RNF220 is an E3 ubiquitin ligase for AMPA receptors to regulate synaptic transmission.

The accurate expression of postsynaptic AMPA receptors (AMPARs) is critical for information processing in the brain, and ubiquitination is a key regulator for this biological process. However, the roles of E3 ubiquitin ligases in the regulation of AMPARs are poorly understood. Here, we find that RNF220 directly interacts with AMPARs to meditate their polyubiquitination, and RNF220 knockout specifically increases AMPAR protein levels, thereby enhancing basal synaptic activity while impairing synaptic plasticity. Moreover, depending on its E3 ubiquitin ligase activity, RNF220 represses AMPAR-mediated excitatory synaptic responses and their neuronal surface expression. Furthermore, learning and memory are altered in forebrain RNF220-deficient mice. In addition, two neuropathology-related RNF220 variants fail to repress excitatory synaptic activity because of the incapability to regulate AMPAR ubiquitination due to their attenuated interaction. Together, we identify RNF220 as an E3 ubiquitin ligase for AMPARs and establish its substantial role in excitatory synaptic transmission and brain function.

A flexible and cost-effective manual droplet operation platform for miniaturized cell assays and single cell analysis.

Herein, we developed a flexible and cost-effective manual droplet operation system (MDOS) for performing miniaturized cell assays as well as single cell analysis. The MDOS consists of a manual x-y-z translation stage for liquid transferring and switching, a high-precision syringe pump for liquid driving and metering, a tapered capillary probe for droplet manipulation, a droplet array chip for droplet loading and reaction, sample/reagent reservoirs for storage, and a microscope for droplet observation, with a total expense of only $4,000. By using the flexible combination of three elementary operations of the x-y-z stage's moving and the pump's aspirating and depositing, the MDOS can manually achieve multiple droplet handling operations in the nanoliter to picoliter range, including droplet generation, assembling, fusion, diluting, and splitting. On this basis, multiple cell-related operations could be performed, such as nanoliter-scale in-droplet cell culture, cell coculture, drug stimulation, cell washing, and cell staining, as well as formation of picoliter single-cell droplets. The feasibility and flexibility of the MDOS was demonstrated in multi-mode miniaturized cell assays, including cell-based drug test, first-pass effect assay, and single-cell enzyme assay. The MDOS with the features of low cost, easy to build and flexible to use, could provide a promising alternative for performing miniaturized assays in routine laboratories, in addition to conventional microfluidic chip-based systems and automated robot systems.

Signal peptide represses GluK1 surface and synaptic trafficking through binding to amino-terminal domain.

Kainate-type glutamate receptors play critical roles in excitatory synaptic transmission and synaptic plasticity in the brain. GluK1 and GluK2 possess fundamentally different capabilities in surface trafficking as well as synaptic targeting in hippocampal CA1 neurons. Here we find that the excitatory postsynaptic currents (EPSCs) are significantly increased by the chimeric GluK1(SPGluK2) receptor, in which the signal peptide of GluK1 is replaced with that of GluK2. Coexpression of GluK1 signal peptide completely suppresses the gain in trafficking ability of GluK1(SPGluK2), indicating that the signal peptide represses receptor trafficking in a trans manner. Furthermore, we demonstrate that the signal peptide directly interacts with the amino-terminal domain (ATD) to inhibit the synaptic and surface expression of GluK1. Thus, we have uncovered a trafficking mechanism for kainate receptors and propose that the cleaved signal peptide behaves as a ligand of GluK1, through binding with the ATD, to repress forward trafficking of the receptor.

The Diffusion of Low-Energy Methyl Group on ITO Film Surface and Its Impact on Optical-Electrical Properties.

