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T cell immunoglobulin and ITIM domain (TIGIT) is a novel immune checkpoint that has been considered as a target in cancer immunotherapy. Current available bioassays for measuring the biological activity of therapeutic antibodies targeting TIGIT are restricted to mechanistic investigations because donor primary T cells are highly variable. Here, we designed a reporter gene assay comprising two cell lines, namely, CHO-CD112-CD3 scFv, which stably expresses CD112 (PVRL2, nectin-2) and a membrane-bound anti-CD3 single-chain fragment variable (scFv) as the target cell, and Jurkat-NFAT-TIGIT, which stably expresses TIGIT as well as the nuclear factor of activated T-cells (NFAT) response element-controlled luciferase gene, as the effector cell. The anti-CD3 scFv situated on the target cells activates Jurkat-NFAT-TIGIT cells through binding and crosslinking CD3 molecules of the effector cell, whereas interactions between CD112 and TIGIT prevent activation. The presence of anti-TIGIT mAbs disrupts their interaction, which in turn reverses the inactivation and luciferase expression. Optimization and validation studies have demonstrated that this assay is superior in terms of specificity, accuracy, linearity, and precision. In summary, this reliable and effective reporter gene assay may potentially be utilized in lot release control, stability assays, screening, and development of novel TIGIT-targeted therapeutic antibodies., Graphical abstract Here we developed one reporter gene assay comprising two cell lines, CHO-CD112-CD3 scFv and Jurkat-NFAT-TIGIT, which can be adopted in the bioactivity determination of anti-TIGIT antibody with high accuracy and specificity.Image 1

Fermentation and chemical methods for industrial cytidine diphosphate choline (CDP-choline) catalytic production both suffer from several disadvantages such as relatively low efficiency and productivity. To overcome these problems, we applied the concept of synthetic biology to develop a new one-pot multi-enzyme system to produce CDP-choline from orotic acid. Enzymes from different sources were selected and optimized as building blocks of the system, and parameters such as oxygen supply were also optimized. This system shows a titer of 37.6 ± 1.1 mM and a reaction rate of 1.6 mM L-1 h-1, both increase 66 % from traditional processes. It also has an efficiency of energy of 25.4%, improves 2-folds. This new one-pot CDP-choline-producing system has a potential for industrial use, and the procedure to design one-pot multi-enzyme system can be applied to build other one-pot system producing energy-rich compounds.

Although enormous success has been achieved with anti-PD-1/PD-L1 and anti-CTLA-4 monoclonal antibodies (mAbs), their unsatisfactory response rate in cancer patients has been driving the research and development of novel immune checkpoint inhibitors (ICIs). Anti-LAG-3 mAbs, as one of the most promising candidates, are now being tested for various human cancers at different stages of clinical trials. Here, we describe the development and validation of a reporter gene assay (RGA) to measure the bioactivity of anti-LAG-3 mAbs. We established the bioassay based on parental Raji cells and a Jurkat cell line stably transfected with human LAG-3 gene and luciferase reporter elements controlled by nuclear factor of activated T cell (NFAT) from the IL-2 promoter. After optimization of key parameters, the established RGA showed excellent precision, specificity, accuracy, and stability. The mechanism of action (MOA) relatedness and the excellent assay performance make the RGA suitable for the characterization, lot release, and stability test of anti-LAG-3 mAbs.

In this work, we studied the kinetics and behavior of wood panel wastes containing ureaformaldehyde resins. For this purpose, we used a TG-FTIR coupled system to analyze the gaseous products of the pyrolysis. With the results, we obtained the models that provide theoretical evidence and data support for the studied reactions. According to our findings, the pyrolysis of the ureaformaldehyde components displayed two stages, and the second stage presented a higher intensity. At a temperature of 900 °C, the gas generation rate for hard UF (UF1) was 32.6% higher than that for soft UF (UF2). When temperature increased from 600 °C to 900 °C, the increase of the gas generation rate was relatively small. In addition, the reaction order was obtained using the CoatsRedfern method, which provided a value for the reaction order of 1.4. The activation energy of UF1 was slightly higher than that of UF2. The FTIR analysis indicated that the main gaseous products of the pyrolysis of the urea-formaldehyde resin samples were CO2, H2O, and other compounds containing the C–H and the N–H bond as well as carbonyl groups. In addition, it was determined that Nitrogen was mainly present in the form of hydronitrogen compounds but not as nitrogen oxides. These last may represent a higher degree of pollution.

The low carbon metallurgy technology based on the high-value utilization of biomass is considered as the key development orientation of green ironmaking. In this paper, a pyrolysis coupling technology of biomass tar and iron ore powder is proposed for the reduction of iron ore. Tar is degraded efficiently due to the synergy effects respectively through oxidation of iron oxides and the catalytic action of reduced iron, and simultaneously the reduction of iron oxide to metallic iron. Representative tar components including vanillin, naphthalene, and catechol were selected as tar model compounds. The various reaction conditions on the reduction degree of iron ores was investigated, which include the type of tar component, the pyrolysis temperature, and the ratio of reactants. According to the results, the optimal relationship between tar degradation and reduction of iron ore was identified. It can be found that under the catalytic effects of iron ore fines, the degradation efficiency of the three model compounds followed the following order: naphthalene > vanillin > catechol. Naphthalene owned the better reduction ability. The highest reduction degree of the product reached to 78.5% at 800 °C for 30 min.

In the present research, TG and FTIR were used to study the effect of chemical activation pretreatment on sludge pyrolysis. The results showed that both acidic and alkaline activation promoted sludge pyrolysis. KOH inhibited the elimination of crystallization water and promoted the release of volatiles. On the other hand, H2SO4 favored the separation of crystallization water and the release of volatiles. Low concentrations of the activator promoted the production of combustible gas and inhibited the generation of CO2. By analyzing the activation energy of the pyrolysis process using the Coats-Redfern method, it was found that both, KOH and H2SO4, were able to reduce the pyrolysis activation energy. When H2SO4 was used, the effect was more significant.

Our previous studies have demonstrated that trametenolic acid B (TAB) extracted from the Laetiporus sulphureus (Fr.) Murrill owned effective anti-proliferation of HepG2/2.215 cells and induced autophagy activity. The present aim was to further investigate its mechanisms involved by proteomic analysis. The iTRAQ of TAB on HepG2/2.215 was carried out and the western blot was used to verify the results of the proteomics analysis. According to the peptide segment quantitative standard (FDR ≤ 1%), a total of 5324 proteins were identified in HepG2/2.215 by proteomic analysis. The results identified that the major up-regulated proteins were HSP90AA4P, MYB, SERPINE1, and down-regulated proteins were Rho C, SERPINA1, and PIK3R4, which were related to PI3K/Akt signaling pathway, cell metastasis, and autophagy. HSP90AA4P and Rho C’s proteomics analysis were further confirmed by the western blot. The proteomic results demonstrated that the anti-hematoma effect of TAB was closely related to the increase of HSP90AA4P protein expressions and autophagy, which may be a critical target of TAB, which was expected to be a candidate drug for the treatment liver cancer.

