RESUMO
Over the past few years, numerous anti-phage defense systems have been discovered in bacteria. Although the mechanism of defense for some of these systems is understood, a major unanswered question is how these systems sense phage infection. To systematically address this question, we isolated 177 phage mutants that escape 15 different defense systems. In many cases, these escaper phages were mutated in the gene sensed by the defense system, enabling us to map the phage determinants that confer sensitivity to bacterial immunity. Our data identify specificity determinants of diverse retron systems and reveal phage-encoded triggers for multiple abortive infection systems. We find general themes in phage sensing and demonstrate that mechanistically diverse systems have converged to sense either the core replication machinery of the phage, phage structural components, or host takeover mechanisms. Combining our data with previous findings, we formulate key principles on how bacterial immune systems sense phage invaders.
Assuntos
Bactérias , Bacteriófagos , Bactérias/genética , Bactérias/virologia , Bacteriófagos/genética , Sistemas CRISPR-Cas , Proteínas Virais/metabolismo , Mutação , Fenômenos Fisiológicos BacterianosRESUMO
Retrons are bacterial genetic elements comprised of a reverse transcriptase (RT) and a non-coding RNA (ncRNA). The RT uses the ncRNA as template, generating a chimeric RNA/DNA molecule in which the RNA and DNA components are covalently linked. Although retrons were discovered three decades ago, their function remained unknown. We report that retrons function as anti-phage defense systems. The defensive unit is composed of three components: the RT, the ncRNA, and an effector protein. We examined multiple retron systems and show that they confer defense against a broad range of phages via abortive infection. Focusing on retron Ec48, we show evidence that it "guards" RecBCD, a complex with central anti-phage functions in bacteria. Inhibition of RecBCD by phage proteins activates the retron, leading to abortive infection and cell death. Thus, the Ec48 retron forms a second line of defense that is triggered if the first lines of defense have collapsed.
Assuntos
Bactérias/genética , Bactérias/imunologia , Bacteriófagos/fisiologia , RNA não Traduzido/genética , DNA Polimerase Dirigida por RNA/genética , Bactérias/virologia , Ilhas de CpG/genética , DNA/metabolismo , Escherichia coli/genética , Escherichia coli/imunologia , Escherichia coli/virologia , Proteínas de Escherichia coli/metabolismo , FilogeniaRESUMO
Retrons are toxin-antitoxin systems protecting bacteria against bacteriophages via abortive infection. The Retron-Eco1 antitoxin is formed by a reverse transcriptase (RT) and a non-coding RNA (ncRNA)/multi-copy single-stranded DNA (msDNA) hybrid that neutralizes an uncharacterized toxic effector. Yet, the molecular mechanisms underlying phage defense remain unknown. Here, we show that the N-glycosidase effector, which belongs to the STIR superfamily, hydrolyzes NAD+ during infection. Cryoelectron microscopy (cryo-EM) analysis shows that the msDNA stabilizes a filament that cages the effector in a low-activity state in which ADPr, a NAD+ hydrolysis product, is covalently linked to the catalytic E106 residue. Mutations shortening the msDNA induce filament disassembly and the effector's toxicity, underscoring the msDNA role in immunity. Furthermore, we discovered a phage-encoded Retron-Eco1 inhibitor (U56) that binds ADPr, highlighting the intricate interplay between retron systems and phage evolution. Our work outlines the structural basis of Retron-Eco1 defense, uncovering ADPr's pivotal role in immunity.
Assuntos
Bacteriófagos , Microscopia Crioeletrônica , NAD , NAD/metabolismo , Bacteriófagos/genética , Bacteriófagos/metabolismo , Bacteriófagos/imunologia , Hidrólise , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/imunologia , Sistemas Toxina-Antitoxina/genética , Escherichia coli/virologia , Escherichia coli/genética , Escherichia coli/imunologia , Escherichia coli/metabolismoRESUMO
Targeted blockade of programmed cell death 1 (PD-1), an immune-checkpoint receptor that inhibits T cell activation, provides clinical benefits in various cancers. However, how PD-1 modulates gene expression in T cells remains enigmatic. Here we investigated how PD-1 affects transcriptome changes induced by T cell receptor (TCR) activation. Intriguingly, we identified a huge variance in PD-1 sensitivity among TCR-inducible genes. When we quantified the half maximal effective concentration (EC50) as the relationship between change in gene expression and TCR signal strength, we found that genes associated with survival and proliferation were efficiently expressed upon TCR activation and resistant to PD-1-mediated inhibition. Conversely, genes encoding cytokines and effector molecules were expressed less efficiently and sensitive to PD-1-mediated inhibition. We further demonstrated that transcription factor binding motifs and CpG frequency in the promoter region affect EC50 and thus the PD-1 sensitivity of genes. Our findings explain how PD-1, dependent on the TCR signal strength, calibrates cellular transcriptomes to shape functional properties of T cell populations.
