Cellular Composition and 5hmC Signature Predict the Treatment Response of AML Patients to Azacitidine Combined with Chemotherapy.

Azacitidine (AZA) is a DNA methyltransferase inhibitor and epigenetic modulator that can be an effective agent in combination with chemotherapy for patients with high-risk acute myeloid leukemia (AML). However, biological factors driving the therapeutic response of such hypomethylating agent (HMA)-based therapies remain unknown. Herein, the transcriptome and/or genome-wide 5-hydroxymethylcytosine (5hmC) is characterized for 41 patients with high-risk AML from a phase 1 clinical trial treated with AZA epigenetic priming followed by high-dose cytarabine and mitoxantrone (AZA-HiDAC-Mito). Digital cytometry reveals that responders have elevated Granulocyte-macrophage-progenitor-like (GMP-like) malignant cells displaying an active cell cycle program. Moreover, the enrichment of natural killer (NK) cells predicts a favorable outcome in patients receiving AZA-HiDAC-Mito therapy or other AZA-based therapies. Comparing 5hmC profiles before and after five-day treatment of AZA shows that AZA exposure induces dose-dependent 5hmC changes, in which the magnitude correlates with overall survival (p = 0.015). An extreme gradient boosting (XGBoost) machine learning model is developed to predict the treatment response based on 5hmC levels of 11 genes, achieving an area under the curve (AUC) of 0.860. These results suggest that cellular composition markedly impacts the treatment response, and showcase the prospect of 5hmC signatures in predicting the outcomes of HMA-based therapies in AML.

Beclin1- and Atg13-dependent autophagy activation and morroniside have synergistic effect on osteoblastogenesis.

Morroniside is known to improve osteoporosis by promoting osteoblastogenesis. The activation of PI3K/Akt/mTOR signaling is a significant mechanism in morroniside-promoted osteoblastogenesis. It is well known that protective autophagy is an important factor in osteoblastogenesis. However, the activation of mTOR signaling can inhibit autophagy. This study aimed to investigate the relationship between mTOR signaling and autophagy in morroniside-regulated osteoblastogenesis. In this study, we investigated the effect of morroniside on the autophagic activity (LC3 conversion rate, LC3-puncta formation, and autophagosome number) of differentiated osteoblast precursors (MC3T3-E1 cells). Then, we identified the roles of mTOR knockdown in morroniside-regulated alterations of autophagy and osteogenic parameters in MC3T3-E1 cells. Next, mTOR knockdown and overexpression were used to observe the roles of mTOR in morroniside-regulated alterations of autophagic molecules (Atg7, Atg13, and Beclin1). Subsequently, the additional value of the above autophagic molecules on morroniside-regulated osteogenic parameters in MC3T3-E1 cells was analyzed based on lentiviral transduction. Finally, combined with morroniside and TAT-Beclin1, the roles of Beclin1 upregulation in the in vivo effects of morroniside was investigated. Our experimental data showed that morroniside promoted both the mTOR activity and autophagy in MC3T3-E1 cells. Morroniside-upregulated autophagic activity and Atg13 or Beclin1 protein level in MC3T3-E1 cells were enhanced by mTOR knockdown. Furthermore, Morroniside-upregulated Atg13 and Beclin1 expression was reversed by mTOR overexpression. Importantly, autophagy upregulation with overexpression of the autophagic gene, Atg13 or BECN1 (gene form of Beclin1), significantly promoted osteoblastogenesis regulated by morroniside. The promotional effect of morroniside on bone microarchitecture, bone mass, and bone parameters (including trabecular bone area and OCN expression in trabecular bone) in ovariectomized (OVX) mice was enhanced by TAT-Beclin1 administration. In conclusion, the autophagy-enhancing drugs related to Beclin1 or Atg13 may be an effective adjuvant therapy in the treatment of osteoporosis with morroniside.

