the proliferation of MSCs. Furthermore, the optimum concentration for cell treatment with S. platensis's extract was investigated.

Overcoming reduced antibiotic susceptibility in intracellular Salmonella enterica serovar Typhimurium using AR-12.

Host-directed therapies (HDTs) could enhance the activity of traditional antibiotics. AR-12 is a promising HDT against intracellular pathogens including Salmonella enterica serovar Typhimurium, and has been shown to act through modulation of autophagy and the Akt kinase pathway. Since AR-12 does not inhibit the growth of planktonic bacteria but only works in conjunction with the infected host-cell, we hypothesized that AR-12 could enhance the activity of antibiotics in less-susceptible strains in the intracellular host environment. We found that repetitive passaging of S. typhimurium in macrophages in the absence of antibiotics led to a 4-fold reduction in their intracellular susceptibility to streptomycin (STR), but had no effect on the bacteria's sensitivity to AR-12. Moreover, when the host-passaged strains were treated with a combined therapy of AR-12 and STR, there was a significant reduction of intracellular bacterial burden compared to STR monotherapy. Additionally, co-treatment of macrophages infected with multi-drug resistant S. typhimurium with AR-12 and STR or ampicillin showed enhanced clearance of the intracellular bacteria. The drug combination did not elicit this effect on planktonic bacteria. Overall, AR-12 enhanced the clearance of less susceptible S. typhimurium in an intracellular environment.

Functional Regulatory Mechanisms Underlying Bone Marrow Mesenchymal Stem Cell Senescence During Cell Passages.

Mesenchymal stem cell (MSC) transplantation is an effective periodontal regenerative therapy. MSCs are multipotent, have self-renewal ability, and can differentiate into periodontal cells. However, senescence is inevitable for MSCs. In vitro, cell senescence can be induced by long-term culture with/without cell passage. However, the regulatory mechanism of MSC senescence remains unclear. Undifferentiated MSC-specific transcription factors can regulate MSC function. Herein, we identified the regulatory transcription factors involved in MSC senescence and elucidated their mechanisms of action. We cultured human MSCs (hMSCs) with repetitive cell passages to induce cell senescence and evaluated the mRNA and protein expression of cell senescence-related genes. Additionally, we silenced the cell senescence-induced transcription factors, GATA binding protein 6 (GATA6) and SRY-box 11 (SOX11), and investigated senescence-related signaling pathways. With repeated passages, the number of senescent cells increased, while the cell proliferation capacity decreased; GATA6 mRNA expression was upregulated and that of SOX11 was downregulated. Repetitive cell passages decreased Wnt and bone morphogenetic protein (BMP) signaling pathway-related gene expression. Silencing of GATA6 and SOX11 regulated Wnt and BMP signaling pathway-related genes and affected cell senescence-related genes; moreover, SOX11 silencing regulated GATA6 expression. Hence, we identified them as pair of regulatory transcription factors for cell senescence in hMSCs via the Wnt and BMP signaling pathways.

A new multi-scratching approach to identify the interaction between grit size and surface roughness on the residual stresses during belt-finishing

The present work investigates the effect of the abrasive grain size and its correlation with the surface roughness during belt-finishing processes of AISI 52100 steel alloy. The belt-finishing operation can be described by the movement of many abrasive grains rubbing the workpiece's surface at the same time. To investigate qualitatively the influence of the grit size and the repetitive passage of abrasive grains on the distribution of the residual stresses, a new multi-abrasive grains approach, based on two-dimensional finite element modeling of progressive scratchings on rough hard-turned profile, is carried out. A theoretical approach is developed to quantify the penetration depth's evolution for every scratching due to the AISI 52100 steel's elastoplastic behavior. The numerical results reveal that, during repeated scratchings on smooth or rough hard-turned surface, increasing the size of the abrasive grains tends to stabilize, quickly, the residual stresses in the interface and to improve the distribution of the compressive residual stresses along the depth. While the smoother is the initial surface, the better is the residual stress field. Moreover, it appears that the residual stresses in the skin and the sub-layer become more compressive, mostly during the additional repeated scratchings on a stabilized scratched surface, which is due to the strain-hardening phenomenon related to the progressive passage of the abrasive grains. These findings are then used to extract simple empirical laws and suggest an efficient way to improve the residual stress field that occurs during abrasive superfinishing processes.