Assuntos
Ativação Linfocitária , Linfócitos do Interstício Tumoral/metabolismo , Neoplasias/metabolismo , Receptor de Morte Celular Programada 1/metabolismo , Linfócitos T/metabolismo , Transcriptoma , Animais , Apoptose , Sítios de Ligação , Proliferação de Células , Técnicas de Cocultura , Ilhas de CpG , Citocinas/genética , Citocinas/metabolismo , Regulação Neoplásica da Expressão Gênica , Genes Codificadores dos Receptores de Linfócitos T , Células HEK293 , Humanos , Células Jurkat , Linfócitos do Interstício Tumoral/imunologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/patologia , Receptor de Morte Celular Programada 1/deficiência , Receptor de Morte Celular Programada 1/genética , Regiões Promotoras Genéticas , Transdução de Sinais , Linfócitos T/imunologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ativação TranscricionalRESUMO
Immune checkpoint therapy has limited efficacy for patients with bone-metastatic castration-resistant prostate cancer (bmCRPC). To improve immunotherapy for bmCRPC, we aimed to identify the mechanism of bmCRPC-induced changes in the immune microenvironment. Among bmCRPC patients, higher levels of a 32-gene M2-like macrophage signature in bone metastasis samples correlated with shorter overall survival. Immunohistochemistry showed that CD206-positive (CD206+) macrophages were enriched in bmCRPC bone biopsy specimens compared with primary tumors or lymph node metastases. In preclinical osteogenic prostate cancer (Pca) xenograft models, CD206+ macrophages were recruited to areas with tumor-induced bone. RNA sequencing (RNAseq) analysis showed higher expression of an M2-like gene signature, with activated canonical and noncanonical Wnt pathways, in tumor-associated macrophages isolated from osteogenic tumors (bone-TAMs) than in TAMs isolated from nonosteogenic tumors (ctrl-TAMs). Mechanistic studies showed that endothelial cells (ECs) that had undergone EC-to-osteoblast (EC-to-OSB) transition, the precursors of tumor-induced OSBs, produced paracrine factors, including Wnts, CXCL14, and lysyl oxidase, which induced M2 polarization and recruited M2-like TAMs to the bone-tumor microenvironment (bone-TME). Bone-TAMs suppressed CD8+ T cells' proliferation and cytolytic activity, and these effects were partially reversed by treating bone-TAMs with Wnt inhibitors. Genetic or pharmacological inhibition of Pca-induced EC-to-OSB transition reduced the levels of M2-like macrophages in osteogenic tumors. Our study demonstrates that Pca-induced EC-to-OSB transition drives immunosuppression in the bone-TME, suggesting that therapies that reduce Pca-induced bone formation may improve immunotherapeutic outcomes for bmCRPC.
Assuntos
Neoplasias Ósseas , Células Endoteliais , Macrófagos , Osteoblastos , Microambiente Tumoral , Via de Sinalização Wnt , Masculino , Microambiente Tumoral/imunologia , Humanos , Neoplasias Ósseas/imunologia , Neoplasias Ósseas/secundário , Neoplasias Ósseas/patologia , Neoplasias Ósseas/metabolismo , Animais , Camundongos , Macrófagos/metabolismo , Macrófagos/imunologia , Células Endoteliais/metabolismo , Células Endoteliais/imunologia , Osteoblastos/metabolismo , Osteoblastos/imunologia , Neoplasias de Próstata Resistentes à Castração/imunologia , Neoplasias de Próstata Resistentes à Castração/patologia , Neoplasias de Próstata Resistentes à Castração/metabolismo , Linhagem Celular Tumoral , Neoplasias da Próstata/patologia , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/metabolismo , Macrófagos Associados a Tumor/metabolismo , Macrófagos Associados a Tumor/imunologiaRESUMO
The relationship of embryonal carcinoma (EC) cells, the stem cells of germ cell- or embryo-derived teratocarcinoma tumors, to early embryonic cells came under intense scrutiny in the early 1970s when mouse chimeras were produced between EC cells and embryos. These chimeras raised tantalizing possibilities and high hopes for different areas of research. The normalization of EC cells by the embryo lent validity to their use as in vitro models for embryogenesis and indicated that they might reveal information about the relationship between malignancy and differentiation. Chimeras also showed the way for the potential introduction of genes, selected in EC cells in vitro, into the germ line of mice. Although EC cells provided material for the elucidation of early embryonic events and stimulated many studies of early molecular differentiation, after years of intense scrutiny, they fell short as the means of genetic manipulation of the germ line, although arguably they pointed the way to the development of embryonic stem (ES) cells that eventually fulfilled this goal.
