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Enzymes that produce second messengers are highly regulated. Revealing the mechanisms underlying such regulation is critical to understanding both how cells achieve specific signaling outcomes and return to homeostasis following a particular stimulus. Pooled genome-wide CRISPR screens are powerful unbiased approaches to elucidate regulatory networks, their principal limitation being the choice of phenotype selection. Here, we merge advances in bioorthogonal fluorescent labeling and CRISPR screening technologies to discover regulators of phospholipase D (PLD) signaling, which generates the potent lipid second messenger phosphatidic acid. Our results reveal glycogen synthase kinase 3 as a positive regulator of protein kinase C and PLD signaling. More generally, this work demonstrates how bioorthogonal, activity-based fluorescent tagging can expand the power of CRISPR screening to uncover mechanisms regulating specific enzyme-driven signaling pathways in mammalian cells.

Low temperature and overcast rain are harmful to directly seeding early rice, it can hinder rice growth and lower rice biomass during the seedling stage, which in turn lowers rice yield. Farmers usually use N to help rice recuperate after stress and minimize losses. However, the effect of N application on the growth recovery for rice seedlings after such low temperature stress and its associated physiological changes remain unclearly. Two temperature settings and four post-stress N application levels were used in a bucket experiment to compare B116 (strong growth recovery after stress) with B144 (weak growth recovery). The results showed that the stress (average daily temperature at 12°C for 4 days) inhibited the growth of rice seedlings. Compared to the zero N group, the N application group’s seedling height, fresh weight and dry weight significantly increased after 12 days. In particular, the increases in all three growth indicators were relatively higher than that of N application at normal temperature, indicating the importance of N application to rice seedlings after low temperature stress. The antioxidant enzyme activity of rice seedlings increased significantly after N application, which reduced the damaging effect of ROS (reactive oxygen species) to rice seedlings. At the same time, the soluble protein content of seedlings showed a slow decrease, while the H 2 O 2 and MDA (malondialdehyde) content decreased significantly. Nitrogen could also promote nitrogen uptake and utilization by increasing the expression of genes related to NH 4 + and NO 3 − uptake and transport, as well as improving the activity of NR (nitrate reductase) and GS (glutamine synthetase) in rice. N could affect GA 3 (gibberellin A3) and ABA (abscisic acid) levels by regulating the anabolism of GA 3 and ABA. The N application group maintained high ABA levels as well as low GA 3 levels from day 0 to day 6, and high GA 3 levels as well as low ABA levels from day 6 to day 12. The two rice varieties showed obvious characteristics of accelerated growth recovery and positive physiological changes by nitrogen application after stress, while B116 generally showed more obvious growth recovery and stronger growth-related physiological reaction than that of B144. The N application of 40 kg hm -2 was more conducive to the rapid recovery of rice growth after stress. The above results indicated that appropriate N application promoted rice seedling growth recovery after low temperature stress mainly by increasing the activities of antioxidant enzymes and nitrogen metabolizing enzymes as well as regulating the levels of GA 3 and ABA. The results of this study will provide a reference for the regulation of N on the recovery of rice seedling growth after low temperature and weak light stress.

N6-methyladenosine (m6A) is the most prevalent internal modification present in the mRNAs of all higher eukaryotes. However, the role of the m6A methylomes in rice is still poorly understood. With the development of the MeRIP-seq technique, the in-depth identification of mRNAs with m6A modification has become feasible. A study suggested that m6A modification is crucial for posttranscriptional regulation related to Cd2+-induced malignant transformation, but the association between m6A modification in plants and Cd tolerance has not been reported. We investigated the m6A methylomes in the roots of a cadmium (Cd)-treated group and compared them with the roots in the control (CK) group by m6A sequencing of cv. 9311 and cv. Nipponbare (NIP) plants. The results indicated that Cd leads to an altered modification profile in 3,406 differential m6A peaks in cv. 9311 and 2,065 differential m6A peaks in cv. NIP. KEGG pathway analysis of the genes with differentially modified m6A peaks indicated that the “phenylalanine”, “tyrosine and tryptophan biosynthesis”, “glycine”, “adherens junctions”, “glycerophospholipid metabolism” and “threonine metabolism” signalling pathways may be associated with the abnormal root development of cv. 9311 rice due to exposure to Cd. The “arginine”, “proline metabolism”, “glycerolipid”, and “protein processing in endoplasmic reticulum” metabolism pathways were significantly enriched in genes with differentially modified m6A peaks in cv. NIP. Unlike that in Arabidopsis, the m6A-modified nucleotide position on mRNAs (m6A peak) distribution in rice exhibited a preference towards both the stop codon and 3′ untranslated regions (3′ UTRs). These findings provide a resource for plant RNA epitranscriptomic studies and further increase our knowledge on the function of m6A modification in RNA in plants.

