Decreasing hydrophobicity or shielding hydrophobic areas of CH2 attenuates low pH-induced IgG4 aggregation.

Protein aggregation is a major challenge in the development of therapeutic monoclonal antibodies (mAbs). Several stressors can cause protein aggregation, including temperature shifts, mechanical forces, freezing-thawing cycles, oxidants, reductants, and extreme pH. When antibodies are exposed to low pH conditions, aggregation increases dramatically. However, low pH treatment is widely used in protein A affinity chromatography and low pH viral inactivation procedures. In the development of an IgG4 subclass antibody, mAb1-IgG4 showed a strong tendency to aggregate when temporarily exposed to low pH conditions. Our findings showed that the aggregation of mAb1-IgG4 under low pH conditions is determined by the stability of the Fc. The CH2 domain is the least stable domain in mAb1-IgG4. The L309E, Q311D, and Q311E mutations in the CH2 domain significantly reduced the aggregation propensity, which could be attributed to a reduction in the hydrophobicity of the CH2 domain. Protein stabilizers, such as sucrose and mannose, could also attenuate low pH-induced mAb1-IgG4 aggregation by shielding hydrophobic areas and increasing protein stability. Our findings provide valuable strategies for managing the aggregation of protein therapeutics with a human IgG4 backbone.

Efficient short time pretreatment on lignocellulosic waste using an isolated fungus Trametes sp. W-4 for the enhancement of biogas production.

Biological pretreatment to the lignocellulosic waste prior to anaerobic digestion is a popular method to increase biogas production. However, the long time needed for the pretreatment is not suitable to the practical application. A fungus strain, which could produce many kinds of lignocellulosic enzymes including CMCase, FPase, xylanase and laccase, was isolated from the soil of Tibet in this study. The fungus was identified as Trametes sp. W-4 by morphological and molecular characterization. The optimum culture temperature was 30 °C and the optimum nitrogen source was peptone. Under the optimum fermentation condition, the activity of CMCase, FPase, xylanase and laccase could reach 2.73 U/mL, 0.41 U/mL, 0.29 U/mL, and 1.11 U/mL, respectively. The results of pretreatment of Trametes sp. W-4 on the mixtures of high land barley straw, cow manure and pig manure for enhancement of biogas production showed that a very short time pretreatment of 3 days could obtain the highest cumulative methane production of 111.51 mL/g-VS, which was 63.81% higher than that of the control group of 68.07 mL/g-VS. The finding indicated that Trametes sp. W-4 pretreatment could be a candidate for the improving of biogas production from lignocellulosic waste.

Wuling San and Xiao Chaihu Decoction affect airway inflammatory response and airway smooth muscle cell proliferation in mice with allergic asthma via miR-486-5p/AQP5 axis.

OBJECTIVE: This study aimed to investigate the effects of Wuling San and Xiao Chaihu Decoction on allergic asthma, and elucidate the potential mechanism of Wuling San and Xiao Chaihu Decoction for ameliorating allergic asthma. METHODS: BALB/c mice were intraperitoneally injected with ovalbumin (OVA) to establish animal model of allergic asthma. Transforming growth factor beta 1 (TGF-ß1) was used to induce the proliferation of airway smooth muscle cells (ASMCs) in order to establish the cell model. Quantitative real-time polymerase chain reaction (qRT-PCR) was applied to quantify the expression levels of miR-486-5p and aquaporin-5 (AQP5) in cells and tissues. Dual-luciferase reporter assay was used to verify the targeting relationship between miR-486-5p and AQP5. MTT assay and flow cytometry were carried out to evaluate cell proliferation and apoptosis, respectively. Enzyme-linked immunosorbent assay (ELISA) was conducted to measure the levels of interleukin-4 (IL-4), IL-5 and IL-13 in the bronchoalveolar lavage fluid (BALF). Hematoxylin and eosin (HE) staining and Masson staining were used to detect the recruitment of eosinophils and collagen deposition. RESULTS: In both in vivo and in vitro experiments, Wuling San and Xiao Chaihu Decoction significantly reduced the number of eosinophils, the levels of inflammatory factors in the BALF of asthmatic mice, and the deposition of collagen in lung tissues, and they also significantly inhibited the proliferation of ASMCs and accelerated their apoptosis (all P<0.05). Wuling San and Xiao Chaihu Decoction significantly upregulated the expression of AQP5 while inhibited the expression of miR-486-5p; additionally, miR-486-5p negatively regulated the expression of AQP5 (all P<0.05). Overexpression of miR-486-5p or silencing AQP5 can partially reverse the therapeutic effect of Wuling San and Xiao Chaihu Decoction on allergic asthma in mice and the inhibitory effect on the abnormal proliferation of ASMCs (all P<0.05). CONCLUSION: Wuling San and Xiao Chaihu Decoction can influence the proliferation and apoptosis of ASMCs and the expression of inflammatory factors in mice with allergic asthma through inhibiting the expression of miR-486-5p and upregulating the expression of AQP5.

