A 62.2dB SNDR Event-Driven Level-Crossing ADC with SAR-Assisted Delay Compensation Loop for Time-Sparse Biomedical Signal Acquisition.

This paper proposed an event-driven clockless level-crossing ADC (LC-ADC) suitable for biomedical applications. Thanks to the LC loop, the sampling rate of the converter automatically adapts to the input activities. Activity-dependent power consumption and data compression can thus be realized, saving system power, especially during time-sparse signal acquisition. Meanwhile, a SAR-assisted loop is exploited to resolve the loop-delay-induced distortion in conventional LC-ADC. Therefore, the resolution and power efficiency of the LC-ADC are improved effectively while maintaining the event-driven feature. Implemented in a 55nm process, the proposed LC-ADC achieves a scalable power consumption and a peak SNDR of 62.2dB for a 20kHz input. It also achieves a Walden FoM of 29.7fJ/conv.-step and a Schreier FoM of 158.6dB, which is best in class, without using off-chip calibration. Sub µW power is realized when the input frequency is below 1.5kHz. The proposed LC-ADC is also verified by simulated electrocardiogram (ECG), neural spike, and electromyogram (EMG) signals. It provides a ~7X data compression for ECG input, providing an attractive solution for time-sparse signal acquisition in biomedical applications.

DNAJC1 facilitates glioblastoma progression by promoting extracellular matrix reorganization and macrophage infiltration.

BACKGROUND: Glioblastoma (GBM) is a high-grade and heterogeneous subtype of glioma that presents a substantial challenge to human health, characterized by a poor prognosis and low survival rates. Despite its known involvement in regulating leukemia and melanoma, the function and mechanism of DNAJC1 in GBM remain poorly understood. METHODS: Utilizing data from the TCGA, CGGA, and GEO databases, we investigated the expression pattern of DNAJC1 and its correlation with clinical characteristics in GBM specimens. Loss-of-function experiments were conducted to explore the impact of DNAJC1 on GBM cell lines, with co-culture experiments assessing macrophage infiltration and functional marker expression. RESULTS: Our analysis demonstrated frequent overexpression of DNAJC1 in GBM, significantly associated with various clinical characteristics including WHO grade, IDH status, chromosome 1p/19q codeletion, and histological type. Moreover, Kaplan‒Meier and ROC analyses revealed DNAJC1 as a negative prognostic predictor and a promising diagnostic biomarker for GBM patients. Functional studies indicated that silencing DNAJC1 impeded cell proliferation and migration, induced cell cycle arrest, and enhanced apoptosis. Mechanistically, DNAJC1 was implicated in stimulating extracellular matrix reorganization, triggering the epithelial-mesenchymal transition (EMT) process, and initiating immunosuppressive macrophage infiltration. CONCLUSIONS: Our findings underscore the pivotal role of DNAJC1 in GBM pathogenesis, suggesting its potential as a diagnostic and therapeutic target for this challenging disease.

Identification of functional sgRNA mutants lacking canonical secondary structure using high-throughput FACS screening.

Coexpressing multiple identical single guide RNAs (sgRNAs) in CRISPR-dependent engineering triggers genetic instability and phenotype loss. To provide sgRNA derivatives for efficient DNA digestion, we design a high-throughput digestion-activity-dependent positive screening strategy and astonishingly obtain functional nonrepetitive sgRNA mutants with up to 48 out of the 61 nucleotides mutated, and these nonrepetitive mutants completely lose canonical secondary sgRNA structure in simulation. Cas9-sgRNA complexes containing these noncanonical sgRNAs maintain wild-type level of digestion activities in vivo, indicating that the Cas9 protein is compatible with or is able to adjust the secondary structure of sgRNAs. Using these noncanonical sgRNAs, we achieve multiplex genetic engineering for gene knockout and base editing in microbial cell factories. Libraries of strains with rewired metabolism are constructed, and overproducers of isobutanol or 1,3-propanediol are identified by biosensor-based fluorescence-activated cell sorting (FACS). This work sheds light on the remarkable flexibility of the secondary structure of functional sgRNA.

Species-specific design of artificial promoters by transfer-learning based generative deep-learning model.

