The Role of TGF-ß/SMAD Signaling in Hepatocellular Carcinoma: from Mechanism to Therapy and Prognosis.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, with high incidence and mortality, accounting for approximately 90% of liver cancer. The development of HCC is a complex process involving the abnormal activation or inactivation of multiple signaling pathways. Transforming growth factor-ß (TGF-ß)/Small mothers against decapentaplegic (SMAD) signaling pathway regulates the development of HCC. TGF-ß activates intracellular SMADs protein through membrane receptors, resulting in a series of biological cascades. Accumulating studies have demonstrated that TGF-ß/SMAD signaling plays multiple regulatory functions in HCC. However, there is still controversy about the role of TGF-ß/SMAD in HCC. Because it involves different pathogenic factors, disease stages, and cell microenvironment, as well as upstream and downstream relationships with other signaling pathways. This review will summary the regulatory mechanism of the TGF-ß/SMAD signaling pathway in HCC, involving the regulation of different pathogenic factors, different disease stages, different cell populations, microenvironments, and the interaction with microRNAs. In addition, we also introduced small molecule inhibitors, therapeutic vaccines, and traditional Chinese medicine extracts based on targeting the TGF-ß/SMAD signaling pathway, which will provide future research direction for HCC therapy targeting the TGF-ß/SMAD signaling pathway.

Changes in China's food security driven by nutrition security and resource constraints.

Food security and the utilization of natural resources in a sustainable manner are vital to the expansion of China's agricultural system. The relationship between environmental pressure and dietary structure has influenced the quantity and spatial distribution of China's food supply and demand, but it has not been evaluated. Our research centered on the security of China's food nutrition-resources-food (NRF) system, considering the inherent relationship between food security, nutritional health, and resource security. The following are the study's findings: (1) The Chinese population is rapidly changing from a diet focused on grains to a more diverse diet. Between 1990 and 2019, the dietary quality and nutritional status of Chinese individuals have vastly improved. In terms of nutrient levels, discrepancies between urban and rural resident persist, with urban residents consuming a diet that is closer to the ideal structure. However, the structure of rural residents' food consumption is diversifying, and the gap between urban and rural residents is gradually narrowing. (2) From 2000 to 2019, the pressure, status, and response indices of China's NRF system all show an upward trend, and the security of the NRF system has steadily grown. The magnitude of change in the response index exceeded that of the state index, which exceeded that of the pressure index. This indicates that the increase in the pressure and state indices of the NRF system was primarily attributable to the effectiveness of policy efforts.

Full-length circular RNA profiling by nanopore sequencing with CIRI-long.

Circular RNAs (circRNAs) have important roles in regulating developmental processes and disease progression. As most circRNA sequences are highly similar to their cognate linear transcripts, the current short-read sequencing-based methods rely on the back-spliced junction signal for distinguishing circular and linear reads, which does not allow circRNAs' full-length structure to be effectively reconstructed. Here we describe a long-read sequencing-based protocol, CIRI-long, for the detection of full-length circular RNAs. The CIRI-long protocol combines rolling circular reverse transcription and nanopore sequencing to capture full-length circRNA sequences. After poly(A) tailing, RNase R treatment, and size selection of polymerase chain reaction products, CIRI-long achieves an increased percentage (6%) of circular reads in the constructed library, which is 20-fold higher compared with previous Illumina-based strategies. This method can be applied in cell lines or tissue samples, enabling accurate detection of full-length circRNAs in the range of 100-3,000 bp. The entire protocol can be completed in 1 d, and can be scaled up for large-scale analysis using the nanopore barcoding kit and PromethION sequencing device. CIRI-long can serve as an effective and user-friendly protocol for characterizing full-length circRNAs, generating direct and convincing evidence for the existence of detected circRNAs. The analytical pipeline offers convenient functions for identification of full-length circRNA isoforms and integration of multiple datasets. The assembled full-length transcripts and their splicing patterns provide indispensable information to explore the biological function of circRNAs.