Honey‐incorporated nanofibre reduces replicative senescence of umbilical cord‐derived mesenchymal stem cells

Umbilical cord-derived mesenchymal stem cells (UCDMSC) are attractive candidates for cell-based regenerative medicine. However, they are susceptible to replicative senescence during repetitive passaging for in-vitro expansion and induced senescence in an oxidative, inflammatory microenvironment in vivo. Aim of this study is to investigate if honey-incorporated matrices can be employed to reduce senescence of UCDMSC. Matrices were prepared by electrospinning solutions of honey with poly-vinyl alcohol (PVA). PVA:honey matrices exhibited free radical scavenging activity. Culture of UCDMSC on PVA:honey matrices showed improvement in cell proliferation compared to pure PVA nanofibres. Expression of vimentin indicated that mesenchymal phenotype is preserved after culturing on these matrices. Further, UCDMSC were serially subcultured and cells of two passages (P2 and P6) were evaluated for reactive oxygen species (ROS) load and senescence parameters. P6 cells showed a higher ROS load and β-galactosidase (β-gal) positive senescent cells compared to P2. However, culturing on PVA:honey substrates significantly reduced both ROS and β-gal markers compared to cells on PVA substrates. Honey contains several antioxidant and anti-inflammatory components, which can reduce the ROS-related senescence. Thus, it is concluded that honey containing nanofibres can be effective substrates for stem cell-based wound healing and regenerative medicine.

The Effect of Thiol Structure on Allyl Sulfide Photodegradable Hydrogels and their Application as a Degradable Scaffold for Organoid Passaging.

Intestinal organoids are useful in vitro models for basic and translational studies aimed at understanding and treating disease. However, their routine culture relies on animal-derived matrices that limit translation to clinical applications. In fact, there are few fully defined, synthetic hydrogel systems that allow for the expansion of intestinal organoids. Here, an allyl sulfide photodegradable hydrogel is presented, achieving rapid degradation through radical addition-fragmentation chain transfer (AFCT) reactions, to support routine passaging of intestinal organoids. Shear rheology to first characterize the effect of thiol and allyl sulfide crosslink structures on degradation kinetics is used. Irradiation with 365 nm light (5 mW cm-2 ) in the presence of a soluble thiol (glutathione at 15 × 10-3 m), and a photoinitiator (lithium phenyl-2,4,6-trimethylbenzoylphosphinate at 1 × 10-3 m), leads to complete hydrogel degradation in less than 15 s. Allyl sulfide hydrogels are used to support the formation of epithelial colonies from single intestinal stem cells, and rapid photodegradation is used to achieve repetitive passaging of stem cell colonies without loss in morphology or organoid formation potential. This platform could support long-term culture of intestinal organoids, potentially replacing the need for animal-derived matrices, while also allowing systematic variations to the hydrogel properties tailored for the organoid of interest.

Involvement of ERK1/2 activation in the gene expression of senescence-associated secretory factors in human hepatic stellate cells.

