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Severe conjunctival diseases can cause significant conjunctival scarring, which seriously limits eye movement and affects patients' vision. Conjunctival reconstruction remains challenging due to the lack of efficient methods for stem cells enrichment. This study indicated that p75 positive conjunctival epithelial cells (CjECs) were mainly located in the basal layer of human conjunctival epithelium and showed an immature differentiation state in vivo. The p75 strongly positive (p75++) CjECs enriched by immuno‐magnetic beads exhibited high expression of stem cell markers and low expression of differentiated keratins. During continuous cell passage cultivation, p75++ CjECs showed the strongest proliferation potential and were able to reconstruct the conjunctiva in vivo with the most complete structure and function. Exogenous addition of NGF promoted the differentiation of CjECs by increasing nuclear localization of SALL2 in p75++ CjECs while proNGF played an opposite role. Altogether, p75++ CjECs present stem cell characteristics and exhibit the strongest proliferation potential so can be used as seed cells for conjunctival reconstruction, and NGF‐p75‐SALL2 signaling pathway was involved in regulating the differentiation of CjECs., p75++ CjECs(conjunctival epithelial cells) were enriched by immuno‐magnetic beads and their stem cell properties were detected. After 10 days of culture on amniotic membrane, conjunctiva constructed by p75++/− CjECs was transplanted to a rabbit model of conjunctival defect to test the repair effect. Exogenous addition of NGF promoted the differentiation of CjECs by increasing nuclear localization of SALL2 in p75++ CjECs.

Neovascularization during ocular tissue repair can cause severe visual loss in the optical axis and is therefore an issue of considerable concern to ophthalmologists. Here, we introduced a cholesterol-modified siRNA delivery system targeting stromal cell-derived factor 1 (SDF-1) to treat ocular angiogenesis in vivo. SDF-1 expression was analyzed in rat endothelial progenitor cells (EPCs) and bone marrow mesenchymal stem cells (BMSCs) using quantitative PCR (qPCR). Migration ability of BMSC and HUVEC were assessed through transwell assay. The proliferation effect of chol-siSDF1 on HUVEC was measured by colony formation assay. In vivo anti-angiogenic effects of chol-siSDF1 were tested in a cornea alkali burn model and the area of cornea neovascularization was measured using computer-imaging analysis system. Then phosphorylated Akt and total Akt protein levels were measured through western blot. Results turned out that rat EPCs and BMSCs showed high SDF-1 mRNA expression, which can be down-regulated by using chol-siSDF-1. Chol-siSDF-1 could significantly inhibit migration of BMSC and HUVEC. In addition, chol-siSDF1 also could inhibit HUVEC proliferation and exert a significant anti-angiogenic effect in corneal alkali burn model. As for the mechanism, chol-siSDF1 may inhibit the neovascularization, proliferation and metastasis through inhibiting the Akt signaling pathway. Thus, cholesterol modification of siRNA targeting SDF-1 displays an effective inhibition of migration and angiogenesis, with a much longer duration of inhibition effect.

Treating corneal endothelial diseases tends to be challenging as human corneal endothelial cells (CECs) do not proliferate in vivo. The pathogenesis or mechanisms underlying injured CECs need further studies. The abnormal expression of PAX6, which is an essential transcription factor for corneal homeostasis, exhibits corneal endothelial defects. However, the effects of PAX6 protein involved in corneal endothelial wound process are still unknown. Here, we found the upregulated protein levels of PAX6 in human corneal endothelial monolayer after injury; the expression of PAX6 also increased in murine and rat corneal endothelium injury models. Enforced PAX6 expression could alleviate the damages to CECs via regulating permeability by prompting cellular tight junction. In addition, SUMOylation mainly happened on both K53 and K89 residues of 48-kD PAX6 (the longest and main isoform expressed in cornea), and de-SUMOylation promoted the stability of PAX6 protein in vitro. In CECs of SENP1+/− mice, increased SUMOylation levels leading to instability and low expression of PAX6, delayed the repair of CECs after injury. Furthermore, overexpression of PAX6 accelerated the rate of corneal endothelial repair of SENP1+/− mice. Our findings indicate that SENP1-mediated de-SUMOylation improving the stability of PAX6, amplifies the protective effects of PAX6 on corneal endothelial injuries, highlighting potentials of PAX6 and/or SUMOylation to be used as a treatment target for corneal endothelial disorders.

This study aimed to describe a cohort of patients with cryptophthalmos (CO), characterize associated oculofacial abnormalities, and expand the classification to summarize surgical strategies for managing CO.A retrospective, interventional case series was conducted on 86 patients (124 eyes) with CO. The study proposed further classifying complete and incomplete CO into cyst, microphthalmia, anophthalmia, and normal eyeball based on globe structures and then modifying surgery accordingly. The demography, ophthalmic features, systemic anomalies, operation methods, and treatment outcomes were reviewed.CO was complete in seven eyes (5.6%) and incomplete in eight eyes (6.5%). A total of 109 eyes (87.9%) of abortive CO were encountered. Among 15 eyes (13 patients) of complete and incomplete types, 9 (60.0%) eyeballs were identified as cysts, 3 (20.0%) as microphthalmia, 1 (6.7%) as anophthalmia, and 2 (13.3%) as normal eyeballs. Cyst reduction was performed in eight eyes and one patient underwent enucleation with hydroxyapatite implantation. The socket was fit with an ocular prosthesis or a conformer after fornix and eyelid reconstruction. Microphthalmia was enucleated, and hydroxyapatites were implanted; patients were fit with ocular prosthesis or conformer after fornix and eyelid reconstruction. A complete CO with normal eyeball was reported with the eyesight of hand movement after ocular surface reconstruction. The upper eyelid contour and adequate fornix were maintained after coloboma repair and fornix reconstruction in all patients with abortive CO.This study demonstrates the clinical manifestations of different types of CO and expands the manifestation spectrum, proposing a refined classification of CO and modifying surgical strategies accordingly.