Retrovectors packaged in CHO cells to generate GLP-1-Fc stable expression CHO cell lines

Background: Chinese hamster ovary (CHO) cells are the most dependable mammalian cells for the production of recombinant proteins. Replication-incompetent retroviral vector (retrovector) is an efficient tool to generate stable cell lines. Multiple copies of integrated genes by retrovector transduction results in improved recombinant protein yield. HEK-293 and their genetic derivatives are principal cells for retrovector production. Retrovectors packaged in HEK-293 cells pose a risk of infectious agent transmission, such as viruses and mycoplasmas, from serum and packaging cells. Results: In this report, retrovectors were packaged in CHO cells cultured in chemically defined (CD) media. The retrovectors were then used to transduce CHO cells. This method can block potential transmission of infectious agents from serum and packaging cells. With this method, we generated glucagon-like protein-1 Fc fusion protein (GLP-1-Fc) stable expression CHO cell lines. Productivity of GLP-1-Fc can reach 3.15 g/L. The GLP-1-Fc protein produced by this method has comparable bioactivity to that of dulaglutide (Trulicity). These stable cell lines retain 95­100% of productivity after 40 days of continuous culture (~48­56 generations). Conclusions: Suspension CHO cells are clean, safe, and reliable cells for retrovector packaging. Retrovectors packaged from this system could be used to generate CHO stable cell lines for recombinant protein expression.

Retrovectors packaged in CHO cells to generate GLP-1-Fc stable expression CHO cell lines.

BACKGROUND: Chinese hamster ovary (CHO) cells are the most dependable mammalian cells for the production of recombinant proteins. Replication-incompetent retroviral vector (retrovector) is an efficient tool to generate stable cell lines. Multiple copies of integrated genes by retrovector transduction results in improved recombinant protein yield. HEK-293 and their genetic derivatives are principal cells for retrovector production. Retrovectors packaged in HEK-293 cells pose a risk of infectious agent transmission, such as viruses and mycoplasmas, from serum and packaging cells. RESULTS: In this report, retrovectors were packaged in CHO cells cultured in chemically defined (CD) media. The retrovectors were then used to transduce CHO cells. This method can block potential transmission of infectious agents from serum and packaging cells. With this method, we generated glucagon-like protein-1 Fc fusion protein (GLP-1-Fc) stable expression CHO cell lines. Productivity of GLP-1-Fc can reach 3.15 g/L. The GLP-1-Fc protein produced by this method has comparable bioactivity to that of dulaglutide (Trulicity). These stable cell lines retain 95-100% of productivity after 40 days of continuous culture (~ 48-56 generations). CONCLUSIONS: Suspension CHO cells are clean, safe, and reliable cells for retrovector packaging. Retrovectors packaged from this system could be used to generate CHO stable cell lines for recombinant protein expression.How to cite: Li J, Wei S, Cao C, et al. Retrovectors packaged in CHO cells to generate GLP-1-Fc stable expression CHO cell lines. Electron J Biotechnol 2019;41. https://doi.org/10.1016/j.ejbt.2019.07.002.

