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Scutellaria Linn., which belongs to the family Lamiaceae, is a commonly used medicinal plant for heat clearing and detoxification. In particular, the roots of S. baicalensis and the entire herb of S. barbata have been widely used in traditional medicine for thousands of years. The main active components of Scutellaria, including: baicalein, wogonin, norwogonin, scutellarein, and their glycosides have potential or existing drug usage. However, the wild resources of Scutellaria plants have been overexploited, and degenerated germplasm resources cannot fulfill the requirements of chemical extraction and clinical usage. Metabolic engineering and green production via microorganisms provide alternative strategies for greater efficiency in the production of natural products. Here, we review the progress of: pharmacological investigations, multi-omics, biosynthetic pathways, and metabolic engineering of various Scutellaria species and their active compounds. In addition, based on multi-omics data, we systematically analyze the phylogenetic relationships of Scutellaria and predict candidate transcription factors related to the regulation of active flavonoids. Finally, we propose the prospects of directed evolution of core enzymes and genome-assisted breeding to alleviate the shortage of plant resources of Scutellaria. This review provides important insights into the sustainable utilization and development of Scutellaria resources.

Septic cardiomyopathy is a life-threatening heart dysfunction caused by severe infection. Considering the complexity of the pathogenesis and high mortality, it was necessary to identify efficient biomarkers to guide the clinical practice. Based on the muti-microarray analysis, this study aimed to explore the pathogenesis of septic cardiomyopathy and the related immune landscape. The results showed that septic cardiomyopathy was organ dysfunction after extreme pro- and anti-inflammation. In this process, KLRG1, PRF1, BCL6, GAB2, MMP9, IL1R1, JAK3, IL6ST, and SERPINE1 were identified as the hub genes regulated the immune landscape of septic cardiomyopathy. Nine transcription factors regulated their expression: SRF, STAT1, SP1, RELA, PPARG, NFKB1, PPARA, SMAD3, and STAT3. Hub genes activated the Th17 cell differentiation pathway, JAK-STAT signaling pathway, and Cytokine-cytokine receptor interaction pathway. These were mainly involved in regulating inflammatory response, adaptive immune response, leukocyte-mediated immunity, cytokine-mediated immunity, immune effector process, myeloid cell differentiation, and T-helper cell differentiation. These nine hub genes can be seen as biomarkers for the early prediction of septic cardiomyopathy.

Anti-PD-1 treatment has shown unprecedented clinical success in the treatment of non-small-cell lung cancer (NSCLC), but the underlying mechanisms remain incompletely understood. Here, we performed temporal single-cell RNA and paired T-cell receptor sequencing on 47 tumor biopsies from 36 patients with NSCLC following PD-1-based therapies. We observed increased levels of precursor exhausted T (Texp) cells in responsive tumors after treatment, characterized by low expression of coinhibitory molecules and high expression of GZMK. By contrast, nonresponsive tumors failed to accumulate Texp cells. Our data suggested that Texp cells were unlikely to be derived from the reinvigoration of terminally exhausted cells; instead, they were accumulated by (1) local expansion and (2) replenishment by peripheral T cells with both new and pre-existing clonotypes, a phenomenon we named clonal revival. Our study provides insights into mechanisms underlying PD-1-based therapies, implicating clonal revival and expansion of Texp cells as steps to improve NSCLC treatment.

