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Recombinant adeno-associated virus (rAAV) has emerged as a prominent vector for in vivo gene therapy, owing to its distinct advantages. Accurate determination of the rAAV genome titer is crucial for ensuring the safe and effective administration of clinical doses. The evolution of the rAAV genome titer assay from quantitative PCR (qPCR) to digital PCR (dPCR) has enhanced accuracy and precision, yet practical challenges persist. This study systematically investigated the impact of various operational factors on genome titration in a single-factor manner, aiming to address potential sources of variability in the quantitative determination process. Our findings revealed that a pretreatment procedure without genome extraction exhibits superior precision compared with titration with genome extraction. Additionally, notable variations in titration results across different brands of dPCR instruments were documented, with relative standard deviation (RSD) reaching 23.47% for AAV5 and 11.57% for AAV8. Notably, optimal operations about DNase I digestion were identified; we thought treatment time exceeding 30 min was necessary, and there was no need for thermal inactivation after digestion. And we highlighted that thermal capsid disruption before serial dilution substantially affected AAV genome titers, causing a greater than ten-fold decrease. Conversely, this study found that additive components of dilution buffer are not significant contributors to titration variations. Furthermore, we found that repeated freeze–thaw cycles significantly compromised AAV genome titers. In conclusion, a comprehensive dPCR titration protocol, incorporating insights from these impact factors, was proposed and successfully tested across multiple serotypes of AAV. The results demonstrate acceptable variations, with the RSD consistently below 5.00% for all tested AAV samples. This study provides valuable insights to reduce variability and improve the reproducibility of AAV genome titration using dPCR.

Of all of the components in SARS-CoV-2 inactivated vaccines, nucleocapsid protein (N) is the most abundant and highly conserved protein. However, the function of N in these vaccines, especially its influence on the targeted spike protein’s response, remains unknown. In this study, the immunization of mice with the N protein alone was shown to reduce the viral load, alleviating pulmonary pathological lesions after challenge with the SARS-CoV-2 virus. In addition, co-immunization and pre-immunization with N were found to induce higher S-specific antibody titers rather than compromise them. Remarkably, the same trend was also observed when N was administered as the booster dose after whole inactivated virus vaccination. N-specific IFN-γ-secreting T cell response was detected in all groups and exhibited a certain relationship with S-specific IgG antibody improvements. Together, these data indicate that N has an independent role in vaccine-induced protection and improves the S-specific antibody response to inactivated vaccines, revealing that an interplay mechanism may exist in the immune responses to complex virus components.

Aquifer diffusivity is a basic physical parameter used in hydrogeological calculations and is important for the evaluation and rational utilization of water resources, pollution prevention, and wetland protection. In this study, with the assumptions of aquifer isotropy, i.e., no vertical flow and constant saturated aquifer thickness, by using the Fourier transform and convolution theorem, a 1D analytical solution of the phase spectrum for an unconfined aquifer system was derived, which subsequently led to a phase spectrum solution for aquifer diffusivity. To test the efficacy, the proposed method was applied to a study site in the middle reach of the Hutuo River in the North China Plain. The estimated aquifer diffusivity ranged from 1.9 × 103 to 4.9 × 104 m2/d, with a mean of 2.2 × 104 m2/d, which was consistent with the results obtained using power spectral analysis, pumping tests, and inverse numerical models. The phase spectral approach proposed in this paper can estimate the aquifer properties on a larger scale. If long time series of hydraulic heads are available, it can estimate hydrogeological parameters accurately and quickly. Considering the similarity of the linearized governing equations, it can also be applied to the river–aquifer system and the confined aquifer system.

NaOH, dolomite and NiCl2 were used as catalysts to examine their effects on co–pyrolysis with waste bicycle tires (WT) and waste engine oil (WEO). The pyrolysis behaviors with catalysts were investigated by thermogravimetric analysis. The activation energy of the catalytic main reaction stage was derived by the Kissinger–Akahira–Sunose (KAS) method under four different heating rates conditions. The calculations show that all three catalysts can reduce the activation energy of the reaction. Co–pyrolysis of WT and WEO with different catalysts was performed in a self–made lab bench at 600 °C to explore the impact on the distribution of three–phase products. The properties of gas and oil products were characterized by FTIR and Py–GC/MS (Agilent 7890B, Santa Clara, CA, USA). With the mixing of catalysts, activation energy (Eα) decreased by 15–30% in the main reaction process. NaOH and dolomite increased the yield of gas by 7% and 10%. NaOH can significantly improve the yield of CH4. The proportion of limonene in pyrolysis oil increased to 19.65% with 10% NaOH. This article provides a new method for efficiently producing limonene by mixing WT and WEO with NaOH.

