Autophagy is an important homeostatic cellular recycling mechanism responsible for degrading injured or dysfunctional subcellular organelles and proteins in all living cells. The process of autophagy can be divided into three relatively independent steps: the initiation of phagophore, the formation of autophagosome and the maturation/degradation stage. Different morphological characteristics and molecular marker changes can be observed at these stages. Morphological approaches are useful to produce novel knowledge that would not be achieved through other experimental methods. Here we summarize the morphological methods in monitoring autophagy, the principles in data interpretation and the cautions that should be considered in the study of autophagy.

Autophagy is a crucial biological process of eukaryotes, which is involved in cell growth, survival and energy metabolism, while the premise of the autophagy function is activated autophagic flux. It has been confirmed that impaired autophagic flux promotes pathogenesis of many chronic inflammatory diseases, especially cancer, neurodegenerative disease and tissue fibrosis, therefore the analysis of autophagic flux state is important for revealing autophagy function and the mechanism of autophagy related diseases. Given that autophagy is a dynamic process with multiple steps, it is very hard to observe the real state of autophagic flux. Summarized here is the novel concept and current approach to detect autophagic flux. This knowledge is crucial for the researching of the biological function of autophagy, and may provide some strategies for developing autophagy-related drug.

Objective To study the immuno-regulatory roles of antibiotics and their implication for pulmonary fibrosis.Methods The regulatory roles of antibiotics on the phenotypes of dendrite cells(DCs) and maturation of B lymphocytes in vitro were detected with flow cytometry.The roles of nystatin on pulmonary fibrosis in vivo were determined with bleomycin-induced acute injury model of mouse.Results Multiple antibiotics including nystatin apparently promoted the maturation of DCs and B cells,as well as subsequently promoted the polarization of Th2 cells through DCs.In addition,nystatin intensified bleomycin-induced pulmonary fibrosis.Conclusion Multiple antibiotics including nystatin induce suppressive immune responses and might aggravate pulmonary fibrosis like nystatin.

To investigate the protective effects and possible mechanism of Mycelium of Hirsutella hepiali Chen et Shen (MHCS) on metabolic syndromes, free fatty acid and MHCS-treated hepatocytes were used for detecting autophagy-related LC3, p62 and lipid accumulation. Moreover, high fat diet fed mice were used to establish metabolic syndromes model. 50-weeks age mice were randomly divided into: control group, model group and MHCS group. At 80-weeks age, 15 mice were randomly chosen from each group separately for examining oral glucose tolerance, serum insulin, insulin-like growth factor 1 (IGF-1), hepatic LC3, p62, p-NF-kappaB p65, NF-kappaB p65, IL-6 and CXCL-8. Moreover, insulin resistance index (IRI) was calculated. Hepatic pathological changes, including vacuoles, lipids accumulation and fibrosis were observed. Remaining mice were fed with diet separately to 110 weeks-age for statistics of mortality. MHCS promoted autophagy of free fatty acid treated hepatocytes. Mice fed with high fat plus MHCS diet exhibited improved oral glucose tolerance, insulin resistance, hepatic pathology, inflammation, mortality and activated autophagy. The protective effects of MHCS against metabolic syndroms might be through the activation of hepatic autophagy.

The surface electromyography (sEMG) is the noninvasive method which can record and measure the changes of local muscle activities. The sEMG's amplitude and frequence signal will change with muscular movement. sEMG application in present study on muscle fatigue is a reliable predictor of muscle functional level. The researches on the sEMG signals changes of limb muscles of hemiplegic patients will take a important role in providing scientific evidence for the neural rehabilitation training after stroke.

Autophagy is an active research area in the biomedical field as its role has been identified in many physiological and pathological processes. Accordingly, there is a growing demand to identify, quantify and manipulate the process accurately. Meanwhile, there is great interest in identifying compounds that modulate autophagy because they may have applications in the treatment of a variety of autophagy-related diseases. In this review, we summarize the current status of autophagy screening systems to facilitate identification of autophagy modulators.