Native prokaryotic promoters share common sequence patterns, but are species dependent. For understudied species with limited data, it is challenging to predict the strength of existing promoters and generate novel promoters. Here, we developed PromoGen, a collection of nucleotide language models to generate species-specific functional promoters, across dozens of species in a data and parameter efficient way. Twenty-seven species-specific models in this collection were finetuned from the pretrained model which was trained on multi-species promoters. When systematically compared with native promoters, the Escherichia coli- and Bacillus subtilis-specific artificial PromoGen-generated promoters (PGPs) were demonstrated to hold all distribution patterns of native promoters. A regression model was developed to score generated either by PromoGen or by another competitive neural network, and the overall score of PGPs is higher. Encouraged by in silico analysis, we further experimentally characterized twenty-two B. subtilis PGPs, results showed that four of tested PGPs reached the strong promoter level while all were active. Furthermore, we developed a user-friendly website to generate species-specific promoters for 27 different species by PromoGen. This work presented an efficient deep-learning strategy for de novo species-specific promoter generation even with limited datasets, providing valuable promoter toolboxes especially for the metabolic engineering of understudied microorganisms.

The role of ST3GAL4 in glioma malignancy, macrophage infiltration, and prognostic outcomes.

Background: Glioma, a prevalent malignancy of the brain and spinal cord, poses a considerable threat to human health. The association between aberrant sialic acid modification and glioma progression has been suggested, but the precise mechanism is still elusive. ST3GAL4, a sialoglycosyltransferase, is implicated in increased metastatic potential and poor prognosis in various cancers; however, its specific role in glioma requires further elucidation. Methods: We evaluated ST3GAL4 expression levels and their clinical relevance using the TCGA database, and we assessed immune infiltration via the Tumor Immune Evaluation Resource (TIMER) database. In vitro experiments were performed to determine the effects of ST3GAL4 knockdown on glioma cell malignancy, with additional co-culture assays to assess its impact on macrophage phenotype. Results: ST3GAL4 expression was markedly elevated in glioma tissues compared to normal brain tissues, with a strong correlation to glioma patient clinical characteristics. Survival analyses and receiver operating characteristic (ROC) curves suggested that ST3GAL4 is a feasible diagnostic and prognostic biomarker for glioma. Knockdown studies revealed that ST3GAL4 inhibition reduces glioma cell line proliferation, migration, and invasion, while causing G1 phase cell cycle arrest. ST3GAL4 appears to mediate glioma progression through extracellular matrix reorganization and EMT signaling pathway activation, further contributing to M2 macrophage polarization and infiltration within the tumor microenvironment. Conclusion: Our research highlights the critical role of ST3GAL4 in glioma development, positioning it as a promising candidate for diagnostic and therapeutic interventions.

Concentration Recognition-Based Auto-Dynamic Regulation System (CRUISE) Enabling Efficient Production of Higher Alcohols.

Microbial factories lacking the ability of dynamically regulating the pathway enzymes overexpression, according to in situ metabolite concentrations, are suboptimal, especially when the metabolic intermediates are competed by growth and chemical production. The production of higher alcohols (HAs), which hijacks the amino acids (AAs) from protein biosynthesis, minimizes the intracellular concentration of AAs and thus inhibits the host growth. To balance the resource allocation and maintain stable AA flux, this work utilizes AA-responsive transcriptional attenuator ivbL and HA-responsive transcriptional activator BmoR to establish a concentration recognition-based auto-dynamic regulation system (CRUISE). This system ultimately maintains the intracellular homeostasis of AA and maximizes the production of HA. It is demonstrated that ivbL-driven enzymes overexpression can dynamically regulate the AA-to-HA conversion while BmoR-driven enzymes overexpression can accelerate the AA biosynthesis during the HA production in a feedback activation mode. The AA flux in biosynthesis and conversion pathways is balanced via the intracellular AA concentration, which is vice versa stabilized by the competition between AA biosynthesis and conversion. The CRUISE, further aided by scaffold-based self-assembly, enables 40.4 g L-1 of isobutanol production in a bioreactor. Taken together, CRUISE realizes robust HA production and sheds new light on the dynamic flux control during the process of chemical production.