Purpose To investigate the proliferation of umbilical cord blood-derived endothelial progenitor cells (UCB EPCs) and the differentiation efficiency toward corneal endothelial cell (CEC)-like cells induced by rho-associated protein kinase (ROCK) inhibitor Y-27632 and to determine the most effective strategy for repairing corneal endothelium injuries in rabbits. Methods UCB EPCs were cultured in Endothelial Cell Growth Medium-2 (EGM-2) media or conditioned media (CM) from human CECs, with and without the addition of Y-27632. Bromo-deoxyuridine (BrdU) immunocytochemistry and cell counting kit-8 assays were used to examine the proliferation of the cells. Real-time polymerase chain reaction, western blot, and immunocytochemistry were used to detect the CEC markers. Nd:YAG laser was used to establish an appropriate endothelium injury model based on rabbit corneas. The following intracameral injections were then performed to repair the model: 100 μL Opti-MEM I reduced serum medium (model group), 2 × 105 UCB EPCs diluted in 100 μL Opti-MEM I reduced serum medium (EPC group), 100 μM Y-27632 diluted in 100 μL Opti-MEM I reduced serum medium (Y-27632 group), and 2 × 105 UCB EPCs supplemented with 100 μM Y-27632 (final volume 100 μL, EPC/Y-27632 group). The follow-up tests focused on corneal transparency, central corneal thickness, intraocular pressure, and in vivo confocal microscopy, which were performed to evaluate the healing of the wounds. Results Culturing UCB EPCs in CM supplemented with 10 μM Y-27632 resulted in higher proliferation rates compared with EGM-2 media and CM. There were significantly improved protein levels of Zona Occludens 1, N-cadherin, Na+-K+-ATPase α1, Na+-K+-ATPase β1, and Pax6 and improved mRNA levels of collagen type IV and VIII and AQP1. The combined intracameral injection of Y-27632 and UCB EPCs accelerated the recovery of corneal transparency, regression of corneal edema, and healing of the corneal endothelium compared with the injections of Y-27632 and UCB EPCs on their own. Conclusions Y-27632 not only promotes the proliferation of UCB EPCs but also contributes to differentiation of UCB EPCs toward CECs in the presence of CM. The intracameral injection of Y-27632 itself promotes the healing of corneal endothelium wounds. On this basis, supplementing UCB EPCs with Y-27632 accelerates the healing of corneal endothelium wounds.

Purpose To develop a new rabbit model of corneal endothelial injury using a neodymium-doped yttrium aluminum garnet (Nd:YAG) laser. Methods The corneal endothelia of 2 groups of New Zealand white rabbits were treated with an Nd:YAG laser in a uniformly scattered fashion. Rabbits in group A underwent laser burns on the whole corneal endothelium, including the limbus area, whereas rabbits in group B were subjected to laser burns in the central 9-mm diameter zone of the endothelium. Slit-lamp biomicroscopy, optical coherence tomography, applanation tonometry, confocal microscopy, scanning electron microscopy, and histological examinations were performed during 4 weeks of follow-up. Results In both groups, dotted or focal corneal endothelium defects were directly observed. The stroma was intact. Right after laser application, a series of clinical manifestations appeared, including subepithelial and stromal edema, increased central corneal thickness, and corneal opacity. Laser burn had more notable effects in group A than in group B. In both groups, we observed no damage to the intraocular structures, and intraocular pressure was normal after laser treatment. Conclusions Nd:YAG laser treatment in a 9-mm diameter zone of the endothelium can effectively induce bullous keratopathy in a rabbit, whereas treatment for the entire corneal endothelium maintains bullous keratopathy for a longer period. The procedure is simple and reproducible, and it retains normal intraocular structures. This study provided a promising model for future research into endothelial cell damage and for the development of new therapies.

