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 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 ; Homeostasis ; Humans ; Organelles ; Phagosomes

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Colorectal cancer remains a major threat to people’s health around the world. Researchers have paid more and more attention to colorectal cancer epigenetics. From two main aspects of colorectal cancer epigenetics: DNA methylation and histone modiifcation, this article analyzes the similarities and differences between patients with colorectal cancer in Eastern and Western countries. This review brielfy introduces epigenetic modiifcation of genes that were used to be biomarkers and therapeutic targets. Although there are some common features of colorectal cancer in the world, analysis has showed that some obvious epigenetic differences do exist in different races. For example, it had been conifrmed in the studies that there are differences in speciifc gene methylation, histone modiifcation sites and the degree of methylation and acetylation among countries, which provide the basis for speciifc diagnosis, treatment and prognosis of colorectal cancer in different ethnic groups. With improved research methods and increased sample size, more and more special molecular targets of colorectal cancer tissues will be found, and then personalized therapy for colorectal cancer can be achieved.

Study the infect of child anorexia granule on serum ghrelin and leptin of anorexia children and its clinical efficacy. Selected 81 cases of anorexia children aged 1-6 years old into treatment group (42 cases) and control group (39 cases), in addition, 30 case healthy children as healthy control group. The control group children were treated with domperidone suspension 0.3 mg x kg(-1) x d(-1), tid, orally 30 minutes before meals. Treatment group were treated with child anorexia granule, 1-3 years 1 package, bid; 4-6 years 1 package, tid; po, 4 weeks as a course of treatment. Study the change of serum ghrelin and leptin before and after therapy. The study demonstrates that before treatment, the serum ghrelin level of disease group was lower than healthy group (P < 0.01), and the serum leptin level was higher than healthy group (P < 0.01). After treatment, the serum ghrelin level both increase, and the serum leptin decline. And the change of treatment group was significantly different with control group (P < 0.01). And the clinical effective rate are 95.23% and 74.35% (P < 0.01). After 6 months of follow-up visit, the children weight significantly increase in treatment group (P < 0.01). Results indicate that child anorexia granule can facilitate secretion of ghrelin, and inhibit secretion of leptin, so as to work up an appetite. And the molecular mechanism is its infect on serum ghrelin, leptin.
Anorexia ; drug therapy ; metabolism ; physiopathology ; Appetite Regulation ; drug effects ; Body Weight ; drug effects ; Child ; Child, Preschool ; Drugs, Chinese Herbal ; administration & dosage ; Female ; Ghrelin ; metabolism ; Humans ; Infant ; Leptin ; metabolism ; Male

This study is to determine the preventive effect and mechanism of targeting IL-17A on pulmonary inflammation and fibrosis after acute lung injury. Mice were treated with anti-IL-17A antibody on the day 7 and sacrificed on the day 14 after bleomycin lung injury. The pulmonary inflammatory status and the deposition of collagen were measured by HE and Sirius stains staining. The contents of hydroxyproline and collagen were measured by using commercial kits. The survival rate of mice was calculated by Kaplan-Meier methods. The inflammatory cytokines in bronchoalveolar lavage fluid were measured by ELISA and the expressions of inflammation-related molecules were detected by Western blotting assay. Targeting of IL-17A could prevent the development of lung inflammation, decrease collagen deposition and the contents of hydroxyproline, and protect against the development of pulmonary fibrosis, which together led to an increase in the animal survival. Moreover, blocking IL-17A decreased the expression ofpro-fibrotic cytokines such as IL-17A, TGF-beta1 and IL-13; increased the expression of anti-fibrotic or anti-inflammatory factors such as IFN-gamma, COX-2, 5-LOX, 15-LOX. Indeed, IL-17A antagonism suppressed the activation of pro-inflammatory p65NF-kappaB but enhanced the activation of pro-resolving p50NF-kappaB. In conclusion, that blockade of IL-17A prevents the development of pulmonary fibrosis from acute lung injury, is because blocking IL-17A may prevent acute inflammation converting to chronic inflammation.

With the development of therapeutic antibodies over the past decade, they have become the treatment options for immunity and inflammation diseases. Major limitations of mouse antibodies as therapeutic agents - immunogenicity, lack of effectors' functions and short serum half-life -- were subsequently identified and largely overcome by the advent of humanized and fully human antibody technologies. The therapeutic antibodies for immunity and inflammatory diseases are primarily utilized in the treatment of allograft rejection, autoimmune disease, autoinflammatory syndromes, allergies and other chronic inflammation. The action mechanisms of therapeutic antibody include blocking ligands or receptors, regulating receptor activity, clearing the target cells or activating receptor. Strategies for generating the antibody drugs with high efficacy and low side effects can be realized by modulation of Fc-mediated activities and optimization of antigen-binding domains.

Objective To construct an Lentiviral expression vector of pLVX‐IRES‐ZsGreen1‐MIA2 targeting to MIA2 and in‐vestigate its effect on the expression of MIA2 and growth of HCC cell line HepG2 in vitro ,observe MIA2 changes and the influence on apotheosis ,thus to provide preliminary experimental fundament for successive researching on the role of MIA2 in the pathogene‐sis of HCC .Methods The sequence of pLVX‐IRES‐ZsGreen1‐MIA2 was designed and synthesized .The pLVX‐IRES‐ZsGreen1‐MIA2 Lentiviral expression vector was constructed and then transiently transfected into HepG2 HCC cells in vitro .The proportion of pLVX‐IRES‐ZsGreen1‐MIA2 positive cells was observed under the fluorescence microscope .Then ,the expression level of MIA2 was detected by real time PCR .Moreover ,the proliferation of HepG2 cells was observed by MTT assay and colony formation as‐say .Finally ,the migration of HepG2 cells in vitro was also determined by Scratch assay .Results pLVX‐IRES‐ZsGreen1‐MIA2 Lentiviral expression vector was successfully constructed .Compared with control group (NC) ,the expression level of MIA2 was significantly decreased in transfected groups(P<0 .05);MTT assay showed that the proliferation of HepG2 cells was dramatically reduced in pIRES2‐ZsGreen1‐MIA2transfected groups(P< 0 .05);furthermore ,the number of both colony forming and migrating cells were also remarkably reduced in transfected groups(P<0 .05) .Conclusion The pIRES2‐ZsGreen1‐MIA2 can significantly re‐duce the expression level of MIA2 and inhibit the proliferation and migration of the HepG2 HCC cells in vitro .

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.