Carcinoma, Renal Cell ; metabolism ; pathology ; surgery ; Diagnosis, Differential ; Humans ; Keratin-18 ; metabolism ; Keratin-19 ; metabolism ; Keratin-8 ; metabolism ; Kidney Neoplasms ; metabolism ; pathology ; surgery ; Male ; Middle Aged ; Nephrectomy ; Neprilysin ; metabolism ; Racemases and Epimerases ; metabolism

Objective To investigate the expression of VIT-1 gene in melanocytes of patients with vitiligo, and to analyze the difference of its sequence. Methods The skin from the foreskins of healthy men by circumcision and from the non-lesional area on the buttocks of 5 patients were digested by dispase, then the epidermis and dermis were separated, and the melanocytes were isolated. Then we cultured the melanocytes from the controls in TICVA medium and those from the patients in TICVA medium supplemented with basic fibroblast growth factor (bFGF) and endothelin-1 ( ET-1). The expression of VIT-1 gene was measured by RT-PCR, the full-length cDNA of VIT-1 ORF was cloned and sequenced, and sequence difference was analyzed by CLUSTAL W ( 1.83 ) software. Results The expression levels of VIT-1 gene were significantly lower in melanocytes from the patients than in those from the controls. An 81 bp-intron was found in the VIT-1 ORF. VIT-1 was a fragment of FBXO11, located at its 3' end. Conclusion VIT-1 gene is not a new gene, but a fragment of FBXO11, and a member of F-box protein family.

Full-length NS5A gene of the hepatitis C virus was amplified by PCR using plasmid pBAC25 containing HCV nonstructural gene as template. The amplified fragment (about 1.34 kb) was cloned into plasmid pQE32, and the recombinant plasmid pQENS5A was expressed in JM109 strain. The NS5A protein was purified by NiSO4 metal chelating resin, and characterized by Western-blot. Its antigenecity was determined by ELISA. The positive detection rate of anti-NS5A was 75% (69/92) in ninety-two clinic sera. The positive rate of anti-NS5A was 82.5% (33/40) in fourty positive standand sera, and the negative rate of anti-NS5A was 100% (40/40) in fourty negative standand sera. The results showed that the Full-length NS5A proteinn had the higher sensitivity and specificity in the detection of HCV antibody in sera, we suggested that NS5A protein was a useful antigen for blood screening.

Objective:To identify and differentiate cell subsets in the epidermis and dermis of vitiligo skin lesions using single-cell RNA sequencing technology, and to study the relationship between them.Methods:Skin samples were collected from 2 healthy people without immune or systemic diseases and 2 patients with stable non-segmental vitiligo in Department of Dermatology in the Third People′s Hospital of Hangzhou in September 2019. Single-cell transcriptome sequencing was performed on 11 000 cells in all the skin samples by using 10 × Genomics single-cell RNA-Seq technology. Cell subsets were analyzed, screened and counted by using Seurat software.Results:Cluster analysis of gene expression in the 2 normal skin tissues revealed several cell subsets, including keratinocytes, fibroblasts, nerve cells and melanocytes, endothelial cells, tissue stem cells, and immune cells mainly consisting of dendritic cells and T cells. In the 2 vitiligo lesions, abnormal differentiation and quantity were observed in fibroblasts and 4 keratinocyte subpopulations. The proportion of fibroblasts was significantly lower in vitiligo lesions than in normal skin tissues (0 vs. 0.4%) , while the proportions of keratinocyte subpopulations 5, 6, 10 and 12 (8.03%, 7.36%, 3.52%, 0.91%, respectively) in vitiligo lesions were significantly higher than those in the normal skin tissues (4.47%, 3.53%, 2.69%, 0.28%, respectively, all P < 0.01) . Moreover, the above keratinocyte subpopulations were at the end of cell differentiation, and expressed very significant and specific marker genes, which were mainly closely related to cell-cell interactions and cell homeostasis. GO and KEGG analysis showed that keratinocyte subpopulations 5 and 6 were mainly related to intercellular connection, cell adhesion and cytoskeleton function, while the keratinocyte subpopulation 10 was closely related to cell homeostasis. Conclusion:The single-cell sequencing technology was firstly used to study the transcriptional expression profile of vitiligo lesions in China, and preliminary analysis revealed 4 groups of keratinocytes with different quantity and functions, suggesting that abnormal differentiation and dysfunction of keratinocyte subpopulations may affect the occurrence and development of vitiligo.

