A regulatory loop involving miR-29c and Sp1 elevates the TGF-ß1 mediated epithelial-to-mesenchymal transition in lung cancer.

Specificity protein1 (Sp1) is required for TGF-ß-induced epithelial-to-mesenchymal transition (EMT) which has been demonstrated to aggravate the progression of cancer including lung cancer. microRNA-29c (miR-29c) is identified to inhibit EMT, but the correlation between miR-29c and Sp1 in human lung cancer remain incompletely clarified. Here, we confirmed decreased expression of miR-29c and enhanced expression of Sp1 in lung cancer tissues (n = 20) and found that Sp1 could be targeted and inhibited by miR-29c. Besides, the expression of miR-29c was down-regulated in high-metastatic lung cancer cell lines and TGF-ß1-treated cells. The inhibition of miR-29c or overexpression of Sp1 in 95C and A549 cells dramatically enhanced the cell migration and invasion, and also induced the decrease in the expression of epithelial markers, e.g. thyroid transcription factor 1 (TTF-1) and E-cadherin, together with an increase in mesenchymal markers including vimentin, α-smooth muscle actin (α-SMA), which could be restored by overexpression of miR-29c mimics during the TGF-ß-induced EMT. Moreover, dual-luciferase reporter assay was performed and the results indicated that miR-29c/Sp1 could form an auto-regulatory loop with TGF-ß1, which impaired TGFB1 transcription. Furthermore, miR-29c overexpression could abrogate the tumor progression and inhibit the Sp1/TGF-ß expressions in vivo, indicating that miR-29c could be a tumor suppressor and repress the Sp1/TGF-ß axis-induced EMT in lung cancer.

[Mutation analysis of the PAH gene in children with phenylketonuria from the Qinghai area of China].

OBJECTIVE: To study the mutation characteristics of the phenylalanine hydroxylase (PAH) gene in children with phenylketonuria (PKU) from the Qinghai area of China, in order to provide basic information for genetic counseling and prenatal diagnosis. METHODS: Mutations of the PAH gene were detected in the promoter and exons 1-13 and their flanking intronic sequences of PAH gene by PCR and DNA sequencing in 49 children with PKU and their parents from the Qinghai area of China. RESULTS: A total of 30 different mutations were detected in 80 out of 98 mutant alleles (82%), including 19 missense (63%), 5 nonsense (17%), 3 splice-site (10%) and 3 deletions (10%). Most mutations were detected in exons 3, 6, 7, 11 and intron 4 of PAH gene. The most frequent mutations were p.R243Q (19%), IVS4-1G>A (9%), p.Y356X (7%) and p.EX6-96A>G(5%). Two novel mutations p.N93fsX5 (c.279-282delCATC) and p.G171E (c.512G>A) were found. p.H64fsX9(c.190delC) was documented for the second time in Chinese PAH gene. The mutation spectrum of the gene PAH in the Qinghai population was similar to that in other populations in North China while significantly different from that in the populations from some provinces in southern China, Japan and Europe. CONCLUSIONS: The mutations of PAH gene in the Qinghai area of China demonstrate a unique diversity, complexity and specificity.

Identification of synthetic peptides that inhibit lipopolysaccharide (LPS) binding to myeloid differentiation protein-2 (MD-2).

Many studies have suggested that the synergic effect of myeloid differential protein-2 (MD-2) on bacterial lipopolysaccharide (LPS) stimulation of toll-like receptor 4 (TLR4) may be a critical step during the LPS-TLR4 response signaling pathway. We performed a bioinformatic analysis on the MD-2 protein and identified the amino acid sequence NH2-FSKGKYKCV-COOH (K128-132) as a possible key sequence involved in the binding between MD-2 and LPS. We then screened a random phage display peptide library using this sequence as bait in order to identify antagonistic peptides. After 3 rounds of selection, 3 positive clones were identified. All 3 peptides were shown to inhibit, in a dose-dependent manner the production of tumor necrosis factor-α and interleukin-6 in human U937 and THP-1 cell lines as well as human peripheral blood monocytes stimulated by LPS. Only 2 of the 3 peptides were able to bind MD-2 directly as shown by sulfo-SBED biotin label transfer experiments. BALB/C mice were used to estimate the protection of these peptides from LPS challenge, and 2 of the 3 peptides (Lys-Thr-Val-Pro-Asp-Asn-His and Ile-Gly-Lys-Phe-Leu-Tyr-Arg) reduced mortality of the challenged mice from 100% to 53.8%. This study has demonstrated that interfering with the binding between MD-2 and LPS might be a potential therapeutic strategy for treating LPS-induced sepsis, and in doing so has identified 2 potential peptide candidates.

