We developed large-scale preparation of phycoerythrin from Porphyra haitanensis, a main economic red algae in China. Firstly, P. haitanensis thallus was broken by using "swelling and smash" method. Then times of grads ammonium sulfate precipitation applied to the crude extraction were compared. Desalted solution was further purified with one-step chromatography using hydroxyapatite and properties on spectrum and molecular weight were identified finally. The results indicated that after four times of ammonium sulfate precipitation (15%, 50%, 10% and 40%), the absorption spectrum purity of P. haitanensis achieved 0.9 (A564/A280), and 507.82 mg phycoerythrin (A564/A280 > 3.2) was obtained from 7 kg fresh algae after further hydroxyapatite chromatography. This research provides a potential way for preparation of phycoerythrin in large sclae.
Ammonium Sulfate ; chemistry ; Chromatography ; methods ; Phycoerythrin ; isolation & purification ; Porphyra ; chemistry

Animals ; Biocatalysis ; Cattle ; Enteropeptidase ; chemistry ; metabolism ; Humans ; Models, Molecular ; Oryzias ; metabolism ; Structure-Activity Relationship ; Substrate Specificity

Objective To explore the automated identification and diameter measurement methods for inferior vena cava (IVC) based on clinical ultrasound images of IVC. Methods An automated identification and localization method based on topology and automatic tracking algorithm was proposed. Tracking algorithm was used for identifying and continuously locating to improve the efficiency and accuracy of measurement. Tests were conducted on 18 sets of ultrasound data collected from 18 patients in intensive care unit (ICU),with clinicians' measurements as the gold standard. Results The recognition accuracy of the automated method was 94.44％ (17/18),and the measurement error of IVC diameter was within the range of±1.96s (s was the standard deviation). The automated method could replace the manual method. Conclusion The proposed IVC automated identification and localization algorithm based on topology and automatic tracking algorithm has high recognition success rate and IVC diameter measurement accuracy. It can assist clinicians in identifying and locating IVC,so as to improve the accuracy of IVC measurement.

Objectives:To investigate the effect of the expression of low-density lipoprotein receptor associated protein (LDLR) on the vascular abnormalities in hepatocellular carcinoma (HCC) and its mechanisms.Methods:Based on the information of Oncomine Cancer GeneChip database, we analyzed the correlation between the expression level of LDLR and the expression level of carcinoembryonic antigen (CEA) and CD31 in hepatocellular carcinoma tissues. Lentiviral transfection of short hairpin RNA target genes was used to construct LDLR-knockdown MHCC-97H and HLE hepatocellular carcinoma cells. The differential genes and their expression level changes in LDLR-knockdown hepatocellular carcinoma cells were detected by transcriptome sequencing, real-time fluorescence quantitative polymerase chain reaction, and protein immunoblotting. The gene-related signaling pathways that involve LDLR were clarified by enrichment analysis. The effect of LDLR on CEA was assessed by the detection of CEA content in conditioned medium of hepatocellular carcinoma cells. Angiogenesis assay was used to detect the effect of LDLR on the angiogenic capacity of human umbilical vein endothelial cells, as well as the role of CEA in the regulation of angiogenesis by LDLR. Immunohistochemical staining was used to detect the expression levels of LDLR in 176 hepatocellular carcinoma tissues, and CEA and CD31 in 146 hepatocellular carcinoma tissues, and analyze the correlations between the expression levels of LDLR, CEA, and CD31 in the tissues, serum CEA, and alanine transaminase (ALT).Results:Oncomine database analysis showed that the expressions of LDLR and CEA in the tissues of hepatocellular carcinoma patients with portal vein metastasis were negatively correlated ( r=-0.64, P=0.001), whereas the expressions of CEA and CD31 in these tissues were positively correlated ( r=0.46, P=0.010). The transcriptome sequencing results showed that there were a total of 1 032 differentially expressed genes in the LDLR-knockdown group and the control group of MHCC-97H cells, of which 517 genes were up-regulated and 515 genes were down-regulated. The transcript expression level of CEACAM5 was significantly up-regulated in the cells of the LDLR-knockdown group. The Gene Ontology (GO) function enrichment analysis showed that the differential genes were most obviously enriched in the angiogenesis function. The Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway enrichment analysis showed that the relevant pathways involved mainly included the cellular adhesion patch, the extracellular matrix receptor interactions, and the interactions with the extracellular matrix receptors. The CEA content in the conditioned medium of the LDLR-knockdown group was 43.75±8.43, which was higher than that of the control group (1.15±0.14, P＜0.001). The results of angiogenesis experiments showed that at 5 h, the number of main junctions, the number of main segments, and the total area of the lattice formed by HUVEC cells cultured with the conditioned medium of MHCC-97H cells in the LDLR-knockdown group were 295.3±26.4, 552.5±63.8, and 2 239 781.0±13 8211.9 square pixels, which were higher than those of the control group (113.3±23.5, 194.8±36.5, and 660 621.0±280 328.3 square pixels, respectively, all P＜0.01).The number of vascular major junctions, the number of major segments, and the total area of the lattice formed by HUVEC cells cultured in conditioned medium with HLE cells in the LDLR-knockdown group were 245.3±42.4, 257.5±20.4, and 2 535 754.5±249 094.2 square pixels, respectively, which were all higher than those of the control group (113.3±23.5, 114.3±12.2, and 1 565 456.5±219 259.7 square pixels, respectively, all P＜0.01). In the conditioned medium for the control group of MHCC-97H cells,the number of main junctions, the number of main segments, and the total area of the lattice formed by the addition of CEA to cultured HUVEC cells were 178.9±12.0, 286.9±12.3, and 1 966 990.0±126 249.5 spixels, which were higher than those in the control group (119.7±22.1, 202.7±33.7, and 1 421 191.0±189 837.8 square pixels, respectively). The expression of LDLR in hepatocellular carcinoma tissues was not correlated with the expression of CEA, but was negatively correlated with the expression of CD31 ( r=-0.167, P=0.044), the level of serum CEA ( r=-0.061, P=0.032), and the level of serum ALT (r=-0.147, P=0.05). The expression of CEA in hepatocellular carcinoma tissues was positively correlated with the expression of CD31 ( r=0.192, P=0.020). The level of serum CEA was positively correlated with the level of serum ALT ( r=0.164, P=0.029). Conclusion:Knocking down LDLR can promote vascular abnormalities in HCC by releasing CEA.