Engineering amino acid residues of pentacyclic triterpene synthases for improving the activity.

Pentacyclic triterpenoids exhibit a wide range of biological activities which have wide applications in the food, cosmetics, and pharmaceutical industries. High-performance chassis strains have been developed for the production of various pentacyclic triterpenoids, e.g., lupane-type and oleanane-type triterpenoids. The production of common pentacyclic triterpenes and their derivatives is limited by the poor activity of typical pentacyclic triterpene synthases (PTSs). However, a general strategy applicable to typical PTSs is still lacking. As typical pentacyclic triterpenes are derived from the baccharenyl cation, engineering the non-active-site residues in the MXXXXR motif might be beneficial for the catalytic efficiencies of typical PTSs by the stabilization of the baccharenyl cation. Here, we develop a general strategy for improving the activity of typical PTSs. As a proof of concept, the activity of three PTSs such as lupeol synthase, ß-amyrin synthase, and α-amyrin synthases was significantly increased up to 7.3-fold by site-directed saturation mutagenesis. This strategy could be applied to improve the activity of various typical PTSs. KEY POINTS: • The strategy could be applied to typical PTSs for improving the activity. • The catalytic activity of typical PTSs was significantly increased.

Inquiry in uncertainty-nursing students' learning experience in challenge-based learning: A qualitative study.

BACKGROUND: Currently, nursing students face a significant lack of preparedness in efficiently addressing complex issues. The use of challenge-based learning, a student-centred situational learning method that utilizes practice scenarios to solve complex problems, can help alleviate the challenges in nurse education. However, there remains to be more extensive exploration on the application of challenge-based learning in nurse practice education. OBJECTIVES: This study aims to explore the learning experiences of nursing students in challenge-based learning to gain a deeper understanding of the ways and mechanisms through which challenge-based learning enhances specific learning abilities. DESIGN/METHODS: This study utilized a hermeneutic phenomenological design, employing semi-structured interviews and thematic analysis. SETTINGS: A university in Hunan, China, hosted the nursing skills competition based on the principles of challenge-based learning, targeting senior students from nursing schools in Hunan province. PARTICIPANTS: A total of 24 fourth-year students from six colleges, including one male and twenty-three female students. RESULTS: Two themes and seven sub-themes emerged from the study. Theme 1 Constructing a knowledge system in complex scenarios with sub-themes: Case-based education centred on comprehensive knowledge promotes knowledge linkage; Reverse reasoning promotes knowledge differentiation; Making mistakes helps finding the correct path of knowledge; Traps inspire identification of potential problems. Theme 2 Inquiry in uncertainty with sub-themes: Unexpected changes promote exploration of general principles; Combining knowledge with diverse case scenarios promotes flexible thinking; The uncertainty of knowledge fosters independent thinking. CONCLUSIONS: The complexity and variability inherent in challenging learning situations promote the construction of students' knowledge systems and the cultivation of an inquisitive spirit. The identification of these mechanisms contributes to the optimization of instructional environment design, the development of a culture of continuous learning and innovation, and effectively tackling complex issues within an ever-evolving global context.

Self-assembly systems to troubleshoot metabolic engineering challenges.

Enzyme self-assembly is a technology in which enzyme units can aggregate into ordered macromolecules, assisted by scaffolds. In metabolic engineering, self-assembly strategies have been explored for aggregating multiple enzymes in the same pathway to improve sequential catalytic efficiency, which in turn enables high-level production. The performance of the scaffolds is critical to the formation of an efficient and stable assembly system. This review comprehensively analyzes these scaffolds by exploring how they assemble, and it illustrates how to apply self-assembly strategies for different modules in metabolic engineering. Functional modifications to scaffolds will further promote efficient strategies for production.

Tumor-immune microenvironment and NRF2 associate with clinical efficacy of PD-1 blockade combined with chemotherapy in lung squamous cell carcinoma.

