Regulation of the Degradation Properties of Tyrosinase-Catalyzed Crosslinking Silk Membranes for Superficial Wound Repair.

Appropriate biodegradability to meet the demands of wound repair is critical for superficial wound repair membrane applications. Tyrosinase-catalyzed crosslinking SF (c-SF) membranes were constructed and regulated the degradation behavior in this study. The crosslinking degree of the c-SF membranes could be adjusted by reaction ratios of tyrosinase against SF (TYR/SF). Upon reaching a TYR/SF ratio of 20/6000, the degree of crosslinking increased to 88.17 ± 0.20%, without obvious changes in the crystal structure. The degradation behavior was regulated by the TYR/SF ratio and the degradation environment. All c-SF membranes remained stable after immersion without collagenase but showed an adjustable degradation behavior in the presence of collagenase. As the TYR/SF ratio increased, the residual weights increased from 23.31 ± 1.35% to 60.12 ± 0.82% after 7 days of degradation, occurring with low increased amounts of ß-sheet structure and free amino acids. This work provides a new c-SF membrane with controllable rapid degradability and favorable cytocompatibility, which can help to meet requirements for biodegradable superficial wound repair membranes.

Bifunctional interfacial engineering enabled efficient and stable carbon-based CsPbIBr2 perovskite solar cells.

Carbon-based inorganic CsPbIBr2 perovskite solar cells (C-IPSC) have attracted widespread attention due to their low cost and excellent thermal stability. Unfortunately, due to the soft ion crystal nature of perovskite, inherent bulk defects and energy level mismatch at the CsPbIBr2/carbon interface limit the performance of the device. In this study, we introduced aromatic benzyltrimethylammonium chloride (BTACl) as a passivation layer to passivate the surface and grain boundaries of the CsPbIBr2 film. Due to the reduction of perovskite defects and better energy level arrangement, carrier recombination is effectively suppressed and hole extraction is improved. The champion device achieves a maximum power conversion efficiency (PCE) of 11.30% with reduces hysteresis and open circuit voltage loss. In addition, unencapsulated equipment exhibits excellent stability in ambient air.

LiNAC100 contributes to linalool biosynthesis by directly regulating LiLiS in Lilium 'Siberia'.

MAIN CONCLUSION: The transcription factor LiNAC100 has a novel function of regulating floral fragrance by directly regulating linalool synthase gene LiLiS. Lilium 'Siberia', an Oriental hybrid, is renowned as both a cut flower and garden plant, prized for its color and fragrance. The fragrance comprises volatile organic compounds (VOCs), primarily monoterpenes found in the plant. While the primary terpene synthases in Lilium 'Siberia' were identified, the transcriptional regulation of these terpene synthase (TPS) genes remains unclear. Thus, understanding the regulatory mechanisms of monoterpene biosynthesis is crucial for breeding flower fragrance, thereby improving ornamental and commercial values. In this study, we isolated a nuclear-localized LiNAC100 transcription factor from Lilium 'Siberia'. The virus-induced gene silencing (VIGS) of LiNAC100 was found to down-regulate the expression of linalool synthase gene (LiLiS) and significantly inhibit linalool synthesis. Conversely, transient overexpression of LiNAC100 produced opposite effects. Additionally, yeast one-hybrid and dual-luciferase assays confirmed that LiNAC100 directly activates LiLiS expression. Our findings reveal that LiNAC100 plays a key role in monoterpene biosynthesis in Lilium 'Siberia', promoting linalool synthesis through the activation of LiLiS expression. These results offer insights into the molecular mechanisms of terpene biosynthesis in Lilium 'Siberia' and open avenues for biotechnological enhancement of floral scent.

Small-Molecule Copper Chloride Modulating the Buried Interfaces of Perovskite Solar Cells.

In perovskite solar cells (PSCs), tin dioxide (SnO2) is a highly effective electron transport material. On the other hand, the low intrinsic conductivity of SnO2, the high trap-state density on the surface and bulk of SnO2, and inadequate interface contacts between SnO2 and perovskite significantly impact device performance. Herein, small-molecule copper(II) chloride (CuCl2) is introduced into the SnO2 dispersion, which inhibits the agglomeration of SnO2 colloids and improves the quality of the electron transport layer. Furthermore, the introduction of CuCl2 optimizes the energy-level array between the ETL and perovskite layer (PVK) and passivates the anion/cation defects in SnO2, perovskite, and their interface, realizing the systematic modulation of the photoelectronic properties of the ETLs and PVKs as well as the PVK/ETL. As a result, the CuCl2-opmized PSC exhibits an impressive power conversion efficiency of 23.71%, along with improved stability.