We performed high-sensitivity mapping observations of several complex organic molecules around Sagittarius B2 with ARO 12m telescope at 3-mm wavelength. Based on their spatial distribution, molecules can be classified as either "extended" that detected not only in Sgr B2(N) and Sgr B2(M), or "compact" that only detected toward or near to Sgr B2(N) and Sgr B2(M). The "extended" molecules including glycolaldehyde (CH2OHCHO), methyl formate (CH3OCHO), formic acid (t-HCOOH), ethanol (C2H5OH) and methyl amine (CH3NH2), while the "compact" molecules including dimethyl ether (CH3OCH3), ethyl cyanide (C2H5CN), and amino acetonitrile (H2NCH2CN). These "compact" molecules are likely produced under strong UV radiation, while "extended" molecules are likely formed under low-temperature, via gas-phase or grain surface reactions. The spatial distribution of "warm" CH2OHCHO at 89 GHz differ from the spatial distribution of "cold" CH2OHCHO observed at 13 GHz. We found evidence for an overabundance of CH2OHCHO compared to that expected from the gas-phase model, which indicates that grain-surface reactions are necessary to explain the origin of CH2OHCHO in Sagittarius B2. Grain-surface reactions are also needed to explain the correlation between the abundances of "cold" CH2OHCHO and C2H5OH. These results demonstrate the importance of grain-surface chemistry in the production of complex organic molecules., Comment: 11 pages, accepted by MNRAS

The effects of phloridzin (PHL), main component of Malus hupehensis (MH) tea leaves, on blood glucose (BG) and glucose-6-phosphatase (G-6-Pase) were investigated to provide a basis for finding a scheme of stabilizing BG. Glucose uptake of insulin resistant HepG2 cells was measured by glucose oxidase method. Glucose tolerance, fasting BG (FBG) and postprandial BG (PBG) were determined by BG test strips. The expression of G-6-Pase was detected by Western blot. The results showed that glucose uptake was enhanced and the expression of G-6-Pase was inhibited by PHL in insulin resistant HepG2 cells. Glucose tolerance was enhanced, FBG level was increased and PBG level was decreased by PHL in mice. The expression of G-6-Pase in the liver was enhanced under fasting state, and was inhibited by the low and medium dose under postprandial state. It indicated that PHL has a positive effect on stabilizing BG in mice, which is related to bidirectional regulation of G-6-Pase activity. PRACTICAL APPLICATIONS: Malus hupehensis, edible and medicinal plant, which has been proved by long-term application and experiments that it has a good effect on stabilizing blood glucose, preventing diabetes and adjuvant treatment. Its effect is closely related to its main component PHL. Thus, MH can be used as a dietary regulating drink for daily life to maintain blood glucose. Its main ingredient is PHL, which can be developed as a candidate drug for diabetes treatment.

Malus hupehensis (Pamp.) Rehd (MH) is a dual-purpose plant used as medicine and food, which possesses various biological activities including hepatoprotective effect. In this study, the hepatoprotective activity of extracts of MH (MHE) against RFP + INH-induced liver injury was investigated. Animals in model group were given saline and INH + RFP. Animals in MHE groups were given MHE and INH + RFP. Animals in normal control group were given equal volume of saline. Subsequently, blood and liver samples were collected and assessed. MHE significantly alleviated the liver injury, as evidenced by decreased activities of the ALT, AST, TBIL, MDA and GSH-ST, and increased levels of SOD and GSH. MHE also effectively reduced the pathological tissue damage. Moreover, the CYP450 levels, and expression of CYP3A4, CYP2C9 and NADPH4 protein were inhibited by MHE. MHE exerts a protective effect against RFP + INH-induced liver injury in mice, and could be developed as a liver protectant for treatment of DILI.

CTLA-4 is an important immune checkpoint for the regulation of T cell activation, and anti-CTLA-4 monoclonal antibodies (mAbs) are being developed as mono- or combination therapy for various tumors with reliable clinical efficacy. Ipilimumab is the first approved inhibitor of immune checkpoint, and many other anti-CTLA-4 mAbs, including ipilimumab biosimilars, are in different stages of clinical trials. However, due to the immunomodulating nature of the mAbs targeting CTLA-4, mode of action (MoA) and cell-based bioassay to determine their bioactivities as the lot release or stability test has been a great challenge to quality control laboratories. In this study, we have developed and validated a reporter gene assay (RGA), in which two kinds of cell lines were engineered to measure the bioactivity of anti-CTLA-4 mAbs. Raji cells were stably transfected with the membrane-anchored anti-CD3 single chain antibody fragment (scFv) as antigen-presenting cells (APCs, Raji-CD3scFv cells), while Jurkat cells were stably transfected with CTLA-4 with Y201V mutation and NFAT controlled luciferase as the effector cells (Jurkat-CTLA-4-NFAT-luc cells). The ligation of CD80/CD86 on the APCs with CTLA-4 could reduce the luciferase expression accompanied with the activation of effector cells, while the anti-CTLA-4 mAb could reverse the reduction, which resulted in good dose response curve to determine its bioactivity. After optimizing various assay conditions, we performed full validation according to ICH-Q2 (R1), which demonstrated the excellent specificity, accuracy, precision, linearity, and the cell passage stability. The satisfied performance characteristics render the RGA a good bioassay in the bioactivity determination of anti-CTLA-4 mAbs, as applied in characterization, batch release control, stability study, and biosimilar assessment.

Glyphosate is the most widely used herbicide in world agriculture and for general vegetation control in a wide range of situations. Global and often intensive glyphosate selection of very large weedy plant populations has resulted in widespread glyphosate resistance evolution in populations of many weed species. Here, working with a glyphosate-resistant (GR) Echinochloa colona population that evolved in a Western Australia agricultural field, we identified an ATP-binding cassette (ABC) transporter (EcABCC8) that is consistently up-regulated in GR plants. When expressed in transgenic rice, this EcABCC8 transporter endowed glyphosate resistance. Equally, rice, maize, and soybean overexpressing the EcABCC8 ortholog genes were made resistant to glyphosate. Conversely, CRISPR/Cas9-mediated knockout of the EcABCC8 ortholog gene OsABCC8 increased rice susceptibility to glyphosate. Subcellular localization analysis and quantification of glyphosate cellular levels in treated ABCC8 transgenic rice plants and isolated leaf protoplasts as well as structural modeling support that EcABCC8 is likely a plasma membrane–localized transporter extruding cytoplasmic glyphosate to the apoplast, lowering the cellular glyphosate level. This is a report of a membrane transporter effluxing glyphosate in a GR plant species, and its function is likely conserved in crop plant species.

Influenza vaccine potency is determined by the quantification of immunologically active hemagglutinin capable of eliciting neutralizing antibodies upon immunization. Currently, the single radial immunodiffusion (SRID) method is the standard in vitro potency assay used for lot release of seasonal inactivated influenza vaccines. Despite the proven usage of SRID, significant limitations such as the time-consuming preparation of reagents and limited dynamic range warrant the need for the development of alternative potency assays. Such alternative approaches need to discriminate and quantify relevant hemagglutinin material, provide strain identity, and be independent of strain-specific and seasonal reagents. Herein, we present a proof of concept method that combines the capture of conformationally well-folded hemagglutinin via a sialic acid binding step with the resolving power of reversed-phase high-performance liquid chromatography for strain identity and determination. Details of the protocol for the selective capture of receptor-binding hemagglutinin, its release from the receptor, and its relative determination are presented. This approach was found to provide flexibility for the reagents to be used and was adaptable to varying strain compositions of influenza vaccines. This proof of concept approach was developed as an antibody-independent methodology.