The RATIONALE-307 study (ClinicalTrials.gov: NCT03594747) demonstrates prolonged progression-free survival (PFS) with first-line tislelizumab plus chemotherapy versus chemotherapy in advanced lung squamous cell carcinoma (LUSC; N = 360). Here we describe an immune-related gene expression signature (GES), composed of genes involved in both innate and adaptive immunity, that appears to differentiate tislelizumab plus chemotherapy PFS benefit versus chemotherapy. In contrast, a tislelizumab plus chemotherapy PFS benefit is observed regardless of programmed death ligand 1 (PD-L1) expression or tumor mutational burden (TMB). Genetic analysis reveals that NRF2 pathway activation is enriched in PD-L1positive and TMBhigh patients. NRF2 pathway activation is negatively associated with PFS, which affects efficacy outcomes associated with PD-L1 and TMB status, impairing their predictive potential. Mechanistic studies demonstrate that NRF2 directly mediates PD-L1 constitutive expression independent of adaptive PD-L1 regulation in LUSC. In summary, the GES is an immune signature that might identify LUSC patients likely to benefit from first-line tislelizumab plus chemotherapy.

PLEKHA4 promotes glioblastoma progression through apoptosis inhibition, tumor cell migration, and macrophage infiltration.

BACKGROUND: Glioblastoma(GBM) has a profound impact on human health, making the identification of reliable prognostic biomarkers pivotal. While PLEKHA4 has been associated with tumor genesis and development, its role in gliomas is still uncertain. METHODS: We analyzed PLEKHA4 expression in tumor tissues using the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. Additionally, we utilized TCGA data to investigate its impact on prognosis, pathway enrichment, and immune infiltration. In vitro loss-of-function experiments were conducted to elucidate the effect of PLEKHA4 silencing on GBM cell behavior. RESULTS: TCGA and GEO data sets revealed increased levels of PLEKHA4 expression in glioma tissues. Furthermore, we identified a correlation between PLEKHA4 expression and higher disease classification, pathological grading, and poorer prognosis. Silencing PLEKHA4 in vitro resulted in decreased glioma cell migration and increased apoptosis. It also reduced macrophage infiltration and hindered M2 polarization of macrophages. CONCLUSION: Our findings highlight the pivotal role of PLEKHA4 in GBM pathogenesis and suggest its potential as a diagnostic and therapeutic target for GBM.

[Tiaoshen acupuncture for primary insomnia: a pilot randomized controlled trial].

OBJECTIVE: To observe the effects of Tiaoshen (regulating the spirit) acupuncture on cognitive function and sleep quality in patients with primary insomnia (PI). METHODS: Sixty patients with PI were randomly divided into an observation group (30 cases, 2 cases dropped off) and a control group (30 cases, 2 cases dropped off, 1 case was excluded). The patients in the observation group were treated with acupuncture at Baihui (GV 20), Shenting (GV 24), Sishencong (EX-HN 1), and bilateral Benshen (GB 13), Shenmen (HT 7), Neiguan (PC 6), Sanyinjiao (SP 6). The patients in the control group were treated with shallow needling at non-effective points. Each treatment was provided for 30 min, once every other day, 3 treatments per week for 4 weeks. The Montreal cognitive assessment (MoCA), digit span test (DST), trail making test (TMT)-A, Pittsburgh sleep quality index (PSQI), and fatigue scale-14 (FS-14) were used to assess cognitive function and sleep quality before and after treatment, as well as in follow-up of 4-week after treatment completion. Correlation analysis was conducted between the differences in PSQI scores and differences in MoCA scores before and after treatment in the observation group. RESULTS: Compared with before treatment, the total score, visuospatial and executive function score and delayed memory score of MoCA as well as DST backward score were increased (P<0.01), while TMT-A time, PSQI and FS-14 scores were significantly reduced (P<0.01) after treatment and in follow-up in the observation group. Compared with before treatment, the PSQI score in the control group was reduced (P<0.01, P<0.05). After treatment and in follow-up, the observation group had significantly higher total score, visuospatial and executive function score, delayed memory score of MoCA, and DST backward score compared to the control group (P<0.05, P<0.01). In the observation group, the TMT-A time was significantly shorter than that in the control group (P<0.05, P<0.01), and the PSQI and FS-14 scores were significantly lower than those in the control group (P<0.01). In the observation group, there was a negative correlation between the difference in PSQI scores (post-treatment minus pre-treatment) and the difference in MoCA scores (post-treatment minus pre-treatment) (r=-0.481, P<0.01). A similar negative correlation was found between the difference in PSQI scores (follow-up minus pre-treatment) and the difference in MoCA scores (follow-up minus pre-treatment) (r=-0.282, P<0.05). CONCLUSION: Tiaoshen acupuncture could improve cognitive function, enhance sleep quality, and alleviate daytime fatigue in patients with PI. The improvement in cognitive function in patients with PI is correlated with the improvement in sleep quality.

