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Direct canopy uptake of atmospheric reactive nitrogen (ARN) is an important process, but the magnitude of ARN assimilation in agricultural ecosystems is unclear. We used a combination of a water-culture rice-growing system with a

Colorectal cancer (CRC) is among the most frequent forms of cancer, and new strategies for its prevention and therapy are urgently needed

bRationale:/bDysregulation of signaling that governs self-renewal and differentiation of intestinal stem cells (ISCs) is a major cause of colorectal cancer (CRC) initiation and progression.bMethods:/bqRT-PCR, western blotting,iin situ/ihybridization, immunohistochemistry and immunofluorescence assays were used to detect the expression levels of MEX3A, KLF4 and E2F3 in CRC tissues. The biological functions of MEX3A were studied usingiMex3a/iknockout (KO) and intestinal epithelium specific conditional knockout (cKO) mice, AOM-DSS mouse colorectal tumor model, Apc floxed mouse tumor model and intestinal and tumor organoids. Transcriptomic RNA sequencing (RNA-seq), RNA crosslinking immunoprecipitation (CLIP) and luciferase reporter assays were performed to explore the molecular mechanisms of MEX3A.bResults:/bRNA-binding protein MEX3A, a specific ISC marker gene, becomes ectopically upregulated upon CRC and its levels negatively correlate with patient survival prognosis. MEX3A functions as an oncoprotein that retains cancer cells in undifferentiated and proliferative status and it enhances their radioresistance to DNA damage. Mechanistically, a rate limiting factor of cellular proliferation E2F3 induces MEX3A, which in turn activates WNT pathway by directly suppressing expression of its pro-differentiation transcription factor KLF4. Knockdown ofiMEX3A/iwith siRNA or addition of KLF4 agonist significantly suppressed tumor growth both by increasing differentiation status of cancer cells and by suppressing their proliferation.bConclusions:/bIt identifies E2F3-MEX3A-KLF4 axis as an essential coordinator of cancer stem cell self-renewal and differentiation, representing a potent new druggable target for cancer differentiation therapy.

SummaryThe initiation and progression of cancer are inextricably linked to the tumor microenvironment (TME). Understanding the function of specific cancer-TME interactions poses a major challenge due in part to the complexity of the in vivo microenvironment. Here we predict cancer-TME interactions from single cell transcriptomic maps of both human colorectal cancers (CRCs) and mouse CRC models, ask how these interactions are altered in established, long-term human tumor organoid (tumoroid) cultures, and functionally recapitulate human myeloid-carcinoma interactions in vitro. Tumoroid cultures suppress gene expression programs involved in promoting inflammation and immune cell migration through receptor-ligand interactions, providing a reductive platform for re-establishing carcinoma-immune cell interactions in vitro. Introduction of human monocyte-derived macrophages into tumoroid cultures instructs macrophages to acquire pro-tumorigenic gene expression programs similar to those observed in vivo. This includes hallmark induction of SPP1, encoding Osteopontin, an extracellular CD44 ligand with established oncogenic effects. Taken together, these findings offer a framework for understanding CRC-TME interactions and provide a reductionist tool for modeling specific aspects of these interactions.

The intestinal epithelium exhibits a rapid and efficient regenerative response to injury. Emerging evidence supports a model where plasticity of differentiated cells, particularly those in the secretory lineages, contributes to epithelial regeneration upon ablation of injury-sensitive stem cells. However, such facultative stem cell activity is rare within secretory populations. Here, we ask whether specific functional properties predict facultative stem cell activity. We utilize in vivo labeling combined with ex vivo organoid formation assays to evaluate how cell age and autophagic state contribute to facultative stem cell activity within secretory lineages. Strikingly, we find that cell age (time elapsed since cell cycle exit) does not correlate with secretory cell plasticity. Instead, high autophagic vesicle content predicts plasticity and resistance to DNA damaging injury independently of cell lineage. Our findings indicate that autophagic status prior to injury serves as a lineage-agnostic marker for the prospective identification of facultative stem cells.

Colorectal cancer (CRC) is a leading cause of cancer-related deaths globally, with the majority of cases initiated by inactivation of the APC tumor suppressor. This results in the constitutive transcriptional activation of the canonical WNT signal transduction pathway effector β-Catenin, along with induction of WNT feedback inhibitors, including the extracellular palmitoleoyl-protein carboxylesterase NOTUM. Here, we show that NOTUM retains cell-autonomous tumor suppressive activity in APC-null adenomatous lesions despite constitutive β-Catenin activation. Strikingly, we find that NOTUM becomes an obligate oncogene upon subsequent P53 inactivation during the adenoma-adenocarcinoma transition, and that these phenotypes are WNT-independent, resulting from differential activity of NOTUM upon its enzymatic targets Glypican 1 and 4 in early vs. late-stage disease, respectively. Ultimately, preclinical mouse models of CRC and human tumoroid cultures demonstrate that pharmacological inhibition of NOTUM is highly effective in arresting primary adenocarcinoma growth and inhibiting metastatic colonization of distal organs. The finding that a single agent targeting an extracellular enzyme is effective in treating highly aggressive tumors make NOTUM a novel therapeutic vulnerability in advanced colorectal adenocarcinomas.

