Abstract:Based on the micro-fabrication techniques combining with biochemistry and biophysics, we can get function structures with feature sizes close to the biomacromolecule scale, which promotes the applications of micropatterning in many research fields such as drug screening and discovery, tissue engineering and disease diagnosis. This review summarizes the development of micropatterning techniques in biomedical field and analyzes the advantages, limitations and application scopes of each micropatterning approach including photolithography, soft lithography, stencil-assisted patterning, scanning-probe lithography, jet patterning and laser guided patterning. Photolithography usually includes several steps such as exposure, development, lift-off and so on. Although it has the advantages of high accuracy, high efficiency and accurate alignment system, it depends on super-clean labs and lift-off processes, which means high cost and unsatisfied bio-compatibility. Soft lithography and stencil-assisted patterning methods avoid exposure and lift-off steps by using elastomeric stamps, which can enhance the bio-compatibility and reduce the cost. However, these two methods have deficiencies in alignment accuracy. Different from above methods, scanning-probe lithography is a kind of direct-writing technique, which sacrifices the advantage of high efficiency to improve its accuracy. Jet patterning is developed from industry with the advantages of low complexity and cost. However, its low accuracy of 10 μm scale is the limitation. Two novel laser based micropatterning techniques are also discussed. Although laser-induced transfer method solves the problem of jet patterning technique in the patterning thickness control, the low accuracy is still a problem. Optical tweezers technique offers a substitution for the scanning-probe lithography, although it has a long way to go in terms of liquor environment limitation and efficiency. It is indicated that current micropatterning methods already have the ability to make micro devices featured from nanometer scale to millimeter scale on a variety of surface materials different in geometry, stiffness and so on. The resolution and accuracy, the patterning scale and the processing condition are the bases for choosing micropatterning methods. The development of micropatterning techniques provides a rapid, real time, and accurate study tool in biological mechanism, drug action and biochemical reaction research. Micropatterning methods can enhance the sensitivity, the automation degree and the integration scale biosensors, which will further improve the efficiency of drug screening and diseases diagnosis. Also by micropatterning techniques, we can control the cells action easily, accurately and concurrently, which is helpful to shorten the development cycle. The main trends of micropatterning research are the further physicochemical analyses of the particles on nano-scale based on biochemistry and biophysics, the further enhancement of its bio-compatibility and material adaptability, as well as the development of in vivo microenvironment simulations suitable for micropatterning chips.

Abstract:Epithelial surfaces of the upper alimentary tract and upper respiratory tract are swarming with protein compounds that protect itself from many kinds of damages. These compounds are consisted of innate immune molecules. From the structure and functional prediction, the new member is added, PLUNC family, to the compounds. They are including at least ten members. SPLUNC1 (short palate, lung, and nasal epithelium clone 1) is isolated from human nasopharyngeal epithelia by suppression subtractive hybridization (SSH) and cDNA microarray analysis in our lab. SPLUNC1 is a gene transcript of the PLUNC gene family and also is a member of the BPI (bactericidal permeability increasing protein)/LBP(lipopolysaccharide-binding protein) family with putative bactericidal/bacteriostatic functions. They may function to protect epithelial surfaces from pathogenic micro-organism and harmful gases, the wrong expression will lead to host tissues destruction and tumorigenesis.

Abstract:Autism spectrum disorder (ASD) is a defective mental disease and its core impairments are social function defect, communication defect, restrictive and stereotyped behavior pattern. The paper introduces the genetic basis and neural mechanism of ASD. ASD has high genetic rate, and 5-HT and testosterone of ASD individual are both higher. Neuroimaging studies find that there are some differences between ASD and normal individuals in the structure and function of amygdala, cingulate gyrus, the fusiform gyrus, mirror neurons, prefrontal lobe and other brain areas, but it is inconsistent in the discrepancy direction of some areas’ activation patterns. In addition, the results of functional connectivity studies also confirm the hypothesis that the ASD individuals are under-connection. Future research should focus more on how to use the basic research outcomes to put forward effective treatment and training for clinical research.

Abstract:Aquaporins (AQPs) got their name from the water transport ability, and recently, their multi-transport activities and multiple physiological functions were also frequently reported. This review focused on the analysis of recent progress on physiological functions and subcellular localizations of plant aquaporins. The relationship between the multi-physiological functionality and the multiple intracellular localizations and redistribution of plant aquaporins were also thoroughly discussed, thus raising questions about the current research and research direction for plant aquaporins. To reveal the possible mechanism on the multiple physiological functions, it is necessary and essential to analyze the connections between functions and tissue and subcellular localizations in future.

