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Abstract Background Macrophage-based cell therapy is promising in solid tumors, but the efficient acquisition of macrophages remains a challenge. Induced pluripotent stem cell (iPSC)-induced macrophages are a valuable source, but time-consuming and costly. The application of reprogramming technologies allows for the generation of macrophages from somatic cells, thereby facilitating the advancement of cell-based therapies for numerous malignant diseases. Methods The composition of CD45+ myeloid-like cell complex (MCC) and induced macrophage (iMac) were analyzed by flow cytometry and single-cell RNA sequencing. The engraftment capacity of CD45+ MCC was evaluated by two transplantation assays. Regulation of c-Myc on MafB was evaluated by ChIP-qPCR and promoter reporter and dual luciferase assays. The phenotype and phagocytosis of iMac were explored by flow cytometry and immunofluorescence. Leukemia, breast cancer, and patient-derived tumor xenograft models were used to explore the anti-tumor function of iMac. Results Here we report on the establishment of a novel methodology allowing for reprogramming fibroblasts into functional macrophages with phagocytic activity by c-Myc overexpression. Fibroblasts with ectopic expression of c-Myc in iPSC medium rapidly generated CD45+ MCC intermediates with engraftment capacity as well as the repopulation of distinct hematopoietic compartments. MCC intermediates were stably maintained in iPSC medium and continuously generated functional and highly pure iMac just by M-CSF cytokine stimulation. Single-cell transcriptomic analysis of MCC intermediates revealed that c-Myc up-regulated the expression of MafB, a major regulator of macrophage differentiation, to promote macrophage differentiation. Characterization of the iMac activity showed NF-κB signaling activation and a pro-inflammatory phenotype. iMac cells displayed significantly increased in vivo persistence and inhibition of tumor progression in leukemia, breast cancer, and patient-derived tumor xenograft models. Conclusions Our findings demonstrate that c-Myc alone is enough to reprogram fibroblasts into functional macrophages, supporting that c-Myc reprogramming strategy of fibroblasts can help circumvent long-standing obstacles to gaining “off-the-shelf” macrophages for anti-cancer immunotherapy.

Abstract Background Neuroblastoma (NB) patients with amplified MYCN often face a grim prognosis and are resistant to existing therapies, yet MYCN protein is considered undruggable. KAP1 (also named TRIM28) plays a crucial role in multiple biological activities. This study aimed to investigate the relationship between KAP1 and MYCN in NB. Methods Transcriptome analyses and luciferase reporter assay identified that KAP1 was a downstream target of MYCN. The effects of KAP1 on cancer cell proliferation and colony formation were explored using the loss-of-function assays in vitro and in vivo. RNA stability detection was used to examine the influence of KAP1 on MYCN expression. The mechanisms of KAP1 to maintain MYCN mRNA stabilization were mainly investigated by mass spectrum, immunoprecipitation, RIP-qPCR, and western blotting. In addition, a xenograft mouse model was used to reveal the antitumor effect of STM2457 on NB. Results Here we identified KAP1 as a critical regulator of MYCN mRNA stability by protecting the RNA N6-methyladenosine (m6A) reader YTHDC1 protein degradation. KAP1 was highly expressed in clinical MYCN-amplified NB and was upregulated by MYCN. Reciprocally, KAP1 knockdown reduced MYCN mRNA stability and inhibited MYCN-amplified NB progression. Mechanistically, KAP1 regulated the stability of MYCN mRNA in an m6A-dependent manner. KAP1 formed a complex with YTHDC1 and RNA m6A writer METTL3 to regulate m6A-modified MYCN mRNA stability. KAP1 depletion decreased YTHDC1 protein stability and promoted MYCN mRNA degradation. Inhibiting MYCN mRNA m6A modification synergized with chemotherapy to restrain tumor progression in MYCN-amplified NB. Conclusions Our research demonstrates that KAP1, transcriptionally activated by MYCN, forms a complex with YTHDC1 and METTL3, which in turn maintain the stabilization of MYCN mRNA in an m6A-dependent manner. Targeting m6A modification by STM2457, a small-molecule inhibitor of METTL3, could downregulate MYCN expression and attenuate tumor proliferation. This finding provides a new alternative putative therapeutic strategy for MYCN-amplified NB.