Notch1 plays various roles in cancer development, and Notch1-induced transactivation is controlled by phosphorylation of its cleaved intracellular domain. However, it is unclear whether there are phosphatases capable of dephosphorylating the cleaved Notch1 transmembrane/intracellular region (NTM) to regulate its function. Here, we show that DUSP6 can function as a phosphatase for Notch1, thereby regulating NTM stability and transcriptional activity, thus influencing colorectal cancer (CRC) development. In human CRC cells, elevated DUSP6 expression correlates with increased NTM levels, leading to enhanced CRC cell proliferation both in vitro and in vivo. High tumoral DUSP6 protein expression is associated with poorer overall CRC patient survival. In mice, DUSP6 deficiency results in reduced CRC development. Mechanistically, DUSP6 dephosphorylates phospho-Y2116, which in turn reduces NTM ubiquitination, leading to increased NTM stability and transcriptional activity. As a result, the expression of Notch1-targeted proliferation genes is increased to promote tumour cell growth. Notch1 activation in cancer is regulated through phosphorylation of Notch1 intracellular domain. Here, the authors find that DUSP6 functions as a phosphatase for Notch1 transmembrane/intracellular domain (NTM) to increase NTM protein stability and promote colorectal cancer progression.

Background Rice is a crop that is very sensitive to low temperature, and its morphological development and production are greatly affected by low temperature. Therefore, understanding the genetic basis of cold tolerance in rice is of great significance for mining favorable genes and cultivating excellent rice varieties. However, there have been limited studies focusing on cold tolerance at the bud burst stage; therefore, considerable attention should be given to the genetic basis of cold tolerance at this stage. Results In this study, a natural population consisting of 211 rice landraces collected from 15 provinces in China and other countries was used for the first time to evaluate cold tolerance at the bud burst stage. Population structure analysis showed that this population was divided into two groups and was rich in genetic diversity. Our evaluation results confirmed that japonica rice was more tolerant to cold at the bud burst stage than indica rice. A genome-wide association study (GWAS) was performed with the phenotypic data of 211 rice landraces and a 36,727 SNP dataset under a mixed linear model. Twelve QTLs ( P  < 0.0001) were identified for the seedling survival rate (SR) after treatment at 4 °C, in which there were five QTLs ( qSR2–2 , qSR3–1 , qSR3–2 , qSR3–3 and qSR9 ) that were colocalized with those from previous studies and seven QTLs ( qSR2–1 , qSR3–4 , qSR3–5 , qSR3–6 , qSR3–7 , qSR4 and qSR7 ) that were reported for the first time. Among these QTLs, qSR9 , harboring the most significant SNP, explained the most phenotypic variation. Through bioinformatics analysis, five genes ( LOC_Os09g12440 , LOC_Os09g12470 , LOC_Os09g12520 , LOC_Os09g12580 and LOC_Os09g12720 ) were identified as candidates for qSR9 . Conclusion This natural population consisting of 211 rice landraces combined with high-density SNPs will serve as a better choice for identifying rice QTLs/genes in the future, and the detected QTLs associated with cold tolerance at the bud burst stage in rice will be conducive to further mining favorable genes and breeding rice varieties under cold stress.

