Construction of an Integrated mCherry Red Fluorescent Protein Expression System for Labeling and Tracing in Lactiplantibacillus plantarum WCFS1

Thorough intestinal adhesion and colonization greatly promote the probiotic properties of lactic acid bacteria (LAB). Labeling and tracing with fluorescent proteins are effective and reliable for studying the in vivo physiological activities of LAB including localization, adhesion, and colonization. Lactiplantibacillus plantarum WCFS1 was successfully traced with a red fluorescent protein (RFP), which was expressed by the bacteria-carrying recombinant plasmids. In this study, we aimed to construct a stable RFP mCherry expression system, whose encoding gene was integrated into the bacterial chromosome via double-crossed homologous recombination, and use it for labeling WCFS1 with the goal of avoiding the potential loss of non-chromosomal plasmids along with intestinal growth. First, the constitutive expression of the mCherry protein was improved after adjusting the length of the spacer between the promoter and the gene start codon. Then, the optimized mCherry gene expression cassette was integrated into the chromosome of WCFS1. The resulting strain had normal unimpaired growth and strong fluorescent signals, even after 100 generations, indicating its stability. Furthermore, quantitative polymerase chain reaction (PCR) results revealed a strong positive correlation between the fluorescence intensity of the strain and the number of viable cells, demonstrating its potential usage for the quantification of in vivo WCFS1 cells. Finally, the increased adhesion ability of WCFS1 due to the recombinant expression of the bsh gene was visualized and evaluated using fluorescence intensity, the results of which were consistent with those obtained using the previously established quantification methods. These results suggest that the chromosomal-integrated mCherry labeling system can be extensively used to examine the distribution, colonization, and survival of LAB in vivo in order to determine the mechanism of its probiotic function

Genetic dissection of grain iron concentration in hexaploid wheat (Triticum aestivum L.) using a genome-wide association analysis method

Iron (Fe) is an essential micronutrient of the body. Low concentrations of bioavailable Fe in staple food result in micronutrient malnutrition. Wheat (Triticum aestivum L.) is the most important global food crop and thus has become an important source of iron for people. Breeding nutritious wheat with high grain-Fe content has become an effective means of alleviating malnutrition. Understanding the genetic basis of micronutrient concentration in wheat grains may provide useful information for breeding for high Fe varieties through marker-assisted selection (MAS). Hence, in the present study, genome-wide association studies (GWAS) were conducted for grain Fe. An association panel of 207 accessions was genotyped using a 660K SNP array and phenotyped for grain Fe content at three locations. The genotypic and phenotypic data obtained thus were used for GWAS. A total of 911 SNPs were significantly associated with grain Fe concentrations. These SNPs were distributed on all 21 wheat chromosomes, and each SNP explained 5.79–25.31% of the phenotypic variations. Notably, the two significant SNPs (AX-108912427 and AX-94729264) not only have a more significant effect on grain Fe concentration but also have the reliability under the different environments. Furthermore, candidate genes potentially associated with grain Fe concentration were predicted, and 10 candidate genes were identified. These candidate genes were related to transport, translocation, remobilization, and accumulationof ironin wheat plants. These findings will not only help in better understanding the molecular basis of Fe accumulation in grains, but also provide elite wheat germplasms to develop Fe-rich wheat varieties through breeding

Screening and characterization of the scFv for chimeric antigen receptor T cells targeting CEA-positive carcinoma

