"Cell Disk" DNA Storage System Capable of Random Reading and Rewriting.

DNA has emerged as an appealing material for information storage due to its great storage density and durability. Random reading and rewriting are essential tasks for practical large-scale data storage. However, they are currently difficult to implement simultaneously in a single DNA-based storage system, strongly limiting their practicability. Here, a "Cell Disk" storage system is presented, achieving high-density in vivo DNA data storage that enables both random reading and rewriting. In this system, each yeast cell is used as a chamber to store information, similar to a "disk block" but with the ability to self-replicate. Specifically, each genome of yeast cell has a customized CRISPR/Cas9-based "lock-and-key" module inserted, which allows selective retrieval, erasure, or rewriting of the targeted cell "block" from a pool of cells ("disk"). Additionally, a codec algorithm with lossless compression ability is developed to improve the information density of each cell "block". As a proof of concept, target-specific reading and rewriting of the compressed data from a mimic cell "disk" comprising up to 105 "blocks" are demonstrated and achieve high specificity and reliability. The "Cell Disk" system described here concurrently supports random reading and rewriting, and it should have great scalability for practical data storage use.

Large-scale pathway reconstruction and colorimetric screening accelerate cellular metabolism engineering.

The capability to manipulate and analyze hard-wired metabolic pathways sets the pace at which we can engineer cellular metabolism. Here, we present a framework to extensively rewrite the central metabolic pathway for malonyl-CoA biosynthesis in yeast and readily assess malonyl-CoA output based on pathway-scale DNA reconstruction in combination with colorimetric screening (Pracs). We applied Pracs to generate and test millions of enzyme variants by introducing genetic mutations into the whole set of genes encoding the malonyl-CoA biosynthetic pathway and identified hundreds of beneficial enzyme mutants with increased malonyl-CoA output. Furthermore, the synthetic pathways reconstructed by randomly integrating these beneficial enzyme variants generated vast phenotypic diversity, with some displaying higher production of malonyl-CoA as well as other metabolites, such as carotenoids and betaxanthin, thus demonstrating the generic utility of Pracs to efficiently orchestrate central metabolism to optimize the production of different chemicals in various metabolic pathways. Pracs will be broadly useful to advance our ability to understand and engineer cellular metabolism.

c-Kit signaling potentiates CAR T cell efficacy in solid tumors by CD28- and IL-2-independent co-stimulation.

The limited efficacy of chimeric antigen receptor (CAR) T cell therapy for solid tumors necessitates engineering strategies that promote functional persistence in an immunosuppressive environment. Herein, we use c-Kit signaling, a physiological pathway associated with stemness in hematopoietic progenitor cells (T cells lose expression of c-Kit during differentiation). CAR T cells with intracellular expression, but no cell-surface receptor expression, of the c-Kit D816V mutation (KITv) have upregulated STAT phosphorylation, antigen activation-dependent proliferation and CD28- and interleukin-2-independent and interferon-γ-mediated co-stimulation, augmenting the cytotoxicity of first-generation CAR T cells. This translates to enhanced survival, including in transforming growth factor-ß-rich and low-antigen-expressing solid tumor models. KITv CAR T cells have equivalent or better in vivo efficacy than second-generation CAR T cells and are susceptible to tyrosine kinase inhibitors (safety switch). When combined with CD28 co-stimulation, KITv co-stimulation functions as a third signal, enhancing efficacy and providing a potent approach to treat solid tumors.

Multiphoton non-local quantum interference controlled by an undetected photon.

The interference of quanta lies at the heart of quantum physics. The multipartite generalization of single-quanta interference creates entanglement, the coherent superposition of states shared by several quanta. Entanglement allows non-local correlations between many quanta and hence is a key resource for quantum information technology. Entanglement is typically considered to be essential for creating non-local quantum interference. Here, we show that this is not the case and demonstrate multiphoton non-local quantum interference that does not require entanglement of any intrinsic properties of the photons. We harness the superposition of the physical origin of a four-photon product state, which leads to constructive and destructive interference with the photons' mere existence. With the intrinsic indistinguishability in the generation process of photons, we realize four-photon frustrated quantum interference. This allows us to observe the following noteworthy difference to quantum entanglement: We control the non-local multipartite quantum interference with a photon that we never detect, which does not require quantum entanglement. These non-local properties pave the way for the studies of foundations of quantum physics and potential applications in quantum technologies.

