Factors and Mechanisms Affecting the Secretion of Recombinant Protein in CHO Cells.

The market demand for recombinant therapeutic proteins (RTPs) has promoted the development of various protein expression host and bioprocessing technologies. Since mammalian cells have the unique advantage of being able to direct the correct folding of proteins and provide post-translational processing such as complex glycosylation, the RTPs produced by them currently account for approximately 80% of the approved marketed RTPs. Among them, Chinese hamster ovary (CHO) cells are currently the preferred host cells for the production of RTPs. Production of RTPs in CHO cells involves the synthesis, processing, transport, and secretion of proteins. The secretion process of proteins is one of the key steps, which greatly limits the yield and quality of RTPs. Here, we review the recombinant protein secretion process of CHO cells and its influencing factors, and further discuss the optimization strategy for recombinant protein secretion and expression in CHO cells.

BMP4 is insufficient to differentiate umbilical cord mesenchymal stem cells into germ cell-like cells in vitro.

OBJECTIVES: Mesenchymal stem cell (MSC)-based therapies are expected to restore the fertility of infertile patients. In addition to MSC-derived paracrine effects to improve reproductive function, the differentiation of MSCs into germ cell (GC)-like cells is still a promising method to repair the injured reproductive system. The aim of this study was to examine the effect and potential mechanism of BMP4 in inducing umbilical cord MSC (UcMSC) transdifferentiation into GC-like cells. MATERIAL AND METHODS: UcMSCs were isolated, cultured and identified by flow cytometry and multilineage differentiation assays. After induction with 12.5 ng/mL BMP4 for 21 days, UcMSCs were collected for further examination. Immunofluorescence was used to detect the expression of Prdm1 and Prdm14; RT-PCR and RNA sequencing were used to detect differential gene expression (DEGs). RESULTS: The morphology of UcMSCs became large and flat after treatment with BMP4; the expression of GC-related genes (OCT4, Prdm1, Ifitm3 and Stella) was significantly downregulated, and further immunofluorescence results also confirmed the significant downregulation of Prdm1 in UcMSCs with BMP4 induction, while the expression of Prdm14 was significantly upregulated. The results of RNA sequencing and further analysis revealed no explicit correlation between BMP4 induction and the differentiation of UcMSCs into GC-like cells based on the 662 screened DEGs in UcMSCs with or without BMP4 induction. CONCLUSIONS: The differentiation of MSCs into GC-like cells is rather complex, and BMP4 alone is insufficient to induce UcMSCs to differentiate into GC-like cells, regardless of protein level or gene expression level.

Increase recombinant antibody yields through optimizing vector design and production process in CHO cells.

Chinese hamster ovary (CHO) cells are the most commonly used host cells for the production of recombinant monoclonal antibodies (mAbs) due to their several advantages. Although the yields of recombinant mAbs can be greatly increased by some strategies, such as medium formulation, culture conditions, and cell engineering, most studies focused on either upstream design or downstream processes. In the present study, we first expressed recombinant adalimumab through combination of the vector design and production process optimization in CHO cells. Bicistronic vector, monocistronic vector, and dual promoter vector were constructed, and the production process was optimized using low-temperature and fed-batch culture. The results showed that the dual promoter vector exhibited the highest yield under the transient and stable transfected cells among three different vector systems in CHO cells. In addition, low-temperature and fed-batch culture could further improve the yields of adalimumab. The purified antibody displayed tumor necrosis factor-α (TNF-α) binding activity. In conclusion, combination of expression vector design and production process optimization can achieve higher expression of recombinant mAbs in CHO cells. KEY POINTS: • The dual promoter vector is more effective for expressing recombinant antibodies. • The yields of antibodies are related to the LC chain expression level. • Low-temperature and feed addition can promote antibody production.

Cell Surface Markers and their Targeted Drugs in Breast Cancer.