[Expression of human triggering receptor expressed on myeloid cells 1 in peripheral blood mononuclear cells of patients with acute obstructive suppurative cholangitis].

OBJECTIVE: To investigate the expression pattern of human triggering receptor expressed on myeloid cells 1 (TREM-1) mRNA in peripheral blood mononuclear cells and its clinical significance in acute obstructive suppurative cholangitis (AOSC). METHODS: Peripheral blood mononuclear cells were collected from 36 patients with AOSC and 40 healthy adults. TREM-1 mRNA was determined by semi-quantitative RT-PCR, and TREM-1 protein by immunocytochemistry. Enzyme-linked immunosorbent assay (TNF-alpha) was used to detect the level of tumor necrosis factor-alpha (TNF-alpha), and immunoturbidimetry employed to detect C reactive protein. RESULTS: The expression of TREM-1 mRNA relative to beta-actin was 1.007-/+0.252 in patients with AOSC, significantly higher than that in the healthy adults (0.457-/+0.053, P<0.05). The two groups also showed significantly different TREM protein expression (P<0.01). The AOSC patients exhibited significantly higher levels of TNF-alpha and C reactive protein than the healthy adults (P<0.01). CONCLUSION: The expression of human TREM-1 in peripheral blood mononuclear cells is up-regulated obviously in early stage of AOSC, probably suggesting an important role of TREM-1 in the development of AOSC.

Up-regulation of IRAK-M is essential for endotoxin tolerance induced by a low dose of lipopolysaccharide in Kupffer cells.

BACKGROUND: Endotoxin tolerance (ET) is an important mechanism to maintain the homeostasis of Kupffer cells (KCs), because KCs are continually exposed to various pathogen-associated molecular patterns including lipopolysaccharide (LPS). ET involves multiple changes in cell signal transduction pathways; however, not all signaling pathways are down-regulated and some proteins are up-regulated. The latter proteins may be counter regulatory, including interleukin-1 receptor-associated kinase M (IRAK-M) expression. The aim of this study is to clarify weather or not IRAK-M is involved in the mechanisms of ET in KCs through dampening nuclear factor-kappa B (NF-kappaB) mediated pathway. MATERIALS AND METHODS: KCs isolated from male C57BL/6J mice were seeded in 24-well plates at 1 x 10(6) cells/well and cultured overnight prior to transfection, were randomly divided into two groups: the pIRAK-M-short hairpin RNA (shRNA) group (transfected with IRAK-M shRNA) and the control group (transfected with control vector); 24 h after transfection, the two groups were further randomly divided into two subgroups: non-endotoxin pretreatment group (incubation in Dulbecco's modified Eagle's medium [Invitrogen, Carlsbad, CA] with 10% fetal bovine serum) and endotoxin pretreatment group (incubation in the same medium containing 10 ng/mL LPS), named pIRAK-M-EP, pIRAK-M-NEP, pCV-EP, and pCV-NEP, respectively. Each subgroup contained 6 wells; 24 h later, fresh media containing LPS (100 ng/mL) was added to each subgroup and incubated for an additional 3 h. The expression of IRAK-M gene and protein level were determined by reverse transcription-polymerase chain reaction and Western blot, the activities of NF-kappaB were estimated by electrophoretic mobility shift assay and enzyme-linked immunosorbent assay, and the supernatant tumor necrosis factor-alpha levels were analyzed by enzyme-linked immunosorbent assay. RESULTS: The recombinant plasmid of pIRAK-M-shRNA specifically inhibited IRAK-M expression after it was transfected into KCs. At 3 h after 100 ng/mL LPS was added to the medium, IRAK-M expression was significantly induced in pCV-EP than that in pCV-NEP; however, there was no difference between pIRAK-M-NEP and pIRAK-M-EP, accompanied with lowest level of NF-kappaB activation and tumor necrosis factor-alpha levels in pCV-EP, and a dramatic enhancement in the other three groups (P < 0.01). CONCLUSIONS: Although a primary low dose of LPS stimulation obviously attenuated KCs response to the second LPS stimulation, the inhibitive influences were partly refracted in pIRAK-M-EP than in pCV-EP, indicating that the absence of IRAK-M caused abnormal enhancement of inflammatory effects. IRAK-M negatively regulates toll-like receptors signaling and involves in the mechanisms of ET in KCs through dampening NF-kappaB mediated pathway; therefore it may be a key component of this important control system, and a new target for the clinical treatment of sepsis.