Development of a novel tyrosine-based selection system for generation of recombinant Chinese hamster ovary cells.

Efficiently expanding Chinese hamster ovary (CHO) cells, which serve as the primary host cells for recombinant protein production, have gained increasing industrial significance. A significant hurdle in stable cell line development is the low efficiency of the target gene integrated into the host genome, implying the necessity for an effective screening and selection procedure to separate these stable cells. In this study, the genes of phenylalanine hydroxylase (PAH) and pterin 4 alpha carbinolamine dehydratase 1 (PCBD1), which are key enzymes in the tyrosine synthesis pathway, were utilized as selection markers and transduced into host cells together with the target genes. This research investigated the enrichment effect of this system and advanced further in understanding its benefits for cell line development and rCHO cell culture. A novel tyrosine-based selection system that only used PCBD1 as a selection marker was designed to promote the enrichment effect. Post 9 days of starvation, positive transductants in the cell pool approached 100%. Applied the novel tyrosine-based selection system, rCHO cells expressing E2 protein were generated and named CHO TS cells. It could continue to grow, and the yield of E2 achieved 95.95 mg/L in a tyrosine-free and chemically-defined (CD) medium. Herein, we introduced an alternative to antibiotic-based selections for the establishment of CHO cell lines and provided useful insights for the design and development of CD medium.

Poly(3-hexylthiophene)/perovskite Heterointerface by Spinodal Decomposition Enabling Efficient and Stable Perovskite Solar Cells.

The best research-cell efficiency of perovskite solar cells (PSCs) is comparable with that of mature silicon solar cells (SSCs); However, the industrial development of PSCs lags far behind SSCs. PSC is a multiphase and multicomponent system, whose consequent interfacial energy loss and carrier loss seriously affect the performance and stability of devices. Here, by using spinodal decomposition, a spontaneous solid phase segregation process, in situ introduces a poly(3-hexylthiophene)/perovskite (P3HT/PVK) heterointerface with interpenetrating structure in PSCs. The P3HT/PVK heterointerface tunes the energy alignment, thereby reducing the energy loss at the interface; The P3HT/PVK interpenetrating structure bridges a transport channel, thus decreasing the carrier loss at the interface. The simultaneous mitigation of energy and carrier losses by P3HT/PVK heterointerface enables n-i-p geometry device a power conversion efficiency of 24.53% (certified 23.94%) and excellent stability. These findings demonstrate an ingenious strategy to optimize the performance of PSCs by heterointerface via Spinodal decomposition.

Development of stable HEK293T cell pools expressing CSFV E2 protein: A potential antigen expression platform.

Large quantities of antigens are required since protective antigens, such as classical swine fever virus (CSFV) E2 protein, are widely used in diagnostic reagents and subunit vaccines. Compared to clonal cell lines and transient gene expression, stable cell pools provide a potential alternative platform to rapidly produce large amounts of antigens. In this work, firstly, Human embryonic kidney 293 T (HEK293T) cell pools expressing E2 protein were developed by transduction of lentiviral vectors. On the one hand, the SP7 was selected from 7 well-performing signal peptides to remarkably increase the production of E2 protein. On the other hand, it was found that high MOI could improve the expression of E2 protein by increasing gene copy numbers. Moreover, the HEK293T cell pools were evaluated for stability by passages and batch cultures, demonstrating that the cell pools were stable for at least 90 days. And then, the performance of the cell pools in batch, fed-batch, and semi-perfusion was studied. Among them, the titer of E2 protein was up to 2 g/L in semi-perfusion, which is currently the highest to the authors' knowledge. Finally, the aggregations and immunogenicity of the E2 protein were analyzed by SDS-PAGE and immunization of mice, respectively. There was no significant difference in aggregations and antibody titers of E2 protein in three culture methods. These results suggest that stable HEK293T cell pools are a promising and robust platform for rapid and efficient production of recombinant proteins.