RESUMO
CaV1.2 channels are critical players in cardiac excitation-contraction coupling, yet we do not understand how they are affected by an important therapeutic target of heart failure drugs and regulator of blood pressure, angiotensin II. Signaling through Gq-coupled AT1 receptors, angiotensin II triggers a decrease in PIP2, a phosphoinositide component of the plasma membrane (PM) and known regulator of many ion channels. PIP2 depletion suppresses CaV1.2 currents in heterologous expression systems but the mechanism of this regulation and whether a similar phenomenon occurs in cardiomyocytes is unknown. Previous studies have shown that CaV1.2 currents are also suppressed by angiotensin II. We hypothesized that these two observations are linked and that PIP2 stabilizes CaV1.2 expression at the PM and angiotensin II depresses cardiac excitability by stimulating PIP2 depletion and destabilization of CaV1.2 expression. We tested this hypothesis and report that CaV1.2 channels in tsA201 cells are destabilized after AT1 receptor-triggered PIP2 depletion, leading to their dynamin-dependent endocytosis. Likewise, in cardiomyocytes, angiotensin II decreased t-tubular CaV1.2 expression and cluster size by inducing their dynamic removal from the sarcolemma. These effects were abrogated by PIP2 supplementation. Functional data revealed acute angiotensin II reduced CaV1.2 currents and Ca2+ transient amplitudes thus diminishing excitation-contraction coupling. Finally, mass spectrometry results indicated whole-heart levels of PIP2 are decreased by acute angiotensin II treatment. Based on these observations, we propose a model wherein PIP2 stabilizes CaV1.2 membrane lifetimes, and angiotensin II-induced PIP2 depletion destabilizes sarcolemmal CaV1.2, triggering their removal, and the acute reduction of CaV1.2 currents and contractility.
Assuntos
Angiotensina II , Acoplamento Excitação-Contração , Células Cultivadas , Angiotensina II/metabolismo , Transdução de Sinais , Miócitos Cardíacos/metabolismo , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismoRESUMO
Mutations in ubiquitously expressed presenilin genes (PSENs) lead to early-onset familial Alzheimer's disease (FAD), but patients carrying the mutation also suffer from heart diseases. To elucidate the cardiac myocyte specific effects of PSEN ΔE9, we studied cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs) from patients carrying AD-causing PSEN1 exon 9 deletion (PSEN1 ΔE9). When compared with their isogenic controls, PSEN1 ΔE9 cardiomyocytes showed increased sarcoplasmic reticulum (SR) Ca2+ leak that was resistant to blockage of ryanodine receptors (RyRs) by tetracaine or inositol-3-reseceptors (IP3Rs) by 2-ABP. The SR Ca2+ leak did not affect electrophysiological properties of the hiPSC-CMs, but according to experiments and in silico simulations the leak induces a diastolic buildup of [Ca2+] near the perinuclear SR and reduces the releasable Ca2+ during systole. This demonstrates that PSEN1 ΔE9 induced SR Ca2+ leak has specific effects in iPSC-CMs, reflecting their unique structural and calcium signaling features. The results shed light on the physiological and pathological mechanisms of PSEN1 in cardiac myocytes and explain the intricacies of comorbidity associated with AD-causing mutations in PSEN1.
Assuntos
Sinalização do Cálcio , Cálcio , Células-Tronco Pluripotentes Induzidas , Mutação , Miócitos Cardíacos , Presenilina-1 , Canal de Liberação de Cálcio do Receptor de Rianodina , Retículo Sarcoplasmático , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/metabolismo , Presenilina-1/genética , Presenilina-1/metabolismo , Retículo Sarcoplasmático/metabolismo , Cálcio/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/genéticaRESUMO
Dysferlin is a 237 kDa membrane-associated protein characterised by multiple C2 domains with a diverse role in skeletal and cardiac muscle physiology. Mutations in DYSF are known to cause various types of human muscular dystrophies, known collectively as dysferlinopathies, with some patients developing cardiomyopathy. A myriad of in vitro membrane repair studies suggest that dysferlin plays an integral role in the membrane repair complex in skeletal muscle. In comparison, less is known about dysferlin in the heart, but mounting evidence suggests that dysferlin's role is similar in both muscle types. Recent findings have shown that dysferlin regulates Ca2+ handling in striated muscle via multiple mechanisms and that this becomes more important in conditions of stress. Maintenance of the transverse (t)-tubule network and the tight coordination of excitation-contraction coupling are essential for muscle contractility. Dysferlin regulates the maintenance and repair of t-tubules, and it is suspected that dysferlin regulates t-tubules and sarcolemmal repair through a similar mechanism. This review focuses on the emerging complexity of dysferlin's activity in striated muscle. Such insights will progress our understanding of the proteins and pathways that regulate basic heart and skeletal muscle function and help guide research into striated muscle pathology, especially that which arises due to dysferlin dysfunction.
