Chimeric Small Molecules for Detouring Drugs into Mitochondria to Engender Apoptosis in Cancer Cells.

Mitochondrion has appeared as one of the important targets for anti-cancer therapy. Subsequently, small molecule anti-cancer drugs are directed to the mitochondria for improved therapeutic efficacy. However, simultaneous imaging and impairing mitochondria by a single probe remained a major challenge. To address this, herein Chimeric Small Molecules (CSMs) encompassing drugs, fluorophore and mitochondria homing moiety were designed and synthesized through a concise strategy. Screening of the CSMs in a panel of cancer cell lines (HeLa, MCF7, A549, and HCT-116) revealed that one of the CSMs comprising Indomethacin V exhibited remarkable cervical cancer cell (HeLa) killing (IC50 =0.97 µM). This lead CSM homed into the mitochondria of HeLa cells within 1 h followed by mitochondrial damage and reactive oxygen species (ROS) generation. This novel Indomethacin V-based CSM-mediated mitochondrial damage induced programmed cell death (apoptosis). We anticipate these CSMs can be used as tools to understand the drug effects in organelle chemical biology in diseased states.

Comparative studies on the antioxidant, anticancer and anti-inflammatory activities of green tea, orthodox black tea and CTC black tea.

Tea is a natural dietary supplement rich in polyphenols and based on the manufacturing process, their polyphenol content also varies. In the present study, we have compared the in vitro antioxidant, anticancer and anti-inflammatory activities of green tea (GT), orthodox black tea (oBT) and CTC black tea (cBT). The analysis was carried out in 50:50 ethanol:water extracts. The total antioxidant capacity, total polyphenol content and free radical scavenging activity were found to be high in GT samples. HPLC profiling indicated a higher percentage of polyphenols like catechin, epicatechin, epigallocatechin and epigallocatechin-gallate in GT when compared to other samples. The comparison of the anticancer potential was done in breast cancer MDA MB-231 cells and it was found that GT has a higher percentage of cell growth inhibition than oBT and cBT. Anti-inflammatory effects were done in LPS stimulated RAW264.7 macrophage cells and here also GT showed maximum effects. This was confirmed by the lower production of iNOS, reduced level of ROS generation and proinflammatory cytokines such as MCP-1, IL-1ɑ, and IL-6 by GT. To conclude, the order for the biological effectiveness of different teas tested is in the order GT > oBT > cBT.

EcTracker: Tracking and elucidating ectopic expression leveraging large-scale scRNA-seq studies.

Dramatic genomic alterations, either inducible or in a pathological state, dismantle the core regulatory networks, leading to the activation of normally silent genes. Despite possessing immense therapeutic potential, accurate detection of these transcripts is an ever-challenging task, as it requires prior knowledge of the physiological gene expression levels. Here, we introduce EcTracker, an R-/Shiny-based single-cell data analysis web server that bestows a plethora of functionalities that collectively enable the quantitative and qualitative assessments of bona fide cell types or tissue-specific transcripts and, conversely, the ectopically expressed genes in the single-cell ribonucleic acid sequencing datasets. Moreover, it also allows regulon analysis to identify the key transcriptional factors regulating the user-selected gene signatures. To demonstrate the EcTracker functionality, we reanalyzed the CRISPR interference (CRISPRi) dataset of the human embryonic stem cells differentiated into endoderm lineage and identified the prominent enrichment of a specific gene signature in the SMAD2 knockout cells whose identity was ambiguous in the original study. The key distinguishing features of EcTracker lie within its processing speed, availability of multiple add-on modules, interactive graphical user interface and comprehensiveness. In summary, EcTracker provides an easy-to-perform, integrative and end-to-end single-cell data analysis platform that allows decoding of cellular identities, identification of ectopically expressed genes and their regulatory networks, and therefore, collectively imparts a novel dimension for analyzing single-cell datasets.

The Cellular basis of loss of smell in 2019-nCoV-infected individuals.