Application of proteomics to determine the mechanism of ozone on sweet cherries (Prunus avium L.) by time-series analysis.

This research investigated the mechanism of ozone treatment on sweet cherry (Prunus avium L.) by Lable-free quantification proteomics and physiological traits. The results showed that 4557 master proteins were identified in all the samples, and 3149 proteins were common to all groups. Mfuzz analyses revealed 3149 candidate proteins. KEGG annotation and enrichment analysis showed proteins related to carbohydrate and energy metabolism, protein, amino acids, and nucleotide sugar biosynthesis and degradation, and fruit parameters were characterized and quantified. The conclusions were supported by the fact that the qRT-PCR results agreed with the proteomics results. For the first time, this study reveals the mechanism of cherry in response to ozone treatment at a proteome level.

Targeting T-cell metabolism to boost immune checkpoint inhibitor therapy.

Immune checkpoint inhibitors (ICIs) have shown promising therapeutic effects in the treatment of advanced solid cancers, but their overall response rate is still very low for certain tumor subtypes, limiting their clinical scope. Moreover, the high incidence of drug resistance (including primary and acquired) and adverse effects pose significant challenges to the utilization of these therapies in the clinic. ICIs enhance T cell activation and reverse T cell exhaustion, which is a complex and multifactorial process suggesting that the regulatory mechanisms of ICI therapy are highly heterogeneous. Recently, metabolic reprogramming has emerged as a novel means of reversing T-cell exhaustion in the tumor microenvironment; there is increasing evidence that T cell metabolic disruption limits the therapeutic effect of ICIs. This review focuses on the crosstalk between T-cell metabolic reprogramming and ICI therapeutic efficacy, and summarizes recent strategies to improve drug tolerance and enhance anti-tumor effects by targeting T-cell metabolism alongside ICI therapy. The identification of potential targets for altering T-cell metabolism can significantly contribute to the development of methods to predict therapeutic responsiveness in patients receiving ICI therapy, which are currently unknown but would be of great clinical significance.

Next-generation immunotherapy for solid tumors: combination immunotherapy with crosstalk blockade of TGFß and PD-1/PD-L1.

INTRODUCTION: In solid tumor immunotherapy, less than 20% of patients respond to anti-programmed cell death 1 (PD-1)/programmed cell death 1 ligand 1 (PD-L1) agents. The role of transforming growth factor ß (TGFß) in diverse immunity is well-established; however, systemic blockade of TGFß is associated with toxicity. Accumulating evidence suggests the role of crosstalk between TGFß and PD-1/PD-L1 pathways. AREAS COVERED: We focus on TGFß and PD-1/PD-L1 signaling pathway crosstalk and the determinant role of TGFß in the resistance of immune checkpoint blockade. We provide the rationale for combination anti-TGFß and anti-PD-1/PD-L1 therapies for solid tumors and discuss the current status of dual blockade therapy in preclinical and clinical studies. EXPERT OPINION: The heterogeneity of tumor microenvironment across solid tumors complicates patient selection, treatment regimens, and response and toxicity assessment for investigation of dual blockade agents. However, clinical knowledge from single-agent studies provides infrastructure to translate dual blockade therapies. Dual TGFß and PD-1/PD-L1 blockade results in enhanced T-cell infiltration into tumors, a primary requisite for successful immunotherapy. A bifunctional fusion protein specifically targets TGFß in the tumor microenvironment, avoiding systemic toxicity, and prevents interaction of PD-1+ cytotoxic cells with PD-L1+ tumor cells.

Regional CAR T cell therapy: An ignition key for systemic immunity in solid tumors.

The success of chimeric antigen receptor (CAR) T cell therapy in solid tumors, unlike in hematologic malignancies, is limited by inadequate tumor infiltration and T cell dysfunction and exhaustion. Regional delivery of CAR T cells in patients with solid tumors is safe and feasible; promotes infiltration, proliferation, and trafficking; and ignites functionally persisting systemic immunity.