Abstract:The risk factors of human cardiovascular diseases are related to plasma cholesterol levels. The major carrier of plasma cholesterol is lipoprotein. Lipoprotein structure-function relationship provides important clues that help identify the role of lipoproteins in cardiovascular disease. The structure determination of lipoprotein is challenging by using traditional approaches, such as X-ray crystallography, nuclear magnetic resonance spectroscopy, mass spectrometry and cryo-electron microscopy. The compositional and conformational heterogeneity of lipoproteins are major barriers to the identification of lipoprotein structures. Recently, Ren et al. reported an optimized negative-staining protocol to directly visualize the structure of each individual lipoprotein particle. The statistical analysis validated this protocol in examining lipoprotein structures. The high-quality image of lipoprotein provides a basis for three-dimensional structure determination of a single lipoprotein. Moreover, this protocol can be used as a general protocol to examine and screen molecular drugs for treating human diseases.

Abstract:MicroRNAs (miRNAs) are a class of small non-coding RNAs that play important roles in post-transcriptional regulation of gene expression[1]. A large number of miRNAs have been found to be involved in a broad spectrum of biological functions such as regulation of innate and adaptive immunity, cell differentiation and development as well as disease pathogenesis, especially in cancer[2-8]. However, the molecular mechanisms remain largely unknown, which severely blocks the miRNA therapeutic and preventative strategies. Some recent studies have provided valuable information to further unveil the mysteries of miRNA.
High-throughput miRNA microarray and small RNA sequencing have identified a large set of miRNAs involved in development of mammal and plant[6,9]. Dysregulation of the miRNA pathway have great impact on neuronal and glial development in the mammalian brain and could lead to neurodevelopmental and neurodegenerative diseases[4]. Furthermore, wide-ranging functions for miRNAs have been identified using gain- and loss-of-function studies, which provide new perspectives regarding regulation mechanisms of miRNAs in heart development and disease[5]. In plants, miRNAs are predicted to be involved in nearly all biological processes, such as cell development, differentiation, and stress responses[9].
Deregulated expression pattern of large set of miRNAs in cancer and their interactions with oncogenes and tumor suppressor genes demonstrate these miRNAs may be involved in tumorigenesis[10-13]. Recent study from He et al. have shown that chromosome gain of miR-151 is a crucial stimulus for tumour invasion and metastasis of hepatocellular carcinoma[11]. Another example, miR-181b is highly expressed in acute myeloid leukemia (AML) and contributed to proliferation of AML cells by targeting MLK2[12]. Furthermore, several differentially expressed miRNAs may be involved in uveal melanoma pathogenesis, and may serves as informative biomarkers for uveal melanoma[13]. Notably, miRNAs have been found to be encoded by animal and plant viral genomes, and many virus-encoded miRNAs have been found to be involved in the cancer development[14-15]. Marek's disease virus (MDV)-encoded miRNAs play key roles in lytic replication, latent infection, T-lymphocyte transformation and tumorigenesis[14]. Lan et al. found a miRNA, miR-K12-11, encoded by Kaposi's sarcoma-associated herpesvirus (KSHV), that is critical for the modulation of IFN signaling by targeting IκB kinase ε and can contribute to maintenance of KSHV latency[3]. All the above studies give us great insights into the pathogenesis of miRNA related cancer.
The advantages of high-throughput sequencing (HTS) technology provide us the abilities to illustrate the complexity of genome and transcriptome more comprehensively and generate an unprecedented landscape of miRNA for various species[16-18]. By virtue of the HTS technique, "competitive endogenous RNA" (ceRNA) hypothesis were propose recently[19], which consider messenger RNAs and long non-coding RNAs, a kind of RNA with regulation[20-21], could communicate each other through a new "language" mediated by microRNA-binding sites. Notably, some previous bioinformatic analysis conducted by Zhao et al. have provide direct evidence for ceRNA hypothesis[22-23], but which were lack of experimental validation that have been done by several research groups recently. Although the throughput and reproducibility of methods for analyzing miRNAome has improved at a rapid rate, many challenges remain. For example, it is difficult to simultaneously measure miRNA and their targets using HTS technology, and the interpretation of miRNA network involved in various diseases is still be to resolved. In all, the combination of biology, medicine and bioinformatics would greatly facilitate the development of miRNA study in future.

Abstract:The epithelial-mesenchymal transition (EMT) is an essential component in tumor metastasis. However, the molecular mechanism of EMT in renal cancer is unclear. IL-8 is an important chemokine in tumor microenvironment. Studies have shown that renal cancer cells can secrete IL-8，whether it could induce EMT in renal cancer cells is unknown. Here we found that IL-8 could induce EMT in renal cancer cells. Upon the stimulation of IL-8，the expression of E-cadherin was up-regulated，while the expression of N-cadherin was down-regulated; IL-8 promoted the invasion of renal cancer cells, but there was no obvious impact on cell proliferation. In addition, IL-8 could activate ERK through PKC. Therefore, we believe that IL-8 may promote renal cancer EMT through PKC / ERK signaling pathway, which may be one of the important mechanisms of renal cancer metastasis.