Strategies for improving the production of bio-based vanillin.

Vanillin (4-hydroxy-3-methoxybenzaldehyde) is one of the most popular flavors with wide applications in food, fragrance, and pharmaceutical industries. However, the high cost and limited yield of plant extraction failed to meet the vast market demand of natural vanillin. Vanillin biotechnology has emerged as a sustainable and cost-effective alternative to supply vanillin. In this review, we explored recent advances in vanillin biosynthesis and highlighted the potential of vanillin biotechnology. In particular, we addressed key challenges in using microorganisms and provided promising approaches for improving vanillin production with a special focus on chassis development, pathway construction and process optimization. Future directions of vanillin biosynthesis using inexpensive precursors are also thoroughly discussed.

Development of meso-Five-Membered Heterocycle BODIPY-Based AIE Fluorescent Probes for Dual-Organelle Viscosity Imaging.

Fluorescent rotors with aggregation-induced emission (AIE) and organelle-targeting properties have attracted great attention for sensing subcellular viscosity changes, which could help understand the relationships of abnormal fluctuations with many associated diseases. Despite the numerous efforts spent, it remains rare and urgent to explore the dual-organelle targeting probes and their structural relationships with viscosity-responsive and AIE properties. Therefore, in this work, we reported four meso-five-membered heterocycle-substituted BODIPY-based fluorescent probes, explored their viscosity-responsive and AIE properties, and further investigated their subcellular localization and viscosity-sensing applications in living cells. Interestingly, the meso-thiazole probe 1 showed both good viscosity-responsive and AIE (in pure water) properties and could successfully target both mitochondria and lysosomes, further imaging cellular viscosity changes by treating lipopolysaccharide and nystatin, attributing to the free rotation and potential dual-organelle targeting ability of the meso-thiazole group. The meso-benzothiophene probe 3 with a saturated sulfur only showed good viscosity-responsive properties in living cells with the aggregation-caused quenching effect and no subcellular localization. The meso-imidazole probe 2 showed the AIE phenomenon without an obvious viscosity-responsive property with a C═N bond, while the meso-benzopyrrole probe 4 displayed fluorescence quenching in polar solvents. Therefore, for the first time, we investigated the structure-property relationships of four meso-five-membered heterocycle-substituted BODIPY-based fluorescent rotors with viscosity-responsive and AIE properties, and among these, 1 with a C═N bond and a saturated sulfur on the meso-thiazole, potentially contributing to their corresponding AIE and viscosity-responsive properties, served as a sensitive AIE fluorescent rotor for imaging dual-organelle viscosity in both mitochondria and lysosomes.

A tRNAModification-based strategy for Identifying amiNo acid Overproducers (AMINO).

Amino acids have a multi-billion-dollar market with rising demand, prompting the development of high-performance microbial factories. However, a general screening strategy applicable to all proteinogenic and non-proteinogenic amino acids is still lacking. Modification of the critical structure of tRNA could decrease the aminoacylation level of tRNA catalyzed by aminoacyl-tRNA synthetases. Involved in a two-substrate sequential reaction, amino acids with increased concentration could elevate the reduced aminoacylation rate caused by specific tRNA modification. Here, we developed a selection system for overproducers of specific amino acids using corresponding engineered tRNAs and marker genes. As a proof-of-concept, overproducers of five amino acids such as L-tryptophan were screened out by growth-based and/or fluorescence-activated cell sorting (FACS)-based screening from random mutation libraries of Escherichia coli and Corynebacterium glutamicum, respectively. This study provided a universal strategy that could be applied to screen overproducers of proteinogenic and non-proteinogenic amino acids in amber-stop-codon-recoded or non-recoded hosts.