Indium tin oxide (ITO) film is one of the ideal candidates for transparent conductive cathode in methylammonium lead halide perovskite solar cells. Thus, the diffusion of methyl group in ITO film is inevitable, which could deteriorate the optical-electrical property of ITO film. In this study, ITO films with and without (100) preferred orientation were bombarded by the low-energy methyl group beam. After the bombardment, the optical-electrical property of ITO film without (100) preferred orientation deteriorated. The bombardment of methyl group had little influence on the optical-electrical property of ITO film with (100) preferred orientation. Finally, combining the crystallographic texture and chemical bond structure analysis, the diffusion mechanism of low-energy methyl group on ITO lattice and grain boundary, as well as the relation between the optical-electrical property and the diffusion of the methyl group, were discussed systematically. With the above results, ITO film with (100) preferred orientation could be an ideal candidate for transparent conductive cathode in methylammonium lead halide perovskite solar cells.

A Microfluidic Droplet Array System for Cell-Based Drug Combination Screening.

In the last few decades, drug combination therapy has been widely applied in oncology and in other complex diseases. Due to its potential advantage of lower drug toxicity and higher therapeutic efficacy, drug combination treatment has been more and more studied in fundamental labs and pharmacy companies. In this chapter, we report cell-based drug combination screening using a microfluidic droplet system based on a sequential operation droplet array (SODA) technique. In this system, an oil-covered two-dimensional droplet array chip was used as the platform for cell culture and analysis. This chip was fixed in an x-y-z translation stage under control of a computer program. A tapered capillary connected with a syringe pump was coupled with the droplet array chip to achieve multiple droplet manipulations including liquid metering, aspirating, depositing, mixing, and transferring. Complex multistep operations for drug combination screening involving long-term cell culture, medium changing, schedule-dependent drug dosage and stimulation, and cell viability testing were achieved in parallel using the present system. The drug consumption for each screening test was substantially decreased to 5 ng-5 µg, corresponding to 10- to 1000-fold reductions compared with traditional drug screening systems with 96- or 384-well plates.

Three-Dimensional Cell Culture and Drug Testing in a Microfluidic Sidewall-Attached Droplet Array.

Three-dimensional (3D) cell culture provides an effective way over conventional two-dimensional (2D) monolayer culture to more closely imitate the complex cellular organization, heterogeneity, and interactions as well as tissue microenvironments in vivo. Here we present a novel droplet-based 3D cell culture method by using droplet array attached on the sidewall of a PDMS piece. Such an arrangement not only avoids cells from adhering on the chip surface for achieving 3D cell culture in nanoliter-scale droplets, but also facilitates performing multiple operations to cells in droplets, including cell suspension droplet generation, drug treatment, and cell staining with a capillary-based liquid handling system, as well as in situ observation and direct scanning with a confocal laser scanning microscope. We optimized the system by studying the effects of various conditions to cell culture including droplet volume, cell density and fabrication methods of the PDMS pieces. We have applied this system in the 3D culture of HepG2 cells and the stimulation testing of an anticancer drug, doxorubicin, to 3D cell spheroids.

Microdroplet chain array for cell migration assays.

Establishing cell migration assays in multiple different microenvironments is important in the study of tissue repair and regeneration, cancer progression, atherosclerosis, and arthritis. In this work, we developed a miniaturized and massive parallel microfluidic platform for multiple cell migration assays combining the traditional membrane-based cell migration technique and the droplet-based microfluidic technique. Nanoliter-scale droplets are flexibly assembled as building blocks based on a porous membrane to form microdroplet chains with diverse configurations for different assay modes. Multiple operations including in-droplet 2D/3D cell culture, cell co-culture and cell migration induced by a chemoattractant concentration gradient in droplet chains could be flexibly performed with reagent consumption in the nanoliter range for each assay and an assay scale-up to 81 assays in parallel in one microchip. We have applied the present platform to multiple modes of cell migration assays including the accurate cell migration assay, competitive cell migration assay, biomimetic chemotaxis assay, and multifactor cell migration assay based on the organ-on-a-chip concept, for demonstrating its versatility, applicability, and potential in cell migration-related research.

