Phytohormonal regulation determines the organization pattern of shoot aerenchyma in greater duckweed (Spirodela polyrhiza).

Airspace or aerenchyma is crucial for plant development and acclimation to stresses such as hypoxia, drought, and nutritional deficiency. Although ethylene-mediated signaling cascades are known to regulate aerenchyma formation in stems and roots under hypoxic conditions, the precise mechanisms remain unclear. Moreover, the cellular dynamics underlying airspace formation in shoots are poorly understood. We investigated the stage-dependent structural dynamics of shoot aerenchyma in greater duckweed (Spirodela polyrhiza), a fast-growing aquatic herb with well-developed aerenchyma in its floating fronds. Using X-ray micro-computed tomography and histological analysis, we showed that the spatial framework of aerenchyma is established before frond volume increases, driven by cell division and expansion. The substomatal cavity connecting aerenchyma to stomata formed via programmed cell death (PCD) and was closely associated with guard cell development. Additionally, transcriptome analysis and pharmacological studies revealed that the organization of aerenchyma in common duckweed is determined by the interplay between PCD and proliferation. This balance is governed by spatiotemporal regulation of phytohormone signaling involving ethylene, abscisic acid, and salicylic acid. Overall, our study reveals the structural dynamics and phytohormonal regulation underlying aerenchyma development in duckweed, improving our understanding of how plants establish distinct architectural arrangements. These insights hold the potential for wide-ranging application, not only in comprehending aerenchyma formation across various plant species but also in understanding how airspaces are formed within the leaves of terrestrial plants.

PRMT1 promotes pancreatic cancer development and resistance to chemotherapy.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal types of cancer, and novel treatment regimens are direly needed. Epigenetic regulation contributes to the development of various cancer types, but its role in the development of and potential as a therapeutic target for PDAC remains underexplored. Here, we show that PRMT1 is highly expressed in murine and human pancreatic cancer and is essential for cancer cell proliferation and tumorigenesis. Deletion of PRMT1 delays pancreatic cancer development in a KRAS-dependent mouse model, and multi-omics analyses reveal that PRMT1 depletion leads to global changes in chromatin accessibility and transcription, resulting in reduced glycolysis and a decrease in tumorigenic capacity. Pharmacological inhibition of PRMT1 in combination with gemcitabine has a synergistic effect on pancreatic tumor growth in vitro and in vivo. Collectively, our findings implicate PRMT1 as a key regulator of pancreatic cancer development and a promising target for combination therapy.

Targeted deletion of CD244 on monocytes promotes differentiation into anti-tumorigenic macrophages and potentiates PD-L1 blockade in melanoma.

BACKGROUND: In the myeloid compartment of the tumor microenvironment, CD244 signaling has been implicated in immunosuppressive phenotype of monocytes. However, the precise molecular mechanism and contribution of CD244 to tumor immunity in monocytes/macrophages remains elusive due to the co-existing lymphoid cells expressing CD244. METHODS: To directly assess the role of CD244 in tumor-associated macrophages, monocyte-lineage-specific CD244-deficient mice were generated using cre-lox recombination and challenged with B16F10 melanoma. The phenotype and function of tumor-infiltrating macrophages along with antigen-specific CD8 T cells were analyzed by flow cytometry and single cell RNA sequencing data analysis, and the molecular mechanism underlying anti-tumorigenic macrophage differentiation, antigen presentation, phagocytosis was investigated ex vivo. Finally, the clinical feasibility of CD244-negative monocytes as a therapeutic modality in melanoma was confirmed by adoptive transfer experiments. RESULTS: CD244fl/flLysMcre mice demonstrated a significant reduction in tumor volume (61% relative to that of the CD244fl/fl control group) 14 days after tumor implantation. Within tumor mass, CD244fl/flLysMcre mice also showed higher percentages of Ly6Clow macrophages, along with elevated gp100+IFN-γ+ CD8 T cells. Flow cytometry and RNA sequencing data demonstrated that ER stress resulted in increased CD244 expression on monocytes. This, in turn, impeded the generation of anti-tumorigenic Ly6Clow macrophages, phagocytosis and MHC-I antigen presentation by suppressing autophagy pathways. Combining anti-PD-L1 antibody with CD244-/- bone marrow-derived macrophages markedly improved tumor rejection compared to the anti-PD-L1 antibody alone or in combination with wild-type macrophages. Consistent with the murine data, transcriptome analysis of human melanoma tissue single-cell RNA-sequencing dataset revealed close association between CD244 and the inhibition of macrophage maturation and function. Furthermore, the presence of CD244-negative monocytes/macrophages significantly increased patient survival in primary and metastatic tumors. CONCLUSION: Our study highlights the novel role of CD244 on monocytes/macrophages in restraining anti-tumorigenic macrophage generation and tumor antigen-specific T cell response in melanoma. Importantly, our findings suggest that CD244-deficient macrophages could potentially be used as a therapeutic agent in combination with immune checkpoint inhibitors. Furthermore, CD244 expression in monocyte-lineage cells serve as a prognostic marker in cancer patients.