Glycine attenuates endotoxin-induced liver injury by downregulating TLR4 signaling in Kupffer cells.

BACKGROUND: Several experimental studies have observed better outcomes after glycine treatment in patients with endotoxin-induced liver injuries, but its molecular mechanism is not yet fully understood. The purpose of this study was to evaluate the hypothesis that glycine attenuates endotoxin-induced liver injury by affecting endotoxin signal transduction in liver macrophages. METHODS: An animal model of endotoxin-induced liver injury was established by intraperitoneally injecting mice with 10 mg/kg body weight endotoxin fed a pretreatment diet with or without 5% (w/w) glycine. Blood and liver samples were obtained for analysis of liver morphology and to determine concentrations of alanine aminotransferase, endotoxin receptor Toll-like receptor 4 (TLR4), tumor necrosis factor-alpha (TNF-alpha), and interleukin (IL)-10 at various time points after injection. To investigate the effect of glycine on liver macrophages, Kupffer cells (KCs) were isolated and challenged by LPS (100 ng/mL), with or without glycine (4 mmol/l) pretreatment, and the expressions of TLR4, IL-10, and TNF-alpha were assayed at mRNA and protein levels. DNA-binding activity of nuclear factor-kappa B (NF-kappaB) was also analyzed using enzyme-linked immunosorbent assay. RESULTS: Dietary glycine significantly improved the survival rate of endotoxemic mice (P < .05), whereas serum alanine aminotransferase and TNF-alpha levels were significantly decreased at different time points (P < .05); IL-10 levels were increased (P < .05). Concurrently, LPS-induced hepatic tissue injury was attenuated as indicated by morphologic analysis; secretion of IL-10 in liver tissue (P < .05) was enhanced; and expression of TLR4 and TNF-alpha in liver tissue was downregulated (P < .05). Consistent with these in vivo experiments, enhanced secretion of IL-10 and inhibited expression of TLR4 and TNF-alpha caused by glycine pretreatment were also observed in LPS-stimulated KCs. NF-kappaB DNA-binding activity was also significantly inhibited by glycine (P < .05, respectively). CONCLUSIONS: Dietary glycine improved survival rates and liver function in endotoxemic mice by regulating the production of proinflammatory or anti-inflammatory cytokines in liver. It attenuated liver injury by deactivating KCs through inhibiting TNF-alpha secretion and increasing IL-10 production. The downregulative effect of glycine on the endotoxin signaling pathway and TLR4/NF-kappaB/TNF-alpha may be a novel potential mechanism by which glycine inhibits KC activity.

A discovery of low hydraulic resistance channel along meridians.

A hydro-mechanic model was put forward to study the fundamental nature of acupuncture meridians. The basic state of low hydraulic resistance was tested on humans and mini pigs using three methods. The first, a modified Guyton's method, proved that there was lower hydraulic resistance on meridians compared with nonmeridians. The second scanning method involved a single pressure transducer that can find the lowest resistance point in tissue, and the third method used two transducers and provided a more stable measurement. Using the latter method, low hydraulic resistance points were found very close to low impedance points along meridians. The transmission of artificial interstitial fluid pressure waves was measured to examine their connection to the low resistance points, with the result that a good connection between the points was confirmed. This means the points form channels along the meridians that we refer to as low hydraulic resistance channels. The channel was imaged through isotopic tracing and a migration of isotope (99m)Te could be found along the channel. The layer of the channel was detected by injecting Alcian blue and the track was found beneath the skin. All of the above experiments suggest the existence of a new type of channel in living tissues that has not yet been described in modern science, but coincides quite well with the Qi channel theory of traditional Chinese medicine.