Sludge gasification for the production of hydrogen-rich gas is a promising technology. In this paper, a pilot study on the hydrogen-rich gas production by sludge gasification using waste heat of blast furnace slag was carried out, and the mass and energy balance of gasification process using waste heat from blast furnace slag were evaluated. The results show that the higher the gasification temperature, the higher the hydrogen content in the gas. When the gasification temperature reaches 880 °C, the hydrogen content in the gas reaches the maximum,35.3%. The technology of sludge gasification combined with waste heat recovery of high furnace slag is feasible. Its efficiency of heat recovery can reach up to 64.35%, and the gasification efficiency and energy consumption ratio can reach to 42.30% and 3.67, respectively.

RANKL (receptor activator of nuclear factor κB ligand) plays a key role in the differentiation, activation and survival of osteoclasts. Denosumab, which targets RANKL, is approved for osteoporosis or bone loss that has a high risk for fracture and bone metastases from solid tumors. Bioactivity determination is essential for the safety and efficacy of therapeutic antibodies. At present, the mechanism of action (MOA) based bioassay for anti-RANKL monoclonal antibodies (mAbs) is the measurement of tartrate resistant acid phosphatase (TRAP) activity, which takes about five days and has complex operation and relatively high variation. In this study, we developed a reporter gene assay (RGA) based on a RAW264.7 cell line stably expressing luciferase reporter under the control of nuclear factor-κB (NF-κB) response elements. After optimizing the key parameters, the validation results based on ICH-Q2 not only show superior specificity, precision, linearity, accuracy and passage stability, but also a short duration and simple operation. These results demonstrate the RGA based on the RANKL-RANK-NF-κB pathway can be an excellent alternative for measuring the bioactivity of anti-RANKL mAbs.

Biocatalysis has become the main approach to produce cytidine diphosphate choline (CDP-choline), which has been applied for treatment of acute craniocerebral injury and consciousness after brain surgery. However, salt accumulates with the production and inhibits enzyme activity, and eventually reduces yield and product accumulation rate. Our work provided a possible solution to this problem by applying a computational designed halophilic choline kinase. The halotolerant CKI (choline kinase) was designed following a unique strategy considering the most variable residue positions on the protein surface among target enzymes from different sources. The basic and neutral surface residues were replaced with acidic ones. This approach was enlightened by features of natural halophilic enzymes. Mutants in the work represented higher catalytic activities and IC50 (inhibit activity by 50%) at high salt concentrations (over 1200 mM). Furthermore, when the mutant was used in fed-batch production, the CDP-choline accumulation rate doubled comparing with process using wild-type CKI at acetate concentration of over 700 mM. The maximum titer was 151 ± 3.2 mM, the productivity was 5.8 ± 0.1 mM·L−1 h−1, and molar yield to CMP and utilization efficiency of energy were 85.3 and 63.5%. The idea of computational design in our work can also be applied to modify other enzymes in industry, and sheds light on alleviating effect of salt accumulation during industrial manufacturing process.

AP25 is an anti-tumor peptide with a high affinity for integrins. It exerts its anti-tumor activity by inhibiting angiogenesis and by directly inhibiting the growth of tumor cells. Its half-life time in vivo is only about 50 minutes, which limits its clinical application. In order to prolong the half-life time of AP25 while preserving its anti-tumor activity, several fusion proteins of AP25 and IgG4 Fc were designed and expressed; their anti-tumor activity and pharmacokinetics properties were evaluated. Firstly, four AP25-Fc fusion protein sequences were designed, and the corresponding proteins were expressed and purified. Based on the results of HUVEC migration inhibition assay, HUVEC and tumor cell proliferation inhibition assay and yields of expression by HEK293 cells, the fusion protein designated PSG4R was selected for further evaluation. The anti-tumor effect of PSG4R was then evaluated in vivo on HCT-116 nude mice xenograft model. And the pharmacokinetics properties of PSG4R were investigated in rats. The results showed that PSG4R could inhibit the growth of xenografts of human colon cancer cell line HCT-116 in nude mice by intravenous administration of 40 mg/kg once every two days. The half-life time of PSG4R was 56.270 ± 15.398 h. This study showed that the construction of AP25-Fc fusion protein could significantly prolong the half-life of AP25 while retaining its anti-tumor activity, which provides a new direction for new drug development of AP25.

We studied Phellinus lonicerinus to determine the cytotoxic effect and the dual estrogenic activities of methyl-hispolon and their relation to estrogen signals in vivo and in vitro. The Glide scores of methyl-hispolon-estrogen receptor α (ERα) and methyl-hispolon-ERβ docked complexes were -7.29 kcal/mol and -6.68 kcal/mol in docking simulations. Methyl-hispolon had a significant antiproliferative effect for estrogen-sensitive ER(+) MCF-7 cells in the absence of estrogen, and it exhibited dual estrogen activities. Methyl-hispolon increased the serum E2 in rats with premature ovarian failure and fulfilled the estrogenic function in the uterus and ovary. Methyl-hispolon significantly inhibited the expression of Ras, API, ERα, C-myc, and cyclinDl, as well as their gene transcription in RL95-2 cells. The phosphorylation of ERK1/2 was inhibited by methyl-hispolon. Thus, methyl-hispolon has potential use in treating estrogen deficiency-related diseases, with good antitumor effects and estrogenic activity.

The aim of this study was to elucidate the gastroprotective activity and possible mechanism of involvement of araloside A (ARA) against ethanol- and aspirin-induced gastric ulcer in mice. The experimental mice were randomly divided into control, model, omeprazole (20 mg/kg, orally) and ARA (10, 20 and 40 mg/kg, orally). Gastric ulcer in mice was induced by intragastric administration of 80% ethanol (10 mL/kg) containing 15 mg/mL aspirin 4 h after drug administration on day 7. The results indicated that ARA could significantly raise gastric juice volume and acidity; ameliorate gastric mucosal blood flow, gastric binding mucus volume, ulcer index and ulcer inhibition rate; suppress H+/K+-ATPase activity, which was confirmed by computer-aided docking simulations; inhibit the release of mitochondrial cytochrome c into the cytoplasm; inhibit caspase-9 and caspase-3 activities and down-regulate mRNA expression levels; down-regulate the mRNA and protein expressions of apoptosis protease-activating factor-1 and protein expression of cleaved poly(ADP ribose) polymerase-1; and up-regulate Bcl-2 mRNA and protein expressions and down-regulate Bax mRNA and protein expressions, thus elevating the Bcl-2/Bax ratio in a dose-dependent manner. Histopathological observations further provided supportive evidence for the aforementioned results. The results demonstrated that ARA exerted beneficial gastroprotective effects on alcohol- and aspirin-induced gastric ulcer in mice, which was related to suppressing H+/K+-ATPase activity as well as pro-apoptotic protein expression, and promoting anti-apoptotic protein expression, thus alleviating gastric mucosal injury and cell death.