Breast cancer is the most common cancer affecting women's health, and its incidence rate is continuously increasing. With the development of immunohistochemistry and gene expression microarray technology, the study on breast cancer has gradually advanced, contributing to the development of targeted therapy for breast cancer. At present, the popular breast cancer cell surface markers includeG protein-coupled estrogen receptor 1 (GPER-1), human epidermal growth factor receptor 2 (HER-2), epidermal growth factor receptor (EGFR), c-mesenchymal-epithelial transition factor (CMet), folate receptor-α (FRα), integrin, programmed death-ligand 1 (PD-L1), trophoblast cell surface antigen 2 (Trop-2), etc. Targeted drugs for breast cancer cell surface markers mainly include antibody drugs and small molecule inhibitor drugs, which exert anti-tumor activity by targeting receptors or ligands. This review summarizes the surface markers of breast cancer cells and their targeted drugs, hoping to provide new ideas for breast cancer targeted therapy.

Generation and Characterization of Induced Pluripotent Stem Cells from Mononuclear Cells in Schizophrenic Patients.

OBJECTIVE: Schizophrenia (SZ) is a mental disorder in which psychotic symptoms are the main problem. The pathogenesis of SZ is not fully understood, partly because of limitations in current disease models and technology. The development of induced pluripotent stem cell (iPSC) technology has opened up the possibility of elucidating disease mechanisms in neurodegenerative diseases. Here, we aimed to obtain iPSCs from peripheral blood mononuclear cells (PBMCs) of normal and schizophrenic individuals and analyze the inflammatory response in these iPSCs. MATERIALS AND METHODS: In this experimental study, we isolated PBMCs from whole blood of healthy individuals and SZ patients and reprogrammed them into iPSCs by transfection of recombinant lentiviruses that contained Yamanaka factors (Oct4, Sox2, Klf4 and c-Myc). We calculated the numbers of iPSC clones and stained them with alkaline phosphatase (ALP), Nanog, SSEA4, Nestin, Vimentin, and AFP to confirm their efficiency and pluripotency. The iPSCs were analyzed by real-time quantitative polymerase chain reaction (qRT-PCR) for the expressions of inflammatory factors. RESULTS: iPSCs from schizophrenic patients (SZ-iPSCs) exhibited typical morphology and highly expressed pluripotent markers. These iPSCs retained their normal karyotype and differentiated in vitro to form embryoid bodies (EBs) that expressed markers of all 3 germ layers. However, iPSCs from the SZ-iPSCs group had a weak capacity to differentiate into ectoderm compared to the normal iPSCs (Con-iPSC). An elevated, stronger inflammatory response existed in iPSCs from schizophrenic individuals. CONCLUSION: We successfully obtained iPSCs from PBMCs of schizophrenic patients without genetic operation and analyzed the expressions of pluripotent markers and inflammatory factors between the Con-iPSC and SZ-iPSC groups. Taken together, our results may assist to explain the pathogenesis of SZ and develop new strategies for clinical diagnosis and treatment.

Umbilical Cord Mesenchymal Stem Cells Conditioned Medium Promotes Aß25-35 phagocytosis by Modulating Autophagy and Aß-Degrading Enzymes in BV2 Cells.

Mesenchymal stem cell (MSC) therapy is a promising prospect for the treatment of Alzheimer's disease (AD); however, the underlying mechanisms by which MSCs mediate positive effects are still unclear. We speculated that MSCs mediate microglial autophagy and enhance the clearance of Aß. To test this hypothesis, we cultured BV2 microglial cells with umbilical cord mesenchymal stem cells conditioned medium (ucMSCs-CM) in the presence or absence of Aß25-35 oligomers. We investigated BV2 cell proliferation, cell death, and Aß25-35 phagocytosis as well as protein expression levels of LC3, Beclin-1, p62, insulin-degrading enzyme (IDE), and neprilysin (Nep) with western blotting. The results showed that ucMSCs-CM inhibited the proliferation and decreased cell death of BV2 cells induced by Aß25-35. ucMSCs-CM also promoted the phagocytosis of Aß25-35 by BV2 cells and changed the expression of autophagy-related proteins LC3, Beclin-1, and p62. Treatment also upregulated the expression of Aß-degrading enzymes IDE and Nep. Furthermore, the culture medium in BV2 cells with Aß25-35 and ucMSCs-CM prevented neuronal cell SH-SY5Y from cell death compared to control medium without ucMSCs-CM. Altogether, these data suggested that ucMSCs-CM protect microglial and neuronal cells from Aß25-35-induced cell death and promote Aß phagocytosis by modulating autophagy and enhancing the expression of Aß-degrading enzymes in microglia.