Objective:To investigate the role of folliculin in apoptosis of and chemokine secretion by melanocytes mediated by interferon-γ (IFN-γ) .Methods:Normal primary melanocytes were isolated from circumcised foreskin tissues from a healthy male child, and primary vitiliginous melanocytes were isolated from normally pigmented suction-blistered epidermis from patients with vitiligo after suction blister epidermal grafting. Western blot analysis was performed to determine the folliculin protein expression in normal primary melanocytes, primary vitiliginous melanocytes and a human primary melanocyte line PIG1. PIG1 cells stimulated with 10 ng/ml IFN-γ for 48 hours served as induction group, and untreated PIG1 cells served as control group. Real-time quantitative RCR (qRT-PCR) was performed to determine the mRNA expression of folliculin, autophagy-related microtubule-associated protein 1 light chain 3 (LC3) -Ⅱ and Beclin genes, and Western blot analysis to determine the protein expression of folliculin, Beclin1 and LC3Ⅱ/Ⅰ, as well as phosphorylation levels of adenosine monophosphate-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) in the above cells. Furthermore, the melanocytes stimulated with 10 ng/ml IFN-γ for 48 hours were divided into several groups: negative control group infected with an empty lentiviral vector, folliculin inhibition group infected with a folliculin-inhibiting lentivirus, autophagy enhancement group infected with a folliculin-inhibiting lentivirus followed by 2-hour treatment with a mTOR inhibitor, autophagy inhibition group infected with a folliculin-inhibiting lentivirus followed by 2-hour treatment with an AMPK inhibitor. Then, flow cytometry was conducted to detect apoptosis of PIG1 cells, and enzyme-linked immunosorbent assay to measure the concentration of chemokines CXCL10 and CCL20 in the culture supernatant of PIG1 cells in the above groups. Measurement data were compared among multiple groups by using one-way analysis of variance, and multiple comparisons were carried out by using least significant difference- t test. Results:The relative protein expression level of folliculin significantly differed among the normal primary melanocytes (0.850 ± 0.120) , primary vitiliginous melanocytes (1.507 ± 0.170) and PIG1 cells (0.697 ± 0.130; F = 50.09, P < 0.001) , and was significantly higher in the primary vitiliginous melanocytes than in the normal primary melanocytes and PIG1 cells ( t = 4.06, 5.89, respectively, both P < 0.01) . Compared with the control group, the induction group showed significantly increased relative mRNA and protein expression levels of folliculin (both P < 0.01) , but significantly decreased relative mRNA and protein expression levels of LC3Ⅱ and Beclin (all P < 0.01) ; moreover, the induction group showed significantly decreased LC3Ⅱ/Ⅰ levels (0.72 ± 0.02) and AMPK phosphorylation levels (0.714 ± 0.023) in the PIG1 cells compared with the control group (1.13 ± 0.02, 1.176 ± 0.002, t = 7.34, 6.67, respectively, both P < 0.01) , but significantly increased mTOR phosphorylation levels (1.051 ± 0.023) compared with the control Group (0.451 ± 0.016, t = 3.81, P = 0.009) . There were significant differences in the PIG1 cell apoptosis rate and concentrations of CXCL10 and CCL20 among the control group, induction group and other treatment groups (all P < 0.001) ; specifically, the PIG1 cell apoptosis rate and concentrations of CXCL10 and CCL20 were significantly higher in the induction group than in the control group, lower in the folliculin inhibition group than in the negative control group, lower in the autophagy enhancement group than in the folliculin inhibition group, and higher in the autophagy inhibition group than in the folliculin inhibition group (all P < 0.05) . Conclusions:Folliculin is highly expressed in vitiliginous melanocytes. Folliculin expression and downstream signaling pathways are regulated by IFN-γ, and folliculin may participate in IFN-γ-mediated melanocyte apoptosis and chemokine secretion via regulating autophagy.

OBJECTIVETo investigate the correlation of the changes in CD8(+)CD28(-) T cell percentage with platelet (PLT) and D-dimer (D-D) levels in patients with multiple injuries (MI).