Acupuncture around the greater tuberosity of the femur combined with acupuncture at Xuehai acupoint alleviates the postoperative pain of elderly patients with intertrochanteric fracture.

OBJECTIVE: To determine the efficacy of acupuncture around the greater tuberosity of the femur (AGTF) combined with acupuncture at Xuehai acupoint for postoperative pain in elderly patients with intertrochanteric fracture. METHODS: A total of 97 elderly patients with intertrochanteric fracture treated by proximal femoral nail antirotation (PFNA) were enrolled and randomly assigned into an observation group (Obs group, n=48) and a control group (Con group, n=49). The Obs group was treated by aspirin and AGTF combined with acupuncture at Xuehai acupoint for analgesia, while the Con group was treated by aspirin alone for analgesia. Both groups were treated for 7 consecutive days. The two groups were compared in pain degree (visual analog scale (VAS) score) after operation and hip joint function (Harris score), daily living ability (modified Barthel index (MBI) score), bone metabolism-related indexes, and inflammatory factors before and after treatment. RESULTS: At 1-7 d after operation, both groups had gradually lower VAS scores, and at 5 and 7 d after operation, the Obs group had a lower VAS score than the Con group (both P<0.05). Additionally, at 2 months after operation, both groups had higher Harris scores and MBI scores, and the scores of the Obs group were both higher than those of the Con group (both P<0.05). At 7 d after operation, both groups showed a decrease in serum beta collagen degradation products (ß-CTx) and an increase in procollagen type I amino-terminal propeptide (PINP) (both P<0.05), but the differences between the two groups in ß-CTx and PINP were insignificant (P>0.05). Moreover, at 7 d after operation, both groups showed a decrease in C-reactive protein (CRP) and tumor necrosis factor-α (TNF-α), and the two levels in the Obs group were lower than those in the Con group (both P<0.05). CONCLUSION: For elderly patients with intertrochanteric fracture, AGTF combined with acupuncture at the Xuehai acupoint can more effectively relieve their postoperative pain and postoperative inflammation and more strongly promote their postoperative recovery of hip joint function.

The loss of RNA N6-adenosine methyltransferase Mettl14 in tumor-associated macrophages promotes CD8+ T cell dysfunction and tumor growth.

Tumor-associated macrophages (TAMs) can dampen the antitumor activity of T cells, yet the underlying mechanism remains incompletely understood. Here, we show that C1q+ TAMs are regulated by an RNA N6-methyladenosine (m6A) program and modulate tumor-infiltrating CD8+ T cells by expressing multiple immunomodulatory ligands. Macrophage-specific knockout of an m6A methyltransferase Mettl14 drives CD8+ T cell differentiation along a dysfunctional trajectory, impairing CD8+ T cells to eliminate tumors. Mettl14-deficient C1q+ TAMs show a decreased m6A abundance on and a higher level of transcripts of Ebi3, a cytokine subunit. In addition, neutralization of EBI3 leads to reinvigoration of dysfunctional CD8+ T cells and overcomes immunosuppressive impact in mice. We show that the METTL14-m6A levels are negatively correlated with dysfunctional T cell levels in patients with colorectal cancer, supporting the clinical relevance of this regulatory pathway. Thus, our study demonstrates how an m6A methyltransferase in TAMs promotes CD8+ T cell dysfunction and tumor progression.

N 6-methyladenosine of chromosome-associated regulatory RNA regulates chromatin state and transcription.

N 6-methyladenosine (m6A) regulates stability and translation of messenger RNA (mRNA) in various biological processes. In this work, we show that knockout of the m6A writer Mettl3 or the nuclear reader Ythdc1 in mouse embryonic stem cells increases chromatin accessibility and activates transcription in an m6A-dependent manner. We found that METTL3 deposits m6A modifications on chromosome-associated regulatory RNAs (carRNAs), including promoter-associated RNAs, enhancer RNAs, and repeat RNAs. YTHDC1 facilitates the decay of a subset of these m6A-modified RNAs, especially elements of the long interspersed element-1 family, through the nuclear exosome targeting-mediated nuclear degradation. Reducing m6A methylation by METTL3 depletion or site-specific m6A demethylation of selected carRNAs elevates the levels of carRNAs and promotes open chromatin state and downstream transcription. Collectively, our results reveal that m6A on carRNAs can globally tune chromatin state and transcription.