Optical Properties and Concentration Quenching Mechanism of Er3+ Heavy Doped Gd2(MoO4)3 Phosphor for Green Light-Emitting Diode.

Upconversion materials capable of converting low-energy excitation photons into high-energy emission photons have attracted considerable interest in recent years. However, the low upconversion luminescence seriously hinders the application of upconversion phosphors. Heavy lanthanide doping without concentration quenching represents a direct and effective method to enhance the emission intensity. In this study, Er3+ heavy doped Gd2(MoO4)3 phosphor with a monoclinic phase was prepared by a sol-gel process. Under excitation at 976 nm, Gd2(MoO4)3:Er3+ phosphor emitted remarkably intense green emission, and Er3+ concentration up to 20 mol% did not cause concentration quenching. Here, we discuss the upconversion mechanism and concentration quenching. When the Er3+ concentration was in the range of 30-60 mol%, the concentration quenching was governed by the electric dipole-dipole interaction, and when the concentration was greater than 60 mol%, the concentration quenching was controlled by the exchange interactions. The result provides a schematic basis for identifying a phosphor host with heavy lanthanide doping.

Metabolic reprogramming and alteration of the redox state in hyper-productive MDCK cells for influenza a virus production.

Influenza is a global public health issue leading to widespread morbidity and mortality with devastating economic loss annually. Madin-Darby Canine Kidney (MDCK) cell line has been a major cell line for influenza vaccine applications. Though many details of the host metabolic responses upon influenza A virus (IAV) infection have been documented, little is known about the metabolic reprogramming features of a hyper-productive host for IAV vaccine production. In this study, a MDCK cell clone H1 was shown to have a particular high productivity of 30 × 103 virions/cell. The glucose and amino acid metabolism of H1 were evaluated, indicating that the high producer had a particular metabolic reprogramming phenotype compared to its parental cell line (P): elevated glucose uptake, superior tricarboxylic acid cycle flux, moderate amino acid consumption, and better regulation of reactive oxygen species. Combined with the stronger mitochondrial function and mild antiviral and inflammatory responses characterized previously, our results indicated that the high producer had a sufficient intracellular energy supply, and balanced substrate distribution for IAV and host protein synthesis as well as the intracellular redox status. Understanding of these metabolic alterations paves the way for the rational cell line development and reasonable process optimization for high-yield influenza vaccine production.

Designing a novel E2-IFN-γ fusion protein against CSFV by immunoinformatics and structural vaccinology approaches.

Subunit vaccines with high purity and safety are gradually becoming a main trend in vaccinology. However, adjuvants such as interferon-gamma (IFN-γ) are required to enhance immune responses of subunit vaccines due to their poor immunogenicity. The conjugation of antigen with adjuvant can induce more potent immune responses compared to the mixture of antigen and adjuvant. At the same time, the selection of linker, indispensable in the construction of the stable and bioactive fusion proteins, is complicated and time-consuming. The development of immunoinformatics and structural vaccinology approaches provides a means to address the abovementioned problem. Therefore, in this study, a E2-IFN-γ fusion protein with an optimal linker (E2-R2-PIFN) was designed by bioinformatics approaches to improve the immunogenicity of the classical swine fever virus (CSFV) E2 subunit vaccine. Moreover, the E2-R2-PIFN fusion protein was expressed in HEK293T cells and the biological effects of IFN-γ in E2-R2-PIFN were confirmed in vitro via Western blotting. Here, an alternative method is utilized to simplify the design and validation of the antigen-adjuvant fusion protein, providing a potential subunit vaccine candidate against CSFV. KEY POINTS: • An effective and simple workflow of antigen-adjuvant fusion protein design and validation was established by immunoinformatics and structural vaccinology. • A novel E2-IFN-γ fusion protein with an optimal linker was designed as a potential CSFV vaccine. • The bioactivity of the newly designed fusion protein was preliminarily validated through in vitro experiments.

Arsenic Induces Continuous Inflammation and Regulates Th1/Th2/Th17/Treg Balance in Liver and Kidney In Vivo.