Objectives We aimed to evaluate the global burden and trends of lower respiratory infections (LRIs) attributable to non-optimal temperatures between 1990 and 2021, focusing on age, period, and cohort effects as well as health inequalities to inform targeted public health policies. Methods Using the Global Burden of Disease 2021 database, we obtained the age-standardized mortality rate (ASMR) and disability-adjusted life-years rate (ASDR) for LRIs related to non-optimal temperatures. We calculated estimated annual percentage changes (EAPC) to assess LRIs burden trends and applied age-period-cohort modeling to quantify age, period, and cohort effects. Health inequalities were evaluated using the slope index of inequality and the concentration index. Results In 2021, the highest ASDR for LRIs due to high temperatures occurred in children under 5 (347.66/100,000), whereas the highest ASMR for LRIs due to low temperatures occurred in adults aged ≥ 65 (338.49/100,000). Globally, the LRIs burden from non-optimal temperatures declined (EAPC: ASMR –2.48; ASDR –3.33). However, among the five climate zones, the LRIs burden in the boreal zone due to high temperatures increased (EAPC: ASMR 24.14; ASDR 45.14), whereas all other climate zones showed decreasing trends. In lower Sociodemographic Index (SDI) regions, the high-temperature–related LRIs burden was more pronounced. Relative inequities driven by non-optimal temperatures worsened in low-SDI regions. Conclusion From 1990 to 2021, the global burden of LRIs attributable to non-optimal temperatures declined overall; however, high-temperature–related LRIs increased in boreal zones. These health inequalities underscore the urgent need for targeted climate adaptation policies, such as providing international assistance, improving infrastructure, offering healthcare resources, and promoting vaccine coverage, particularly for vulnerable populations in low-SDI regions and boreal zones.

Stroke is associated with a high disability and fatality rate, and adversely affects the quality of life of patients and their families. Traditional Chinese Medicine (TCM) has been used effectively in the treatment of stroke for more than 2000 years in China and surrounding countries and regions, and over the years, this field has gleaned extensive clinical treatment experience. The Phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT) pathway is important for regulation of cell migration, proliferation, differentiation, and apoptosis, and plays a vital role in vascularization and oxidative stress in stroke. Current Western medicine treatment protocols for stroke include mainly pharmacologic or mechanical thrombectomy to restore blood flow. This review collates recent advances in the past 5 years in the TCM treatment of stroke involving the PI3K/AKT pathway. TCM treatment significantly reduces neuronal damage, inhibits cell apoptosis, and delays progression of stroke via various PI3K/AKT-mediated downstream pathways. In the future, TCM can provide new perspectives and directions for exploring the key factors, and effective activators or inhibitors that affect occurrence and progression of stroke, thereby facilitating treatment.

Jian-Pi-Yi-Shen formula (JPYSF), a traditional Chinese medicine, has been recommended to treat renal fibrosis for decades. Previous studies had shown that JPYSF could inhibit epithelial–mesenchymal transition (EMT), an important regulatory role in renal fibrosis. However, the mechanism of JPYSF action is largely unknown. In this study, network pharmacology and experimental verification were combined to elucidate and identify the potential mechanism of JPYSF against renal fibrosis by suppressing EMT at molecular and pathway levels. Network pharmacology was first performed to explore the mechanism of JPYSF against renal fibrosis targeting EMT, and then a 5/6 nephrectomy (5/6 Nx)-induced rat model of renal fibrosis was selected to verify the predictive results by Masson’s trichrome stains and western blot analysis. Two hundred and thirty-two compounds in JPYSF were selected for the network approach analysis, which identified 137 candidate targets of JPYSF and 4,796 known therapeutic targets of EMT. The results of the Gene Ontology (GO) function enrichment analysis included 2098, 88, and 133 GO terms for biological processes (BPs), molecular functions (MFs), and cell component entries, respectively. The top 10 enrichment items of BP annotations included a response to a steroid hormone, a metal ion, oxygen levels, and so on. Cellular composition (CC) is mainly enriched in membrane raft, membrane microdomain, membrane region, etc. The MF of JPYSF analysis on EMT was predominately involved in proximal promoter sequence-specific DNA binding, protein heterodimerization activity, RNA polymerase II proximal promoter sequence-specific DNA binding, and so on. The involvement signaling pathway of JPYSF in the treatment of renal fibrosis targeting EMT was associated with anti-fibrosis, anti-inflammation, podocyte protection, and metabolism regulation. Furthermore, the in vivo experiments confirmed that JPYSF effectively ameliorated interstitial fibrosis and inhibited the overexpression of α-SMA, Wnt3a, and β-catenin, and increased the expression of E-cadherin by wnt3a/β-catenin signaling pathway in 5/6 Nx-induced renal fibrosis rats. Using an integrative network pharmacology-based approach and experimental verification, the study showed that JPYSF had therapeutic effects on EMT by regulating multi-pathway, among which one proven pathway was the Wnt3a/β-catenin signaling pathway. These findings provide insights into the renoprotective effects of JPYSF against EMT, which could suggest directions for further research of JPYSF in attenuating renal fibrosis by suppressing EMT.

