Objective To explore the value of time-dependent diffusion MRI(td-dMRI)parameters for differentiating invasive breast cancer(IBC)with ductal carcinoma in situ(DCIS)(IBC-DCIS)from simple IBC.Methods A total of 19 patients with IBC-DCIS(IBC-DCIS group)and 53 patients with simple IBC(IBC group)confirmed by surgery and postoperation pathology were retrospectively enrolled.Breast td-dMRI acquired with oscillating gradient spin-echo(OGSE)and pulsed gradient spin-echo(PGSE)sequences before operation were interpreted,and apparent diffusion coefficient(ADC)and microstructure parameters,including OGSE-ADC value,PGSE-ADC value,cellularity,cell diameter,intracellular volume fraction and extracellular diffusion coefficient were obtained and compared between groups.Receiver operating characteristic(ROC)curves of parameters being significantly different between groups were plotted,and the area under the curve(AUC)was calculated to evaluate the efficacy of these parameters for differentiating IBC-DCIS from IBC.Results Significant differences of OGSE-ADC value,PGSE-ADC value,cellularity,cell diameter,intracellular volume fraction and extracellular diffusion coefficient were found between groups(all P＜0.05).The AUC of the above parameters for differentiating IBC-DCIS from IBC was 0.81,0.79,0.78,0.68,0.77 and 0.81,respectively.Conclusion td-dMRI parameters could be used to noninvasively and effectively differentiate IBC-DCIS from simple IBC.

Objective:To construct a sizeable naive human Fab phage display antibody library to screen high-affinity specific antibodies in vitro. Methods:Total RNA was extracted from peripheral blood mononuclear cells (PBMCs) of 126 healthy individuals, subsequently reverse-transcribed into cDNA, and used as a template. PCR amplification was performed to obtain the V H from IgG, IgM and light chain κ, λ, separately, with the initial PCR products serving as templates for a second round of PCR. Overlap extension PCR was employed to generate fragments of the κ and λ light chains. These fragments were ligated with the phage vector pNC3, which harbors the variable region 1 of the heavy chain, to construct a recombinant phage plasmid. This plasmid was then electroporated into competent Escherichia Coli TG1 cells to establish a naive human Fab phage display antibody library. One hundred clones were randomly selected for identification and sequencing, and antibody gene polymorphisms were analyzed using the IMGT database and MAFFT software. Recombinant α-hemolysin from Staphylococcus aureus was utilized to screen Fab antibody fragments through biopanning of the antibody library, followed by random selection of phage ELISA-identified clones. The positive clones (antigen A450∶blank control A450≥2.1) were sequenced. Results:Two large naive Fab phage display antibody libraries were successfully constructed, in which the capacity of κ and λ chain antibody libraries were 1.25×10 11 and 1.54×10 11, respectively. The titers for two antibody libraries were 6.04×10 13 CFU/ml and 3.50×10 13 CFU/ml. The positive transformation insertion rates for κ and λ chain antibody libraries were 96% (96/100) and 100% (100/100), respectively. Sequence analysis revealed that all antibody sequences were unique. The amino acid sequences in the skeletal region were relatively conserved. In contrast, significant variations in the length of the complementarity determining region (CDR) were found, and the diversity of amino acid sequence of the complementary determining region was high, especially the CDR3. Analysis using the IMGT database indicated that the sequences exhibited a broad distribution across variable-diversity-joining gene families. After six rounds of panning, specific phage antibodies enrichment targeting α-hemolysin were achieved. A total of 142 monoclonal antibodies were sequenced, yielding 8 distinct Fab antibody sequences. Conclusion:This study successfully constructed two naive human Fab phage display antibody libraries with large capacity and good diversity, which can be used for screening human antibodies for serum epidemiology.