Integrating atomic layer deposition and ultra-high vacuum physical vapor deposition for in situ fabrication of tunnel junctions.

Atomic Layer Deposition (ALD) is a promising technique for growing ultrathin, pristine dielectrics on metal substrates, which is essential to many electronic devices. Tunnel junctions are an excellent example which require a leak-free, ultrathin dielectric tunnel barrier of typical thickness around 1 nm between two metal electrodes. A challenge in the development of ultrathin dielectric tunnel barriers using ALD is controlling the nucleation of dielectrics on metals with minimal formation of native oxides at the metal surface for high-quality interfaces between the tunnel barrier and metal electrodes. This poses a critical need for integrating ALD with ultra-high vacuum (UHV) physical vapor deposition. In order to address these challenges, a viscous-flow ALD chamber was designed and interfaced to an UHV magnetron sputtering chamber via a load lock. A sample transportation system was implemented for in situ sample transfer between the ALD, load lock, and sputtering chambers. Using this integrated ALD-UHV sputtering system, superconductor-insulator-superconductor (SIS) Nb-Al/Al2O2/Nb Josephson tunnel junctions were fabricated with tunnel barriers of thickness varied from sub-nm to ~1 nm. The suitability of using an Al wetting layer for initiation of the ALD Al2O3 tunnel barrier was investigated with ellipsometry, atomic force microscopy, and electrical transport measurements. With optimized processing conditions, leak-free SIS tunnel junctions were obtained, demonstrating the viability of this integrated ALD-UHV sputtering system for the fabrication of tunnel junctions and devices comprised of metal-dielectric-metal multilayers.

Cell-based drug combination screening with a microfluidic droplet array system.

We performed cell-based drug combination screening using an integrated droplet-based microfluidic system based on the sequential operation droplet array (SODA) technique. In the system, a tapered capillary connected with a syringe pump was used for multistep droplet manipulations. An oil-covered two-dimensional droplet array chip fixed in an x-y-z translation stage was used as the platform for cell culture and analysis. Complex multistep operations for drug combination screening involving long-term cell culture, medium changing, schedule-dependent drug dosage and stimulation, and cell viability testing were achieved in parallel in the semiopen droplet array, using multiple droplet manipulations including liquid metering, aspirating, depositing, mixing, and transferring. Long-term cell culture as long as 11 days was performed in oil-covered 500 nL droplets by changing the culture medium in each droplet every 24 h. The present system was applied in parallel schedule-dependent drug combination screening for A549 nonsmall lung cancer cells with the cell cycle-dependent drug flavopiridol and two anticancer drugs of paclitaxel and 5-fluorouracil. The highest inhibition efficiency was obtained with a schedule combination of 200 nM flavopiridol followed by 100 µM 5-fluorouracil. The drug consumption for each screening test was substantially decreased to 5 ng-5 µg, corresponding to 10-1000-fold reductions compared with traditional drug screening systems with 96-well or 384-well plates. The present work provides a novel and flexible droplet-based microfluidic approach for performing cell-based screening with complex and multistep operation procedures.

[Prediction and identification of B-cell linear epitopes of hepatitis B e antigen].

OBJECTIVE: To predict and identify B-cell linear epitopes of hepatitis B e antigen (HBeAg). METHODS: The B-cell linear epitopes of HBeAg were predicted using the software provided by NCBI Database and Immune Epitope Database (IEDB) and synthesized by a solid-phase method followed by conjugation with keyhole limpet hemocyanin (KLH). The KLH conjugates were used for immunization of New Zealand white rabbits, and the immune response of the rabbits was monitored by direct ELISA using a bovine serum albumin conjugate of the predicted epitopes. RESULTS Four new B-cell linear epitopes of HBeAg were identified, namely (1)MDIDPYKEFG(10), (37)LYREALESPEHCSP(50), (74)SNLEDPAS(81) and (127)RTPPAYRPPNAPIL(140). The rabbits immunized with the KLH conjugate showed an antibody titer over 1:512 000. The antisera of B-cell linear epitopes collected could specifically react with HBeAg as shown by ELISA. CONCLUSION: Four B-cell linear epitopes of HBeAg have been confirmed using bioinformatics methods, which provides new evidence for further functional studies of HBeAg in hepatitis B.

