Near-cognate tRNAs increase the efficiency and precision of pseudouridine-mediated readthrough of premature termination codons.

Programmable RNA pseudouridylation has emerged as a new type of RNA base editor to suppress premature termination codons (PTCs) that can lead to truncated and nonfunctional proteins. However, current methods to correct disease-associated PTCs suffer from low efficiency and limited precision. Here we develop RESTART v3, which uses near-cognate tRNAs to improve the readthrough efficiency of pseudouridine-modified PTCs. We show an average of ~5-fold (range: 2.1- to 9.5-fold) higher editing efficiency than RESTART v2 in cultured cells and achieve functional PTC readthrough in disease cell models of cystic fibrosis and Hurler syndrome. Furthermore, RESTART v3 enables accurate incorporation of the original amino acid for nearly half of the PTC sites, considering the naturally occurring frequencies of sense-to-nonsense codons, without affecting normal termination codons. Although off-target sites were detected, we did not observe changes to the coding information or the expression level of transcripts, and the overall natural tRNA abundance remained constant.

GLP-1 receptor agonists alleviate colonic inflammation by modulating intestinal microbiota and the function of group 3 innate lymphoid cells.

Glucagon-like peptide-1 receptor agonists (GLP-1RAs), which are drugs used for treating type 2 diabetes, have been reported to exert anti-inflammatory effects on inflammatory bowel disease (IBD), the mechanism of which remains elusive. Here, we report that GLP-1RAs ameliorate dextran sulfate sodium (DSS)-induced colitis in both wild-type and T/B-cell-deficient mice through modulating group 3 innate lymphoid cells (ILC3s), a subset of innate lymphoid cells that regulate intestinal immunity. GLP-1RAs promote IL-22 production by ILC3, and the protective effect of GLP-1RAs on DSS-induced colitis was abrogated in ILC3-deficient RORgtgfp/gfp mice. Furthermore, the treatment effect of GLP-RAs on colitis, as well as the generation of IL-22-producing ILC3s by GLP-RAs, is dependent on the gut microbiota. GLP-1RAs increase the abundance of Firmicutes and Proteobacteria in the gut, particularly beneficial bacteria such as Lactobacillus reuteri, and decrease the abundance of enteropathogenic Staphylococcus bacteria. The untargeted gas chromatography (GC)/liquid chromatography (LC)-mass spectrometry (MS) of faecal metabolites further revealed enrichment of N,N-dimethylsphingosine (DMS), an endogenous metabolite derived from sphingosine, in the GLP-1RA-treated group. Strikingly, DMS ameliorates colitis while promoting intestinal IL-22-producing ILC3s. Taken together, our findings show that GLP-1RAs exert a therapeutic effect on colitis possibly by regulating the microbiota-DMS-IL-22+ILC3 axis, highlighting the potential beneficial role of GLP-RAs in inflammatory intestinal disorders with diabetes complications.

SIRT3 and SIRT4 double-genes remodeled the mitochondrial network to induce hepatocellular carcinoma cell line differentiation and suppress malignant phenotypes.

Tumor cells with damaged mitochondria undergo metabolic reprogramming, but gene therapy targeting mitochondria has not been comprehensively reported. In this study, plasmids targeting the normal hepatocyte cell line (L-O2) and hepatocellular carcinoma cell line were generated using three genes SIRT3, SIRT4, and SIRT5. These deacetylases play a variety of regulatory roles in cancer and are related to mitochondrial function. Compared with L-O2, SIRT3 and SIRT4 significantly ameliorated mitochondrial damage in HCCLM3, Hep3B and HepG2 cell lines and regulated mitochondrial biogenesis and mitophagy, respectively. We constructed double-gene plasmid for co-express SIRT3 and SIRT4 using the internal ribosome entry site (IRES). The results indicated that the double-gene plasmid effectively expressed SIRT3 and SIRT4, significantly improved mitochondrial quality and function, and reduced mtDNA level and oxidative stress in HCC cells. MitoTracker analysis revealed that the mitochondrial network was restored. The proliferation, migration capabilities of HCC cells were reduced, whereas their differentiation abilities were enhanced. This study demonstrated that the use of IRES-linked SIRT3 and SIRT4 double-gene vectors induced the differentiation of HCC cells and inhibited their development by ameliorating mitochondrial dysfunction. This intervention helped reverse metabolic reprogramming, and may provide a groundbreaking new framework for HCC treatment.

