USP8-governed GPX4 homeostasis orchestrates ferroptosis and cancer immunotherapy.

Ferroptosis is an iron-dependent type of regulated cell death resulting from extensive lipid peroxidation and plays a critical role in various physiological and pathological processes. However, the regulatory mechanisms for ferroptosis sensitivity remain incompletely understood. Here, we report that homozygous deletion of Usp8 (ubiquitin-specific protease 8) in intestinal epithelial cells (IECs) leads to architectural changes in the colonic epithelium and shortens mouse lifespan accompanied by increased IEC death and signs of lipid peroxidation. However, mice with heterozygous deletion of Usp8 in IECs display normal phenotype and become resistant to azoxymethane/dextran sodium sulfate-induced colorectal tumorigenesis. Mechanistically, USP8 interacts with and deubiquitinates glutathione peroxidase 4 (GPX4), leading to GPX4 stabilization. Thus, USP8 inhibition destabilizes GPX4 and sensitizes cancer cells to ferroptosis in vitro. Notably, USP8 inhibition in combination with ferroptosis inducers retards tumor growth and enhances CD8+ T cell infiltration, which potentiates tumor response to anti-PD-1 immunotherapy in vivo. These findings uncover that USP8 counteracts ferroptosis by stabilizing GPX4 and highlight targeting USP8 as a potential therapeutic strategy to boost ferroptosis for enhancing cancer immunotherapy.

Comparative transcriptome analysis on candidate genes associated with fruiting body growth and development in Lyophyllum decastes.

Lyophyllum decastes is a mushroom that is highly regarded for its culinary and medicinal properties. Its delectable taste and texture make it a popular choice for consumption. To gain a deeper understanding of the molecular mechanisms involved in the development of the fruiting body of L. decastes, we used RNA sequencing to conduct a comparative transcriptome analysis. The analysis encompassed various developmental stages, including the vegetative mycelium, primordial initiation, young fruiting body, medium-size fruiting body, and mature fruiting body stages. A range of 40.1 to 60.6 million clean reads were obtained, and de novo assembly generated 15,451 unigenes with an average length of 1,462.68 bp. Functional annotation of transcriptomes matched 76.84% of the unigenes to known proteins available in at least one database. The gene expression analysis revealed a significant number of differentially expressed genes (DEGs) between each stage. These genes were annotated and subjected to Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses. Highly differentially expressed unigenes were also identified, including those that encode extracellular enzymes, transcription factors, and signaling pathways. The accuracy of the RNA-Seq and DEG analyses was validated using quantitative PCR. Enzyme activity analysis experiments demonstrated that the extracellular enzymes exhibited significant differences across different developmental stages. This study provides valuable insights into the molecular mechanisms that underlie the development of the fruiting body in L. decastes.

An Improved Active Damping Method for Enhancing Robustness of LCL-Type, Grid-Tied Inverters under Weak Grid Conditions.

The conventional proportional-gain-feedback link can only obtain the smallest effective damping region (EDR) due to the control delay among all the active damping methods regarding the capacitor current feedback. The digitally controlled system tends to be unstable when the system resonant frequency reaches the critical frequency caused by the grid impedance variation. To weaken the adverse effect on the system caused by the control delay, phase-lead feedback links are applied along the feedback path to provide phase compensation. By taking the simplicity and reliability of the feedback links into account, this paper proposes an alternative to an ideal differentiator, which consists of the Tustin discrete form of 's' and a digital low-pass filter. This proposed method has an identical phase frequency characteristic as an ideal differentiator but a better magnitude frequency characteristic, and its EDR can reach [0, fs/3]. The system stability analysis is conducted under different resonant frequencies, and under the condition of a weak grid, the co-design approach of the active damper and digital controller is presented. Finally, the experimental results are shown to verify the proposed method.

[Construction of NKG2D CAR-NK92 cells and its killing effect on multiple myeloma cells].