[Effect of recombinant human erythropoietin on bcl-2 protein expression in the retina in a rabbit model of acute high intraocular pressure].

OBJECTIVE: To investigate the effect of recombinant human erythropoietin (rhEPO) on the expression of bcl-2 protein in the retina of rabbits with acute high intraocular pressure and explore the mechanism underlying the protective effect of rhEPO on the retina against ischemia-reperfusion injury. METHODS: rhEPO was injected subcutaneously in the ear of a rabbit model of acute high intraocular pressure induced by physiological saline perfusion into the anterior chamber. Bcl-2 protein expression in the retina of the rabbits was observed by immunohistochemical staining on days 1, 3, 7, and 14 after retinal ischemia-reperfusion and compared with that in normal rabbits and untreated rabbit models. RESULTS: bcl-2-positive cells were observed in the retina of normal rabbits with a mean positive cell number of 10.5-/+1.2 in each high-power visual field. Compared with that in the normal control group, the number of the positive cells decreased significantly in both the model group and EPO group (P<0.05, P<0.01), but the latter group showed a significantly greater number than the former (P<0.05 at day 7 and P<0.01 at day 14). CONCLUSION: Systemic administration of rhEPO can up-regulate the expression of bcl-2 protein in the retina of rabbits with acute high intraocular pressure, which is probably one of the mechanisms for the protective effect of rhEPO on the retina against ischemia-reperfusion injury.

Hepatocellular carcinoma-associated protein markers investigated by MALDI-TOF MS.

Hepatocellular carcinoma (HCC) is one of the most common types of cancer worldwide. The initial hepatocellular alterations that precede the appearence of HCC include chronic viral hepatitis/cirrhosis, foci of phenotypically altered hepatocytes and, subsequently, dysplastic hepatocytes that form foci and nodules. These changes cause a discrepancy in the microenvironment of liver cells, which may result in changes in the protein expression profile of the cells. The aim of the present study was to investigate differences between the protein expression profiles at various stages of liver disease in order to better understand the mechanisms of HCC and to identify potential biomarkers for its early diagnosis. The proteins of specific cells were obtained from HCC tissue sections and pre-cancerous lesions using a manual microdissection technique, and were investigated by a two dimensional gel electrophoresis (2-DE) MALDI-TOF MS proteomics approach. Select identified proteins were reconfirmed by immunohistochemistry. A total of 95 differentially expressed proteins, with an over 2-fold disparity in expression levels between cells of varying morphology during the stages of hepatocarcinogenesis, were detected by 2-DE. Among these 95 proteins, 80 were determined to be involved in numerous cell functions, including cell growth and proliferation, protein synthesis and metabolism, apoptosis and signal transduction. These identified proteins, which include stratifin (14-3-3), transgelin 2, heat-shock protein (HSP)70, HSP27, manganese superoxide dismutase, prohibitin, DJ1, α-enolase, peroxiredoxin 6, aldo-keto reductase family member B10, phosphoglycerate kinase 1, α-1-antitrypsin and nm23-H1, may play a role in the development of HCC. Protein expression profiles differed markedly between the HCC tissue samples and pre-cancerous lesions, suggesting that alterations in protein expression occurred frequently during the process of hepatocarcinogenesis. Analysis of the differential expression of proteins related to the development of HCC may help elucidate the molecular mechanisms of the disease. These proteins may also serve as candidate biomarkers for early HCC diagnosis.

[Effects of rAAV-mediated rhBDNF gene transfection on BDNF gene expression in the retina of a rabbit model of acute high intraocular pressure].