METHODSTwenty-six patients with MI, 31 with a single injury (SI group) and 26 healthy individuals were examined for peripheral blood CD8(+)CD28(-) T cells and intracellular transformation growth factor-β1 (TGF-β1) and interleukin 10 (IL-10) contents using flow cytometry at 24, 48, and 72 h after the injuries. PLT and D-dimer levels were compared among the 3 groups.

RESULTSCD8(+)CD28(-) T cells, TGF-β1 and IL-10 were significantly higher in MI group than in SI group and healthy control group (P<0.05) without significant differences between the latter 2 groups. The levels of PLT and D-D differed significantly among the 3 groups, the highest in MI group and the lowest in the control group. In MI group, CD8(+)CD28(-) T cells, TGF-β1 and IL-10 significantly increased at 48 h after the injury (P<0.05) but decreased significantly at 72 h (P<0.05) compared with the measurements at 24 h. The levels of PLT and D-D trended to decrease with time after the injuries and showed significant differences among the 3 groups at any of the 3 time points (P<0.05). CD8(+)CD28(-) T cells, TGF-β1 and IL-10 were all positively correlated with the levels of PLT and D-D in MI patients (r>0.70, P<0.05 for all comparisons).

CONCLUSIONIn MI patients, CD8(+)CD28(-) T cell percentage and their cytokines tend to increase early after the injury but decrease significantly at 72 h in close relation with the changes of the coagulation function following the injuries.

CD28 Antigens ; metabolism ; CD8 Antigens ; metabolism ; Case-Control Studies ; Fibrin Fibrinogen Degradation Products ; metabolism ; Flow Cytometry ; Humans ; Interleukin-10 ; metabolism ; Multiple Trauma ; immunology ; T-Lymphocyte Subsets ; cytology ; Transforming Growth Factor beta1 ; metabolism

OBJECTIVETo study the therapeutic molecular mechanism of the warm-hot drugs treating cold syndrome.

METHODA brother and his sister with deficiency-cold syndrome were chosen and treated with appropriate Chinese formula consisted chiefly of warm-hot drugs for 45 days. Then microarray technique was applied for comparing the gene expression difference of sister who had significant effect, the data was dialed with multiple analysis method and the results were mined though gene function and pathway.

RESULT276 differential genes were obtained, which were related to metabolism and 18 pathways.

CONCLUSIONWarm-hot drugs work on the gene expression of metabolism. It may be exerting the curative action by gene network and there is distinct difference between gene expression of curative effect and syndrome.

Drugs, Chinese Herbal ; therapeutic use ; Female ; Gene Expression Profiling ; Humans ; Kidney Diseases ; drug therapy ; genetics ; Male ; Medicine, Chinese Traditional ; Oligonucleotide Array Sequence Analysis ; Syndrome ; Yang Deficiency ; drug therapy ; genetics

OBJECTIVETo establish a nude mouse model of orthotopic engineered gastric tumor for in vivo fluorescence imaging studies.

METHODSAn engineered gastric tumor was constructed in vitro using collagen as the scaffold and the human gastric cancer cell line BGC823-EGFP cells expressing enhanced green fluorescence protein (EGFP) as the seed cells. The engineered tumor was then implanted into the stomach of nude mice, and the tumor growth was observed with in vivo fluorescence imaging. The nude mice were sacrificed 6 weeks after the transplantation to assess the tumor growth and metastasis, and the tumor histology was evaluated.

RESULTSThe tumor cells in the engineered tumor model grew well in three-dimensional culture. The success rate of orthotopic gastric tumor implantation was 100% (10/10) in nude mice with metastasis in the abdominal organs. The isolated tumor mass, weighing 1.719∓0.349 g, showed a histological characteristic of poorly differentiated adenocarcinoma. In vivo fluorescence imaging detected EGFP-expressing tumors in the abdominal cavity of the nude mice, but not accurately.

CONCLUSIONThe nude mouse model bearing orthotopic engineered gastric tumor provides a simple animal model for the study of gastric cancer, but a stronger fluorescence than green fluorescence is more desirable for more effective observation in in vivo fluorescence imaging.