Senescent hepatic stellate cells (senescent HSCs) are found in patients with liver cirrhosis and have been thought to be involved in the development of hepatocellular carcinoma (HCC) in mice via the senescence-associated secretory proteins. However, in humans, which secretory proteins are involved and what regulate their expression remain unclear. In the current study, we characterized senescence-associated β-galactosidase-positive senescent human HSCs (hHSCs) induced by repetitive passaging. They exhibited enhanced expression of 14 genes for secretory protein and persistent phosphorylation of ERK1/2 protein but not JNK or p38 MAPK proteins. Enhanced nuclear ERK1/2 phosphorylation was observed in senescent hHSCs. Treatment of the senescent hHSCs with ERK1/2 inhibitor, SCH772984, significantly decreased the levels of angiopoietin like 4 (ANGPTL4), C-C motif chemokine ligand 7 (CCL7), Interleukin-8 (IL-8), platelet factor 4 variant 1 (PF4V1), and TNF superfamily member 15 (TNFSF15) mRNA levels in a dose-dependent manner. The enhanced phosphorylation of ERK1/2 and expression of ANGPTL4, IL-8 and PF4V1 genes were observed in both of senescent human dermal fibroblasts and X-ray-induced senescent hHSCs. However, transient ERK1/2 activation induced by epidermal growth factor could not mimic the gene profile of the senescent hHSCs. These results revealed involvement of ERK1/2 signaling in the regulation of senescence-associated secretory factors, suggesting that simultaneous induction of ANGPTL4, IL-8, and PF4V1 genes is a marker of hHSC senescence. This study will contribute to understanding roles of senescent hHSCs in liver diseases.

Amustaline (S-303) treatment inactivates high levels of Zika virus in red blood cell components.

The potential for Zika virus (ZIKV) transfusion-transmission (TT) has been demonstrated in French Polynesia and Brazil. Pathogen inactivation (PI) of blood products is a proactive strategy to inactivate TT pathogens including arboviruses. Inactivation of West Nile, dengue, Zika, and chikungunya viruses was previously demonstrated by photochemical treatment with amotosalen and ultravioletA (UVA) illumination. In this study, we evaluated ZIKV inactivation in red blood cell (RBC) components by a chemical approach that uses amustaline (S-303) and glutathione (GSH). RBC components were spiked with a high titer of ZIKV. Viral titers (infectivity) and ZIKV RNA loads (reverse transcription-polymerase chain reaction) were measured in spiked RBCs before and after S-303 and GSH treatment and confirmed using repetitive passages in cell culture. A mock-treated arm validated the approach by demonstrating stability of the virus (infectivity and RNA load) during the process. The mean ZIKV infectivity titer and RNA load in RBCs were 5.99±0.2 log 50% tissue culture infectious dose (TCID50 )/mL and 7.75±0.16 log genomic equivalents/mL before inactivation. No infectivity was detected immediately after S-303 and GSH treatment and after five serial passages in cell culture. Complete ZIKV inactivation of more than 5.99 log TCID50 /mL in RBCs was achieved using S-303 and GSH at levels higher than those found in asymptomatic ZIKV-infected blood donors. Therefore, the S-303 and GSH PI system is promising for mitigating the risk of ZIKV TT.

Restoration of Mesenchymal RPE by Transcription Factor-Mediated Reprogramming.

Transforming growth factor β-mediated epithelial-to-mesenchymal transition (EMT) is a major component of the wound healing response and a negative determinant of retinal pigment epithelium (RPE) differentiation. We have shown previously that inhibition of TGFβ signaling restored the capacity of mesenchymal RPE to differentiate; however, the potential lessens with extensive passaging. We investigated TGFβ-independent mechanisms that regulate RPE differentiation following repetitive passaging. Retinal pigment epithelium-EMT was induced by repetitive passaging of fetal RPE at subconfluence. Suppression of EMT was achieved by the addition of A-83-01, a TGFβ receptor kinase inhibitor. Transcriptomic analysis was used to identify potential TGFβ independent processes that prevent differentiation after extensive passage. Downregulated transcription factors were identified and transduced into highly passaged RPE to restore cell differentiation. Restoration was evaluated according to morphology, expression of RPE/mesenchymal markers, transcriptomic analysis, cell doubling time, and senescence-associated β-galactosidase (SA-β-gal) activity. A-83-01-treated RPE failed to differentiate after 7 passages (P7). This failure was concomitant with downregulation of RPE genes, misregulation of cell cycle genes, a decline in proliferative potential, and cell senescence. Exogenous expression of MYCN and OTX2 in conjunction with A-83-01 restored P7-RPE differentiation to a status similar to minimally passaged RPE. Moreover, the treatment allowed cells to maintain their differentiation capacity after extended passaging. Retinal pigment epithelium subjected to chronic wound stimulus undergoes TGFβ-mediated EMT, loss of expression of signature RPE genes, and senescence. Targeting these aspects with a TGFβ receptor kinase inhibitor, a RPE transcription factor, and a cell cycle regulator restores the capacity of highly passaged RPE cells to regenerate and differentiate.