Hepatocyte-Specific Smad4 Deficiency Alleviates Liver Fibrosis via the p38/p65 Pathway.

Liver fibrosis is a wound-healing response caused by the abnormal accumulation of extracellular matrix, which is produced by activated hepatic stellate cells (HSCs). Most studies have focused on the activated HSCs themselves in liver fibrosis, and whether hepatocytes can modulate the process of fibrosis is still unclear. Sma mothers against decapentaplegic homologue 4 (Smad4) is a key intracellular transcription mediator of transforming growth factor-ß (TGF-ß) during the development and progression of liver fibrosis. However, the role of hepatocyte Smad4 in the development of fibrosis is poorly elucidated. Here, to explore the functional role of hepatocyte Smad4 and the molecular mechanism in liver fibrosis, a CCl4-induced liver fibrosis model was established in mice with hepatocyte-specific Smad4 deletion (Smad4Δhep). We found that hepatocyte-specific Smad4 deficiency reduced liver inflammation and fibrosis, alleviated epithelial-mesenchymal transition, and inhibited hepatocyte proliferation and migration. Molecularly, Smad4 deletion in hepatocytes suppressed the expression of inhibitor of differentiation 1 (ID1) and the secretion of connective tissue growth factor (CTGF) of hepatocytes, which subsequently activated the p38 and p65 signaling pathways of HSCs in an epidermal growth factor receptor-dependent manner. Taken together, our results clearly demonstrate that the Smad4 expression in hepatocytes plays an important role in promoting liver fibrosis and could therefore be a promising target for future anti-fibrotic therapy.

Enhancement of recombinant human IL-24 (rhIL-24) protein production from site-specific integrated engineered CHO cells by sodium butyrate treatment.

Interleukin-24 (IL-24) has specific inhibitory effects on the proliferation of various tumor cells with almost no toxicity to normal cells. The antitumor activity of recombinant human IL-24 protein produced in mammalian cells is much higher than that of bacteria, but its expression level is extremely low. Sodium butyrate (NaBu) was utilized as a media additive to increase protein expression in Chinese hamster ovary cells. The site-specific integrated engineered cells FCHO/IL-24 were treated with NaBu under different culture conditions (10% and 0.5% serum adherent culture, 0.5% serum suspension culture). First, 3 days of 1 mmol/L NaBu treatment significantly increased rhIL-24 expression level in FCHO/IL-24 cells by 119.94 ± 1.5% (**p < 0.01), 57.49 ± 2.4% (**p < 0.01), and 20.17 ± 3.03% (*p < 0.05) under the above culture conditions. Second, NaBu has a time- and dose-dependent inhibitory effect on FCHO/IL-24 proliferation and induces G0/G1 phase arrest. Under 10% and 0.5% serum adherent culture, G0/G1 phase cells were increased by 11.3 ± 0.5% (**p < 0.01) and 15.0 ± 2.6% (**p < 0.01), respectively. No induction of apoptosis was observed under a high dosage of NaBu treatment. These results suggest that NaBu increases rhIL-24 secretion via inhibiting cell cycle progression, thereby trapping cells in the highly productive G0/G1 phase. Finally, with increasing NaBu dose, glucose concentration increased (**p < 0.01) while lactic acid and ammonia concentrations reduced significantly (**p < 0.01) in 10% and 0.5% serum adherent culture supernatant. RNA-seq showed that NaBu treatment affected multiple tumor and immune-related pathways. In conclusion, NaBu treatment dramatically promoted rhIL-24 production in engineered FCHO/IL-24 cells by altering downstream pathways and inducing G0/G1 cell arrest with little effect on apoptosis.

Adaptation process of engineered cell line FCHO/IL-24 stably secreted rhIL-24 in serum-free suspension culture.