Author Correction: Single-cell RNA-seq highlights intra-tumoral heterogeneity and malignant progression in pancreatic ductal adenocarcinoma.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Single-cell RNA-seq highlights intra-tumoral heterogeneity and malignant progression in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer featured with high intra-tumoral heterogeneity and poor prognosis. To comprehensively delineate the PDAC intra-tumoral heterogeneity and the underlying mechanism for PDAC progression, we employed single-cell RNA-seq (scRNA-seq) to acquire the transcriptomic atlas of 57,530 individual pancreatic cells from primary PDAC tumors and control pancreases, and identified diverse malignant and stromal cell types, including two ductal subtypes with abnormal and malignant gene expression profiles respectively, in PDAC. We found that the heterogenous malignant subtype was composed of several subpopulations with differential proliferative and migratory potentials. Cell trajectory analysis revealed that components of multiple tumor-related pathways and transcription factors (TFs) were differentially expressed along PDAC progression. Furthermore, we found a subset of ductal cells with unique proliferative features were associated with an inactivation state in tumor-infiltrating T cells, providing novel markers for the prediction of antitumor immune response. Together, our findings provide a valuable resource for deciphering the intra-tumoral heterogeneity in PDAC and uncover a connection between tumor intrinsic transcriptional state and T cell activation, suggesting potential biomarkers for anticancer treatment such as targeted therapy and immunotherapy.

Author Correction: Anti-tumour immunity controlled through mRNA m6A methylation and YTHDF1 in dendritic cells.

In this Letter, a citation to 'Fig. 1e' has been corrected to 'Fig. 1d' in the sentence starting "By contrast, the anti-tumour response…". This has been corrected online.

Anti-tumour immunity controlled through mRNA m6A methylation and YTHDF1 in dendritic cells.

There is growing evidence that tumour neoantigens have important roles in generating spontaneous antitumour immune responses and predicting clinical responses to immunotherapies1,2. Despite the presence of numerous neoantigens in patients, complete tumour elimination is rare, owing to failures in mounting a sufficient and lasting antitumour immune response3,4. Here we show that durable neoantigen-specific immunity is regulated by mRNA N6-methyadenosine (m6A) methylation through the m6A-binding protein YTHDF15. In contrast to wild-type mice, Ythdf1-deficient mice show an elevated antigen-specific CD8+ T cell antitumour response. Loss of YTHDF1 in classical dendritic cells enhanced the cross-presentation of tumour antigens and the cross-priming of CD8+ T cells in vivo. Mechanistically, transcripts encoding lysosomal proteases are marked by m6A and recognized by YTHDF1. Binding of YTHDF1 to these transcripts increases the translation of lysosomal cathepsins in dendritic cells, and inhibition of cathepsins markedly enhances cross-presentation of wild-type dendritic cells. Furthermore, the therapeutic efficacy of PD-L1 checkpoint blockade is enhanced in Ythdf1-/- mice, implicating YTHDF1 as a potential therapeutic target in anticancer immunotherapy.

Enhanced mammalian genome editing by new Cas12a orthologs with optimized crRNA scaffolds.

CRISPR-Cas12a/Cpf1, a single RNA-guided endonuclease system, provides a promising tool for genome engineering. However, only three Cas12a orthologs have been employed for mammalian genome editing, and the editing efficiency as well as targeting coverage still requires improvements. Here, we harness six novel Cas12a orthologs for genome editing in human and mouse cells, some of which utilize simple protospacer adjacent motifs (PAMs) that remarkably increase the targeting range in the genomes. Moreover, we identify optimized CRISPR RNA (crRNA) scaffolds that can increase the genome editing efficiency of Cas12a.