Medicinal trees provide a main resource for diverse medicinal compounds. However, the biosynthesis of tree metabolites and their pathway evolution has gained limited understanding. Horse chestnut (Aesculus chinensis) is an important medicinal tree and its seeds are rich in aescins, barrigenol-type triterpenoid saponins (BAT), and aesculin, a coumarin glycoside, which are effective in the therapy of chronic venous insufficiency and asthenopia (eye strain). To understand the biosynthesis of these compounds, herein, we assembled a 470.04-Mb high-quality horse chestnut genome and characterized an Aesculus-specific whole-genome duplication (WGD) event. Spatial metabolome imaging, co-expression, and biosynthetic gene cluster analyses indicated that the Aesculus-specific WGD event led to the formation of two gene clusters (BGCs) including oxidosqualene cyclase, cytochrome P450 monooxygenase, cellulose synthase-derived glycotransferases, and BADH acyltransferases. Further biochemical investigation revealed the roles of AcOCS6, AcCYP716A278, AcCYP716A275, AcCSL1, and AcBAHD3 genes distributed between these two BGCs in catalyzing the formation of aescins. To understand the evolution of BAT pathways, the collinearity analysis showed the collinear BGC segments could be traced back to early-diverging angiosperms, then the essential gene-encoding enzymes necessary for the BAT biosynthesis were recruited before the split of Aesculus, Acer, and Xanthoceras. Meanwhile, we identified three UDP-glucosyltransferases and demonstrated their involvement in the biosynthesis of aesculin via a de novo synthesis. Taken together, these findings provide important information in understanding the evolution of gene clusters associated with medicinal tree metabolites.

Laportea bulbifera, a Miao medicine grown in karst areas, has exerts a unique curative effect on skin itching in the elderly, with an annual sales of > 100 million Yuan. Owing to the shortage of resources and large morphological variations in L. bulbifera, it is difficult to identify the species correctly using only traditional methods, which seriously affects the safety of drug usage for patients. This study obtained the complete high-quality L. bulbifera chloroplast (cp) genome, using second- and third-generation high-throughput sequencing. The cp genome was 149,911 bp in length, with a typical quadripartite structure. A total of 127 genes were annotated, including 83 protein-coding genes, 36 tRNA genes, and 8 rRNA genes. There was an inverted small single copy (SSC) structure in the L. bulbifera cp genome, one large-scale rearrangement of ~ 39 kb excised in the SSC and IR regions. The complete cp genome sequence used as a super-barcode and the highly variable regions (ycf1, matK, and ndhD), especially matK, can be used as specific barcodes to accurately distinguish L. bulbifera from counterfeits and closely related species. This study is important for the identification of L. bulbifera and lays a theoretical foundation for elucidating the phylogenetic relationship of the species.

The increasingly wide application of chloroplast (cp) genome super‐barcode in taxonomy and the recent breakthrough in cp genetic engineering make the development of new cp gene resources urgent and significant. Corydalis is recognized as the most genotypes complicated and taxonomically challenging plant taxa in Papaveraceae. However, there currently are few reports about cp genomes of the genus Corydalis. In this study, we sequenced four complete cp genomes of two endangered lithophytes Corydalis saxicola and Corydalis tomentella in Corydalis, conducted a comparison of these cp genomes among each other as well as with others of Papaveraceae. The cp genomes have a large genome size of 189,029–190,247 bp, possessing a quadripartite structure and with two highly expanded inverted repeat (IR) regions (length: 41,955–42,350 bp). Comparison between the cp genomes of C. tomentella, C. saxicola, and Papaveraceae species, five NADH dehydrogenase‐like genes (ndhF, ndhD, ndhL, ndhG, and ndhE) with psaC, rpl32, ccsA, and trnL‐UAG normally located in the SSC region have migrated to IRs, resulting in IR expansion and gene duplication. An up to 9 kb inversion involving five genes (rpl23, ycf2, ycf15, trnI‐CAU, and trnL‐CAA) was found within IR regions. The accD gene was found to be absent and the ycf1 gene has shifted from the IR/SSC border to the SSC region as a single copy. Phylogenetic analysis based on the sequences of common CDS showed that the genus Corydalis is quite distantly related to the other genera of Papaveraceae, it provided a new clue for recent advocacy to establish a separate Fumariaceae family. Our results revealed one special cp genome structure in Papaveraceae, provided a useful resources for classification of the genus Corydalis, and will be valuable for understanding Papaveraceae evolutionary relationships., Our results revealed one special cp genome structure in Papaveraceae, provided a useful resources for classification of the genus Corydalis, and will be valuable for understanding Papaveraceae evolutionary relationships.