Objective This study aims to construct and identify the chimeric antigen receptor NK92 (CAR-NK92) cells targeting NKG2D ligand (NKG2DL) (secreting IL-15Ra-IL-15) and verify the killing activity of NKG2D CAR-NK92 cells against multiple myeloma cells. Methods The extracellular segment of NKG2D was employed to connect 4-1BB and CD3Z, as well as IL-15Ra-IL-15 sequence to obtain a CAR expression framework. The lentivirus was packaged and transduced into NK92 cells to obtain NKG2D CAR-NK92 cells. The proliferation of NKG2D CAR-NK92 cells was detected by CCK-8 assay, IL-15Ra secretion was detected by ELISA and killing efficiency was detected by lactate dehydrogenase (LDH) assay. The molecular markers of NKp30, NKp44, NKp46, the ratio of apoptotic cell population, CD107a, and the secretion level of granzyme B and perforin were detected using flow cytometry. In addition, the cytotoxic mechanism of NKG2D CAR-NK92 cells on the tumor was verified by measuring the degranulation ability. Moreover, after NKG2D antibody inhibited effector cells and histamine inhibited tumor cells, LDH assay was utilized to detect the effect on cell-killing efficiency. Finally, the multiple myeloma tumor xenograft model was constructed to verify its anti-tumor activity in vivo. Results Lentiviral transduction significantly increased NKG2D expression in NK92 cells. Compared with NK92 cells, the proliferation ability of NKG2D CAR-NK92 cells was weaker. The early apoptotic cell population of NKG2D CAR-NK92 cells was less, and NKG2D CAR-NK92 cells had stronger cytotoxicity to multiple myeloma cells. Additionally, IL-15Ra secretion could be detected in its culture supernatant. NKp44 protein expression in NKG2D CAR-NK92 cells was clearly increased, demonstrating an enhanced activation level. Inhibition test revealed that the cytotoxicity of CAR-NK92 cells to MHC-I chain-related protein A (MICA) and MICB-positive tumor cells was more dependent on the interaction between NKG2D CAR and NKG2DL. After stimulating NKG2D CAR-NK92 cells with tumor cells, granzyme B and perforin expression increased, and NK cells obviously upregulated CD107α. Furthermore, multiple myeloma tumor xenograft model revealed that the tumors of mice treated with NKG2D CAR-NK92 cells were significantly reduced, and the cell therapy did not sensibly affect the weight of the mice. Conclusion A type of CAR-NK92 cell targeting NKG2DL (secreting IL-15Ra-IL-15) is successfully constructed, indicating the effective killing of multiple myeloid cells.

Development of Nectin4/FAP-targeted CAR-T cells secreting IL-7, CCL19, and IL-12 for malignant solid tumors.

Background: Chimeric antigen receptor T (CAR-T) cell therapy has made significant advances for hematological malignancies but encounters obstacles in the treatment of solid tumors mainly due to tumor immunosuppressive microenvironment. Methods: Immunohistochemistry analysis was performed to examine the cellular expression of nectin cell adhesion molecule-4 (Nectin4) and fibroblast activation protein (FAP) in a variety of malignant solid tumors. Then, we engineered the fourth-generation Nectin4-targeted CAR-T (Nectin4-7.19 CAR-T) and FAP-targeted CAR-T (FAP-12 CAR-T) cells to evaluate their safety and efficacy in vitro and in vivo. Results: In our study, we firstly demonstrated the aberrant overexpression of Nectin4 on both primary and metastatic solid tumors and FAP on cancer-associated fibroblasts. Then, we found that our fourth-generation Nectin4-7.19 CAR-T cells expressed IL-7 and CCL19 efficiently and exhibited superior proliferation, migration, and cytotoxicity compared to the second-generation Nectin4 CAR-T cells, while FAP-12 CAR-T cells exerted their ability of targeting both murine and human FAP effectively in vitro. In a fully immune-competent mouse model of metastatic colorectal cancer, lymphodepletion pretreated mice achieved complete remission with human Nectin4-targeted murine CAR-T (Nectin4 mCAR-T) cells. In the NSG mouse model of lung metastases, Nectin4-7.19 CAR-T cells eradicated metastatic tumors and prolonged survival in combination with FAP-12 CAR-T cells. Conclusions: These findings showed that Nectin4-7.19 CAR-T cells had potential therapeutic efficacy and exerted a synergistic role with FAP-12 CAR-T cells, further demonstrating that Nectin4 and FAP were able to serve as promising targets for safe and effective CAR-T therapy of malignant solid tumors.

Overexpression of SgGH3.1 from Fine-Stem Stylo (Stylosanthes guianensis var. intermedia) Enhances Chilling and Cold Tolerance in Arabidopsis thaliana.