Dynamic Forces at Square and Inclined Rail Joints: Field Experiments

AbstractInsulated rail joints (IRJs) are widely used in signaling and broken rail identification systems. Track deterioration adjacent to IRJ is frequent due to excessive dynamic forces generated at IRJs by the repetitive passage of the ongoing traffic. Hence, they exhibit low service life and are considered high-risk elements and maintained through high standards. With a view to increase operational speed and the annual operational throughput, many improved structural designs have been proposed, of which inclined IRJs are the focus of this paper. To compare noise, vibration, and adjacent sleepers’ vertical displacement of square and 30° and 45° cut joints, a series of field tests have been carried out in the Tehran-Karaj urban metro track. Results show that sleepers in the vicinity of 45° cut joint have less vertical displacements compared to that of 30° and square cut joints. Peak root-mean square (RMS) values of acceleration signatures of 30 and 45° cut joints are almost half the value for square joint...

Repetition analysis function (ReAF) I

Abstract Repetitions are relevant for several aspects of historical philology. With regard to Avestan, they may allow for the identification of ceremonial frames or opening and closing sections revealing the compositional structure of a ceremony. In case of manuscript comparison, the question arises whether a variant appears only once or in all of its repetitive passages. Furthermore, by analysing the compositional structure we may be able to detect ceremonial structures different to the practice of today. A secondary aspect relates to the interpretation of the grammaticality of Young Avestan passages. The repetition analysis provides evidence that passages which are hitherto considered ill-formed actually follow the rules of Avestan grammar. The scope of this study is to investigate computational means for detecting repetitive sequences. It represents a case study of the manuscript J2 by means of tools that were set up in the LOEWE priority programme Digital Humanities at the Goethe University Frankfurt am Main: a digital lexicon, a letter discrimination matrix for Avestan, and the programme Repetition Analysis Function. The article ReAF I offers some basic observations on repetitive sequences in the manuscript J2 and lays the foundation for ReAF II (Jügel forthc.), where the results of the repetition analyses will be used to discuss the compositional structure of the Yasna.

Cyclin-Dependent Kinase 5–Mediated Hyperphosphorylation of Sirtuin-1 Contributes to the Development of Endothelial Senescence and Atherosclerosis

Endothelial senescence represents one of the major characteristics of vascular aging and promotes the development of atherosclerosis. Sirtuin-1 (SIRT1) is an NAD-dependent deacetylase possessing antiaging activities. During the occurrence of endothelial senescence, both the expression and activity of SIRT1 are downregulated. The present study was designed to investigate the molecular mechanisms contributing to the loss-of-SIRT1 function in senescent endothelial cells. After repetitive passages, primary cultures of porcine aortic endothelial cells exhibited a severe senescence phenotype. Western blotting revealed that phosphorylation of SIRT1 at serine 47 (S47) was significantly enhanced in senescent endothelial cells. S47 phosphorylation was stimulated by agents promoting senescence and attenuated by drugs with antisenescence properties. Mutation of S47 to nonphosphorable alanine (S47A) enhanced whereas replacing S47 with phospho-mimicking aspartic acid (S47D) abolished the antisenescent, growth-promoting, and LKB1-downregulating actions of SIRT1. Phosphorylation at S47 was critically involved in the nuclear retention of SIRT1 but abolished its association with the telomeric repeat-binding factor 2-interacting protein 1. Cyclin-dependent kinase 5 (CDK5) was identified as an SIRT1 kinase modulating S47 phosphorylation. Knockdown or inhibition of CDK5 reduced the number of senescent endothelial cells, promoted nuclear exportation of SIRT1, and attenuated the expression of inflammatory genes in porcine aortic endothelial cells. The truncated regulatory subunit of CDK5, P25, accumulated in senescent porcine aortic endothelial cells and atherosclerotic aortas. Long-term treatment with roscovitine, a CDK5 inhibitor, blocked the development of cellular senescence and atherosclerosis in aortas of hypercholesterolemic apolipoprotein E-deficient mice. CDK5-mediated hyperphosphorylation of SIRT1 facilitates the development of endothelial senescence and atherosclerosis.