Karst leakage is the key problem that restricts the construction of reservoir areas. In this article, the hydrogeochemical origin and hydraulic connection of the river water, pore water, fissure water, and karst water in Jinghe Dongzhuang Reservoir, which is located in a karst area, are analyzed to determine the possibility of karst leakage in the reservoir area. Piper diagram, Gibbs diagram, ion proportion coefficient, and cluster analysis were comprehensively used to systematically study the hydrogeochemical characteristics and formation mechanism of the study area. The research results show that the water in the study area is weakly alkaline, with complex hydrogeochemical types, including SO4−Na, HCO3·SO4−Na, and HCO3·SO4−Na·Mg. Affected by evaporation and concentration, Jinghe River and shallow pore water have high TDS content, and the content of Na+(including K+), Cl− and SO42− is significantly higher than that of fissure water and karst water. Fissure water and karst water are significantly weathered by rocks, and their Ca2+ and Mg2+ mainly come from carbonate rock dissolution. In the process of groundwater evolution, cation exchange occurs more or less in the three groundwater bodies, resulting in different cation contents in different water bodies. In general, Jinghe River is similar to most of the pore water, but its hydrogeochemical characteristics are obviously different from those of fissure water and karst water, so it has little hydraulic connection with fissure water and karst water, indicating that the leakage in the reservoir area is not significant.

Landscape connectivity refers to the degree of continuity between the spatially structured units of a landscape. Ecological connectivity can characterise the degree to which ecological functional areas are connected in terms of function and ecological processes. In this study, the landscape pattern index and ecosystem service values were used to evaluate the ecological functional resistance of each landscape type, taking the Three Parallel Rivers Natural World Heritage Site as an example and the habitat distribution and population size of the Yunnan snub-nosed monkey as a reference. The minimum cost distance model, combined with the barrier impact index (BEI) and ecological connectivity index (ECI), was used to determine the degree of barrier impact on the study area and the ecological connectivity of the core reserve of the heritage site in both 2000 and 2020. The resistances of the different land types and landscape heterogeneity to the ecological function of species migration between the core protected areas of the heritage site were, in descending order, those of the forest, shrubs and grass, water, unused land, cultivated land, and built-up land. In 2020, the study area had a large BEI, with areas such as built-up areas, major roads, the sides of large rivers, and arable land being significant contributors to the blockage of landscape connectivity. The overall landscape connectivity in the study area was generally low, with clear spatial differentiation and a three-column parallel distribution pattern influenced by the topography and landscape. With the adjustment of the core reserve boundaries of the heritage site, the proportion of areas with high connectivity (ECI = 4–5) increased from 11.31% in 2000 to 34.36% in 2020. This increased landscape connectivity was conducive to the migration and reproduction of large terrestrial animals, such as the Yunnan snub-nosed monkey, with increasing numbers of populations and individuals. This study provides theoretical and methodological insights into the delineation and conservation of natural heritage sites and landscape connectivity.

Alopecurus japonicus has been a serious weed across China and developed resistance to the acetyl-CoA carboxylase (ACCase)-inhibiting herbicide. The A. japonicus ACCase gene mutations accounting for target-site resistance (TSR) have been clarified, while non-target-site resistance (NTSR) is not distinct. Here, dose–response testing indicated that an A. japonicus population (R) was resistant to four ACCase-inhibiting herbicides fenoxaprop-P-ethyl, sethoxydim, clethodim, and pinoxaden. Compared with herbicide-susceptible A. japonicus (S), no known resistant mutations for TSR in ACCase were identified in the R population using sequencing. To investigate the NTSR mechanism, increased metabolism of fenoxaprop-P-ethyl was detected in the R population using high-performance liquid chromatography (HPLC) analysis. Notably, resistance cannot be reversed by P450 and GST inhibitors. RNA-seq was performed to further explore the resistance mechanisms, and eight candidate contigs (four glycosyl transferases (GT) and four ATP-binding cassette (ABC) transporters) were chosen and their expression patterns were validated using RT-qPCR. Three GT and three ABC transporter contigs were constitutively upregulated in the R population. In short, six contigs expressed highly in the R population causing enhanced fenoxaprop-P-ethyl metabolism appear to be involved in fenoxaprop-P-ethyl resistance.

Real-time matching of millimeter wave (mmWave) narrow beams is a great challenge in dynamic vehicle to infrastructure (V2I) environments due to the mobility of vehicles. In this paper, a novel beam search strategy based on spectrum-environment awareness is proposed. Combining the technique of label iterative optimization with three-dimensional (3D) grid encoding, the strategy treats the optimal beam pair indexes (BPIs) as labels and encodes the environments as features. Three-dimensional grid encoding is a symmetry-based environmental coding technology. A new Convolutional Neural Network (CNN) model is also constructed, which is trained by the features. The situational beam search of actual vehicles is performed under the trained CNN model. As a result, real-time mmWave narrow beam matching can be achieved. Simulation results demonstrate that the proposed strategy can effectively reduce the beam search overhead and improve the efficiency while guaranteeing the matching accuracy.