Safety assessment of crude saponins from Chenopodium quinoa willd. husks: 90-day oral toxicity and gut microbiota & metabonomics study in rats.

The subchronic toxicity of saponins of Chenopodium quinoa Willd. husks in healthy adult Sprague-Dawley (SD) rats was explored. Female and male rats were randomly divided into 0, 5, 50, and 500 mg/kg body weight (BW)/day groups. Subchronic general toxicity, metabonomics and gut microbiota were assessed. The rats treated with saponins weighed less and had lower blood sugar levels (P < 0.05). Thirty-two differential metabolites were found in female rats and 23 in male rats. Saponins also led to changes in metabonomics. Slight necrosis was observed in the intestinal mucosa, which was associated with an increase in the gut microbiota diversity of female rats in the high-dose saponin treatment group and metabolic changes in the liver and kidney. In conclusion, the toxic effect of quinoa saponins is sex-dependent; however, the no-observed-adverse-effect level for quinoa saponins was evaluated to be under 50 mg/kg BW/day for both sexes in the current study.

Do Engineered Nanomaterials Affect Immune Responses by Interacting With Gut Microbiota?

Engineered nanomaterials (ENMs) have been widely exploited in several industrial domains as well as our daily life, raising concern over their potential adverse effects. While in general ENMs do not seem to have detrimental effects on immunity or induce severe inflammation, their indirect effects on immunity are less known. In particular, since the gut microbiota has been tightly associated with human health and immunity, it is possible that ingested ENMs could affect intestinal immunity indirectly by modulating the microbial community composition and functions. In this perspective, we provide a few pieces of evidence and discuss a possible link connecting ENM exposure, gut microbiota and host immune response. Some experimental works suggest that excessive exposure to ENMs could reshape the gut microbiota, thereby modulating the epithelium integrity and the inflammatory state in the intestine. Within such microenvironment, numerous microbiota-derived components, including but not limited to SCFAs and LPS, may serve as important effectors responsible of the ENM effect on intestinal immunity. Therefore, the gut microbiota is implicated as a crucial regulator of the intestinal immunity upon ENM exposure. This calls for including gut microbiota analysis within future work to assess ENM biocompatibility and immunosafety. This also calls for refinement of future studies that should be designed more elaborately and realistically to mimic the human exposure situation.

Purification of Ethyl Linoleate from Foxtail Millet (Setaria italica) Bran Oil via Urea Complexation and Molecular Distillation.

Foxtail millet (Setaria italica) bran oil is rich in linoleic acid, which accounts for more than 60% of its lipids. Ethyl linoleate (ELA) is a commercially valuable compound with many positive health effects. Here, we optimized two ELA processing steps, urea complexation (UC) and molecular distillation (MD), using single-factor and response surface analyses. We aimed to obtain a highly concentrated ELA at levels that are permitted by current regulations. We identified the optimal conditions as follows: 95% ethanol-to-urea ratio = 15:1 (w/w), urea-to-fatty acid ratio = 2.5:1 (w/w), crystallization time = 15 h, and crystallization temperature = -6 °C. Under these optimal UC conditions, ELA concentration reached 45.06%. The optimal MD purification conditions were established as follows: distillation temperature = 145 °C and vacuum pressure = 1.0-5.0 × 10-2 mbar. Under these conditions, ELA purity increased to 60.45%. Together, UC and MD were effective in improving the total concentration of ELA in the final product. This work shows the best conditions for separating and purifying ELA from foxtail millet bran oil by UC and MD.

Stem-leaves of Panax as a rich and sustainable source of less-polar ginsenosides: comparison of ginsenosides from Panax ginseng, American ginseng and Panax notoginseng prepared by heating and acid treatment.