Background: Brown adipose tissue (BAT) dissipates chemical energy as heat and has the potential to be a protective strategy to prevent obesity. microRNAs (miRNAs) are emerging as important posttranscriptional factors affecting the thermogenic function of BAT. However, the regulatory mechanism underlying miRNA-mediated energy metabolism in BAT is not fully understood. Here, we explored the roles of miR-22 in BAT thermogenesis and energy metabolism. Methods: Using global and conditional knockout mice as in vivo models and primary brown adipocytes as an in vitro system, we investigated the function of miR-22 in BAT thermogenesis in vivo and in vitro. Results: miR-22 expression was upregulated in BAT in response to cold exposure and during brown preadipocyte differentiation. Both global and conditional knockout mice displayed BAT whitening, impaired cold tolerance, and decreased BAT thermogenesis. Moreover, we found that miR-22 deficiency impaired BAT glycolytic capacity, which is critical for thermogenesis. The mechanistic results revealed that miR-22 activated the mTORC1 signaling pathway by directly suppressing Tsc1 and concomitantly directly suppressing Hif1an, an inhibitor of Hif1α, which promotes glycolysis and maintains thermogenesis. Conclusions: Our findings identify miR-22 as a critical regulator in the control of thermogenesis in BAT and as a potential therapeutic target for human metabolic disorders.

Mammary and extramammary Paget's Diseases (PD) are a malignant skin cancer characterized by the appearance of Paget cells. Although easily diagnosed, its pathogenesis remains unknown. Here, single-cell RNA-sequencing identified distinct cellular states, novel biomarkers, and signaling pathways - including mTOR, associated with extramammary PD. Interestingly, we identified MSI1 ectopic overexpression in basal epithelial cells of human PD skin, and show that Msi1 overexpression in the epidermal basal layer of mice phenocopies human PD at histopathological, single-cell and molecular levels. Using this mouse model, we identified novel biomarkers of Paget-like cells that translated to human Paget cells. Furthermore, single-cell trajectory, RNA velocity and lineage-tracing analyses revealed a putative keratinocyte-to-Paget-like cell conversion, supporting the in situ transformation theory of disease pathogenesis. Mechanistically, the Msi1-mTOR pathway drives keratinocyte-Paget-like cell conversion, and suppression of mTOR signaling with Rapamycin significantly rescued the Paget-like phenotype in Msi1-overexpressing transgenic mice. Topical Rapamycin treatment improved extramammary PD-associated symptoms in humans, suggesting mTOR inhibition as a novel therapeutic treatment in PD.

Background Colorectal cancer (CRC) is the third leading cause of cancer in the United States, and inflammatory bowel disease patients have an increased risk of developing CRC due to chronic intestinal inflammation with it being the cause of death in 10% to 15% of inflammatory bowel disease patients. TIPE2 (TNF-alpha-induced protein 8-like 2) is a phospholipid transporter that is highly expressed in immune cells and is an important regulator of immune cell function. Methods The azoxymethane/dextran sulfate sodium murine model of colitis-associated colon cancer (CAC) was employed in Tipe2 –/– and wild-type mice, along with colonoid studies, to determine the role of TIPE2 in CAC. Results Early on, loss of TIPE2 led to significantly less numbers of visible tumors, which was in line with its previously described role in myeloid-derived suppressor cells. However, as time went on, loss of TIPE2 promoted tumor progression, with larger tumors appearing in Tipe2 –/– mice. This was associated with increased interleukin-22/STAT3 phosphorylation signaling. Similar effects were also observed in primary colonoid cultures, together demonstrating that TIPE2 also directly regulated colonocytes in addition to immune cells. Conclusions This work demonstrates that TIPE2 has dual effects in CAC. In the colonocytes, it works as a tumor suppressor. However, in the immune system, TIPE2 may promote tumorigenesis through suppressor cells or inhibit it through IL-22 secretion. Going forward, this work suggests that targeting TIPE2 for CRC therapy requires cell- and pathway-specific approaches and serves as a cautionary tale for immunotherapy approaches in general in terms of colon cancer, as intestinal inflammation can both promote and inhibit cancer.

Colorectal cancer (CRC) is the 2nd-leading cause of cancer-related deaths worldwide and metastatic disease remains a major unmet clinical need. Nevertheless, the molecular underpinnings of metastatic spread and colonization of distant sites remain elusive. To systematically identify functional genes contributing to metastasis in a physiologically relevant model of CRC, we developed a novel platform enabling robust, pooled CRISPR/Cas9 screening in primary colonic organoids engineered with common CRC driver mutations in Apc, Kras, and Tp53. We performed parallel in vitro invasion and in vivo metastasis gain-of-function CRISPR/Cas9 knockout screens with high-content gRNA libraries to uncover novel suppressors of metastasis. We confirmed resulting hits as bona-fide metastatic suppressors using both pooled validation screens and single-gene approaches. Genes scoring in both in vitro and in vivo screens included previously identified regulators of migration and invasion, such as Ctnna1, and genes scoring only in the in vivo screen included novel negative regulators of metastasis such as Bcl2l13. These hits in the murine system were supported by human TCGA data, indicating that low expression of these genes is associated with worse 5-year survival in colon and rectal adenocarcinoma patients (COAD and READ). Utilizing transwell and live-imaging assays, we found that sgRNAs targeting Ctnna1 markedly enhanced invasion and migration of CRC organoids while sgRNAs targeting Bcl2l13 had no effect, an observation consistent with our in vitro screen. Therefore, while loss of Ctnna1 enhances metastasis by augmenting invasion and migration, loss of Bcl2l13 acts through alternative mechanisms. Single cell RNA sequencing of primary and metastatic lesions derived from Bcl2l13 knockout and control CRC tumors identified novel putative pathways promoting metastatic spread via migration-independent mechanisms, including upregulation of hypoxia signalling and recruitment of pro-tumorigenic macrophages. Thus, our CRISPR/Cas9 screening pipeline is a viable approach for the identification of novel regulators of CRC metastasis in vitro and in vivo, uncovering both motility and migration-dependent and independent paths to metastatic success. These findings will help untangle the complex web of molecular events underlying CRC metastasis and provide clinically-relevant insights into the process of human CRC metastasis. Citation Format: Zvi Cramer, Xin Wang, Nicolae Adrian Leu, Keara Monaghan, Joshua H. Rhoades, Yuhua Tian, Joshua Rico, Diego Mendez, Ricardo Petroni, Melissa S. Kim, Ning Li, Christopher J. Lengner, M. Andres Blanco. In vitro and in vivo CRISPR screens in colorectal cancer organoids identify suppressors of metastasis [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr PR004.

