The chelating reaction between glutathione peptides and divalent cadmium ion was used as a typical model for investigating the coordination chemistry of SH-containing peptides and heavy metal ions, which was essential to understand the mechanism of intracellular cadmium detoxification.Here, the mutant (CM113R)7 αHL protein nanopore equipped with a first synthesized per-6-quaternary ammonium-β-cyclodextrin (p-QABCD) was used as nanoreactor and detector to investigate the single-molecule reaction of GSH molecules and Cd2+ ions.Different reaction pathways, intermediates, and products could be recognized.Cd2+-GSH reaction was highly dependent on the solution pH.The Cd(GSH)2 was formed at pH 7.4, while the Cd(GSH)2 and Cd2(GSH)2 were formed at pH 9.0.Cd2(GSH)2 was formed by two possible pathways: (1) A Cd2+ ion primarily coordinated with the thiol group of two GSH molecules to form Cd(GSH)2, and then the second Cd2+ ion quickly incorporated with the deprotonated amino group of Cd(GSH)2 to form Cd2(GSH)2;(2) Two Cd2+ ions separately coordinated with the thiol and deprotonated amino group of one GSH molecule to form Cd2(GSH)1, and the second GSH molecule quickly bounded to Cd2+ ions to form Cd2(GSH)2.This work studied the chelating reaction between metal ions and bioactive glutathione at single-molecule level without labeling and chemical modification, and would be important to further understand intracellular mechanisms of detoxification of heavy metals and greatly expand the research field of nanopore single-molecule technique.

Objective To acquire high spatial resolution two-dimension pulse pressure distribution for objectifying research of pulse diagnosis in traditional Chinese medicine.Methods A sensor array with flexible packing was designed and implemented.The array was formed on a circle substrate with 37 silicon piezoresistive pressure sensors,which were distributed as equilateral triangles with 1.95 mm pitch.The diameter of the substrate was 15 mm.By using flexible packing,the array had the hardness similar to that of human finger,which made this method closer to the traditional Chinese diagnosis.After differential amplifying,37-way pulse pressure signals were digitalized and reconstructed to obtain the pulse pressure distribution.Results With the application of the sensor array,the pulse pressure signals were measured and transformed into pulse pressure distribution.Conclusions The experimental result contains the relevant characteristics of traditional Chinese pulse diagnosis' description,providing a new way for objective study of traditional Chinese pulse diagnosis and further study.

Objective To clone the human Islet neogenesis associated protein(rhINGAP)gene,express the gene extraeellulary in Pichia yeast for.further study on biological function and animal test on INGAP.Methods INGAP gene Was amplified with PCR and inserted into the recombinant plasmidα/pUC18.Then,the fusion gene of α and INGAP was digested and inserted into the expression plasmid pPIC9K.The positive recombinant plasmid which integrated INGAP Was confirmed by restriction enzyme digestion and sequencing,and it Was linearized with Sal Ⅰ digestion and transfered into the yeast host strain GS115 through electroporation.The yeast transformants that harbor the desired gene INGAP with high copy were selected by the auxotroph mediam G418,and verified by PCR.The condition of hake-flask culture was optimized,and the recombinant human INGAP Was induced expression with methanol as the only Carbone source.The antigen activity of the desired protein Was detected by Western blotting and ELISA method.Results Recombinant plasmid αINGAP/pPIC9K were successfully constructed and three positive Pichia yeast transformants were obtained.The expressed protein had satisfactory antigen activity,which Was confirmed by the Western blotting and ELISA method.Conclusions Pichia yeast expressing human Islet neogenesis associated protein (rhINGAP)gene was successfully constructed.

Objective To develop and test a high precision medical device which allows measurement of bioimpedance in regions below skin surface,and to enable the assessment of strained muscle's situation and instruct patient's rehabilitation exercise.Methods The system was based on the four-electrode impedance measurement mode and designed by using impedance converter network analyzer AD5933 and monolithic operational amplifier AD844.The hardware consisted of an excitation current source,a response signal adjustment circuit,a digital demodulation circuit and a shield driving circuit.This hardware circuit was combined with a LCD screen and a keyboard,which turned it into a portable precision bioimpedance testing device.Results Using an model of three components as test target,a comparison between the device and the Agilent 4294A precision impedance analyzer was presented.In the range of 1 kHz to 100 kHz,the device showed the similar relative error of the complex impedance's modulus and 1 degree absolute error of the complex impedance's phase.The strained muscle's bioelectrical impedance changes in a clinical case during the convalescent were presented and discussed with the results proven to be clinical meaningful.Conclusions The device is accurate,simple and convenient,which is proved qualified on helping doctors to assess the injury states of the strained muscle and to instruct patient's rehabilitation exercise.

