Genome editing tools such as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated system (Cas) have been widely used to modify genes in model systems including animal zygotes and human cells, and hold tremendous promise for both basic research and clinical applications. To date, a serious knowledge gap remains in our understanding of DNA repair mechanisms in human early embryos, and in the efficiency and potential off-target effects of using technologies such as CRISPR/Cas9 in human pre-implantation embryos. In this report, we used tripronuclear (3PN) zygotes to further investigate CRISPR/Cas9-mediated gene editing in human cells. We found that CRISPR/Cas9 could effectively cleave the endogenous β-globin gene (HBB). However, the efficiency of homologous recombination directed repair (HDR) of HBB was low and the edited embryos were mosaic. Off-target cleavage was also apparent in these 3PN zygotes as revealed by the T7E1 assay and whole-exome sequencing. Furthermore, the endogenous delta-globin gene (HBD), which is homologous to HBB, competed with exogenous donor oligos to act as the repair template, leading to untoward mutations. Our data also indicated that repair of the HBB locus in these embryos occurred preferentially through the non-crossover HDR pathway. Taken together, our work highlights the pressing need to further improve the fidelity and specificity of the CRISPR/Cas9 platform, a prerequisite for any clinical applications of CRSIPR/Cas9-mediated editing.
Blastocyst ; CRISPR-Cas Systems ; Hemoglobins, Abnormal ; genetics ; metabolism ; Humans ; Zygote

In the present study, the regulation of Vitreoscilla hemoglobin (VHb) on astragaloside IV biosynthesis was investigated. An intermediate expression vector consisting of the CaMV35S promoter fused to the vgb and nopaline synthase terminator was transferred into Astragalus membranaceus via Agrobacterium rhizogenes. The transgenic hairy roots were confirmed by PCR amplification and Southern blot hybridization. The expression of vgb in transgenic hairy roots was confirmed by RT-PCR. After 15 days cultivation, the dry weight and growth rate of transgenic hairy roots were higher than that of the non-transgenic hairy root. ELSD-HPLC analysis showed that astragaloside IV content of transgenic hairy roots was 5 to 6 times of non-transgenic hairy root control and 10 to 12 times of Radix Astragali from Shanxi Province. These results suggested that the expression of vgb promoted the growth of transgenic hairy roots, and increased the content of astragaloside IV.
Astragalus membranaceus ; genetics ; growth & development ; metabolism ; Bacterial Proteins ; genetics ; metabolism ; Plant Roots ; growth & development ; metabolism ; Plants, Genetically Modified ; genetics ; growth & development ; metabolism ; Plants, Medicinal ; genetics ; growth & development ; metabolism ; Saponins ; analysis ; biosynthesis ; Triterpenes ; analysis ; Truncated Hemoglobins ; genetics ; metabolism ; Vitreoscilla ; genetics

BACKGROUND: Hemoglobin concentration reportedly is positively associated with muscle strength, for example, handgrip strength. However, hemoglobin cannot repair muscle directly, but is beneficial only in a supportive role. Since hepatocyte growth factor (HGF) regulates muscle satellite cell production and differentiation, which is stimulated by organ injury, the supportive effect of hemoglobin should thus be stronger for participants with high HGF than for those with low HGF. However, the association between hemoglobin concentration and handgrip strength in relation to HGF levels remains unknown. METHODS: We conducted a cross-sectional study of 255 Japanese elderly men aged 60-69 years who participated in annual health check-ups in 2014-2015. The study population was categorized on the basis of a median value of HGF of 300.6 pg/mL. RESULTS: Among present study population, 128 participants showed low HGF. For participants with low HGF, hemoglobin concentration showed no significant association with handgrip strength (standardized parameter estimate (β) = 0.03, p = 0.767), but for those with high HGF, hemoglobin concentration was significantly positively associated with handgrip strength (β = 0.23, p = 0.014). CONCLUSIONS A significant positive association between hemoglobin level and handgrip strength was established for elderly Japanese men aged 60-69 years with high HGF but not for participants with low HGF. Our finding indicates that HGF levels could determine the relationship of hemoglobin concentration with handgrip strength in elderly Japanese men aged 60-69 years. This result can be expected to serve as an effective tool for the clarification of the roles played by HGF and hemoglobin concentration in maintenance of muscle strength.
Aged ; Cross-Sectional Studies ; Hand Strength ; physiology ; Hemoglobins ; metabolism ; Hepatocyte Growth Factor ; genetics ; metabolism ; Humans ; Japan ; Male ; Middle Aged

