Background Pecan ( Carya illinoinensis ) and Chinese hickory ( C. cathayensis ) are important commercially cultivated nut trees in the genus Carya (Juglandaceae), with high nutritional value and substantial health benefits. Results We obtained >187.22 and 178.87 gigabases of sequence, and ∼288× and 248× genome coverage, to a pecan cultivar (“Pawnee”) and a domesticated Chinese hickory landrace (ZAFU-1), respectively. The total assembly size is 651.31 megabases (Mb) for pecan and 706.43 Mb for Chinese hickory. Two genome duplication events before the divergence from walnut were found in these species. Gene family analysis highlighted key genes in biotic and abiotic tolerance, oil, polyphenols, essential amino acids, and B vitamins. Further analyses of reduced-coverage genome sequences of 16 Carya and 2 Juglans species provide additional phylogenetic perspective on crop wild relatives. Conclusions Cooperative characterization of these valuable resources provides a window to their evolutionary development and a valuable foundation for future crop improvement.

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Hickory (Carya cathayensis) kernel is rich in powerful bioactive flavonoids, which can remove excess free radicals in the human body and play an important role in regulating the physiological metabolism of the plant. This study investigated the changes of flavonoids in hickory exocarp and embryo during development. In this study, 72 DEGs involved in the regulation of flavonoid biosynthesis in fruits were identified, and TT4, CCoAOMT1, UGT71D1, C4H, F3H, TT8, FLS1, and LDOX were identified as the core genes of flavonoid biosynthesis. A total of 144 flavonoid-related metabolites were detected by metabolite analysis. Transcriptome and metabolome analysis combined to construct the flavonoid biosynthesis regulatory pathway in the development stage of hickory fruit. Our results provide a theoretical basis for the exploration and regulation of functional genes related to flavonoid biosynthesis and metabolism in hickory and other plants and the breeding of new walnut varieties.

This study was conducted to investigate epigenetic landscape across multiple species and identify transcription factors (TFs) and their roles in controlling cell fate decision events during early embryogenesis. We made a comprehensively joint-research of chromatin accessibility of five species during embryogenesis by integration of ATAC-seq and RNA-seq datasets. Regulatory roles of candidate early embryonic TFs were investigated. Widespread accessible chromatin in early embryos overlapped with putative cis-regulatory sequences. Sets of cell-fate-determining TFs were identified. YOX1, a key cell cycle regulator, were found to homologous to clusters of TFs that are involved in neuron and epidermal cell-fate determination. Our research provides an intriguing insight into evolution of cell-fate decision during early embryogenesis among organisms.

e16261 Background: Tumor-infiltrating myeloid cells (TIMs) are abundant in tumor stroma and emerging evidence indicate that the presence of these cells influences clinical outcomes in many cancer types. TIMs play important roles in tumor progression and specific subsets may exhibit divergent anti-tumor or tumor-promoting functions. Currently, the molecular characteristics of TIMs in pancreatic ductal adenocarcinoma (PDAC) largely remain unknown. Like other programmed cell death procedures such as ferroptosis and pyroptosis, necroptosis has recently emerged as an important cellular event that modulates tumorigenesis and tumor progression. Combination of necroptosis and TIMs is of great interest to study the molecular mechanism of clinical outcomes for PDAC patients. Methods: This study enrolled 16 PDAC patients. The scRNA-seq data were obtained from a published dataset and each patient included primary tumor, metastatic tumor, primary tumor-adjacent and PBMC samples. All myeloid cells comprising granulocytes, monocytes, macrophages, and dendritic cells from enrolled patients were extracted for further analysis. We performed comprehensive analysis about the characteristics of myeloid lineages, differentially expressed genes, pathway enrichment analysis and novel cell subtype identification. The necroptosis-associated novel TIMs cell sub-clusters were elucidated in detail. Results: To comprehensively analyze the heterogeneous TIMs microenvironment in PDAC patients using scRNA-seq dataset, we firstly obtained consistently upregulated genes among granulocytes, monocytes, macrophages, and dendritic cell types between tumor samples and non-tumor samples. Those uniquely expressed genes represent different functions in necroptosis-associated TIMs cells. In total, we identified 10 necroptosis-associated upregulated genes in PDAC tumors and 5 upregulated genes in tumor-adjacent and peripheral blood samples, respectively. Based on the cells expressed those necroptosis-related genes, we identified three novel cell populations, including GLUL-SQSTM1- RTM, HSP90AA1+HSP90AB1+ mast cells, and JAK3+TLR4+ CD16 monocytes, which were regulators of immunity either tumor-promoters or tumor-suppressors. Clinical outcomes further supported their function roles in the PDAC tumor microenvironment. Conclusions: This study comprehensively studied the heterogeneity of myeloid microenvironment in PDAC by scRNA-Seq. Several necroptosis-associated genes and myeloid cell sub-clusters were identified and they have prognostic clinical values in PDAC. These preliminary results were helpful for further understanding the molecular mechanism of PDAC treatment strategies.

