Single-cell Transcriptional Landscape of Temporal Neutrophil Response to Burn Wound in Larval Zebrafish.

Neutrophils accumulate early in tissue injury. However, the cellular and functional heterogeneity of neutrophils during homeostasis and in response to tissue damage remains unclear. In this study, we use larval zebrafish to understand neutrophil responses to thermal injury. Single-cell transcriptional mapping of myeloid cells during a 3-d time course in burn and control larvae revealed distinct neutrophil subsets and their cell-cell interactions with macrophages across time and conditions. The trajectory formed by three zebrafish neutrophil subsets resembles human neutrophil maturation, with varying transition patterns between conditions. Through ligand-receptor cell-cell interaction analysis, we found that neutrophils communicate more in burns in a pathway and temporal manner. Finally, we identified the correlation between zebrafish myeloid signatures and human burn severity, establishing GPR84+ neutrophils as a potential marker of early innate immune response in burns. This work builds a comparative single-cell transcriptomic framework to identify neutrophil markers of tissue damage using model organisms.

Effects of Berberine on Lipid Metabolism, Antioxidant Status, and Immune Response in Liver of Tilapia (Oreochromis niloticus) under a High-Fat Diet Feeding.

Berberine, a natural alkaloid found abundantly in various medicinal plants, exhibits antioxidative, anti-inflammatory, and lipid metabolism-regulatory properties. Nonetheless, its protective effects and the molecular mechanisms underlying liver injury in fish have not been fully elucidated. The aims of this study were to investigate the antioxidative, anti-inflammatory, and lipid metabolism-regulating effects of berberine against high-fat diet (HFD)-induced liver damage and to clarify the underlying molecular mechanisms. Tilapia were fed diets containing two doses of berberine (50 and 100 mg/kg diet) alongside high fat for 60 days. The results showed that berberine treatments (50 and/or 100 mg/kg) significantly reduced elevated aminotransferases, triglycerides (TG), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-c) in the plasma. In the liver, berberine treatments significantly increased the expression of peroxisome proliferator-activated receptor α (pparα) and carnitine palmitoyltransferase 1 (cpt-1) genes, leading to a reduction in lipid accumulation. Meanwhile, berberine treatment suppressed lipid peroxidation formation and enhanced antioxidant capacity. Berberine upregulated the mRNA levels of erythroid 2-related factor 2 (nrf2) and its downstream genes including heme oxygenase 1 (ho-1) and glutathione-S-transferase (gstα). Additionally, berberine attenuated the inflammation by inhibiting the expression of toll-like receptor 2 (tlr2), myeloid differential protein-88 (myd88), relb, and inflammatory cytokines such as interleukin-1ß (il-1ß), tumor necrosis factor-α (tnf-α), and il-8. In summary, this study suggested that berberine offers protection against HFD-induced liver damage in tilapia via regulating lipid metabolism, antioxidant status, and immune response. This protective effect may be attributed to the modulation of the Nrf2, TLR2/MyD88/NF-κB, and PPARα signaling pathways.

Multi-Omics Analysis to Understand the Effects of Dietary Proanthocyanidins on Antioxidant Capacity, Muscle Nutrients, Lipid Metabolism, and Intestinal Microbiota in Cyprinus carpio.

Proanthocyanidins (Pros), a natural polyphenolic compound found in grape seed and other plants, have received significant attention as additives in animal feed. However, the specific mechanism by which Pros affect fish health remains unclear. Therefore, the aim of this study was to investigate the potential effects of dietary Pro on common carp by evaluating biochemical parameters and multi-omics analysis. The results showed that Pro supplementation improved antioxidant capacity and the contents of polyunsaturated fatty acids (n-3 and n-6) and several bioactive compounds. Transcriptomic analysis demonstrated that dietary Pro caused an upregulation of the sphingolipid catabolic process and the lysosome pathway, while simultaneously downregulating intestinal cholesterol absorption and the PPAR signaling pathway in the intestines. Compared to the normal control (NC) group, the Pro group exhibited higher diversity in intestinal microbiota and an increased relative abundance of Cetobacterium and Pirellula. Furthermore, the Pro group had a lower Firmicutes/Bacteroidetes ratio and a decreased relative abundance of potentially pathogenic bacteria. Collectively, dietary Pro improved antioxidant ability, muscle nutrients, and the diversity and composition of intestinal microbiota. The regulation of lipid metabolism and improvement in muscle nutrients were linked with changes in the intestinal microbiota.

