Discovery of phage determinants that confer sensitivity to bacterial immune systems.

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.

Bacterial Retrons Function In Anti-Phage Defense.

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.

Retron-Eco1 assembles NAD+-hydrolyzing filaments that provide immunity against bacteriophages.

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.

PD-1 Imposes Qualitative Control of Cellular Transcriptomes in Response to T Cell Activation.

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.

Prostate cancer-induced endothelial-cell-to-osteoblast transition drives immunosuppression in the bone-tumor microenvironment through Wnt pathway-induced M2 macrophage polarization.

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.

Mouse embryos, chimeras, and embryonal carcinoma stem cells-Reflections on the winding road to gene manipulation.

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.

Acute phosphatidylinositol 4,5 bisphosphate depletion destabilizes sarcolemmal expression of cardiac L-type Ca2+ channel CaV1.2.

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.

Presenilin-1 ΔE9 mutation associated sarcoplasmic reticulum leak alters [Ca2+]i distribution in human iPSC-derived cardiomyocytes.

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.

On the role of dysferlin in striated muscle: membrane repair, t-tubules and Ca2+ handling.

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.

Role of endothelial PDGFB in arterio-venous malformations pathogenesis.

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.

Human leukocyte antigen class I antibody-activated endothelium promotes CD206+ M2 macrophage polarization and MMP9 secretion through TLR4 signaling and P-selectin in a model of antibody-mediated rejection and allograft vasculopathy.

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.

Cellular calcium handling and electrophysiology are modulated by chronic physiological pacing in human induced pluripotent stem cell-derived cardiomyocytes.

Electric pacing of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) has been increasingly used to simulate cardiac arrhythmias in vitro and to enhance cardiomyocyte maturity. However, the impact of electric pacing on cellular electrophysiology and Ca2+-handling in differentiated hiPSC-CM is less characterized. Here we studied the effects of electric pacing for 24h or 7d at a physiological rate of 60 bpm on cellular electrophysiology and Ca2+-cycling in late-stage, differentiated hiPSC-CM (>90% troponin+, >60d post differentiation). Electric culture pacing for 7d did not influence cardiomyocyte cell size, apoptosis or generation of reactive oxygen species in differentiated hiPSC-CM compared to 24h pacing. However, epifluorescence measurements revealed that electric pacing for 7d improved systolic Ca2+-transient amplitude and Ca2+-transient upstroke, which could be explained by elevated sarcoplasmic reticulum Ca2+-load and SERCA activity. Diastolic Ca2+-leak was not changed in line-scanning confocal microscopy suggesting that the improvement in systolic Ca2+-release was not associated with a higher open probability of RyR2 during diastole. While bulk cytosolic Na+-concentration and NCX activity were not changed, patch-clamp studies revealed that chronic pacing caused a slight abbreviation of the action potential duration (APD) in hiPSC-CM. We found in whole-cell voltage-clamp measurements that chronic pacing for 7d led to a decrease in late Na+-current, which might explain the changes in APD. In conclusion, our results show that chronic pacing improves systolic Ca2+-handling and modulates the electrophysiology of late-stage, differentiated iPSC-CM. This study might help to understand the effects of electric pacing and its numerous applications in stem cell research including arrhythmia simulation.

Phosphoproteomic profiling identifies DNMT1 as a key substrate of beta IV spectrin-dependent ERK/MAPK signaling in suppressing angiogenesis.

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.

Circ_0001944 depletion inhibits glycolysis and esophageal cancer progression by binding to miR-338-5p to reduce PDK1 expression.

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.

Deep learning and targeted metabolomics-based monitoring of chewing insects in tea plants and screening defense compounds.

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.

Insertion of YFP at P5CS1 and AFL1 shows the potential, and potential complications, of gene tagging for functional analyses of stress-related proteins.

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.

NY-ESO-1-specific T cell receptor-engineered T cells and Tranilast, a TRPV2 antagonist bivalent treatment enhances the killing of esophageal cancer: a dual-targeted cancer therapeutic route.

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.

An update of clinical value of circulating tumor DNA in esophageal cancer: a systematic review and meta-analysis.

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.

Hypoxia-induced SHMT2 protein lactylation facilitates glycolysis and stemness of esophageal cancer cells.

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.

Endothelial TIE1 Restricts Angiogenic Sprouting to Coordinate Vein Assembly in Synergy With Its Homologue TIE2.

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.