Angiogenesis-on-a-chip coupled with single-cell RNA sequencing reveals spatially differential activations of autophagy along angiogenic sprouts.

Several functions of autophagy associated with proliferation, differentiation, and migration of endothelial cells have been reported. Due to lack of models recapitulating angiogenic sprouting, functional heterogeneity of autophagy in endothelial cells along angiogenic sprouts remains elusive. Here, we apply an angiogenesis-on-a-chip to reconstruct 3D sprouts with clear endpoints. We perform single-cell RNA sequencing of sprouting endothelial cells from our chip to reveal high activation of autophagy in two endothelial cell populations- proliferating endothelial cells in sprout basements and stalk-like endothelial cells near sprout endpoints- and further the reciprocal expression pattern of autophagy-related genes between stalk- and tip-like endothelial cells near sprout endpoints, implying an association of autophagy with tip-stalk cell specification. Our results suggest a model describing spatially differential roles of autophagy: quality control of proliferating endothelial cells in sprout basements for sprout elongation and tip-stalk cell specification near sprout endpoints, which may change strategies for developing autophagy-based anti-angiogenic therapeutics.

Elucidation of molecular basis of osteolytic bone lesions in advanced multiple myeloma.

Osteolytic bone lesion is a major cause of decreased quality of life and poor prognosis in patients with multiple myeloma (MM), but molecular pathogenesis of the osteolytic process in MM remains elusive. Fms-like tyrosine kinase 3 ligand (FLT3L) was reported to be elevated in bone marrow and blood of patients with advanced MM who often show osteolysis. Here, we investigated a functional link of FLT3L to osteolytic process in MM. We recruited 86, 306 and 52 patients with MM, acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), respectively. FLT3L levels of patients with hematologic malignancies were measured in bone marrow-derived plasma and found to be significantly elevated in MM than in AML or ALL that rarely show osteolysis. FLT3L levels were further elevated in MM patients with bone lesion compared with patients without bone lesion. In vitro cell-based assays showed that the administration of FLT3L to HEK293T, HeLa and U2OS cells led to an increase in the DKK1 transcript level through STAT3 phosphorylation at tyrosine 705. WNT reporter assay showed that FLT3L treatment reduced WNT signaling, and nuclear translocation of ß-catenin. These results collectively show that FLT3L-STAT3-DKK1 pathway inhibits WNT signaling-mediated bone formation in MM, which can cause osteolytic bone lesion. Finally, transcriptomic profiles revealed that FLT3L and DKK1 were predominantly elevated in the hyperdiploidy subtype of MM. Taken together, FLT3L can serve as a promising biomarker for predicting osteolytic bone lesion and also a potential therapeutic target to prohibit the progression of osteolytic process in MM with hyperdiploidy.