Numerous studies on arsenic-induced hepatonephric toxicity including cancer have been reported. Given that chronic inflammatory response and immune imbalance are associated with oncogenesis, we investigated whether arsenic could influence the hepatic and nephritic expression of inflammatory factors and the differentiation of T cells. Mice were exposed to NaAsO2 (0, 25, and 50 mg/L) for 1 and 3 months. Our data showed the destruction of the structure and inflammatory infiltration in the liver. The arsenic markedly increased the activity of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST). The myeloperoxidase (MPO) activities increased in the liver at 25 and 50 mg/L arsenic for 3 months as well as in the kidney at both 1 and 3 months. An increased expression of inflammatory indicators (IL-1ß, IL-12, and TNF-α) at 25 and 50 mg/L arsenic for 1 and 3 months in the liver and kidney, as well as IL-1ß in the liver for 3 months and in the kidney at 50 mg/L for 1 and 3 months were demonstrated in our experiments. Besides, a definite tendency toward Th1/Th17 cytokines in the liver while Th2/Th17 cytokines in kidney was also observed by arsenic. Moreover, arsenic enhanced the expression of MAPK/Nrf2/NF-κB signaling molecules. In conclusion, the results of the study suggested that arsenic induces continuous immune-inflammatory responses in the liver and kidney.

Transcriptomic Characterization Reveals Attributes of High Influenza Virus Productivity in MDCK Cells.

The Madin-Darby Canine Kidney (MDCK) cell line is among the most commonly used cell lines for the production of influenza virus vaccines. As cell culture-based manufacturing is poised to replace egg-based processes, increasing virus production is of paramount importance. To shed light on factors affecting virus productivity, we isolated a subline, H1, which had twice the influenza virus A (IAV) productivity of the parent (P) through cell cloning, and characterized H1 and P in detail on both physical and molecular levels. Transcriptome analysis revealed that within a few hours after IAV infection, viral mRNAs constituted over one fifth of total mRNA, with several viral genes more highly expressed in H1 than P. Functional analysis of the transcriptome dynamics showed that H1 and P responded similarly to IAV infection, and were both subjected to host shutoff and inflammatory responses. Importantly, H1 was more active in translation and RNA processing intrinsically and after infection. Furthermore, H1 had more subdued inflammatory and antiviral responses. Taken together, we postulate that the high productivity of IAV hinges on the balance between suppression of host functions to divert cellular resources and the sustaining of sufficient activities for virus replication. Mechanistic insights into virus productivity can facilitate the process optimization and cell line engineering for advancing influenza vaccine manufacturing.

Alkali Metal Fluoride-Modified Tin Oxide for n-i-p Planar Perovskite Solar Cells.

The practical applications of perovskite solar cells (PSCs) are limited by further improvement of their stability and performance. Additive engineering and interface engineering are promising medicine to cure this stubborn disease. Herein, an alkali metal fluoride as an additive is introduced into the tin oxide (SnO2) electron transport layer (ETL). The formation of coordination bonds of F- ions with the oxygen vacancy of Sn4+ ions decreases the trap-state density and improves the electron mobility; the hydrogen bond interaction between the F ion and amine group (FA+) of perovskite inhibits the diffusion of organic cations and promotes perovskite (PVK) stability. Meanwhile, the alkali metal ions (K+, Rb+, and Cs+) permeated into PVK fill the organic cation vacancies and ameliorate the crystal quality of PVK films. Consequently, a SnO2-based planar PSC exhibits a power conversion efficiency (PCE) of 20.24%, while the PSC modified by CsF achieves a PCE of 22.51%, accompanied by effective enhancement of stability and negligible hysteresis. The research results provide a typical example for low-cost and multifunctional additives in high-performance PSCs.

Phthalide and 1-Iodooctadecane Synergistic Optimization for Highly Efficient and Stable Perovskite Solar Cells.

The carrier non-radiative recombination and instability of device caused by the inherent defects are main factors limiting development of perovskite solar cells (PSCs). During the fabrication process of a PSC device, perovskite films often produce Pb0 and I0 defects. This paper reports a strategy for synergistic optimization of perovskite films by defects passivation and surface modification. The doping of phthalide (PT) in the Pb-rich (CH(NH2 )2 )1-x (CH3 NH3 )x PbI3 film can passivate lead cation defects, and the modification of 1-iodooctadecane (1-IO) can reduce halogen anion defects and improve stability of PSCs owing to its hydrophobicity. The PT and 1-IO optimized device achieves a power conversion efficiency (PCE) of 22.27%. The optimized PSCs remain 93.2% of the initial PCE when placed in air environment (relative humidity of 10%, 25 °C) more than 70 days. The PT and 1-IO synergistic optimization provides a novel strategy for improving the performance and stability of PSCs.