IntroductionChimeric antigen receptor T (CAR-T) cell therapy presents a promising treatment option for various cancers, including solid tumors. Carcinoembryonic antigen (CEA) is an attractive target due to its high expression in many tumors, particularly gastrointestinal cancers, while limited expression in normal adult tissues. In our previous clinical study, we reported a 70% disease control rate with no severe side effects using a humanized CEA-targeting CAR-T cell. However, the selection of the appropriate single-chain variable fragment (scFv) significantly affects the therapeutic efficacy of CAR-T cells by defining their specific behavior towards the target antigen. Therefore, this study aimed to identify the optimal scFv and investigate its biological functions to further optimize the therapeutic potential of CAR-T cells targeting CEA-positive carcinoma.MethodsWe screened four reported humanized or fully human anti-CEA antibodies (M5A, hMN-14, BW431/26, and C2-45), and inserted them into a 3rd-generation CAR structure. We purified the scFvs and measured the affinity. We monitored CAR-T cell phenotype and scFv binding stability to CEA antigen through flow cytometry. We performed repeated CEA antigen stimulation assays to compare the proliferation potential and response of the four CAR-T cells, then further evaluated the anti-tumor efficacy of CAR-T cells ex vivo and in vivo.ResultsM5A and hMN-14 CARs displayed higher affinity and more stable CEA binding ability than BW431/26 and C2-45 CARs. During CAR-T cell production culture, hMN-14 CAR-T cells exhibit a larger proportion of memory-like T cells, while M5A CAR-T cells showed a more differentiated phenotype, suggesting a greater tonic signal of M5A scFv. M5A, hMN-14, and BW431/26 CAR-T cells exhibited effective tumor cell lysis and IFN-γ release when cocultured with CEA-positive tumor cells in vitro, correlating with the abundance of CEA expression in target cells. While C2-45 resulted in almost no tumor lysis or IFN-γ release. In a repeat CEA antigen stimulation assay, M5A showed the best cell proliferation and cytokine secretion levels. In a mouse xenograft model, M5A CAR-T cells displayed better antitumor efficacy without preconditioning.DiscussionOur findings suggest that scFvs derived from different antibodies have distinctive characteristics, and stable expression and appropriate affinity are critical for robust antitumor efficacy. This study highlights the importance of selecting an optimal scFv in CAR-T cell design for effective CEA-targeted therapy. The identified optimal scFv, M5A, could be potentially applied in future clinical trials of CAR-T cell therapy targeting CEA-positive carcinoma

Adaptive Tracking Constrained Controller Design for Solid Oxide Fuel Cells Based on a Wiener-Type Neural Network

In order to solve the control problem of the solid oxide fuel cell(SOFC), a novel adaptive tracking constrained control strategy based on a Wiener-type neural network is proposed in this paper. The working principle of SOFC is introduced, and the dynamical model of SOFC is studied. Besides, a Wiener model formulation for SOFC is proposed to approximate the nonlinear dynamics of the system, and an adaptive Wiener model identification method is utilized to identify the parameters of the model. Moreover, an adaptive exponential PID controller is designed to keep the stack output voltage stable. Meanwhile, the saturation problem is considered in the paper including input magnitude and rate constraints. Additionally, an anti-windup compensator is employed to eliminate the abominable influence of the saturation problem. Then, the stability of the control plant is analyzed and proven via the Lyapunov function. Finally, the simulation based on the MATLAB/Simulink environment is carried out, and the conventional PID controller is added and simulated as a contrast to verify the control performance of the proposed control algorithm. The results indicate that the proposed control algorithm possesses favorable control performance when dealing with nonlinear systems with complex dynamics

Adaptive-Observer-Based Data Driven Voltage Control in Islanded-Mode of Distributed Energy Resource Systems

In this paper, an adaptive observer based data driven control scheme is proposed for the voltage control of dispatchable distributed energy resource (DER) systems which work in islanded operation. In the design procedure of the proposed control scheme, we utilize the novel transformation and linearization technique for the islanded DER system dynamics, which is proper for the proposed data driven control algorithm. Moreover, the pseudo partial derivative (PPD) parameter matrix can be estimated online by multiple adaptive observers. Then, the adaptive constrained controller is designed only based on the online identification results derived from the input/output (I/O) data of the controlled DER system. It is theoretically proven that all the signals in the closed-loop control system are uniformly ultimately bounded based on the Lyapunov stability analysis approach. In addition, the results of the simulation comparison are given to verify the voltage control effect of the proposed control scheme