Accuracy and eligibility of Bonwill⁃Hawley arch form established by CBCT image for dental crowding measurement: a comparative study with the conventional brass wire and caliper methods.

OBJECTIVES: The purpose of this study was to develop a novel Bonwill⁃Hawley method (Bonwill⁃Hawley arch form based on CBCT image) for the assessment of dental crowding, and to investigate and compare the accuracy and eligibility with the conventional brass wire and caliper methods under different crowding conditions. MATERIAL AND METHODS: Sixty patients with the pair of plaster casts and CBCT data were collected. All the casts were marked and transformed into digital models using iTero scanner, and imported into OrthoCAD software to measure the required space. Using the conventional brass wire (M1) and caliper methods (M2), the available space and dental crowding were measured and calculated basing on digital models, respectively. Correspondingly, the axial planes in the level of dental arches were oriented and captured from the CBCT images to draw the Bonwill⁃Hawley arch forms (M3), which were used to measure and calculate the available space and dental crowding. For each method, intra and inter-examiner reliabilities were evaluated with intra-class correlation coefficients (ICCs). Wilcoxon test and Kruskal-Wallis test were performed for statistically analyzing the discrepancy among different groups. RESULTS: Both intra- and inter-examiner reliability were generally excellent for all parameters obtained by the three methods, except for the dental crowding measured using M1(ICC: 0.473/0.261). The dental crowding measured using M2 were significantly increased in mild, moderate and severe-crowding groups compared with M1. However, no significant difference was detected between M1 and M3 in severe-crowding group (maxilla, p = 0.108 > 0.05; mandible, p = 0.074 > 0.05). With the deterioration of crowding condition, the discrepancy of dental crowding between M1 and M2, or M1 and M3 were significantly decreased (maxilla, M2-M1, mild VS serve, p = 0.003 < 0.05; maxilla, M3-M1, mild VS serve, p = 0.003 < 0.05; mandible, M2-M1, mild VS serve, p = 0.000 < 0.001; mandible, M3-M1, mild VS serve, p = 0.043 < 0.05). CONCLUSION: Dental crowding measured using the novel Bonwill⁃Hawley method was relatively greater than the caliper method, but not exceeding the brass wire method, which wound gradually come close to the brass wire method with the deterioration of crowding condition. CLINICAL RELEVANCE: The Bonwill⁃Hawley method basing on CBCT image proved to be a reliable and acceptable choice for orthodontists to analyze the dental crowding.

Biomechanical study of internal fixation methods for femoral neck fractures based on Pauwels angle.

Objective: To select the most appropriate internal fixation method based on the Pauwels angle, in order to provide a new concept for clinical accurate treatment of femoral neck fractures (FNFs). Methods: FNFs models of Pauwels 30 ° ; 40 ° ; 50 ° ; 60 ° were created respectively. For Pauwels ≤ 50 ° , 1, 2 and 3 Cannulated Compression Screws (CCS) and Porous Tantalum Screws (PTS) were used to fix the fracture for the models. For Pauwels 60 ° , 3CCS and Medial Buttress Plate (MBP) combined with 1, 2 and 3CCS were used to fix the fracture. Based on the results of the finite element (FE) analysis, the biomechanical properties of each model were compared by analyzing and evaluating the following four parameters: maximal stress of the bone (MBS), maximal stress of the implants (MIS), maximal displacement of bone (MBD), interfragmentary motion (IFM). Results: At Pauwels 30 ° , the larger parameters were found in 1CCS, which was 94.8 MPa (MBS), 307.7 MPa (MIS), 0.86 mm (MBD) and 0.36 mm (IFM). In 2CCS group, the parameters were 86.1 MPa (MBS), 254.4 MPa (MIS), 0.73 mm (MBD) and 0.27 mm (IFM), which were similar to those of PTS. At Pauwels 40 ° ; 50 ° , with the increase of the number of used CCS, accordingly, the parameters decreased. Particularly, the MIS (Pauwels 50 ° ) of 1CCS was 1,195.3 MPa, but the other were less than the yield range of the materials. At Pauwels 60 ° , the MBS of 3CCS group was 128.6 Mpa, which had the risk of failure. In 2CCS + MBP group, the parameters were 124.2 MPa (MBS), 602.5 MPa (MIS), 0.75 mm (MBD) and 0.48 mm (IFM), The model stability was significantly enhanced after adding MBP. Conclusion: Pauwels type Ⅰ (< 30 ° ) fractures can reduce the number of CCS, and PTS is an appropriate alternative treatment. For Pauwels type Ⅱ fractures ( 30 ° ∼ 50 ° ), the 3CCS fixation method is still recommended. For Pauwels type Ⅲ fractures (> 50 ° ), it is recommended to add MBP to the medial femoral neck and combine with 2CCS to establish a satisfactory fracture healing environment.