Comparative study of reactor performance and microbial community in psychrophilic and mesophilic biogas digesters under solid state condition.

Psychrophilic (15°C) and mesophilic (35°C) reactor performance and microbial community dynamics were compared when the biogas fermenters were performed at high altitude and solid state condition using animal manure and highland barley straw as substrate. Longer biogas fermentation time, higher peak methane content and lower volatile fatty acids (VFA) accumulation were found at psychrophilic condition compared to that of at mesophilic condition although the biogas production in both temperature conditions was similar. The cumulative biogas production at 35°C and 15°C were 246 (±5) and 225 (±7) ml/g volatile solids, respectively. The highest total VFA concentration under 35°C was 10,796 (±310) mg/kg total solid, while it only reached to 2346 (±87) mg/kg total solid at the condition of 15°C. Additionally, the variation of pH, soluble chemical oxygen demand and total ammonia nitrogen during the anaerobic digestion under psychrophilic condition were much smaller than that of under mesophilic condition. Polymerase chain reaction and denaturing gradient gel electrophoresis analysis followed by 16S rDNA sequencing showed that bacteria of genera Bacillus and Clostridium and archaea of genera Methanosarcina and Methanosaeta played a pivotal role during the biogas production.

Differential Expression of Toll-Like Receptor Signaling Pathway Is Associated with Microscopic Polyangiitis in Peripheral Blood Neutrophils.

Constitutive or excessive activation of Toll-like receptor (TLR) signaling pathway can disrupt the body's immune tolerance to autoantigen, thus promoting the development of autoimmune disease. However, the expression profile of TLR signaling pathway in peripheral blood neutrophils in the pathogenesis of microscopic polyangiitis (MPA) remains unclear. Thus, improved understanding of the pathobiology of this disease may aid in the development of therapeutic targets for patients with MPA. In the present study, we assessed the expression of TLR signaling pathway-related genes in peripheral blood neutrophils in patients with MPA. PCR array analysis was performed on 20 patients with MPA and 12 healthy controls. Gene expression profile was performed using the human TLR for autoimmunity and inflammation PCR array of Genecopoeia, containing 84 genes related to TLR signaling pathway and six house-keeping genes. We then used quantitative real-time PCR to validate the array test. The array results identified 13 upregulated genes and 5 genes which were downregulated. The resulting qRT-PCR was consistent with the findings by PCR array. Our results suggest that peripheral blood neutrophils display changes in the expression of TLR signaling pathway-related genes associated with the pathogenesis of microscopic polyangiitis.

Arbuscular Mycorrhizal Fungus Rhizophagus irregularis Increased Potassium Content and Expression of Genes Encoding Potassium Channels in Lycium barbarum.

Potassium in plants accounts for up to 10% dry weight, and participates in different physiological processes. Under drought stress, plant requires more potassium but potassium availability in soil solutes is lowered by decreased soil water content. Forming symbiosis with arbuscular mycorrhizal (AM) fungi not only enlarges exploration range of plant for mineral nutrients and water in soil, but also improves plant drought tolerance. However, the regulation of AM fungi on plant root potassium uptake and translocation from root to shoot was less reported. In current study, the effect of an AM fungus (Rhizophagus irregularis), potassium application (0, 2, and 8 mM), and drought stress (30% field capacity) on Lycium barbarum growth and potassium status was analyzed. Ten weeks after inoculation, R. irregularis colonized more than 58% roots of L. barbarum seedlings, and increased plant growth as well as potassium content. Potassium application increased colonization rate of R. irregularis, plant growth, potassium content, and decreased root/shoot ratio. Drought stress increased colonization rate of R. irregularis and potassium content. Expression of two putative potassium channel genes in root, LbKT1 and LbSKOR, was positively correlated with potassium content in root and leaves, as well as the colonization rate of R. irregularis. The increased L. barbarum growth, potassium content and genes expression, especially under drought stress, suggested that R. irregularis could improve potassium uptake of L. barbarum root and translocation from root to shoot. Whether AM fungi could form a specific mycorrhizal pathway for plant potassium uptake deserves further studies.