Species belonging to the order Ranunculales have attracted much attention because of their phylogenetic position as a sister group to all other eudicot lineages and their ability to produce unique yet diverse benzylisoquinoline alkaloids (BIAs). The Papaveraceae family in Ranunculales is often used as a model system for studying BIA biosynthesis. Here, we report the chromosome-level genome assembly of Corydalis tomentella, a species of Fumarioideae, one of the two subfamilies of Papaveraceae. Based on comparisons of sequenced Ranunculalean species, we present clear evidence of a shared whole-genome duplication (WGD) event that has occurred before the divergence of Ranunculales but after its divergence from other eudicot lineages. The C. tomentella genome enabled us to integrate isotopic labeling and comparative genomics to reconstruct the BIA biosynthetic pathway for both sanguinarine biosynthesis shared by papaveraceous species and the cavidine biosynthesis that is specific to Corydalis. Also, our comparative analysis revealed that gene duplications, especially tandem gene duplications, underlie the diversification of BIA biosynthetic pathways in Ranunculales. In particular, tandemly duplicated berberine bridge enzyme-like genes appear to be involved in cavidine biosynthesis. In conclusion, our study of the C. tomentella genome provides important insights into the occurrence of WGDs during the early evolution of eudicots, as well as into the evolution of BIA biosynthesis in Ranunculales.

Scutellaria baicalensis Georgi is a common kind of traditional Chinese medicine; it promotes heat-clearing and detoxification and has been used to treat coronavirus disease 2019. Scutellaria baicalensis contains a unique group of flavonoids that are largely distributed within the Scutellaria species. As such, there is significant interest in research focused on the metabolic pathways that promote endogenous flavonoid production and methods that might be used for their in vitro biosynthesis. However, as is the case with various Chinese medicinal herbs, there are still many challenges to overcome including shortage of wild resources, degradation associated with the cultivated germplasm, and instability leading to uneven product quality. The use of a multiomics approach will provide genetic information as well as insights into the endogenous mechanisms of flavonoid biosynthesis and their regulation, thus promoting frontier research and practical application of traditional Chinese medicine. In this study, we presented a systematic review of genomics, transcriptomics, proteomics, and metabolomics associated with S. baicalensis ; our current understanding of the functional genes that promote biosynthesis in vivo as well as the methods that facilitate the in vitro biosynthesis of active compounds are summarized. We also discussed the critical factors associated with further development of S. baicalensis including the cultivation of enriched varieties through genome-assisted breeding, regulatory mechanisms associated with plant growth and development, and optimization of conditions that promote biosynthesis of active compounds both in vivo and in vitro . This review provides a foundation for determining effective solutions for issues associated with the cultivation of S. baicalensis and for the production of high quality products.