Accurate determination of biological activity is essential in quality control of recombinant human brain natriuretic peptide (rhBNP). In previous study, we successfully developed a genetically modified cell line 293GCAC3-based ELISA assay for rhBNP. But ELISA procedure is still tedious, so this study was aimed to develop a rapid and simple bioassay for rhBNP using GloSensor technology, which provides a platform of flexible luciferase-based biosensors for real-time detection of signaling events in live cells, including cGMP production. A reporter cell line 293GCAGlo-G1 was constructed by transfecting pGloSensor™ 40 F plasmid into 293GCAC3. The reporter assay based on 293GCAGlo-G1 showed high precision with intra-assay CV being 8.3% and inter-assay CV being 14.1%; high accuracy with 80%, 100% and 120% recovery rate being 99.2%, 102.4% and 99.0% respectively; and great linearity with R2 of linear fitting equation being 0.99. Besides, no significant difference was found in test results of reporter assay and 293GCAC3-based ELISA assay (paired t test, p = 0.630). All these results suggested that the reporter assay was a viable assay for biological determination of rhBNP. Keywords: RhBNP, cGMP, GloSensor technology, Reporter assay

Calcitonin gene-related peptide (CGRP) is critical for the pathophysiology of migraine, and four therapeutic antibodies targeting CGRP and its corresponding receptors have been approved by the Food and Drug Administration (FDA), while many others are in the different stages of clinical trials. Bioactivity determination is essential for the quality control and clinical application of therapeutic monoclonal antibodies (mAbs). However, no bioassay has been reported to date. In this study, we developed a reporter gene assay (RGA) based on SK-N-MC cells stably expressing firefly luciferase driven by cAMP response element (CRE). The key assay parameters were optimized according to signal-to-noise (SNR), the response value, and the fitted dose-response curve. Validation of the RGA in accordance with ICH-Q2 guidelines showed that the method had good specificity, accuracy, linearity, and precision. The established RGA can be utilized as a reference method for release testing and stability studies of relevant antibodies.

Background Exploring herbicide resistance mechanisms in weeds is regarded as an important part of designing resistance management strategies and rationalizing the use of herbicides. Beckmannia syzigachne is one of the most important agricultural weeds in China. Long-term use of acetyl-CoA carboxylase (ACCase)-inhibiting herbicides has led to evolution of herbicide resistance in B. syzigachne. ACCase-inhibiting herbicides consist of three chemical families, including the aryloxyphenoxypropionates (APPs), cyclohexanediones (CHDs) and phenylpyraxoline (DENs). Results Based on whole-plant dose-response experiments, a B. syzigachne population (BS-R) was confirmed to be 20- and 12-fold resistant to the APP herbicides clodinafop-propargyl and quizalofop-P-ethyl; 2.2-, 2.8-, and 2.8-fold resistant to fenoxaprop-P-ethyl, the CHD herbicide sethoxydim and PPZ herbicide pinoxaden, respectively, compared to the susceptible counterpart (BS-S). Resistance to clodinafop-propargyl in the BS-R population could not be reversed by the known cytochrome P450 (CYP450) inhibitor malathion and glutathione S-transferase (GST) inhibitor 4-chloro-7-nitrobenzoxadiazole. In addition, no difference in CYP450 and GST activity was confirmed in BS-R and BS-S population. ACCase gene sequencing revealed an Ile-2041-Val mutation in the BS-R population. A derived cleaved amplified polymorphic sequence (dCAPS) marker was developed for rapid detection of the specific Ile-2041-Val mutation. Correlation quantification of resistance in homo- and hetro-resistant versus WT plants showed that the resistance to clodinafop-propargyl in this population is conferred by the Ile-2041-Val mutation. Conclusion Unlike the previous reports on the unique cross-resistance pattern by the 2041 mutation, this study demonstrates that the BS-R population confers resistance to certain ACCase-inhibiting APP, CHD and PPZ herbicides in B. syzigachne. This article is protected by copyright. All rights reserved.

The tumor necrosis factor alpha (TNF-α)/nuclear factor-kappa B (NF-κB) signaling pathway plays a crucial role in the pathogenesis of inflammatory diseases. Several therapeutic monoclonal antibodies (mAbs) and biosimilars against TNF-α have been developed to treat patients who suffer from inflammatory diseases caused by disordered expression of TNF-α. Hence, quality control of biopharmaceuticals is crucial during research and development. The high-order structure of these complex molecules cannot be entirely identified by physiochemical attributes; however, they can be inferred by observing biological activities. Thus, we developed a U937-based bioassay to determine the biological activities of mAbs and biosimilars against TNF-α using a low-basal NF-κB-inducible lentiviral reporter gene. The reporter gene assay (RGA) can be induced with a high signal-to-noise ratio (SNR) in a short time by TNF-α. Validation of the RGA showed accuracy (% relative standard deviation [RSD] = 4.64%), linearity (r2 = 0.9856), and precision (Interday RSD = 4.6%, between analysts RSD = 3.51%) as well as reasonable specificity and robustness. The measured potency values of a biosimilar to adalimumab were between 90% and 110%. Results showed our RGA is suitable for mAb quality control and lot release, and for evaluation of the biological activity similarity of the biosimilar.

OX40 plays a prominent role in the onset and development of solid tumors, and OX40-targeted monoclonal antibodies (mAbs) have entered clinical trials for various tumors. Bioactivity determination of therapeutic mAbs is of great significance in product quality, however, mechanism of action-based bioassays to determine the bioactivity of anti-OX40 mAbs is still lacking. Here, we established a reporter gene assay system based on two cell lines, namely Jurkat-OX40-NFκB-Luc which stably expresses NFκB-controlled luciferase, and Raji cells which inherently express FcγRs. In the model, FcγRs on Raji cells could crosslink the Fc of anti-OX40 mAbs, which leads to the further crosslinking between Fab of anti-OX40 mAbs and OX40 on Jurkat-OX40-NFκB-Luc cells. OX40 crosslinking could activate Jurkat-OX40-NFκB-Luc cells, and induce the expression of NFκB-controlled luciferase, the extent of which could reflect the bioactivity of anti-OX40 mAbs in a dose-dependent manner. After the optimization of various assay conditions, the validation of the cell-based bioassay showed good assay performance characteristics, including specificity, accuracy, precision, linearity, and stability. This innovative assay that is based on the OX40-NFκB pathway can be a powerful pool to measure the bioactivity of OX40-targeted mAbs.

Objective This work designs an in vitro multi-enzyme system to produce CDP-choline form D-ribose, and develop an optimization procedure for one-pot multi-enzyme catalytic system. Results The entire process integrated ten enzymes, and an efficient acetate kinase/acetyl phosphate-based ATP regeneration module was applied. Then some optimizations to this system were made including selecting optimum enzyme building blocks and improving expression parameters. The process improved the final yield of CDP-choline form 0.2g/L to 6g/L (CDP-choline titer 12.2 mM). Conclusions This new one pot CDP-choline producing system has a potential for industrial use, and the optimization procedure shed light on improving other one pot multi-enzyme system for industrial production of energy rich compounds. This article is protected by copyright. All rights reserved.

Type 2 inflammatory cytokines, including IL-4, IL-5 and IL-13, contribute considerably to the pathogenesis of asthma. Anti-IL-4R monoclonal antibody (mAb) has been approved for the therapeutic treatment of asthma, and many mAbs with the same target are in the different stages of R&D and clinical trials. Bioactivity determination is required to ensure the quality control of mAbs. However, current ELISA and SPR assays or cell-based anti-proliferation assays for IL-4R mAbs are either not mechanism-of-action (MOA) representative or tedious and time consuming. Therefore, we developed a reporter gene assay (RGA) based on the HEK-293 cell line that stably expressed signal transducer and activator of transcription 6 (STAT6) and the luciferase reporter controlled by STAT6 binding elements. Anti-4R mAb could bind to IL-4R, and block the interaction between IL-4 and IL-4R, resulting in the reduction of IL-4 induced STAT6 controlled luciferase expression. After careful optimization of the experiment parameters, the RGA method demonstrated optimal dose-response curve between anti-IL-4R mAb concentration and luciferase expression level. Validation according ICH-Q2 proved the excellent assay performance characteristics of the established RGA, including specificity, accuracy, precision, linearity and range. The established transgenic cell line was stable for the bioactivity determination of anti-IL-4R mAb up to 46 generations, and the RGA was also suitable for the bioactivity determination of anti-IL-4 mAbs, and potentially of anti-IL-13 mAbs. The established RGA could be adopted to determine the bioactivity during the development, characterization, lot release, stability, and comparability studies of anti-IL-4R mAbs.