Interleukin-24 (IL-24) displays tumor cell-specific proliferation inhibition in vitro and in vivo. Recombinant human IL-24 (rhIL-24) has significantly higher activity, yet significantly lower expression level in mammalian cells than in bacteria. To further realize therapeutic potential of IL-24, we enhanced rhIL-24 expression in mammalian cell systems by adapting engineered Flp-InTMCHO/IL-24 (FCHO/IL-24) cells (adherent cultured in Ham's F12 medium with 10% serum) to serum-free suspension culture. First, MTT assay showed that among four different media (F12, DMEM/F12, 1640 and DMEM), DMEM/F12 medium was the most suitable media for lower-serum adherent culture. Then, cells were adherently cultured in DMEM/F12 with serum concentration reduced from 10% to 0.5% in a gradient manner. Compared to cells in 10% serum, cells in 0.5% serum displayed significantly lower relative cell viability by 40%, increased G0/G1 phase arrest (8.5 ± 2.4%, p < 0.05), decreased supernatant rhIL-24 concentration by 73%, and altered metabolite profiles, such as glucose, lactate and ammonia concentration. Next, the cells were directly adapted to 0.5% serum suspension culture in 125 mL shake flask at 119 rpm with the optimal cell seeding density of 5 × 105 cells/mL (3.3 times higher than that of adherent culture), under which the concentration of rhIL-24 in culture medium was stable at 3.5 ng/mL. Finally, cells adapted to 0.5% serum proliferated better in serum-free medium Eden™-B300S with higher rhIL-24 expression level compared to CDM4CHO. The successful adaptation of engineered cells FCHO/IL-24 laid foundation for adapting cells from adherent culture to suspension serum-free culture to mass produce rhIL-24 protein for therapeutic purposes.

The Mitochondrial tRNAPhe 625G>A Mutation in Three Han Chinese Families With Cholecystolithiasis.

In this study, we assessed three Chinese families with inherited cholecystolithiasis and conducted the clinical, genetic, and molecular characterization of these subjects. Eight of eighteen matrilineal relatives had a clinical phenotype in these three families. Sequence analysis of complete mitochondrial genomes in these probands identified the homoplasmic tRNAPhe 625 G > A mutation and distinct sets of mtDNA polymorphisms belonging to haplogroups H2, F4b, and M10a. The 625G > A mutation disturbed the classic G-C base-pairings at a highly conserved position 49 in the T-stem of mitochondrial tRNAs. Molecular dynamics simulation showed that the structure of tRNAphe with 625 G > A mutation was noticeably remodeled while compared with the isoform of the wild type. The occurrence of tRNAPhe 625 G > A mutation in these various genetically unrelated subjects strongly indicates that this mutation is involved in the pathogenesis of cholecystolithiasis. This is the first evidence that tRNA mutations are associated with cholecystolithiasis, and it provided more insights into the genetic mechanism of cholecystolithiasis.

MyD88 in hepatic stellate cells promotes the development of alcoholic fatty liver via the AKT pathway.

Myeloid differentiation primary response gene 88 (MyD88), an adaptor protein in the Toll-like receptors (TLRs) signalling pathway, is expressed in various liver cells including hepatocytes, Kupffer cells and hepatic stellate cells (HSCs). And yet, the functional role of MyD88 in HSCs is poorly elucidated in alcoholic fatty liver (AFL). Here, to study the functional role of MyD88 in HSCs and the molecular mechanism related to the development of AFL, chronic-binge ethanol mouse models were established in mice with specific MyD88 knockout in quiescent (MyD88GFAP-KO) and activated HSCs (MyD88SMA-KO), respectively. Our results clearly showed an elevated expression of MyD88 in liver tissues of ethanol treated mouse model which harbours the wild type. Intriguingly, ethanol treatment profoundly inhibited inflammation in both MyD88GFAP-KO and MyD88SMA-KO mice, but the suppression of lipogenesis was only observed in MyD88GFAP-KO mice. Molecularly, our study indicated that MyD88 induced osteopontin (OPN) secretion in HSCs, which consequently resulted in activation of AKT signalling pathway and accumulation of fat in hepatocytes. Additionally, our data also suggested that OPN promoted inflammation by activating p-STAT1. Thus, targeting MyD88 may be a potentially represent a promising strategy for the prevention and treatment of AFL. KEY MESSAGES: The expression of MyD88 in HSCs was significantly increased in ethanol-induced liver tissues of wild-type mice. MyD88 deficiency in quiescent HSCs inhibited inflammation and lipogenesis under the ethanol feeding condition. MyD88 deficiency in activated HSCs only inhibited inflammation under the ethanol feeding condition. MyD88 promoted the OPN secretion of HSCs, which further activated the AKT signalling pathway of hepatocytes and upregulated lipogenic gene expression to promote fat accumulation. OPN also promotes inflammation by activating p-STAT1.