BACKGROUND: Ginsenosides, which have strong biological activities, can be divided into polar or less-polar ginsenosides. METHODS: This study evaluated the phytochemical diversity of the saponins in Panax ginseng (PG) root, American ginseng (AG) root, and Panax notoginseng (NG) root; the stem-leaves from Panax ginseng (SPG) root, American ginseng (SAG) root, and Panax notoginseng (SNG) root as well as the saponins obtained following heating and acidification [transformed Panax ginseng (TPG), transformed American ginseng (TAG), transformed Panax notoginseng (TNG), transformed stem-leaves from Panax ginseng (TSPG), transformed stem-leaves from American ginseng (TSAG), and transformed stem-leaves from Panax notoginseng (TSNG)]. The diversity was determined through the simultaneous quantification of the 16 major ginsenosides. RESULTS: The content of ginsenosides in NG was found to be higher than those in AG and PG, and the content in SPG was greater than those in SNG and SAG. After transformation, the contents of polar ginsenosides in the raw saponins decreased, and contents of less-polar compounds increased. TNG had the highest levels of ginsenosides, which is consistent with the transformation of ginseng root. The contents of saponins in the stem-leaves were higher than those in the roots. The transformation rate of SNG was higher than those of the other samples, and the loss ratios of total ginsenosides from NG (6%) and SNG (4%) were the lowest among the tested materials. In addition to the conversion temperature, time, and pH, the crude protein content also affects the conversion to rare saponins. The proteins in Panax notoginseng allowed the highest conversion rate. CONCLUSION: Thus, the industrial preparation of less-polar ginsenosides from SNG is more efficient and cheaper.

In vivo acute toxicity and mutagenic analysis of crude saponins from Chenopodium quinoa Willd husks.

Background: As a functional food factor, quinoa saponins are valuable as additives and in medical care, pharmaceutical development, cosmetics and other fields. However, few studies have investigated the toxicity of saponins. The main purpose of this study was to evaluate the toxicity of crude saponins extracted from quinoa husks. Thus, acute toxicity and excretion experiments were carried out in rats. The Ames test, micronucleus test and mouse sperm aberration test were carried out in mice. Results: In the acute toxicity study, the obtained LD50 was more than 10 g per kg per bw for both sexes, the food intake of all rats decreased over a period of time, and some rats developed diarrhea. In the case of large-dose gavage, the saponin excretion time in rats was approximately four days. When the dosage was 10 mg kg-1, quinoa saponins were hydrolyzed into aglycone within 24 hours and excreted out of the body. The results of the mutagenicity experiment showed that saponins had no mutagenicity in mice. Conclusion: This work has demonstrated that quinoa saponins have limited acute toxicity effects, which provides a theoretical basis for their rational utilization.

Label-free quantitative proteomics analysis of jujube (Ziziphus jujuba Mill.) during different growth stages.

Chinese jujube (Zizyphus jujuba Mill.), a member of the Rhamnaceae family with favorable nutritional and flavor quality, exhibited characteristic climacteric changes during its fruit growth stage. Therefore, fruit samples were harvested at four developmental stages on days 55 (young fruits), 76 (white-mature fruits), 96 (half-red fruits), and 116 (full-red fruits) after flowering (DAF). This study then investigated those four growth stage changes of the jujube proteome using label-free quantification proteomics. The results identified 4762 proteins in the samples, of which 3757 proteins were quantified. Compared with former stages, the stages examined were designated as "76 vs. 55 DAF" group, "96 vs. 76 DAF" group, and "116 vs. 96 DAF" group. Gene Ontology (GO) and KEGG annotation and enrichment analysis of the differentially expressed proteins (DEPs) showed that 76 vs. 55 DAF group pathways represented amino sugar, nucleotide sugar, ascorbate, and aldarate metabolic pathways. These pathways were associated with cell division and resistance. In the study, the jujube fruit puffing slowed down and attained a stable growth stage in the 76 vs. 55 DAF group. However, fatty acid biosynthesis and phenylalanine metabolism was mainly enriched in the 96 vs. 76 DAF group. Fatty acids are precursors of aromatic substances and fat-soluble pigments in fruit. The upregulation of differential proteins at this stage indicates that aromatic compounds were synthesized in large quantities at this stage and that fruit would enter the ripening stage. During the ripening stage, 55 DEPs were identified to be involved in photosynthesis and flavonoid biosynthesis in the 116 vs. 96 DAF group. Also, the fruit entered the mature stage, which showed that flavonoids were produced in large quantities. Furthermore, the color of jujube turned red, and photosynthesis was significantly reduced. Hence, a link was established between protein profiles and growth phenotypes, which will help improve our understanding of jujube fruit growth at the proteomic level.