PurposeThe objective of this study is to evaluate and grade the extent and severity of ocular involvement in Tessier number 10 cleft.Patients and methodsA retrospective, noncomparative, interventional case series was conducted between January 2006 and December 2015. Clinical data were reviewed from 59 patients (85 eyes) with Tessier number 10 clefts. Detailed medical history and ophthalmic examination of patients with a confirmed diagnosis of Tessier number 10 cleft were recorded on an itemized data collection form. Ocular manifestations were categorized as upper eyelid defect, symblepharon with cutaneous pterygium (skin growing onto the globe), corneal complications, and lower eyelid ectropion; components were evaluated and graded on a scale from 0 to 3, according to their severity.ResultsMore than half of the cases (43 eyes, 53.8%) had severe upper eyelid defect, and severe symblepharon with cutaneous pterygium were observed in 38 eyes (47.5%). Nearly half of the cases (40 eyes, 50.0%) have severe corneal complications, and lower eyelid ectropion was found in 34 eyes (42.5%). The severity of symblepharon, corneal complications, and lower eyelid ectropion were significantly correlated with the upper eyelid defect; the correlation coefficient (r) ranged from 0.844 to 0.629 (P

Recent research has demonstrated critical roles of a number of microRNAs (miRNAs) in stem cell proliferation and differentiation. miRNA-9 (miR-9) is a brain-enriched miRNA. Whether miR-9 has a role in retinal progenitor cell (RPC) proliferation and differentiation remains unknown. In this study, we show that miR-9 plays an important role in RPC fate determination. The expression of miR-9 was inversely correlated with that of the nuclear receptor TLX, which is an essential regulator of neural stem cell self-renewal. Overexpression of miR-9 downregulated the TLX levels in RPCs, leading to reduced RPC proliferation and increased neuronal and glial differentiation, and the effect of miR-9 overexpression on RPC proliferation and differentiation was inhibited by the TLX overexpression; knockdown of miR-9 resulted in increased TLX expression as well as enhanced proliferation of RPCs. Furthermore, inhibition of endogenous TLX by small interfering RNA suppressed RPC proliferation and promoted RPCs to differentiate into retinal neuronal and glial cells. These results suggest that miR-9 and TLX form a feedback regulatory loop to coordinate the proliferation and differentiation of retinal progenitors.

Polyethylene/paraffin oil blend sheets with different molecular weights of polyethylene were prepared by thermally induced phase separation. Isothermal and non-isothermal crystallization behaviors of blend sheets were investigated by differential scanning calorimetry (DSC). Isothermal DSC curves were analyzed by Avrami equation, whereas non-isothermal DSC curves were analyzed by Jeziorny method and Mo method. Effective activation energy (ΔE) of isothermal and non-isothermal crystallization was calculated by Friedman method. Under isothermal condition, value of n in Avrami equation hovered at 2.1, and lgZ increased with the decrease of crystallization temperature. lgZ and ΔE of blend sheets with a larger molecular weight of polyethylene was smaller than that of blend sheets with smaller molecular weight. Under non-isothermal condition, value of n obtained by Jeziorny method hovered at 2.4, close to n of isothermal condition. lgZ c increased with the increase of cooling rate and decrease of molecular weight of polyethylene. ΔE of different blend sheets were close to each other. Crystal structures of blend sheets formed under non-isothermal condition were analyzed by X-ray diffraction (XRD) analysis. XRD analysis showed that molecular weight of polyethylene and cooling rate had slight influence on crystal structure and crystallinity of polyethylene/paraffin oil blend sheet.

Purpose Excessive accumulation of extracellular matrix (ECM) in the trabecular meshwork (TM) reduces aqueous humor outflow, which likely contributes to elevation of IOP in primary open-angle glaucoma (POAG). Salidroside, a phenolic glycoside isolated from Rhodiola rosea is reported to prevent profibrotic responses by inhibiting Smad signaling pathway activated by TGF-β in liver, lung, and kidney tissues. We tested if salidroside can (1) inhibit TGF-β2-induced ECM expression in cultured human TM cells, and (2) lower TGF-β2-induced ocular hypertension in the mouse. Methods Cultured human TM cells stimulated with 5 ng/mL TGF-β2 for 48 hours were treated with salidroside for 24 hours. The expressions of fibronectin (FN), collagen type IV (COL-IV), and laminin (LN) were evaluated by quantitative PCR, Western blot, and immunocytochemistry. BALB/cJ mice were injected intravitreally with an adenoviral vector encoding a bioactive mutant of TGF-β2 (Ad.hTGF-β2226/228) in one eye to induce ocular hypertension, with the uninjected contralateral or Ad.Empty-injected eyes serving as controls. Mice were treated with a daily intraperitoneal injection of 40 mg/kg salidroside. Conscious mouse IOP values were measured using a TonoLab rebound tonometer. Results In cultured human TM cells, treatment with TGF-β2 increased expressions of FN, COL-IV, and LN, as assessed by quantitative PCR, Western blotting, and immunocytochemistry, all of which were significantly and completely ameliorated by 30 μM salidroside. Daily intraperitoneal injections of salidroside (40 mg/kg), starting either at day 0 (same day as Ad.hTGF-β2226/228 injection) or at day 14, significantly lowered TGF-β2-induced ocular hypertension in the mouse. In contrast, salidroside did not affect IOP of control eyes. Conclusions These results demonstrated that salidroside is capable of minimizing TGF-β2-induced ECM expression in cultured human TM cells. It also reduced TGF-β2-induced ocular hypertension in the mouse. These findings indicate that this phenolic glycoside may be useful as a novel treatment for POAG.