Natural products are important resources for drug discovery and have marvelous roles in the treatment of various diseases, such as malaria, cancers, and viral infections. Crocins are the colored apocarotenoids derived from the stigmas of Crocus sativus and often used as a spice/colorant in the food industry. Pharmacologically, crocins confer protection to the central nervous system (especially in the treatment of Alzheimer’s disease) and prevent cardiovascular/cerebrovascular diseases. Unfortunately, the demand for crocins based on their pharmacological benefits combined with a complex harvesting process of C. sativus has driven the retail price of C. sativus stigmas up to 2000–7000 €/kg. Thus, their expense and limited supply greatly restrict the development and clinical application of crocin-based pharmaceuticals. It is therefore urgent to develop an alternative, sustainable way to bolster the supply of crocins. In recent years, synthetic biology has been widely used to overcome shortages of plant-based natural products, including artemisinic acid, etoposide aglycone, opioids, cannabinoids, breviscapine, and ginsenosides. As such, the genetic tools required for crocin biosynthesis have attracted a great deal of attention. The crocin biosynthetic pathway includes three stages: Stage (I), the precursor geranylgeranyl pyrophosphate (GGPP) is generated by the methylerythritol-4-phosphate (MEP) biosynthetic pathway; stage (II), C40-carotenoids are produced via the carotenoid biosynthetic pathway; stage (III), crocins are synthesized by three key catalytic steps, namely, carotenoid cleavage, aldehyde oxidation, and glucosyl group transfer. A series of studies have successfully decoded the crocin biosynthetic pathway and led to its reconstitution in Saccharomyces cerevisiae and Escherichia coli . Hereby, we summarized the chemical characterization of crocins using spectroscopy and mass spectrometry, and elucidated stage (III) of the crocin biosynthetic pathway. Carotenoid cleavage dioxygenases (CCDs) degrade carotenoids. CsCCD2, CaCCD2 ( Crocus ancyrensis ), BdCCD4.1 ( Buddleja davidii ), BdCCD4.3 and GjCCD4a ( Gardenia jasminoides ) can cleave the 7,8 (7′,8′) double bond of zeaxanthin to yield crocetin dialdehyde. Subsequently, crocetin dialdehyde was oxidized to crocetin by the catalysis of aldehyde dehydrogenases (ALDHs), including CsALDH54788, CsALDH3898, CsALDH20158, CsALDH11367, CsALDH3I1, CsALDH3, and GjALDH2C3. Finally, the biosynthesis of crocins from crocetin was catalyzed by UDP-glycosyltransferases (UGTs), including CsUGT74AD1, GjUGT74F8, GjUGT75L6, GjUGT94E5, GjUGT94E13, Bs-GT ( Bacillus subtilis 168), and Bc-GTA ( Bacillus cereus WQ9-2). And then, we displayed the research progress on the synthetic biology of crocins. With the rapid updating of genetic tools, we have the potential to generate more types of crocins. The in vivo production of crocins is highlighted in this review. The engineered E. coli with GjUGT74F8 and GjUGT94E13 can catalyze crocetin into five types of crocins. Furthermore, we put forward some suggestions that might help to improve the bioproduction of crocins, such as the exploration of evolutionary mechanisms of the key enzymes and the optimization of reaction conditions, etc. Taken together, this review will facilitate the industrial production of crocins and the development of crocin-based pharmaceuticals, providing a theoretical basis and method guidance for the sustainable development of crocin-based traditional Chinese medicine.

Scutellaria baicalensis and Scutellaria barbata, common medicinal plants of the Lamiaceae family, produce specific flavonoid compounds with antioxidant and antitumor activities, including baicalein, scutellarein, norwogonin, wogonin, and their glycosides. Here, we reported two chromosome-level genome assemblies of S. baicalensis and S. barbata with significant quantitative chromosomal variation (2n = 18 and 2n = 26, respectively). The divergence of S. baicalensis and S. barbata occurred far earlier than previously reported, and a whole-genome duplication event was identified. The insertion of long terminal repeat elements after speciation might be responsible for the observed chromosomal expansion and rearrangement. The comparative genome analysis of congeneric species elucidated the species-specific evolution of chrysin and apigenin biosynthetic genes, such as the S. baicalensis-specific tandem duplication of the phenylalanine ammonia lyase (PAL) and chalcone synthase (CHS) genes, and the S. barbata-specific duplication of 4-CoA ligase (4CL) genes. In addition, the paralogous duplication, collinearity, and expression diversity of CYP82D subfamily members revealed the functional divergence of flavone hydroxylase genes between S. baicalensis and S. barbata. These Scutellaria genomes highlight the common and species-specific evolution of flavone biosynthetic genes, promoting the development of molecular breeding and the study of the biosynthesis and regulation of bioactive compounds.

