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Recombinant proteins are gaining increasing popularity for treating human diseases. The clinical effectiveness of recombinant proteins is directly related to their biological activity, which is an important indicator in drug development and quality control. However, certain recombinant proteins have unclear or complex signal pathways, making detecting their activity in vitro difficult. For instance, recombinant human endostatin (endostatin), a new antitumor drug developed in China, lacks a sensitive and stable assay for its biological activity since being market approval. To address this issue, we performed a genome‐wide screening of immortalized human umbilical vein endothelial cells (HUVECs) using a CRISPR/Cas9 knockout library containing 20,000 targeted genes. We identified two potential endostatin‐resistant genes, NEPSPP and UTS2, and successfully constructed a highly sensitive cell line, HUVEC‐UTS2‐3#, by knocking down the UTS2 gene. Based on the optimized parameters of HUVEC‐UTS2‐3# cells, we established a new method for detecting the biological activity of endostatin. The method was validated, and it produced results consistent with primary HUVEC cells but with higher sensitivity and more stable data. The use of gene‐editing technology provides a novel solution for detecting the biological activity of recombinant proteins that other methods cannot detect. [ABSTRACT FROM AUTHOR]

Hepatitis-hydropericardium syndrome (HHS), caused by fowl adenovirus serotype 4 (FAdV-4), has been widely spread across China, resulting in great financial losses in the poultry industry. Therefore, efficient vaccines against this disease urgently need to be developed. In our study, the fiber-2 and penton base proteins derived from the FAdV-4 JS strain were expressed in a prokaryotic system (E. coli) in a soluble form. Then, the efficacy of the two recombinant proteins formulated with cheap and widely used adjuvants (Marcol™ 52 white oil) were respectively tested, and the minimum immune doses and safety of the above proteins were also determined. It was indicated that the fiber-2 (20 µg/bird, 200 µg/bird) and penton base (200 µg/bird) could provide complete protection against the highly pathogenic FAdV-4 and suppress its replication and shedding. Unfortunately, only the fiber-2 protein could induce complete protection (10/10) at a low dose (10 µg/bird). In addition, we confirmed that the fiber-2 subunit vaccine formulated with oil adjuvants was safe for vaccinated chickens. Conclusively, all of our results suggest that we successfully prepared an efficient and cheap fiber-2 subunit vaccine with few side effects. [ABSTRACT FROM AUTHOR]

Pseudorabies virus (PRV) is an acute infectious disease characterized by neurological and respiratory symptoms. In order to have a better understanding of the current prevalence of PRV in northeastern China, a strain of PRV was isolated by us. Then, protein structure analysis and pathogenicity testing of the virus were performed to give insight into the characterization of the isolated PRV strains. In this study, the PRV strain named CH/HLJPRVJ/2023 was isolated and identified. Genome-wide phylogenetic analysis shows CH/HLJPRVJ/2023 and HeN1 have higher homology. The CH/HLJPRVJ/2023 strain had the highest homology with HeN1 strain (97.3%) and the lowest homology with Bartha-K61 (89.2%). Recombinant evolution analysis shows CH/HLJPRVJ/2023 shows many variants in OBP, AN, UL21, UL17, VP11/12, and VP1/2 fragments, which predict its unique genetically. VP1/2, an effector protein of capsid transport and neuroinvasion, has mutations and deletions in its amino acids, which cause changes in the protein conformation of CH/HLJPRVJ/2023. Besides the typical neurologic and respiratory lesions, infection with highly pathogenic CH/HLJPRVJ/2023 can lead to damage to the colonic villi and colonic barrier in piglets. This study will provide a basis for knowledge about the prevalence, genetic evolution, and vaccine optimization of endemic PRV strains in northeastern China. [ABSTRACT FROM AUTHOR]