[Heterogeneity changes in secretion ability of monocyte/macrophage and its significance after trauma hemorrhage in rat].

OBJECTIVE: To investigate the relationship between the heterogeneity in secretion ability of monocyte (Mo)/macrophage and the immune dysfunction after severe trauma. METHODS: Twenty-four healthy Wistar rats were randomly divided into normal control group and trauma hemorrhage 1, 4 and 7 days groups. The contents of interleukin-6 (IL-6) and IL-10 secretion of Mo/macrophage from different anatomical regions were determined by radioimmunoassay. RESULTS: (1)In normal rats, the ability to secrete IL-6 and IL-10 was different among alveolar macrophages (AM), peritoneal macrophages (PM) and Mo. PM showed the highest ability to secrete IL-10 while Mo had the highest ability to secrete IL-6. (2)After trauma hemorrhage, the secretion of IL-6 and IL-10 by AM were increased dramatically. On the contrary, the secretion of IL-6 by PM was declined from the 1st day to the 4th day, then increased even over that of the normal control group on the 7th day. However, the secretion of IL-10 by PM was significantly elevated on the 1st day after trauma hemorrhage, peaking on the 4th day, and only slight lowering was found on the 7th day. The secretion of IL-6 by Mo was declined gradually all the time, reaching the lowest point on the 7th day. On the contrary, the secretion of IL-10 by Mo was increasing, reaching its peak on the 7th day. CONCLUSION: The heterogeneity of secretion ability of Mo/macrophage obtained from different anatomical regions is present under normal condition, and is more obvious following a severe injury. This change may play an important role in the immune dysfunction and the development of complications after trauma.

[Application of proteomic technology in the study on infection].

Proteomics is aimed to illuminate the expression and function pattern of global protein in given organism, tissue, cell or subcellular structure. And its technical platform consists of high throughout protein separation and identification technology combined with bioinformatic technology. In many research fields, proteomic technology provided comprehensive, dynamic, and reticular proteome information critical for the understanding of the molecular mechanism of disease processes or life phenomena. As infection is one of the most basic etiological factors, the strategy and technology of proteomics would be very useful in isolation and identification of pathogen proteome, host immune cellular proteome, infection-related proteins, vaccine candidate antigens, biomarkers and drug target proteins, which would then obviously promote the processes of infection-related research, such as pathogen, host response, mechanism of infection as well as the prophylaxis, diagnosis, and therapy of infection.

[The role of myeloid differential protein-2 in innate immunity].

As typical PRRs (pattern-recognition receptors), TLRs (Toll-like receptors) play an important role during innate immunity recognition. MD-2 (myeloid differential protein-2) may contain distinct functional domains that can separately and simultaneously bind TLRs (TLR4 or TLR2) and TLR ligands, such as lipopolysaccharide (LPS). The special structure of MD-2 may result in its three main functions: (1) An association with TLR4 that amplifies TLR4 responsiveness to ligands, especially LPS. (2) Enabling TLR2-mediated responses to LPS and enhancing TLR2-mediated responses to bacteria and their cell wall components. (3) Increasing the expression of TLR2 and TLR4 and possibly influencing the correct intracellular distribution of TLR4. Importantly, while MD-2 regulation of TLR expression and distribution is well established, determining whether the interaction is direct or not will require further study. Thus, MD-2 is not only an assistant molecule of TLR4 but is also a key regulatory molecule in innate immunity, and may play important roles during infection, inflammation, immune responses and many other pathologic and physiologic processes.

[The use of gene expression differential display analysis technics in trauma research].

Gene is the regulation core of cell proliferation, differentiation and maturation. It is also the decisive factor of occurrence, progression and prognosis of many diseases. The changes in gene expression will inevitably lead to many kinds of abnormal in cells, tissue, organs even in whole biological organism. Trauma, as well as other stimulators, may bring a series complications through the abnormal expression of genes following injury. With the development of bioinformatics and molecular biology, a series of assays (so called gene expression differential display analysis technics, such as DNA microarray) have been established, which have been used to analyze differentially expressed genes effectively. These methods have been widely used in the research of tumor and other disease. This review will focus to some usage of the ways in traumatology.