Morc2a variants cause hydroxyl radical-mediated neuropathy and are rescued by restoring GHKL ATPase.

Mutations in the Microrchidia CW-Type Zinc Finger 2 (MORC2) GHKL ATPase module cause a broad range of neuropathies, such as Charcot-Marie-Tooth disease type 2Z; however, the aetiology and therapeutic strategy are not fully understood. Previously, we reported that the Morc2a p.S87L mouse model exhibited neuropathy and muscular dysfunction through DNA damage accumulation. In the present study, we analysed the gene expression of Morc2a p.S87L mice and designated the primary causing factor. We investigated the pathological pathway using Morc2a p.S87L mouse embryonic fibroblasts and human fibroblasts harbouring MORC2 p.R252W. We subsequently assessed the therapeutic effect of gene therapy administered to Morc2a p.S87L mice. This study revealed that Morc2a p.S87L causes a protein synthesis defect, resulting in the loss of function of Morc2a and high cellular apoptosis induced by high hydroxyl radical levels. We considered the Morc2a GHKL ATPase domain as a therapeutic target because it simultaneously complements hydroxyl radical scavenging and ATPase activity. We used the adeno-associated virus (AAV)-PHP.eB serotype, which has a high central nervous system transduction efficiency, to express Morc2a or Morc2a GHKL ATPase domain protein in vivo. Notably, AAV gene therapy ameliorated neuropathy and muscular dysfunction with a single treatment. Loss of functional characteristics due to protein synthesis defects in Morc2a p.S87L was also noted in human MORC2 p.S87L or p.R252W variants, indicating the correlation between mouse and human pathogenesis. In summary, CMT2Z is known as an incurable genetic disorder, but the present study demonstrated its mechanisms and treatments based on established animal models. This study demonstrates that the Morc2a p.S87L variant causes hydroxyl radical-mediated neuropathy, which can be rescued through AAV-based gene therapy.

Adnp-mutant mice with cognitive inflexibility, CaMKIIα hyperactivity, and synaptic plasticity deficits.

ADNP syndrome, involving the ADNP transcription factor of the SWI/SNF chromatin-remodeling complex, is characterized by developmental delay, intellectual disability, and autism spectrum disorders (ASD). Although Adnp-haploinsufficient (Adnp-HT) mice display various phenotypic deficits, whether these mice display abnormal synaptic functions remain poorly understood. Here, we report synaptic plasticity deficits associated with cognitive inflexibility and CaMKIIα hyperactivity in Adnp-HT mice. These mice show impaired and inflexible contextual learning and memory, additional to social deficits, long after the juvenile-stage decrease of ADNP protein levels to ~10% of the newborn level. The adult Adnp-HT hippocampus shows hyperphosphorylated CaMKIIα and its substrates, including SynGAP1, and excessive long-term potentiation that is normalized by CaMKIIα inhibition. Therefore, Adnp haploinsufficiency in mice leads to cognitive inflexibility involving CaMKIIα hyperphosphorylation and excessive LTP in adults long after its marked expressional decrease in juveniles.

DDM1-mediated gene body DNA methylation is associated with inducible activation of defense-related genes in Arabidopsis.

BACKGROUND: Plants memorize previous pathogen attacks and are "primed" to produce a faster and stronger defense response, which is critical for defense against pathogens. In plants, cytosines in transposons and gene bodies are reported to be frequently methylated. Demethylation of transposons can affect disease resistance by regulating the transcription of nearby genes during defense response, but the role of gene body methylation (GBM) in defense responses remains unclear. RESULTS: Here, we find that loss of the chromatin remodeler decrease in DNA methylation 1 (ddm1) synergistically enhances resistance to a biotrophic pathogen under mild chemical priming. DDM1 mediates gene body methylation at a subset of stress-responsive genes with distinct chromatin properties from conventional gene body methylated genes. Decreased gene body methylation in loss of ddm1 mutant is associated with hyperactivation of these gene body methylated genes. Knockout of glyoxysomal protein kinase 1 (gpk1), a hypomethylated gene in ddm1 loss-of-function mutant, impairs priming of defense response to pathogen infection in Arabidopsis. We also find that DDM1-mediated gene body methylation is prone to epigenetic variation among natural Arabidopsis populations, and GPK1 expression is hyperactivated in natural variants with demethylated GPK1. CONCLUSIONS: Based on our collective results, we propose that DDM1-mediated GBM provides a possible regulatory axis for plants to modulate the inducibility of the immune response.