Patients with dyskeratosis congenita (DC) and related telomeropathies resulting from premature telomere dysfunction suffer from multi-organ failure. In the liver, DC patients present with nodular hyperplasia, steatosis, inflammation, and cirrhosis. We model DC liver pathologies using isogenic human induced pluripotent stem (iPS) cells harboring a causal DC mutation in DKC1, or a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-corrected control allele. Differentiation of these iPS cells into hepatocytes or hepatic stellate cells followed by generation of genotype-admixed hepatostellate organoids revealed a dominant phenotype in the parenchyma, with DC hepatocytes eliciting a pathogenic hyperplastic response in stellate cells independent of stellate cell genotype. Pathogenic phenotypes could be rescued via suppression of AKT activity, a central regulator of MYC-driven hyperplasia downstream of DKC1 mutation. Thus, isogenic iPS-derived admixed hepatostellate organoids offer insight into the liver pathologies in telomeropathies and provide a framework for evaluating emerging therapies.

Positive Allosteric Modulators Transient receptor potential vanilloid 1 (TRPV1) ion channel is critically involved in pain sensation. Positive allosteric modulators (PAMs) of TRPV1 are de novo designed based on the structure of TRPV1. Surface plasmon resonance and patch‐clamp recording demonstrate that the PAMs bind to TRPV1 and allosterically enhance its activities. The PAM exhibits analgesic effects without changing body temperature in rats. More details can be found in article number 2101716 by Yuhua Tian, Jie Zheng, Fan Yang, and co‐workers. [Image: see text]

MicroRNA-mediated post-transcriptional regulation plays key roles in stem cell self-renewal and tumorigenesis. However, the in vivo functions of specific microRNAs in controlling mammary stem cell (MaSC) activity and breast cancer formation remain poorly understood. Here we show that miR-31 is highly expressed in MaSC-enriched mammary basal cell population and in mammary tumors, and is regulated by NF-κB signaling. We demonstrate that miR-31 promotes mammary epithelial proliferation and MaSC expansion at the expense of differentiation in vivo. Loss of miR-31 compromises mammary tumor growth, reduces the number of cancer stem cells, as well as decreases tumor-initiating ability and metastasis to the lung, supporting its pro-oncogenic function. MiR-31 modulates multiple signaling pathways, including Prlr/Stat5, TGFβ and Wnt/β-catenin. Particularly, it activates Wnt/β-catenin signaling by directly targeting Wnt antagonists, including Dkk1. Importantly, Dkk1 overexpression partially rescues miR31-induced mammary defects. Together, these findings identify miR-31 as the key regulator of MaSC activity and breast tumorigenesis.

The purpose of this study is to investigate the role of the long noncoding RNA metastasis associated lung adenocarcinoma transcript 2 (MALAT2) in the prognosis of stage II/III gastric cancer (GC) patients. The expression of MALAT2 was evaluated in cancer tissues from 146 stage II/III GC patients undergoing radical resection and 60 paired normal samples using quantitative real-time reverse transcriptase PCR. Differences in the expression of MALAT2 between 23 GC and paired normal colonic mucosa samples were analysed with the Wilcoxon test. Relationships between the expression level of MALAT2, patient clinico-pathological parameters and disease-free survival and overall survival were analysed using the uni-variate Kaplan-Meier method and the multivariate COX regression model. The quantitative polymerase chain reaction results showed that MALAT2 was frequently over-expressed in cancer tissues and this over-expression was found to significantly correlate with lymph node metastasis and tumor stage. The ectopic expression of MALAT2 contributed to the migration of human GC SGC-7901 cells, whereas knockdown of MALAT2 inhibited the migration of the SGC-7901 cells in vitro. Further investigation into the underlying mechanisms responsible for the migratory effects revealed that MALAT2 induced the epithelial-mesenchymal transition (EMT) through an MEK/extracellular signal-regulated kinase-dependent mechanism as treatment with the MEK inhibitor, U0126, decreased migration and reversed the EMT in the MALAT2 over-expressed SGC-7901 cells. The expression of MALAT2 is upregulated in GC tissues, and a higher expression level of MALAT2 might serve as a negative prognostic marker in stage II/III GC patients which implies the potential application of MALAT2 in the therapeutic treatment of GC.Cancer Gene Therapy advance online publication, 27 February 2015; doi:10.1038/cgt.2015.6.

The nociceptive transient receptor potential vanilloid 1 (TRPV1) ion channel is a polymodal receptor for multiple painful stimuli, hence actively pursued as a target for analgesic drugs. We identified a small peptide toxin RhTx2 from the Chinese red-headed centipede that strongly modulates TRPV1 activities. RhTx2, a 31-amino-acid peptide, is similar to a TRPV1-activating toxin RhTx we have previously discovered but with four extra amino acids at the N terminus. We observed that, like RhTx, RhTx2 activated TRPV1, but RhTx2 rapidly desensitized the channel upon prolonged exposure. Desensitization was achieved by reducing both the open probability and the single-channel conductance. RhTx2 is not only a tool to study the desensitization mechanism of TRPV1, but also a promising starting molecule for developing novel analgesics.

