Lithocarpus litseifolius is rich in the chalcones phloridzin and trilobatin, the biosynthesis pathways of which have not been fully demonstrated. Chalcone synthase(CHS) is the first key rate-limiting enzyme in the biosynthesis of flavonoids in plants. To explore the functions of CHS gene family in chalcone synthesis of L. litseifolius, this study screened out two CHS genes(LlCHS1 and LlCHS2) from the transcriptome data of this plant, and then bioinformatics analysis and functional characterization were performed for the two genes. The bioinformatics analysis showed that LlCHS1 and LlCHS2 were acidic hydrophilic stable proteins with no transmembrane domain, composed of 395 and 390 amino acid residues, respectively. Both of them contained the characteristic amino acid sequence "WGVLFGFGPGL" and highly conserved active sites(Cys-164, Phe-215, His-303, and Asn-336) of the CHS family. The phylogenetic tree showed that LlCHS1 shared the same clade with similar genes in Aquilaria sinensis, and LlCHS2 was closely related to similar genes in Malus domestica. Under exogenous addition of phloretic acid, co-expression of LlCHS1 or LlCHS2 with Aa4CL from Aromatoleum aromaticum in Escherichia coli catalyzed the production of phloretin from phloretic acid. This study laid a theoretical foundation for revealing the functions of CHS in plants and provided new enzymatic modules for producing phloretin by synthetic biology.
Acyltransferases/chemistry* ; Phylogeny ; Plant Proteins/chemistry* ; Cloning, Molecular ; Amino Acid Sequence

Cardiovascular diseases (CVDs) are the most common cardiovascular system disorders. Cellular senescence is a key mechanism associated with dysfunction of aged vascular endothelium. Hyaluronic acid and proteoglycan link protein 1 (HAPLN1) has been known to non-covalently link hyaluronic acid (HA) and proteoglycans (PGs), and forms and stabilizes HAPLN1-containing aggregates as a major component of extracellular matrix. Our previous study showed that serum levels of HAPLN1 decrease with aging. Here, we found that the HAPLN1 gene expression was reduced in senescent human umbilical vein endothelial cells (HUVECs). Moreover, a recombinant human HAPLN1 (rhHAPLN1) decreased the activity of senescence-associated β-gal and inhibited the production of senescence-associated secretory phenotypes, including IL-1β, CCL2, and IL-6. rhHAPLN1 also downregulated IL-17A levels, which is known to play a key role in vascular endothelial senescence. In addition, rhHAPLN1 protected senescent HUVECs from oxidative stress by reducing cellular reactive oxygen species levels, thus promoting the function and survival of HUVECs and leading to cellular proliferation, migration, and angiogenesis. We also found that rhHAPLN1 not only increases the sirtuin 1 (SIRT1) levels, but also reduces the cellular senescence markers levels, such as p53, p21, and p16. Taken together, our data indicate that rhHAPLN1 delays or inhibits the endothelial senescence induced by various aging factors, such as replicative, IL-17A, and oxidative stress-induced senescence, thus suggesting that rhHAPLN1 may be a promising therapeutic for CVD and atherosclerosis.

Hair loss is a common condition that can have a negative impact on an individual’s quality of life. The severe side effects and the low efficacy of current hair loss medications create unmet needs in the field of hair loss treatment. Hyaluronan and Proteoglycan Link Protein 1 (HAPLN1), one of the components of the extracellular matrix, has been shown to play a role in maintaining its integrity. HAPLN1 was examined for its ability to impact hair growth with less side effects than existing hair loss treatments. HAPLN1 was predominantly expressed in the anagen phase in three stages of the hair growth cycle in mice and promotes the proliferation of human hair matrix cells. Also, recombinant human HAPLN1 (rhHAPLN1) was shown to selectively increase the levels of transforming growth factor-β receptor II in human hair matrix cells. Furthermore, we observed concomitant activation of the ERK1/2 signaling pathway following treatment with rhHAPLN1. Our results indicate that rhHAPLN1 elicits its cell proliferation effect via the TGF-β2-induced ERK1/2 pathway. The prompt entering of the hair follicles into the anagen phase was observed in the rhHAPLN1-treated group, compared to the vehicle-treated group. Insights into the mechanism underlying such hair growth effects of HAPLN1 will provide a novel potential strategy for treating hair loss with much lower side effects than the current treatments.