Crocins, enriched in Gardenia jasminoides fruits, have a pharmacological activity against central nervous system diseases, cardiovascular diseases, and cancer cell growth. The biosynthesis of crocins has been widely explored, but its regulatory mechanism remains unknown. Here, the basic helix-loop-helix (bHLH) transcription factors related to crocin biosynthesis were systematically identified on the basis of the genome of G. jasminoides. A total of 95 GjbHLH transcription factor genes were identified, and their phylogenetic analysis indicated that they could be classified into 23 subfamilies. The combination of gene-specific bHLH expression patterns, the coexpression analysis of biosynthesis genes, and the analysis of promoter sequences in crocin biosynthesis pathways suggested that nine bHLHs in G. jasminoides might negatively regulate crocin biosynthesis. This study laid a foundation for understanding the regulatory mechanism of crocin biosynthesis and the improvement and breeding of G. jasminoides varieties.

An atlas of cancer-associated T cells The tumor microenvironment contains many different kinds of immune cells, the composition, function, and roles of which are unclear. Using single-cell RNA sequencing of T cells in 21 cancer types from more than 300 patients, Zheng et al . identified differences in transcript composition that could be used to catalog different T cell types (see the Perspective by van der Leun and Schumacher). These annotations identified the different roles of specific types of CD4 + and CD8 + T cells among the different tumor types. Some of these clusters revealed evidence for two developmental paths for T cells, one of which shows a trajectory toward the “exhausted” T cell state, knowledge of which may be useful in developing future cancer immunotherapies. —LMZ

The tumor microenvironment (TME) is connected to immunotherapy responses, but it remains unclear how cancer cells and host tissues differentially influence the immune composition within TME. Here, we performed single-cell analyses for autologous samples from liver metastasized colorectal cancer to disentangle factors shaping TME. By aligning CD45

Background A patient’s ability to cough is important for assessing a patient’s airway condition and likely mechanical ventilation outcome,there is still a lack of data comparing patients’ initial cough ability and outcomes. Methods The study is a prospective, observational trial which includes 144 patients from Xinjiang Medical University. After a patients is assigned a cough strength score, the cough intensity assessments will be implemented every other day for a week. The primary endpoint is whether the patient requires endotracheal intubation (including tracheostomy). Secondary endpoints include time spent under mechanical ventilation (excluding noninvasive ventilation), ICU and hospital lengths of stay, hospital expenses and in-hospital 30-day mortality. Discussion Anecdotally in our practice, we found that patients with a high Cough Reflex Intensity Score don't require endotracheal intubation, while patients with a low score always need to be intubated. This trial will test to what degree cough intensity is correlated to patient’s outcomes. Trial registration: Chinese Clinical Trial Registry,ChiCTR1900028265. Registered 16 December 2019

Distant species producing the same secondary metabolites is an interesting and common phenomenon in nature. A classic example of this is scutellarein whose derivatives have been used clinically for more than 30 years. Scutellarein occurs in significant amounts in species of two different orders, Scutellaria baicalensis and Erigeron breviscapus, which diverged more than 100 million years ago. Here, according to the genome-wide selection and functional identification of 39 CYP450 genes from various angiosperms, we confirmed that only seven Scutellaria-specific CYP82D genes and one Erigeron CYP706X gene could perform the catalytic activity of flavone 6-hydroxylase (F6H), suggesting that the convergent evolution of scutellarein production in these two distant species was caused by two independently evolved CYP450 families. We also identified seven Scutellaria-specific CYP82D genes encoding flavone 8-hydroxylase (F8H). The evolutionary patterns of CYP82 and CYP706 families via kingdom-wide comparative genomics highlighted the evolutionary diversity of CYP82D and the specificity of CYP706X in angiosperms. Multi-collinearity and phylogenetic analysis of CYP82D in Scutellaria confirmed that the function of F6H evolved from F8H. Furthermore, the SbaiCYP82D1

In this study, cytometric beads array(CBA) was used to determine the immunoglobulin content in humoral immunity evaluation of biomedical materials. The bovine-derived acellular dermal matrix was selected as a test sample and implanted into Balb/C mice subcutaneously to 4 weeks according to the high, medium and low dose groups. Four weeks later, IgG1, IgG2a, IgG2b, IgG3, IgA, and IgM were measured by CBA. The data of the test group and the control group were analyzed statistically. The results showed that compared with the negative control group, there was no significant difference in the IgG3, IgA content in the positive control group, while the IgG1, IgG2a, IgG2b, and IgM contents were significantly higher than the negative control group; no significant differences were seen in the sample groups. The results show that the method is suitable for analysis of immunoglobulin content in humoral immunity evaluation of biomedical materials.