The application of biotechnology on the enhancing of biogas production from lignocellulosic waste.

Anaerobic digestion of lignocellulosic waste is considered to be an efficient way to answer present-day energy crisis and environmental challenges. However, the recalcitrance of lignocellulosic material forms a major obstacle for obtaining maximum biogas production. The use of biological pretreatment and bioaugmentation for enhancing the performance of anaerobic digestion is quite recent and still needs to be investigated. This paper reviews the status and perspectives of recent studies on biotechnology concept and investigates its possible use for enhancing biogas production from lignocellulosic waste with main emphases on biological pretreatment and bioaugmentation techniques.

Expression and bioactivity of recombinant human serum albumin and dTMP fusion proteins in CHO cells.

The 14-amino acid (IEGPTLRQWLAARA) thrombopoietin mimetic peptide (TMP) shares no sequence homology with native thrombopoietin (TPO). When dimerized, it displays a high-binding affinity for the TPO receptor and has equipotent bioactivity with recombinant human TPO (rhTPO) in stimulating proliferation and maturation of megakaryocytes in vitro. However, TMP is limited for clinical usage because of its short half-life in vivo. In this study, fusion proteins that composed of tandem dimer of TMP (dTMP) genetically fused at the C- or N-terminus of human serum albumin (HSA) were separately expressed in Chinese hamster ovary (CHO) cells. In vitro bioactivity assays showed that purified fusion proteins promoted the proliferation of megakaryocytes in a dose-dependent manner and activated signal transducer and activator of transcription (STAT) pathway in TPO receptor-dependent manner. Following subcutaneous administration, both HSA-dTMP and dTMP-HSA significantly elevated peripheral platelet counts in normal mice in a dose-dependent manner. In addition, fusion with HSA successfully prolonged dTMP half-life in mice. However, when HSA was fused at the C-terminus of dTMP, the bioactivity of dTMP-HSA was about half of that of HSA-dTMP. In conclusion, these results suggested that HSA/dTMP fusion proteins might be potential drugs for thrombocytopenia and, when HSA was fused at the N-terminus of dTMP, the fusion protein had a higher activity.

Functional expression of human serum albumin-tandem thrombopoietin mimetic peptide fusion protein as a novel thrombopoietin analog in Pichia pastoris.

OBJECTIVES: To develop a novel thrombopoietin (TPO) analog by fusing the tandem TPO mimetic peptide (TMP-TMP) to human serum albumin (HSA) and performing functional expression of recombinant fusion protein HSA-TMP-TMP. RESULTS: After optimizing the fusion orientation in shake-flask culture, HSA-TMP-TMP was expressed at 0.4 g/l in Pichia pastoris grown in a 20 l bioreactor, during which pH was controlled at 5 by addition of NH4OH and citric acid. The fusion protein significantly activated signal transducer and activator of transcription-mediated transcription in TPO receptor-dependent manner, which was demonstrated by a luciferase reporter assay. Following subcutaneous administration, HSA-TMP-TMP effectively stimulated the platelet production in healthy mice in a dose-dependent manner. CONCLUSION: Successful expression of HSA-TMP-TMP fusion protein in P. pastoris was achieved and the recombinant HSA-TMP-TMP is a promising TPO analog.

[Methanotrophs and their applications in environment treatment: a review].