Identification of MYH9 as a key regulator for synoviocyte migration and invasion through secretome profiling.

OBJECTIVES: 'Invasive pannus' is a pathological hallmark of rheumatoid arthritis (RA). This study aimed to investigate secretome profile of synovial fibroblasts of patients with RA (RA-FLSs), a major cell type comprising the invasive pannus. METHODS: Secreted proteins from RA-FLSs were first identified using liquid chromatography-tandem mass spectrometry analysis. Ultrasonography was performed for affected joints to define synovitis severity at the time of arthrocentesis. Expression levels of myosin heavy chain 9 (MYH9) in RA-FLSs and synovial tissues were determined by ELISA, western blot analysis and immunostaining. A humanised synovitis model was induced in immuno-deficient mice. RESULTS: We first identified 843 proteins secreted from RA-FLSs; 48.5% of the secretome was associated with pannus-driven pathologies. Parallel reaction monitoring analysis of the secretome facilitated discovery of 16 key proteins related to 'invasive pannus', including MYH9, in the synovial fluids, which represented synovial pathology based on ultrasonography and inflammatory activity in the joints. Particularly, MYH9, a key protein in actin-based cell motility, showed a strong correlation with fibroblastic activity in the transcriptome profile of RA synovia. Moreover, MYH9 expression was elevated in cultured RA-FLSs and RA synovium, and its secretion was induced by interleukin-1ß, tumour necrosis factor α, toll-like receptor ligation and endoplasmic reticulum stimuli. Functional experiments demonstrated that MYH9 promoted migration and invasion of RA-FLSs in vitro and in a humanised synovitis model, which was substantially inhibited by blebbistatin, a specific MYH9 inhibitor. CONCLUSIONS: This study provides a comprehensive resource of the RA-FLS-derived secretome and suggests that MYH9 represents a promising target for retarding abnormal migration and invasion of RA-FLSs.

ANT2 Accelerates Cutaneous Wound Healing in Aged Skin by Regulating Energy Homeostasis and Inflammation.

An effective healing response is critical to healthy aging. In particular, energy homeostasis has become increasingly recognized as a factor in effective skin regeneration. ANT2 is a mediator of adenosine triphosphate import into mitochondria for energy homeostasis. Although energy homeostasis and mitochondrial integrity are critical for wound healing, the role played by ANT2 in the repair process had not been elucidated to date. In our study, we found that ANT2 expression decreased in aged skin and cellular senescence. Interestingly, overexpression of ANT2 in aged mouse skin accelerated the healing of full-thickness cutaneous wounds. In addition, upregulation of ANT2 in replicative senescent human diploid dermal fibroblasts induced their proliferation and migration, which are critical processes in wound healing. Regarding energy homeostasis, ANT2 overexpression increased the adenosine triphosphate production rate by activating glycolysis and induced mitophagy. Notably, ANT2-mediated upregulation of HSPA6 in aged human diploid dermal fibroblasts downregulated proinflammatory genes that mediate cellular senescence and mitochondrial damage. This study shows a previously uncharacterized physiological role of ANT2 in skin wound healing by regulating cell proliferation, energy homeostasis, and inflammation. Thus, our study links energy metabolism to skin homeostasis and reports, to the best of our knowledge, a previously unreported genetic factor that enhances wound healing in an aging model.