Simple Summary: This study developed an indirect ELISA (GMD-ELISA) to detect the Streptococcus suis (S. suis) antibody. The antigen—antibody response was optimized using checkerboard titration. This method has strong specificity and can detect the main epidemic strains of S. suis in China—type 2, type 7, and type 9—compared with using the commercial Streptococcus suis ELISA type 2 kit. The GMD-ELISA method has high sensitivity and good repeatability. The novel GMD-ELISA method provides technical support for rapid diagnosis and epidemiological investigation. S. suis is an important zoonotic pathogen from sick and recessive carrier pigs that poses a serious threat to animal husbandry production and public health. It usually causes horizontal transmission among pigs. The morbidity and mortality of this disease are very high. Human infection is caused through direct or indirect contact with sick pigs. The two large-scale outbreaks in China were due to the outbreak of S. suis on pig farms, which spread to human infection; thus, detecting S. suis in pig herds is crucial. At present, the commercial S. suis ELISA type 2 kits on the market can only detect single serotypes, high probabilities of interaction reactions, and biosafety risks when using inactivated S. suis as an antigen. Phosphate-3-glyceraldehyde dehydrogenase (GAPDH), muramidase-released protein (MRP), and dihydrolipoamide dehydrogenase (DLDH) are important S. suis type 2, S. suis type 7, and S. suis type 9 protective antigens. This study purified the GMD protein (B-cell-dominant epitopes of GAPDH, MRP, and DLDH antigens) and used a diverse combination of dominant epitopes of the multiple different antigens as coated antigens, improving the sensitivity and safety of the indirect ELISA experiments. An indirect ELISA method (GMD-ELISA) was developed for detecting S. suis antibodies. The antigen—antibody response was optimized using checkerboard titration. The results of testing using ELISA for Salmonella enterica (S. enterica), Escherichia coli (E. coli), Staphylococcus aureus (SA), and Streptococcus pyogenes (S. pyogenes) were all negative, indicating that this method had strong specificity. The results were still positive when the dilution ratio of S. suis-positive serum reached 1:6, 400, thus indicating that the method had high sensitivity. The results of the reproducibility assay for indirect ELISA showed that the intra-assay coefficient of variation and the inter-assay coefficient of variation were less than 10%, indicating that the method had good repeatability. We investigated the seroprevalence of S. suis in 167 serum samples collected in East China, and 33.5% of the samples were positive for antibodies against S. suis, indicating that the prevalence of S. suis is high in pig farms in Eastern China. The novel GMD-ELISA is a convenient, sensitive, and specific diagnostic method that provides technical support for rapid diagnosis and epidemiological investigation. [ABSTRACT FROM AUTHOR]

Simple Summary: The pink bollworm, Pectinophora gossypiella, is one of the most damaging pests of cotton worldwide. Cotton has been genetically engineered to produce insect-killing proteins from the bacterium Bacillus thuringiensis (Bt) to control major lepidopteran pests, including the pink bollworm. The Bt proteins in genetically engineered crops are not toxic to people, other vertebrates, or most beneficial insects. Advantages of Bt crops can include pest suppression, improved yields, increased farmer profits, and decreased use of conventional insecticides. In the United States, Bt cotton, sterile moth releases, and other tactics were used to eradicate the pink bollworm. For more than 20 years, Bt cotton has been effective against pink bollworm in China. However, the benefits of Bt crops are reduced when pests evolve resistance, as exemplified by pink bollworm resistance to Bt cotton in India. For each of the two Bt proteins used widely in Bt cotton, the genetic basis of resistance is similar between resistance selected in the lab versus the field, regardless of the country of origin. The results suggest that lab selection can be useful for identifying genes likely to be important in field-evolved resistance to Bt crops and that differences in management practices among countries caused different outcomes. Transgenic crops producing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) control some important insect pests. However, evolution of resistance by pests reduces the efficacy of Bt crops. Here we review resistance to Bt cotton in the pink bollworm, Pectinophora gossypiella, one of the world's most damaging pests of cotton. Field outcomes with Bt cotton and pink bollworm during the past quarter century differ markedly among the world's top three cotton-producing countries: practical resistance in India, sustained susceptibility in China, and eradication of this invasive lepidopteran pest from the United States achieved with Bt cotton and other tactics. We compared the molecular genetic basis of pink bollworm resistance between lab-selected strains from the U.S. and China and field-selected populations from India for two Bt proteins (Cry1Ac and Cry2Ab) produced in widely adopted Bt cotton. Both lab- and field-selected resistance are associated with mutations affecting the cadherin protein PgCad1 for Cry1Ac and the ATP-binding cassette transporter protein PgABCA2 for Cry2Ab. The results imply lab selection is useful for identifying genes important in field-evolved resistance to Bt crops, but not necessarily the specific mutations in those genes. The results also suggest that differences in management practices, rather than genetic constraints, caused the strikingly different outcomes among countries. [ABSTRACT FROM AUTHOR]