A tessellated lymphoid network provides whole-body T cell surveillance in zebrafish.

Homeostatic trafficking to lymph nodes allows T cells to efficiently survey the host for cognate antigen. Non-mammalian jawed vertebrates lack lymph nodes but maintain similarly diverse T cell pools. Here, we exploit in vivo imaging of transparent zebrafish to investigate how T cells organize and survey for antigen in an animal devoid of lymph nodes. We find that naïve-like T cells in zebrafish organize into a previously undescribed whole-body lymphoid network that supports streaming migration and coordinated trafficking through the host. This network has the cellular hallmarks of a mammalian lymph node, including naïve T cells and CCR7-ligand expressing non-hematopoietic cells, and facilitates rapid collective migration. During infection, T cells transition to a random walk that supports antigen presenting cell interactions and subsequent activation. Our results reveal that T cells can toggle between collective migration and individual random walks to prioritize either large-scale trafficking or antigen search in situ . This novel lymphoid network thus facilitates whole-body T cell trafficking and antigen surveillance in the absence of a lymph node system. Significance Statement: In mammals, lymph nodes play a critical role in the initiation of adaptive immune responses by providing a dedicated place for T cells to scan antigen-presenting cells. Birds, reptiles, amphibians, and fish all maintain diverse repertoires of T cells but lack lymph nodes, raising questions about how adaptive immunity functions in lower jawed vertebrates. Here, we describe a novel network of lymphocytes in zebrafish that supports whole-body T cell trafficking and provides a site for antigen search, mirroring the function of mammalian lymph nodes. Within this network, T cells can prioritize large-scale trafficking or antigen scanning by toggling between two distinct modes of migration. This network provides valuable insights into the evolution of adaptive immunity.

Increased mitochondrial fission drives the reprogramming of fatty acid metabolism in hepatocellular carcinoma cells through suppression of Sirtuin 1.

BACKGROUND: Mitochondria are dynamic organelles that constantly change their morphology through fission and fusion processes. Recently, abnormally increased mitochondrial fission has been observed in several types of cancer. However, the functional roles of increased mitochondrial fission in lipid metabolism reprogramming in cancer cells remain unclear. This study aimed to explore the role of increased mitochondrial fission in lipid metabolism in hepatocellular carcinoma (HCC) cells. METHODS: Lipid metabolism was determined by evaluating the changes in the expressions of core lipid metabolic enzymes and intracellular lipid content. The rate of fatty acid oxidation was evaluated by [3 H]-labelled oleic acid. The mitochondrial morphology in HCC cells was evaluated by fluorescent staining. The expression of protein was determined by real-time PCR, iimmunohistochemistry and Western blotting. RESULTS: Activation of mitochondrial fission significantly promoted de novo fatty acid synthesis in HCC cells through upregulating the expression of lipogenic genes fatty acid synthase (FASN), acetyl-CoA carboxylase1 (ACC1), and elongation of very long chain fatty acid protein 6 (ELOVL6), while suppressed fatty acid oxidation by downregulating carnitine palmitoyl transferase 1A (CPT1A) and acyl-CoA oxidase 1 (ACOX1). Consistently, suppressed mitochondrial fission exhibited the opposite effects. Moreover, in vitro and in vivo studies revealed that mitochondrial fission-induced lipid metabolism reprogramming significantly promoted the proliferation and metastasis of HCC cells. Mechanistically, mitochondrial fission increased the acetylation level of sterol regulatory element-binding protein 1 (SREBP1) and peroxisome proliferator-activated receptor coactivator 1 alpha (PGC-1α) by suppressing nicotinamide adenine dinucleotide (NAD+)/Sirtuin 1 (SIRT1) signaling. The elevated SREBP1 then upregulated the expression of FASN, ACC1 and ELOVL6 in HCC cells, while PGC-1α/PPARα suppressed the expression of CPT1A and ACOX1. CONCLUSIONS: Increased mitochondrial fission plays a crucial role in the reprogramming of lipid metabolism in HCC cells, which provides strong evidence for the use of this process as a drug target in the treatment of this malignancy.

