Biomedical subjects
Publications and source records attributed to Yun Huang.
The preview discusses the scalable platform for methylation-based trait mapping published in Cell Genomics by Goldberg et al.1 This work represents not only a methodological advance but also marks a conceptual shift toward scalable and high-throughput functional, targeted, and context-sensitive epigenomic screening.
We present a generative model, Lux, to quantify DNA methylation modifications from any combination of bisulfite sequencing approaches, including reduced, oxidative, TET-assisted, chemical-modification assisted, and methylase-assisted bisulfite sequencing data. Lux models all cytosine modifications (C, 5mC, 5hmC, 5fC, and 5caC) simultaneously together with experimental parameters, including bisulfite conversion and oxidation efficiencies, as well as various chemical labeling and protection steps. We show that Lux improves the quantification and comparison of cytosine modification levels and that Lux can process any oxidized methylcytosine sequencing data sets to quantify all cytosine modifications. Analysis of targeted data from Tet2-knockdown embryonic stem cells and T cells during development demonstrates DNA modification quantification at unprecedented detail, quantifies active demethylation pathways and reveals 5hmC localization in putative regulatory regions.
A highly sensitive and specific method, based on high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS), was developed for simultaneous detection of eleven steroidal saponins and one phytoecdysone in extracts from the rhizomes of thirteen PARIS species and four prepared Chinese medicines (PCMs). The HPLC experiments were performed by means of a reversed-phase C18 column (4.6 mm x 150 mm, 5 microm) and mobile phase system consisting of 0.1 % aqueous formic acid and acetonitrile under gradient elution conditions. The most intensive electrospray ionization signals were found in the negative ion spectra due to HCOO- adducts. The limits of detection (LODs) for the saponins were lower than 40 ng/mL in selected ion monitoring (SIM) mode. The identification of the saponins in the extracts of PARIS species and PCMs was confirmed using their retention times and mass spectral comparisons to standard compounds. The validated method was successfully applied for simultaneous detection of twelve standard compounds in the analytes so that it provided a new means for quality evaluation of Rhizoma Paridis and Chinese multiherb remedies that contain Rhizoma Paridis. The results showed that most species of PARIS contain steroidal saponins, which provided evidence for expanding the botanical origin of Rhizoma Paridis.
OBJECTIVE: To compare the reparative effects between the acellular small intestinal submucosa and the acellular amnion as dressings for traumatic skin defects. METHODS: Three full-thickness skin defects, which were close to the vertebral column of the pig, were created on both sides of the dorsum. The skin defects were randomly divided into three groups. In each group, the following different materials were used to cover the skin defects: the acellular amnion in Group A, the acellular small intestinal submucosa (SIS) in Group B, and the physiological saline gauze in Group C (the control group). The specimens from the skin defects were harvested for a histological evaluation and for determination of the hydroxyproline content at 10 (2 pigs), 20 (2 pigs), and 30 days (3 pigs). We observed the healing process of the wound and its healing rate, counted the inflammatory cells, vascular endothelial cells, and proliferating cells, and determined the hydroxyproline content. RESULTS: The acellular amnion in Group A and the acellular SIS in Group B adhered to the wound tightly, but they did not adhere to the dressing; when the dressing was changed, the wound did not bleed. The saline gauze in Group C adhered to the wound tightly, but when the dressing was changed, the wound bled until 22 days after operation. Under the microscope, the collagen in the tissue below the epithelium was arranged more regularly and there were fewer cells concerned with inflammation in Groups A and B than in Group C at 10, 20, and 30 days after operation. At 10, 20, and 30 days after operation, the wound healing rate was greater in Groups A and B than in Group C. The number of the inflammatory cells and the proliferating cells were greater in Group C than in Groups A and B. There was a statistically significant difference (P < 0.05), At 20 and 30 days after operation, the content of hydroxyproline was greater in Group C than in Group A and B. There was a statistically significant difference (P < 0.05). However, there was no statistically significant difference between Group A and Group B in the wound healing rate, the numbers of the inflammatory cells, vascular endothelial cells and proliferating cells, and the content of hydroxyproline (P > 0.05). There was no statistically significant difference among the three groups in the number of the vascular endothelial cells. CONCLUSION: Compared with Group C, the wound healing rate is improved, the collagen in the tissue below the epithelium is arranged more regularly, and the inflammatory cells, bleeding, and effusion are reduced in Groups A and B. The reparative effects of the acellular amnion and the acellular small intestinal submucosa as dressings on the skin defects are almost the same.