Assuntos
Cálcio , Disferlina , Humanos , Cálcio/metabolismo , Disferlina/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Proteínas Musculares/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/fisiologia , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiologia , Músculo Estriado/metabolismo , Músculo Estriado/fisiologiaRESUMO
Arterial-venous malformations (AVMs) are direct connections between arteries and veins without an intervening capillary bed. Either familial inherited or sporadically occurring, localized pericytes (PCs) drop is among the AVMs' hallmarks. Whether impaired PC coverage triggers AVMs or it is a secondary event is unclear. Here we evaluated the role of the master regulator of PC recruitment, Platelet derived growth factor B (PDGFB) in AVM pathogenesis. Using tamoxifen-inducible deletion of Pdgfb in endothelial cells (ECs), we show that disruption of EC Pdgfb-mediated PC recruitment and maintenance leads to capillary enlargement and organotypic AVM-like structures. These vascular lesions contain non-proliferative hyperplastic, hypertrophic and miss-oriented capillary ECs with an altered capillary EC fate identity. Mechanistically, we propose that PDGFB maintains capillary EC size and caliber to limit hemodynamic changes, thus restricting expression of Krüppel like factor 4 and activation of Bone morphogenic protein, Transforming growth factor ß and NOTCH signaling in ECs. Furthermore, our study emphasizes that inducing or activating PDGFB signaling may be a viable therapeutic approach for treating vascular malformations.
Assuntos
Células Endoteliais , Doenças Vasculares , Humanos , Proteínas Proto-Oncogênicas c-sis/metabolismo , Células Endoteliais/metabolismo , Doenças Vasculares/metabolismo , Capilares/metabolismo , Pericitos/metabolismoRESUMO
HLA donor-specific antibodies (DSA) elicit alloimmune responses against the graft vasculature, leading to endothelial cell (EC) activation and monocyte infiltration during antibody-mediated rejection (AMR). AMR promotes chronic inflammation and remodeling, leading to thickening of the arterial intima termed transplant vasculopathy or cardiac allograft vasculopathy (CAV) in heart transplants. Intragraft-recipient macrophages serve as a diagnostic marker in AMR; however, their polarization and function remain unclear. In this study, we utilized an in vitro Transwell coculture system to explore the mechanisms of monocyte-to-macrophage polarization induced by HLA I DSA-activated ECs. Anti-HLA I (IgG or F(ab')2) antibody-activated ECs induced the polarization of M2 macrophages with increased CD206 expression and MMP9 secretion. However, inhibition of TLR4 signaling or PSGL-1-P-selectin interactions significantly decreased both CD206 and MMP9. Monocyte adherence to Fc-P-selectin coated plates induced M2 macrophages with increased CD206 and MMP9. Moreover, Fc-receptor and IgG interactions synergistically enhanced active-MMP9 in conjunction with P-selectin. Transcriptomic analysis of arteries from DSA+CAV+ rejected cardiac allografts and multiplex-immunofluorescent staining illustrated the expression of CD68+CD206+CD163+MMP9+ M2 macrophages within the neointima of CAV-affected lesions. These findings reveal a novel mechanism linking HLA I antibody-activated endothelium to the generation of M2 macrophages which secrete vascular remodeling proteins contributing to AMR and CAV pathogenesis.
Assuntos
Receptor 4 Toll-Like , Doenças Vasculares , Humanos , Metaloproteinase 9 da Matriz , Selectina-P , Macrófagos , Endotélio , Antígenos HLA , Aloenxertos , Imunoglobulina GRESUMO
ßIV-spectrin is a membrane-associated cytoskeletal protein that maintains the structural stability of cell membranes and integral proteins such as ion channels and transporters. Its biological functions are best characterized in the brain and heart, although recently we discovered a fundamental new role in the vascular system. Using cellular and genetic mouse models, we reported that ßIV-spectrin acts as a critical regulator of developmental and tumor-associated angiogenesis. ßIV-spectrin was shown to selectively express in proliferating endothelial cells (EC) and suppress VEGF/VEGFR2 signaling by enhancing receptor internalization and degradation. Here we examined how these events impact the downstream kinase signaling cascades and target substrates. Based on quantitative phosphoproteomics, we found that ßIV-spectrin significantly affects the phosphorylation of epigenetic regulatory enzymes in the nucleus, among which DNA methyltransferase 1 (DNMT1) was determined as a top substrate. Biochemical and immunofluorescence results showed that ßIV-spectrin inhibits DNMT1 function by activating ERK/MAPK, which in turn phosphorylates DNMT1 at S717 to impede its nuclear localization. Given that DNMT1 controls the DNA methylation patterns genome-wide, and is crucial for vascular development, our findings suggest that epigenetic regulation is a key mechanism by which ßIV-spectrin suppresses angiogenesis.