Renal foreign-body is rare in clinical practice. This article reports a case of 12-year-old male patient who presented with intermittent right lumbar stabbing pain for one year. Physical and laboratory examinations showed no significant abnormalities. Enhanced CT and three-dimensional angiography indicated a metal object in the anterior superior pole of the right kidney. The patient had a history of consuming vermicelli frequently over the past year. Considering the medical history and clinical examinations, it was hypothesized that a foreign body had penetrated the duodenum and lodged into the kidney. A foreign body extraction was performed via 3D laparoscopy, confirming the object to be a 25 mm long metallic needle. The patient was discharged on the third postoperative day. Follow-up at six months showed the patient to be in good condition.

OBJECTIVETo study the clinicopathologic features and differential diagnosis of small cell variant of peripheral T-cell lymphoma, not otherwise specified (PTCL, NOS).

METHODSThe clinicopathologic features of 5 cases of small cell variant of PTCL, NOS were retrospectively reviewed, with immunohistochemical study, T-cell receptor (TCR) gene rearrangement analysis and evaluation for Epstein-Barr virus (EBV) status.

RESULTSAll the 5 patients were males. The mean age was 52.6 years. The median duration before diagnosis was 1 month. Clinically, 3 patients presented in stage IV and 2 in stage III. Four of them had generalized lymphadenopathy and splenomegaly. Hepatomegaly and massive effusion were found in 1 and 2 cases, respectively. Marrow involvement was detected in 3 of the 4 patients with bone marrow biopsy performed and one of them also accompanied by lymphocytosis. Histologically, the involved lymph nodes showed partial or complete effacement of nodal architecture and replacement by a monomorphous population of small lymphoid cells. Scanty large lymphoid cells were also identified in 4 cases. Increase in number of blood vessels was noticed in two of them as well. Immunohistochemically, the lymphoma cells in all cases expressed two or more of the T-cell markers and CD43. The staining for CD20, TdT, CD56 and granzyme B was negative. CD99 expression was noted in 3 of the 4 cases. The Ki-67 index ranged from 5% to 15%. Clonal TCRgamma gene rearrangement was detected in the 4 cases studied and one of them also showed TCRbeta gene rearrangement. In-situ hybridization for EBV-encoded RNA was negative in the 4 cases studied. Follow up information was available in 3 of the 5 cases. All of the 3 patients died of the disease, with an average survival of 21.7 months.

CONCLUSIONSmall cell variant of PTCL, NOS represents a rare disease entity which often presents in advanced tumor stage and carries a poor prognosis.

12E7 Antigen ; Adult ; Aged ; Antigens, CD ; metabolism ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; CD3 Complex ; metabolism ; Cell Adhesion Molecules ; metabolism ; Cyclophosphamide ; therapeutic use ; Doxorubicin ; therapeutic use ; Follow-Up Studies ; Gene Rearrangement, gamma-Chain T-Cell Antigen Receptor ; Humans ; Immunophenotyping ; Leukosialin ; metabolism ; Lymphatic Metastasis ; Lymphoma, T-Cell, Peripheral ; drug therapy ; genetics ; metabolism ; pathology ; Male ; Middle Aged ; Neoplasm Staging ; Prednisone ; therapeutic use ; Retrospective Studies ; Survival Rate ; Vincristine ; therapeutic use

OBJECTIVETo analyze the clinicopathologic features of extranodal NK/T cell lymphoma, nasal type (ENKTCL-N), to explore the expression of NK cell-associated receptors in ENKTCL-N and the relationship with prognosis, and to establish a prognostic model.

METHODSOne hundred and twenty-six cases of ENKTCL-N were selected from the files of the Department of Pathology, West China Hospital of Sichuan University. The relevant clinical and follow-up data were collected, and the histopathology was reviewed. All specimens were stained immunohistochemically for CD16, ICAM-1 and LFA-1. RT-PCR was used to detect the expression of CD94, NKG2 and KIR. The relationship between the prognosis of ENKTCL-N, clinical features, histopathological characteristics and expression of these markers were also analyzed.

RESULTSENKTCL-N mainly occurred in middle-age and young patients (median age, 41 years). The male to female ratio was 3.2:1. Sites more commonly involved were the nose and upper aerodigestive tract whereas those for the non-nasal type were the skin and gut. Only six cases involved two or more extranodal sites. Most (86.5%, 109/126) of the patients were in clinical stages I/II. The tumors showed predominately medium-sized tumor cells and large-sized tumor cells accounted for only 9.5% (12/126). Coagulative necrosis was present in all cases. The expression rates of CD56, CD16, CD94, LFA-1 and ICAM-1 were 82.6% (95/115), 15.1% (19/126), 55.4% (41/74), 40.5% (51/126) and 0, respectively. The expression rate of NKG2 receptor was 90.5% (67/74) overall. NKG2 receptor expression was independent of CD94. The overall expression rate of KIR receptor was 33.8% (25/74) and KIR receptor restriction was not detected in 20.8% (5/24) of the cases. Follow-up data was available in all patients, with median and average survival time being 15 months and 20.2 months, respectively. Survival analysis showed that prognostic factors included the gender, age, disease type, extranodal involvement, stage, the expression of CD16, LFA-1 and CD94. Cox's proportional hazard regression analysis revealed four factors, age, involved site, stage and CD16 expression, were independent prognostic factors.