Objectives:To investigate the effect of the expression of low-density lipoprotein receptor associated protein (LDLR) on the vascular abnormalities in hepatocellular carcinoma (HCC) and its mechanisms.Methods:Based on the information of Oncomine Cancer GeneChip database, we analyzed the correlation between the expression level of LDLR and the expression level of carcinoembryonic antigen (CEA) and CD31 in hepatocellular carcinoma tissues. Lentiviral transfection of short hairpin RNA target genes was used to construct LDLR-knockdown MHCC-97H and HLE hepatocellular carcinoma cells. The differential genes and their expression level changes in LDLR-knockdown hepatocellular carcinoma cells were detected by transcriptome sequencing, real-time fluorescence quantitative polymerase chain reaction, and protein immunoblotting. The gene-related signaling pathways that involve LDLR were clarified by enrichment analysis. The effect of LDLR on CEA was assessed by the detection of CEA content in conditioned medium of hepatocellular carcinoma cells. Angiogenesis assay was used to detect the effect of LDLR on the angiogenic capacity of human umbilical vein endothelial cells, as well as the role of CEA in the regulation of angiogenesis by LDLR. Immunohistochemical staining was used to detect the expression levels of LDLR in 176 hepatocellular carcinoma tissues, and CEA and CD31 in 146 hepatocellular carcinoma tissues, and analyze the correlations between the expression levels of LDLR, CEA, and CD31 in the tissues, serum CEA, and alanine transaminase (ALT).Results:Oncomine database analysis showed that the expressions of LDLR and CEA in the tissues of hepatocellular carcinoma patients with portal vein metastasis were negatively correlated ( r=-0.64, P=0.001), whereas the expressions of CEA and CD31 in these tissues were positively correlated ( r=0.46, P=0.010). The transcriptome sequencing results showed that there were a total of 1 032 differentially expressed genes in the LDLR-knockdown group and the control group of MHCC-97H cells, of which 517 genes were up-regulated and 515 genes were down-regulated. The transcript expression level of CEACAM5 was significantly up-regulated in the cells of the LDLR-knockdown group. The Gene Ontology (GO) function enrichment analysis showed that the differential genes were most obviously enriched in the angiogenesis function. The Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway enrichment analysis showed that the relevant pathways involved mainly included the cellular adhesion patch, the extracellular matrix receptor interactions, and the interactions with the extracellular matrix receptors. The CEA content in the conditioned medium of the LDLR-knockdown group was 43.75±8.43, which was higher than that of the control group (1.15±0.14, P＜0.001). The results of angiogenesis experiments showed that at 5 h, the number of main junctions, the number of main segments, and the total area of the lattice formed by HUVEC cells cultured with the conditioned medium of MHCC-97H cells in the LDLR-knockdown group were 295.3±26.4, 552.5±63.8, and 2 239 781.0±13 8211.9 square pixels, which were higher than those of the control group (113.3±23.5, 194.8±36.5, and 660 621.0±280 328.3 square pixels, respectively, all P＜0.01).The number of vascular major junctions, the number of major segments, and the total area of the lattice formed by HUVEC cells cultured in conditioned medium with HLE cells in the LDLR-knockdown group were 245.3±42.4, 257.5±20.4, and 2 535 754.5±249 094.2 square pixels, respectively, which were all higher than those of the control group (113.3±23.5, 114.3±12.2, and 1 565 456.5±219 259.7 square pixels, respectively, all P＜0.01). In the conditioned medium for the control group of MHCC-97H cells,the number of main junctions, the number of main segments, and the total area of the lattice formed by the addition of CEA to cultured HUVEC cells were 178.9±12.0, 286.9±12.3, and 1 966 990.0±126 249.5 spixels, which were higher than those in the control group (119.7±22.1, 202.7±33.7, and 1 421 191.0±189 837.8 square pixels, respectively). The expression of LDLR in hepatocellular carcinoma tissues was not correlated with the expression of CEA, but was negatively correlated with the expression of CD31 ( r=-0.167, P=0.044), the level of serum CEA ( r=-0.061, P=0.032), and the level of serum ALT (r=-0.147, P=0.05). The expression of CEA in hepatocellular carcinoma tissues was positively correlated with the expression of CD31 ( r=0.192, P=0.020). The level of serum CEA was positively correlated with the level of serum ALT ( r=0.164, P=0.029). Conclusion:Knocking down LDLR can promote vascular abnormalities in HCC by releasing CEA.