This paper presents a Chirplet Wigner–Ville Distribution (CWVD) that is free for cross-term that usually occurs in Wigner–Ville distribution (WVD). By transforming the signal with frequency rotating operators, several mono-frequency signals without intermittent are obtained, WVD is applied to the rotated signals that is cross-term free, then some frequency shift operators corresponding to the rotating operator are utilized to relocate the signal′s instantaneous frequencies (IFs). The operators′ parameters come from the estimation of the IFs which are approached with a polynomial functions or spline functions. What is more, by analysis of error, the main factors for the performance of the novel method have been discovered and an effective signal extending method based on the IFs estimation has been developed to improve the energy concentration of WVD. The excellent performance of the novel method was manifested by applying it to estimate the IFs of some numerical signals and the echolocation signal emitted by the Large Brown Bat.

AIM To explore the effects of conditioned media on the proliferation of corneal endothelial cells (CECs) and to compare the efficiency of different conditioned media (CM). METHODS Rat CECs, corneal stromal cells (CSCs), bone marrow-derived endothelial progenitor cells (BEPCs), and bone marrow-derived mesenchymal stem cells (BMSCs) were isolated and cultured in vitro. CM was collected from CSCs, BEPCs, and BMSCs. CECs were cultivated in different culture media. Cell morphology was recorded, and gene and protein expression were analyzed. RESULTS After grown in CM for 5d, CECs in each experimental group remained polygonal, in a cobblestone-like monolayer arrangement. Immunocytofluorescence revealed positive expression of Na(+)/K(+)-ATP, aquaporin 1 (AQP1), and zonula occludens 1 (ZO-1). Based on quantitative polymerase chain reaction (qPCR) analysis, Na(+)/K(+)-ATP expression in CSC-CM was notably upregulated by 1.3-fold (±0.036) (P

This paper presents a novel multichannel fusion approach based on coupled hidden Markov models (CHMMs) for rolling element bearing fault diagnosis. Different from a hidden Markov model (HMM), a CHMM contains multiple state sequences and observation sequences, and hence has powerful potential for multichannel fusion. In this study, a two-chain CHMM is employed to integrate the two-channel vibration signals collected from bearings, i.e. the horizontal and vertical vibration signals. Efficient probabilistic inference and parameter estimation algorithms are developed for the model. An experiment was carried out to validate the proposed approach. Normalized wavelet packet energy and wavelet packet energy entropy are extracted as features for classification respectively. Then, the results of the proposed approach are compared with those of the currently used approach based on HMMs and one-channel signals. The results show that the proposed approach is feasible and effective to improve the classification rate.

The vibration signals of rolling element bearings are random cyclostationary when they have faults. Also, statistical properties of the signals change periodically with time. The accurate analysis of time-varying signals is an essential pre-requisite for the fault diagnosis and hence safe operation of rolling element bearings. The Wigner distribution is probably most widely used among the Cohen’s class in order to describe how the spectral content of a signal changes over time. However, the basic nature of such signals causes significant interfering cross-terms, which do not permit a straightforward interpretation of the energy distribution. To overcome this difficulty, the Wigner–Ville distribution (WVD) based on the cyclic spectral density (CSD) is discussed in this article. It is shown that the improved WVD, based on CSD of a long time series, can render the time–frequency distribution less susceptible to noise, and restrain the cross-terms in the time–frequency domain. Simulation and experiment of the rolling element-bearing fault diagnosis are performed, and the results indicate the validity of WVD based on CSD in time–frequency analysis for bearing fault detection.

In order to overcome the limitation of traditional near-field acoustical holography (NAH), that the sound field on one side of the holographic surface must be free, a sound field separation technique based on single holographic surface and wave superposition method (WSM) is proposed. According to the WSM, the field on and near the measurement surface may be approximated by the field produced by virtual source points placed on a surface inside the structure. The source strengths are evaluated by applying boundary conditions on the measurement surface. Here, the ‘pseudo’ sound pressure of the reconstruction surface is first obtained based on the principle of sound field mirror image and WSM, then the sound pressure of the target sound source acting on the holographic surface is separated by the sound pressures of the holographic surface and the reconstruction surface, and the sound field separation is realized. The technique requires the inversion of the Green's function matrix, which may be ill-conditioned. The Tikhonov regularization method is used to invert it, and the value of the regularization parameter is determined by the L-curve criteria. Through the numerical simulation and experiment, the results show the validity and efficiency of this technique.

Sound field visualization is a helpful design and analysis tool for the study of sound radiation and dispersion problems. It can help to comprehend deeply about noise transmission mechanism, monitor environment noise, evaluate sound quality, and even diagnose the machinery faults based on mechanical noise. The well-known near-field acoustic holography is an accurate sound field visualization technique. However, this technique has disadvantages such as strict measurement requirements and the need of an enormous number of microphones, which limits its extended applications. In order to visualize the sound field with a small number of microphones for measurements, the regeneration method of the radiated field by using the wave superposition algorithm is attempted in this study. It is based on the principle of equivalent source: the sound field radiated by an arbitrarily shaped radiator is substituted by the distributed point sources (monopole or dipole) constrained inside the actual source surface. For suppressing the adverse effect of measurement noise, the Tikhonov regularization strategy is adopted to work together with the wave superposition algorithm to give an accurate solution. Numerical simulations were performed based on a two-pulse-ball model to evaluate the accuracy of the combined algorithm of the wave superposition and the Tikhonov regularization strategy. In addition, an integrated sound field visualization system is designed and implemented. The functions include acoustic signal acquisition and processing, sound field reconstruction, and results visualization. The performance of the presented system was tested by experiments in a semi-anechoic chamber by using two sound boxes to simulate the sound sources. As concerning practical measurement microphones, there exist phase mismatches between the channels. Results will go wrong if the sound field reconstruction is performed directly with these uncalibrated measurement data. Therefore, a calibration procedure is applied to eliminate them. Experimental results indicate that the phase mismatches between the channels after calibration decay to 0.1°. Both the numerical simulations and experimental results accurately reconstructed the exterior sound field of the models. It is shown that the wave superposition algorithm together with the Tikhonov regularization strategy can exactly reconstruct the exterior sound field of radiators, which makes a base to its applications in practice. This sound field visualization system will make an operator's experimental work much easier.