Abstract Background Cancer stem-like cell is a key barrier for therapeutic resistance and metastasis in various cancers, including breast cancer, yet the underlying mechanisms are still elusive. Through a genome-wide lncRNA expression profiling, we identified that LINC00115 is robustly upregulated in chemoresistant breast cancer stem-like cells (BCSCs). Methods LncRNA microarray assay was performed to document abundance changes of lncRNAs in paclitaxel (PTX)-resistant MDA-MB-231 BCSC (ALDH+) and non-BCSC (ALDH−). RNA pull-down and RNA immunoprecipitation (RIP) assays were performed to determine the binding proteins of LINC00115. The clinical significance of the LINC00115 pathway was examined in TNBC metastatic lymph node tissues. The biological function of LINC00115 was investigated through gain- and loss-of-function studies. The molecular mechanism was explored through RNA sequencing, mass spectrometry, and the CRISPR/Cas9-knockout system. The therapeutic potential of LINC00115 was examined through xenograft animal models. Results LINC00115 functions as a scaffold lncRNA to link SETDB1 and PLK3, leading to enhanced SETDB1 methylation of PLK3 at both K106 and K200 in drug-resistant BCSC. PLK3 methylation decreases PLK3 phosphorylation of HIF1α and thereby increases HIF1α stability. HIF1α, in turn, upregulates ALKBH5 to reduce m6A modification of LINC00115, resulting in attenuated degradation of YTHDF2-dependent m6A-modified RNA and enhanced LINC00115 stability. Thus, this positive feedback loop provokes BCSC phenotypes and enhances chemoresistance and metastasis in triple-negative breast cancer. SETDB1 inhibitor TTD-IN with LINC00115 ASO sensitizes PTX-resistant cell response to chemotherapy in a xenograft animal model. Correlative expression of LINC00115, methylation PLK3, SETDB1, and HIF1α are prognostic for clinical triple-negative breast cancers. Conclusions Our findings uncover LINC00115 as a critical regulator of BCSC and highlight targeting LINC00115 and SETDB1 as a potential therapeutic strategy for chemotherapeutic resistant breast cancer.

Abstract The factors driving glioma progression remain poorly understood. Here, the epigenetic regulator TRIM24 is identified as a driver of glioma progression, where TRIM24 overexpression promotes HRasV12 anaplastic astrocytoma (AA) progression into epithelioid GBM (Ep‐GBM)‐like tumors. Co‐transfection of TRIM24 with HRasV12 also induces Ep‐GBM‐like transformation of human neural stem cells (hNSCs) with tumor protein p53 gene (TP53) knockdown. Furthermore, TRIM24 is highly expressed in clinical Ep‐GBM specimens. Using single‐cell RNA‐sequencing (scRNA‐Seq), the authors show that TRIM24 overexpression impacts both intratumoral heterogeneity and the tumor microenvironment. Mechanically, HRasV12 activates phosphorylated adaptor for RNA export (PHAX) and upregulates U3 small nucleolar RNAs (U3 snoRNAs) to recruit Ku‐dependent DNA‐dependent protein kinase catalytic subunit (DNA‐PKcs). Overexpressed TRIM24 is also recruited by PHAX to U3 snoRNAs, thereby facilitating DNA‐PKcs phosphorylation of TRIM24 at S767/768 residues. Phosphorylated TRIM24 induces epigenome and transcription factor network reprogramming and promotes Ep‐GBM‐like transformation. Targeting DNA‐PKcs with the small molecule inhibitor NU7441 synergizes with temozolomide to reduce Ep‐GBM tumorigenicity and prolong animal survival. These findings provide new insights into the epigenetic regulation of Ep‐GBM‐like transformation and suggest a potential therapeutic strategy for patients with Ep‐GBM.

Abstract ZF2001, a protein subunit vaccine against coronavirus disease 2019 (COVID-19), contains recombinant tandem repeat of dimeric receptor-binding domain (RBD) protein of the SARS-CoV-2 spike protein with an aluminium-based adjuvant. During the development of this vaccine, two nonclinical studies were conducted to evaluate female fertility, embryo-fetal development, and postnatal developmental toxicity in Sprague‒Dawley rats according to the ICH S5 (R3) guideline. In Study 1 (embryo-fetal developmental toxicity, EFD), 144 virgin female rats were randomly assigned into four groups and received three doses of vaccine (25 μg or 50 μg RBD protein/dose, containing the aluminium-based adjuvant), the aluminium-based adjuvant or a sodium chloride injection administered intramuscularly on days 21 and 7 prior to mating and on gestation day (GD) 6. In Study 2 (pre- and postnatal developmental toxicity, PPND), ZF2001 at a dose of 25 μg RBD protein/dose or sodium chloride injection was administered intramuscularly to female rats (n = 28 per group) 7 days prior to mating and on GD 6, GD 20 and postnatal day (PND) 10. There were no obvious adverse effects in dams, except for local injection site reactions related to the aluminium-based adjuvant (yellow nodular deposits in the interstitial muscle fibres). There were also no effects of ZF2001 on the mating performance, fertility or reproductive performance of parental females, embryo-fetal development, postnatal survival, growth, physical development, reflex ontogeny, behavioural and neurofunctional development, or reproductive performance of the offspring. The strong immune responses associated with binding and neutralising antibodies were both confirmed in dams and fetuses or offspring in these two studies. These results would support clinical trials or the use of ZF2001 in maternal immunisation campaigns, including those involving women with childbearing potential, regardless of pregnancy status.