BACKGROUND Asia minor bluegrass (Polypogon fugax) is one of the main weeds invading Chinese canola fields. The P. fugax resistant population SC-R, which survived quizalofop-p-ethyl at the field-recommended rate (67.5 g ha-1 ), was collected from a canola field in Qingsheng County in China. The present study aimed to (1) characterize the SC-R resistance pattern to acetyl-CoA carboxylase (ACCase)-inhibiting herbicides, and (2) investigate the mechanism of quizalofop-p-ethyl resistance in this population. RESULTS Dose-response studies showed that resistance to quizalofop-p-ethyl and haloxyfop occurred in the SC-R population. Four transcripts/genes encoding the plastidic ACCase carboxyl-transferase domain were isolated from the P. fugax plants. No mutations in the four ACCase genes were detected in the SC-R population compared to the SC-S population. Pre-treatment with the known glutathione S-transferase (GST) inhibitor 4-chloro-7-nitrobenzoxadiazole (NBDCl), reversed resistance to quizalofop-p-ethyl and partially reversed resistance to haloxyfop-R-methyl in the resistant population (SC-R). However, the cytochrome P450 inhibitor malathion did not reverse the resistance. There was no difference in basal GST activity (using CDNB as a substrate), but there was higher inducible GST activity in SC-R relative to SC-S. Two GST genes, GST2c and GSTL3, were constitutively overexpressed in the resistant SC-R population. CONCLUSION This study confirmed that resistance to quizalofop-p-ethyl in the resistant P. fugax population is likely nontarget-site based involving GST, and this resistance mechanism also partially confers haloxyfop-R-methyl resistance. © 2020 Society of Chemical Industry.

Environmental disturbances may result in dysregulation of interleukin-23 (IL-23), which is a crucial modulator of immunity. Several therapeutic monoclonal antibodies (mAbs) have been developed for treating IL-23-related autoimmune inflammation, such as ustekinumab, guselkumab, tildrakizumab, and risankizumab. Accurate bioactivity determination of therapeutic mAbs is essential for their quality control and clinical application. However, the current methods are tedious and complicated. In the present study, we employed low-background lentivirus carrying sis-inducible element (SIE)-driven firefly luciferase to generate a stable DB-SIE-Luc cell line that expresses endogenous IL-23 receptors and developed a sensitive and straightforward reporter gene assay (RGA) based on DB-SIE-Luc cells. After the optimization of various assay parameters, we set up a bioassay with the best fit of a four-parameter model and an appropriate signal-to-noise ratio (SNR) for bioactivity determination of guselkumab. We further verified the excellent assay performance characteristics of our RGA, including specificity, linearity, accuracy, precision, and stability, according to ICH-Q2. Taken together, we established a reliable and robust cell-based RGA, which potentially serves as a valubale alternative bioactivity determination assay for the release control and stability study of anti-IL-23 mAbs.

In this study, the estrogenic effect of GL-1, a component of the Ganoderma lucidum, was studied, and the possible mechanism was discussed preliminarily. The binding ability of GL-1 to estrogen receptor was calculated by computer aided simulation. The effects of GL-1 on the proliferation of estrogen sensitive estrogen receptor (ER) (+) MCF-7 cells and estrogen insensitive ER (−) MDA-MB-231 cells were detected by MTT method. The effects of GL-1 on the proliferation of estrogen-induced MCF-7 cells, and the effects of the estrogen receptor inhibitor ICI182780 on the proliferation of GL-1-induced MCF-7 cells were detected by MTT assay. The expression of ERα and ERβ monoclonal antibody were detected by Western blot. The results showed that GL-1 has a good binding ability to estrogen receptor β, and has estrogen-like effect, which might be related to secretion of estrogen and expression of ERβ by binding to ERs.

We have introduced a new method of estimating the electron temperature and density of H II regions by using single dish observations. In this method, multiple hydrogen radio recombination lines of different bands are computed under the assumption of low optical depth. We use evolutionary hydrodynamical models of H II regions to model hydrogen recombination line emission from a variety of H II regions and assess the reliability of the method. According to the simulated results, the error of the estimated temperature is commonly < 13%, and that of the estimated density is < 25% for a < 1% uncertainty of the observed line fluxes. A reasonable estimated value of electron density can be achieved if the uncertainty of the line fluxes are lower than 3%. In addition, the estimated values are more representative of the properties in the relatively high-density region if the gas density gradient is present in the H II region. Our method can be independent of the radio continuum observations. But the accuracy will be improved if a line-to-continuum ratio at millimeter wavelengths is added to the estimation. Our method provides a way to measure the temperature and density in ionized regions without interferometers., 17 pages, 12 figures

To overcome the lack of availability of fresh human whole blood for pyrogen detection, we explored the feasibility of utilizing cryopreserved pooled human blood to detect the responses of the pro-inflammatory cytokines IL-6 and IL-1β to LPS. Whole blood was obtained from five donors and incubated with LPS. The quantities of pro-inflammatory cytokines were measured using ELISA, and the results were compared among the samples. After the blood was cryopreserved with Dimethyl sulfoxide (DMSO) (10% v/v) and stored for 4 mo at –196℃, the detection limits of the IL-6/IL-1β responses to LPS were 0.2/0.4 endotoxin units (EU)/ml, respectively, and IL-6/IL-1β release increased in response to LPS in a dose-dependent manner. When these experiments were performed in three separate laboratories, the within-laboratory reproducibility of the IL-6/IL-1β responses was 100%/86.7%, 93.3%/100%, and 86.7%/80%, and the inter-laboratory reproducibility was 92.9%/85.7%, 64.3%/63.6%, and 57.1%/66.7%, respectively. The sensitivity (the probability of correctly classifying positive samples) and specificity (the probability of correctly classifying negative samples) of the IL-6/IL-1β tests were 81.7%/82.5% and 100%/100%, respectively. The results of this study suggest that cryopreserved pooled blood is a convenient and viable alternative for evaluating in vitro pyrogenicity. Additionally, maintaining cryopreserved pooled blood promotes safety for the user because it is released only after pretesting for infection parameters and has lower variation than fresh donations from a variety of donors.