Ergosterol is a principal sterol of fungi. It is a raw material for production of vitamin D2, hydrocortisone, progesterone and brassinolide. Synthesis of ergosterol requires molecular oxygen, and low oxygen tensions was reported to dramatically reduce ergosterol concentration. Vitreoscilla Hemoglobin Gene (vgb), a homodimeric hemoglobin gene from Gram-negative obligate aerobic bacterium Vitreoscilla, enables a higher specific cellular oxygen uptake rate, it also improves the oxygen transportation. In this study, recombinant plasmid pVgb-kanMX4 containing Vitreoscilla Hemoglobin Gene (vgb) and geneticin (G418) was constructed and transformed into Saccharomyces cerevisiae 1190 for enhanced ergosterol production. With sufficient oxygen supply, the ergosterol contents of recombinant and wild type strains grown in shake flasks were 1.07% and 0.573%, respectively. Under oxygen limitation condition, ergosterol contents in recombinant and wild type strains were reduced to 0.39% and 0.25%, respectively. In a 30 hours fermentation study conducted in a 5 liter fermentor, 15.1 g/L Cell Dry Weight (CDW) containing 1.38% ergosterol was obtained from growth of the recombinant strains; Only 14.8 g/L CDW containing 0.9% ergosterol was produced by the wild type strain. These results demonstrated that vgb played a role in enhancing ergosterol production.
Bacterial Proteins ; biosynthesis ; genetics ; physiology ; Cloning, Molecular ; Ergosterol ; biosynthesis ; genetics ; Fermentation ; Recombinant Proteins ; biosynthesis ; genetics ; Saccharomyces cerevisiae ; genetics ; metabolism ; Truncated Hemoglobins ; biosynthesis ; genetics ; physiology

Yarrowia lipolytica lipase Lip2 (YlLip2) is an important industrial enzyme with many potential applications. To alleviate the dissolved oxygen (DO) limitation and improve YlLip2 production during high-cell density fermentation, the YlLip2 gene lip2 and Vitreoscilla hemoglobin (VHb) gene vgb were co-expressed in Pichiapastoris under the control of AOX1 and PsADH2 promoter, respectively. The PsADH2 promoter from Pichia stipitis could be activated under oxygen limitation. The SDS-PAGE and CO-difference spectrum analysis indicated that VHb and YlLip2 had successfully co-expressed in recombinant strains. Compared with the control cells (VHb-, GS115/9Klip2), the expression levels of YlLip2 in VHb-expressing cells (VHb+, GS115/9Klip2-pZPVT) under oxygen limitation were improved 25% in shake-flask culture and 83% in a 10 L fermentor. Moreover, the VHb+ cells displayed higher biomass than VHb- cells at lower DO levels in a 10 L fermentor. In this study, we also achieved a VHb-expressing clone harboring multicopy lip2 gene (GS115/9Klip2-pZPVTlip2 49#), which showed the maximum lipolytic activity of 33 900 U/mL in a 10 L fermentor under lower DO conditions. Therefore, it can be seen that expression of VHb with PsADH2 promoter in P. pastoris combined with increasing copies of lip2 gene is an effective strategy to improve YlLip2 production.
Bacterial Proteins ; biosynthesis ; genetics ; Fermentation ; Fungal Proteins ; biosynthesis ; genetics ; Lipase ; biosynthesis ; genetics ; Oxygen ; analysis ; pharmacology ; Pichia ; metabolism ; Protein Engineering ; Recombinant Proteins ; biosynthesis ; genetics ; Truncated Hemoglobins ; biosynthesis ; genetics

The expression of the vgb gene in vivo could improve the fermentation density and then contribute the extracellular secretion of the product of bxn gene. Constructed the recombination plasmid pPIC9K-vgbbxn and transformed into Pichia pastoris GS115. The results of PCR and SDS-PAGE indicate that the vgb gene and bxn gene had integrated into the genome of Pichia pastoris GS115 and expressed in efficient level. Also, the protein activity of their products had been verified respectively. Shake flask fermentation experiments showed that the presence of VHb in yeast Pichia pastoris efficiently enhanced cell growth and secretive expression of bxn gene under hypoxic habitats.
Aminohydrolases ; genetics ; metabolism ; Bacterial Proteins ; genetics ; physiology ; Electrophoresis, Polyacrylamide Gel ; Pichia ; genetics ; Plasmids ; Polymerase Chain Reaction ; Recombinant Proteins ; biosynthesis ; Truncated Hemoglobins ; genetics ; physiology

Energy metabolism plays an important role in life survival for species living in high altitude hypoxia condition. Air-breathing organisms require oxygen to create energy. Tibetans are the well-adapted highlanders in Qinghai-Tibetan Plateau. It was thought that different metabolic approaches could lead to different adaptation traits to high altitude hypoxia. Recently identified hypoxia inducible factors pathway regulators, endothelial PAS domain protein1 (EPAS1)/HIF-2a and PPARA, were involved in decreasing hemoglobin concentrations in Tibetans. Because EPAS1 and PPARA also modulated the energy metabolism during hypoxia, we hypothesized that positive selected EPAS1 and PPARA genes were also involved in unique energy metabolisms in Tibetans. In this brief review, we take a look into genetic determinations to energy metabolisms for hypoxia adaptations traits in Tibetans and mal-adaptive conditions such as high altitude diseases.
Acclimatization ; genetics ; Altitude ; Basic Helix-Loop-Helix Transcription Factors ; metabolism ; Energy Metabolism ; Hemoglobins ; analysis ; Humans ; Hypoxia ; metabolism ; Oxygen ; metabolism ; Phenotype ; Tibet