Abstract:The adipocyte development process of C3H10T1/2 pluripotent stem cells includes two stages: the commitment stage and the differentiation stage．There are evidences that BMP4 commits C3H10T1/2 cells into preadipocytes．Previous studies have shown that mice lacking adipocyte Lrp1(adLrp1-/-) displays reduced body weight and smaller fat stores which highlights the important role of Lrp1 in adipogenesis．However, little is known about the role of Lrp1 in preadipocyte commitment．RNAi was used to study the effect of low density lipoprotein receptor-related protein 1(Lrp1) on commitment of C3H10T1/2 to preadipocyte．Through microscopic examination, Oil red O staining, Western blotting, it is demonstrated that Lrp1 RNAi inhibits C3H10T1/2 adipogenesis in commitment stage, rather than in lipid maturation period．BMP4 signals through Smad1/5/8 and Lrp1 RNAi significantly suppresses the BMP4-triggered phosphorylation of Smad1/5/8．These data suggest that Lrp1 RNAi has inhibitory effects on the BMP4-induced of C3H10T1/2 preadipocyte commitment via down-regulating Smad signaling.

Abstract:Heat shock protein 90 (Hsp90) is essential for folding, maturation and stabilization of many important proteins, which are involved in cell cycle regulation, signal transduction, and cell growth regulation. The highly conserved N-terminal domain contains an ATP binding cleft and thus is responsible for the catalytic activity of Hsp90. In order to further study the function and structure of Hsp90, the N-terminal of the human Hsp90 was cocrystallized with AMPPNP and ATPγS. The cocrystallization experiments were carried out at 277K using the hanging drop vapor-diffusion method, X-ray diffraction data were collected on beamline 17U at the SSRF and the structures were solved by molecular replacement. The densities of the two nucleotides were captured and the interactions between Hsp90^N and nucleotides were clearly described. We confirmed that the γ-phosphate of ATPγS was not hydrolyzed by Hsp90^N. The position of S1 and ATP lid in human Hsp90^N-AMPPNP differs significantly from that of the structure of yeast Hsp90-AMPPNP. By analyzing the structure of human Hsp90^N-AMPPNP, we found that the interactions of E18-K100 and N40-D127 block the moving of S1 and ATP lid, and then prevent the dimerization of Hsp90^N. This reflects the complexity and coordination of Hsp90 on the regulation of the function.

Abstract:The chromatin-associated proteins play important roles in regulating DNA duplication, gene transcription expression etc. in eukaryotic system. Previously, Arabidopsis LFR (leaf and flower related) protein localized in nucleus, and its loss-of-function mutants were reported to have pleiotropic phenotypes in leaf, flower and anther development, but the molecular characteristics of LFR protein should be detected further. Firstly, the fusion protein of GAL4 DNA-binding domain (GBD) with full-length of LFR had transactivation activity in yeast Y190 by yeast one-hybrid assay, and at least 2 ARM-repeat domains in the C terminal and full N teminal of LFR were necessary to its transactivation activity. Next, we further analyzed GBD-LFR transactivation activity in Arabidopsis protoplast system, it showed that the transactivation of full-length LFR was not obvious compared with that of a classical transcription activity domain of VP16. In protoplast transient expression system, fluorescence microscopy was used to observed subcellular localization of YFP fusion proteins of truncated or site-mutation LFR, and the data showed that the N terminal 1～25 amino acids of LFR, particular lysine 22 and arginine 4, 23, 25, was responsible for its nuclear localization. In transient co-expressing onion epidermal cell, LFR obviously co-localized with chromatin stucture protein, histone H4, and chromatin-binding protein, HMGA, in nucleus. All these data suggested that LFR might be as one of chromatin-associated proteins to function in Arabidospis development.

Abstract:Dual-color two-photon laser scanning microscopy is a useful method for simultaneously studying the expression, localization and trafficking of two different proteins in tissues. Because most two-photon microscopes only use a single wavelength excitation laser, simultaneously exciting multiple fluorescent proteins remains a challenge. Here, we present mAmetrine and mKate2, which can be used as a novel fluorescent protein pair in dual-color two-photon imaging by taking advantage of the large Stokes shift of mAmetrine and high brightness of mKate2. Both proteins have high two-photon absorption efficiencies and can be simultaneously excited at an optical wavelength of 765 nm. Dual-color two-photon imaging using this protein pair is highly effective in living cells.

Abstract:Digital PCR is an absolute DNA quantification technique to determine the copy number of target DNA. PCR reaction solution is divided into aliquot throughout numerous partitions and amplifies independently. The copy number of target DNA is estimated by the statistical analysis of positive signals. Random and independent distribution of the target DNA molecules throughout the partitions of the digital panel and successful amplification from single molecules are critical to the validity of this approach for estimating target DNA copy number. In this review, we discuss the advances in development of digital PCR, the differences between digital PCR and real time quantitative PCR. We further show the advances in application of digital PCR in the clinical diagnosis, GMO analysis, single cell expression, environmental microbiology and next generation sequencing. The prospects for the applications of digital PCR are discussed.