The increasingly wide application of chloroplast(cp) genome super-barcode in taxonomy and the recent breakthrough in cp genetic engineering make the development of new cp gene resources urgent and significant. Corydalis is recognized as the most genotypes complicated and taxonomically challenging plant taxa in Papaveraceae. However, no complete cp genome for this genus has been reported to date. In this study, we sequenced four complete cp genomes of two endangered lithophytes Corydalis saxicola and C. tomentellav in Corydalis, conducted a comparative genomics study on them, and a highly variable cp genome structure was found. The cp genomes have a large genome size of 189,029 to 190,247 bp, possessing a quadripartite structure and with two highly expanded inverted repeat (IR) regions (length: 41,955 to 42,350 bp). Comparison between the cp genomes of C. tomentella, C. saxicola and Papaveraceae species, five NADH dehydrogenase-like genes (ndhF, ndhD, ndhL, ndhG, ndhE) with psaC, rpl32, ccsA and trnL-UAG normally located in the SSC region have migrated to IRs, resulting in IR expansion and gene duplication. An up to 9 kb inversion involving five genes (rpl23, ycf2, ycf15, trnI-CAU and trnL-CAA) was found within IR regions. The accD gene was found to be absent and the ycf1 gene has shifted from the IR/SSC border to the SSC region as a single copy. Phylogenetic analysis based on the sequences of common CDS showed that the genus Corydalis is quite distantly related to the other genera of Papaveraceae, it provided a new clue for recent advocacy to establish a separate Fumariaceae family. Our results revealed one special cp genome structure in Papaveraceae, provided a useful resources for classification of the genus Corydalis, and will be valuable for understanding Papaveraceae evolutionary relationships.

Camptotheca acuminata produces camptothecin (CPT), a monoterpene indole alkaloid (MIA) that is widely used in the treatment of lung, colorectal, cervical, and ovarian cancers. Its biosynthesis pathway has attracted significant attention, but the regulation of CPT biosynthesis by the APETALA2/ethylene-responsive factor (AP2/ERF) transcription factors (TFs) remains unclear. In this study, a systematic analysis of the AP2/ERF TFs family in C. acuminata was performed, including phylogeny, gene structure, conserved motifs, and gene expression profiles in different tissues and organs (immature bark, cotyledons, young flower, immature fruit, mature fruit, mature leaf, roots, upper stem, and lower stem) of C. acuminata. A total of 198 AP2/ERF genes were identified and divided into five relatively conserved subfamilies, including AP2 (26 genes), DREB (61 genes), ERF (92 genes), RAV (18 genes), and Soloist (one gene). The combination of gene expression patterns in different C. acuminata tissues and organs, the phylogenetic tree, the co-expression analysis with biosynthetic genes, and the analysis of promoter sequences of key enzymes genes involved in CPT biosynthesis pathways revealed that eight AP2/ERF TFs in C. acuminata might be involved in CPT synthesis regulation, which exhibit relatively high expression levels in the upper stem or immature bark. Among these, four genes (CacAP2/ERF123, CacAP2/ERF125, CacAP2/ERF126, and CacAP2/ERF127) belong to the ERF-B2 subgroup; two genes (CacAP2/ERF149 and CacAP2/ERF152) belong to the ERF-B3 subgroup; and two more genes (CacAP2/ERF095 and CacAP2/ERF096) belong to the DREB-A6 subgroup. These results provide a foundation for future functional characterization of the AP2/ERF genes to enhance the biosynthesis of CPT compounds of C. acuminata.