Assuntos
DNA (Citosina-5-)-Metiltransferase 1 , Sistema de Sinalização das MAP Quinases , Proteômica , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/genética , Animais , Proteômica/métodos , Camundongos , Fosforilação , Humanos , Neovascularização Fisiológica , Espectrina/metabolismo , Espectrina/genética , Fosfoproteínas/metabolismo , Neovascularização Patológica/metabolismo , Neovascularização Patológica/genética , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais/metabolismo , AngiogêneseRESUMO
Circular RNA (circRNA) plays multiple roles in the development of esophageal cancer (EC). Herein, we investigate the function of circ_0001944 in EC progression and the related mechanism. Expression of circ_0001944, microRNA-338-5p (miR-338-5p), pyruvate dehydrogenase kinase 1 (PDK1), E-cadherin and N-cadherin was analyzed by quantitative real-time polymerase chain reaction, Western blotting or immunohistochemistry assay. Cell viability, proliferation, apoptosis, invasion and migration were investigated by cell counting kit-8 (CCK-8), 5-Ethynyl-2'-deoxyuridine (EdU), flow cytometry, transwell invasion and wound-healing assays, respectively. Glucose consumption was detected by Glucose Assay Kit. Lactate production was analyzed by Lactate Assay Kit. ATP/ADP ratio was determined by ADP/ATP ratio Assay Kit. The associations among circ_0001944, miR-338-5p and PDK1 were identified by dual-luciferase reporter and RNA pull-down assays. Xenograft mouse model assay was used to explore the role of circ_0001944 on tumor tumorigenesis in vivo. Circ_0001944 and PDK1 expression were significantly upregulated, while miR-338-5p was downregulated in EC tissues and cells in contrast with normal esophageal tissues and cells. Circ_0001944 knockdown inhibited EC cell proliferation, invasion, migration and glycolysis but induced apoptosis. Meanwhile, circ_0001944 depletion suppressed tumor tumorigenesis in vivo. Mechanistically, circ_0001944 bound to miR-338-5p, and miR-338-5p targeted PDK1. In addition, miR-338-5p inhibitors attenuated circ_0001944 depletion-induced effects in EC cells. The regulation of miR-338-5p on EC progression involved the downregulation of PDK1. Further, circ_0001944 controlled PDK1 expression through miR-338-5p. Circ_0001944 knockdown inhibited EC development and glycolysis by regulating the miR-338-5p/PDK1 pathway, providing a promising target for EC therapy.
Assuntos
Neoplasias Esofágicas , MicroRNAs , Humanos , Animais , Camundongos , Neoplasias Esofágicas/genética , Carcinogênese , Glicólise , Proliferação de Células , Modelos Animais de Doenças , Glucose , Lactatos , Trifosfato de Adenosina , MicroRNAs/genéticaRESUMO
Tea is an important cash crop that is often consumed by chewing pests, resulting in reduced yields and economic losses. It is important to establish a method to quickly identify the degree of damage to tea plants caused by leaf-eating insects and screen green control compounds. This study was performed through the combination of deep learning and targeted metabolomics, in vitro feeding experiment, enzymic analysis and transient genetic transformation. A small target damage detection model based on YOLOv5 with Transformer Prediction Head (TPH-YOLOv5) algorithm for the tea canopy level was established. Orthogonal partial least squares (OPLS) was used to analyze the correlation between the degree of damage and the phenolic metabolites. A potential defensive compound, (-)-epicatechin-3-O-caffeoate (EC-CA), was screened. In vitro feeding experiments showed that compared with EC and epicatechin gallate, Ectropis grisescens exhibited more significant antifeeding against EC-CA. In vitro enzymatic experiments showed that the hydroxycinnamoyl transferase (CsHCTs) recombinant protein has substrate promiscuity and can catalyze the synthesis of EC-CA. Transient overexpression of CsHCTs in tea leaves effectively reduced the degree of damage to tea leaves. This study provides important reference values and application prospects for the effective monitoring of pests in tea gardens and screening of green chemical control substances.