Activation of neural progenitor cells in human eyes with proliferative vitreoretinopathy

In addition to the ability for self-renewal and functional differentiation, neural stem/progenitor cells (NSCs) can respond to CNS injuries by targeted migration. In lower vertebrates, retinal injury is known to activate NSCs in the ciliary marginal zone (CMZ). Cells expressing markers of NSCs are also present in the ciliary body epithelium (CE) and in Müller glia in the peripheral retina (PR) of the adult human eye. However, these cells seem to be quiescent in the adult human eye and recent reports have shown that CE cells have limited properties of NSCs. In order to further clarify whether NSCs exist in the adult human eye, we tested whether NSC-like cells could be activated in eyes with proliferative vitreoretinopathy (PVR). The PR and CE were studied for NSC-associated markers in human enucleated control eyes and eyes with confirmed PVR, as well as in a mouse model of PVR. Furthermore, cells isolated from vitreous samples obtained during vitrectomies for retinal detachment were directly fixed or cultured in a stem cell-promoting medium and compared to cells cultured from the post-mortem retina and CE. In situ characterization of the normal eyes revealed robust expression of markers present in NSCs (Nestin, Sox2, Pax6) only around peripheral cysts of the proximal pars plana region and the PR, the latter population also staining for the glial marker GFAP. Although there were higher numbers of dividing cells in the CE of PVR eyes than in controls, we did not detect NSC-associated markers in the CE except around the proximal pars plana cysts. In the mice PVR eyes, Nestin activation was also found in the CE. In human PVR eyes, proliferation of both non-glial and glial cells co-staining NSC-associated markers was evident around the ora serrata region. Spheres formed in 7/10 vitreous samples from patients with PVR compared to 2/15 samples from patients with no known PVR, and expressed glial – and NSC-associated markers both after direct fixation and repetitive passages. In conclusion, the adult human eye may harbor two different populations of neuroepithelial stem/progenitor cells; a non-glial population located in the proximal pars plana around peripheral cysts in addition to a population with Müller glia characteristics. Yet, we only found that the glial population was able to respond to retinal injury by targeted migration into the vitreous.

Characterization of neuroepithelial progenitor cells in patients with proliferative vitreoretinopathy

AbstractPurpose Proliferative vitreoretinopathy (PVR) is an important cause of retinal diseases such as retinal detachment (RD). In lower vertebrates, retinal damage is known to activate neuroepithelial stem cells (NSCs) in the ciliary margin in an attempt to regenerate the neuroretina. Cells expressing some markers of NSCs are also present in the retina and the ciliary body epithelium (CB) of the adult human eye. We hypothesized that if NSCs exist in the adult human eye, they should be activated by PVR formation.Methods Cells isolated from vitreous samples (n=25) obtained during vitrectomies for RD were directly fixed or cultured in a stem cell‐promoting medium, and compared to cells isolated from post mortem CB and peripheral retina (PR) using sphere‐forming assay, immunohistochemistry, molecular biology and electron microscopy. Markers of NSCs were also studied in whole retinal control/PVR sections obtained from enucleations.Results Spheres formed in 7/10 vitreous samples from patients with PVR compared to 2/15 samples from patients with no known PVR. These spheres stained for markers of NSCs both in vivo and after repetitive passages. Their mRNA and immunohistochemical profile resembled sphere‐forming cells from the PR with only a few characteristics of CB cells. In situ characterization of the CB revealed that although there were higher numbers of dividing cells in PVR eyes than in controls, we did not detect markers of NSCs. Interestingly, markers of NSCs were evident around PR cysts with some evidence of activation following PVR formation.Conclusion A population of NSC‐like cells are found in the vitreous of patients with PVR. These cells seem to be originated from the retina itself and not the CB.