Background: To evaluate the effectiveness of individualized-dose polyethylene glycol recombinant human growth hormone (PEG-rhGH) for short stature. Methods: This real-world study enrolled children with short stature in 19 hospitals throughout China. They were treated with PEG-rhGH for 6 months. The starting dosage ranged from 0.10 to 0.20 mg/kg/week. The primary outcome was the change in height standard deviation score (ΔHt SDS). Results: Five hundred and ten patients were included and grouped based on dosage as A (0.10–0.14 mg/kg/week), B (0.15–0.16 mg/kg/week), C (0.17–0.19 mg/kg/week), and D (0.20 mg/kg/week). The mean 6-month ΔHt SDS for the total cohort was 0.49 ± 0.27, and the means differed among the four dose groups (P = 0.002). The ΔHt SDS was lower in group A than in groups B (LSM difference [95%CI], -0.09 [-0.17, -0.01]), C (LSM difference [95%CI], -0.10 [-0.18, -0.02]), and D (LSM difference [95%CI], -0.13 [-0.21, -0.05]) after adjusting baseline covariates. There were no significant differences among groups B, C, and D. When the baseline IGF-1 was < -2 SDS or > 0 SDS, the △Ht SDS was not different among the four groups (P = 0.931 and P = 0.400). In children with baseline IGF-1 SDS of -2 ~ 0 SDS, a higher dosage was associated with a better treatment effect (P = 0.003), and the △Ht SDS was lower in older children than in younger ones (P < 0.001). Conclusions: PEG-rhGH could effectively increase height in prepubertal short children. When the baseline IGF-1 was < -2 SDS, 0.10 mg/kg/week could be a starting dose. In other IGF-1 statuses, 0.15–0.20 mg/kg/week might be preferred. Trial registration: ClinicalTrials.gov: NCT03249480, retrospectively registered. [ABSTRACT FROM AUTHOR]

Objective: Allergic disease caused by airborne pollen is a major health problem in China. Intensive study on pollen allergens can be of great help for preventing and treating pollinosis. Four aspects of the study on pollen allergens in China including major allergic pollen in our country, analysis and purification of pollen allergen composition, recombinant pollen allergens and clinical application of pollen allergens are described in this paper. [Copyright &y& Elsevier]

Odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) play a fundamental role in insect olfaction. Galeruca daurica (Joannis) is a new pest with outbreak status in the Inner Mongolia grasslands, northern China. In this study, six olfactory protein genes (GdauOBP1 , GdauOBP6 , GdauOBP10 , GdauOBP15 , GdauCSP4 , and GdauCSP5) were cloned by RACE and expressed by constructing a prokaryotic expression system. Their binding affinities to 13 compounds from host volatiles (Allium mongolicum) were determined by fluorescence-binding assay. In order to further explore the olfactory functions of GdauOBP15 and GdauCSP5 , RNA interference (RNAi) and electroantennogram (EAG) experiments were conducted. Ligand-binding assays showed that the binding properties of the six recombinant proteins to the tested volatiles were different. GdauOBP6, GdauOBP15, GdauCSP4, and GdauCSP5 could bind several tested ligands of host plants. It was suspected that GdauOBP6, GdauOBP15, GdauCSP4, and GdauCSP5 were related to the host location in G. daurica. We also found that there were different EAG responses between males and females when the GdauOBP15 and GdauCSP5 genes were silenced by RNAi. The EAG response of G. daurica females to 2-hexenal was significantly decreased in dsRNA-OBP15-injected treatment compared to the control, and the dsRNA-CSP5-treated females significantly reduced EAG response to eight tested host volatiles (1,3-dithiane, 2-hexenal, methyl benzoate, dimethyl trisulfide, myrcene, hexanal, 1,3,5-cycloheptatriene, and p-xylene). However, the EAG response had no significant difference in males. Both GdauOBP15 and GdauCSP5 may have different functions between males and females in G. daurica and may play more important roles in females searching for host plants. [ABSTRACT FROM AUTHOR]

Hyperthermostable proteins are highly resistant to various extreme conditions. Many factors have been proposed to contribute to their ultrahigh structural stability. Some thermostable proteins have larger oligomeric size when compared to their mesophilic homologues. The formation of compact oligomers can minimize the solvent accessible surface area and increase the changes of Gibbs free energy for unfolding. Similar to mesophilic proteins, hyperthermostable proteins also face the problem of unproductive aggregation. In this research, we investigated the role of high-order oligomerization in the fight against aggregation by a hyperthermostable superoxide dismutase identified from Tengchong, China (tcSOD). Besides the predominant tetramers, tcSOD could also form active high-order oligomers containing at least eight subunits. The dynamic equilibrium between tetramers and high-order oligomers was not significantly affected by pH, salt concentration or moderate temperature. The secondary and tertiary structures of tcSOD remained unchanged during heating, while cross-linking experiments showed that there were conformational changes or structural fluctuations at high temperatures. Mutational analysis indicated that the last helix at the C-terminus was involved in the formation of high-order oligomers, probably via domain swapping. Based on these results, we proposed that the reversible conversion between the active tetramers and high-order oligomers might provide a buffering system for tcSOD to fight against the irreversible protein aggregation pathway. The formation of active high-order oligomers not only increases the energy barrier between the native state and unfolded/aggregated state, but also provides the enzyme the ability to reproduce the predominant oligomers from the active high-order oligomers. [ABSTRACT FROM AUTHOR]