Rheumatoid arthritis (RA) is a multifactorial immune-mediated disease, the pathogenesis of which involves different cell types. T-cell activation plays an important role in RA. Therefore, inhibiting T-cell activation is one of the current therapeutic strategies.Cytotoxic T-lymphocyte antigen 4-immunoglobulin (CTLA4-Ig), also known as abatacept, reduces cytokine secretion by inhibiting T-cell activation. To achieve a homeostatic therapeutic effect, CTLA4-Ig has to be administered repeatedly over several weeks, which limits its applicability in RA treatment. To overcome this limitation, we increased the number of sialic acid-capped antennas by genetically engineering the CTLA4 region to increase the therapeutic effect of CTLA4-Ig. N-acetylglucosaminyltransferase (GnT) and α2,6-sialyltransferase (α2,6-ST) were co-overexpressed in Chinese hamster ovary (CHO) cells to generate a highly sialylated CTLA4-Ig fusion protein, named ST6. The therapeutic and immunogenic effects of ST6 and CTLA4-Ig were compared. ST6 dose-dependently decreased paw edema in a mouse model of collagen-induced arthritis and reduced cytokine levels in a co-culture cell assay in a similar manner to CTLA4-Ig. ST6- and CTLA4-Ig-induced T cell-derived cytokines were examined in CD4 T cells isolated from peripheral blood mononuclear cells after cell killing through irradiation followed by flow- and magnetic-beadassisted separation. Interestingly, compared to CTLA4-Ig, ST6 was substantially less immunogenic and more stable and durable. Our data suggest that ST6 can serve as a novel, less immunogenic therapeutic strategy for patients with RA.

【Objective】 To study the level of occult hepatitis B virus methylation and replication related genes, and to explore the effect of the former on the latter. 【Methods】 The cases in control group (healthy control, n=3), occult hepatitis B group (occult HBV group, n=3) and hepatitis B group (HBV group, n=3) were detected by Illumina methylation 850k chip. The difference analysis, GO analysis and KEGG analysis were carried out. The methylation and virus replication related genes DNMT1, DNMT2, Dnmt3a and ZHX2 were screened for RT-PCR. 【Results】 The methylation level of occult HBV group and HBV group was significantly higher than that of the control group. Difference analysis showed that there were 1 050 differential methylation sites in occult HBV group with the methylation level greater than non-methylation level, and 1 340 differential methylation sites as the opposite compared with the control group. In HBV group, there were 1 008 differential methylation sites with methylation level greater than non-methylation level, and 1 242 differential methylation sites as the opposite. Go analysis showed that compared with the control group, the differential gene expression in occult HBV group and HBV group was significantly related to many anabolic processes in biological process (BP), cell composition (CC) and molecular function (MF). The enrichment analysis of KEGG pathway between the control group and the occult HBV group showed that the differential genes were mainly involved in adhesion junction, basal cell carcinoma, endometrial carcinoma, EB virus infection, hepatocellular carcinoma and other signal pathways. The enrichment analysis of KEGG pathway in occult HBV group and HBV group showed that the differential genes were mainly involved in AMPK signal pathway, cell cycle, endometrial cancer, hepatitis C, hepatocellular carcinoma and other signal pathways. DNMT1 and DNMT3a in occult HBV group and HBV group were significantly higher while ZHX2 was significantly lower than those in control group. 【Conclusion】 The methylation level of occult HBV group and HBV group increased significantly while ZHX2 decreased significantly. Hypermethylation inhibited the expression of ZHX2 and changed the replication of hepatitis B virus. Hepatitis B virus DNA methylation provides a theoretical basis for the replication mechanism of hepatitis B virus and a new method for the treatment of hepatitis B virus.

Ceramide metabolism is known to be an essential etiology for various diseases, such as atopic dermatitis and Gaucher disease. Glucosylceramide synthase (GCS) is a key enzyme for the synthesis of glucosylceramide (GlcCer), which is a main ceramide metabolism pathway in mammalian cells. In this article, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to determine GCS activity using synthetic non-natural sphingolipid C8-ceramide as a substrate. The reaction products, C8-GlcCer for GCS, could be separated on a C18 column by reverse-phase high-performance liquid chromatography (HPLC). Quantification was conducted using the multiple reaction monitoring (MRM) mode to monitor the precursor-to-product ion transitions of m/z 588.6 → 264.4 for C8-GlcCer at positive ionization mode. The calibration curve was established over the range of 0.625–160 ng/mL, and the correlation coefficient was larger than 0.999. This method was successfully applied to detect GCS in the human hepatocellular carcinoma cell line (HepG2 cells) and mouse peripheral blood mononuclear cells. We also evaluated the inhibition degree of a known GCS inhibitor 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) on GCS enzymatic activity and proved that this method could be successfully applied to GCS inhibitor screening of preventive and therapeutic drugs for ceramide metabolism diseases, such as atopic dermatitis and Gaucher disease.
Animals ; Calibration ; Carcinoma, Hepatocellular ; Cell Line ; Chromatography, Liquid ; Dermatitis, Atopic ; Gaucher Disease ; Humans ; Mass Screening ; Mass Spectrometry ; Metabolism ; Methods ; Mice

OBJECTIVETo summarize and analyze the compatibility laws for acupoint selection of acupuncture in treating diabetic gastroparasis (DGP) in modern literatures of clinical researches.