Trichosanthin alleviates streptozotocin-induced type 1 diabetes mellitus in mice by regulating the balance between bone marrow-derived IL6+ and IL10+ MDSCs.

Myeloid-derived suppressor cells (MDSCs) occupy a pivotal role in the intricate pathogenesis of the autoimmune disorder, Type 1 diabetes mellitus (T1DM). Since our previous work demonstrated that trichosanthin (TCS), an active compound of Chinese herb medicine Tian Hua Fen, regulated immune response, we aimed to clarify the efficacy and molecular mechanism of TCS in the treatment of T1DM. To this end, T1DM mouse model was established by streptozotocin (STZ) induction. The mice were randomly divided into normal control group (Ctl), T1DM group (STZ), TCS treated diabetic group (STZ + TCS) and insulin-treated diabetic group (STZ + insulin). Our comprehensive evaluation encompassed variables such as blood glucose, glycosylated hemoglobin, body weight, pertinent biochemical markers, pancreatic histopathology, and the distribution of immune cell populations. Furthermore, we meticulously isolated MDSCs from the bone marrow of T1DM mice, probing into the expressions of genes pertaining to the advanced glycation end product receptor (RAGE)/NF-κB signaling pathway through RT-qPCR. Evidently, TCS exhibited a substantial capacity to effectively counteract the T1DM-induced elevation in random blood glucose, glycosylated hemoglobin, and IL-6 levels in plasma. Pathological scrutiny underscored the ability of TCS to mitigate the damage incurred by islets. Intriguingly, TCS interventions engendered a reduction in the proportion of MDSCs within the bone marrow, particularly within the IL-6+ MDSC subset. In contrast, IL-10+ MDSCs exhibited an elevation following TCS treatment. Moreover, we observed a significant down-regulation of relative mRNA of pro-inflammatory genes, including arginase 1 (Arg1), inducible nitric oxide synthase (iNOS), RAGE and NF-κB, within MDSCs due to the influence of TCS. It decreases total MDSCs and regulates the balance between IL-6+ and IL-10+ MDSCs thus alleviating the symptoms of T1DM. TCS also down-regulates the RAGE/NF-κB signaling pathway, making it a promising alternative therapeutic treatment for T1DM. Collectively, our study offered novel insights into the underlying mechanism by which TCS serves as a promising therapeutic intervention for T1DM.

GLORI for absolute quantification of transcriptome-wide m6A at single-base resolution.

N6-methyladenosine (m6A) is the most abundant posttranscriptional chemical modification in mRNA, involved in regulating various physiological and pathological processes throughout mRNA metabolism. Recently, we developed GLORI, a sequencing method that enables the production of a globally absolute-quantitative m6A map at single-base resolution. Our technique utilizes the glyoxal- and nitrite-based chemical reaction, which selectively deaminates unmethylated adenosines while leaving m6A intact. The RNA library can then be prepared using a modified library construction protocol from enhanced UV crosslinking and immunoprecipitation (eCLIP) or commercial kits. Here we provide a detailed protocol for proper RNA sample handling and provide further guidelines for the use of a tailored bioinformatics pipeline (GLORI-tools) for subsequent data analysis. Compared with current methods, this new method is both exceptionally sensitive and robust, capable of identifying ~80,000 m6A sites with 50 Gb sequencing data in mammalian cells. It also provides a quantitative readout for m6A sites at single-base resolution. We hope the technique will provide a precise and unbiased tool for investigating m6A biology across various fields. Basic expertise in molecular biology and knowledge of bioinformatics are required for the protocol. The entire procedure can be completed within a week, with the library construction process taking ~4 d.

Carotenoids in maternal and cord blood, breast milk and their association with maternal dietary intake: a longitudinal study in Shanghai, China.