Highly efficient base editing in Staphylococcus aureus using an engineered CRISPR RNA-guided cytidine deaminase.

Novel therapeutic means against Staphylococcus aureus infections are urgently needed due to the emergence of drug-resistant S. aureus. We report the development of a CRISPR RNA-guided cytidine deaminase (pnCasSA-BEC), enabling highly efficient gene inactivation and point mutations in S. aureus. We engineered a fusion of a Cas9 nickase (Cas9D10A) and a cytidine deaminase (APOBEC1) that can be guided to a target genomic locus for gene inactivation via generating a premature stop codon. The pnCasSA-BEC system nicks the non-edited strand of the genomic DNA, directly catalyzes the conversion of cytidine (C) to uridine (U), and relies on DNA replication to achieve C → T (G → A) conversion without using donor repair templates. The development of the base-editing system will dramatically accelerate drug-target exploration in S. aureus and provides critical insights into the development of base-editing tools in other microbes.

A novel single-stranded RNA virus isolated from the rice-pathogenic fungus Magnaporthe oryzae with similarity to members of the family Tombusviridae.

Here, we report a novel virus isolated from rice blast fungus, Magnaporthe oryzae, an important plant pathogen. This virus has an RNA genome of 3246 nucleotides. Its genome possesses two in-frame open reading frames (ORFs). The smaller ORF1 encodes a protein with significant similarity to a protein encoded by the ssRNA mycovirus Diaporthe ambigua RNA virus 1 (DaRV1). The larger ORF2 encodes a protein with similarity to RNA-dependent RNA polymerases (RdRp) of DaRV1 and other plant viruses of the family Tombusviridae. In silico analysis and comparisons with DaRV1 genome expression suggest that ORF2 is translated via a readthrough mechanism together with ORF1. Based upon results of this study, this virus, for which the provisional name Magnaporthe oryzae virus A (MoVA) is proposed, belongs to a new virus species. Furthermore, MoVA along with DaRV1 belong to a new taxon of mycoviruses that are evolutionarily related to plant viruses belonging to the family Tombusviridae.

Genome sequence of a novel mycovirus of Rhizoctonia solani, a plant pathogenic fungus.

Here we present the genome sequence of a novel dsRNA virus we designed as Rhizoctonia solani RNA virus HN008 (RsRV-HN008) from a filamentous fungus R. solani. Its genome (7596 nucleotides) contains two non-overlapping open reading frames (ORF1 and ORF2). ORF1 encoded a 128 kDa protein that showed no significant identity to any other virus sequence in the NCBI database. ORF2 encoded a protein with a molecular weight of 140 kDa and shared a low percentage of sequence identity to the RdRps of unclassified dsRNA viruses. Sequence analysis revealed that RsRV-HN008 may be a member of a novel unclassified family of mycoviruses.

Genome sequence of a novel endornavirus from the phytopathogenic fungus Alternaria brassicicola.

In an effort to discover new mycoviruses from phytopathogenic fungi, a dsRNA molecule of 10,290 nt, resembling those associated with the viruses belonging to the family Endornaviridae, was isolated from Alternaria brassicicola, one of the causal agents of rapeseed black spot disease. Genome analysis revealed the presence of a single open reading frame coding for a polyprotein of 3400 aa containing conserved viral methyltransferase (MTR), viral RNA helicase 1 (Hel-1), and RNA-dependent RNA polymerase (RdRp) domains. In addition, a cysteine-rich region (CRR) with conserved CXCC motifs, shared among several endornaviruses, was also identified between the MTR and Hel-1 domains. Phylogenetic analysis based on the RdRp sequence strongly suggested that the virus infecting A. brassicicola should be considered a representative of a novel endornavirus species, and this virus was designated as Alternaria brassicicola endornavirus 1 (AbEV1).