Abstract Purpose The objective of this study was to investigate the molecular characteristics and potential resistance mechanisms of linezolid-resistant (LZR) Staphylococcus capitis isolates from a tertiary hospital in China. Methods S. capitis isolates were obtained from clinical patient specimens; three of the isolates came from blood cultures and one from the hydrothorax. The agar dilution and E-test methods were used to identify antibiotic resistance. The chloramphenicol-florfenicol resistance (cfr) gene carrier status of the strains was determined by PCR. Whole-genome sequencing (WGS) was used to identify point mutations and L3, L4, and L22 mutations and to study the genetic environment of the cfr gene and the relationships between strains. Results The 4 isolates obtained in this study were all linezolid-resistant Staphylococcus strains. A similar of susceptibility profile pattern was observed in all four S. capitis strains, each of which exhibited a multidrug-resistant phenotype. A potentially novel mutation, C2128T, was identified, and the cfr genes of S. capitis strains were all positive. Additionally, the same mutations (C2128T and G2600T) were identified in all 23S rRNA sequences of the isolates, whereas mutations were lacking in the L3, L4, and L22 ribosomal proteins. The genetic environments surrounding cfr were identical in all four isolates. A schematic diagram of the phylogenetic tree showed that they were closely related to AYP1020, CR01, and TW2795, and a total of seven drug resistance genes were identified in these strains. Conclusions The study indicated that the resistance of the Staphylococcus capitis strains to linezolid was caused by multiple mechanisms, and a potential novel mutation, C2128T, that may have an impact on bacterial resistance was identified.

Dendrobium officinale Kimura et Migo (D. officinale) is a traditional medicinal and food homologous plant that has been used for thousands of years in folk medicine and nutritious food. Recent studies have shown that polysaccharide is one of the main biologically active components in D. officinale. D. officinale polysaccharides possess several biological activities, such as anti-oxidant, heptatoprotective, immunomodulatory, gastrointestinal protection, hypoglycemic, and anti-tumor activities. In the past decade, polysaccharides have been isolated from D. officinale by physical and enzymatic methods and have been subjected to structural characterization and activity studies. Progress in extraction, purification, structural characterization, bioactivity, structure-activity relationship, and possible bioactivity mechanism of polysaccharides D. officinale were reviewed. In order to provide reference for the in-depth study of D. officinale polysaccharides and the application in functional food and biomedical research.

Phosphatidylinositol 3-kinase catalyzes the reaction from PI(4,5)P2 to PI(3,4,5)P3 and is encoded by the age-1 gene known to regulate lifespan. Here the researchers found that the metabolite myo-inositol, which can be converted to PI(3,4,5)P3 extends worm lifespan and alleviates worm as well as mouse health decline during aging.

Although the new coronavirus disease 2019 (COVID-19) outbreak occurred in late 2019, it is still endemic worldwide, and has become a global public health problem. Vaccination against SARS-CoV-2 is considered to be the most effective intervention to prevent the spread of COVID-19. ZF2001 is a recombinant protein vaccine based on SARS-CoV-2 receptor-binding domain (RBD) subunit which contains aluminum adjuvant. In order to advance our research on ZF2001 into clinical trial, we investigated the general toxicity and immunogenicity of ZF2001 in cynomolgus monkeys and assessed the possible target organs for vaccine-induced toxicity. In the present research, we observed no significant systemic toxicities and abnormal cardiovascular and respiratory events following four times injections of intramuscular ZF2001 in cynomolgus monkeys. Histological examination revealed recoverable inflammatory changes in quadricep muscle and adjacent lymph node at the vaccine injection site. As expected, the vaccine can produce a strongly specific binding antibody and neutralizing antibodies in cynomolgus monkeys after inoculation. Taken together, our regulatory toxicology research proves the safety and immunogenicity of the ZF2001 vaccine, supporting its entry into large scale clinical trials.