Assuntos
Camellia sinensis , Aprendizado Profundo , Lepidópteros , Animais , Camellia sinensis/metabolismo , Insetos , Chá/química , Chá/metabolismoRESUMO
Crispr/CAS9-enabled homologous recombination to insert a tag in frame with an endogenous gene can circumvent difficulties such as context-dependent promoter activity that complicate analysis of gene expression and protein accumulation patterns. However, there have been few reports examining whether such gene targeting/gene tagging (GT) can alter expression of the target gene. The enzyme encoded by Δ1-pyrroline-5-carboxylate synthetase 1 (P5CS1) is key for stress-induced proline synthesis and drought resistance, yet its expression pattern and protein localisation have been difficult to assay. We used GT to insert YFP in frame with the 5' or 3' ends of the endogenous P5CS1 and At14a-Like 1 (AFL1) coding regions. Insertion at the 3' end of either gene generated homozygous lines with expression of the gene-YFP fusion indistinguishable from the wild type allele. However, for P5CS1 this occurred only after selfing and advancement to the T5 generation allowed initial homozygous lethality of the insertion to be overcome. Once this was done, the GT-generated P5CS1-YFP plants revealed new information about P5CS1 localisation and tissue-specific expression. In contrast, insertion of YFP at the 5' end of either gene blocked expression. The results demonstrate that GT can be useful for functional analyses of genes that are problematic to properly express by other means but also show that, in some cases, GT can disrupt expression of the target gene.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Plantas Geneticamente Modificadas , Regulação da Expressão Gênica de Plantas , Estresse Fisiológico/genética , Mutagênese Insercional/genética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismoRESUMO
BACKGROUND: Esophageal cancer (EC) is a global canker notorious for causing high mortality due to its relentless incidence rate, convoluted with unyielding recurrence and metastasis. However, these intricacies of EC are associated with an immoderate expression of NY-ESO-1 antigen, presenting a lifeline for adoptive T cell therapy. We hypothesized that naturally isolated higher-affinity T cell receptors (TCRs) that bind to NY-ESO-1 would allow T lymphocytes to target EC with a pronounced antitumor response efficacy. Also, targeting TRPV2, which is associated with tumorigenesis in EC, creates an avenue for dual-targeted therapy. We exploited the dual-targeting antitumor efficacy against EC. METHODS: We isolated antigen-specific TCRs (asTCRs) from a naive library constructed with TCRs obtained from enriched cytotoxic T lymphocytes. The robustness of our asTCRs and their TCR-T cell derivatives, Tranilast (TRPV2 inhibitor), and their bivalent treatment were evaluated with prospective cross-reactive human-peptide variants and tumor cells. RESULTS: Our study demonstrated that our naive unenhanced asTCRs and their TCR-Ts perpetuated their cognate HLA-A*02:01/NY-ESO-1(157-165) specificity, killing varying EC cells with higher cytotoxicity compared to the known affinity-enhanced TCR (TCRe) and its wild-type (TCR0) which targets the same NY-ESO-1 antigen. Furthermore, the TCR-Ts and Tranilast bivalent treatment showed superior EC killing compared to any of their monovalent treatments of either TCR-T or Tranilast. CONCLUSION: Our findings suggest that dual-targeted immunotherapy may have a superior antitumor effect. Our study presents a technique to evolve novel, robust, timely therapeutic strategies and interventions for EC and other malignancies.
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BACKGROUND: Esophageal cancer (EC) is a deadly disease with limited therapeutic options. Although circulating tumor DNA (ctDNA) could be a promising tool in this regard, the availiable evidence is limited. We performed a systematic review and meta-analysis to summarize the clinical applicability of the next-generation sequencing (NGS) and droplet digital polymerase chain reaction (ddPCR) technology on the ctDNA detection of the EC and listed the current challenges. METHODS: We systematically searched MEDLINE (via PubMed), Embase (via OVID), ISI Web of Science database and Cochrane Library from January, 2000 to April, 2023. Progression-free survival (PFS) and overall survival (OS) were set as primary outcome endpoints. Pathologic response was evaluated by tumor regression grade (TRG), according to the eighth edition of the American Joint Committee on Cancer (AJCC). Major pathologic regression (MPR) was defined as TRG 1 and 2. The MPR was set as secondary endpoint. Hazard rate (HR) and associated 95% CI were used as the effect indicators the association between ctDNA and prognosis of EC. MPR rates were also calculated. Fixed-effect model (Inverse Variance) or random-effect model (Mantel-Haenszel method) was performed depending on the statistically heterogeneity. RESULTS: Twenty-two studies, containing 1144 patients with EC, were included in this meta-analysis. The results showed that OS (HR = 3.87; 95% CI, 2.86-5.23) and PFS (HR = 4.28; 95% CI, 3.34-5.48) were shorter in ctDNA-positive patients. In the neoadjuvant therapy, the sensitivity analysis showed the clarified HR of ctDNA-positive was 1.13(95% CI, 1.01-1.28). We also found that TP53, NOTCH1, CCND1 and CNKN2A are the most frequent mutation genes. CONCLUSIONS: Positive ctDNA is associated with poor prognosis, which demonstrated clinical value of ctDNA. Longitudinal ctDNA monitoring showed potential prognostic value in the neoadjuvant therapy. In an era of precision medicine, ctDNA could be a promising tool to individualize treatment planning and to improve outcomes in EC. PROSPERO REGISTRATION NUMBER: CRD42023412465.