Curcumin, the major bioactive polyphenol derived from the edible rhizome turmeric (Curcuma longa L.), is recognized for its health-promoting properties. Despite well-documented antioxidant effects, its molecular mechanisms, particularly those involving post-transcriptional regulation, remain incompletely understood. This in vitro study identifies a novel microRNA-mediated pathway contributing to the antioxidant activity of curcumin in human hepatic LO2 cells. Curcumin treatment downregulated the stress-responsive microRNA miR-22-3p. Bioinformatics analysis and a dual-luciferase reporter assay identified malonyl-CoA-acyl carrier protein transacylase (MCAT), a mitochondrial enzyme, as a direct target of miR-22-3p. Modulation of this axis reduced intracellular reactive oxygen species (ROS), enhanced total reducing capacity, increased activities of key antioxidant enzymes (SOD, CAT, GPx), and improved mitochondrial bioenergetics without altering membrane potential. Crucially, siRNA-mediated knockdown of MCAT attenuated the ROS-scavenging effect of curcumin. These findings reveal a mechanistic pathway wherein curcumin downregulates miR-22-3p, resulting in upregulation of MCAT and enhanced mitochondrial antioxidant defense. This work broadens the understanding of curcumin’s bioactivity from direct radical scavenging to include the post-transcriptional fine-tuning of mitochondrial metabolism. The study establishes a molecular framework for further exploration of curcumin’s potential in alleviating oxidative stress.

Traditional Chinese medicine (TCM) is an important complementary and alternative branch of chronic kidney disease (CKD) therapy. Jian-Pi-Yi-Shen formula (JPYSF) is a TCM formula used for treating CKD with good efficacy. However, the underlying mechanisms of JPYSF in treating CKD remain to be elucidated. The purpose of the present study was to investigate the renoprotective effect and potential mechanism of JPYSF in treating CKD. CKD rat model was induced by feeding a diet containing 0.75% w/w adenine for 4 weeks. JPYSF was given by gavage every day, starting from the 3rd week of the adenine-containing diet and continuing for 4 weeks at the dose of 10.89 g/kg. Renal injury was evaluated by serum creatinine (Scr), blood urea nitrogen (BUN), histopathology, and fibrotic markers expression. Serum levels of tryptophan metabolites were detected by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Aryl hydrocarbon receptor (AHR) signaling was tested by Western blot analysis. The results found that JPYSF treatment significantly lowered Scr and BUN levels, improved renal pathological injury, and down-regulated fibrotic markers expression in CKD rats. Furthermore, JPYSF significantly reduced the levels of 10 tryptophan metabolites in the serum of CKD rats and restored the level of tryptophan. Additionally, the kidney expression of AHR signaling was enhanced in CKD rats and was further suppressed in JPYSF treated rats. These results suggested that JPYSF protected against adenine-induced CKD via modulating tryptophan metabolism and AHR activation.