The Hippo-YAP signaling pathway plays an essential role in epithelial cells during intestinal regeneration and tumorigenesis. However, the molecular mechanism linking stromal signals to YAP-mediated intestinal regeneration and tumorigenesis is poorly defined. Here, we report a stroma-epithelium ISLR-YAP signaling axis essential for stromal cells to modulate epithelial cell growth during intestinal regeneration and tumorigenesis. Specifically, upon inflammation and in cancer, an oncogenic transcription factor ETS1 in stromal cells induces expression of a secreted protein ISLR that can inhibit Hippo signaling and activate YAP in epithelial cells. Deletion of Islr in stromal cells in mice markedly impaired intestinal regeneration and suppressed tumorigenesis in the colon. Moreover, the expression of stromal cell-specific ISLR and ETS1 significantly increased in inflamed mucosa of human IBD patients and in human colorectal adenocarcinoma, accounting for the epithelial YAP hyperactivation. Collectively, our findings provide new insights into the signaling crosstalk between stroma and epithelium during tissue regeneration and tumorigenesis.

The transient receptor potential vanilloid 1 (TRPV1) channel is a heat-activated cation channel that plays a crucial role in ambient temperature detection and thermal homeostasis. Although several structural features of TRPV1 have been shown to be involved in heat-induced activation of the gating process, the physiological significance of only a few of these key elements has been evaluated in an evolutionary context. Here, using transient expression in HEK293 cells, electrophysiological recordings, and molecular modeling, we show that the pore turret contains both structural and functional determinants that set the heat activation thresholds of distinct TRPV1 orthologs in mammals whose body temperatures fluctuate widely. We found that TRPV1 from the bat Carollia brevicauda exhibits a lower threshold temperature of channel activation than does its human ortholog and three bat-specific amino acid substitutions located in the pore turret are sufficient to determine this threshold temperature. Furthermore, the structure of the TRPV1 pore turret appears to be of physiological and evolutionary significance for differentiating the heat-activated threshold among species-specific TRPV1 orthologs. These findings support a role for the TRPV1 pore turret in tuning the heat-activated threshold, and they suggest that its evolution was driven by adaption to specific physiological traits among mammals exposed to variable temperatures.

In China, the current high reliance on chemical nitrogen (N) in agriculture has resulted in low N use efficiency and high N loss. Reducing N fertilizer input is necessary for environmental protection, but it has not been attractive to Chinese farmers because of insufficient increase in net income. Reduced use of N fertilizer might be attractive if reduced N combined with organic fertilizer could increase rice yield. Therefore, a three-year field trial and a 15N micro-plot experiment were conducted to study the effect of organic fertilizer combined with chemical N on rice yield, NH3 loss and the fate of 15N in an intensive rice cropping system in Taihu Lake region. Six treatments were used: control (0 N, zero N application), organic N alone (OrgN, 41 kg N ha−1), local farmers’ N practice (FN, 300 kg N ha−1), reduced N (RN, 225 kg N ha−1), organic fertilizer combined with FN (FN + OrgN) and organic fertilizer combined with RN (RN + OrgN). Compared to chemical N, organic fertilizer markedly increased soil organic matter content, promoted grain N accumulation, and improved rice production. Organic fertilizer treatments showed 11%–13% higher yield and 4%–5% higher net economic benefit over FN for three years. Organic fertilizer produced high NH4+ in the floodwater and increased NH3 loss. Although FN + OrgN had significantly higher 15N-NH3 loss than FN, it increased soil residual 15N and decreased 15N loss by 29%. There was no significant difference of yield and NEB between FN + OrgN and RN + OrgN, while RN + OrgN was superior to FN + OrgN because of the lower NH3 loss and higher N recovery efficiency (NRE). Therefore, organic fertilizer is an economically attractive practice to increase NRE and rice yield without increasing chemical N input.

Wound healing is essential for skin repair after injury, and it consists of hemostasis, inflammation, re-epithelialization, and remodeling phases. Successful re-epithelialization, which relies on proliferation and migration of epidermal keratinocytes, requires a reduction in tissue inflammation. Therefore, understanding the molecular mechanism underlying the transition from inflammation to re-epithelialization will help to better understand the principles of wound healing. Currently, the in vivo functions of specific microRNAs in wound healing are not fully understood. We observed that miR-31 expression is strongly induced in wound edge keratinocytes, and is directly regulated by the activity of NF-κB and signal transducer and activator of transcription 3 signaling pathways during the inflammation phase. We used miR-31 loss-of-function mouse models to demonstrate that miR-31 promotes keratinocyte proliferation and migration. Mechanistically, miR-31 activates the Ras/mitogen-activated protein kinase signaling by directly targeting Rasa1, Spred1, Spred2, and Spry4, which are negative regulators of the Ras/mitogen-activated protein kinase pathway. Knockdown of these miR-31 targets at least partially rescues the delayed scratch wound re-epithelialization phenotype observed in vitro in miR-31 knockdown keratinocytes. Taken together, these findings identify miR-31 as an important cell-autonomous mediator during the transition from inflammation to re-epithelialization phases of wound healing, suggesting a therapeutic potential for miR-31 in skin injury repair.