CONCLUSIONSThe age, disease type, stage and CD16 expression are independent prognostic factors. Establishment of a prognostic model based on the above four factors can be more accurate in the prognostication of ENKTCL-N. The differences in the clinical features, prognosis, and expression of NK cell-associated receptors are obvious between nasal NK-cell lymphoma and non-nasal NK-cell lymphoma.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; CD56 Antigen ; metabolism ; Child ; Female ; Follow-Up Studies ; Humans ; Intercellular Adhesion Molecule-1 ; metabolism ; Lymphocyte Function-Associated Antigen-1 ; metabolism ; Lymphoma, Extranodal NK-T-Cell ; metabolism ; pathology ; Male ; Middle Aged ; NK Cell Lectin-Like Receptor Subfamily D ; metabolism ; Neoplasm Staging ; Nose Neoplasms ; metabolism ; pathology ; Prognosis ; Proportional Hazards Models ; Receptors, IgG ; metabolism ; Receptors, KIR ; metabolism ; Receptors, NK Cell Lectin-Like ; metabolism ; Survival Rate ; Young Adult

OBJECTIVETo clone the recombinant human islet neogenesis-associated protein (rhINGAP) gene for its secretory expression in Pichia pastoris.

METHODSINGAP gene was amplified with PCR and inserted between Xho I and EcoR I downstream sites of the alpha factor of the recombinant plasmid alpha/pUC18. The fusion gene of alpha factor and INGAP was subsequently inserted between BamH I and EcoR I sites of the plasmid pPIC9K of P. pastoris. After confirmation with restriction enzyme digestion and sequencing, the positive recombinant plasmid that integrated INGAP gene was linearized with Sal I digestion and transformed into the yeast host strain GS115 through electroporation. The yeast transformants that harbored the INGAP gene with high copies were selected with the auxotroph medium and G418, followed then by PCR verification of the positive transformants, from which the expression of recombinant human INGAP was induced with methanol as the only carbone source. The antigenic activity of the desired protein was then detected using Western blotting and enzyme-linked immunosorbent assay (ELISA).

RESULTS AND CONCLUSIONThe recombinant expression plasmid INGAP/pPIC9K was successfully constructed, and 3 positive transformants were obtained. The expressed protein showed good antigenic activity as confirmed by Western blotting and ELISA.

Antigens, Neoplasm ; genetics ; metabolism ; Biomarkers, Tumor ; genetics ; metabolism ; Blotting, Western ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; Gene Expression ; Humans ; Islets of Langerhans ; metabolism ; Lectins, C-Type ; genetics ; metabolism ; Pancreatitis-Associated Proteins ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; metabolism

Animals ; Bone Marrow Cells ; cytology ; Carbon Tetrachloride ; Carbon Tetrachloride Poisoning ; Cell Differentiation ; Cells, Cultured ; Genetic Therapy ; methods ; Hepatocyte Growth Factor ; genetics ; pharmacology ; Hepatocytes ; cytology ; Liver Cirrhosis, Experimental ; therapy ; Male ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells ; cytology ; Rats ; Rats, Wistar

Objective:To explore the correlation between the affinity while donor's HLA type recognizing different cytomegalovirus (CMV) antigen peptide and the occurrence of CMV infection after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in children.Methods:To investigate the relationship between CMV reactivation, CMV infection or CMV related tissue/organ diseases and the different HLA-type recognition antigen peptide of the donors, we retrospectively analyzed the clinical data of 146 children with CMV infection for 6 months since from the time they underwent transplantation in Wuhan Children's Hospital.Results:Among 146 patients, the HLA type of 82 (56.16%) cases had high affinity with PP65 alone, and 34 cases of CMV infection occurred after transplantation (41.46%) . None of 5 cases that had a high affinity with IE-1 alone got CMV infection. None of 2 cases with no clear high-affinity peptide had CMV infection. Three of 5 cases that had a high affinity with PP65 and PP50 had CMV infection. Thirteen of 52 cases that had a high affinity with PP65 and IE-1 had CMV infection (25.00%) . HLA with exclusive PP50 high affinity was not encountered. Donors with a high-affinity HLA locus associated with IE-1 showed a lower incidence of CMV infection after HSCT compared to those carrying only the PP65 high-affinity allele (22.81% vs 41.46%, P=0.029) . Conclusion:HLA type with PP65 and IE-1 high-affinity covers approximately 99.8% of the donors. Stem cells generated from HLA donors with high affinity with the CMV antigen peptide IE-1 can reduce the risk of post-transplantation CMV-activated infection in children.