Pigment epithelial cells isolated from human peripheral iridectomies have limited properties of retinal stem cells

The identification of cells with properties of retinal progenitor cells (RPCs) in the adult human ciliary margin (CM) prompted a number of studies of their proliferative and differentiation potential. One of the remaining challenges is to find a feasible method of isolating RPCs from the patient's eye. In the human CM, only the iris pigment epithelium (IPE) is easily obtained by a minimally invasive procedure. In the light of recent studies questioning the existence of RPCs in the adult mammalian CM, we wanted to assess the potential of the adult human IPE as source of RPCs. The IPE were isolated from peripheral iridectomies during glaucoma surgery, and IPE and ciliary body (CB) epithelium were also isolated from post-mortem tissue. Cells were cultivated in sphere-promoting conditions or as monolayers. Whole-tissue samples, undifferentiated and differentiated cells were studied by immunocytochemistry, RT-PCR and transmission electron microscopy. The adult human IPE, like the CB, expressed markers of RPCs such as Pax6, Sox2 and Nestin in vivo. Both sphere-promoting and monolayer cultures preserved this phenotype. However, both IPE/CB cultures expressed markers of differentiated epithelial cells such as Claudin, microphtalmia-associated transcription factor (MITF) and Cytokeratin-19. Ultrastructurally, IPE spheres displayed epithelial-like junctions and contained mature melanosomes. After induced differentiation, IPE-derived cells showed only partial neuronal differentiation expressing β-III-tubulin, Map-2 and Rhodopsin, whereas no mature glial markers were found. Proliferative cells with some properties of RPCs can be isolated from the adult human IPE by peripheral iridectomies. Yet, many cells retain properties of differentiated epithelial cells and lack central properties of somatic stem cells.

SIRT1 Promotes Proliferation and Prevents Senescence Through Targeting LKB1 in Primary Porcine Aortic Endothelial Cells

Endothelial senescence causes endothelial dysfunction, promotes atherogenesis and contributes to age-related vascular disorders. SIRT1 is a conserved NAD(+)-dependent deacetylase possessing beneficial effects against aging-related diseases, despite that the detailed functional mechanisms are largely uncharacterized. The present study is designed to evaluate the protective effects of SIRT1 on endothelial senescence and to elucidate the underlying mechanisms. An in vitro senescence model was established by prolonged culture of primary endothelial cells isolated from porcine aorta. The freshly isolated "young" cells gradually underwent senescence during 1 month of repetitive passages. Both mRNA and protein expressions of SIRT1 were progressively decreased. In contrast, the protein levels of LKB1, a serine/threonine kinase and tumor suppressor, and the phosphorylation of its downstream target AMPK(Thr172) were dramatically increased in senescent cells. Overexpression of LKB1 promoted cellular senescence and retarded endothelial proliferation, which could be blocked by increasing SIRT1 levels. Knocking down of SIRT1 induced senescence and elevated the protein levels of LKB1 and phosphorylated AMPK(Thr172). Regardless of the nutritional status, hyperactivation of AMPK was able to induce endothelial senescence. SIRT1 antagonized LKB1-dependent AMPK activation through promoting the deacetylation, ubiquitination and proteasome-mediated degradation of LKB1. The survival signaling of Akt was also found to be modulated by SIRT1 and LKB1, and could cross-regulate AMPK activity. SIRT1 and LKB1/AMPK are the 2 key sensor systems for regulating endothelial cell survival, proliferation and senescence. The protective activities of SIRT1 may be achieved at least in part by fine tuning the acetylation/deacetylation status and stabilities of LKB1 protein.