Towards integrated production of an influenza A vaccine candidate with MDCK suspension cells.

Seasonal influenza epidemics occur both in northern and southern hemispheres every year. Despite the differences in influenza virus surface antigens and virulence of seasonal subtypes, manufacturers are well-adapted to respond to this periodical vaccine demand. Due to decades of influenza virus research, the development of new influenza vaccines is relatively straight forward. In similarity with the ongoing coronavirus disease 2019 pandemic, vaccine manufacturing is a major bottleneck for a rapid supply of the billions of doses required worldwide. In particular, egg-based vaccine production would be difficult to schedule and shortages of other egg-based vaccines with high demands also have to be anticipated. Cell culture-based production systems enable the manufacturing of large amounts of vaccines within a short time frame and expand significantly our options to respond to pandemics and emerging viral diseases. In this study, we present an integrated process for the production of inactivated influenza A virus vaccines based on a Madin-Darby Canine Kidney (MDCK) suspension cell line cultivated in a chemically defined medium. Very high titers of 3.6 log10 (HAU/100 µl) were achieved using fast-growing MDCK cells at concentrations up to 9.5 × 106 cells/ml infected with influenza A/PR/8/34 H1N1 virus in 1 L stirred tank bioreactors. A combination of membrane-based steric-exclusion chromatography followed by pseudo-affinity chromatography with a sulfated cellulose membrane adsorber enabled full recovery for the virus capture step and up to 80% recovery for the virus polishing step. Purified virus particles showed a homogenous size distribution with a mean diameter of 80 nm. Based on a monovalent dose of 15 µg hemagglutinin (single-radial immunodiffusion assay), the level of total protein and host cell DNA was 58 µg and 10 ng, respectively. Furthermore, all process steps can be fully scaled up to industrial quantities for commercial manufacturing of either seasonal or pandemic influenza virus vaccines. Fast production of up to 300 vaccine doses per liter within 4-5 days makes this process competitive not only to other cell-based processes but to egg-based processes as well.

High cell density perfusion process for high yield of influenza A virus production using MDCK suspension cells.

Similar to the recent COVID-19 pandemic, influenza A virus poses a constant threat to the global community. For the treatment of flu disease, both antivirals and vaccines are available with vaccines the most effective and safest approach. In order to overcome limitations in egg-based vaccine manufacturing, cell culture-based processes have been established. While this production method avoids egg-associated risks in face of pandemics, process intensification using animal suspension cells in high cell density perfusion cultures should allow to further increase manufacturing capacities worldwide. In this work, we demonstrate the development of a perfusion process using Madin-Darby canine kidney (MDCK) suspension cells for influenza A (H1N1) virus production from scale-down shake flask cultivations to laboratory scale stirred tank bioreactors. Shake flask cultivations using semi-perfusion mode enabled high-yield virus harvests (4.25 log10(HAU/100 µL)) from MDCK cells grown up to 41 × 106 cells/mL. Scale-up to bioreactors with an alternating tangential flow (ATF) perfusion system required optimization of pH control and implementation of a temperature shift during the infection phase. Use of a capacitance probe for on-line perfusion control allowed to minimize medium consumption. This contributed to a better process control and a more economical performance while maintaining a maximum virus titer of 4.37 log10(HAU/100 µL) and an infectious virus titer of 1.83 × 1010 virions/mL. Overall, this study clearly demonstrates recent advances in cell culture-based perfusion processes for next-generation high-yield influenza vaccine manufacturing for pandemic preparedness. KEY POINTS: • First MDCK suspension cell-based perfusion process for IAV produciton was established. • "Cell density effect" was overcome and process was intensified by reduction of medium use and automated process control. • The process achieved cell density over 40 × 106 cells/mL and virus yield over 4.37 log10(HAU/100 µL).

Exploring the Potential Mechanism of Chuanxiong Rhizoma Treatment for Migraine Based on Systems Pharmacology.