Unfavourable intrauterine environment contributes to abnormal gut microbiome and metabolome in twins.

OBJECTIVE: Fetal growth restriction (FGR) is a devastating pregnancy complication that increases the risk of perinatal mortality and morbidity. This study aims to determine the combined and relative effects of genetic and intrauterine environments on neonatal microbial communities and to explore selective FGR-induced gut microbiota disruption, metabolic profile disturbances and possible outcomes. DESIGN: We profiled and compared the gut microbial colonisation of 150 pairs of twin neonates who were classified into four groups based on their chorionicity and discordance of fetal birth weight. Gut microbiota dysbiosis and faecal metabolic alterations were determined by 16S ribosomal RNA and metagenomic sequencing and metabolomics, and the long-term effects were explored by surveys of physical and neurocognitive development conducted after 2~3 years of follow-up. RESULTS: Adverse intrauterine environmental factors related to selective FGR dominate genetics in their effects of elevating bacterial diversity and altering the composition of early-life gut microbiota, and this effect is positively related to the severity of selective FGR in twins. The influence of genetic factors on gut microbes diminishes in the context of selective FGR. Gut microbiota dysbiosis in twin neonates with selective FGR and faecal metabolic alterations features decreased abundances of Enterococcus and Acinetobacter and downregulated methionine and cysteine levels. Correlation analysis indicates that the faecal cysteine level in early life is positively correlated with the physical and neurocognitive development of infants. CONCLUSION: Dysbiotic microbiota profiles and pronounced metabolic alterations are associated with selective FGR affected by adverse intrauterine environments, emphasising the possible effects of dysbiosis on long-term neurobehavioural development.

MyD88 in hepatic stellate cells enhances liver fibrosis via promoting macrophage M1 polarization.

During liver fibrosis, quiescent HSCs (qHSCs) are activated to become activated HSCs (aHSCs)/myofibroblasts. The signal adapter MyD88, an essential component of TLR signaling, plays an important role in liver fibrosis. However, far less is known about the specific effects of MyD88 signaling in both qHSCs and aHSCs in the progress of liver fibrosis. Here, we used a CCl4-induced mouse fibrosis model in which MyD88 was selectively depleted in qHSCs (GFAPMyD88-/- mice) or aHSCs (α-SMAMyD88-/- mice). MyD88 deficiency in qHSCs or aHSCs attenuated liver fibrosis in mice and inhibited α-SMA-positive cell activation. Inhibition of MyD88 in HSCs decreased α-SMA and collagen I levels, inflammatory cell infiltration, and pro-inflammatory gene expression. Furthermore, MyD88 signaling in HSCs increased the secretion of CXCL10, which promoted macrophage M1 polarization through CXCR3, leading to activation of the JAK/STAT1 pathway. Inhibition of CXCL10 attenuated macrophage M1 polarization and reduced liver fibrosis. Thus, MyD88 signaling in HSCs crucially contributes to liver fibrosis and provides a promising therapeutic target for the prevention and treatment of liver fibrosis.

An Extremely Streamlined Macronuclear Genome in the Free-Living Protozoan Fabrea salina.