Bone marrow mesenchymal stem cells accelerate the hyperglycemic refractory wound healing by inhibiting an excessive inflammatory response.

The aim of the present study was to evaluate the healing effect of bone marrow-derived mesenchymal stem cells administered to hyperglycemia model mice with skin wounds, and to explore the underlying mechanism contributing to their effects in promoting refractory wound healing. A full­thickness skin wound mouse model was established, and refers to a wound of the skin and subcutaneous tissue. The mice were randomly divided into three groups: Blank control group, hyperglycemic group and a hyperglycemic group treated with stem cells. Wound healing was monitored and the wound­healing rate was determined at 3, 6, 9, and 12 days following trauma. The structure of the organization of new skin tissue was observed by hematoxylin and eosin staining, and expression levels of the inflammatory cytokines interleukin (IL)­6 and tumor necrosis factor (TNF)­α were determined from 1 to 6 days following trauma. The wound healing of the hyperglycemic group was slower than that of the blank group, and the hyperglycemic mice treated with stem cells presented faster healing than the hyperglycemia group. The horny layer and granular layer of the skin were thinner and incomplete in the new skin tissue of the hyperglycemic group, whereas the new skin wound tissue basal layer was flat and demonstrated better fusion with the wound edge in the other two groups. The expression of inflammatory cytokines (IL­6 and TNF­α) was significantly increased in all three groups, with continuously higher expression in the hyperglycemic group and decreased expression in the other two groups over time. Hyperglycemia refractory wounds are likely related to the excessive expression of inflammatory cytokines surrounding the wound area. Stem cells may be able to alleviate the excessive inflammatory reaction in the wound tissue of hyperglycemic mice, so as to promote wound healing.

Shewanella gelidii sp. nov., isolated from the red algae Gelidium amansii, and emended description of Shewanella waksmanii.

A novel Gram-stain-negative, straight or slightly curved rod-shaped, non-spore-forming, facultatively anaerobic bacterium with a single polar flagellum, designated RZB5-4T, was isolated from a sample of the red algae Gelidium amansii collected from the coastal region of Rizhao, PR China (119.625° E 35.517° N). The organism grew optimally between 24 and 28 °C, at pH 7.0 and in the presence of 2-3 % (w/v) NaCl. The strain required seawater or artificial seawater for growth, and NaCl alone did not support growth. Strain RZB5-4T contained C16 : 1ω7c and/or C16 : 1ω6c, C16 : 0 and iso-C15 : 0 as the dominant fatty acids. The respiratory quinones detected in strain RZB5-4T were ubiquinone 7, ubiquinone 8, menaquinone 7 and methylmenaquinone 7. The polar lipids of strain RZB5-4T comprised phosphatidylethanolamine, phosphatidylglycerol, phosphatidylmonomethylethanolamine, one unidentified glycolipid, one unidentified phospholipid and one unknown lipid. The DNA G+C content of strain RZB5-4T was 47 mol %. Phylogenetic analysis based on 16S rRNA and gyrase B (gyrB) gene sequences showed that strain RZB5-4T belonged to the genus Shewanella, clustering with Shewanella waksmanii ATCC BAA-643T. Strain RZB5-4T exhibited the highest 16S rRNA gene sequence similarity value (96.6 %) and the highest gyrB gene sequence similarity value (80.7 %), respectively, to S. waksmanii ATCC BAA-643T. On the basis of polyphasic analyses, strain RZB5-4T represents a novel species of the genus Shewanella, for which the name Shewanella gelidii sp. nov. is proposed. The type strain is RZB5-4T (=JCM 30804T=KCTC 42663T=MCCC 1K00697T).

Pseudofulvibacter marinus sp. nov., isolated from seawater.