Carotenoids are important bioactive substances in breast milk, the profile of which is seldom studied. This study aimed to explore the profile of carotenoids in breast milk and maternal/cord plasma of healthy mother-neonate pairs in Shanghai, China, and their correlation with dietary intake. Maternal blood, umbilical cord blood and breast milk samples from five lactation stages (colostrum, transitional milk and early-, mid- and late-term mature milk) were collected. Carotenoid levels were analysed by HPLC. Carotenoid levels in breast milk changed as lactation progressed (P < 0·001). ß-Carotene was the primary carotenoid in colostrum. Lutein accounted for approximately 50 % of total carotenoids in transitional milk, mature milk and cord blood. Positive correlations were observed between five carotenoids in umbilical cord blood and maternal blood (P all < 0·001). ß-Carotene levels were also correlated between maternal plasma and three stages of breast milk (r = 0·605, P < 0·001; r = 0·456, P = 0·011, r = 0·446; P = 0·013, respectively). Dietary carotenoid intakes of lactating mothers also differed across lactation stages, although no correlation with breast milk concentrations was found. These findings suggest the importance of exploring the transport mechanism of carotenoids between mothers and infants and help guide the development of formulas for Chinese infants as well as the nutritional diets of lactating mothers.

Single-Cell Profiling of CD8+ T Cells in Acute Myeloid Leukemia Reveals a Continuous Spectrum of Differentiation and Clonal Hyperexpansion.

Comprehensive investigation of CD8+ T cells in acute myeloid leukemia (AML) is essential for developing immunotherapeutic strategies beyond immune checkpoint blockade. Herein, we performed single-cell RNA profiling of CD8+ T cells from 3 healthy bone marrow donors and 23 newly diagnosed (NewlyDx) and 8 relapsed/refractory (RelRef) patients with AML. Cells coexpressing canonical exhaustion markers formed a cluster constituting <1% of all CD8+ T cells. We identified two effector CD8+ T-cell subsets characterized by distinct cytokine and metabolic profiles that were differentially enriched in NewlyDx and RelRef patients. We refined a 25-gene CD8-derived signature correlating with therapy resistance, including genes associated with activation, chemoresistance, and terminal differentiation. Pseudotemporal trajectory analysis supported enrichment of a terminally differentiated state in CD8+ T cells with high CD8-derived signature expression at relapse or refractory disease. Higher expression of the 25-gene CD8 AML signature correlated with poorer outcomes in previously untreated patients with AML, suggesting that the bona fide state of CD8+ T cells and their degree of differentiation are clinically relevant. Immune clonotype tracking revealed more phenotypic transitions in CD8 clonotypes in NewlyDx than in RelRef patients. Furthermore, CD8+ T cells from RelRef patients had a higher degree of clonal hyperexpansion associated with terminal differentiation and higher CD8-derived signature expression. Clonotype-derived antigen prediction revealed that most previously unreported clonotypes were patient-specific, suggesting significant heterogeneity in AML immunogenicity. Thus, immunologic reconstitution in AML is likely to be most successful at earlier disease stages when CD8+ T cells are less differentiated and have greater capacity for clonotype transitions.

Regulation and functions of non-m6A mRNA modifications.

Nucleobase modifications are prevalent in eukaryotic mRNA and their discovery has resulted in the emergence of epitranscriptomics as a research field. The most abundant internal (non-cap) mRNA modification is N6-methyladenosine (m6A), the study of which has revolutionized our understanding of post-transcriptional gene regulation. In addition, numerous other mRNA modifications are gaining great attention because of their major roles in RNA metabolism, immunity, development and disease. In this Review, we focus on the regulation and function of non-m6A modifications in eukaryotic mRNA, including pseudouridine (Ψ), N6,2'-O-dimethyladenosine (m6Am), N1-methyladenosine (m1A), inosine, 5-methylcytidine (m5C), N4-acetylcytidine (ac4C), 2'-O-methylated nucleotide (Nm) and internal N7-methylguanosine (m7G). We highlight their regulation, distribution, stoichiometry and known roles in mRNA metabolism, such as mRNA stability, translation, splicing and export. We also discuss their biological consequences in physiological and pathological processes. In addition, we cover research techniques to further study the non-m6A mRNA modifications and discuss their potential future applications.

Single-Cell Profiling of CD8+ T Cells in Acute Myeloid Leukemia Reveals a Continuous Spectrum of Differentiation and Clonal Hyperexpansion.