Melon (Cucumis melo L.) is an important vegetable crop that ranks second in salt tolerance among the Cucurbitaceae. Previous studies on the two muskmelon cultivars 'Bing XueCui' (BXC) and 'Yu Lu' (YL) revealed that they had different characteristics under salt stress, but the molecular basis underlying their different physiological responses is unclear. Here, we combined a physiological study with a genome-wide transcriptome analysis to understand the molecular basis of genetic variation that responds to salt stress in the melon. BXC performed better under salt stress than YL in terms of biomass and photosynthetic characteristics, because it exhibited less reduction in transpiration rate, net photosynthesis rate, and stomatal conductance under 150-mM NaCl stress than YL. A transcriptome comparison of the leaves of the cultivars revealed that 1171 genes responded to salt stress in BXC while 1487 genes were identified as salt-stress-responsive in YL. A real-time polymerase chain reaction analysis of 12 of the responsive genes revealed that there was a strong, positive correlation with RNA sequencing data. The genes were involved in several pathways, including photosynthesis, the biosynthesis of secondary metabolites, metabolism, and plant hormone signal transduction, and their expression levels differed between the two cultivars in response to salt stress. This study provides a molecular perspective of two melon cultivars in response to salt stress, and its results could be used to investigate the complex molecular mechanisms underlying salt tolerance in the melon.

Biocompatible polymer scaffolds are promising as potential carriers for the delivery of retinal progenitor cells (RPCs) in cell replacement therapy for the repair of damaged or diseased retinas. The primary goal of the present study was to investigate the effects of blended electrospun nanofibrous membranes of silk fibroin (SF) and poly(L-lactic acid-co-ε-caprolactone) (PLCL), a novel scaffold, on the biological behaviour of RPCs in vitro. To assess the cell-scaffold interaction, RPCs were cultured on SF/PLCL scaffolds for indicated durations. Our data revealed that all the SF/PLCL scaffolds were thoroughly cytocompatible and the SF:PLCL (1:1) scaffolds yielded the best RPC growth. The in vitro proliferation assays showed that RPCs proliferated more quickly on the SF:PLCL (1:1) than on the other scaffolds and the control. Quantitative polymerase chain reaction (qPCR) and immunocytochemistry analyses demonstrated that RPCs grown on the SF:PLCL (1:1) scaffolds preferentially differentiated toward retinal neurons, including, most interestingly, photoreceptors. In summary, we demonstrated that the SF:PLCL (1:1) scaffolds can not only markedly promote RPC proliferation with cytocompatibility for RPC growth but also robustly enhance RPCs’ differentiation toward specific retinal neurons of interest in vitro, suggesting that SF:PLCL (1:1) scaffolds may have potential applications in retinal cell replacement therapy in the future.

Corneal transparency is maintained by the corneal endothelium through its barrier and ionic pump function. However, this function could be compromised with age and variety of diseases and trauma, leading to cornea dycompensation, corneal edema, bullous keratopathy and even loss of visual acuity. So far, a lot of measures have been proposed to solve the problem through promoting the corneal endothelial cells (CECs) proliferation both in vivo and in vitro. However, the exact molecular mechanism regarding the proliferation potential as well as associated phenotype maintenance of CECs has not been well clarified. Accordingly, we will review the studies outlined the signal transduction pathways that were involved in the process of CECs proliferation, which is an important and relatively seldom touched research direction for future new therapies of corneal endothelium dysfunction. By operating the crucial signaling molecular in these pathways, we anticipate to activate or block the signaling pathways and thus help engineering CEC monolayer for clinical transplantation.