In order to improve the expression of recombinant human atrial natriuretic peptide (ANP), a new plasmid (pET28a(+)/ANP₃) containing 3 tandem ANP genes with lysine codon as the interval linker, was constructed. Target gene was transformed into Escherichia coli BL21 (DE3) and induced by IPTG, about 60% of the total-cell-protein was the target protein, His₆-ANP₃. After denaturation and refolding, it was digested by Endoproteinase Lys-C and Carboxypeptidase B (CPB) and then purified by a series of purification processes, about 16 mg purified ANP monomer could be obtained from one liter bacteria broth of shaking culture. Ultimately, the purity of protein was above 90% determined by UPLC and Tricine SDS-PAGE, its molecular weight was 3 080 Da according to LC-MS identification and it was proved to be equivalent to the reference product by ELISA. The use of tandem gene expression can provide a new possible model for the expression of other peptide drugs.
Atrial Natriuretic Factor ; biosynthesis ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; metabolism ; Gene Expression ; Humans ; Metalloendopeptidases ; Peptides ; Plasmids ; genetics ; Recombinant Fusion Proteins ; biosynthesis

Background Silicosis is an occupational disease mainly characterized by pulmonary progressive fibrosis induced by the accumulation of free silica (SiO₂) in the lungs due to long-term exposure to SiO₂ dust. It has been shown that neutrophil extracellular traps (NETs) are increased in the lung tissues of silicotic mice after 28 d SiO₂ exposure, but it is unclear how the levels of NETs change throughout entire progression of silicosis in mice. Objective To observe the levels of NETs and pathological changes in the lungs of silicotic mice after different duration of SiO₂ exposure, and to confirm the possible role and significance of NETsin the development of SiO₂-induced pulmonary fibrosis. Methods A total of 28 SPF male C57BL/6J mice were randomly divided into a control group, and a model group, and the model group was subdivided into, a 2 d model group, a 7 d model group, and a 28 d model group, with 7 mice in each group. The mice in the model groups were given intratracheal instillation with 10 mg SiO₂ suspension (50 μL), and the mice in the control group were received same volume of saline. Mice were sacrificed and samples were collected at designed time points. The pathological changes of lung tissues of mice were observed after hematoxylin-eosin (HE) and Van Gieson (VG) staining. Immunofluorescence was used to observe the NETs markers citrullination histone H3 (CitH3) and myeloperoxidase (MPO) in bronchoalveolar lavage fluid (BALF), and the percentage of NETs-positive cells was calculated. PicoGreen fluorescent dye kit was used to detect the content of extracelluar DNA (ex-DNA) in mouse BALF, and the expression levels of fibrosis-related proteins α-smooth muscle actin (α-SMA) and fibronectin (FN) and NETs marker CitH3 in lung tissues of mice were detected by Western blot (WB). Results Compared with the control group, inflammatory cells accumulation, alveolar wall thickening, and collagen deposition were obviously observed in the lungs of the silicosis model groups, and a large number of silicone nodules were recorded in the lung tissues in the 28 d group. Compared with the control group, the expressions of α-SMA and FN in the lung tissue of the 28 d group were significantly increased (P<0.05). The percentages of NETs in BALF increased significantly in the 2 d and the 7 d model group, then decreased in the 28 d model group (P<0.05). Compared with the control group (7.434±0.258) ng·mL⁻¹, the ex-DNA levels in BALF of mice in the 2 d [(35.110±6.331) ng·mL⁻¹], the 7 d [(39.491±6.948) ng·mL⁻¹], and the 28 d [(23.360±4.809) ng·mL⁻¹] model groups were increased (P<0.05), and the increase of ex-DNA in the 2 d and the 7 d model groups were statistically significant (P<0.05). In comparison with the control group, the protein level of CitH3 was significantly increased in the lung tissues of mice in the 7 d model group (P<0.05). Conclusion The content of NETs increases significantly and reaches a peak in the early inflammatory stage of silicosis, and decreases as the disease progresses to the fibrotic stage, suggesting that NETs may play a role in early stage of silicosis.