Assuntos
DNA Tumoral Circulante , Neoplasias Esofágicas , Humanos , DNA Tumoral Circulante/genética , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/terapia , Bases de Dados Factuais , Biblioteca Gênica , Genes cdcRESUMO
Esophageal cancer (EC) is a familiar digestive tract tumor with highly lethal. The hypoxic environment has been demonstrated to be a significant factor in modulating malignant tumor progression and is strongly associated with the abnormal energy metabolism of tumor cells. Serine hydroxymethyl transferase 2 (SHMT2) is one of the most frequently expressed metabolic enzymes in human malignancies. The study was designed to investigate the biological functions and regulation mechanisms of SHMT2 in EC under hypoxia. We conducted RT-qPCR to assess SHMT2 levels in EC tissues and cells (TE-1 and EC109). EC cells were incubated under normoxia and hypoxia, respectively, and altered SHMT2 expression was evaluated through RT-qPCR, western blot, and immunofluorescence. The biological functions of SHMT2 on EC cells were monitored by performing CCK-8, EdU, transwell, sphere formation, glucose uptake, and lactate production assays. The SHMT2 protein lactylation was measured by immunoprecipitation and western blot. In addition, SHMT2-interacting proteins were analyzed by bioinformatics and validated by rescue experiments. SHMT2 was notably upregulated in EC tissues and cells. Hypoxia elevated SHMT2 protein expression, augmenting EC cell proliferation, migration, invasion, stemness, and glycolysis. In addition, hypoxia triggered lactylation of the SHMT2 protein and enhanced its stability. SHMT2 knockdown impeded the malignant phenotype of EC cells. Further mechanistic studies disclosed that SHMT2 is involved in EC progression by interacting with MTHFD1L. Hypoxia-induced SHMT2 protein lactylation and upregulated its protein level, which in turn enhanced MTHFD1L expression and accelerated the malignant progression of EC cells.
RESUMO
BACKGROUND: Vascular growth followed by vessel specification is crucial for the establishment of a hierarchical blood vascular network. We have shown that TIE2 is required for vein development while little is known about its homologue TIE1 (tyrosine kinase with immunoglobulin-like and EGF [epithelial growth factor]-like domains 1) in this process. METHODS: We analyzed functions of TIE1 as well as its synergy with TIE2 in the regulation of vein formation by employing genetic mouse models targeting Tie1, Tek, and Nr2f2, together with in vitro cultured endothelial cells to decipher the underlying mechanism. RESULTS: Cardinal vein growth appeared normal in TIE1-deficient mice, whereas TIE2 deficiency altered the identity of cardinal vein endothelial cells with the aberrant expression of DLL4 (delta-like canonical Notch ligand 4). Interestingly, the growth of cutaneous veins, which was initiated at approximately embryonic day 13.5, was retarded in mice lack of TIE1. TIE1 deficiency disrupted the venous integrity, displaying increased sprouting angiogenesis and vascular bleeding. Abnormal venous sprouts with defective arteriovenous alignment were also observed in the mesenteries of Tie1-deleted mice. Mechanistically, TIE1 deficiency resulted in the decreased expression of venous regulators including TIE2 and COUP-TFII (chicken ovalbumin upstream promoter transcription factor, encoded by Nr2f2, nuclear receptor subfamily 2 group F member 2) while angiogenic regulators were upregulated. The alteration of TIE2 level by TIE1 insufficiency was further confirmed by the siRNA-mediated knockdown of Tie1 in cultured endothelial cells. Interestingly, TIE2 insufficiency also reduced the expression of TIE1. Combining the endothelial deletion of Tie1 with 1 null allele of Tek resulted in a progressive increase of vein-associated angiogenesis leading to the formation of vascular tufts in retinas, whereas the loss of Tie1 alone produced a relatively mild venous defect. Furthermore, the induced deletion of endothelial Nr2f2 decreased both TIE1 and TIE2. CONCLUSIONS: Findings from this study imply that TIE1 and TIE2, together with COUP-TFII, act in a synergistic manner to restrict sprouting angiogenesis during the development of venous system.