Ciliated protists are among the oldest unicellular organisms with a heterotrophic lifestyle and share a common ancestor with Plantae. Unlike any other eukaryotes, there are two distinct nuclei in ciliates with separate germline and somatic cell functions. Here, we assembled a near-complete macronuclear genome of Fabrea salina, which belongs to one of the oldest clades of ciliates. Its extremely minimized genome (18.35 Mb) is the smallest among all free-living heterotrophic eukaryotes and exhibits typical streamlined genomic features, including high gene density, tiny introns, and shrinkage of gene paralogs. Gene families involved in hypersaline stress resistance, DNA replication proteins, and mitochondrial biogenesis are expanded, and the accumulation of phosphatidic acid may play an important role in resistance to high osmotic pressure. We further investigated the morphological and transcriptomic changes in the macronucleus during sexual reproduction and highlighted the potential contribution of macronuclear residuals to this process. We believe that the minimized genome generated in this study provides novel insights into the genome streamlining theory and will be an ideal model to study the evolution of eukaryotic heterotrophs.

Mutations in Sdh Gene Subunits Confer Different Cross-Resistance Patterns to SDHI Fungicides in Alternaria alternata Causing Alternaria Leaf Spot of Almond in California.

Alternaria leaf spot caused by Alternaria alternata and A. arborescens is a common disease of almond in California. Succinate dehydrogenase inhibitors (SDHIs) are widely used for its management; however, we observed reduced performance of SDHI fungicides at some field sites. Thus, we evaluated the sensitivity to boscalid of 520 isolates of the main pathogen A. alternata collected from major production areas between 2006 and 2019, and also evaluated the sensitivity of a subset of 204 isolates to six members of the SDHIs belonging to six subgroups. Additionally, 97 isolates (14 sensitive and 83 with reduced sensitivity) of the 204 were used to determine the molecular mechanisms of resistance. A wide range of in vitro concentrations to effectively inhibit mycelial growth by 50% (EC50 values) was determined for each fungicide using the spiral gradient dilution method. Some isolates were highly resistant (EC50 values >10 µg/ml) to boscalid (a pyridine-carboxamide), pyraziflumid (a pyrazine-carboxamide), and fluxapyroxad (a pyrazole-4-carboxamide), but not to fluopyram (a pyridinyl-ethyl-benzamide), isofetamid (a phenyl-oxo-ethyl thiophene amide), and pydiflumetofen (a N-methoxy-(phenyl-ethyl)-pyrazole-carboxamide). There was no strong cross resistance among the fungicides tested, including for the two pyrazole-4-carboxamides fluxapyroxad and penthiopyrad (tested for 33 of the 204 isolates). The comparison of EC50 values for fluopyram and isofetamid resulted in the highest coefficient of determination (R2 = 0.582) among 10 pairwise comparisons between subgroups. Sequence analyses of the 97 isolates revealed five mutations in SdhB, SdhC, or SdhD subunits of the Sdh target gene among 73 isolates with reduced sensitivity to at least one SDHI. No mutations were detected in the 14 sensitive isolates and in 10 of the 83 isolates with reduced sensitivity. The most common mutation (59 isolates) was H134R in SdhC. Other mutations included H277Y (eight isolates) and H277L (two isolates) in SdhB, as well as G79R (two isolates) and S135R (two isolates) in SdhC. Mutations H277Y in SdhB and S135R in SdhC were only present in isolates collected in 2012 or earlier. Both conferred mostly high levels of resistance to boscalid and also reduced sensitivity to pyraziflumid, fluxapyroxad, and isofetamid with intermediate EC50 levels. Mutations H277L in SdhB, as well as H134R and G79R in SdhC, found in isolates obtained after 2012 had very similar resistance phenotypes with different levels of resistance to boscalid, pyraziflumid, and fluxapyroxad, whereas sensitivity to fluopyram, isofetamid, and pydiflumetofen was mostly less affected. Our data for SDHI fungicides do not support the classical concept of positive cross resistance within a single mode of action. Because some mutations conferred resistance to multiple SDHI subgroups, however, resistance management needs to consider all SDHIs as a homogenous group that should be mixed or rotated with other modes of action to delay development of resistance.