Junzhao Chen,1,* Chenxi Yan,1,* Mengyu Zhu,1,* Qinke Yao,1 Chunyi Shao,1 Wenjuan Lu,1 Jing Wang,2 Xiumei Mo,2 Ping Gu,1 Yao Fu,1 Xianqun Fan1 1Department of Ophthalmology, Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, 2Biomaterials and Tissue Engineering Laboratory, College of Chemistry and Chemical Engineering and Biotechnology, Donghua University, Shanghai, People’s Republic of China *These authors contributed equally to this work Background: Cornea transplant technology has progressed markedly in recent decades, allowing surgeons to replace diseased corneal endothelium by a thin lamellar structure. A thin, transparent, biocompatible, tissue-engineered substratum with corneal endothelial cells for endothelial keratoplasty is currently of interest. Electrospinning a nanofibrous structure can simulate the extracellular matrix and have beneficial effects for cell culture. Silk fibroin (SF) has good biocompatibility but poor mechanical properties, while poly(L-lactic acid-co-Ɛ-caprolactone) (P(LLA-CL)) has good mechanical properties but poor biocompatibility. Blending SF with P(LLA-CL) can maintain the advantages of both these materials and overcome their disadvantages. Blended electrospun nanofibrous membranes may be suitable for regeneration of the corneal endothelium. The aim of this study was to produce a tissue-engineered construct suitable for endothelial keratoplasty.Methods: Five scaffolds containing different SF:P(LLA-CL) blended ratios (100:0, 75:25, 50:50, 25:75, 0:100) were manufactured. A human corneal endothelial (B4G12) cell line was cultured on the membranes. Light transmission, speed of cell adherence, cell viability (live-dead test), cell proliferation (Ki-67, BrdU staining), and cell monolayer formation were detected on membranes with the different blended ratios, and expression of some functional genes was also detected by real-time polymerase chain reaction.Results: Different blended ratios of scaffolds had different light transmittance properties. The 25:75 blended ratio membrane had the best transmittance among these scaffolds. All electrospun nanofibrous membranes showed improved speed of cell adherence when compared with the control group, especially when the P(LLA-CL) ratio increased. The 25:75 blended ratio membranes also had the highest cell proliferation. B4G12 cells could form a monolayer on all scaffolds, and most functional genes were also stably expressed on all scaffolds. Only two genes showed changes in expression.Conclusion: All blended ratios of SF:P(LLA-CL) scaffolds were evaluated and showed good biocompatibility for cell adherence and monolayer formation. Among them, the 25:75 blended ratio SF:P(LLA-CL) scaffold had the best transmittance and the highest cell proliferation. These attributes further the potential application of the SF:P(LLA-CL) scaffold for corneal endothelial transplantation. Keywords: silk fibroin, poly(L-lactic acid-co-Ɛ-caprolactone), B4G12, corneal endothelium, regeneration

MicroRNAs manifest significant functions in brain neural stem cell (NSC) self-renewal and differentiation through the post-transcriptional regulation of neurogenesis genes. Let-7b is expressed in the mammalian brain and regulates NSC proliferation and differentiation by targeting the nuclear receptor TLX, which is an essential regulator of NSC self-renewal. Whether let-7b and TLX act as important regulators in retinal progenitor cell (RPC) proliferation and differentiation remains unknown. Here, our data show that let-7b and TLX play important roles in controlling RPC fate determination in vitro. Let-7b suppresses TLX expression to negatively regulate RPC proliferation and accelerate the neuronal and glial differentiation of RPCs. The overexpression of let-7b downregulates TLX levels in RPCs, leading to reduced RPC proliferation and increased neuronal and glial differentiation, whereas antisense knockdown of let-7b produces robust TLX expression,enhanced RPC proliferation and decreased differentiation. Moreover, the inhibition of endogenous TLX by small interfering RNA suppresses RPC proliferation and promotes RPC differentiation. Furthermore, overexpression of TLX rescues let-7b-induced proliferation deficiency and weakens the RPC differentiation enhancement caused by let-7b alone. These results suggest that let-7b, by forming a negative feedback loop with TLX, provides a novel model to regulate the proliferation and differentiation of retinal progenitors in vitro.

Corneal endothelial dysfunction involves progressive corneal edema and loss of visual acuity, which result in the need for corneal transplantation. The global shortage of donor corneas limits the development of the surgery. Reconstruction of a bioengineered corneal endothelium might resolve this problem. Various scaffolds have been used, but poor biocompatibility and degradation limit their applications. In this study, a novel method of targeted cellular transplantation without permanent residence of cell carriers in the host was proposed. Human umbilical cord blood endothelial progenitor cells (UCB EPCs) were labeled with CD34 immunomagnetic nanoparticles. The efficiency of the magnet attraction was evaluated in vitro with a simple device simulating the anterior chamber. The UCB EPCs labeled with nanoparticles were transplanted into the anterior chamber of rabbits with magnet attraction. The results indicated that labeling the nanoparticles did not affect the proliferation of the UCB EPCs. The in vitro study indicated that the magnet could directionally attract UCB EPCs labeled with nanoparticles. The in vivo study indicated that the corneas in rabbits transplanted with UCB EPCs labeled with nanoparticles and magnet attraction became relatively transparent with little edema. These results showed that UCB EPCs labeled with CD34 immunomagnetic nanoparticles could be attracted directionally by a magnet and could repair corneal endothelial defects, providing a promising cell therapy for corneal endothelial dysfunction.