M1 polarization enhances the antitumor activity of chimeric antigen receptor macrophages in solid tumors.

BACKGROUND: Chimeric antigen receptor macrophage (CAR-M) therapy is a novel cancer immunotherapy approach that integrates CAR structure and macrophage functions. CAR-M therapy has shown unique and impressive antitumor effects in immunotherapy for solid tumors. However, the polarization state of macrophages can affect the antitumor effect of CAR-M. We hypothesized that the antitumor activity of CAR-Ms may be further improved after inducing M1-type polarization. METHODS: In this report, we constructed a novel HER2-targeting CAR-M, which was composed of humanized anti-HER2 scFv, CD28 hinge region and FcγRI transmembrane domain and intracellular domain. Phagocytosis, tumor-killing capacities, and cytokine release of CAR-Ms were detected with or without M1-polarization pretreatment. Several syngeneic tumor models were used to monitor the in vivo antitumor activity of M1-polarized CAR-Ms. RESULTS: After polarization with LPS combined with interferon-γ in vitro, we found that the phagocytic and tumor-killing capacities of CAR-Ms against target cells were significantly enhanced. The expression of costimulatory molecules and proinflammatory cytokines was also significantly increased after polarization. By establishing several syngeneic tumor models in vivo, we also demonstrated that infusing polarized M1-type CAR-Ms could effectively suppress tumor progression and prolong the survival of tumor-bearing mice with enhanced cytotoxicity. CONCLUSIONS: We demonstrated that our novel CAR-M can effectively eliminate HER2-positive tumor cells both in vitro and in vivo, and M1 polarization significantly enhanced the antitumor ability of CAR-M, resulting in a stronger therapeutic effect in solid cancer immunotherapy.

Simplified Construction of Engineered Bacillus subtilis Host for Improved Expression of Proteins Harboring Noncanonical Amino Acids.

The UAG-based genetic code expansion (GCE) enables site-specific incorporation of noncanonical amino acids (ncAAs) harboring novel chemical functionalities in specific target proteins. However, most GCE studies were done in several whole-genome engineered chassis cells whose hundreds of UAG stop codons were systematically edited to UAA to avoid readthrough in protein synthesis in the presence of GCE. The huge workload of removing all UAG limited the application of GCE in other microbial cell factories (MCF) such as Bacillus subtilis, which has 607 genes ended with UAG among its 4245 coding genes. Although the 257 essential genes count only 6.1% of the genes in B. subtilis, they transcribe 12.2% of the mRNAs and express 52.1% of the proteins under the exponential phase. Here, we engineered a strain named Bs-22 in which all 22 engineerable UAG stop codons in essential genes were edited to UAA via CRISPR/Cas9-mediated multiple-site engineering to minimize the negative effect of GCE on the expression of essential genes. Besides the process of constructing GCE-compatible B. subtilis was systematically optimized. Compared with wild-type B. subtilis (Bs-WT), the fluorescence signal of the eGFP expression could enhance 2.25-fold in Bs-22, and the production of protein tsPurple containing l-(7-hydroxycoumarin-4-yl) ethylglycine (Cou) was increased 2.31-fold in Bs-22. We verified that all purified tsPurple proteins from Bs-22 contained Cou, indicating the excellent fidelity of the strategy. This proof-of-concept study reported efficient overexpression of ncAA-rich proteins in MCF with minimized engineering, shedding new light on solving the trade-off between efficiency and workload.