OBJECTIVE: To observe the changes in the expression of brain derived neurotrophic factor (BDNF) gene in the retina of rabbits with acute high intraocular pressure (IOP) after injection of recombinant adeno-associated virus (rAAV) vector containing human BDNF gene (rAAV-hBDNF), and investigate the neuroprotective mechanism of rAAV-hBDNF. METHODS: The unilateral eyes of 24 white rabbits were randomly chosen as the model group with high IOP induced by saline perfusion into the anterior chamber, and the contralateral eyes served as the control group without treatment. In another 24 white rabbits, 10 microl rAAV-BDNF was injected into the vitreous body of one of the eyes 3 days before induction of high IOP. On days 1, 3, 7, and 14 after perfusion, the bilateral eyes of 6 rabbits were excised for immunohistochemistry for the expression of endogenous BDNF gene in the retina. RESULTS: The number of BDNF-positive cells in the retina decreased after induction of high IOP, and injection of rAAV-hBDNF resulted in a significant increase in BDNF-positive cells as compared with the positive cell number in the high IOP model and control groups (P<0.05, P<0.01). CONCLUSION: rAAV-mediated BDNF gene transfection can increase endogenous BDNF expression in the retina of rabbits with acute high IOP. Intravitreous injection is an effective pathway for rAAV-hBDNF gene transfection into the retina.

[Neuroprotective effect of rAAV-mediated rhBDNF gene transfection on rabbit retina against acute high intraocular pressure].

OBJECTIVE: To investigate the neuroprotective effect of human brain-derived neurotrophic factor gene transfection into rabbit retina against acute high intraocular pressure (HIOP). METHODS: Acute HIPO was induced in one eye of 24 white rabbits via saline perfusion into the anterior chamber (model group), and the contralateral eye without treatment served as the control group. In another 24 rabbits, 10 microl recombinant adeno-associated virus (rAAV) vector containing human BDNF gene (rAAV-BDNF) was injected into the vitreous body of one of the eyes 3 days before the operation for HIPO (BDNF group). At 1, 3, 7, and 14 days after HIOP model establishment, 6 eyes in each group were excised to observe the number of retinal ganglion cells (RGCs) and the thickness of the inner retina layer. For the eyes dissected on day 14, electroretinogram b (ERG-b) wave was detected 30 min before (baseline) and on days 1, 3, 7 and 14 after HIOP. Another 5 rabbits were used for ultrastructural observation of the RGCs using transmission electron microscopy, including 1 without treatment, 2 with unilateral HIOP and 2 with rAAV-BDNF transfection before HIOP. RESULTS: The amplitude of ERG-b wave showed no significant difference between the 3 groups before HIOP (P>0.05). In HIOP model group and BDNF group, the amplitude decreased to the lowest at 1 day after HIOP and failed to recover the baseline level at 14 days (P<0.01); at the end of the observation, the amplitude was significantly higher in BDNF group than in the model group (P<0.01). Decreased number of RGCs and thickness of inner retina layer occurred in the model group, but these changes were milder in BDNF group (P<0.05, P<0.01). Electron microscopy revealed ultrastructural changes in the RGCs following acute HIOP, and transfection with rAAV-BDNF ameliorated these changes. CONCLUSION: rAAV-BDNF transfection protects the retinal structure and improves the amplitude of ERG-b wave after acute high IOP suggesting its neuroprotective effects.

[Histomorphology and proteomics analysis of human fetus liver].

OBJECTIVE: To investigate the histomorphology and protein expression profiles of human fetal liver at different developmental stages. METHODS: The protein expression patterns of human fetus livers at early and late stages were profiled by two-dimensional gel electrophoresis (2-DE) combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis. The differentially expressed proteins were identified by searching the protein databases with Mascot or ProFound softwares. RESULTS: The obvious differences between early and late fetus liver were not only in morphology but also in protein expression profiles. Compared with protein expression pattern of hepatocellular carcinoma, 32 differential or similar protein spots were analyzed by MALDI-TOF MS, and 26 proteins, including proteins involved in cell proliferation, apoptosis and metabolism, were identified. CONCLUSION: The data suggest that the hepatic cancer cells regain some characteristics of fetal hepatic cells during the process of carcinogenesis.