Mineral Composition and Graphitization Structure Characteristics of Contact Thermally Altered Coal

Contact metamorphism in coal is usually characterized by a rapid, brief, and exotherm reaction that can change the geothermal gradient. In this process, coal adjacent to the intrusive body can form thermally altered coal-based graphite (TACG). In order to further study the structural changes of TACG at different distances from the intrusive body, four TACG samples were collected in the Zhuji coal mine in the Huainan Coalfield, North China, and their vitrinite reflectance and Raman spectra were measured using polarizing microscopy and Raman spectroscopy. The results showed that: (1) affected by the temperature and stress of magmatic hydrothermal intrusion, the clay minerals in the coal seams appeared distributed in strips; the occurrence of ankerite and pyrite in the coal seams near the magmatic intrusions could be due to a late magmatic hydrothermal mineralization; (2) the Rmax − Rmin correlation for the TACG samples under study showed that thermal metamorphism was the main factor leading to the graphitization of the TACG samples, without an obvious pressure effect; (3) with the increase of the graphitization process, the D- and G-band showed some similar changes, specifically, their peak positions shifted to lower wave numbers, and the full width at half maximum (FWG and FWD) gradually decreased; the difference was that the intensity of the G-band increased, while that of the D-band decreased; (4) the graphitization degree of the TACG samples increased with the increase of the transverse size of the crystals, while the FWG and FWD values of the G- and D-band decreased; (5) in comparison to natural graphite, the TACG still presented structural defects

PCL/Collagen/UA Composite Biomedical Dressing with Ordered Microfiberous Structure Fabricated by a 3D Near-Field Electrospinning Process

In this work, a functionalized polycaprolactone (PCL) composite fiber combining calf-type I collagen (CO) and natural drug usnic acid (UA) was prepared, in which UA was used as an antibacterial agent. Through 3D near-field electrospinning, the mixed solution was prepared into PCL/CO/UA composite fibers (PCUCF), which has a well-defined perfect arrangement structure. The influence of electrospinning process parameters on fiber diameter was investigated, the optimal electrospinning parameters were determined, and the electric field simulation was conducted to verify the optimal parameters. The addition of 20% collagen made the composite fiber have good hydrophilicity and water absorption property. In the presence of PCUCF, 1% UA content significantly inhibited the growth rate of Gram-positive and negative bacteria in the plate culture. The AC-PCUCF (after crosslinking PCUCF) prepared by crosslinking collagen with genipin showed stronger mechanical properties, water absorption property, thermal stability, and drug release performance. Cell proliferation experiments showed that PCUCF and AC-PCUCF had no cytotoxicity and could promote cell proliferation and adhesion. The results show that PCL/CO/UA composite fiber has potential application prospects in biomedical dressing

Starch Bio-Synthetic Pathway Genes Contribute to Resistant Starch Content Differentiation in Bread Wheat

Resistant starch (RS) is a special group of starches which are slowly degraded and rarely digested in the gastrointestinal tract. It was recognized as a new type of dietary fiber that improved cardiovascular, cerebrovascular, and intestinal health. Breeding high-RS-content wheat is one of the most efficient and convenient approaches for providing an adequate amount of RS for a healthy diet. However, studies which aim to genetically illustrate RS content in wheat are still rare. In the present study, a panel of 207 wheat varieties were collected world-wide and planted under three locations. The RS content of each variety was measured, and 14 additive genetic loci were found to stably exist under more than two environments. Meanwhile, four genes were recognized as the putative candidates with annotated functions of β-amylase, α-1,4 glucan phosphorylase, sucrose transporter, and NAC domain protein. A kompetitive allele-specific PCR (KASP) marker was developed from the SNP AX-94546744, representing the genetic locus of β-amylase located. The AX-94546744-T allele can significantly increase the RS content compared to the AX-94546744-C allele. The genetic loci and KASP marker associated with RS content may be useful for wheat germplasm cultivation and variety breeding with a high RS content, further helping to improve the nutritional quality in wheat