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Phenotypic diversity and evolutionary innovation ultimately trace to variation in genomic sequence and rewiring of regulatory networks. Here, we constructed a pan-genome of the Gossypium genus using ten representative diploid genomes. We document the genomic evolutionary history and the impact of lineage-specific transposon amplification on differential genome composition. The pan-3D genome reveals evolutionary connections between transposon-driven genome size variation and both higher-order chromatin structure reorganization and the rewiring of chromatin interactome. We linked changes in chromatin structures to phenotypic differences in cotton fiber and identified regulatory variations that decode the genetic basis of fiber length, the latter enabled by sequencing 1,005 transcriptomes during fiber development. We showcase how pan-genomic, pan-3D genomic and genetic regulatory data serve as a resource for delineating the evolutionary basis of spinnable cotton fiber. Our work provides insights into the evolution of genome organization and regulation and will inform cotton improvement by enabling regulome-based approaches.

Symbiosis between legumes and rhizobia results in the formation of nitrogen-fixing root nodules. Endoreduplication is essential for nodule development and efficient nitrogen fixation; however, the cellular mechanism by which rhizobial infection causes endoreduplication in symbiotic nodules and the roles of the resulting polyploid cells in nitrogen fixation remain largely unknown. Here, we developed a series of different approaches to separate infected cells (ICs) and uninfected cells (UCs) and determined their ploidy levels in soybean (Glycine max) developing nodules. We demonstrated that 4C nuclei exist in both UCs and ICs of developing nodules and that these 4C cells are primarily invaded by rhizobia and subsequently undergo endoreduplication. Furthermore, RNA-sequencing analysis of nuclei with different ploidy levels from soybean nodules at 12 d post-infection (dpi) and 20 dpi showed that 4C cells are predominantly ICs in 12-dpi nodules but UCs in 20-dpi nodules. We conclude that the infection of 4C cells by rhizobia is critical for initiating endoreduplication. These findings provide significant insight into rhizobial infection, nodule endoreduplication and nitrogen fixation in symbiotic nodules.

Interpreting the functional impacts of genetic variants (GVs) is an important challenge for functional genomic studies in crops and next-generation breeding. Previous studies in rice (Oryza sativa) have focused mainly on the identification of GVs, whereas systematic functional annotation of GVs has not yet been performed. Here, we present a functional impact map of GVs in rice. We curated haplotype information for 17 397 026 GVs from sequencing data of 4726 rice accessions. We quantitatively evaluated the effects of missense mutations in coding regions in each haplotype based on the conservation of amino acid residues and obtained the effects of 918 848 non-redundant missense GVs. Furthermore, we generated high-quality chromatin accessibility (CA) data from six representative rice tissues and used these data to train deep convolutional neural network models to predict the impacts of 5 067 405 GVs for CA in regulatory regions. We characterized the functional properties and tissue specificity of the GV effects and found that large-effect GVs in coding and regulatory regions may be subject to selection in different directions. Finally, we demonstrated how the functional impact map could be used to prioritize causal variants in mapping populations. This impact map will be a useful resource for accelerating gene cloning and functional studies in rice, and can be freely queried in RiceVarMap V2.0 ( http://ricevarmap.ncpgr.cn ).