Objective:To construct a sizeable naive human Fab phage display antibody library to screen high-affinity specific antibodies in vitro. Methods:Total RNA was extracted from peripheral blood mononuclear cells (PBMCs) of 126 healthy individuals, subsequently reverse-transcribed into cDNA, and used as a template. PCR amplification was performed to obtain the V H from IgG, IgM and light chain κ, λ, separately, with the initial PCR products serving as templates for a second round of PCR. Overlap extension PCR was employed to generate fragments of the κ and λ light chains. These fragments were ligated with the phage vector pNC3, which harbors the variable region 1 of the heavy chain, to construct a recombinant phage plasmid. This plasmid was then electroporated into competent Escherichia Coli TG1 cells to establish a naive human Fab phage display antibody library. One hundred clones were randomly selected for identification and sequencing, and antibody gene polymorphisms were analyzed using the IMGT database and MAFFT software. Recombinant α-hemolysin from Staphylococcus aureus was utilized to screen Fab antibody fragments through biopanning of the antibody library, followed by random selection of phage ELISA-identified clones. The positive clones (antigen A450∶blank control A450≥2.1) were sequenced. Results:Two large naive Fab phage display antibody libraries were successfully constructed, in which the capacity of κ and λ chain antibody libraries were 1.25×10 11 and 1.54×10 11, respectively. The titers for two antibody libraries were 6.04×10 13 CFU/ml and 3.50×10 13 CFU/ml. The positive transformation insertion rates for κ and λ chain antibody libraries were 96% (96/100) and 100% (100/100), respectively. Sequence analysis revealed that all antibody sequences were unique. The amino acid sequences in the skeletal region were relatively conserved. In contrast, significant variations in the length of the complementarity determining region (CDR) were found, and the diversity of amino acid sequence of the complementary determining region was high, especially the CDR3. Analysis using the IMGT database indicated that the sequences exhibited a broad distribution across variable-diversity-joining gene families. After six rounds of panning, specific phage antibodies enrichment targeting α-hemolysin were achieved. A total of 142 monoclonal antibodies were sequenced, yielding 8 distinct Fab antibody sequences. Conclusion:This study successfully constructed two naive human Fab phage display antibody libraries with large capacity and good diversity, which can be used for screening human antibodies for serum epidemiology.

Objective To explore the value of time-dependent diffusion MRI(td-dMRI)parameters for differentiating invasive breast cancer(IBC)with ductal carcinoma in situ(DCIS)(IBC-DCIS)from simple IBC.Methods A total of 19 patients with IBC-DCIS(IBC-DCIS group)and 53 patients with simple IBC(IBC group)confirmed by surgery and postoperation pathology were retrospectively enrolled.Breast td-dMRI acquired with oscillating gradient spin-echo(OGSE)and pulsed gradient spin-echo(PGSE)sequences before operation were interpreted,and apparent diffusion coefficient(ADC)and microstructure parameters,including OGSE-ADC value,PGSE-ADC value,cellularity,cell diameter,intracellular volume fraction and extracellular diffusion coefficient were obtained and compared between groups.Receiver operating characteristic(ROC)curves of parameters being significantly different between groups were plotted,and the area under the curve(AUC)was calculated to evaluate the efficacy of these parameters for differentiating IBC-DCIS from IBC.Results Significant differences of OGSE-ADC value,PGSE-ADC value,cellularity,cell diameter,intracellular volume fraction and extracellular diffusion coefficient were found between groups(all P＜0.05).The AUC of the above parameters for differentiating IBC-DCIS from IBC was 0.81,0.79,0.78,0.68,0.77 and 0.81,respectively.Conclusion td-dMRI parameters could be used to noninvasively and effectively differentiate IBC-DCIS from simple IBC.