AIM: To study the anti-tumor bioactive steroid saponins of Paris vietnamensis (Takht.). METHODS: The constituents were isolated and purified by chromatography and their structures were identified by spectral analysis and physicochemical properties. The cytotoxic bioactivities of the constituents were determined by MTT. RESULTS: Eleven compounds were obtained and identified as 3beta, 5alpha,6alpha-trihydroxy-7(8)-en-isospirostanol-3-O-beta-D-glucopyranosyl(1-->3) [alpha-L-rhamnopyranosyl(1-->2)]-beta-D-glucopyranoside (1), which was named as parisvietnaside A, 25 (R) diosgenin-3-O-alpha-L-arabinofuranosyl(1-->4)-beta-D-glucopyranoside (2), 25(R) diosgenin-3-O-alpha-L-rhamnopyranosyl(1-->2)-beta-D-glucopyranoside (3), 25 (R) diosgenin-3-O-alpha-L-arabinofuranosyl (1-->4) [alpha-L-rhamnopyranosyl (1-->2)]-beta-D-glucopyranoside (4), 25 (R) diosgenin-3-O-beta-D-glucopyranosyl (1-->3) [alpha-L-rhamnopyranosyl (1-->2)]-beta-D-glucopyranoside (5), 25 (R) diosgenin-3-O-alpha-L-rhamnopyranosyl (1-->4)-alpha-L-rhamnopyranosyl(1-->4) [alpha-L-rhamnopyranosyl-(1-->2)]-beta-D-glucopyranoside (6), 25 (R) pennogenin-3-O-alpha-L-arabinofuranosyl(1-->4)-beta-D-glucopyranoside (7), 25 (R) pennogenin-3-O-alpha-L-rhamnopyranosyl (1-->2)-beta-D-glucopyranoside (8), 25 (R) pennogenin-3-O-alpha-L-arabinofuranosyl (1-->4) [alpha-L-rhamnopyranosyl-(1-->2)]-beta-D-glucopyranoside (9), 25 (R) pennogenin-3-O-beta-D-glycopyranosyl (1-->3) [alpha-L-rhamnopyranosyl(1-->2)-beta-D-glucopyranoside (10) and 25 (R) pennogenin-3-O-alpha-L-rhamnopyranosyl (1-->4)-alpha-L-rhamnopyranosyl(1-->4) [alpha-L-rhamnopyranosyl-(1-->2)]-beta-D-glucopyranoside (11). Some constituents had cytotoxic bioactivities. CONCLUSION: Compound 1 is a new spirostanol saponin, and compounds 2, 3, 6-11 were obtained from Paris vietnamensis (Takht.) for the first time. Compounds 3, 4, 6, 8 had cytotoxic bioactivities against tumor cells HepG2 and SGC-7901.
OBJECTIVE: The several species of the genus Paris called as Rhizoma Paridis were famous traditional Chinese medica. To develop the quantitative analysis method of the steroidal saponins in some species of the genus Paris and commercially available Rhizoma Paridis samples by HPLC-ELSD. METHOD: The contents of 11 steroidal saponins in Rhizoma Paridis samples were dectected with a Kromasil C18(4.6 mm x 150 mm, 5 microm) column which was deluted with acetonitrile-water (30:70-60:40) at a flow rate of 1 mL x min(-1) by HPLC-ELSD. RESULT: All the authentic samples could be separated and calibration curves of 11 saponins were prepared. 11 steroidal saponins in 16 Rhizoma Paridis samples were detected in 30 min. The recovery for the assay of saponins was between 95% and 97%. The precision and stability of samples (RSD) were below 3%. CONCLUSION: The method was shown to be accurate and convenient, and suitable for the quantitative analysis of these 11 steroidal saponins in the commercially available Rhizoma Paridis samples.
Activation of the receptor tyrosine kinase DDR1 by collagen results in robust and sustained phosphorylation, however little is known about its downstream mediators. Using phosphopeptide mapping and site-directed mutagenesis, we here identified multiple tyrosine phosphorylation sites within DDR1. We found that Nck2 and Shp-2, two SH2 domain-containing proteins, bind to DDR1 in a collagen-dependent manner. The binding site of Shp-2 was mapped to tyrosine-740 of DDR1 within an ITIM-consensus sequence. Lastly, ablation of DDR1 in the mouse mammary gland resulted in delocalized expression of Nck2, suggesting that defects observed during alveologenesis are caused by the lack of the DDR1-Nck2 interaction.