EV PD-L1 Contributes to Immunosuppressive CD8+ T Cells in Peripheral Blood of Pediatric Wilms Tumor.

Wilms tumor (WT) is the most common renal cancer and the most prevalent abdominal cancer in children. Children with recurrent or progressive forms of WT could benefit from novel immune-targeted approaches. While the immune status of these patients, especially the immunosuppression of peripheral T cells, was rarely reported. The present study enrolled a consecutive series of 14 Chinese WT children and 14 age- and gender-matched healthy controls. We demonstrated that plasma extracellular vesicular (EV) PD-L1 levels significantly increased in WT patients than in healthy controls. EV PD-L1 significantly inhibited the activation of human CD8+ T cells by down-regulating the cell surface CD69 expression and the intracellular IFNγ and TNFα production in vitro. In peripheral CD8+ T cells of WT patients, the intracellular IFNγ and TNFα production significantly decreased than healthy controls. The level of plasma EV PD-L1 significantly correlated with the intracellular TNFα production in peripheral CD8+ T cells of WT patients. In conclusion, the significantly increased plasma EV PD-L1 in WT patients contributed to the immunosuppression of peripheral CD8+ T cells. Monitoring the level of plasma EV PD-L1 will be helpful for the selection of immune-targeted therapies for WT patients.

Cellular diversity of the regenerating caudal fin.

Zebrafish faithfully regenerate their caudal fin after amputation. During this process, both differentiated cells and resident progenitors migrate to the wound site and undergo lineage-restricted, programmed cellular state transitions to populate the new regenerate. Until now, systematic characterizations of cells comprising the new regenerate and molecular definitions of their state transitions have been lacking. We hereby characterize the dynamics of gene regulatory programs during fin regeneration by creating single-cell transcriptome maps of both preinjury and regenerating fin tissues at 1/2/4 days post-amputation. We consistently identified epithelial, mesenchymal, and hematopoietic populations across all stages. We found common and cell type-specific cell cycle programs associated with proliferation. In addition to defining the processes of epithelial replenishment and mesenchymal differentiation, we also identified molecular signatures that could better distinguish epithelial and mesenchymal subpopulations in fish. The insights for natural cell state transitions during regeneration point to new directions for studying this regeneration model.

SIK2 enhances synthesis of fatty acid and cholesterol in ovarian cancer cells and tumor growth through PI3K/Akt signaling pathway.

Salt-inducible kinase 2 (SIK2) has been established as a regulator of diverse biological processes including cell metabolism. A recent study has reported that SIK2 is required for adipocyte-induced ovarian cancer (OC) survival through facilitating fatty acid oxidation. However, whether SIK2 also plays a role in the lipid synthesis in OC cells remains elusive. Here, we showed that SIK2 significantly promoted the lipid synthesis in OC cells. On the one hand, SIK2 enhanced fatty acid synthesis through upregulating the expression of sterol regulatory element binding protein 1c (SREBP1c) and thus the transcription of major lipogenic enzyme FASN. On the other hand, SIK2 promoted cholesterol synthesis through upregulating the expression of sterol regulatory element binding protein 2 (SREBP2) and thus the transcription of major cholesterol synthesis enzymes HMGCR. Moreover, PI3K/Akt signaling pathway was found to be involved in the upregulation of SREBP1c and SREBP2 in OC cells. Moreover, in vitro and in vivo assays indicated that the SIK2-regulated fatty acid and cholesterol synthesis played a critical role in the growth of OC cells. Our findings demonstrate that SIK2 is a critical regulator of lipid synthesis in OC cells and thus promotes OC growth, which provides a strong line of evidence for this molecule to be used as a therapeutic target in the treatment of this malignancy.