Methanotrophs can oxidize methane, playing an important role in regulating methane emission, and gaining increasing attention by the researchers around the world. Two biological pathways are involved in methane oxidation, i.e., anaerobic oxidation and aerobic oxidation, which are governed by anaerobic and aerobic methanotrophs, respectively. In this paper, the research advances about methanotrophs were summarized, with the focus on the phylogeny and taxonomy of methanotrophs, the key enzymes responsible for the aerobic oxidation of methane, the microorganisms involved in the anaerobic oxidation of methane, and the mechanisms of microbial methane consumption. The application prospects of the two methane oxidizers in greenhouse gases removal, pollutants degradation, biological denitrification, and recovery of metals and sulfur compounds were also analyzed.

[Characteristics of microbial community in biohydrogen production from alkali pretreated sludge].

In order to reveal the characteristics of microbial community in biohydrogen production from alkali pretreated sludge, the biohydrogen fermentation was conducted under acidic condition (pH 5) and alkali condition (pH 11) using alkali pretreated sludge from three wastewater treatment plants with different process, respectively. The results indicate that, although the sludge from different sources, protein is main component in the soluble organic matter released from the three sludge samples by the alkali pretreatment and carbohydrate is only 15% -16% of protein. A high hydrogen yield occurs at the condition of initial pH 11, a maximal hydrogen yield of up to 31.9 mL/g, while the hydrogen yield decreases and hydrogen consumption occurs at the condition of initial pH 5. The analysis using PCR-DGGE technique based on 16S rDNA sequences with the universal primers (F338GC and R534) show that the microbial community in the biohydrogen production process from various sludge structure is significant different, and the microbial population appear a phenomenon of turnover growth and decline and the amount of dominant microbial community present a increasing trend.

Lack of cross-resistance to Mtx1 from Bacillus sphaericus in B. sphaericus-resistant Culex quinquefasciatus (Diptera: Culicidae).

The toxicities of Mtx1 toxin against dipteran and lepidopteran species have been evaluated in this study. It was shown that Mtx1 has little or no toxicity to the tested lepidopteran species, but has moderate-level toxicity to Aedes albopictus Skuse (Diptera: Culicidae) and high-level toxicity to both susceptible and binary toxin-resistant Culex quinquefasciatus Say (Diptera: Culicidae). The LC(50) values of Mtx1 against a susceptible C. quinquefasciatus colony SLCq and two resistant colonies RLCq1/C3-41 and RLCq2/IAB59 selected in the laboratory with Bacillus sphaericus (Mayer & Neide) strains C3-41 and IAB59 respectively were 0.508, 0.854 and 0.675 mg L(-1) respectively. The data indicate that Mtx1 has a different mode of action from the binary toxin, and that it could be an alternative toxin to delay or overcome resistance development to binary toxin in C. quinquefasciatus.

Mosquitocidal toxin from Bacillus sphaericus induces stronger delayed effects than binary toxin on Culex quinquefasciatus (Diptera: Culicidae).

We studied the toxicity and delayed effects of a mosquitocidal toxin (Mtx1) and a binary toxin (Bin) produced in Escherchia coli E-TH21 and Bacillus thuringiensis B-CW1, respectively, on Culex quinquefasciatus (Diptera: Culicidae). Bioassay results showed that both E-TH21 powder and B-CW1 sporulated culture were highly toxic against susceptible Cx. quinquefasciatus, with LC50 values of 0.65 and 1.70 mg/liter against third and fourth instars at 48 h, respectively. After initial 48-h exposure of larvae to different concentrations of Mtx1 and Bin, significant continued mortality could be observed in larval, pupal, and emergence stages of Cx. quinquefasciatus. Importantly, the Mtx1 could induce higher cumulative larval and preadult mortalities than Bin toxin on the target mosquito. This finding is important for understanding the mode of action of Mtx1 and Bin toxins and for developing a new bioassay procedure for evaluation of toxicity of Bacillus sphaericus Neide, some strains of which produce Mtx1 and Bin, in the laboratory and field.