Comprehensive investigation of CD8+ T cells in acute myeloid leukemia (AML) is essential for developing immunotherapeutic strategies beyond immune checkpoint blockade. Herein, we performed single-cell RNA profiling of CD8+ T cells from 3 healthy bone marrow donors and 23 newly diagnosed (NewlyDx) and 8 relapsed/refractory (RelRef) AML patients. Cells co-expressing canonical exhaustion markers formed a cluster constituting <1% of all CD8+ T cells. We identified two effector CD8+ T cell subsets characterized by distinct cytokine and metabolic profiles that were differentially enriched in NewlyDx and RelRef patients. We refined a 25-gene CD8-derived signature correlating with therapy resistance, including genes associated with activation, chemoresistance, and terminal differentiation. Pseudotemporal trajectory analysis supported enrichment of a terminally differentiated state in CD8+ T cells with high CD8-derived signature expression at relapse or refractory disease. Higher expression of the 25-gene CD8 AML signature correlated with poorer outcomes in previously untreated AML patients, suggesting that the bona fide state of CD8+ T cells and their degree of differentiation are clinically relevant. Immune clonotype tracking revealed more phenotypic transitions in CD8 clonotypes in NewlyDx than in RelRef patients. Furthermore, CD8+ T cells from RelRef patients had a higher degree of clonal hyperexpansion associated with terminal differentiation and higher CD8-derived signature expression. Clonotype-derived antigen prediction revealed that most previously unreported clonotypes were patient-specific, suggesting significant heterogeneity in AML immunogenicity. Thus, immunologic reconstitution in AML is likely to be most successful at earlier disease stages when CD8+ T cells are less differentiated and have greater capacity for clonotype transitions.

Aberrant alteration of peripheral B lymphocyte subsets in hepatocellular carcinoma patients.

Although B lymphocytes are widely known to participate in the immune response, the conclusive roles of B lymphocyte subsets in the antitumor immune response have not yet been determined. Single-cell data from GEO datasets were first analyzed, and then a B cell flow cytometry panel was used to analyze the peripheral blood of 89 HCC patients and 33 healthy controls recruited to participate in our research. Patients with HCC had a higher frequency of B10 cells and a lower percentage of MZB cells than healthy controls. And the changes in B cell subsets might occur at an early stage. Moreover, the frequency of B10 cells decreased after surgery. Positively correlated with B10 cells, the elevated IL-10 level in HCC serum may be a new biomarker in HCC identification. For the first time, our results suggest that altered B cell subsets are associated with the development and prognosis of HCC. Increased B10 cell percentage and IL-10 in HCC patients suggest they might augment the development of liver tumors. Hence, B cell subsets and related cytokines may have predictive value in HCC patients and could be potential targets for immunotherapy in HCC.

CRISPR-free, programmable RNA pseudouridylation to suppress premature termination codons.

Nonsense mutations, accounting for >20% of disease-associated mutations, lead to premature translation termination. Replacing uridine with pseudouridine in stop codons suppresses translation termination, which could be harnessed to mediate readthrough of premature termination codons (PTCs). Here, we present RESTART, a programmable RNA base editor, to revert PTC-induced translation termination in mammalian cells. RESTART utilizes an engineered guide snoRNA (gsnoRNA) and the endogenous H/ACA box snoRNP machinery to achieve precise pseudouridylation. We also identified and optimized gsnoRNA scaffolds to increase the editing efficiency. Unexpectedly, we found that a minor isoform of pseudouridine synthase DKC1, lacking a C-terminal nuclear localization signal, greatly improved the PTC-readthrough efficiency. Although RESTART induced restricted off-target pseudouridylation, they did not change the coding information nor the expression level of off-targets. Finally, RESTART enables robust pseudouridylation in primary cells and achieves functional PTC readthrough in disease-relevant contexts. Collectively, RESTART is a promising RNA-editing tool for research and therapeutics.

WZ35 inhibits gastric cancer cell metastasis by depleting glutathione to promote cellular metabolic remodeling.