In this work, novel cyclic-brush polymers with high grafting densities were successfully constructed via the grafting onto approach by combining a light-induced Diels–Alder ring-closure approach and activated ester chemistry as a post-modification method. The cyclic activated ester containing polymer precursor [poly(pentafluorophenyl 4-vinylbenzoate), c-PPF4VB4.0k] was synthesized by combining reversible addition–fragmentation chain transfer (RAFT) polymerization and the light-induced Diels–Alder click reaction, which was fully characterized by NMR, SEC, MALDI TOF mass and UV-Vis spectroscopy. Using activated ester chemistry, this cyclic polymer (c-PPF4VB4.0k), bearing a pentafluorophenyl group, was post-modified with PEG-NH2 to obtain a cyclic-brush polymer (c-PPF4VB4.0k-g-PEG) with hydrophilic grafting polymer chains and a hydrophobic cyclic polymer backbone. Meanwhile, c-PPF4VB4.0k was post-functionalized using propargyl amine to obtain a clickable cyclic polymer (c-P1). The cyclic polymer (c-P1) was further coupled with an azide-containing polymeric chain (l-PS-N3) to produce the cyclic-brush polymer (c-P1-g-PS). This work illustrated a concept for constructing cyclic-brush polymers by combining the ring-closure strategy and activated ester chemistry. Notably, these two cyclic-brush polymers exhibited high grafting densities (almost quantitatively) and narrow molecular weight distribution (Mw/Mn < 1.10).

Eosinophilic asthma is characterized by the eosinophilic inflammation with the allergen independent activation of Th2 lymphocytes. Since IL-5 plays an important role in the maturation, survival and migration of eosinophils, hence the pathogenesis of eosinophilic asthma, biotherapeutics targeting IL-5/IL-5Rα have been developed and/or marketed, including Mepolizumab, Reslizumab, and Benralizumab. Accurate determination of bioactivity is crucial for the safety and efficacy of therapeutic antibodies. The current mode of action (MOA) based method used in the quality control and stability tests for anti-IL-5 mAbs is anti-proliferation assay, which is tedious with long duration and high variation. We describe here the development and validation of a reporter gene assay (RGA), based on an IL-5-dependent TF-1 cell line variant we established that stably expresses the luciferase reporter under the control of STAT5 response elements. After careful optimization, we demonstrate the excellent specificity, precision, accuracy and linearity of the established RGA. Our study also proves that the assay is superior on precision, sensitivity and assay simplicity to the anti-proliferation assay. The established RGA is also applicable to another anti-IL-5Rα mAb. These results show for the first time that this novel RGA, based on the IL-5-IL-5R-STAT5 pathway, can be a valuable supplement to the anti-proliferation assay and employed in the bioactivity determination of anti-IL-5/anti-IL-5Rα biotherapeutics.

Arthrobacter sp. CGMCC 3584 is able to produce high yields of extracellular cyclic adenosine monophosphate (cAMP), which plays a vital role in the field of treatment of disease and animal food, during aerobic fermentation. However, the molecular basis of cAMP production in Arthrobacter species is rarely explored. Here, for the first time, we report the comparative transcriptomic and proteomic study of Arthrobacter cells to elucidate the higher productivity of cAMP under high oxygen supply. We finally obtained 14.1% and 19.3% of the Arthrobacter genome genes which were up-regulated and down-regulated notably, respectively, with high oxygen supply, and identified 54 differently expressed proteins. Our results revealed that high oxygen supply had two major effects on metabolism: inhibition of glycolysis, pyruvate metabolism, nitrogen metabolism, and amino acid metabolism (histidine, branched-chain amino acids and glutamate metabolism); enhancement of the tricarboxylic acid cycle and purine metabolism. We also found that regulation of adenylate cyclase and phosphodiesterase was not significant under high oxygen supply, suggesting efficient cAMP export might be important in cAMP production. These findings may contribute to further understanding of capacities of Arthrobacter species and would be highly useful in genetic regulation for desirable production.

More than 100 monoclonal antibodies (mAbs) have been approved by FDA. The mechanism of action (MoA) involves in neutralization of a specific target via the Fab region and Fc effector functions through Fc region, while the latter include complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). ADCP has been recognized one of the most important MoAs, especially for anti-cancer mAbs in recent years. However, traditional bioassays measuring ADCP always introduced primary macrophages and flow cytometry, which are difficult to handle and highly variable. In this study, we engineered a monoclonal Jurkat/NFAT/CD32a-FceRIγ effector cell line that stably expresses CD32a-FceRIγ chimeric receptor and NFAT-controlled luciferase. The corresponding mAb could bind with the membrane antigens on the target cells with its Fab fragment and CD32a-FceRIγ on the effector cells with its Fc fragment, leading to the crosslinking of CD32a-FceRIγ and the resultant expression of subsequent NFAT-controlled luciferase, which represents the bioactivity of ADCP based on the MoA of the mAb. With rituximab as the model mAb, Raji cells as the target cells, and Jurkat/NFAT/CD32a-FceRIγ cells as the effector cells, we adopted the strategy of Design of Experiment (DoE) to optimize the bioassay. Then we fully validated the established bioassay according to ICH-Q2(R1), which proved the good assay performance characteristics of the bioassay, including specificity, accuracy, precision, linearity, stability and robustness. This RGA can be applied to evaluate the -ADCP bioactivity for anti-CD20 mAbs in lot release, stability testing as well as biosimilar comparability. The engineered cells may also potentially be used to evaluate the ADCP bioactivity of mAbs with other targets.

Peptide bonds, as the molecular bridges that connect amino acids, are crucial to the formation of proteins. Searches and studies of molecules with embedded peptide-like bonds are thus important for the understanding of protein formation in space. Here we report the first tentative detection of propionamide (C2H5CONH2), the largest peptide-like molecule detected in space toward Sagittarius B2(N1) at a position called N1E that is slightly offset from the continuum peak. A new laboratory measurements of the propionamide spectrum were carried out in the 9-461 GHz, which provide good opportunity to check directly for the transition frequencies of detected interstellar lines of propionamide. Our observing result indicates that propionamide emission comes from the warm, compact cores in Sagittarius B2, in which massive protostellars are forming. The column density of propionamide toward Sgr B2(N1E) was derived to be 1.5\times 10^{16} cm^-2, which is three fifths of that of acetamide, and one nineteenth of that of formamide. This detection suggests that large peptide-like molecules can form and survive during star-forming process and may form more complex molecules in the interstellar medium. The detection of propionamide bodes well for the presence of polypeptides, as well as other complex prebiotic molecules in the interstellar medium., 49 pages, 13 figures, accepted by ApJ

Seasonal inactivated quadrivalent influenza vaccines are currently formulated to include antigens from two strains of influenza A and a strain from each of the two circulating influenza B virus lineages. However, the applicability of the potency assay currently required for the release of vaccines has been hindered due to cross-reactivity between the two B strains. In this study, a reversed-phase high-performance liquid chromatography method previously developed for the separation and quantitative determination of the hemagglutinin content in trivalent influenza vaccine preparations was further extended and found to be adaptable for the assessment of all four hemagglutinin antigens present in quadrivalent influenza vaccines. Vaccines prepared from monovalent bulks and commercial quadrivalent products from the past three vaccination seasons in the Northern Hemisphere were tested with the new method. The results showed excellent resolution of all four hemagglutinins from frequently interfering formulation agents such as surfactants. This method provides a simple approach for fast evaluation of quality and hemagglutinin strain identification in influenza vaccines. It is also the only physicochemical method capable of distinguishing the B strains in quadrivalent influenza vaccines.