e18042 Background: Cytokine-induced memory-like (CIML) NK cells are attractive for adoptive cell therapeutic approaches due to their key characteristics which include anti-tumor responses, prolonged proliferation and persistence in vivo. Several NK cell subsets have been reported in human and mice, but their heterogeneity and trajectories in human peripheral blood remains poorly characterized. We aimed to evaluate the developmental trajectories and prognosis outcomes of several CIML NK subsets between HPV+ and HPV- HNSC patients. Methods: A publicly available scRNA-seq dataset (only CD45+PBMC data sets were used) (Kürten et al., 2021) was obtained and stratified by two definitions of CIML NKs: (1) increased granzyme B mRNA expression and (2) preactivated with iterleukin-12 (IL-12), IL-15 and IL-18. Single-cell analysis was performed, followed by sub-clustering of NK populations. Results: 4,794 NK cells from 6 HPV+ and 11 HPV- HNSC patients were analyzed in our study. Expression profiles showed that these NK cells were CD56dimCD16+CD57-NKs. Further investigation showed that these cell populations exhibit a cytokine-induced memory-like phenotype, including increased granzyme B mRNA and preactivated with IL-12, IL-15 and IL-18. Transcriptomic profiles demonstrated that CIML NK cells can be classified into three subsets, including Bl1, Bl2, and Bl3, where Bl1 and Bl3 were found to be consistent with the previous studies, Bl2 was a newly identified NK cluster in this study. Pseudotime analyses suggested that CIML NK cells transitioned from Bl3 (is in cytolytic state) to Bl2, and then (maintains intermediate cell states) to Bl1 (supports cytotoxic activity), regardless of HPV status. There were 20, 60, and 26 Up-regulated genes in Bl, Bl2, and Bl3 in HPV+ patient compared to HPV- patients, respectively. Biocarta pathway analyses showed that NK cell pathway was highly enriched in all NK subsets from HPV+ patients, instead of HPV- patients, indicating a developmental independent characteristic. Survival analyses showed poor prognosis outcomes for high expressed CIML NK signatures in no matter HPV+ or HPV- patients. Conclusions: We investigated CIML NK cells in human peripheral blood and found that Bl1, Bl2, and Bl3 NK clusters showing the developmental patterns of increased cytotoxicity independent of HPV status. The Bl2 is a novel NK cell cluster which presents intermediate cell states. The cluster signatures were correlated with poor clinical outcomes, highlighting its’ potential clinical values for CIML NK-targeted immunotherapies.