Stylosanthes (stylo) species are commercially significant tropical and subtropical forage and pasture legumes that are vulnerable to chilling and frost. However, little is known about the molecular mechanisms behind stylos' responses to low temperature stress. Gretchen-Hagen 3 (GH3) proteins have been extensively investigated in many plant species for their roles in auxin homeostasis and abiotic stress responses, but none have been reported in stylos. SgGH3.1, a cold-responsive gene identified in a whole transcriptome profiling study of fine-stem stylo (S. guianensis var. intermedia) was further investigated for its involvement in cold stress tolerance. SgGH3.1 shared a high percentage of identity with 14 leguminous GH3 proteins, ranging from 79% to 93%. Phylogenetic analysis classified SgGH3.1 into Group Ⅱ of GH3 family, which have been proven to involve with auxins conjugation. Expression profiling revealed that SgGH3.1 responded rapidly to cold stress in stylo leaves. Overexpression of SgGH3.1 in Arabidopsis thaliana altered sensitivity to exogenous IAA, up-regulated transcription of AtCBF1-3 genes, activated physiological responses against cold stress, and enhanced chilling and cold tolerances. This is the first report of a GH3 gene in stylos, which not only validated its function in IAA homeostasis and cold responses, but also gave insight into breeding of cold-tolerant stylos.

SgRVE6, a LHY-CCA1-Like Transcription Factor From Fine-Stem Stylo, Upregulates NB-LRR Gene Expression and Enhances Cold Tolerance in Tobacco.

Stylosanthes species are economically important tropical and subtropical forage legumes which are generally vulnerable to chilling and frost. Fine-stem stylo (S. guianensis var. intermedia) has the most superior cold tolerance among all stylo species. A REVEILLE (RVE) gene, SgRVE6, was cloned from fine-stem stylo. Bioinformatic analysis suggests that SgRVE6 encodes a transcription factor of 292 amino acid residues, which belongs to the LATE ELONGATED HYPOCOTYL/CIRCADIAN CLOCK ASSOCIATED 1-LIKE (LCL) subgroup of RVE family and contains a SHAQKYF-class MYB domain and a LCL domain. SgRVE6 is universally expressed in root, stem and leaf tissues of fine-stem stylo and is rapidly up-regulated in all tested tissues under cold stress. Over-expressing SgRVE6 affects expression of 21 circadian clock genes, up-regulates expression of 6 nucleotide binding domain leucine-rich repeats (NB-LRR) encoding genes associated with tobacco cold tolerance, improves physiological responses to low temperature, and endows the transgenic tobaccos with higher tolerance to cold stress. This is the first time a study investigates the biological function of RVE6 in cold responses of plant species.

Epigenetic modification of ESP, encoding a putative long noncoding RNA, affects panicle architecture in rice.

Epigenetic variants broaden phenotypic diversity in eukaryotes. Epialleles may also provide a new genetic source for crop breeding, but very few epialleles related to agricultural traits have been identified in rice. Here, we identified Epi-sp, a gain-of-function epiallele of the rice ESP (Epigenetic Short Panicle, Os01g0356951), which encodes a putative long noncoding RNA. The Epi-sp plants show a dense and short panicle phenotype, an agronomically important phenotypes that is inherited in a semidominant manner. We did not find any nucleotide sequence variation in ESP. Instead, we found hypomethylation in the transcriptional termination region (TTR) of ESP gene, which caused ectopic expression of ESP in Epi-sp plants. Bisulfite analysis revealed that the methylation status of 26 CGs and 13 CHGs within a continuous 313-bp region is essential for the regulation of ESP expression. Thus, our work identified a unique rice epiallele and demonstrated that epigenetic modification of ESP is associated with the regulation of panicle architecture in rice.

Rice OsPEX1, an extensin-like protein, affects lignin biosynthesis and plant growth.