The interaction mechanisms of a series of bisabolonalone hydrazone carboxamides as H+,K+-ATPase reversible inhibitors were studied by molecular docking, QM/MM calculation, and MM/GBSA binding free energy analysis methods. The correlation coefficient, R, between calculated ΔG bind (the binding free energy with H+,K+-ATPase homology model) and experimental pIC50 (negative logarithms of 50% inhibition concentration) of ligands is −0.9386. A suitable molecular volume of ligand is needed to dock into the whole binding pocket, which includes Gln127–Leu141 in the loop of TM1–TM2; Arg328–Ala335 in the loop of TM4; Tyr799–Thr815 in the loop of TM5–TM6; Trp899–Phe917 in the loop of TM7–TM8; Leu921–Tyr928 in the loop of TM8; and Asn986–Asn989 in the loop of TM9–TM10. By the decomposed energy comparisons of residues in binding sites, the naphthyl group substituting benzene ring in bisabolonalone hydrazone carboxamides could increase the hydrophobic interaction with Cys813, which is a very important binding site for enhancing the inhibition activity. In addition, Asp137 is another key residue binding with ligand through the hydrogen bond and electrostatic interactions. The results of this analysis could help in further rational design of novel H+,K+-ATPase reversible inhibitors.

To develop a new one-pot polyphosphate kinase (PPK) system with low cost and high efficiency for ATP regeneration in industrial CTP production. We developed a new one-pot PPK system by applying a three-enzyme cascade (CMK, NDK and PPK) with an in vitro polyP-based ATP regeneration system. The PPK was selected from twenty sources, and was made solvable by fusion expressing with soluble protein and constructing polycistronic plasmids, or co-expressing with molecular chaperones GroES/EL. Activities of other enzymes were optimized by employing fusion expression, tac-pBAD system, Rosetta host and codon optimization. After 24 h, the concentration of CDP and CTP reached 3.8 ± 0.2 and 6.9 ± 0.3 mM l−1 respectively with a yield of approximately 79%. The molar conversion rate of CTP was 51%, and its yield and conversion rate increased 100% from the traditional system. A new one-pot ATP regeneration system applying polyphosphate kinase for CTP production was developed.

Excessive activation of macrophages has been implicated in various types of inflammatory injury. Suppression of macrophage activation would have therapeutic benefits, leading to the alleviation of the progression of inflammatory diseases. Eburicoic acid (EA) is one of main bioactive components isolated from Laetiporus sulphureus (Bull.:Fr.) Murrill. In our previous study, we found that EA possessed anti-inflammatory activities. However, the cellular and molecular mechanisms underlying its anti-inflammatory activities remain to be poorly understood. The present study aimed to further evaluate its effect on lipopolysaccharide (LPS)-induced inflammatory responses in RAW264.7 macrophage cells. We investigated the anti-inflammatory effect by modulating LPS-induced activation of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR)/nuclear transcription factor-κB (NF-κB) pathway in RAW264.7 cells. The results showed that EA caused no obvious cytotoxicity, and its suitable concentrations on RAW264.7 cells were in the range from 0.02 to 0.08 μM. EA significantly inhibited the releases of inflammatory mediators, nitrite oxide (NO) and prostaglandin E2 (PGE2); suppressed mRNA and protein expression levels of inducible nitrite oxide synthase (iNOS) and cyclooxygenase-2 COX-2 and pro-inflammatory cytokine TNF-α, IL-6, and IL-1β; and reduced levels of phosphorylated PI3K, Akt, mTOR, and NF-κBp65 in LPS-induced RAW264.7 cells in dose- and time-dependent manners. These aforementioned results indicated that EA executed anti-inflammatory effect on LPS-induced RAW264.7 cells, and this effect might be achieved via suppressing the PI3K/Akt/mTOR/NF-κB signaling pathway and inhibiting the LPS-induced productions of inflammatory mediators and pro-inflammatory cytokines.

Phytoestrogen has previously been proposed as an alternative to hormone replacement therapy. Hispolon has been found to have phytoestrogenic properties. However, the possible effects of hispolon on estrogen receptors and other related molecules remain to be determined. This study was performed mainly to confirm the estrogenic function of hispolon as it relates to estrogen receptors, aromatase, and cyclooxygenase 2 (COX-2). Hispolon was shown to increase the serum 17β-estradiol in vivo. Immunohistochemical staining methods showed that hispolon exhibited a biphasic effect on ERα/β and aromatase expression in MCF-7 cells. Hispolon could also significantly inhibit aromatase activity, assessed using the enzyme-linked immunosorbent assay. Western blotting showed that COX-2 and aromatase could be inhibited by hispolon. These results further prove the phytoestrogenic features of hispolon and explore some pharmacological mechanisms that suggest that hispolon could be useful in the treatment of breast cancers or other gynecologic diseases.

ObjectivesOur previous studies indicated that the triterpenes from the fruits of Chaenomeles speciosa (Sweet) Nakai (TCS) owned effectively therapeutic effects on gastric ulcer patients and animals, but its mechanisms have not been fully understood. The current study was to further investigate its protective effect on indomethacin (IND)-damaged RGM-1 cells and rats, as well as its mechanisms involved.MethodsThe gastroprotection of TCS was evaluated with IND-induced gastric lesions model in RGM-1 cells and rats. In vitro, the proliferation, migration, mitochondrial viability and apoptosis were assessed. In vivo, ulcer index, ulcer inhibition rate, gastric juice acidity, gastric wall mucus (GWM) and histopathology of gastric mucosa were detected. The gastroprotective effects of TCS through the TFF1-mediated EGF/EGFR and apoptotic pathways were measured by qRT-PCR and Western blot assays.Key findingsThe results demonstrated that TCS had gastroprotective function, which was related to the amelioration in promoting IND-damaged RGM-1 cell proliferation and migration, hoisting gastric juice acidity and GWM, improving ulcer index and ulcer inhibition rate, attenuating the haemorrhage, oedema, epithelial cell loss and inflammatory cell infiltration of gastric mucosa, upregulating PCNA, Bcl-2, Bcl-xl mRNA and TFF1, EGF, p-EGFR, p-Src, pro-caspase-3, pro-caspase-9 protein expressions, mitochondrial viability, mitochondrial cytochrome c concentration and p-EGFR/EGFR, p-Src/Src, Bcl-2/Bax, Bcl-xl/Bad ratioes, downregulating Bax, Bad, Apaf-1 mRNA and cleaved-caspase-3, cleaved-caspase-9, cleaved PARP-1 protein expressions and cytosol cytochrome c concentration.ConclusionsOur present study demonstrated that TCS’s gastroprotective effect was closely connected with boosting TFF1 expression, activating TFF1-mediated EGF/EGFR pathway, thus restraining mitochondrial-dependent apoptosis, which provided new insights into interpreting its underlying mechanism and promised to act as a candidate drug to treat gastric mucosal injury.

To investigate the pharmacokinetics of PEGylated recombinant human endostatin (M2ES) in rhesus monkey. M2ES was administered to rhesus monkeys by intravenous bolus injection, and serum M2ES concentration was determined by a self-developed ELISA assay. Pharmacokinetic parameters were calculated using a non-compartmental model of WinNonlin V2.1A software. The standard curve of self-developed ELISA assay was fitted by four-parameter method. The limit of detection (LOD) and LOQ were 0.3050 ng/mL and 0.9140 ng/mL, respectively. Following IV infusions of M2ES at 0.3, 1, and 3 mg/kg in rhesus monkeys, the serum M2ES concentration-time curve was fitted with a non-compartment model. The pharmacokinetic parameters were evaluated as follows: Terminal elimination half-life (T1/2) of M2ES were 3.30 ± 0.70, 29.50 ± 18.80 and 24.60 ± 8.90 h. Systemic clearance (CLsys) of M2ES were 339.60 ± 66.30, 161.40 ± 18.20 and 260.10 ± 43.70 mL/h/kg. AUC(0-∞) values of M2ES were 909.30 ± 199.60, 6251.00 ± 739.60 and 11758.00 ± 2010.10 ng∙h/mL. The dosage was positively correlated with AUC, and the correlation coefficient r2 = 0.9327. Self-developed ELISA assay could meet the requirements of serum M2ES concentration detection. PEGylation modification substantially expands the circulation time of recombinant human endostatin and effectively improves its pharmacokinetic behavior.