Summary Safflower (Carthamus tinctorius L.), a member of the Asteraceae, is a popular crop due to its high linoleic acid (LA) and flavonoid (such as hydroxysafflor yellow A) contents. Here, we report the first high‐quality genome assembly (contig N50 of 21.23 Mb) for the 12 pseudochromosomes of safflower using single‐molecule real‐time sequencing, Hi‐C mapping technologies and a genetic linkage map. Phyloge nomic analysis showed that safflower diverged from artichoke (Cynara cardunculus) and sunflower (Helianthus annuus) approximately 30.7 and 60.5 million years ago, respectively. Comparative genomic analyses revealed that uniquely expanded gene families in safflower were enriched for those predicted to be involved in lipid metabolism and transport and abscisic acid signalling. Notably, the fatty acid desaturase 2 (FAD2) and chalcone synthase (CHS) families, which function in the LA and flavonoid biosynthesis pathways, respectively, were expanded via tandem duplications in safflower. CarFAD2‐12 was specifically expressed in seeds and was vital for high‐LA content in seeds, while tandemly duplicated CarFAD2 genes were up‐regulated in ovaries compared to CarFAD2‐12, which indicates regulatory divergence of FAD2 in seeds and ovaries. CarCHS1, CarCHS4 and tandem‐duplicated CarCHS5˜CarCHS6, which were up‐regulated compared to other CarCHS members at early stages, contribute to the accumulation of major flavonoids in flowers. In addition, our data reveal multiple alternative splicing events in gene families related to fatty acid and flavonoid biosynthesis. Together, these results provide a high‐quality reference genome and evolutionary insights into the molecular basis of fatty acid and flavonoid biosynthesis in safflower.

Targeting inhibitory immune checkpoint molecules has significantly altered cancer treatment regimens. T cell immunoglobulin and mucin domain 3 (TIM3) is one of the major inhibitory immune checkpoints expressed on T cells. Blocking the engagement of TIM3 and its inhibitory ligand galectin-9 may potentiate the effects of immunotherapy or overcome the adaptive resistance to the therapeutic blockade of programmed cell death protein 1, cytotoxic T-lymphocyte-associated protein 4, B- and T-lymphocyte attenuator and lymphocyte-activation gene 3, amongst others, as each of these immune checkpoints harbors unique properties that set it apart from the rest. Heavy chain variable fragment (VH)-derived single-domain antibodies (sdAbs) represent a class of expanding drug candidates. These sdAbs have unique advantages, including their minimal size in the antibody class, ease of expression, broad scope for modular structure design and re-engineering, and excellent tumor penetration. In the present study, two sdAbs, TIM3-R23 and TIM3-R53, were generated by immunizing rabbits with the recombinant extracellular domain of TIM3 and applying phage display technology. These sdAbs were easily expressed in mammalian cells. The purified sdAbs were able to bind to both recombinant and cell surface TIM3, and blocked it from binding to the ligand galectin-9. In vivo studies demonstrated that TIM3-R53 was able to potentiate the antitumor activity of chimeric antigen receptor T cells that targeted mesothelin. In conclusion, the results of the present study suggested that TIM3-R53 may be a novel and attractive immune checkpoint inhibitor against TIM3, which is worthy of further investigation.

Reproductive transition of grasses is characterized by switching the pattern of lateral branches, featuring the suppression of outgrowth of the subtending leaves (bracts) and rapid formation of higher-order branches in the inflorescence (panicle). However, the molecular mechanisms underlying such changes remain largely unknown. Here, we show that bract suppression is required for the reproductive branching in rice. We identified a pathway involving the intrinsic time ruler microRNA156/529, their targets SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL) genes, NECK LEAF1 (NL1), and PLASTOCHRON1 (PLA1), which regulates the bract outgrowth and thus affects the pattern switch between vegetative and reproductive branching. Suppression of the bract results in global reprogramming of transcriptome and chromatin accessibility following the reproductive transition, while these processes are largely dysregulated in the mutants of these genes. These discoveries contribute to our understanding of the dynamic plant architecture and provide novel insights for improving crop yields.

ATAC-seq (Assay for Transposase-Accessible Chromatin with high-throughput sequencing) provides an efficient way to analyze nucleosome-free regions and has been applied widely to identify transcription factor footprints. Both applications rely on the accurate quantification of insertion events of the hyperactive transposase Tn5. However, due to the presence of the PCR amplification, it is impossible to accurately distinguish independently generated identical Tn5 insertion events from PCR duplicates using the standard ATAC-seq technique. Removing PCR duplicates based on mapping coordinates introduces increasing bias towards highly accessible chromatin regions. To overcome this limitation, we establish a UMI-ATAC-seq technique by incorporating unique molecular identifiers (UMIs) into standard ATAC-seq procedures. UMI-ATAC-seq can rescue about 20% of reads that are mistaken as PCR duplicates in standard ATAC-seq in our study. We demonstrate that UMI-ATAC-seq could more accurately quantify chromatin accessibility and significantly improve the sensitivity of identifying transcription factor footprints. An analytic pipeline is developed to facilitate the application of UMI-ATAC-seq, and it is available at https://github.com/tzhu-bio/UMI-ATAC-seq., Tao Zhu et al. present UMI-ATAC-seq, an improved ATAC-seq technique that uses unique molecular identifiers to avoid filtering out true independent - but identical - Tn5 insertion events that would otherwise be treated as PCR duplicates. They apply UMI-ATAC-seq to rice panicle samples and show that their approach improves the quantification of chromatin accessibility and footprinting.