Migraine is a disease whose aetiology and mechanism are not yet clear. Chuanxiong Rhizoma (CR) is employed in traditional Chinese medicine (TCM) to treat various disorders. CR is effective for migraine, but its active compounds, drug targets, and exact molecular mechanism remain unclear. In this study, we used the method of systems pharmacology to address the above issues. We first established the drug-compound-target-disease (D-C-T-D) network and protein-protein interaction (PPI) network related to the treatment of migraine with CR and then established gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. The results suggest that the treatment process may be related to the regulation of inflammation and neural activity. The docking results also revealed that PTGS2 and TRPV1 could directly bind to the active compounds that could regulate them. In addition, we found that CR affected 11 targets that were more highly expressed in the liver or heart but were the lowest in the whole brain. It also expounds the description of CR channel tropism in TCM theory from these angles. These findings not only indicate that CR can be developed as a potential effective drug for the treatment of migraine but also demonstrate the application of systems pharmacology in the discovery of herbal-based disease therapies.

Additive Engineering by Bifunctional Guanidine Sulfamate for Highly Efficient and Stable Perovskites Solar Cells.

High efficiency and good stability are the challenges for perovskite solar cells (PSCs) toward commercialization. However, the intrinsic high defect density and internal nonradiative recombination of perovskite (PVK) limit its development. In this work, a facile additive strategy is devised by introducing bifunctional guanidine sulfamate (GuaSM; CH6 N3 + , Gua+ ; H2 N-SO3 - , SM- ) into PVK. The size of Gua+ ion is suitable with Pb(BrI)2 cavity relatively, so it can participate in the formation of low-dimensional PVK when mixed with Pb(BrI)2 . The O and N atoms of SM- can coordinate with Pb2+ . The synergistic effect of the anions and cations effectively reduces the trap density and the recombination in PVK, so that it can improve the efficiency and stability of PSCs. At an optimal concentration of GuaSM (2 mol%), the PSC presents a champion power conversion efficiency of 21.66% and a remarkably improved stability and hysteresis. The results provide a novel strategy for highly efficient and stable PSCs by bifunctional additive.

Chronic arsenic exposure induces the time-dependent modulation of inflammation and immunosuppression in spleen.

BACKGROUND: Arsenic exposure has become a matter of worldwide concern, which is associated with immune-related diseases. However, little is known about its effect on inflammatory immune-related homeostasis. The purpose of our study was to understand the potential tuning of above responses exerted by chronic arsenic exposure. METHODS: Kunming mice were treated with 25 and 50 mg/L sodium arsenite for 1, 3 and 12 months via drinking water. At different endpoints of arsenic exposure, all animals and the whole spleen of the mice were weighed. The total arsenic levels of spleen were determined by the HPLC-HG-AFS method. Splenic NF-κB, MAPK and NRF2 protein levels by treatment of 25 mg/L NaAsO2 for 1, 3 and 12 months and 25 mg/L and 50 mg/L NaAsO2 for 12 months were assessed by western blot. Total RNA of spleen was isolated and relative mRNA levels of Foxp3, Il-10, Tnf-α, Il-6, Ifn-γ, Il-1ß and Il-12 were measured by real-time PCR. RESULTS: Our results shown that NF-κB were continuously activated with treatment of 25 mg/L arsenic from 1, 3 to 12 months and 50 mg/L arsenic for 12 months. The transcription factor Foxp3 increased at 1 month but decreased at 3 and 12 months no matter 25 or 50 mg/L arsenic exposure. However, cytokine Il-10 always showed increased trend in mice treated with 25 or 50 mg/L arsenic for 1, 3 and 12 months. The transcriptional profiles of Tnf-α, Il-1ß, Il-6, Ifn-γ and Il-12 revealed transient elevation at 1 and 3 months but shown significant decrease at 12 months on the whole. In addition, the sustained activation of inflammatory MAPK and anti-oxidative Nrf2 signaling pathways were observed in mice exposed to arsenic for 1, 3 and 12 months. CONCLUSION: In summary, our experiment in vivo suggested chronic arsenic exposure induces the time-dependent modulation of the inflammation and immunosuppression in spleen, which may be related to the activation of Tregs induced by MAPK/NF-κB as well as the increased transcription level of Foxp3 and Il-10.