Purpose: To investigate the effect of the ATP-P2Y2-PI3K/Akt signaling axis on promoting rabbit corneal endothelial cell (RCEC) proliferation in vitro. Methods: Five concentrations of adenosine triphosphate (ATP; 1, 10, 25, 50 and 100 μM) were added to RCECs, and the cell proliferation was detected using Cell Counting Kit-8 (CCK8) and Ki67 immunohistochemical staining. Other P2Y2 receptor agonists and antagonists were added to the cells, and the proliferation effect was evaluated using CCK8 to determine the involvement of the P2Y2 receptor. Changes in the expression of phosphorylated Akt in RCECs treated with different concentrations of extracellular ATP and the duration of extracellular ATP on Akt phosphorylation were investigated using Western blotting. The pharmacological profiles with or without the PI3K/Akt pathway inhibitors were also determined using Western blotting. Results: We found that 10 μM ATP strongly promoted RCEC proliferation in vitro. Additionally, 25 μM ATP had a proliferation effect, whereas other concentrations (1, 50 and 100 μM) had no effect compared with the control group. Selective P2Y2 receptor agonists (UTP, ATPγS and Ap4A) showed the same promotion effect, while P2Y2 antagonists and PI3K/Akt inhibitors inhibited the effect of ATP. Moreover, phosphorylated Akt could be induced by the addition of extracellular ATP at all five concentrations and lasted for 1 h. This phosphorylation was prevented by PI3K/Akt inhibitors and a P2Y2 antagonist. Conclusions: These findings showed that 10 μM ATP markedly promoted RCEC proliferation via the P2Y2-PI3K/Akt signaling axis.

In this paper, using the information theory and the statistics analysis method, we have established the theory and method of faults diagnosis based on the multi-index fusion, including the information theory of multi-index diagnosis, diagnosis ability testing, indexes selecting, and Bayesian diagnosis modelling. And then, we applied the theories and methods to analyse the piston–liner wear condition of a diesel engine. In that, satisfactory results are achieved.

In this paper, some non-linear diagnostic methods for rotating machinery are introduced and evaluated from the view of diagnostic practice. The methods are pseudo-phase portrait, singular spectrum analysis and correlation dimension. The pseudo-phase portrait is simple, easy to draw, and is sensitive to some fault types. Singular spectrum analysis can reveal the complexity of a signal. By means of singular spectrum analysis one can reduce the noise of a signal. Correlation dimension can provide some intrinsic information of an underlying dynamical system, and can be used to classify different faults intelligently. Examples show that all these methods are advantageous in the field of non-linear diagnostics. Therefore, it is quite reasonable for effective fault diagnosis to use non-linear diagnostic methods in addition to the methods currently used, such as orbit portrait, FFT spectra, time–frequency analysis, etc.

This paper reports on the application of the correlation dimension in large rotating machinery fault diagnosis. The Grassberger-Procaccia algorithm and its modified version are introduced. Some important influencing factors relating directly to the computational precision of the correlation dimension are discussed. Industrial vibration signals measured from large rotating machinery with different faults are researched using the above-mentioned methods. The results show that the correlation dimension can provide some intrinsic information on an underlying dynamic system and can be used to classify different faults intelligently.

To investigate the dynamic changes of tear fluid after cosmetic transcutaneous lower blepharoplasty.Prospective, observational case series study.A total of 60 eyes of 30 patients with transcutaneous lower blepharoplasty were involved in the study. All subjects completed the Ocular Surface Disease Index questionnaire and underwent tear film break-up time measurements and the Schirmer test before surgery and 1 week, 1 month, and 3 months after surgery. Tear meniscus parameters measured by optical coherence tomography were recorded. Lower lid margin reflex distance was also measured. At each time point after surgery, these parameters were compared with the baseline parameters. The incidence of dry eye and chemosis after surgery was assessed.The Ocular Surface Disease Index score increased significantly 1 week after surgery (P.01). The Schirmer test values were significantly decreased 1 week postoperatively (P.01), and they returned to baseline by 3 months. There was no significant change in tear film break-up time before and after surgery. The tear meniscus height, depth, and cross section area values were significantly increased at 1 week postoperatively, decreased at 1 month (P.01), and returned to baseline at 3 months (P.05). The cornea-lower eyelid angle significantly decreased at 1 week postoperatively (P.01), increased at 1 month (P.01), and returned to baseline by 3 months (P.05). The lower lid margin reflex distance was increased 1 week after surgery (P.01), and gradually recovered but had not returned to the baseline 3 months after surgery (P.01). The increased tear meniscus height had a positive correlation with lower lid margin reflex distance. The incidence of dry eye and chemosis at 1 week postoperatively was 16.7% and 15%, respectively.Cosmetic transcutaneous lower blepharoplasty affects ocular surface and tear fluid, which leads to dryness, tearful eyes, and chemosis. However, this influence is temporary after surgery, and the symptoms resolve within 3 months. A positive correlation between tear meniscus height and lower lid margin reflex distance was observed. Assessment of the tear meniscus with optical coherence tomography provided a quick, noninvasive, and quantitative method to investigate the dynamic changes of the tear fluid after blepharoplasty.