A novel Gram-stain-negative, rod-shaped, non-spore-forming, non-flagellated, strictly aerobic strain, designated RZW2-1T, was isolated from coastal seawater of the Yellow Sea in China (35.475° N 119.613° E). The organism grew optimally at 24 °C, at pH 7.0 and in the presence of 3.0 % (w/v) NaCl. The strain requires seawater or artificial seawater for growth and NaCl alone does not support growth. Strain RZW2-1T contained MK-6 as the only respiratory quinone and iso-C15 : 0, iso-C15 : 1 G and 10-methyl C16 : 0 and/or iso-C17 : 1ω9c as the dominant fatty acids. The polar lipids of strain RZW2-1T were four unidentified phospholipids (PL1-PL4), two unknown lipids (L1, L2) and one unidentified aminolipid (AL1). The DNA G+C content of strain RZW2-1T was 32 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that the novel strain was most closely related to the type strain of the only described species of genus Pseudofulvibacter, Pseudofulvibacter geojedonensis YCS-9T, with 95.1 % 16S rRNA gene sequence similarity. On the basis of polyphasic analyses, strain RZW2-1T represents a novel species of the genus Pseudofulvibacter, for which the name Pseudofulvibacter marinus sp. nov. is proposed. The type strain is RZW2-1T ( = JCM 30826T = MCCC 1K00695T).

Ox-LDL promotes migration and adhesion of bone marrow-derived mesenchymal stem cells via regulation of MCP-1 expression.

Bone marrow-derived mesenchymal stem cells (bmMSCs) are the most important cell source for stem cell transplant therapy. The migration capacity of MSCs is one of the determinants of the efficiency of MSC-based transplant therapy. Our recent study has shown that low concentrations of oxidized low-density lipoprotein (ox-LDL) can stimulate proliferation of bmMSCs. In this study, we investigated the effects of ox-LDL on bmMSC migration and adhesion, as well as the related mechanisms. Our results show that transmigration rates of bmMSCs and cell-cell adhesion between bmMSCs and monocytes are significantly increased by treatments with ox-LDL in a dose- and time-dependent manner. Expressions of ICAM-1, PECAM-1, and VCAM-1 as well as the levels of intracellular Ca(2+) are also markedly increased by ox-LDL in a dose-dependent manner. Cytoskeleton analysis shows that ox-LDL treatment benefits to spreading of bmMSCs and organization of F-actin fibers after being plated for 6 hours. More interestingly, treatments with ox-LDL also markedly increase expressions of LOX-1, MCP-1, and TGF- ß ; however, LOX-1 antibody and MCP-1 shRNA markedly inhibit ox-LDL-induced migration and adhesion of bmMSCs, which suggests that ox-LDL-induced bmMSC migration and adhesion are dependent on LOX-1 activation and MCP-1 expression.

Kineococcus glutineturens sp. nov., isolated from soil in Yunnan, south-west China.

An orange-coloured, non-spore-forming, motile and coccus-shaped actinobacterium, designated YIM 75677(T), was isolated from a soil sample collected from a dry-hot river valley in Dongchuan county, Yunnan Province, south-west China and its taxonomic position was investigated. Growth of strain YIM 75677(T) occurred at 12-55 °C, pH 6.0-9.0 and NaCl tolerance up to 2 % (w/v). Cells adhered to agar media and were agglutinated tightly together. The peptidoglycan contained meso-diaminopimelic acid, alanine and glutamic acid. The whole-cell hydrolysates mainly contained glucose, galactose, mannose and ribose. The predominant menaquinone was MK-9 (H(2)) and the major fatty acids were anteiso-C(15:0) and iso-C(15:0). Mycolic acids were not present. The DNA G+C content of strain YIM 75677(T) was 74.8 mol%. Phylogenetic analyses based on 16S rRNA gene sequence comparisons clearly revealed that strain YIM 75677(T) represents a novel member of the genus Kineococcus and is closely related to Kineococcus xinjiangensis S2-20(T) (level of similarity, 98.6 %). Meanwhile, the result of DNA-DNA hybridization between strain YIM 75677(T) and K. xinjiangensis S2-20(T) demonstrated that this isolate represented a different genomic species in the genus Kineococcus. On the basis of phylogenetic, phenotypic and chemotaxonomic characteristics, strain YIM 75677(T) represents a novel species of the genus Kineococcus, for which the name Kineococcus glutineturens sp. nov. is proposed. The type strain is YIM 75677(T) (=CCTCC AA 209075(T) = JCM 18126(T)).