Ecosystem services valuation in China: A meta-analysis.

Although the number of studies on ecosystem service value (ESV) has steadily increased, large variations and inconsistent patterns in the estimated results have motivated us to systematically explore the factors underlying such discrepancies. Therefore, this study aims to explore the role of different ecosystems, ESs, valuation methods, and economic development in the ESV by employing a meta-analysis of valuation research conducted on China's ES based on 3356 observations from 140 studies. The results show that wetlands ecosystem has the highest value among the seven major ecosystems, and regulation of water flows service is more valuable than the other services. We also provide a matrix of monetary values for different categories of ecosystems and their services, which can be used as a quick tool to predict ESVs in China and assess the value changes caused by land-use changes. We find that the ESVs estimated following the equivalent factor method are different from those estimated by the other methods, indicating that researchers should be very careful when selecting valuation methods for evaluating ESs. The economic development level has different impacts on different ESs, that is, gross domestic product (GDP) per capita has a high, positive correlation with the recreational service value, but has no correlation with the habitat service value.

MyD88 in myofibroblasts regulates aerobic glycolysis-driven hepatocarcinogenesis via ERK-dependent PKM2 nuclear relocalization and activation.

Hepatic stellate cells (HSCs) and cancer-associated fibroblasts (CAFs) play critical roles in liver fibrosis and hepatocellular carcinoma (HCC). MyD88 controls the expression of several key modifier genes in liver tumorigenesis; however, whether and how MyD88 in myofibroblasts contributes to the development of fibrosis-associated liver cancer remains elusive. Here, we used an established hepatocarcinogenesis mouse model involving apparent liver fibrogenesis in which MyD88 was selectively depleted in myofibroblasts. Myofibroblast MyD88-deficient (Fib-MyD88 KO) mice developed significantly fewer and smaller liver tumor nodules. MyD88 deficiency in myofibroblasts attenuated liver fibrosis and aerobic glycolysis in hepatocellular carcinoma tissues. Mechanistically, MyD88 signaling in myofibroblasts increased the secretion of CCL20, which promoted aerobic glycolysis in cancer cells. This process was dependent on the CCR6 receptor and ERK/PKM2 signaling. Furthermore, liver tumor growth was greatly relieved when the mice were treated with a CCR6 inhibitor. Our data revealed a critical role for MyD88 in myofibroblasts in the promotion of hepatocellular carcinoma by affecting aerobic glycolysis in cancer cells and might provide a potential molecular therapeutic target for HCC. © 2021 The Pathological Society of Great Britain and Ireland.

Silencing of SBF2-AS1 inhibits cell growth and invasion by sponging microRNA-338-3p in serous ovarian carcinoma.

Long noncoding RNA SET-binding factor 2 (SBF2) antisense RNA 1 (AS1) is associated with the growth and metastasis of multiple cancer types, but its biological roles in serous ovarian carcinoma (SOC) remain unclear. In this study, the aberrant upregulation of SBF2-AS1 is detected in SOC after analysis of differentially expressed genes between SOC tissues and normal fallopian tubes from the public Gene Expression Omnibus (GEO) database. We determine that knockdown of SBF2-AS1 inhibits SOC cell proliferation and invasion by sponging miR-338-3p. MiR-338-3p acts as a tumor suppressor in SOC, and E26 transformation specific-1 (ETS1) is identified as a potential target of miR-338-3p regulation. Furthermore, SBF2-AS1 could modulate ETS1 by operating as a competing endogenous RNA for miR-338-3p. This finding elucidates a new mechanism for SBF2-AS1 in SOC development and provides a potential target for SOC therapeutic intervention.

Grassland ecological compensation policy in China improves grassland quality and increases herders' income.