This study aimed at elucidating the crosstalk between redox reaction and metabolic remodeling through uncovering the mechanism underlying WZ35-mediated reactive oxygen species (ROS) production and regulation of amino acid metabolism to inhibit gastric cancer (GC) cell metastasis. The activity and biosafety of curcumin analog, WZ35, were verified in vitro and in vivo. The potential molecular mechanism underlying WZ35-mediated enhanced radiotherapeutic sensitivity by reduced Glutathione (GSH) depletion was elucidated by RNA sequencing, single-cell sequencing (scRNA-seq), metabolic mass spectrometry, and other molecular experiments. Compared to curcumin, WZ35 proved more potent anti-proliferative and anti-metastasis properties. Importantly, we demonstrated that WZ35 could consume GSH in multiple ways, including by reduction of raw materials and consumption reserves, inhibition of reformation, and enhanced decomposition. Mechanistically, we identify that WZ35 maintains the GSH depletion phenotype through the ROS-YAP-AXL-ALKBH5-GLS2 loop, further backing the relevance of metabolic remodeling in the tumor microenvironment with tumor metastasis and the role of m6A in tumor metastasis. Collectively, our study identified WZ35 as a novel GSH depletion agent and a previously undiscovered GSH depletion loop mechanism in GC cell metastasis.

Absolute quantification of single-base m6A methylation in the mammalian transcriptome using GLORI.

N6-methyladenosine (m6A) is the most abundant RNA modification in mammalian cells and the best-studied epitranscriptomic mark. Despite the development of various tools to map m6A, a transcriptome-wide method that enables absolute quantification of m6A at single-base resolution is lacking. Here we use glyoxal and nitrite-mediated deamination of unmethylated adenosines (GLORI) to develop an absolute m6A quantification method that is conceptually similar to bisulfite-sequencing-based quantification of DNA 5-methylcytosine. We apply GLORI to quantify the m6A methylomes of mouse and human cells and reveal clustered m6A modifications with differential distribution and stoichiometry. In addition, we characterize m6A dynamics under stress and examine the quantitative landscape of m6A modification in gene expression regulation. GLORI is an unbiased, convenient method for the absolute quantification of the m6A methylome.

CCN1 upregulates IL-36 via AKT/NF-κB and ERK/CEBP ß-mediated signaling pathways in psoriasis-like models.

Psoriasis is a chronic skin disorder characterized by epidermal keratinocyte hyperproliferation and inflammatory infiltration. CCN1 (also termed CYR61 or cysteine-rich angiogenic inducer 61) is an extracellular matrix-associated protein that is involved in multiple physiological functions. In psoriasis, we recently demonstrated that the overexpression of CCN1 promoted keratinocyte proliferation and activation. Furthermore, CCN1 was highly expressed in psoriatic skin lesions from psoriasis vulgaris patients. Here, we dissect the underlying molecular mechanism in imiquimod (IMQ) and interleukin (IL)-23-induced psoriasis-like models. Our results demonstrate that CCN1 can significantly upregulate IL-36 production in the murine skin of IMQ and IL-23-induced psoriasis-like models. Injection of CCN1-neutralizing antibody improved epidermal acanthosis and significantly reduced IL-36 production in vivo. These results suggest that CCN1 can be a critical upstream pro-inflammatory factor in psoriasis. In primary normal human epidermal keratinocytes, we demonstrated that CCN1 can selectively induced the production of IL-36α and IL-36γ through the activation of the protein kinase B (AKT)/nuclear factor kappa light chain enhancer of activated B cells (NF-κB) and extracellular-regulated kinase (ERK)/CCAAT/enhancer binding protein ß (CEBPß) signaling pathways via integrin receptor α6ß1 in vitro. Our results suggest that targeting CCN1 can be a potential therapeutic strategy for psoriasis.

Setd2 supports GATA3+ST2+ thymic-derived Treg cells and suppresses intestinal inflammation.

Treg cells acquire distinct transcriptional properties to suppress specific inflammatory responses. Transcription characteristics of Treg cells are regulated by epigenetic modifications, the mechanism of which remains obscure. Here, we report that Setd2, a histone H3K36 methyltransferase, is important for the survival and suppressive function of Treg cells, especially those from the intestine. Setd2 supports GATA3+ST2+ intestinal thymic-derived Treg (tTreg) cells by facilitating the expression and reciprocal relationship of GATA3 and ST2 in tTreg cells. IL-33 preferentially boosts Th2 cells rather than GATA3+ Treg cells in Foxp3Cre-YFPSetd2 flox/flox mice, corroborating the constraint of Th2 responses by Setd2 expression in Treg cells. SETD2 sustains GATA3 expression in human Treg cells, and SETD2 expression is increased in Treg cells from human colorectal cancer tissues. Epigenetically, Setd2 regulates the transcription of target genes (including Il1rl1) by modulating the activity of promoters and intragenic enhancers where H3K36me3 is typically deposited. Our findings provide mechanistic insights into the regulation of Treg cells and intestinal immunity by Setd2.