Recent studies have shown difficulties in balancing convergence and diversity for many-objective optimization problems with various types of Pareto fronts. This paper proposes an adaptive reference vector based evolutionary algorithm for many-objective optimization, termed as ARVEA. The ARVEA develops a reference vector adaptation method, which can adapt different types of Pareto fronts by adjusting the distribution of reference vectors. Besides, this algorithm adopts Pareto dominance as the first selection criterion, and the achievement scalarizing function (ASF) is introduced as the secondary selection criterion. The empirical results demonstrate that the proposed ARVEA has good performance for solving problems with various types of Pareto fronts, surpassing several state-of-the-art evolutionary algorithms designed for many-objective optimization.

To study the lubrication and wear reduction characteristics of the QAL9-4 and 17-4pH textured friction pair during water lubrication, the dynamic pressure bearing and cavitation characteristics were analyzed using a computational fluid dynamics method. Then, based on 13 groups of samples with different depths, diameters, and area ratios, relevant frictional and wear tests were carried out. The results showed that the texture affected the pressure distribution in the textured region and improved the liquid film bearing capacity, which increased gradually as the equivalent Reynolds number increased. Under test conditions, the texture parameters had a significant influence on lubrication and wear reduction performance. The optimum texture diameter, the depth, and the area ratio were 300 µm, 40 µm, and 10%, respectively. The wear tracks on the textured specimens were small in size and shallow in depth compared to those on the non-textured specimens. The wear tracks on the textured specimens were regional, and there were more wear tracks in the area without micro-dimples. In summary, it was feasible to apply surface texture to the QAL9-4 and 17-4PH friction pair to improve the lubrication and wear reduction characteristics with water lubrication.

Vertical tanks are commonly used appliances for liquids, and its capacity is very important for quantitative liquid ratio and liquid trade. In order to measure the capacity of vertical tanks more conveniently, this paper proposes a vertical tank capacity measurement method based on Monte Carlo Method. The method arranges a plurality of sensor points on the inner surface of the tank, and then performs Monte Carlo tests by generating a large number of random sample points, and finally calculates the capacity by counting the sample points that meet the criterion. The criterion for whether a sample point is located in the tank, which is the core issue, is established with the coordinates of sensor points and the distance between different sensor points along the surface of the tank. The results show that the absolute error of the measurement results of the proposed method does not exceed ±0.0003[m3], and the absolute error of capacity per unit volume has a linear relationship with the height of the vertical tank, and has little effect with the radial size of the vertical tank.

Combinatorial effects of epigenetic modifications on transcription activity have been proposed as "histone codes". However, it is unclear whether there also exist inter-nucleosomal communications among epigenetic modifications at single nucleosome level, and if so, what functional roles they play. Meanwhile, how clear nucleosome patterns, such as nucleosome phasing and depletion, are formed at functional regions remains an intriguing enigma. To address these questions, we developed a Bayesian network model for interactions among different histone modifications across neighboring nucleosomes, based on the framework of dynamic Bayesian network (DBN). From this model, we found that robust inter-nucleosomal interactions exist around transcription start site (TSS), transcription termination sites (TTS) or around CTCF binding sites; and these inter-nucleosomal interactions are often involved in transcription regulation. In addition to these general principles, DBN also uncovered a novel specific epigenetic interaction between H2A.Z and H4K20me1 on neighboring nucleosomes, involved in nucleosome free region (NFR) and nucleosome phasing establishment or maintenance. The level of negative correlation between neighboring H2A.Z and H4K20me1 strongly correlate with the size of NFR and the strength of nucleosome phasing around TSS. Our study revealed inter-nucleosomal communications as important players in signal propagation, chromatin remodeling and transcription regulation.

Summary: Multiple sampling strategies to cover different or dynamic stages of malignant continuum with organoid cultures provide a valuable platform for epithelium homeostasis, transformation, and cancer progression. Here, we present a protocol to initiate, culture, passage, and characterize organoids from normal and cancer-prone human esophageal tissues. We describe steps for multiple sampling of malignant continuum and the initiation and maintenance of multi-stage organoids. We then detail procedures for the histological characterization of organoids and co-culture systems based on organoids and stromal cells.For complete details on the use and execution of this protocol, please refer to Chen et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.