METHODSRetrieved were literatures related to treating DGP by acupuncture or acupuncture combined other therapies from PubMed, CNKI, and WF from 1982 to 2014. Analyzed were frequency of acupoint use, meridians selected acupoints belonged to, regions selected, association laws of selected compatible acupoints.

RESULTSRetrieved were 35 with compatibility frequencies more than 15 listed as follows: compatibility frequency of Zusanli (ST36) and Zhongwan (CV12) was 33 (94.29%); compatibility frequency of Zusanli (ST36) and Neiguan (PC6) was 23 (65.71%), compatibility frequency of Zhongwan (CV12) and Neiguan (PC6) was 22 (62.86%), compatibility frequency of Zusanli (ST36), Zhongwan (CV12), and Neiguan (PC6) was 22 (62.86%); compatibility frequency of Zusanli (ST36) and Sanyinjiao (SP6) was 16 (45.71%); compatibility frequency of Zhongwan (CV12) and Sanyinjiao (SP6) was 16 (45.71%); compatibility frequency of Zusanli (ST36), Zhongwan (CV12), and Sanyinjiao (SP6) was 15 (42.86%). Meridians selected acupoints belonged to were sequenced as Foot Yangming Stomach channel, Ren channel, Foot Taiyang Bladder channel, and so on. Acupoints selected were mainly in lower limbs, chest and abdomen, waist and back. The compatibility of Zusanli (ST36) and Zhongwan (CV12) was the most often used with the highest frequency.

CONCLUSIONSThe compatibility laws for acupoint selection of acupuncture in treating DGP were mainly dominated as upper-lower selection, three regions selection, local selection, anterior-posterior selection. The compatibility laws for acupoint selection of acupuncture along meridians were mainly dominated as the convergence points and exterior-interior meridian points.

Acupuncture Points ; Acupuncture Therapy ; Diabetes Complications ; drug therapy ; Diabetes Mellitus ; drug therapy ; Humans ; Meridians ; Stomach ; physiopathology ; Stomach Diseases ; drug therapy

Anemia, Aplastic ; therapy ; Bone Marrow Transplantation ; statistics & numerical data ; China ; Cooperative Behavior ; Family ; Hematopoietic Stem Cell Transplantation ; statistics & numerical data ; Hemoglobinopathies ; therapy ; Hong Kong ; Humans ; Immunologic Deficiency Syndromes ; therapy ; Leukemia ; therapy ; Lymphoma ; therapy ; Malaysia ; Myelodysplastic Syndromes ; therapy ; Peripheral Blood Stem Cell Transplantation ; statistics & numerical data ; Philippines ; Singapore ; Thailand ; Tissue Donors ; statistics & numerical data ; Transplantation, Autologous ; statistics & numerical data ; Transplantation, Homologous ; statistics & numerical data

Paraquat dichloride (N,N-dimethyl-4-4'-bipiridinium, PQ) is an extremely toxic chemical that is widely used in herbicides. PQ generates reactive oxygen species (ROS) and causes multiple organ failure. In particular, PQ has been reported to be an immunotoxic agrochemical compound. PQ was shown to decrease the number of macrophages in rats and suppress monocyte phagocytic activity in mice. However, the effect of PQ on macrophage cell viability remains unclear. In this study, we evaluated the cytotoxic effect of PQ on the mouse macrophage cell line, RAW264.7 and its possible mechanism of action. RAW264.7 cells were treated with PQ (0, 75, and 150 muM), and cellular apoptosis, mitochondrial membrane potential (MMP), and intracellular ROS levels were determined. Morphological changes to the cell nucleus and cellular apoptosis were also evaluated by DAPI and Annexin V staining, respectively. In this study, PQ induced apoptotic cell death by dose-dependently decreasing MMP. Additionally, PQ increased the cleaved form of caspase-3, an apoptotic marker. In conclusion, PQ induces apoptosis in RAW264.7 cells through a ROS-mediated mitochondrial pathway. Thus, our study improves our knowledge of PQ-induced toxicity, and may give us a greater understanding of how PQ affects the immune system.
Animals ; Annexin A5 ; Apoptosis* ; Caspase 3 ; Cell Death ; Cell Line ; Cell Nucleus ; Cell Survival ; Herbicides ; Immune System ; Macrophages ; Membrane Potential, Mitochondrial ; Mice ; Mitochondria ; Monocytes ; Multiple Organ Failure ; Paraquat* ; Rats ; Reactive Oxygen Species

No abstract available.
Asian Continental Ancestry Group* ; Humans ; Keratin-17* ; Mutation, Missense* ; Steatocystoma Multiplex*