e15548 Background: Cancer-associated fibroblasts (CAFs) play prominent and diverse tumor-promoting function roles in tumor microenvironment (TME) and influence cancer hallmarks, but without systematic investigation between left-sided and right-sided CRC (colorectal cancer). Methods: We collected a public scRNA-seq dataset (Pelka et al., 2021) with 21 samples from left-sided CRC and 79 from right-sided CRC. Only CAFs were extracted for downstream analysis. Seurat (Stuart et al., 2019), Monocle2 (den Berge et al., 2020) and pySCENIC (Aibar et al., 2017) were used to perform dimensionality reductions, differential analysis, pseudo-time trajectory analysis, and the transcriptional regulation analysis, respectively. Results: Firstly, we performed differential analysis on 2,049 cells from 62 of 100 samples that contained CAFs. The results showed that highly expressed genes including IGFBP1, IGFBP2, LCN2, ALDH1A1 and CASP1 in left-sided CRC were mainly enriched in immune response pathway, while up-regulated genes in right-sided CRC were neutrophil-migration-chemotaxis-related such as PPBP1, PF4V1, CCL7, CXCL3 and CXCL8. Then, we classified 2,049 CAFs into four subclusters based on the markers of CCL8+ CAF, CXCL14+ CAF, GREM1+ CAF and MMP3+ CAF. The proportion of CXCL14+ CAF was higher in left-sided CRC while MMP3+ CAF was preferentially enriched in right-sided CRC, which was also independently validated by 33 TCGA cohorts. Further investigation on those subclusters showed that CXCL14+ CAF was myCAF, while the rest (CCL8+ CAF, MMP3+ CAF and GREM1+ CAF) were iCAF. After investigating the heterogeneity of CAFs, we focused on the transcriptional regulatory networks (TRNs). The results showed that the dominant regulons in left-sided CRC ( HOXD9, HSF2, MYBL1, HES6, XRCC4, and ZNF585B) and right-sided CRC ( CREB5, ZNF274, IRF7, TFEC, NR2F1, and PITX3) were distinct, which highlighted the heterogeneity and plasticity of CAF-based TRNs between left-sided and right-sided CRC. Conclusions: Collectively, although further experimental verification is required to establish the functional roles of each CAF cluster and the diverse origins of CAFs, this study may facilitate better understanding molecular mechanism of CAF-targeted therapy strategies in CRC.

e15550 Background: Myeloid cells play critical roles in the growth and metastasis of malignant tumors with both protumor and antitumor capabilities. Plenty of studies have reported they participated in modulating tumor inflammation and angiogenesis, and therapeutic strategies targeting myeloid cells are ongoing in pre-clinical and clinical trials. The functions of myeloid cells and their subtypes in left-sided and right-side CRC remain unclear. Methods: A published scRNA-seq dataset (Pelka et al., 2021) with 21 samples from left-sided CRC and 79 from right-sided CRC (colorectal cancer) was obtained. Seurat (Stuart et al., 2019) was used to perform dimensionality reduction and differential expression analysis, and the cell subtypes in the myeloid cells were identified to study the associations between clinical outcomes and different tumor tissue sites of CRC. We also obtained an immunotherapy dataset from TISMO to identify associations between cell subtypes and immunotherapy outcomes. Results: Systematic comparisons of myeloid cells (n = 39,167) from left-sided and right-sided CRC revealed that highly expressed genes from right-sided CRC showed protumor capability and those from left-sided CRC showed antitumor activity. Specifically, left-sided CRC harbored a significant (p = 0.018) higher proportion of ACKR1+ DCs compared to right-sided CRC. ACKR1+ DCs might be immunological as its marker genes were significantly positively correlated with CD8+T cells and highly enriched in APC(antigen processing presentation)-related pathways. TCGA survival analysis revealed that the higher expression of the marker genes in ACKR1+ DC was associated with better overall survival (OS, p = 0.0088). Furthermore, immunotherapy biopsy showed ACKR1+ DCs subtype significantly enriched in immunotherapy responsive group compared to non-responsive group in colon cancer (p = 0.019), melanoma (p = 0.0058) and renal adenocarcinoma (p = 3.3e-06) among the obtained TISMO dataset. Conclusions: We identified an immunological ACKR1+ DCs subtype, which was highly enriched in left-sided CRC and was correlated with better clinical outcomes and immunotherapy responses. Those findings might provide potential therapeutic target and help better understand the mechanism of treatment strategies in CRC.