Backgroud: Corydalis DC., the largest genus of Papaveraceae, is recognized as one of the most taxonomically challenging plant taxa. However, no complete chloroplast (cp) genome for this genus has been reported to date. Results: We sequenced four complete cp genomes of two affinities Corydalis saxicola and C. tomentellav of the genus Corydalis, compared these cp genomes with each other and others from Papaveraceae, and analyzed the phylogenetic relationships based on the sequences of common CDS. The cp genomes are 189,029 to 190,247 bp in length, possessing a quadripartite structure and with two highly expanded inverted repeat (IR) regions (length: 41,955 to 42,350 bp). Comparison between the cp genomes of C. tomentella, C. saxicola and Papaveraceae species revealed high variability in genome sizes, genome structures, gene content, and gene arrangements. Five NADH dehydrogenase-like genes with psaC, rpl32, ccsA and trnL-UAG normally located in the SSC region have migrated to IRs resulting in IR expansion and gene duplication. An up to 9 kb inversion involving five genes (rpl23, ycf2, ycf15, trnI-CAU and trnL-CAA) was found within IR regions. In addition, the accD gene was found to be absent. The ycf1 gene has shifted from the IR/SSC border to the SSC region as a single copy. Phylogenetic analysis showed that genus Corydalis is quite distantly related to the other genera of Papaveraceae, supporting for recent advocacy to establish a separate Fumariaceae family. Conclusions: Our results provide a useful resource for classification of this taxonomically complicated genus, and will be valuable for understanding Papaveraceae evolutionary relationships.

Understanding of the functional states and clonal dynamics of T cells after immune checkpoint blockade (ICB) is valuable for improving these therapeutic strategies. Here we performed Smart-seq2 single-cell RNA sequencing (scRNA-seq) analysis on 3,110 peripheral T cells of non-small cell lung cancer (NSCLC) patients before and after the initiation of programmed cell death protein 1 (PD-1) blockade. We identified individual peripheral T cell clones based on the full-length T cell receptor (TCR) sequences and monitored their dynamics during immunotherapy. We found a higher cytotoxic activity in the tumor-related CD4+ T cell clones than in the CD8+ T cell clones. Based on a large tumor-related CD4+ T cell clone, we observed a dramatically decreased abundance after progression, as well as a reduction in the percentage of PD-1+ T cells. We also detected 25 genes, such as CXCR4, DUSP2 and ZFP36, that were noticeably upregulated or downregulated following progression. In addition, the pseudotime trajectory of CD8+ T cell clones corresponded to the treatment time points, showing a decreased activity in the "cytokine and cytokine receptor interaction" pathway. These analyses provided an insight into the dynamics of peripheral T cell clones during PD-1 blockade in NSCLC.

Lonicera japonica is a wide-spread member of the Caprifoliaceae (honeysuckle) family utilized in traditional medical practices. This twining vine honeysuckle is also a much-sought ornamental, in part due to its dynamic flower coloration, which changes from white to gold during development. The molecular mechanism underlying dynamic flower coloration in L. japonica was elucidated by integrating whole genome sequencing, transcriptomic analysis, and biochemical assays. Here, we report a chromosome-level genome assembly of L. japonica, comprising nine pseudo-chromosomes with a total size of 843.2 Mb. We also provide evidence for a whole genome duplication event in the lineage leading to L. japonica, which occurred after its divergence from Dipsacales and Asterales. Moreover, gene expression analysis not only revealed correlated expression of the relevant biosynthetic genes with carotenoid accumulation, but also suggested a role for carotenoid degradation in L. japonica’s dynamic flower coloration. The variation of flower color is consistent with not only the observed carotenoid accumulation pattern, but also with the release of volatile apocarotenoids that presumably serve as pollinator attractants. Beyond novel insights into the evolution and dynamics of flower coloration, the high-quality L. japonica genome sequence also provides a foundation for molecular breeding to improve desired characteristics.