Objective To explore the application value of simultaneous multi-slice(SMS)technique in diffusion tensor imaging(DTI)for evaluating brain glioma.Methods Thirty-four brain glioma patients were prospectively enrolled,and brain conventional DTI and SMS-DTI were acquired.The subjective scores of image quality,signal-to-noise ratio(SNR)and contrast-to-noise ratio(CNR)were compared between SMS-DTI and conventional DTI,so were the numbers of whole brain fiber bundles,tumor relative fractional anisotropy(rFA)and relative mean diffusivity(rMD)obtained based on SMS-DTI and conventional DTI.Results Among 34 patients,there were 23 cases of high-grade glioma and 11 cases of low-grade glioma.No significant difference of subjective scores of image quality,tumor edge clarity nor magnetic susceptibility artifacts was found between SMS-DTI and conventional DTI(all P＞0.05).SNR and CNR on SMS-DTI were both lower than those on conventional DTI(both P＜0.05).No significant difference of the numbers of whole brain fiber bundles,rFA nor rMD of gliomas with different pathological grades was detected based on SMS-DTI compared with those on conventional DTI(all P＞0.05).Conclusion SMS applicated in DTI for evaluating brain gliomas was able to shorten acquisition time under the condition of ensuring image quality and quantitative analysis accuracy.

Objective To analyze the differences in immune system between Npc1 gene mutant (Npc1-/ -) and wild-type (Npc1+/ +) mice for better understanding the pathogenesis of Niemann-Pick disease type C1 (NPC1) from an immunological perspective and providing reference for NPC1 treatment in clinic.Methods Body, thymus and spleen weight of Npc1-/ -and Npc1+/ + mice aged (14±2) days, (42±2) days and (63±2) days (Day14±2 , Day42±2 and Day63±2 ) were recorded and the associated organ index were calcu-lated. White blood cell count in peripheral blood of mice aged Day42±2 was examined by routine blood test. Expression of cytokines at mRNA level in mouse peripheral blood was detected by qPCR. Percentages of CD4+, CD8+ and CD19+ lymphocytes in peripheral blood and spleen of mice aged Day42±2 were measured by flow cytometry. Apoptosis and senescence of spleen in mice aged Day63±2 were examined by immunofluores-cence and β-galactosidase staining. Results Compared with Npc1+/ + mice, there was no significant differ-ence in the weight of spleen and thymus in Npc1-/ - mice aged Day14±2; the weight of spleen in Npc1-/ - mice aged Day42±2 significantly increased, but the weight of thymus showed a significant decrease; furthermore, both the weight of spleen and thymus in Npc1-/ - mice aged Day63±2 significantly decreased; and the body weight of Npc1-/ - mice of each age group significantly decreased. Moreover, compared with Npc1+/ + mice, the absolute number of lymphocytes in the peripheral blood of Npc1-/ - mice aged Day42±2 showed no signifi-cant difference, but the percentage in whole white blood cells significantly decreased due to the significantly increased neutrophils. Expression of cytokines ( IL-1, IL-2, IFN-γ, TNF-α, IL-4, granzyme A and granzyme B) at mRNA level in the peripheral blood leukocytes of Npc1-/ - mice aged Day42±2 was abnormal as compared with that in Npc1+/ + mice. The number of T (CD4+ and CD8+) lymphocytes in Npc1-/ - mice aged Day42±2 significantly decreased, while the number of B (CD19+) lymphocytes increased significantly as com-pared with those in the Npc1+/ + mice. Compared with Npc1+/ + mice, apoptosis and senescence of the spleen in Npc1-/ - mice aged Day63±2 aggravated significantly. Conclusion The abnormal lipid metabolism triggered by Npc1 gene mutation causes severe immune dysfunction in Npc1-/ - mice. Therefore, immune dysfunction should be taken into full consideration when treating patients with NPC1, which might help improve the life quality and prolong the survival time.

In modern biology and biotechnology research, recombinant gene expression has been the most popular method to obtain the target protein. In recent years, many foreign genes have been efficiently expressed in Escherichia coli. However, proteins encoded by animal, plant or mesophilic microbial genes often lose activities or become denatured within a few hours at regular growth temperatures for E. coli; some other target proteins are toxic to host cells and therefore difficult to be over-expressed. The new T-vector, pEXC-T, was constructed by combining TA cloning and cold-shock induction to obtain high expression levels with low costs. This paper reports the construction of pEXC-T and optimization of induction techniques for gene expression. Two instable proteins were tested and successfully expressed in soluble form by using pEXC vector. The development of pEXC-T offers a convenient technique for the preparations of recombinant proteins to be used in structure/function studies, or as diagnostic markers and medicinal proteins.
Biotechnology ; Cold Temperature ; Escherichia coli ; genetics ; Gene Expression ; Genetic Vectors ; Plasmids ; genetics ; Recombinant Proteins