Chronic LCMV infection regulates the effector T cell response by inducing the generation of less immunogenic dendritic cells.

Chronic viral infection impairs systemic immunity in the host; however, the mechanism underlying the dysfunction of immune cells in chronic viral infection is incompletely understood. In this study, we studied the lineage differentiation of hematopoietic stem cells (HSCs) during chronic viral infection to elucidate the changes in dendritic cell (DC) differentiation and subsequent impact on T cell functionality using a chronic lymphocytic choriomeningitis virus (LCMV) infection model. We first investigated the lineage differentiation of HSCs in the bone marrow (BM) to elucidate the modulation of immune cell differentiation and found that the populations highly restrained in their differentiation were common myeloid progenitors (CMPs) and common dendritic cell progenitors (CDPs). Of interest, the main immune cells infected with LCMV Clone 13 (CL13) in the BM were CD11b/c+ myeloid DCs. We next characterized CD11b+ DCs that differentiated during chronic LCMV infection. These DCs displayed a less immunogenic phenotype than DCs in naive or acutely infected mice, showing low expression of CD80 but high expression of PD-L1, B7-H4, IDO, TGF-ß, and IL-10. Consequently, these CD11b+ DCs induced less effective CD8+ T cells and more Foxp3+ regulatory T (Treg) cells. Furthermore, CD11b+ DCs generated during CL13 infection could not induce effective CD8+ T cells specific to the antigens of newly invading pathogens. Our findings demonstrate that DCs generated from the BM during chronic viral infection cannot activate fully functional effector CD8+ T cells specific to newly incoming antigens as well as persistent antigens themselves, suggesting a potential cause of the functional alterations in the T cell immune response during chronic viral infection.

Integration of Single-Cell RNA-Seq Datasets: A Review of Computational Methods.

With the increased number of single-cell RNA sequencing (scRNA-seq) datasets in public repositories, integrative analysis of multiple scRNA-seq datasets has become commonplace. Batch effects among different datasets are inevitable because of differences in cell isolation and handling protocols, library preparation technology, and sequencing platforms. To remove these batch effects for effective integration of multiple scRNA-seq datasets, a number of methodologies have been developed based on diverse concepts and approaches. These methods have proven useful for examining whether cellular features, such as cell subpopulations and marker genes, identified from a certain dataset, are consistently present, or whether their condition-dependent variations, such as increases in cell subpopulations in particular disease-related conditions, are consistently observed in different datasets generated under similar or distinct conditions. In this review, we summarize the concepts and approaches of the integration methods and their pros and cons as has been reported in previous literature.

Proteogenomic landscape of human pancreatic ductal adenocarcinoma in an Asian population reveals tumor cell-enriched and immune-rich subtypes.

We report a proteogenomic analysis of pancreatic ductal adenocarcinoma (PDAC). Mutation-phosphorylation correlations identified signaling pathways associated with somatic mutations in significantly mutated genes. Messenger RNA-protein abundance correlations revealed potential prognostic biomarkers correlated with patient survival. Integrated clustering of mRNA, protein and phosphorylation data identified six PDAC subtypes. Cellular pathways represented by mRNA and protein signatures, defining the subtypes and compositions of cell types in the subtypes, characterized them as classical progenitor (TS1), squamous (TS2-4), immunogenic progenitor (IS1) and exocrine-like (IS2) subtypes. Compared with the mRNA data, protein and phosphorylation data further classified the squamous subtypes into activated stroma-enriched (TS2), invasive (TS3) and invasive-proliferative (TS4) squamous subtypes. Orthotopic mouse PDAC models revealed a higher number of pro-tumorigenic immune cells in TS4, inhibiting T cell proliferation. Our proteogenomic analysis provides significantly mutated genes/biomarkers, cellular pathways and cell types as potential therapeutic targets to improve stratification of patients with PDAC.