Additional file 6: Figure S29. Pasta phylogenetic tree of UGTs from different species.

Additional file 2. Review history.

Additional file 3. Detailed methods and results for crocin biosynthesis.

Additional file 7: Figure S30. Pasta phylogenetic tree of ALDHs from different species.

Additional file 4: Figure S27. Pasta phylogenetic tree of NMTs from different species.

Additional file 5: Figure S28. Pasta phylogenetic tree of CCDs from different species.

Additional file 1. Detailed methods and results for genome sequencing.

Additional file 2: Figures S1-S26; Tables S1-S18.

T cells are key elements of cancer immunotherapy1 but certain fundamental properties, such as the development and migration of T cells within tumours, remain unknown. The enormous T cell receptor (TCR) repertoire, which is required for the recognition of foreign and self-antigens2, could serve as lineage tags to track these T cells in tumours3. Here we obtained transcriptomes of 11,138 single T cells from 12 patients with colorectal cancer, and developed single T cell analysis by RNA sequencing and TCR tracking (STARTRAC) indices to quantitatively analyse the dynamic relationships among 20 identified T cell subsets with distinct functions and clonalities. Although both CD8+ effector and ‘exhausted’ T cells exhibited high clonal expansion, they were independently connected with tumour-resident CD8+ effector memory cells, implicating a TCR-based fate decision. Of the CD4+ T cells, most tumour-infiltrating T regulatory (Treg) cells showed clonal exclusivity, whereas certain Treg cell clones were developmentally linked to several T helper (TH) cell clones. Notably, we identified two IFNG+ TH1-like cell clusters in tumours that were associated with distinct IFNγ-regulating transcription factors —the GZMK+ effector memory T cells, which were associated with EOMES and RUNX3, and CXCL13+BHLHE40+ TH1-like cell clusters, which were associated with BHLHE40. Only CXCL13+BHLHE40+ TH1-like cells were preferentially enriched in patients with microsatellite-instable tumours, and this might explain their favourable responses to immune-checkpoint blockade. Furthermore, IGFLR1 was highly expressed in both CXCL13+BHLHE40+ TH1-like cells and CD8+ exhausted T cells and possessed co-stimulatory functions. Our integrated STARTRAC analyses provide a powerful approach to dissect the T cell properties in colorectal cancer comprehensively, and could provide insights into the dynamic relationships of T cells in other cancers. An integrated RNA-sequencing approach demonstrates that CXCL13+ TH1-like cells are preferentially enriched in microsatellite-instable tumours from patients with colorectal cancer, and IGFLR1 is identified as a co-stimulatory molecule.

Background Tumor-reactive CD8+ tumor-infiltrating lymphocytes (TILs) represent a subtype of T cells that can recognize and destroy tumor specifically. Understanding the regulatory mechanism of tumor-reactive CD8+ T cells has important therapeutic implications. Yet the DNA methylation status of this T cell subtype has not been elucidated. Results In this study, we segregate tumor-reactive and bystander CD8+ TILs, as well as naïve and effector memory CD8+ T cell subtypes as controls from colorectal cancer patients, to compare their transcriptome and methylome characteristics. Transcriptome profiling confirms previous conclusions that tumor-reactive TILs have an exhausted tissue-resident memory signature. Whole-genome methylation profiling identifies a distinct methylome pattern of tumor-reactive CD8+ T cells, with tumor-reactive markers CD39 and CD103 being specifically demethylated. In addition, dynamic changes are observed during the transition of naïve T cells into tumor-reactive CD8+ T cells. Transcription factor binding motif enrichment analysis identifies several immune-related transcription factors, including three exhaustion-related genes (NR4A1, BATF, and EGR2) and VDR, which potentially play an important regulatory role in tumor-reactive CD8+ T cells. Conclusion Our study supports the involvement of DNA methylation in shaping tumor-reactive and bystander CD8+ TILs, and provides a valuable resource for the development of novel DNA methylation markers and future therapeutics.