Many countries have undertaken large and high-profile payment-for-ecosystem-services (PES) programs to sustain the use of their natural resources. Nevertheless, few studies have comprehensively examined the impacts of existing PES programs. Grassland Ecological Compensation Policy (GECP) is one of the few pastorally focused PES programs with large investments and long duration, which aim to improve grassland quality and increase herder income. Here we present empirical evidence of the effects of GECP on grassland quality and herder income. Through a thorough and in-depth econometric analysis of remote sensing and household survey data, we find that, although GECP improves grassland quality (albeit to only a small extent) and has a large positive effect on income, it exacerbates existing income inequality among herders within their local communities. The analysis demonstrates that the program has induced herders to change their livestock production behavior. Heterogeneity analysis emphasizes the importance of making sure the programs are flexible and are adapted to local resource circumstances.

Combining Protein Content and Grain Yield by Genetic Dissection in Bread Wheat under Low-Input Management.

The simultaneous improvement of protein content (PC) and grain yield (GY) in bread wheat (Triticum aestivum L.) under low-input management enables the development of resource-use efficient varieties that combine high grain yield potential with desirable end-use quality. However, the complex mechanisms of genotype, management, and growing season, and the negative correlation between PC and GY complicate the simultaneous improvement of PC and GY under low-input management. To identify favorable genotypes for PC and GY under low-input management, this study used 209 wheat varieties, including strong gluten, medium-strong gluten, medium gluten, weak gluten, winter, semi-winter, weak-spring, and spring types, which has been promoted from the 1980s to the 2010s. Allelic genotyping, performed using kompetitive allele-specific polymerase chain reaction (KASP) technology, found 69 types of GY-PC allelic combinations in the tested materials. Field trials were conducted with two growing season treatments (2018-2019 and 2019-2020) and two management treatments (conventional management and low-input management). Multi-environment analysis of variance showed that genotype, management, and growing season had extremely substantial effects on wheat GY and PC, respectively, and the interaction of management × growing season also had extremely significant effects on wheat GY. According to the three-sigma rule of the normal distribution, the GY of wheat varieties Liangxing 66 and Xinmai 18 were stable among the top 15.87% of all tested materials with high GY, and their PC reached mean levels under low-input management, but also stably expressed high GY and high PC under conventional management, which represents a great development potential. These varieties can be used as cultivars of interest for breeding because TaSus1-7A, TaSus1-7B, TaGW2-6A, and TaGW2-6B, which are related to GY, and Glu-B3, which is related to PC, carry favorable alleles, among which Hap-1/2, the allele of TaSus1-7A, and Glu-B3b/d/g/i, the allele of Glu-B3, can be stably expressed. Our results may be used to facilitate the development of high-yielding and high-quality wheat varieties under low-input management, which is critical for sustainable food and nutrition security.

Comprehensive profiling of circular RNAs with nanopore sequencing and CIRI-long.

Reconstructing the sequence of circular RNAs (circRNAs) from short RNA sequencing reads has proved challenging given the similarity of circRNAs and their corresponding linear messenger RNAs. Previous sequencing methods were unable to achieve high-throughput detection of full-length circRNAs. Here we describe a protocol for enrichment and full-length sequencing of circRNA isoforms using nanopore technology. Circular reverse transcription and size selection achieves a 20-fold higher enrichment of circRNAs from total RNA compared to previous methods. We developed an algorithm, called circRNA identifier using long-read sequencing data (CIRI-long), to reconstruct the sequence of circRNAs. The workflow was validated with simulated data and by comparison to Illumina sequencing as well as quantitative real-time RT-PCR. We used CIRI-long to analyze adult mouse brain samples and systematically profile circRNAs, including mitochondria-derived and transcriptional read-through circRNAs. We identified a new type of intronic self-ligated circRNA that exhibits special splicing and expression patterns. Our method takes advantage of nanopore long reads and enables unbiased reconstruction of full-length circRNA sequences.