KEY MESSAGE: Rice leucine-rich repeat extensin-like protein OsPEX1 mediates the intersection of lignin deposition and plant growth. Lignin, a major structural component of secondary cell wall, is essential for normal plant growth and development. However, the molecular and genetic regulation of lignin biosynthesis is not fully understood in rice. Here we report the identification and characterization of a rice semi-dominant dwarf mutant (pex1) with stiff culm. Molecular and genetic analyses revealed that the pex1 phenotype was caused by ectopic expression of a leucine-rich repeat extension-like gene, OsPEX1. Interestingly, the pex1 mutant showed significantly higher lignin content and increased expression levels of lignin-related genes compared with wild type plants. Conversely, OsPEX1-suppresssed transgenics displayed low lignin content and reduced transcriptional abundance of genes associated with lignin biosynthesis, indicating that the OsPEX1 mediates lignin biosynthesis and/or deposition in rice. When OsPEX1 was ectopically expressed in rice cultivars with tall stature that lacks the allele of semi-dwarf 1, well-known green revolution gene, the resulting transgenic plants displayed reduced height and enhanced lodging resistance. Our study uncovers a causative effect between the expression of OsPEX1 and lignin deposition. Lastly, we demonstrated that modulating OsPEX1 expression could provide a tool for improving rice lodging resistance.

Mutation in a putative glycosyltransferase-like gene causes programmed cell death and early leaf senescence in rice.

Leaf senescence is a genetically regulated, highly complex and ordered process. Although it has been extensively studied, the mechanism of leaf senescence is not well understood. In this study, we isolated a rice mutant, designated as premature senescence leaf (psl), which exhibits early senescence and spontaneous lesion mimic phenotype after flowering. The psl mutant displays programmed cell death with elevated accumulation of reactive oxygen species (ROS). Molecular and genetic analyses revealed that the phenotypes were caused by a phenylalanine deletion in the OsPSL (LOC_Os12g42420) that encode a putative core 2/I branching beta-1,6-N-acetylglucosaminyl transferase predicted to be involved in protein glycosylation modification. OsPSL mRNA levels increased as senescence progressed, with maximum accumulation of transcripts at late senescence stages in WT plants. Moreover, remarkedly down-regulated transcriptional levels of O-linked N-acetylglucosamine (O-GlcNAc) transferases (OGTs) genes were observed in psl mutant, supporting the occurrence of impaired O-glycosylation modification. Proteomic analysis showed that ethylene-related metabolic enzymes including S-adenosyl methionine (SAM) synthetase (SAMS) were significantly upregulated in the psl mutant compared with WT. Consistent with the proteomic results, ethylene concentration is higher in psl mutant than in wild-type plants, and transcript levels of ethylene synthesis and signal transduction genes were induced in psl mutant. The early leaf senescence of psl can be partially rescued by ethylene biosynthesis inhibitor aminoethoxyvinylglycine treatment. These results highlight the importance of protein O-glycosylation in PCD and leaf senescence, and suggest a possible role of OsPSL in ethylene signaling.

Genome-wide transcriptome profiling provides insights into panicle development of rice (Oryza sativa L.).

Panicle architecture is an important component of agronomic trait in rice, which is also a key ingredient that could influence yield and quality of rice. In the panicle growth and development process, there are a series of complicated molecular and cellular events which are regulated by many interlinking genes. In this study, to explore the potential mechanism and identify genes and pathways involved in the formation of rice panicle, we compared the transcriptional profile of rice panicles (NIL-GW8 and NIL-gw8Amol) at three different stages of panicle development: In5 (formation of higher-order branches), In6 (differentiation of glumes) and In7 (differentiation of floral organs). A range of 40.5 to 54.1 million clean reads was aligned to 31,209 genes in our RNA-Seq analysis. In addition, we investigated transcriptomic changes between the two rice lines during different stages. A total of 726, 1121 and 2584 differentially expressed genes (DEGs) were identified at stages 1, 2 and 3, respectively. Based on an impact analysis of the DEGs, we hypothesize that MADS-box gene family, cytochrome P450 (CYP) and pentatricopeptide repeat (PPR) protein and various transcription factors may be involved in regulation of panicle development. Further, we also explored the functional properties of DEGs by gene ontology analysis, and the results showed that different numbers of DEGs genes were associated with 53 GO groups. In KEGG pathway enrichment analysis, many DEGs related to biosynthesis of secondary metabolites and plant hormone signal transduction, suggesting their important roles during panicle development. This study provides the first examination of changes in gene expression between different panicle development stages in rice. Our results of transcriptomic characterization provide important information to elucidate the complex molecular and cellular events about the panicle formation in rice or other cereal crops. Also, the findings will be helpful for the further identification of the genes related to panicle development.