Zi Wang,1,* Ming Lin,1,* Qing Xie,1 Hao Sun,1 Yazhuo Huang,1 DanDan Zhang,1 Zhang Yu,1 Xiaoping Bi,1 Junzhao Chen,1 Jing Wang,2 Wodong Shi,1 Ping Gu,1 Xianqun Fan1 1Department of Ophthalmology, Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, 2Biomaterials and Tissue Engineering Laboratory, College of Chemistry & Chemical Engineering and Biotechnology, Donghua University, Shanghai, People’s Republic of China *These authors contributed equally tothis work Background: Tissue engineering has become a promising therapeutic approach for bone regeneration. Nanofibrous scaffolds have attracted great interest mainly due to their structural similarity to natural extracellular matrix (ECM). Poly(lactide-co-ε-caprolactone) (PLCL) has been successfully used in bone regeneration, but PLCL polymers are inert and lack natural cell recognition sites, and the surface of PLCL scaffold is hydrophobic. Silk fibroin (SF) is a kind of natural polymer with inherent bioactivity, and supports mesenchymal stem cell attachment, osteogenesis, and ECM deposition. Therefore, we fabricated hybrid nanofibrous scaffolds by adding different weight ratios of SF to PLCL in order to find a scaffold with improved properties for bone regeneration.Methods: Hybrid nanofibrous scaffolds were fabricated by blending different weight ratios of SF with PLCL. Human adipose-derived stem cells (hADSCs) were seeded on SF/PLCL nanofibrous scaffolds of various ratios for a systematic evaluation of cell adhesion, proliferation, cytotoxicity, and osteogenic differentiation; the efficacy of the composite of hADSCs and scaffolds in repairing critical-sized calvarial defects in rats was investigated.Results: The SF/PLCL (50/50) scaffold exhibited favorable tensile strength, surface roughness, and hydrophilicity, which facilitated cell adhesion and proliferation. Moreover, the SF/PLCL (50/50) scaffold promoted the osteogenic differentiation of hADSCs by elevating the expression levels of osteogenic marker genes such as BSP, Ocn, Col1A1, and OPN and enhanced ECM mineralization. In vivo assays showed that SF/PLCL (50/50) scaffold improved the repair of the critical-sized calvarial defect in rats, resulting in increased bone volume, higher trabecular number, enhanced bone mineral density, and increased new bone areas, compared with the pure PLCL scaffold.Conclusion: The SF/PLCL (50/50) nanofibrous scaffold facilitated hADSC proliferation and osteogenic differentiation in vitro and further promoted new bone formation in vivo, suggesting that the SF/PLCL (50/50) nanofibrous scaffold holds great potential in bone tissue regeneration. Keywords: human adipose-derived mesenchymal stem cells, silk fibrin, poly(lactide-co-ε-caprolactone), proliferation, osteogenesis

Study question What are the implications of multiple alterations of the forkhead box L2 (FOXL2) gene in blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) patients? Summary answer A multi-mutation of FOXL2, consisting of the expansion of the polyalanine tract from 14 to 24 residues (FOXL2-Ala24), an novel Y186C substitution from c.557A>G, and a synonymous variant (c.505G>A), had a cumulative effect on ovarian phenotypes in BPES patients. What is known already Mutations in FOXL2, a gene encoding a forkhead transcription factor cause BPES. Overall, the expansion of the polyalanine tract of FOXL2 from 14 to 24 residues (FOXL2-Ala24) accounts for 30% of intragenic mutations. Study design, size, duration In this study, patients from seven BPES families and six sporadic cases were included. Participants/materials, setting, methods We conducted an extensive clinical, hormonal and functional study in 20 patients carrying the expansion of the polyalanine tract of FOXL2 associated with BPES. A multi-mutation of FOXL2 was detected in one BPES family that showed more severe BPES symptoms. Subcellular localization and transactivation studies were performed for the constructs of FOXL2-Ala24, Y186C and FOXL2-Ala24-Y186C. Main results We described the first multi-mutation of FOXL2 (c. [672_701dup30; 557A>G]) that leads to the polyalanine expansion of +10 residues (FOXL2-Ala24) combined with an Y186C substitution and a synonymous variant in a Chinese BPES family. This multi-mutation genotype was associated with more serious BPES clinical manifestations and the development of esotropia in the right eye. In in vitro studies, the multi-mutation affected the function of FOKL2 on the StAR promoter and DK3, and induced more aggressive aggregation and mislocalization of FOXL2 protein. The synonymous variant, while not affecting amino acid coding, causes a change in the RNA stem-loop structure. Limitations, reasons for caution The multi-mutation of FOXL2 was detected in one BPES family and it needs to be validated further by more BPES subjects. Wider implications of the findings The results of our study contribute new insights into the research field of BPES caused by the multi-mutation of FOXL2. Study funding/competing interests This study was supported by Shanghai Leading Academic Discipline Project (Grant number S30205) and Shanghai Jiao Tong University School of Medicine Doctor Innovation Fund (Grant number 201131). The authors have no competing interests to declare.

Bearing is one of the most important units in rotary machinery, its performance may vary significantly under different working stages. Thus it is critical to choose the most effective features for bearing performance degradation prediction. Linear Discriminant Analysis (LDA) is a useful method in finding few feature's dimensions that best discriminate a set of features extracted from original vibration signals. Another challenge in bearing performance degradation is how to build a model to recognize the different conditions with the data coming from different monitoring channels. In this paper, coupled hidden Markov models (CHMM) is presented to model interacting processes which can overcome the defections of the HMM. Because the input data in CHMM are collected by several sensors, and the interacting information can be fused by coupled modalities, it is more effective than HMM which used only one state chain. The model can be used in estimating the bearing performance degradation states according to several observation data. When becoming degradation pattern recognition, the new observation features should be input into the pre-trained CHMM and calculate the performance index (PI) of the outputs, the changing of PI could be used to describe the different degradation level of the bearings. The results show that PI will decline with the increase of the bearing degradation. Assessment results of the whole life time experimental bearing signals validate the feasibility and effectiveness of this method.