Reducing environmental harm and improving fertilizer use efficiency is critical in Chinese agriculture. Azolla has particular value for paddy rice crop due to its biological nitrogen (N) fixation ability; however, the Chinese rice fields are highly fertilized, which will depress the N fixation of Azolla. Urea deep placement (UDP) reduces floodwater NH4+ and ammonia volatilization losses. To date, few studies have explored the effects of UDP combined with Azolla on the N uptake pattern, N recovery and N loss from 15N-labeled urea. Therefore, a 3-year field experiment was conducted with five treatments (control without urea (CK), urea broadcasting without Azolla (B) or with Azolla (BA), and UDP without Azolla (P) or with Azolla (PA)) in an intensive rice cropping system in the Taihu Region of China. The total 15N recovery (soil-plant system), crop 15N uptake (straw and grain) and 15N loss were assessed, and the ammonia (NH3) loss, rice yield, gross return and net economic benefit were measured. The results showed that the UDP treatments had delayed but long-lasting fertilization effects. The PA treatment resulted in the lowest 15N loss and highest total 15N recovery at harvest due to the prolonged N availability and negligible NH3 loss. The PA, P and BA treatments decreased 15N loss by 47%, 33% and 18% and increased total 15N recovery by 58%, 40% and 22%, respectively, compared to urea broadcasting alone. Almost all labeled Azolla-N was released within two months of which approximately 40% could be absorbed by the crop. Little labeled fertilizer N was absorbed by Azolla during the rice season. In addition, the PA, P and BA treatments increased rice yield and gross return by 13%, 10% and 9%, respectively, and they produced similar net economic benefit compared to urea broadcasting alone. In conclusion, UDP combined with Azolla is a potential strategy to further reduce fertilizer N loss and improve total fertilizer N recovery while maximizing rice yield if it can be widely adopted by Chinese rice farmers. However, widespread adoption will require improved technologies with lower labour requirement for mechanized deep placement of urea and for the propagation of Azolla.

It is urgently necessary to reduce environmental harm while simultaneously ensuring food security from agriculture in China. Fertilizer deep placement, especially urea deep placement (UDP), has been widely recognized as an efficient way to increase rice yield with less N use in flooded rice fields; however, few studies have explored UDP as a way to abate N loss, particularly ammonia (NH3) volatilization, in intensive rice cropping systems. Therefore, a field experiment was performed in the Taihu Region with five treatments (two surface split broadcasting treatments: a current traditional practice with 300 kg N ha−1 (CT) and a reduced N practice with 225 kg N ha−1 (RN), two one-time UDP treatments: a current traditional practice under UDP (CTDP) and a reduced N practice under UDP (RNDP), and a CK treatment with no urea). The NH3 volatilization, grain yield and N use efficiency in terms of the N recovery efficiency (NRE) were investigated during the 2014–2016 rice growing seasons. Additionally, N diffusion and 15N-labeled urea experiments were conducted to explore the N release pattern and the fate of 15N under UDP. The results demonstrated that little NH4+-N could diffuse into surface water under UDP, thus, negligible floodwater NH4+-N was detected. The seasonally cumulative NH3 volatilization of the UDP accounted for only 1% of the total applied N, which decreased by 91% compared to surface broadcasting treatments. As a result, the NH3 intensity (NH3-N loss per crop yield) was reduced by 92% over surface broadcasting. Moreover, the deep placement of urea could supply a higher NH4+-N concentration in the soil during the early growth stage and prolong the duration of N availability for 2 months; as a result, UDP remarkably increased the N uptake by 28% compared with surface broadcasting treatments in the 3 years. The UDP treatments significantly increased the yield and NRE by 10% and 55% under favorable weather conditions (2015 and 2016 rice seasons), respectively. The RNDP treatment obtained the lowest N surplus (36 kg N ha−1), and the plant 15N uptake was 62% higher and the 15N loss was 38% lower with RNDP than surface broadcasting. Therefore, the deep placement of urea can be considered a slow release fertilizer, which better matches the N demand of rice plants and effectively minimizes N losses, especially NH3 volatilization. The RNDP treatment, with a 25% reduction in the N dose, can represent an effective and promising strategy to achieve environmental integrity and food security in intensive rice cropping systems.

The efficient use of nitrogen (N) in crop production is critical for meeting the challenges of food security and environmental integrity. Azolla biofertilizer may be a promising approach to achieve better N use efficiency (NUE) in paddy rice fields due to its great potential for biological N fixation (BNF). The objective of this study was to determine the efficacy of partially substituting Azolla biofertilizer for synthetic N fertilizer to improve NUE, reduce N loss and enhance rice yield in the currently highly fertilized rice cropping systems of China. Therefore, a 3-year field experiment was conducted with five treatments: CK (control without urea), FN (farmers’ N practice), FNA (the farmers’ N combined with Azolla biofertilizer), RN (reducing farmers’ N by 25%) and RNA (substituting Azolla biofertilizer for 25% farmers’ N). The NUE, ammonia (NH3) volatilization, rice yield and net economic benefit (the difference between the value of the harvest grain and the costs of fertilizer and Azolla inputs) were assessed. The results showed that in the RNA and FNA treatments, Azolla biofertilizer produced higher recovery efficiency of fertilizer N by 69% and 59%, provided higher agronomic N use efficiency by 52% and 31% and achieved higher partial factor productivity of applied N by 43% and 13% than FN for the 3 years, respectively. In addition, the RNA and FNA treatments achieved crop 15N recovery that was 64% and 49% higher than the FN treatment, respectively. The improved NUE under the Azolla biofertilizer treatments were attributed to reduced N loss and enhanced N uptake by rice plants. The RNA and FNA treatments significantly reduced 15N loss by 48% and 26%, as well as lowered NH3 loss by 42% and 12% over FN, respectively. In addition, Azolla could fix 52 and 44 kg N ha−1 crop−1 in the RNA and FNA treatments, and thereby, Azolla biofertilizer resulted in higher N uptake that was 17% and 33% higher in the RNA and FNA groups than in FN, respectively. As a result, the RNA and FNA treatments achieved higher rice yield by 8% and 14% over FN, respectively, but they attained similar and higher net economic benefit over FN for the 3 years. Therefore, substituting Azolla biofertilizer for 25% of urea-N provides a financially attractive option for farmers to substantially improve NUE and yield and effectively reduce N loss in intensive rice cropping systems.