Paenibacillus frigoriresistens sp. nov., a novel psychrotroph isolated from a peat bog in Heilongjiang, Northern China.

A novel cold-resistant bacterium, designated YIM 016(T), was isolated from a peat bog sample collected from Mohe County, Heilongjiang Province, Northern China and its taxonomic position was investigated using a polyphasic approach. The strain was Gram-positive, aerobic, endospore-forming, motile and rod-shaped. Phylogenetic analyses based on the 16S rRNA gene sequence clearly revealed that strain YIM 016(T) is a member of the genus Paenibacillus. The strain is closely related to Paenibacillus alginolyticus DSM 5050(T), Paenibacillus chondroitinus DSM 5051(T) and Paenibacillus pocheonensis Gsoil 1138(T) with similarities of 99.0 %, 97.0 % and 96.3 %, respectively. Meanwhile, the low DNA-DNA relatedness levels between strain YIM 016(T) and its closely related phylogenetic neighbours demonstrated that this isolate represents a new genomic species in the genus Paenibacillus. Phenotypic and chemotaxonomic tests showed that growth of strain YIM 016(T) occurred at 4-37 °C, pH 6.0-8.0 and with a NaCl tolerance up to 0.5 % (w/v). The peptidoglycan contained meso-diaminopimelic acid, alanine and glutamic acid. The whole-cell hydrolysates mainly contained glucose, galactose and ribose. The predominant menaquinone was MK-7 and the major fatty acids were anteiso-C(15:0) and iso-C(16:0). The DNA G+C content of strain YIM 016(T) was 51.7 mol %. On the basis of phylogenetic, phenotypic and chemotaxonomic characteristics, strain YIM 016(T) could be clearly distinguished from other species of the genus Paenibacillus. It is therefore concluded that strain YIM 016(T) represents a novel species in the genus Paenibacillus, for which the name Paenibacillus frigoriresistens sp. nov. is proposed. The type strain is YIM 016(T) (= CCTCC AB 2011150(T) = JCM 18141(T)).

Geodermatophilus nigrescens sp. nov., isolated from a dry-hot valley.

A novel actinomycete, designated as strain YIM 75980(T), was isolated from a soil sample collected from a dry-hot river valley in Dongchuan county, Yunnan province, south-west China and was subjected to polyphasic taxonomic characterization. The organism produced circular, smooth, red to black coloured colonies comprising coccoid-shaped cells. Colonies on agar medium lacked mycelia and cells adhered to the agar. Strain YIM 75980(T) contained meso-diaminopimelic acid as the diagnostic diamino acid in the cell-wall peptidoglycan and contained galactose, arabinose and glucosamine as the main sugars in the whole-cell hydrolysates. The predominant menaquinone was MK-9 (H(4)) and the major fatty acids were iso-C(15:0), iso-C(16:0) and C(16:0). The DNA G + C content of strain YIM 75980(T) was 73.1 mol%. Phylogenetic analyses based on 16S rRNA gene sequences clearly showed that strain YIM 75980(T) formed a distinct clade within the genus Geodermatophilus and was closely related to Geodermatophilus obscurus DSM 43160(T) (level of similarity, 97.9%). Furthermore, the result of DNA-DNA hybridization between strain YIM 75980(T) and G. obscurus 43160(T) demonstrated that this isolate represented a different genomic species in the genus Geodermatophilus. Moreover, the physiological and biochemical data showed the differentiation of strain YIM 75980(T) from its closest phylogenetic neighbour. Therefore, it is proposed that strain YIM 75980(T) represents a novel species of the genus Geodermatophilus, for which the name Geodermatophilus nigrescens sp. nov. is proposed. The type strain is YIM 75980(T) (=CCTCC AA 2011015(T) =JCM 18056(T)).

Amycolatopsis dongchuanensis sp. nov., an actinobacterium isolated from soil.