A naturally occurring conditional albino mutant in rice caused by defects in the plastid-localized OsABCI8 transporter.

A wide range of molecules are transported across membranes by the ATP binding cassette (ABC) transporters. Plants possess a collection of ABC proteins bearing similarities to the components of prokaryotic multi subunit ABC transporters, designed as ABC group I. However the functions of most of them are not well understood. Here, we characterized a naturally occurring rice mutant that exhibited albino phenotype under continuous rainy days in the field, but gradually recovered to normal green after the rainy season. Molecular and genetic analyses revealed that the phenotypes were caused by a mutation in the OsABCI8 that encoded a member of the ABCI family. Subcellular localization demonstrated that OsABCI8 is a chloroplast ABC transporter. Expression of OsABCI8 is significantly enhanced in rainy days compared to sunny days. Besides defects in chloroplast development and chlorophyll biosynthesis, the mutant phenotype is accompanied by a higher accumulation of iron, suggesting that OsABCI8 is involved in iron transportation and/or homeostasis in rice. Our results demonstrate that OsABCI8 represents a conserved ABCI protein involved in transition metals transportation and/or homeostasis and suggest an important role of the plastid-localized OsABCI8 for chloroplast development.

ER-localized adenine nucleotide transporter ER-ANT1: an integrator of energy and stress signaling in rice.

Most environmental perturbations have a direct or indirect deleterious impact on photosynthesis, and, in consequence, the overall energy status of the cell. Despite our increased understanding of convergent energy and stress signals, the connections between photosynthesis, energy and stress signals through putative common nodes are still unclear. Here we identified an endoplasmic reticulum (ER)-localized adenine nucleotide transporter1 (ER-ANT1), whose deficiency causes seedling lethality in air but viable under high CO2, exhibiting the typical photorespiratory phenotype. Metabolic analysis suggested that depletion of ER-ANT1 resulted in circadian rhythm disorders in sucrose synthesis and induced sucrose signaling pathways, indicating that the ER is involved in the regulation of vital energy metabolism in plants. In addition, the defect of ER-ANT1 triggers ER stress and activates the unfolded protein response in plant cells, suggesting ER stress and photorespiration are closely linked. These findings provide an important evidence for a key role of ER-localized ER-ANT1 in convergent energy and stress signals in rice. Our findings support the idea that ATP is a central signal involved in the plant response to a variety of stresses.

4-Coumarate-CoA Ligase-Like Gene OsAAE3 Negatively Mediates the Rice Blast Resistance, Floret Development and Lignin Biosynthesis.

Although adenosine monophosphate (AMP) binding domain is widely distributed in multiple plant species, detailed molecular functions of AMP binding proteins (AMPBPs) in plant development and plant-pathogen interaction remain unclear. In the present study, we identified an AMPBP OsAAE3 from a previous analysis of early responsive genes in rice during Magnaporthe oryzae infection. OsAAE3 is a homolog of Arabidopsis AAE3 in rice, which encodes a 4-coumarate-Co-A ligase (4CL) like protein. A phylogenetic analysis showed that OsAAE3 was most likely 4CL-like 10 in an independent group. OsAAE3 was localized to cytoplasm, and it could be expressed in various tissues. Histochemical staining of transgenic plants carrying OsAAE3 promoter-driven GUS (ß-glucuronidase) reporter gene suggested that OsAAE3 was expressed in all tissues of rice. Furthermore, OsAAE3-OX plants showed increased susceptibility to M. Oryzae, and this finding was attributable to decreased expression of pathogen-related 1a (PR1) and low level of peroxidase (POD) activity. Moreover, OsAAE3 over-expression resulted in increased content of H2O2, leading to programmed cell-death induced by reactive oxygen species (ROS). In addition, OsAAE3 over-expression repressed the floret development, exhibiting dramatically twisted glume and decreased fertility rate of anther. Meanwhile, the expressions of lignin biosynthesis genes were significantly decreased in OsAAE3-OX plants, thereby leading to reduced lignin content. Taken together, OsAAE3 functioned as a negative regulator in rice blast resistance, floret development, and lignin biosynthesis. Our findings further expanded the knowledge in functions of AMBPs in plant floret development and the regulation of rice-fungus interaction.