Synthesis, characteristics and medical applications of plant nanomaterials.

MAIN CONCLUSION: The recent preparations of metal nanoparticles using plant extracts as reducing agents are summarized here. The synthesis and characterization of plant-metal nanomaterials and the progress in antibacterial and anti-inflammatory medical applications are detailed, providing a new vision for plant-based medical applications. The medical application of plant-metal nanoparticles is becoming a research hotspot. Compared with traditional preparation methods, the synthesis of plant-metal nanoparticles is less toxic and more eco-friendly, increasing application potential. Highly efficient plant-metal nanoparticles are usually smaller than 100 nm. This review describes the synthesis, characterization and bioactivities of gold- and silver-plant nanoparticles as examples and clearly explained their antibacterial and anticancer mechanisms. An analysis of actual cases shows that the synthetic method and type of plant extract affect the activities of the products.

Luteolin, an aryl hydrocarbon receptor ligand, suppresses tumor metastasis in vitro and in vivo.

Estrogen receptor (ER)­negative breast tumors are associated with low survival rates, which is related to their ability to grow and metastasize into distal organs. The aryl hydrocarbon receptor (AhR), a ligand­activated transcription factor that is involved in several biological processes, is a promising anti­metastatic target. Luteolin, a non­toxic naturally occurring plant flavonoid with diverse biological activities, has been demonstrated to be effective against certain types of cancer, and has also been described as a ligand of AhR. In the present study, various cancer cell lines were first investigated following treatment with luteolin, and luteolin exhibited the lowest IC50 in MDA­MB­231 cells. Then, the efficiency of luteolin in suppressing the metastasis of ER­negative breast cancer in vitro was assessed. MDA­MB­231 cells were treated with luteolin in vitro. Subsequently, MTT assay and flow cytometry were used to detect cell viability, the cell cycle and apoptosis, and a Transwell assay was used to evaluate cell invasion. In addition, reverse transcription­semi­quantitative PCR and western blot were performed to detect the mRNA and protein expression levels of matrix metalloproteinase (MMP)­2 and MMP­9. In addition, the number of surface tumor nodules was measured in vivo, in mice bearing B16­F10 tumors, following treatment with luteolin. Luteolin inhibited the viability and induced the apoptosis of MDA­MB­231 cells, which was accompanied by cell cycle arrest. This was associated with a decrease in the expression of the pro­metastatic markers C­X­C chemokine receptor type 4 (CXCR4), MMP­2 and MMP­9, which was reversed by AhR inhibition. Furthermore, it was identified that luteolin could inhibit the metastasis in a B16F10 mouse xenograft model, and the levels of MMP­9, MMP­2 and CXCR4 were significantly decreased in the lung tissues isolated from tumor­bearing nude mice following luteolin treatment. In conclusion, luteolin is a potential molecule for inhibiting breast cancer invasion and metastasis, which could have promising clinical applications.

Reducing the damage of quinoa saponins on human gastric mucosal cells by a heating process.

Different food processing methods will influence the structure and activity of compounds. In this work, molecular structure and different content crude saponins that were extracted from quinoa, treated with water soaking, water boiling, and water steaming were analyzed by HPLC. Flow cytometry was employed to investigate the effects of the main saponins on the GES-1 cell line. HPLC/MS analysis revealed that water soaking induced an extensive conversion of polar saponin Qc (424.41 ± 21.11 mg/g) to the less polar compound Qf (247.04 ± 15.71 mg/g). After treatment with 100 µg of Qf instead of Qc for 24 hr, the percentage of dead cells increased from 20.1 ± 2.2% to 86.2 ± 4.8%. One major reason of this result is that less polar saponins could damage membrane integrity more easier than polar saponins. The results indicate that saponin toxicity is enhanced after degradation, so it is necessary to avoid degradation before use.

Prominent immune signatures of T cells are specifically associated with indolent B-cell lymphoproliferative disorders and predict prognosis.