A novel actinomycete strain, designated YIM 75904(T), was isolated from a soil sample that had been collected from a dry and hot river valley in Dongchuan county, Yunnan province, south-western China. The taxonomic position of the novel strain was investigated by a polyphasic approach. In phylogenetic analyses based on 16S rRNA gene sequences, strain YIM 75904(T) formed a distinct clade within the genus Amycolatopsis and appeared to be closely related to Amycolatopsis sacchari K24(T) (99.3% sequence similarity). Strain YIM 75904(T) had a type-IV cell wall, with no detectable mycolic acids, and had MK-9(H(4)) as its predominant menaquonine. Its cell wall contained meso-diaminopimelic acid, galactose, glucose and arabinose, and its major cellular fatty acids were iso-C(16:0), iso-C(15:0), anteiso-C(17:0) and anteiso-C(15:0). The genomic DNA G+C content of the novel strain was 68.5 mol%. Based on the results of physiological and biochemical tests and DNA-DNA hybridizations, strain YIM 75904(T) represents a novel species of the genus Amycolatopsis for which the name Amycolatopsis dongchuanensis sp. nov. is proposed. The type strain is YIM 75904(T) (=CCTCC AA 2011016(T) =JCM 18054(T)).

[Mutation analysis of the pathogenic gene in a Chinese family with Brachydactyly type B1].

We identified and characterized a Chinese family with autosomal dominant Brachydactyly type B1 (BDB1). Linkage analysis revealed that the disease gene of the Chinese BDB1 family was linked to ROR2 locus. Mutational hot spot of ROR2 gene was amplified by polymerase chain reaction (PCR) and sequenced directly. A c.2265C>A heterozygous mutation was detected in all of the patients. This mutation led to the change of p.Y755X in protein level and a truncated ROR2 protein losing integrant domains was generated. The mutation was detected in all the patients, but not in all the normal individuals of this family and 50 normal controls. This paper for the first time reported a c.2265C>A mutation in ROR2 gene of a family with BDB1 in China, which enriches ROR2 gene mutation spectrum in Chinese with BDB1.

[The mutation spectrum of phenylalanine hydroxylase gene in patients with phenylketonuria in Henan province].

OBJECTIVE: To investigate the characteristics of the phenylalanine hydroxylase (PAH) gene mutations in patients with phenylketonuria (PKU) in Henan province, China, in order for providing basic information for clinical genetic counseling and prenatal diagnosis. METHODS: All the exons and partial flanking introns of the PAH gene were detected by polymerase chain reaction (PCR) and bi-directional sequencing in 34 patients with PKU from Henan province. RESULTS: A total of 23 different disease-causing mutations were identified which corresponded to 92.65% (63/68) of the PAH alleles, including 12 missense mutations, 4 nonsense mutations, 4 splicing junction mutations, and 3 deletion mutations. Among them, A156P and P69_S70delinsP(delCTT) were novel mutations; IVS2+ 5G to C, G332E, IVS10-14C to G and L367 to Wfs were reported in Chinese population for the first time according to the PAH database (www.pahdb.mcgill.ca). Among all the 13 exons, exon 7 harbored the most type of mutations, exon 11 and exon 5 the second. The most common mutations included R243Q (17.65%, 12/68), V399V (11.76%, 8/68), IVS4-1G to A (8.82%, 6/68), R400T(7.35%, 5/68), Y166X(5.88%,4/68) and G247R(5.88%, 4/68). In addition, 9 other gene variations were found in this study. CONCLUSION: The mutation spectrum and frequency of the PAH gene of patients with phenylketonuria in Henan province were slightly different from those from other parts of China.

Molecular characterization and functional analysis of a protease-related protein in Chang-liver cells.

In this study, the cDNA library of Chang-liver cells was immunoscreened using common ADAMs antibody to obtain ADAM related genes. We found one positive clone that was confirmed as a new gene by Blast, which is an uncharacterized helical and coil protein and processes protease activity, and named protease-related protein 1 (ARP1). The submitted GenBank accession number is AY078070. Molecular characterizations of ARP1 were analyzed with appropriate bioinformatics software. To analyse its expression and function, ARP1 was subcloned into glutathione S-transferase fusion plasmid pGEX-2T and expressed by E. coli system. The in vitro expression product of ARP1 was recognized by common ADAMs antibody with western blot. Interestingly, ARP1 cleaves gelatine at pH9.5, which suggests it is an alkaline protease. Semi-quantitative RT-PCR result indicates that ARP1 mRNA is strongly transcribed in the liver and the treated Chang-liver cells.