Herpes Simplex Type 1 UL43 Multiple Membrane-Spanning Protein Increases Energy Metabolism in Host Cells through Interacting with ARL2.

Non-essential proteins for viral replication affect host cell metabolism, while the function of the UL43 protein of herpes simplex virus 1 (HSV-1) is not clear. Herein, we performed a comprehensive microarray analysis of HUVEC cells infected with HSV-1 and its UL43-deficient mutant and found significant variation in genes associated with cellular energy metabolic pathways. The localization of UL43 protein in host cells and how it affects cellular energy metabolism pathways were further investigated. Internalization analysis showed that the UL43 protein could be endocytosis-mediated by YPLF motif (aa144-147) and localized to mitochondria. At the same time, more ATP was produced by coupling with mitochondrial small G protein ARF-like 2 (ARL2) GTPase, which triggered the phosphorylation of ANT1 (SLC25A4) to affect the opening degree of mitochondrial permeability transition pore (mPTP), and significantly promoted the aerobic oxidation and oxidative phosphorylation of glucose. Our study shows that UL43 mediates the improvement of host cell metabolism after HSV-1 infection. Additionally, UL43 protein could be a valuable ATP-stimulating factor for mammalian cells.

Gallic Acid Ameliorates the Inflammatory State of Periodontal Ligament Stem Cells and Promotes Pro-Osteodifferentiation Capabilities of Inflammatory Stem Cell-Derived Exosomes.

The slow proliferation rate and poor osteodifferentiation ability of inflammatory periodontal membrane stem cells extracted from periodontitis tissues (i-PDLSCs) account for poor efficiency in treating inflammatory bone loss. Exosomes reportedly have inducible and relatively stable components, allowing them to promote inflammatory bone repair, but obtaining i-PDLSCs exosomes with the ability to promote osteodifferentiation is challenging. In the present study, i-PDLSCs were extracted from periodontal membrane tissues of patients with severe periodontitis, and in vitro induction with gallic acid (GA) significantly promoted the proliferative activity of i-PDLSCs at a concentration of 10 mM, with TC0 of 11.057 mM and TC50 of 67.56 mM for i-PDLSCs. After mRNA sequencing, we found that GA could alleviate oxidative stress in i-PDLSCs and increase its mitochondrial membrane potential and glucose aerobic metabolism level, thus promoting the osteodifferentiation of i-PDLSCs. After exosomes of i-PDLSCs after GA induction (i-EXO-GA) were isolated by differential centrifugation, we found that 200 ug/mL of i-EXO-GA could remarkably promote the osteodifferentiation of i-PDLSCs. Overall, our results suggest that GA induction can enhance the proliferation and osteodifferentiation in primary cultures of i-PDLSCs in vitro, mediated by alleviating oxidative stress and glycometabolism levels in cells, which further influences the osteodifferentiation-promoting ability of i-EXO-GA. Overall, we provide a viable cell and exosome induction culture method for treating inflammatory alveolar defects associated with periodontitis.

Clinical and molecular profiling of AML patients with chromosome 7 or 7q deletions in the context of TP53 alterations and venetoclax treatment.

Deletions in chromosome 7 (del(7)) or its long arm (del(7q)) constitute the most common adverse cytogenetic events in acute myeloid leukemia (AML). We retrospectively analyzed 243 treatment-naive patients with AML and del(7) (168/243; 69%) or del(7q) (75/243; 31%) who did not receive any myeloid-directed therapy prior to AML diagnosis. This is the largest comprehensive clinical and molecular analysis of AML patients with del(7) and del(7q). Our results show that relapse-free survival was significantly longer for AML patients with del(7q) compared to del(7), but the overall survival and remission duration were similar. TP53 mutations and del5/5q were the most frequent co-occurring mutations and cytogenetic abnormalities, and conferred worse outcomes in del(7) and del(7q) patients. Venetoclax-based treatments were associated with worse outcomes in TP53 mutated AML patients with del(7) or del(7q), as well as del(7) with TP53 wildtype status, requiring further investigation.