Epidermal growth factor receptor (EGFR) mutation status is the best predictor of patient response to treatments with tyrosine kinase inhibitors in primary lung adenocarcinoma and is typically analyzed by DNA-based techniques, such as direct DNA sequencing and allele-specific PCR. Recently, however, two mutation-specific antibodies against delE746-A750 in exon 19 and L858R in exon 21 have opened the door for a more convenient and more efficient strategy to determine EGFR mutation status. To evaluate the clinical application of a new mutation-specific mouse monoclonal antibody for EGFR (L858R), we performed immunohistochemistry (IHC) studies with tumor samples from primary lung adenocarcinoma in retrospective and validation settings. A total of 215 cases of primary lung adenocarcinoma were examined and compared using a combination of DNA-based techniques (direct DNA sequencing and/or allele-specific PCR) and protein-based IHC. IHC staining was assessed on a 0 to 3+ score scale, and a cutoff value of 2+ was used as positive by IHC. In the retrospective setting, statistical analyses of the data showed that the sensitivity of IHC was 90.9 % and the specificity was 96.8 %. Findings from the validation study demonstrated that the sensitivity and specificity of IHC were 88.2 % and 100 %, respectively. IHC with the novel mutation-specific antibody could be used as a screening method to assess the EGFR L858R mutation status in primary lung adenocarcinoma.

With the increased uses of targeted therapeutics, diagnostic detection of target mutations becomes essential for the effective clinical applications of targeted therapeutics. Currently, there are two types of methods detecting target mutations in clinics: one is based on DNA sequence and the other uses the newly developed mutation-specific antibodies recognizing mutated proteins. Each method has its own advantages and disadvantages. Here, we explored the sensitivity and specificity of a new commercially available BRAF(V600E) mutation-specific mouse monoclonal antibody. Using routine manual immunohistochemistry (IHC), we tested tumor tissues from 38 melanoma patients. For those melanoma tissues with abundant endogenous melanin, we pretreated the tumor tissues with 3 % hydrogen peroxide to remove melanin for reliable signal detection. We also performed DNA sequencing and ARMS-PCR analyses for these 38 tumor samples. Comparing to the results from DNA-based detection methods, the IHC method with this BRAF(V600E) mutation-specific antibody displayed 100 % sensitivity and 92.9 % specificity. Hence, this IHC detection is sensitive for clinic uses as a simple, fast, inexpensive, and reliable method to screen cancer patients for the BRAF(V600E) mutation and could be easily adapted for use in most hospital pathology laboratories.

A new, simple and sensitive method for the quantitative analysis of cytochrome C (Cyt C) based on the reduction wave of guanidine modified Co(II)-Cyt C complex at about -1.74 V (vs. SCE) by single sweep polarography in the solution containing 8 x 10(-6) mol L(-1) CoCl2, 0.04 mol L(-1) guanidine hydrochloride, 0.2 mol L(-1) NaOH and 0.5% Na2SO3. The peak height is linearly proportional to the concentration of Cyt C in the range of 0.005 approximately 1.500 mg L(-1) (correlation coefficient 0.999). Common amino acids, saccharide, organic acid and metal ions of appropriate concentrations have no interference on the Cyt C determination. The released Cyt C in the process of mitochondrial permeability transition of Hong-Lian cytoplasmic male sterile line of rice has been measured by the method, and the result is satisfactory.