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Previously we have found that N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), an alkylating agent, can induce the clustering of cellular surface receptors including tumor necrosis factor receptor (TNFR) and epidermal growth factor receptor (EGFR). Since sphingolipids, especially ceramide, have been suggested as major players in ligand-induced receptor clustering, their involvement in this ligand-independent, chemical-induced receptor clustering was evaluated. It was shown that MNNG-induced EGFR clustering occurred primarily at lipid rafts, as nystatin, which can disrupt lipid raft structure, significantly decreasing MNNG-induced EGFR clustering. Lipidomic studies revealed that MNNG treatment induced profound changes in sphingolipids metabolism, which were not the same as those induced by EGF treatment. Acid sphingomyelinase (ASM) is responsible for hydrolyzing sphingomyelin to generate ceramide, and it was demonstrated that MNNG treatment caused ASM distribution changing from diffused state to concentrated area of cells, which colocalized with lipid rafts. Nystatin treatment also abolished the redistribution of ASM. In addition, blockage of ceramide production by ASM inhibitor imipramine interrupted MNNG-induced receptor clustering. Taken together, these data suggested that sphingolipids are involved in MNNG-induced receptor clustering; however, the specific species involved may be different from those involved in EGF-mediated receptor clustering.
Many environmental factors, such as ultraviolet (UV) and arsenic, can induce the clustering of cell surface receptors, including epidermal growth factor receptor (EGFR). This is accompanied by the phosphorylation of the receptors and the activation of ensuing cellular signal transduction pathways, which are implicated in the various cellular responses caused by the exposure to these factors. In this study, we have shown that N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), an alkylating agent, also induced the clustering of EGFR in human amnion FL cells, which was similar in morphology to that of epidermal growth factor treatment. However, MNNG treatment did not activate Ras, the downstream mediator in EGFR signaling pathway, as compared to EGF treatment. The autophosphorylation of tyrosine residues Y1068 and Y1173 at the intracellular domain of EGFR, which is related to Ras activation under EGF treatment, was also not observed by MNNG exposure. Interestingly, although MNNG did not affect the binding of EGF to EGFR, MNNG can interfere with EGF function. For instance, pre-incubating FL cells with MNNG inhibited the autophosphorylation of EGFR by EGF treatment, as well as the activation of Ras. In addition, the phosphorylation of Y845 on EGFR by EGF, which is mediated through c-Src or related kinases but not autophosphorylation, was also affected by MNNG. Therefore, MNNG may influence the tyrosine kinase activity as well as the phosphorylation of EGFR through its interaction with EGFR.
Systems biology is a new and rapidly developing research area in which, by quantitatively describing the interaction among all the individual components of a cell, a systems-level understanding of a biological response can be achieved. Therefore, it requires high-throughput measurement technologies for biological molecules, such as genomic and proteomic approaches for DNA/RNA and protein, respectively. Recently, a new concept, lipidomics, which utilizes the mass spectrometry (MS) method for lipid analysis, has been proposed. Using this lipidomic approach, the effects of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) on sphingomyelin metabolism, a major class of sphingolipids, were evaluated. Sphingomyelin molecules were extracted from cells and analyzed by matrix-assisted laser desorption ionization-time of flight MS. It was found that MNNG induced profound changes in sphingomyelin metabolism, including the appearance of some new sphingomyelin species and the disappearance of some others, and the concentrations of several sphingomyelin species also changed. This was accompanied by the redistribution of acid sphingomyelinase (ASM), a key player in sphingomyelin metabolism. On the other hand, imipramine, an inhibitor of ASM, caused the accumulation of sphingomyelin. It also prevented some of the effects of MNNG, as well as the redistribution of ASM. Taken together, these data suggested that the lipidomic approach is highly effective for the systematic analysis of cellular lipids metabolism.
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A large number of numerical and structural aberrations were analyzed in human tumor metastatic cells and 13q14 aberrations were frequently detected in some types of metastatic cancers. The rearrangement of 13q14 was identified previously in two lung adenocarcinoma cell lines with the same origin but different metastatic potential AGZY83-a and Anip973. BRI gene showed different expression levels in the cell lines as revealed by mRNA differential display (mRNA DD) in the two cell lines, and located in 13q14. In order to investigate the relationship between 13q14 abnormalities and tumor metastasis, a painting probe (13q) was used to hybridize three G-banded NSCLC cell lines with different metastatic potential. The major abnormality of 13q differs among different cell lines, including 13q32-33 frequent breakpoint in these three cell lines. But low metastatic potential cell lines PAa, SPC-1-A were not found breakpoint in 13q14, while 95D cell line with high metastatic potential had the common breakpoint 13q14 in two cell clones. The results suggested that the breakage at 13q14 may possibly be related to lung cancer metastasis. The affirmative relationship between 13q14 aberration and NSCLC needs further investigation.