PD-L1-directed PlGF/VEGF blockade synergizes with chemotherapy by targeting CD141+ cancer-associated fibroblasts in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) has a poor 5-year overall survival rate. Patients with PDAC display limited benefits after undergoing chemotherapy or immunotherapy modalities. Herein, we reveal that chemotherapy upregulates placental growth factor (PlGF), which directly activates cancer-associated fibroblasts (CAFs) to induce fibrosis-associated collagen deposition in PDAC. Patients with poor prognosis have high PIGF/VEGF expression and an increased number of PIGF/VEGF receptor-expressing CAFs, associated with enhanced collagen deposition. We also develop a multi-paratopic VEGF decoy receptor (Ate-Grab) by fusing the single-chain Fv of atezolizumab (anti-PD-L1) to VEGF-Grab to target PD-L1-expressing CAFs. Ate-Grab exerts anti-tumor and anti-fibrotic effects in PDAC models via the PD-L1-directed PlGF/VEGF blockade. Furthermore, Ate-Grab synergizes with gemcitabine by relieving desmoplasia. Single-cell RNA sequencing identifies that a CD141+ CAF population is reduced upon Ate-Grab and gemcitabine combination treatment. Overall, our results elucidate the mechanism underlying chemotherapy-induced fibrosis in PDAC and highlight a combinatorial therapeutic strategy for desmoplastic cancers.

Data Speak How to Treat Disease: Big data-based precision medicine

Schematic overview of big data-based precision medicine platform.

Aß-induced mitochondrial dysfunction in neural progenitors controls KDM5A to influence neuronal differentiation.

Mitochondria in neural progenitors play a crucial role in adult hippocampal neurogenesis by being involved in fate decisions for differentiation. However, the molecular mechanisms by which mitochondria are related to the genetic regulation of neuronal differentiation in neural progenitors are poorly understood. Here, we show that mitochondrial dysfunction induced by amyloid-beta (Aß) in neural progenitors inhibits neuronal differentiation but has no effect on the neural progenitor stage. In line with the phenotypes shown in Alzheimer's disease (AD) model mice, Aß-induced mitochondrial damage in neural progenitors results in deficits in adult hippocampal neurogenesis and cognitive function. Based on hippocampal proteome changes after mitochondrial damage in neural progenitors identified through proteomic analysis, we found that lysine demethylase 5A (KDM5A) in neural progenitors epigenetically suppresses differentiation in response to mitochondrial damage. Mitochondrial damage characteristically causes KDM5A degradation in neural progenitors. Since KDM5A also binds to and activates neuronal genes involved in the early stage of differentiation, functional inhibition of KDM5A consequently inhibits adult hippocampal neurogenesis. We suggest that mitochondria in neural progenitors serve as the checkpoint for neuronal differentiation via KDM5A. Our findings not only reveal a cell-type-specific role of mitochondria but also suggest a new role of KDM5A in neural progenitors as a mediator of retrograde signaling from mitochondria to the nucleus, reflecting the mitochondrial status.

Novel Online Three-Dimensional Separation Expands the Detectable Functional Landscape of Cellular Phosphoproteome.

Protein phosphorylation is a prevalent post-translational modification that regulates essentially every aspect of cellular processes. Currently, liquid chromatography-tandem mass spectrometry (LC-MS/MS) with an extensive offline sample fractionation and a phosphopeptide enrichment method is a best practice for deep phosphoproteome profiling, but balancing throughput and profiling depth remains a practical challenge. We present an online three-dimensional separation method for ultradeep phosphoproteome profiling that combines an online two-dimensional liquid chromatography separation and an additional gas-phase separation. This method identified over 100,000 phosphopeptides (>60,000 phosphosites) in HeLa cells during 1.5 days of data acquisition, and the largest HeLa cell phosphoproteome significantly expanded the detectable functional landscape of cellular phosphoproteome.