Assuntos
Receptor de TIE-1 , Receptor TIE-2 , Camundongos , Animais , Receptor de TIE-1/genética , Receptor de TIE-1/metabolismo , Receptor TIE-2/genética , Receptor TIE-2/metabolismo , Células Endoteliais/metabolismo , Transdução de Sinais , VeiasRESUMO
Eradicating invasive predators from islands can result in substantial recovery of seabirds, but the mechanisms that drive population changes remain poorly understood. Meta-analyses have recently revealed that immigration is surprisingly important to the recovery of philopatric seabirds, but it is not known whether dispersal and philopatry interact predictably to determine rates of population growth and changes of distribution. We used whole-island surveys and long-term monitoring plots to study the abundance, distribution, and trends of 4 burrowing seabird species on Macquarie Island, Australia, to examine the legacy impacts of invasive species and ongoing responses to the world's largest eradication of multiple species of vertebrates. Wekas (Gallirallus australis) were eradicated in 1988; cats (Felis catus) in 2001; and rabbits (Oryctolagus cuniculus), black rats (Rattus rattus), and mice (Mus mus) in 2011-2014. We compared surveys from 1976-1979 and 2017-2018 and monitoring from the 1990s and 2000s onward. Antarctic prions (Pachyptila desolata) and white-headed petrels (Pterodroma lessonii) increased â¼1% per year. Blue petrels (Halobaena caerulea) and gray petrels (Procellaria cinerea) recolonized following extirpation from the main island in the 1900s but remained spatially and numerically rare in 2018. However, they increased rapidly at 14% and 10% per year, respectively, since cat eradication in 2001. Blue and gray petrel recolonization occurred on steep, dry, west-facing slopes close to ridgelines at low elevation (i.e., high-quality petrel habitat). They overlapped <5% with the distribution of Antarctic prion and white-headed petrels which occurred in suboptimal shallow, wet, east-facing slopes at high elevation. We inferred that the speed of population growth of recolonizing species was related to their numerically smaller starting size compared with the established species and was driven by immigration and selection of ideal habitat.
Patrones de recuperación en aves marinas existentes y extirpadas después de la mayor erradicación mundial de multidepredadores Resumen La erradicación de depredadores invasores en las islas puede derivar en la recuperación sustancial de aves marinas, aunque entendemos muy poco los mecanismos que causan los cambios poblacionales. Los metaanálisis recientes han revelado que la inmigración es de gran importancia para la recuperación de aves marinas filopátricas, aunque no sabemos si la dispersión y la filopatría interactúan de forma predecible para poder determinar las tasas de crecimiento poblacional y los cambios en la distribución. Aplicamos censos de isla completa y parcelas de monitoreo a largo plazo para estudiar la abundancia, distribución y tendencias de cuatro especies de aves marinas cavadoras en la Isla Macquarie, Australia, para analizar los impactos heredados de las especies invasoras y la respuesta continua a la mayor erradicación mundial de varias especies de vertebrados. El rascón weka (Gallirallus australis) se erradicó en 1988; los gatos (Felis catus) en 2001; y los conejos (Oryctolagus cuniculus), ratas (Rattus rattus) y ratones (Mus mus) entre 2011 y 2014. Comparamos los censos de 19761979 y 20172018 y el monitoreo realizado en los 90s y del año 2000 en adelante. El pato petrel antártico (Pachyptila desolata) y el petrel cabeciblanco (Pterodroma lessonii) incrementaron â¼1% por año. El petrel azulado (Halobaena caerulea) y la pardela gris (Procellaria cinerea) recolonizaron la isla después de su extirpación en la década de 1900, pero todavía eran especies raras espacial y numéricamente en 2018. Sin embargo, esta especie incrementó rápidamente en un 14% y 10% por año respectivamente desde que se erradicaron los gatos en 2001. La recolonización ocurrió desde las laderas empinadas, secas y con orientación al oeste en los sistemas montañosos de baja elevación (es decir, hábitats de gran calidad para los petreles). La distribución del petrel azulado y la pardela gris ocurrió en laderas someras subóptimas y húmedas con orientación al este a altas elevaciones. Esta distribución se traslapó menos del 5% con la del pato petrel antártico y la del petrel cabeciblanco. Inferimos que la velocidad del crecimiento poblacional de las especies que recolonizaron estuvo relacionada con el menor tamaño inicial en comparación con las especies establecidas y fue causada por la inmigración y la selección del hábitat ideal.