Increasing rice production to feed the world’s growing population while protecting the environment requires more optimal use of fertilizers. In China, the current high input, high output and high reliance on synthetic nitrogen (N) fertilizer in agriculture has resulted in high N losses, especially ammonia (NH 3 ) emission. Urea combined with green manure (GM) might be a promising approach to improve N fertilizer management. However, few studies have evaluated duckweed in this manner. Duckweed does not require arable land for cultivation and thus avoids competition with food crops. Therefore, a field experiment was conducted for three years with five treatments (CK, no N-fertilizer; CT, conventional practice, urea alone at 300 kg N ha −1 ; CTD, urea combined with duckweed at 300 kg N ha −1 ; RN, urea alone at 225 kg N ha −1 ; and RND, urea combined with duckweed at 225 kg N ha −1 ) in an intensive rice cropping system in the Taihu Region of China. The results for two years showed that urea combined with duckweed cover reduced NH 3 loss by 36–52% over CT. This reduction was attributed primarily to the formation of a physical barrier and the uptake of NH 4 + by duckweed. The 15 N recovery for 15 N balance conducted for one year was 38% higher and the 15 N loss was 16% lower for CTD than that of CT. Furthermore, urea combined with duckweed increased N accumulation in the aboveground plants by 14–25% over CT for the 3 years. As a result, urea combined with duckweed achieved higher rice yield by 9–10%, and higher net economic benefit by 10–11% over CT for the 3 years; however, using the conventional rate of 300 kg N ha −1 did not increase rice yield over using the reduced N rate of 225 kg N ha −1 , with or without duckweed. Thus, duckweed as GM combined with chemical fertilizer application provided an approach for increasing the rice yield without increasing inputs of N fertilizer and thereby provided a financially attractive option for farmers to achieve environmental integrity and ensure food security in rice production.

Transient receptor potential vanilloid 1 (TRPV1) ion channel serves as the detector for noxious temperature above 42 °C, pungent chemicals like capsaicin, and acidic extracellular pH. This channel has also been shown to function as an ionotropic cannabinoid receptor. Despite the solving of high-resolution three-dimensional structures of TRPV1, how endocannabinoids such as anandamide and N-arachidonoyl dopamine bind to and activate this channel remains largely unknown. Here we employed a combination of patch-clamp recording, site-directed mutagenesis, and molecular docking techniques to investigate how the endocannabinoids structurally bind to and open the TRPV1 ion channel. We found that these endocannabinoid ligands bind to the vanilloid-binding pocket of TRPV1 in the "tail-up, head-down" configuration, similar to capsaicin; however, there is a unique interaction with TRPV1 Y512 residue critical for endocannabinoid activation of TRPV1 channels. These data suggest that a differential structural mechanism is involved in TRPV1 activation by endocannabinoids compared with the classic agonist capsaicin.

Transient receptor potential vanilloid 1 (TRPV1) ion channel is a nociceptor critically involved in pain sensation. Direct blockade of TRPV1 exhibits significant analgesic effects but also incurs severe side effects such as hyperthermia, causing failures of TRPV1 inhibitors in clinical trials. In order to selectively target TRPV1 channels that are actively involved in pain‐sensing, peptidic positive allosteric modulators (PAMs) based on the high‐resolution structure of the TRPV1 intracellular ankyrin‐repeat like domain are de novo designed. The hotspot centric approach is optimized for protein design; its usage in Rosetta increases the success rate in protein binder design. It is demonstrated experimentally, with a combination of fluorescence resonance energy transfer (FRET) imaging, surface plasmon resonance, and patch‐clamp recording, that the designed PAMs bind to TRPV1 with nanomolar affinity and allosterically enhance its response to ligand activation as it is designed. It is further demonstrated that the designed PAM exhibits long‐lasting in vivo analgesic effects in rats without changing their body temperature, suggesting that they have potentials for developing into novel analgesics., Transient receptor potential vanilloid 1 (TRPV1) ion channel is critically involved in pain sensation. Positive allosteric modulators (PAMs) of TRPV1 are de novo designed based on the structure of TRPV1. Surface plasmon resonance and patch‐clamp recording demonstrate that the PAMs bind to TRPV1 and allosterically enhance its activities. The PAM exhibits analgesic effects without changing body temperature in rats.

Sensing noxiously high temperatures is crucial for living organisms to avoid heat-induced injury. The TRPV1 channel has long been known as a sensor for noxious heat. However, the mechanism of how this channel is activated by heat remains elusive. Here we found that a series of polyols including sucrose, sorbitol, and hyaluronan significantly elevate the heat activation threshold temperature of TRPV1. The modulatory effects of these polyols were only observed when they were perfused extracellularly. Interestingly, mutation of residues E601 and E649 in the outer pore region of TRPV1 largely abolished the effects of these polyols. We further observed that intraplantar injection of polyols into the hind paws of rats reduced their heat-induced pain response. Our observations not only suggest that the extracellular regions of TRPV1 are critical for the modulation of heat activation by polyols, but also indicate a potential role of polyols in reducing heat-induced pain sensation.

MicroRNA-mediated post-transcriptional regulation plays key roles in stem cell self-renewal and tumorigenesis. However, the in vivo functions of specific microRNAs in controlling mammary stem cell (MaSC) activity and breast cancer formation remain poorly understood. Here we show that miR-31 is highly expressed in MaSC-enriched mammary basal cell population and in mammary tumors, and is regulated by NF-κB signaling. We demonstrate that miR-31 promotes mammary epithelial proliferation and MaSC expansion at the expense of differentiation in vivo. Loss of miR-31 compromises mammary tumor growth, reduces the number of cancer stem cells, as well as decreases tumor-initiating ability and metastasis to the lung, supporting its pro-oncogenic function. MiR-31 modulates multiple signaling pathways, including Prlr/Stat5, TGFβ and Wnt/β-catenin. Particularly, it activates Wnt/β-catenin signaling by directly targeting Wnt antagonists, including Dkk1. Importantly, Dkk1 overexpression partially rescues miR31-induced mammary defects. Together, these findings identify miR-31 as the key regulator of MaSC activity and breast tumorigenesis.