A prominent clinical symptom of 2019-novel coronavirus (nCoV) infection is hyposmia/anosmia (decrease or loss of sense of smell), along with general symptoms such as fatigue, shortness of breath, fever and cough. The identity of the cell lineages that underpin the infection-associated loss of olfaction could be critical for the clinical management of 2019-nCoV-infected individuals. Recent research has confirmed the role of angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2) as key host-specific cellular moieties responsible for the cellular entry of the virus. Accordingly, the ongoing medical examinations and the autopsy reports of the deceased individuals indicate that organs/tissues with high expression levels of ACE2, TMPRSS2 and other putative viral entry-associated genes are most vulnerable to the infection. We studied if anosmia in 2019-nCoV-infected individuals can be explained by the expression patterns associated with these host-specific moieties across the known olfactory epithelial cell types, identified from a recently published single-cell expression study. Our findings underscore selective expression of these viral entry-associated genes in a subset of sustentacular cells (SUSs), Bowman's gland cells (BGCs) and stem cells of the olfactory epithelium. Co-expression analysis of ACE2 and TMPRSS2 and protein-protein interaction among the host and viral proteins elected regulatory cytoskeleton protein-enriched SUSs as the most vulnerable cell type of the olfactory epithelium. Furthermore, expression, structural and docking analyses of ACE2 revealed the potential risk of olfactory dysfunction in four additional mammalian species, revealing an evolutionarily conserved infection susceptibility. In summary, our findings provide a plausible cellular basis for the loss of smell in 2019-nCoV-infected patients.

OdoriFy: A conglomerate of artificial intelligence-driven prediction engines for olfactory decoding.

The molecular mechanisms of olfaction, or the sense of smell, are relatively underexplored compared with other sensory systems, primarily because of its underlying molecular complexity and the limited availability of dedicated predictive computational tools. Odorant receptors (ORs) allow the detection and discrimination of a myriad of odorant molecules and therefore mediate the first step of the olfactory signaling cascade. To date, odorant (or agonist) information for the majority of these receptors is still unknown, limiting our understanding of their functional relevance in odor-induced behavioral responses. In this study, we introduce OdoriFy, a Web server featuring powerful deep neural network-based prediction engines. OdoriFy enables (1) identification of odorant molecules for wildtype or mutant human ORs (Odor Finder); (2) classification of user-provided chemicals as odorants/nonodorants (Odorant Predictor); (3) identification of responsive ORs for a query odorant (OR Finder); and (4) interaction validation using Odorant-OR Pair Analysis. In addition, OdoriFy provides the rationale behind every prediction it makes by leveraging explainable artificial intelligence. This module highlights the basis of the prediction of odorants/nonodorants at atomic resolution and for the ORs at amino acid levels. A key distinguishing feature of OdoriFy is that it is built on a comprehensive repertoire of manually curated information of human ORs with their known agonists and nonagonists, making it a highly interactive and resource-enriched Web server. Moreover, comparative analysis of OdoriFy predictions with an alternative structure-based ligand interaction method revealed comparable results. OdoriFy is available freely as a web service at https://odorify.ahujalab.iiitd.edu.in/olfy/.

Machine-OlF-Action: a unified framework for developing and interpreting machine-learning models for chemosensory research.

SUMMARY: Machine Learning-based techniques are emerging as state-of-the-art methods in chemoinformatics to selectively, effectively and speedily identify biologically relevant molecules from large databases. So far, a multitude of such techniques have been proposed, but unfortunately due to their sparse availability, and the dependency on high-end computational literacy, their wider adaptation faces challenges, at least in the context of G-Protein Coupled Receptors (GPCRs)-associated chemosensory research. Here, we report Machine-OlF-Action (MOA), a user-friendly, open-source computational framework, that utilizes user-supplied SMILES (simplified molecular input line entry system) of the chemicals, along with their activation status, to synthesize classification models. MOA integrates a number of popular chemical databases collectively harboring approximately 103 million chemical moieties. MOA also facilitates customized screening of user-supplied chemical datasets. A key feature of MOA is its ability to embed molecules based on the similarity of their local neighborhood, by utilizing a state-of-the-art model interpretability framework LIME. We demonstrate the utility of MOA in identifying previously unreported agonists for human and mouse olfactory receptors OR1A1 and MOR174-9 by leveraging the chemical features of their known agonists and non-agonists. In summary, here we develop an ML-powered software playground for performing supervisory learning tasks involving chemical compounds. AVAILABILITY AND IMPLEMENTATION: MOA is available for Windows, Mac and Linux operating systems. It's accessible at (https://ahuja-lab.in/). Source code, user manual, step-by-step guide and support is available at GitHub (https://github.com/the-ahuja-lab/Machine-Olf-Action). For results, reproducibility and hyperparameters, refer to Supplementary Notes. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Small molecule NSAID derivatives for impairing powerhouse in cancer cells.