[Effects of recombinant human erythropoietin on hypoxia inducible factor-1alpha expression in the retina of rabbits with acute high intraocular pressure].

OBJECTIVE: To observe the effect of recombinant human erythropoietin (rhEPO) on the expression of hypoxia inducible factor-1alpha (HIF-1alpha) in the retina of rabbits with acute high intraocular pressure and investigate the mechanism of rhEPO in protecting the retina from ischemia-reperfusion injury. METHODS: Acute high intraocular pressure was induced in the rabbits by perfusion of normal saline into the anterior chamber, and rhEPO was injected subcutaneously. The changes in HIF-1alpha protein expression in the retina was observed by immunohistochemistry on days 1, 3, 7, and 14 after retinal ischemia- reperfusion. RESULTS: HIF-1alpha expression was not observed in the retina of the normal control rats, but intense HIF-1alpha expression was found in the model group (P<0.01). In rabbits with rhEPO injection and those in the model group, the patterns of HIF-1alpha expression alterations were similar, but the HIF-1alpha-positive cells in the retina were significantly fewer in rhEPO group (P<0.05). CONCLUSION: rhEPO can down-regulate HIF-1alpha expression in the retina of rabbits with acute high intraocular pressure, which may be one of the mechanisms that rhEPO protects the retina from ischemia-reperfusion injury.

[Study on tetrahydrobiopterin deficiency in Northern Chinese population].

OBJECTIVE: To emphasize early differential diagnosis from patients with hyperphenylalaninemia (HPA) and to evaluate the treatment and long-term outcome of patients with tetrahydrobiopterin synthase (BH4) deficiency in Northern Chinese population. METHODS: From 1992 to 2005, a total of 618 patients with HPA were diagnosed and/or cared for in our outpatient clinic. Urinary pterin analysis, detection of dihydropteridine reductase (DHPR) activity in blood, and then BH4 loading tests were carried out to differentiate BH4 deficiency in these patients from classical phenylketonuria. BH4 deficient patients were treated with BH4, levodopa and 5-hydroxytryptophane (5-HTP) immediately while the diagnosis was done to disease. Patientso blood phenylalanine levels, psychomotor and intelligence development were followed up. RESULTS: A total of 38 cases were diagnosed as BH4 deficiency, all of them were revealed as 6-pyruvoyl-tetrahydropterin synthase (PTPS) deficiency from the extremely decreased urine biopterin, normal DHPR activities and drop down of blood phenylalanine level to normal range within 4 to 8 hours after BH4 loading. The most common manifestations were progressively psychomotor and mental retardation to patients even after taking early dietary treatment. The patients were diagnosed and treated with drugs at the ages of 2.1 months to 13 years. With 4 patients died of pneumonia, 7 patients refused to treatment, only 27 patients were under treatment and followed up. The average full scale development or intelligence quotient (DQ/IQ) of patients who were treated within and after 6 months were 86+/- 10 or 66+/- 7 respectively. Development was not even in different aspects. A significant negative correlation was observed between the level of the DQ and the age of treatment commenced (r was -0.714, P< 0.01). Eleven patients experienced the extrapyramidal movement disorders, 3 of them combined with epilepsy. The extrapyramidal disorders were controlled by administration of levodopa. CONCLUSION: The differential diagnosis for BH4 deficiency should be carried out in all patients with HPA. PTPS deficiency is the most common form of BH4 deficiency in Northern Chinese population. The long-term outcome of these patients benefits from diagnosis and treatment with BH4, levodopa and 5-HTP as early as possible.