Autophagy is an important homeostatic cellular recycling mechanism responsible for degrading injured or dysfunctional cellular organelles and proteins in all living cells. Aging is a universal phenomenon characterized by progressive deterioration of cells and organs due to accumulation of macromolecular and organelle damage. Growing evidences indicate that the rate of autophagosome formation and maturation and the efficiency of autophagosome/lysosome fusion decline with age. Dysfunctional autophagy has also been observed in age-related diseases. Autophagy disruption resulted accumulation of mutated or misfolded proteins is the essential feature of neurodegenerative disorders. However, in cancers, fibroproliferative diseases or cardiovascular diseases, autophagy can play either a protective or destructive role in different types of disease, and even in different stages of the same disease. The review will discuss the cellular and molecular mechanisms of autophagy and its important role in the pathogenesis of aging and age-related diseases, and the ongoing drug discovery strategies for therapeutic intervention.

To prepare large naive phage antibody library, the host bacteria with high transformation efficiency is used in the Cre-LoxP recombination system. The variable regions of immunoglobulin light and heavy genes were amplified from lymphocytes collected from adult peripheral blood and newborn cord blood. The genes were spliced to form the single-chain variable fragments (scFv) by overlap PCR, cloned into pDAN5a vector and then transformed into XL2-blue MRF' with the Hte gene. Compared with XL1-blue strain, the size of the primary library was increased by 3.9 times. The primary library infected Cre recombinase-expressing bacteria, and the genes between phagemids created many new VH/VL combinations. The library was calculated to have a diversity of 1.7 x 10(11) and validated by the selection of antibodies against six different protein antigens. This library provides the basis for further selection of antibody-based drugs. It is the first time to report that XL2-blue MRF' can be used to improve the diversity of the library in the recombination system.

DEDD is a member of the death-effector domain protein family. DEDD inhibits the Smad3 mediated transcriptional activity and participates in the regulation of apoptosis. In this study, how the death-effector domain of DEDD participates in the regulation of Smad3 activity and apoptosis has been further investigated. Immunoblotting, immunofluorescence and immunoprecipitation had been used to detect the effects of the full length DEDD and its two truncated mutants, N-DEDD and C-DEDD on Smad3 subcellular distribution, phosphorylation, and interaction between Smad4. The effects of the full length DEDD and its two truncated mutants on cell apoptosis and proliferation had also been explored by flow cytometry and MTT assay. It showed that DEDD and N-DEDD inhibit TGF-beta1 induced Smad3 nuclear translocation and the formation of Smad3-Samd4 complex. DEDD and its two mutants can induce cell apoptosis and inhibit cell proliferation. These results suggested that DEDD inhibits the activity of Smad3 through its death-effector domain. Both the two truncated mutants of DEDD participate in the regulation of apoptosis and cell proliferation.

TLR2 activity plays an important role in the pathogenesis of autoimmune diseases, tumor carcinogenesis and cardio-cerebrovascular diseases. To establish a TLR2 receptor-based cell screening model, NF-kappaB promoter-driven luciferase reporter plasmids were transfected into human embryonic kidney cells (HEK293) stably expressing human TLR2 and co-receptors CD14, TLR1 and TLR6. Single clones were then isolated and characterized. Using this screening system, a human TLR2-binding peptide C8 was obtained from the Ph.D.-7 Phage Display Peptide Library through biopanning and rapid analysis of selective interactive ligands (BRASIL). The binding characteristic of C8 with human TLR2 was evaluated by ELISA, flow cytometry and immunofluorescence. The NF-kappaB luciferase activity assay showed that C8 could activate the TLR2/TLR1 signaling pathway and induce the production of cytokines TNF-alpha and IL-6. In conclusion, the TLR2 receptor-based cell screening system is successfully established and a new TLR2-binding peptide is identified by using this system.