Mitochondrion emerged as an important therapeutic target for anti-cancer strategy due to its involvement in cancer progression and development. However, progress of novel small molecules for selective targeting of mitochondria in cancer cells remained a major challenge. To address this, herein, through a concise synthetic strategy, we have synthesized a small molecule library of indomethacin and ibuprofen (non-steroidal anti-inflammatory drugs, NSAIDs) derivatives having triarylphosphonium moiety for mitochondria localization. Two of the library members were identified to induce mitochondrial damage through outer membrane permeabilization (MOMP) followed by generation of reactive oxygen species (ROS) leading to the remarkable MCF7 breast cancer cell death through apoptosis. These novel mitochondria targeted NSAID derivatives could open a new direction in understanding mitochondrial biology towards anti-cancer therapeutics in future.

Phenanthrenoid Coelogin Isolated from Coelogyne cristata Exerts Osteoprotective Effect Through MAPK-Mitogen-Activated Protein Kinase Signaling Pathway.

Osteoporosis is a major health problem in postmenopausal women globally. This study determined the mechanism through which coelogin stimulates osteoblastogenesis and its osteoprotective and bone regenerating potential. Coelogin effect on primary calvarial osteoblast cells was determined by measuring alkaline phosphatase activity, mineralization, osteoblast survival, and apoptosis and protein expression studies. The osteoprotective effect of coelogin was also evaluated on osteopenic adult female Swiss mice. At autopsy, bones were collected for dynamic and histomorphometry studies. Serum samples were also collected for assessment of serum parameters. Coelogin treatment led to increased osteoblast proliferation, survival, differentiation, and mineralization in osteoblast cells. Coelogin supplementation to Ovx mice promoted new bone formation, prevented Ovx-induced deterioration of bone microarchitecture, and enhanced bone regeneration. In addition, signaling studies revealed that coelogin treatment activates the ER-Erk and Akt-dependent signaling pathways which stimulate the osteoblastogenesis in osteoblast cells.

Coelogin ameliorates metabolic dyshomeostasis by regulating adipogenesis and enhancing energy expenditure in adipose tissue.

Obesity and associated metabolic disorders are heading up with an alarming rate in developing nations. One of highly sought solution for metabolic disorders is to identify natural molecule with an ability to reduce obesity and increase insulin sensitivity. Coelogin (CLN) is a phenanthrene derivative isolated from the ethanolic extract of Coelogyne cristata. In our constant efforts to identify novel anti-dyslipidemic and anti-adipogenic compounds using CFPMA (common feature pharmacophore model using known anti-adipogenic compounds) model, predicted possible anti-adipogenic activity of CLN. In vitro results showed significant inhibition of adipogenesis in 3T3-L1 and C3H10T1/2 cell by CLN. It arrests the cell cycle in G1 phase of interphase and inhibits mitotic clonal expansion to regulate adipogenesis. CLN elicits insulin sensitizing effect in mature adipocytes. During extracellular flux assessment studies, it increases oxidative respiration and energy expenditure in adipocytes. In vivo, CLN reversed HFD-induced dyslipidemia as well as insulin resistance in C57BL/6 mice. It promoted the expression of genes involved in improved mitochondrial function and fatty acid oxidation in eWAT. CLN restored energy expenditure and increased the capacity of energy utilization in HFD fed mice. Taken together, the study indicated beneficial effects of CLN in combating obesity-associated metabolic complications.

Chebulinic acid inhibits MDA-MB-231 breast cancer metastasis and promotes cell death through down regulation of SOD1 and induction of autophagy.