Fibroblast growth factor 21 (FGF21) is a member of FGF family. It has been demonstrated that FGF21 is an independent, safe and effective regulator of blood glucose levels in vivo. In order to improve the activity of FGF21, we exchanged the beta10-beta12 domain of the human FGF21 with that of the mouse FGF21 to construct a novel FGF21 gene (named hmFGF21), and then subcloned hmFGF21 gene into the SUMO expression vector to create pSUMO-hmFGF21 and transformed it into E. coli Rosetta for expression of the fusion protein SUMO-hmFGF21. Both in vitro and in vivo glucose regulation activity of hmFGF21 was evaluated. The SDS-PAGE result showed that compared with wild-type hFGF21, the soluble expression of hmFGF21 increased about 2-fold. HmFGF21 was more potent in stimulation of glucose uptake in HepG2 cells in vitro. The results of anti-diabetic effect on db/db mice demonstrated that hmFGF21 had better efficacy on controlling the blood glucose of the db/db diabetic animals than wild-type hFGF21. These results suggest that the biological properties of FGF21 are significantly improved by optimization.

Objective To investigate the in vitro effect of interferon-γ (IFN-γ) and ATRA on the morphological transition, proliferation of and apoptosis in a human cutaneous squamous cell carcinoma cell line SCL-1. Methods Cultured SCL-1 cells were divided into 6 groups to be treated with ATRA of 1 μmol/L, various concentrations ( 100, 500, 1000 U/ml) of IFN-γ, the combination of ATRA of 1 μmol/L and IFN-y of 1000 U/ml,respectively, or to remain untreated. MTT assay and flow cytometry were performed to evaluate the cell proliferation and apoptosis. The morphological features of apoptotic cells were observed by a transmission electron microscope (TEM) and inverted phase contrast microscope after 1% propidium iodide staining. Results IFN-γ could inhibit the proliferation of SCL-1 cells in a dose-dependent manner, and the most pronounced inhibitory effect was observed at a dose of 1000 U/ml . ATRA and IFN-γ induced an apoptosis in SCL-1 cells, and the early apoptosis rate was 4.84%, 11.96% and 18.71% in SCL-1 cells after treated with ATRA of 1 μmol/L, IFN-γ of 1000 U/ml and their combination, respectively. A series of morphological changes characteristic of apoptosis,such as bipolar changes, were observed in SCL-1 cells treated with ATRA and IFN-γ, with the presence of many early apoptotic cells, which showed a trend towards benign differentiation. Conclusions Within a certain concentration range, IFN-γcan promote the differentiation, but inhibit the proliferation of SCL-1 cells in a dose-dependent manner, and ATRA could enhance the effects of IFN-γ.

Objective To investigate the expression of survivin and bcl-2 in human squamous cell carcinoma (SCC) lesions and cell line SCL-1. Methods Tissue samples from 60 patients with SCC and 10 normal human controls were immunohistochemically stained to detect the expressions of survivin and bcl-2.Western blot was used to measure the expressions of bcl-2 and survivin proteins in HaCaT human keratinocytes and SCL-1 human squamous cell carcinoma cells. Results In normal control tissues, there was no expressions of survivin or bcl-2, while in SCC, the expression rates of bcl-2 and survivin were 70% and 60%, respectively,and there was no statistical correlation between the expressions of bcl-2 and survivin (P >0.05). Neither the expression of survivin nor that of bcl-2 was correlated to patients' age, gender or lesional site (all P >0.05). A statistical correlation was observed between the pathological stage in patients and expression of bcl-2 as well as between lymph node metastasis and expression of survivin (both P < 0.05). Western blot analysis revealed a significant increase in the expression of survivin and bcl-2 in SCL-1 cells compared with HaCaT cells. Con-clusion In SCC, survivin and bcl-2 seem to play their roles via different anti-apoptotic pathways.