Few studies have examined nitrogen (N) loss and crop yield under urea deep placement combined with organic addition in highly fertilized paddy field. Therefore, a three-year paddy field trial was conducted in Taihu Lake region of China to gain insight into yields, NH 3 volatilization and N leaching. Six treatments were used: control (CK, zero N application), conventional (CT, broadcasting of 300 kg ha −1 N), recommended N (RT, broadcasting of 225 kg ha −1 N), organic addition (RTM, broadcasting of 225 kg ha −1 N and organic N), N deep placement (RTD, deep placement of 225 kg ha −1 N) and organic addition combined with N deep placement (RTDM, organic N combined with deep placement of 225 kg ha −1 N). Compared to the CK treatment, the RTM and RTDM treatments increased soil organic matter by 8%–11% and TN by 9%–14%. NH 3 loss was dominated by floodwater NH 4 + -N concentration and increased with the increase of chemical N use. The RTM treatment had obviously higher floodwater NH 4 + -N concentration and NH 3 loss than the RT treatment. By contrast, NH 3 loss in the RTD and RTDM treatment was 84%–89% less than in the RT treatment, only accounting for 0.2%–1% of the total N application. Compared to NH 3 volatilization, N leaching was low; and NO 3 − -N in the percolation water prevailed over NH 4 + -N and was linearly correlated with floodwater NO 3 − -N concentration. NO 3 − -N and TN leaching between the CT and RTDM treatments differed little (p > 0.05). N loss (NH 3 loss and N leaching) induced by fertilizer was linearly correlated with N surplus. By comparison with the CT treatment, the RTDM treatment decreased N surplus by 73.1%, and increased grain yield by 18.3% and N recovery efficiency by 63.8%. The RTD treatment also had higher rice yield than the CT treatment, whereas unfavorable delayed senescence occurred in it in 2016 and led to a marked decrease of grain yield. The RTD treatment significantly increased plant N uptake (PNU) by rice straw compared to chemical N treatment and had the lowest N harvest index (NHI) and N physiological efficiency (NPE) among the fertilizer treatments. In short, urea deep placement combined with organic addition was superior to N deep placement or organic addition combined with N broadcasting to decrease N loss and increase grain yield, representing an effective and promising practice to achieve food and environmental security.

To increase the rice yield and nitrogen (N) use efficiency (NUE), an integrated agronomic practice, including rapeseed cake addition, increased fertilizer frequency, improved irrigation and crop cultivation management, was adopted in the Taihu Lake region of China. The environmental impacts of the whole rice–wheat rotation system have not yet been fully evaluated. Here, a two-rotation field trail system was used to assess the effects of the improved practice adopted in rice season on crop yield and nitrate (NO3−-N) leaching in the rice–wheat rotation. Four treatment conditions were used—control (CK), conventional (CT), reduced N (RT), and improved (IP). During the rice season, the leaching losses of NO3−-N in the IP treatment group (2.29–3.88 kg N ha−1) were lower than those in the CT treatment group (4.11–5.38 kg N ha−1), and the former had a 28.55% higher crop yield than the latter. Similarly, the RT treatment had lower N leaching loss and a 9.32% higher rice crop yield than the CT treatment. In the wheat seasons, the CT treatment had 41.83% and 38.03% higher NO3−-N leaching than the RT and IP treatments, respectively, despite the three treatments having the same fertilizer application and field management; and N leaching was strongly affected by chemical N surplus in rice season. The RT treatment had the lowest wheat yield, which was 18.72% and 32.06% lower than the CT and IP treatments, respectively; and wheat yield appeared an exponentially increasing response to N surplus that included organic fertilizer N from rice production. These results indicated that management practices applied in rice season had subsequent effect on N leaching and crop yield of the following wheat season. During the whole rice–wheat rotation system, the IP treatment produced 23.56% higher crop yield and 30.87% lower TN leaching than the CT treatment. The IP treatment also increased chemical NUE by 24.67–31.22% and agronomic efficiency of applied chemical N (AEN) by 17.71–33.4% compared with the CT and RT treatments. Thus, compared with traditional practice and reduced N use management, the improved practice management was more in line with the target of food increase and environmental protection.

Accidental contact with caterpillar bristles causes local symptoms such as severe pain, intense heat, edema, erythema, and pruritus. However, there is little functional evidence to indicate a potential mechanism. In this study, we analyzed the biological characteristics of the crude venom from the larval stage of Latoia consocia living in South-West China. Intraplantar injection of the venom into the hind paws of mice induced severe acute pain behaviors in wild type (WT) mice, the responses were much reduced in TRPV1-deficit (TRPV1 KO) mice. The TRPV1-specific inhibitor, capsazepine, significantly attenuated the pain behaviors. Furthermore, the crude venom evoked strong calcium signals in the dorsal root ganglion (DRG) neurons of WT mice but not those of TRPV1 KO mice. Among the pain-related ion channels we tested, the crude venom only activated the TRPV1 channel. To better understand the venom components, we analyzed the transcriptome of the L. consocia sebaceous gland region. Our study suggests that TRPV1 serves as a primary nociceptor in caterpillar-induced pain and forms the foundation for elucidating the pain-producing mechanism.