Soluble glycoprotein 130 kDa (sgp130)-Fc fusion protein, an innovative therapeutic bio-macromolecular drug specifically targeting IL-6 trans-signaling, proved to have good potential for application in the treatment of chronic inflammatory diseases. A simple and quick bioassay for sgp130-Fc was developed in this study. First, a stable reporter cell line was obtained by transfecting CHO-K1 cells with a sis-inducible element (SIE)-driving luciferase reporter gene (CHO/SIE-Luc). Sgp130-Fc could inhibit the expression of luciferase induced by IL-6/sIL-6Rα complex, and the dose−response curve fitted the four-parameter logistic model, with 50% inhibitive concentration (IC50) being about 500 ng/mL and detection range between 40 and 5000 ng/mL. Both the intra-assay and inter-assay coefficient of variation (CV) were below 10.0%, and the accuracy estimates ranged from 94.1% to 106.2%. The assay indicated a good linearity (R² = 0.99) in the range of 50% to 150% of optimized initial concentration. No significant difference was found between the test results of new assay and BAF3/gp130 proliferation assay (unpaired t test, p = 0.4960, n = 6). The dose-response effect and copy number of the luciferase gene was basically unchanged after long-term culture (up to passage 60), demonstrating the stability of CHO/SIE-Luc cells. These results suggested that the new reporter assay was suited to routine potency determination of therapeutic sgp130-Fc.

Five new 2(5H)-furanone-type derivatives, pestalafuranones F–J (compounds 3–7), together with two known compounds, pestalafuranones A (1) and B (2), were isolated from the ethyl acetate extract from the fermentation broth of the endophytic fungus Nigrospora sp. BM-2 in a hypersaline medium. The structures of these metabolites were elucidated by EIMS, HREIMS and NMR spectroscopic data. Compounds 1–7 exhibited no cytotoxic activities against the MDA-MB-231 and Caski cancer cell lines.

The long-acting growth hormone (LAGH) is a promising alternative biopharmaceutical to treat growth hormone (GH) deficiency in children, and it was developed using a variety of technologies by several pharmaceutical companies. Most LAGH preparations, such as Fc fusion protein, are currently undergoing preclinical study and clinical trials. Accurate determination of bioactivity is critical for the efficacy of quality control systems of LAGH. The current in vivo rat weight gain assays used to determine the bioactivity of recombinant human GH (rhGH) in pharmacopoeias are time-consuming, expensive, and imprecise, and there are no recommended bioassays for LAGH bioactivity in pharmacopoeias. Therefore, we developed a cell-based bioassay for bioactivity determination of therapeutic long-acting Fc-fusion recombinant human growth hormone (rhGH-Fc) based on the luciferase reporter gene system, which is involved in the full-length human GH receptor (hGHR) and the SG (SIE and GAS) response element. The established bioassay was comprehensively validated according to the International Council for Harmonization (ICH) Q2 (R1) guidelines and the Chinese Pharmacopoeia, and is highly precise, time-saving, simple, and robust. The validated bioassay could be qualified for bioactivity determination during the research, development, and manufacture of rhGH-Fc, and other LAGH formulations.

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 261–306 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.

The therapeutic recombinant human keratinocyte growth factor 1 (rhKGF-1) was approved by the FDA for oral mucositis resulting from hematopoietic stem cell transplantation for hematological malignancies in 2004. However, no recommended bioassay for rhKGF-1 bioactivity has been recorded in the U.S. Pharmacopoeia. In this study, we developed an rhKGF-1-dependent bioassay for determining rhKGF-1 bioactivity based on HEK293 and HaCat cell lines that stably expressed the luciferase reporter driven by the serum response element (SRE) and human fibroblast growth factor receptor (FGFR2) IIIb. A good responsiveness to rhKGF-1 and rhKGF-2 shared by target HEK293/HaCat cell lines was demonstrated. Our stringent validation was completely focused on specificity, linearity, accuracy, precision, and robustness according to the International Council for Harmonization (ICH) Q2 (R1) guidelines, AAPS/FDA Bioanalytical Workshop and the Chinese Pharmacopoeia. We confirmed the reliability of the method in determining rhKGF bioactivity. The validated method is highly timesaving, sensitive, and simple, and is especially valuable for providing information for quality control during the manufacture, research, and development of therapeutic rhKGF.

60Co γ-ray irradiation-initiated reversible addition–fragmentation chain transfer (RAFT) polymerization at room temperature with 2-cyanoprop-2-yl 1-dithionaphthalate (CPDN) as the chain transfer agent was first applied to acrylonitrile (AN) polymerization, providing a “green” platform for preparing polyacrylonitrile (PAN)-based carbon fibers using an environment-friendly energy source. Various effects of dose rate, molar ratio of the monomer to the chain transfer agent, monomer concentration and reaction time on the AN polymerization behaviors were performed to improve the controllability of molecular the weight and molecular weight distribution of the obtained PAN. The feature of the controlled polymerization was proven by the first-order kinetics, linear increase of the molecular weight with the monomer conversion and a successful chain-extension experiment. The molecular weight and molecular weight distribution of PAN were characterized by size exclusion chromatography (SEC). 1H NMR and Matrix assisted laser desorption ionization/time of flight mass spectra (MALDI-TOF-MS) confirmed the chain-end functionality of PAN, which also was supported by the successful chain-extension experiments of original PANs with acrylonitrile and styrene as the second monomers respectively.