Glycosylation modification is an important post-translational modification of proteins, which participates in regulating protein half-life, biological activity, and immunogenicity, thereby affecting their functions. Glycoproteins expressed in Pichia pastoris predominantly carry high-mannose type glycans, primarily composed of mannose residues, which starkly contrasts with the complex-type glycans synthesized by mammalian cells. This study aims to transform the high mannose glycosylation modification of P. pastoris into a hybrid glycosylation modification similar to that of mammalian cells through genetic engineering technology. We introduced the mannosidase Ⅰ gene (MDSⅠ) from Trichoderma viride and the human β-1,2-N-acetylglucosaminyltransferase I gene (GnTⅠ) into a previously constructed P. pastoris strain (∆och1) capable of producing Man₈GlcNAc₂ glycans. To precisely regulate the expression of MDSⅠ and GnTⅠ, we designed various promoter combinations, including the strong inducible AOX promoter and the constitutive GAP promoter. The receptor-binding domain (RBD, residues 377-588) of the spike protein from the Middle East respiratory syndrome coronavirus (MERS-CoV) was selected as the reporter protein for this investigation (MERS-RBD). The N-glycosylation profile of MERS-RBD was systematically analyzed using PNGase F digestion coupled with mass spectrometry. The results showed that after the knockout of och1 and the introduction of MDSⅠ and GnTⅠ genes with different promoter combinations, P. pastoris strains capable of producing GlcNAcMan₅GlcNAc₂ glycans were successfully generated. When the AOX promoter was used to control the MDSⅠ gene and the GAP promoter was used to control the GnTⅠ gene, the engineered strain exhibited the highest proportion of hybrid-type GlcNAcMan₅GlcNAc₂ glycans, which accounted for 68.38% of the total N-glycosylation. In conclusion, we successfully engineered a P. pastoris strain capable of synthesizing hybrid-type GlcNAcMan₅GlcNAc₂ glycans, establishing a foundation for subsequent research on the biosynthesis of complex-type N-glycans in P. pastoris.
Glycosylation ; Glycoproteins/genetics* ; Polysaccharides/metabolism* ; N-Acetylglucosaminyltransferases/metabolism* ; Pichia/metabolism* ; Humans ; Mannosidases/metabolism* ; Genetic Engineering ; Trichoderma/genetics* ; Recombinant Proteins/genetics* ; Saccharomycetales

This study aimed to characterize the chemical composition of a new sulfated polysaccharide from the red alga Gracilaria chouae and evaluate its activation effects on RAW264.7 macrophages. It showed that the obtained G. chouae polysaccharide (GCP-3A) was a sulfated acidic polysaccharide with a molecular weight of 11.87 kDa. GCP-3A was composed of xylose, galactose, glucose, and mannose with a molar ratio of 3.00:29.28:0.63:0.45, and it contained α,β‍-glycosidic linkages. Scanning electron microscopy (SEM) and a Congo red test showed that it was a heterogeneous polysaccharide with irregular interwoven sheets and rods, and did not have a triple-helix conform‍ation. Furthermore, GCP-3A significantly promoted the proliferation of RAW264.7 macrophages and the secretion of nitric oxide (NO) in tests of 3-‍(4,‍5-dimethylthiahiazo-2-yl)‍-2,‍5-diphenytetrazoliumromide(MTT) and NO.
Gracilaria/chemistry* ; Macrophages ; Molecular Weight ; Polysaccharides/pharmacology* ; Sulfates/pharmacology*

Elsholtiza Bodinieri Vaniot has the food and medicinal value. The whole plant contains aromatic oils, flavonoids, triterpenoids, phenolic constituents, β-carotene, vitamin C and inorganic elements, and is characterized by antibacterial, antioxidant, hypolipidemic and other kinds of activity. However, few researches are on the extraction method and activity of the effective ingredient of Elsholtiza Bodinieri Vaniot. This article reviewed the chemical constituents of Elsholtiza Bodinieri Vaniot, with a purpose to provide references for further study and development.

Objective To investigate the clinical efficacy of Ilizarov bone transport combined with dressing change in the treatment of acute tibial osteomyditis accompanied with massive soft tissue defection.Methods From July 2009 to september 2012, 14 patients with acute tibial osteomyelitis associsted with large-area soft tissue necrosis and defection were treated in Traumatic Orthopaedics Department of the Central Hospital of Binzhou.All cases were Gustillu Ⅲ type open fracture, and Ⅲ C type injury in 11 casas accompanied with vascular injure.Associated with contralateral lower limb in 4 casas.They were 10 male and 4 female,aged from 21 to 45 years old(average age of 35 years old).The duration from injury to surgery ranged from 12-24 days,averaging of 19 days.All cases were treated first with excision of the infected or necrosis bone segment and soft tissue.The wound keep opening, followed by llizarov bone transport combined with dressing change to repair the bone and soft tissue defection.The clinical efficacy was assessed using the Paley's evaluation system at the last follow-up.Results All the patients were followed up for 24 to 36 months (average of 27 months).The bone defection rapaired was from 5 to 1 3 cm(average of 7 cm).The mean duration of distraction was 4 months, range from 2 to 6 months.All cases' gain s.ufficient soft tissue coverage the later period of Ilizarov bone transport.All the fracture and bone defection were cured.Infection were cured in 13 cases, and 1 case converted to chronicle osteomyelitis, and the cure rate was 93％.According to the Paley' s system, the bone union was excellent in 11 cases, good in 2 cases, and fair in 1 Cases, with a good-to-excellent rate was 93％.The functional outcome was excellent in 10 cases,good in 2 cases,and fair in 2 cases,with a good-to-excellent rate of 86％.Conclusion Ilizarov bone transport and dressing change is one reliable and effective treatment of tibial acute infected and soft tissue defection.

Objective To study the optimum extraction process of Compound Ganmaoling Capsules by orthogonal design. Methods The rate of extraction and the content of chlorogenic acid were chosen as the assessment indexes.The amount of water, boiling duration and boiling times were studied to examine the extraction process by orthogonal design. Results The optimum water extraction process conditions were adding ten times amount of water for the first time and eight times amount of water for the second time, 1.5 h each time. Conclusion The preparation method is reasonable and stable.

Objective To establish an assay for the quality control of Chanhoukang Granules. Methods Radix Astragali, Radix Codonopsis, Radix Salviae Miltiorrhiae, Pericarpium Citri Reticulatae, Radix Rehmanniae preparata in Chanhoukang Granules were identified by TLC; paeoniflorin content in Chanhoukang Granules was determined by HPLC. Results The relevant spots in Radix Astragali, Radix Codonopsis, Radix Salviae Miltiorrhiae, Pericarpium Citri Reticulatae, Radix Rehmanniae preparata can be identified by TLC. The content of paeoniflorin in Raidix Paeoniae Alba can be determined by HPLC. The linearity of paeoniflorin was good in the range of 0.044 2～ 0.353 6 ? g(r=0.999 9)The average recovery of paeoniflorin was 97.56 % with RSD=1.18 % . Conclusion The established method is simple, feasible and repeatable, and can be used for the quality supervisory of Chanhoukang Granules.