Circadian clock gene NPAS2 promotes reprogramming of glucose metabolism in hepatocellular carcinoma cells.

Emerging evidences show that dysregulation of circadian genes is closely associated with tumorigenesis. However, whether circadian genes regulate the reprogramming of metabolism in tumor cells is largely unknown. Here, we showed that NPAS2, one of the core circadian molecules, significantly contributed to the reprogramming of glucose metabolism mainly through two mechanisms. On the one hand, NPAS2 upregulated the expression of glycolytic genes GLUT1, HK2, GPI, ALDOA, ENO2, PKM2 and MCT4. On the other hand, NPAS2 downregulated the expression of peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α). Mechanistically, HIF-1α was found to be a direct transcriptional target of NPAS2, which mediated both the upregulation of glycolytic genes and downregulation of mitochondrial biogenesis in HCC cells. In addition, we found that upregulation of NPAS2 expression was mainly due to the downregulation of miR-199b-5p. In vitro and in vivo assays further indicated that HIF-1α-mediated reprogramming of glucose metabolism played a critical role in NPAS2-regulated growth and metastasis of HCC cells. Our findings demonstrate that NPAS2 plays a critical role in glucose metabolism reprogramming, suggesting that NPAS2 may serve as a potential therapeutic target in HCC.

Upregulation of histamine receptor H1 promotes tumor progression and contributes to poor prognosis in hepatocellular carcinoma.

H1 histamine receptor (H1HR) belongs to the family of rhodopsin-like G-protein-coupled receptors. Recent studies have shown that H1HR expression is increased in several types of cancer. However, its functional roles in tumor progression remain largely unknown, especially in hepatocellular carcinoma (HCC). We found that H1HR is frequently unregulated in HCC, which is significantly associated with both recurrence-free survival and overall survival in HCC patients. Functional experiments revealed that H1HR promoted both the growth and metastasis of HCC cells by inducing cell cycle progression, formation of lamellipodia, production of matrix metalloproteinase 2, and suppression of cell apoptosis. Activation of cyclic adenosine monophosphate-dependent protein kinase A was found to be involved in H1HR-mediated HCC cell growth and metastasis. In addition, we found that overexpression of H1HR was mainly due to the downregulation of miR-940 in HCC cells. Moreover, the H1HR inhibitor terfenadine significantly suppressed tumor growth and metastasis in an HCC xenograft nude mice model. Our findings demonstrate that H1HR plays a critical role in the growth and metastasis of HCC cells, which provides experimental evidence supporting H1HR as a potential drug target for the treatment of HCC.

DNMT and HDAC inhibitors induce cryptic transcription start sites encoded in long terminal repeats.

Several mechanisms of action have been proposed for DNA methyltransferase and histone deacetylase inhibitors (DNMTi and HDACi), primarily based on candidate-gene approaches. However, less is known about their genome-wide transcriptional and epigenomic consequences. By mapping global transcription start site (TSS) and chromatin dynamics, we observed the cryptic transcription of thousands of treatment-induced non-annotated TSSs (TINATs) following DNMTi and HDACi treatment. The resulting transcripts frequently splice into protein-coding exons and encode truncated or chimeric ORFs translated into products with predicted abnormal or immunogenic functions. TINAT transcription after DNMTi treatment coincided with DNA hypomethylation and gain of classical promoter histone marks, while HDACi specifically induced a subset of TINATs in association with H2AK9ac, H3K14ac, and H3K23ac. Despite this mechanistic difference, both inhibitors convergently induced transcription from identical sites, as we found TINATs to be encoded in solitary long terminal repeats of the ERV9/LTR12 family, which are epigenetically repressed in virtually all normal cells.