Triple-negative breast cancers (TNBC) are highly aggressive and drug resistant accounting for majority of cases with poor outcome. Purified natural compounds display substantial anticancer activity with reduced cytotoxicity providing a new avenue to combat TNBC. Chebulinic acid (CA), a polyphenol derived from the fruits of various medicinal plants has potent anticancer activity. Here, we demonstrate that CA shows significant cytotoxicity against triple negative MDA-MB-231 cells. CA exhibited cytotoxicity to MDA-MB-231 cells in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Further, CA mitigated MDA-MB-231 cells viability and proliferation as shown by reduced live cell count, crystal violet staining, colony formation assay, soft agar assay and cell cycle analysis. Wound healing assay and trans-well migration assay demonstrated that CA significantly inhibited migration of MDA-MB-231 cells. Also reduced MMP9 expression was observed in CA-treated cells by gelatin zymography. CA negatively regulated mesenchymal characteristics of MDA-MB-231 cells demonstrated by F-actin staining and reduced expression of N-cadherin by confocal microscopy and western blot analysis. Annexin V/propidium iodide (PI) and active caspase-3 staining showed that CA was able to induce apoptosis in MDA-MB-231 cells but did not activate caspase-3. Two-dimensional gel electrophoresis based proteomic analysis demonstrated that CA regulated proteins belonging to the oxidative stress pathway, apoptotic pathway and proteins with antiproliferative activity. Western blot analysis analysis revealed that CA negatively regulated superoxide dismutase 1 (SOD1) and enhanced oxidative stress in MDA-MB-231 cells. SOD1 in-gel activity assay also showed reduced SOD1 activity upon CA treatment. Overexpression studies with GFP-LC3 and tandem tagged RFP-GFP-LC-3 also demonstrated enhanced autophagy upon CA treatment.

Phytoremediation ability of naturally growing plant species on the electroplating wastewater-contaminated site.

The presence of heavy metal in soil and water resources has serious impact on human health. The study was designed to examine the phytoremediation ability of plant species that are growing naturally on the Zn-contaminated site. For the study, six plant species and their rhizospheric soil as well as non-rhizospheric soil samples were collected from different parts of the industrial sites for chemical and biological characterization. Visual observations and highest importance value index (IVI) through biodiversity study revealed potential plants as effective ecological tools for the restoration of the contaminated site. Among the plants, almost all were the most efficient in accumulating Fe, Mn, Cu and Zn in its shoots and roots, while Cynodon dactylon, Chloris virgata and Desmostachya bipinnata were found to be stabilizing Cr, Pb and Cd (bioconcentration factor in root = 7.95, 6.28 and 1.98 as well as translocation factor = 0.48, 0.46 and 0.78), respectively. Thus, the results of this study showed that the naturally growing plant species have phytoremediation potential to remediate the electroplating wastewater-contaminated site. These plant species are successful phytoremediators with their efficient metal stabilizing and well-evolved tolerance to heavy metal toxicity.

Assessment of Ziziphus mauritiana grown on fly ash dumps: Prospects for phytoremediation but concerns with the use of edible fruit.

A field study was carried out on fly ash (FA) dumps of Panki Thermal Power Station to assess the phytoaccumulation of elements in various plant parts of edible fruit tree Ziziphus mauritiana. Of the twelve analyzed elements, the highest concentration was found for Fe followed by Mn > Se > Zn > Mo > Cu > Cr > Pb > Cd >Ni > As > Co in rhizospheric substrate of Z. mauritiana grown on FA dumps. Metal accumulation, bioconcentration factor, and translocation factor for each metal were calculated in various parts of the edible fruit tree. Significant variations of metal accumulations were observed among various plant parts. Accumulation of toxic elements was higher in roots, and it gradually declined toward the aerial parts of the plant corresponding to its distance from the ground. The concentration of some elements in fruit tree was found to be above prescribed limits in edible parts. Therefore, the present study suggested that additional care should be undertaken, if edible fruit trees are considered for phytoremediation or afforestation programs of FA dumps.

Characterization of a splice-site mutation in the tumor suppressor gene FLCN associated with renal cancer.