Animals ; Cell Line, Tumor ; Disease Models, Animal ; Female ; Fluorescence ; Green Fluorescent Proteins ; analysis ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Optical Imaging ; Stomach Neoplasms ; Tissue Engineering

OBJECTIVETo evaluate the clinical value of three-dimensional pseudo-continuous arterial spin labeling (3D pCASL) perfusion magnetic resonance imaging (MRI) and dynamic susceptibility contrast (DSC) enhanced perfusion MRI in the diagnosis of transient ischemic attack (TIA).

METHODSThirty-nine consecutive patients with suspected TIA underwent multi-modal MRI scans including DSC, magnetic resonance angiography (MRA), diffusion-weighted imaging (DWI) and 3D pCASL (post-labeling delay, PLD=1.5 s and 2.5 s) within 24 h of symptom onset. Cerebral blood flow (CBF) from ASL and the time to the maximum of tissue residual function (Tmax) map from DSC were calculated using AW workstation. DWI and MRA were applied to detect acute cerebral infarction and intracranial artery stenosis. Two neuroradilogists who were blinded to the patients' clinical data assessed the presence of perfusion deficit, ischemic lesion and the lesion sites both from 1.5 s, 2.5 s PLD ASL-CBF and DSC-Tmax independently, and then graded them. The differences in the ranking grades between 1.5 s, 2.5 s PLD ASL and DSC were analyzed, and the frequency of lesion detection was compared between ASL-CBF, Tmax and MRA combining DWI method.

RESULTSNo significant differences was found in hypoperfusion grades detected by 3D pCASL (including PLD1.5 s and 2.5 s) CBF and Tmax maps, while significant differences were detected between 1.5 s PLD ASL-CBF and MRA combining DWI method; ASL with PLD 1.5 s CBF detected ischemic lesions and lesion site significantly more frequently than MRA combining DWI method.

CONCLUSIONs Three dimensional pCASL is a non-invasive perfusion method free of radiation exposure, and short PLD ASL is more sensitive than long PLD ASL for detecting ischemic lesions and lesion sites.

Arteries ; physiopathology ; Brain ; physiopathology ; Brain Infarction ; diagnosis ; Brain Ischemia ; diagnosis ; Cerebrovascular Circulation ; Diffusion Magnetic Resonance Imaging ; Humans ; Magnetic Resonance Angiography ; Perfusion ; Perfusion Imaging ; Spin Labels

OBJECTIVERotavirus is the single most common cause of severe dehydrating diarrhea in young children worldwide, but the pathogenesis and immunity against this disease are not completely understood. A prospective study was conducted to assess gene expression of toll-like receptors (TLR) in children with acute rotavirus diarrhea.

METHODSSeventy-five children with acute rotavirus diarrhea and 38 control children were enrolled in this study from Sep. 2004 to Jan. 2005. All the 75 patients had detailed records of clinical characteristics. Rotavirus antigen was detected by ELISA from stools. Peripheral blood mononuclear cells (PBMC) were separated by Ficoll reagent and RNA was extracted by Trizol. The levels of mRNA for five TLRs in PBMC were examined by fluorescent quantitative RT-PCR.

RESULTSPatients with acute rotavirus infection had elevated mean levels of TLR 2, 3, 4, 7, 8 mRNA expressions in PBMC within 3 days since onset of the disease, P less than 0.05. But only TLR 2, 3, 8 mRNA levels remained increased in patients within 7 or 14 days since onset (P less than 0.05). Mean levels of mRNA for TLR 4 in PBMC was higher in patients with more severe diarrhea including longer duration of diarrhea, more episodes of diarrhea per day and higher severity scores (P less than 0.05).

CONCLUSIONManifold TLR may play roles in the start-up and regulation of immune responses in children with acute rotavirus diarrhea. These findings will be helpful to further recognize immune response in Chinese children with rotavirus diarrhea and, consequently, may provide directions and insights that could prove critical to the prevention or treatment of this important disease.

Acute Disease ; Antigens, Viral ; analysis ; Child, Preschool ; Diarrhea ; genetics ; virology ; Enzyme-Linked Immunosorbent Assay ; Feces ; virology ; Gene Expression ; Host-Pathogen Interactions ; Humans ; Infant ; Leukocytes, Mononuclear ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Rotavirus ; immunology ; physiology ; Rotavirus Infections ; genetics ; virology ; Toll-Like Receptors ; genetics