To develop fusion protein of a GnRH Fc fragment and the integrin targeting AP25 antitumor peptide for GnRH receptor-expressing cancer therapy. The LMRAP fusion protein was constructed. A transwell invasion assay was performed. The gene mRNA and protein levels of GnRHR-I, α5β1, and αvβ3 in different cancer cell lines were assessed. Cell proliferation was measured using a cell counting kit-8. An antagonist assay was performed on GnRH receptors. Anti-tumor activity was evaluated with a mouse xenograft tumor model. Immunohistochemistry (IHC) was applied to detect CD31 and CD34 expressions. Pharmacokinetic characteristics were determined with an indirect competition ELISA. The developed bifunctional fusion protein LMRAP not only inhibited HUVEC invasion, but also inhibited proliferation of GnRHR-I, α5β1, and αvβ3 high expression cancer cells. The IC50 for LMRAP in the GnRH receptor was 6.235 × 10−4 mol/L. LMRAP significantly inhibited human prostate cancer cell line 22RV1 proliferation in vivo and in vitro. LMRAP significantly inhibited CD31 and CD34 expressions. The elimination half-life of the fusion protein LMRAP was 33 h in rats. The fusion protein made of a GnRH Fc fragment and the integrin targeting AP25 peptide retained the bifunctional biological activity of GnRHR blocking, angiogenesis inhibition, prolonged half-life and good tolerance., Graphical abstract LMRAP fusion protein, constructed by GnRH Fc fragment and the integrin targeting AP25 antitumor peptide, not only retained the bifunctional activity of GnRH receptor blocking and angiogenesis inhibition, but also prolonged the half-life. This study provides clues for developing a new strategy for GnRH receptor-expressing cancer treatment.Image 1

Trametenolic acid B (TAB) was a lanostane-type triterpenoid isolated from the trametes lactinea (Berk.) Pat. We have previously reported that extract from trametes lactinea (Berk.) Pat and TAB could efficiently improve learning and memory ability of the cerebral ischemia injury rats and suppress mitochondrial-mediated apoptosis in hydrogen peroxide damaged SH-SY5Y cells. However, the potential mechanisms have not been fully understood yet. The current study was to further investigate the protective effect of TAB on oxygen glucose deprivation/reoxygenation (OGD/R)-damaged SH-SY5Y cells and cerebral ischemia/reperfusion (I/R) injury rats, as well as its mechanisms involved. Cell experiments demonstrated that TAB (10, 20 and 40 μg/mL) protected OGD/R-induced SH-SY5Y cell injury by promoting cell proliferation and suppressing LDH leakage; Meanwhile, the results in vivo showed that TAB (20, 40 and 80 mg/kg) might significantly ameliorate the neurological deficit score, cerebral edema, neuronal cell loss and apoptosis, suppress cerebral infarction volume of the cerebral I/R injury rats. Further studies in vitro and in vivo indicated TAB could efficiently reduce OGD/R-damaged SH-SY5Y cell and cerebral I/R rat serum ROS, LDH and MDA levels, elevate SOD, GSH-Px and CAT activities, downregulate miR-10a mRNA and Bax, cytochrome C, cleaved-caspase-3 and cleaved-caspase-9 protein expressions, upregulate p-PIK3CA, p-Akt, p-mTOR, Bcl-2, pro-caspase-9 and pro-caspase-3 protein expressions and p-PIK3CA/PIK3CA, p-Akt/Akt, p-mTOR/mTOR ratios (P

During the past two decades, recombinant human interleukin-12 (rhIL-12) has emerged as one of the most potent cytokines in mediating antitumor activity in a variety of preclinical models and clinical studies. Purity is a critical quality attribute (CQA) in the quality control system of rhIL-12. In our study, rhIL-12 bulks from manufacturer B showed a different pattern in non-reduced SDS-PAGE compared with size-exclusion chromatography (SEC)-HPLC. A small fragment was only detected in non-reduced SDS-PAGE but not in SEC-HPLC. The results of UPLC/MS and N-terminal sequencing confirmed that the small fragment was a 261&ndash, 306 amino acid sequence of a p40 subunit of IL-12. The cleavage occurs between Lys260 and Arg261, a basic rich region. With the presence of 0.2% SDS, the small fragment appeared in both native PAGE and in SEC-HPLC, suggesting that it is bound to the remaining part of the IL-12 non-covalently, and is dissociated in a denatured environment. The results of a bioassay showed that the fractured rhIL-12 proteins had deficient biological activity. These findings provide an important reference for the quality control of the production process and the final products of rhIL-12.

Leukocyte immunoglobulin (Ig)-like receptors (LILRs), also known as CD85 and immunoglobulin-like transcripts (ILTs), play pivotal roles in regulating immune responses. These receptors define an immune checkpoint that immune therapy can target. Through cis or trans interactions with human leukocyte antigen (HLA)-G, the two most abundantly expressed inhibitory LILRs, LILRB1, and LILRB2 (LILRB1/2, also known as CD85j/d and ILT2/4), are involved in immunotolerance in pregnancy and transplantation, autoimmune diseases, and immune evasion by tumors. Although the discrete domains of LILRB1/2 are clear, the assembly mode of the four extracellular Ig-like domains (D1, D2, D3, and D4) remains unknown. Previous data indicate that D1D2 is responsible for binding to HLA class I (HLA-I), but the roles of D3D4 are still unclear. Here, we determined the crystal structure of the four Ig-like domain LILRB2 and four-domain LILRB1 in complex with HLA-G1. The angles between adjacent domains and the staggered assembly of the four domains suggest limited flexibility and limited plasticity of the receptors during ligand binding. The complex structure of four-domain LILRB1 and HLA-G1 supports the model that D1D2 is responsible for HLA-I binding, while D3D4 acts as a scaffold. Accordingly, cis and trans binding models for HLA-I binding to LILRB1/2 are proposed. The geometries of LILRB1/2 in complex with dimeric and monomeric HLA-G1 suggest the accessibility of the dimeric receptor, which in turn, transduces more inhibitory signals. The assembly of LILRB1/2 and its binding to HLA-G1 could aid in the design of immune regulators and benefit immune interference.

Bioassay of recombinant protein products is important tests to ensure protein effectiveness. Some recombinant protein products have no cells used in their bioassay but instead use animal models, while others have no suitable method. Here, we developed a method to obtain responsive cells used in bioassay of proteins. After screening of a CRISPR/Cas9 library, we obtained a responsive cell line that grew faster in the presence of rhEGF (recombinant human epidermal growth factor) than that of control cells. We used this cell line for bioassay of rhEGF. This cell line, compared with the control cells, had a 2 day shorter operation time and had lower interference. The responsive cell line is more suitable for use in bioassay of rhEGF.