The mammalian trematode Paragonimus westermani is a typical digenetic parasite, which can cause paragonimiasis in humans. Host tissues and blood cells are important sources of nutrients for development, growth and reproduction of P. westermani. In this study, a cDNA clone encoding a 47 kDa hemoglobinase of P. westermani was characterized by sequencing analysis, and its localization was investigated immunohistochemically. The phylogenetic tree prepared based on the hemoglobinase gene showed high homology with hemoglobinases of Fasciola hepatica and Schistosoma spp. Moreover, recombinant P. westermani hemoglobinase degradaded human hemoglobin at acidic pH (from 3.0 to 5.5) and its activity was almost completely inhibited by E-64, a cysteine proteinase inhibitor. Immunohistochemical studies showed that P. westermani hemoglobinase was localized in the epithelium of the adult worm intestine implying that the protein has a specific function. These observations suggest that hemoglobinase may act as a digestive enzyme for acquisition of nutrients from host hemoglobin. Further investigations may provide insights into hemoglobin catabolism in P. westermani.
Sequence Alignment ; Recombinant Proteins/biosynthesis/genetics ; Phylogeny ; Paragonimus westermani/*enzymology/genetics ; Molecular Sequence Data ; Hemoglobins/metabolism ; Escherichia coli/enzymology/genetics ; DNA, Complementary/genetics ; Cysteine Endopeptidases/*genetics/immunology/*metabolism ; Astacoidea/parasitology ; Antigens, Helminth/genetics/immunology/metabolism ; Animals ; Amino Acid Sequence

BACKGROUNDDuring the blood stage of malaria infection, parasites internalize in the host red blood cells and degrade massive amounts of hemoglobin for their development. Although the morphology of the parasite's hemoglobin uptake pathway has been clearly observed, little has been known about its molecular mechanisms.

METHODSThe recombinant proteins from Plasmodium falciparum, dynamin like protein 1 (PfDYN1) and 2 (PfDYN2) GTPase domain, were expressed in E.coli and showed GTPase activity. By using a dynamin inhibitor, dynasore, we demonstrated the involvement of PfDYN1 in the hemoglobin uptake pathway.

RESULTSThe GTPase activity of the two recombinant proteins was inhibited by dynasore in vitro. Treatment of parasite cultures with 80 micromol/L dynasore at the ring and early trophozoite stage resulted in substantial inhibition of parasite growth and in an obvious decline of hemoglobin quantum. Furthermore, reduced intracellular hemozoin accumulation and decreased uptake of the FITC-dextran were also observed, together with distinctive changes in the ultrastructure of parasites after the dynasore treatment.

CONCLUSIONSOur results show that PfDYN1 plays an important role in the hemoglobin uptake pathway of P. falciparum and suggest its possibility of being a novel target for malaria chemotherapy.

Animals ; Antimalarials ; pharmacology ; Dynamins ; antagonists & inhibitors ; GTP Phosphohydrolases ; genetics ; metabolism ; Hemoglobins ; metabolism ; Hydrazones ; pharmacology ; Malaria, Falciparum ; metabolism ; Microscopy, Electron, Transmission ; Plasmodium falciparum ; drug effects ; metabolism ; ultrastructure ; Protozoan Proteins ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism

D-amino acid oxidase (DAAO) is one of important industrial enzymes. To increase the solubility and activity of the TvDAAO from Trignoposis variabilis expressed in recombinant Escherichia coli (E. coli), a maltose binding protein (MBP) and Vitreoscilla hemoglobin (VHb) was introduced to fuse with N-terminal of the TvDAAO, respectively. Fusion protein of MBP-TvDAAO was constitutively expressed in JM105/pMKC-DAAO and inductively expressed in JM105/pMKL-DAAO. With respect to the control strain of BL21 (DE3)/pET-DAAO without MBP fusion, the constitutive fusion expression obtained 28% of soluble protein with 3.7 folds of solubility improvement. As for the inductive fusion expression, corresponding results changed to 17% and 1.8 folds, respectively. However, the DAAO activity significantly decreased in the MBP-fusing expression. Fusion protein of VHb-TvDAAO was constructed and inductively expressed in BL21 (DE3)/pET-VDAAO. Its DAAO activity highly reached 3.24 u/mL in flask culture, about 90% increase in contrast to the control without VHb.
Bacterial Proteins ; biosynthesis ; genetics ; Carrier Proteins ; biosynthesis ; genetics ; D-Amino-Acid Oxidase ; biosynthesis ; genetics ; Escherichia coli ; genetics ; metabolism ; Maltose-Binding Proteins ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Truncated Hemoglobins ; biosynthesis ; genetics ; Yeasts ; enzymology ; genetics