Accidental contact with caterpillar bristles causes local symptoms such as severe pain, intense heat, edema, erythema, and pruritus. However, there is little functional evidence to indicate a potential mechanism. In this study, we analyzed the biological characteristics of the crude venom from the larval stage of Latoia consocia living in South-West China. Intraplantar injection of the venom into the hind paws of mice induced severe acute pain behaviors in wild type (WT) mice; the responses were much reduced in TRPV1-deficit (TRPV1 KO) mice. The TRPV1-specific inhibitor, capsazepine, significantly attenuated the pain behaviors. Furthermore, the crude venom evoked strong calcium signals in the dorsal root ganglion (DRG) neurons of WT mice but not those of TRPV1 KO mice. Among the pain-related ion channels we tested, the crude venom only activated the TRPV1 channel. To better understand the venom components, we analyzed the transcriptome of the L. consocia sebaceous gland region. Our study suggests that TRPV1 serves as a primary nociceptor in caterpillar-induced pain and forms the foundation for elucidating the pain-producing mechanism.

The YAP signaling activation in epithelial cells is essential for intestinal regeneration and tumorigenesis. However, the molecular mechanism linking stromal signals to YAP-mediated intestinal regeneration and tumorigenesis is poorly defined. Here we report a stroma-epithelia YAP signaling axis essential for stromal cells to modulate epithelial cell growth during intestinal regeneration and tumorigenesis. Specifically, upon inflammation and in cancer, an oncogenic transcription factor ETS1 in stromal cells induces expression of a secreted protein ISLR that can directly binds to a transmembrane protocadherin FAT1 on the surface of epithelial cells. This binding suppressed the Hippo signaling by disrupting MST1-FAT1 association, resulting in YAP signaling activation. Deletion of Islr in stromal cells in mice markedly impaired intestinal regeneration, and suppressed tumorigenesis in the colon. Moreover, the expression of stromal cell-specific ISLR and ETS1 significantly increased in inflamed mucosa of human IBD patients and in human colorectal adenocarcinoma, accounting for the epithelial YAP hyperactivation. Collectively, our findings uncovered a molecular mechanism governing signals for communication between stroma and epithelium during tissue regeneration and tumorigenesis.

Background and purpose Like chili peppers, gingers produce pungent stimuli by a group of vanilloid compounds that activate the nociceptive transient receptor potential vanilloid 1 (TRPV1) ion channel. How these compounds interact with TRPV1 remains unclear. Experimental approach We used computational structural modelling, functional tests (electrophysiology and calcium imaging), and mutagenesis to investigate the structural mechanisms underlying ligand-channel interactions. Key results The potency of three principal pungent compounds from ginger -shogaol, gingerol, and zingerone-depends on the same two residues in the TRPV1 channel that form a hydrogen bond with the chili pepper pungent compound, capsaicin. Computational modelling revealed binding poses of these ginger compounds similar to those of capsaicin, including a "head-down tail-up" orientation, two specific hydrogen bonds, and important contributions of van der Waals interactions by the aliphatic tail. Our study also identified a novel horizontal binding pose of zingerone that allows it to directly interact with the channel pore when bound inside the ligand-binding pocket. These observations offer a molecular level explanation for how unique structures in the ginger compounds affect their channel activation potency. Conclusions and implications Mechanistic insights into the interactions of ginger compounds and the TRPV1 cation channel should help guide drug discovery efforts to modulate nociception.

Piperine, the principle pungent compound in black peppers, is known to activate the capsaicin receptor TRPV1 ion channel. How piperine interacts with the channel protein, however, remains unclear. Here we show that piperine binds to the same ligand-binding pocket as capsaicin but in different poses. There was no detectable detrimental effect when T551 and E571, two major sites known to form hydrogen bond with capsaicin, were mutated to a hydrophobic amino acid. Computational structural modeling suggested that piperine makes interactions with multiple amino acids within the ligand binding pocket, including T671 on the pore-forming S6 segment. Mutations of this residue could substantially reduce or even eliminate piperine-induced activation, confirming that T671 is an important site. Our results suggest that the bound piperine may directly interact with the pore-forming S6 segment to induce channel opening. These findings help to explain why piperine is a weak agonist, and may guide future efforts to develop novel pharmaceutical reagents targeting TRPV1.

A major challenge in cereal production is achieving the dual goal of closing yield gaps without further undermining environmental benefits by increasing gaseous nitrogen (N) emissions. To address this challenge, we conducted a two-rotation field experiment with four different management practices in the Taihu Lake region to gain insight into crop yields, N use efficiency (NUE), and the emission fluxes of nitrous oxide (N2O), nitric oxide (NO), and ammonia (NH3) from the rice cropping system. The four practices were a control (CK, local practice with zero N-fertilizer), the current traditional practice (CT, local practice with farmers’ N management), an improved practice (IP, which closed the yield gap with a reduced N dose of 25%), and a high-yield practice (HY, which maximized the attainable yield with more nutrient inputs). The HY attained the yield potential that was higher by 40% than current yield from the CT. The IP closed the yield gap, achieving 80% of the yield potential, and increased the NUE by 31% and reduced the N surplus by 57% compared with the CT. The lower N surplus of the IP resulted in a decrease in the N2O and NH3 emissions intensity (the N2O or NH3 emission per unit crop yield) of 40% and 65%, respectively relative to the CT. Low NO emissions concomitant with yield increases incurred the marginal NO emission intensity. Thus, the IP should be a promising strategy to increase yield while simultaneously mitigating the gaseous N emissions intensity. Linear or nonlinear responses of gaseous N emissions (N2O, NO and NH3) by N fertilizer to incremental N surplus suggested that reducing the N surplus by both increasing the crop uptake and optimizing N management should be effective in reducing projected gaseous N emissions.