OBJECTIVE: To identify the relationship between p53-dependent apoptosis associated genes and tumor metastasis. METHODS: mRNA differential display (mRNA DD) was adopted for gene cloning after the different metastatic potential lung cancer cell lines were infected by Adv-p53 (a reconstructed adenovirus encoding wild type p53 gene). RT-PCR, Northern blot and Western blot assays were used to confirm the result from mRNA DD. RESULTS: After induction by p53 gene, the ANNEXIN A2 gene had differential expression in the cell lines; its level was down regulated in all the cells infected by Adv-p53 gene, especially in the Anip973 cell lines with high metastatic potential. RT-PCR, Northern blot and Western blot assays confirmed the consequence. CONCLUSION: The experimental data suggest that the ANNEXIN A2 gene may relate to cellular apoptosis induced by p53 gene. The affirmative relationship between ANNEXIN A2 gene and p53 needs further investigation.
To investigate if microdeletion of chromosome 22q11 is an epidemiologically important cause of congenital heart disease (CHD), we studied 25 cases with CHD phenotypes. Venous blood samples were tested by fluorescence in situ hybridization (FISH) for microdeletion of 22q11. Among 23 cases with simple CHD, 19 were shown not to have microdeletion of 22q11 and the other 4 cases were shown to have 22q11 microdeletion. Microdeletion of 22q11 was found in 2 cases with Tetralogy of Fallot (TOF) accompanied by multiple malformations. The results suggested that microdeletion of 22q11 was associated with CHD.
AIM: To analyze the diagnostic and therapeutic features of extrahepatic bile duct carcinoma (EBDC) without jaundice. METHODS: Between 1985 and 1999, 101 patients underwent surgery for EBDC in Xiangya Hospital. These patients were divided into two groups: 84 jaundiced patients and 17 non-jaundiced patients according to preoperative serum total bilirubin levels. The clinical manifestations, laboratory findings, location, pathology and surgical resectability of the tumors were compared between the two groups. RESULTS: The laboratory parameters such as hemoglobin, serum albumin ALB, AKP, gamma-GT, and sonography appearance were similar between the two groups, and there was no significant difference in tumor location, pathological type and resectability. However, the number of non-jaundiced patients associated with chololithiasis was significantly greater than that of jaundiced patients (P = 0.008). CONCLUSION: The presence of jaundice is not a reliable criterion for the prediction of the resectability and the extent of tumor progression in extrahepatic bile duct carcinoma. Decreased levels of blood hemoglobin and serum albumin, elevated levels of AKP and gamma-GT, and /or abnormal sonography may be suggestive. Biopsy of a stenotic or thickened bile duct is strongly recommended for a correct diagnosis before the appearance of jaundice.
Cellular response to genotoxic stress is a very complex process, and it usually starts with the "sensing" or "detection" of the DNA damage, followed by a series of events that include signal transduction and activation of transcription factors. The activated transcription factors induce expressions of many genes which are involved in cellular functions such as DNA repair, cell cycle arrest, and cell death. There have been extensive studies from multiple disciplines exploring the mechanisms of cellular genotoxic responses, which have resulted in the identification of many cellular components involved in this process, including the mitogen-activated protein kinases (MAPKs) cascade. Although the initial activation of protein kinase cascade is not fully understood, human protein kinases ATM (ataxia-telangiectasia, mutated) and ATR (ATM and Rad3-related) are emerging as potential sensors of DNA damage. Current progresses in ATM/ATR research and related signaling pathways are discussed in this review, in an effort to facilitate a better understanding of genotoxic stress response.
Full activation of naive T cells requires both engagement of the T cell antigen receptor (TCR; signal 1) and costimulatory signaling by CD28 (signal 2). We previously identified two types of rat CD28-specific monoclonal antibodies (mAbs): "conventional," TCR signaling-dependent costimulatory mAbs and "superagonistic" mAbs capable of inducing the full activation of primary resting T cells in the absence of TCR ligation both in vitro and in vivo. Using chimeric rat/mouse CD28 molecules, we show that the superagonists bind exclusively to the laterally exposed C"D loop of the immunoglobulin-like domain of CD28 whereas conventional, costimulatory mAbs recognize an epitope close to the binding site for the natural CD80/CD86 ligands. Unexpectedly, the C"D loop reactivity of a panel of new antibodies raised against human CD28 could be predicted solely on the basis of their superagonistic properties. Moreover, mouse CD28 molecules engineered to express the rat or human C"D loop sequences activated T cell hybridomas without TCR ligation when cross-linked by superagonistic mAbs. Finally, biochemical analysis revealed that superagonistic CD28 signaling activates the nuclear factor kappaB pathway without inducing phosphorylation of either TCRzeta or ZAP70. Our findings indicate that the topologically constrained interactions of anti-CD28 superagonists bypass the requirement for signal 1 in T cell activation. Antibodies with this property may prove useful for the development of T cell stimulatory drugs.