OBJECTIVES: T cells play an essential role in controlling the development of B-cell lymphoproliferative disorders (BLPDs), but the dysfunction of T cells in BLPDs largely remains elusive. METHODS: Using multiplexed flow cytometry, we quantified all major subsets of CD4+ helper T cells (Th) and CD8+ cytotoxic T cells (Tc) in 94 BLPD patients and 66 healthy controls. Statistics was utilised to rank T-cell signatures that distinguished BLPDs from healthy controls and differentially presented between indolent and aggressive categories. RESULTS: By comparing with healthy controls, we found that the indolent but not aggressive type of BLPDs demonstrated a high degree of T-cell activation, showing the increase in type I helper T (Th1) cells and follicular B-helper T (Tfh) cells, both of which strongly associated with the enhanced differentiation of exhaustion-like effector cytotoxic CD8+ T cells expressing PD-1 (Tc exhaustion-like) in indolent BLPDs. Random forest modelling selected a module of T-cell immune signatures best performing binary classification of all BLPD patients. This signature module was composed of low naïve Th cells and high Th1, Tfh and Tc exhaustion-like cells which efficiently identified > 85% indolent cases and was, therefore, assigned as the Indolent Dominant Module of T-cell immune signature. In indolent BLPD patients, a strong bias towards such signatures was found to associate with clinical characteristics of worse prognosis. CONCLUSION: Our study identified a prominent signature of T-cell dysregulation specifically for indolent BLPDs, suggesting Th1, Tfh and Tc exhaustion-like cells represent potential prognostic biomarkers and targets for immunotherapies.

5'-triphosphate siRNA targeting HBx elicits a potent anti-HBV immune response in pAAV-HBV transfected mice.

RNA with 5'-triphosphate (3p-RNA) is recognized by RNA sensor RIG-I (retinoic acid-inducible gene I protein). Previously, we reported that small interfering RNA targeting HBx (3p-siHBx) could confer potent anti-hepatitis B virus (HBV) efficacy via HBx silencing and RIG-I activation. However, the characteristics of innate and adaptive immunity especially exhaustion profiles in the liver microenvironment in response to 3p-siHBx therapy have not been fully elucidated. Here, we observed that 3p-siHBx more significantly inhibited HBV replication in vivo. 3p-siHBx enhanced natural killer (NK) cell activation with KLRG1 and CD69 upregulation and interferon (IFN)-γ secretion. 3p-siHBx significantly reversed the exhaustion phenotype of CD8+ T cells, and augmented CD8+ T cell activation and function. Importantly, 3p-siHBx disrupted the differentiation of myeloid-derived suppressor cells (MDSC) and regulatory T cells (Treg), accompanied by the reduction of the immunosuppressive cytokines interleukin (IL)-10 and transforming growth factor (TGF)-ß. 3p-siHBx also enhanced dendritic cell maturation. Further investigation showed that RIG-I was involved in 3p-siHBx-induced IFN-α, IFN-ß, and IFN-λ production. Moreover, RIG-I activation in HBV+ hepatocytes would improve the recruitment of CD8+ T cells and NK cells. These results reveal that 3p-siHBx therapy can improve the immune microenvironment in HBV-carrier liver and inhibit HBV replication, indicating the potential utility of RIG-I ligands as molecular adjuvants for viral vaccines or candidate drugs.

miR-146a negatively regulates NK cell functions via STAT1 signaling.

It is known that natural killer (NK) cell function is downregulated in chronic hepatitis B (CHB)-infected patients and in hepatic carcinoma (HCC) patients, but the mechanisms underlying this functional downregulation are largely unclear. In this study, microRNA (miR)-146a expression increased in NK cells from CHB and HCC patients compared with NK cells from healthy donors, and miR-146a levels were negatively correlated to NK cell functions. Overexpression of miR-146a reduced NK cell-mediated cytotoxicity and the production of interferon (IFN)-γ and tumor necrosis factor-α, which were reversed upon inhibition of miR-146a. In NK cells, miR-146a expression was induced by interleukin (IL)-10 and transforming growth factor-ß, but reduced after treatment with interleukin-12, IFN-α and IFN-ß. We further revealed that miR-146a regulated NK cell functions by targeting STAT1. Taken together, upregulated miR-146a expression, at least partially, attributes to NK cell dysfunction in CHB and HCC patients. Therefore, miR-146a may become a therapeutic target with great potential to ameliorate NK cell functions in liver disease.