BACKGROUND: Renal cell carcinoma is among the most prevalent malignancies. It is generally sporadic. However, genetic studies of rare familial forms have led to the identification of mutations in causative genes such as VHL and FLCN. Mutations in the FLCN gene are the cause of Birt-Hogg-Dubé syndrome, a rare tumor syndrome which is characterized by the combination of renal cell carcinoma, pneumothorax and skin tumors. METHODS: Using Sanger sequencing we identify a heterozygous splice-site mutation in FLCN in lymphocyte DNA of a patient suffering from renal cell carcinoma. Furthermore, both tumor DNA and DNA from a metastasis are analyzed regarding this mutation. The pathogenic effect of the sequence alteration is confirmed by minigene assays and the biochemical consequences on the protein are examined using TALEN-mediated transgenesis in cultured cells. RESULTS: Here we describe an FLCN mutation in a 55-year-old patient who presented himself with progressive weight loss, bilateral kidney cysts and renal tumors. He and members of his family had a history of recurrent pneumothorax during the last few decades. Histology after tumor nephrectomy showed a mixed kidney cancer consisting of elements of a chromophobe renal cell carcinoma and dedifferentiated small cell carcinoma component. Subsequent FLCN sequencing identified an intronic c.1177-5_-3delCTC alteration that most likely affected the correct splicing of exon 11 of the FLCN gene. We demonstrate skipping of exon 11 to be the consequence of this mutation leading to a shift in the reading frame and the insertion of a premature stop codon. Interestingly, the truncated protein was still expressed both in cell culture and in tumor tissue, though it was strongly destabilized and its subcellular localization differed from wild-type FLCN. Both, altered protein stability and subcellular localization could be partly reversed by blocking proteasomal and lysosomal degradation. CONCLUSIONS: Identification of disease-causing mutations in BHD syndrome requires the analysis of intronic sequences. However, biochemical validation of the consecutive alterations of the resulting protein is especially important in these cases. Functional characterization of the disease-causing mutations in BHD syndrome may guide further research for the development of novel diagnostic and therapeutic strategies.

Chebulinic Acid Isolated From the Fruits of Terminalia chebula Specifically Induces Apoptosis in Acute Myeloid Leukemia Cells.

Chebulinic acid, an ellagitannin found in the fruits of Terminalia chebula, has been extensively used in traditional Indian system of medicine. It has shown to have various biological activities including antitumor activity. The present study aims to investigate the cytotoxic potential of chebulinic acid in human myeloid leukemia cells. Interestingly, chebulinic acid caused apoptosis of acute promyelocytic leukemia HL-60 and NB4 cells but not K562 cells. In vitro antitumor effects of chebulinic acid were investigated by using various acute myeloid leukemia cell lines. Chebulinic acid treatment to HL-60 and NB4 cells induced caspase activation, cleavage of poly(ADP-ribose) polymerase, DNA fragmentation, chromatin condensation, and changes in the mitochondrial membrane permeability. Additionally, inhibition of caspase activation drastically reduced the chebulinic acid-induced apoptosis of acute promyelocytic leukemia cells. Our data also demonstrate that chebulinic acid-induced apoptosis in HL-60 and NB4 cells involves activation of extracellular signal-regulated kinases, which, when inhibited with ERK inhibitor PD98059, mitigates the chebulinic acid-induced apoptosis. Taken together, our findings exhibit the selective potentiation of chebulinic acid-induced apoptosis in acute promyelocytic leukemia cells. Copyright © 2017 John Wiley & Sons, Ltd.

Restoration of red mud deposits by naturally growing vegetation.

Disposal of red mud (RM) poses serious environmental problems such as wind erosion, air and water pollution. To overcome these problems, effective restoration of the disposal land through naturally growing vegetation is a sustainable and economical approach. The present study involved estimation of frequency (F), density (D), abundance (Ab), and important value index (IVI) of natural flora on abandoned RM sites in order to assess their metal toxicity tolerance capacity. Based on visual observations and highest IVI, S. Asper and S. punicea were identified as effective ecological tools for the restoration of barren RM sites. From the study, remarkable differences were observed between non-rhizospheric and rhizospheric RM of both species. These rhizospheric RM analyses confirm the ability of S. asper and S. punicea for enhancing the biological activities of abandoned RM. Translocation factor (TF) of iron was maximum (2.58) in S. asper, and bioconcentration factor (BCF) was found maximum (1.25) in S. punicea, but both TF (2.58) and BCF (1.35) were high in S. asper. Therefore, this plant could be reported as an iron hyperaccumulator plant. These results suggest that these plant species can be exploited for effective restoration of RM deposited land without any inputs or maintenance.

Biomaterials for Targeting Endoplasmic Reticulum in Cancer.

Endoplasmic reticulum (ER) is one of the most important sub-cellular organelles which controls myriads of biological functions including protein biosynthesis with proper functional folded form, protein misfolding, protein transport into Golgi body for secretion, Ca2+ homeostasis and so on. Subsequently, dysregulation in ER function leads to ER stress followed by disease pathology like cancer. Hence, targeting ER in the cancer cells emerged as one of the futuristic strategies for cancer treatment. However, the major challenge is to selectively and specifically target ER in the sub-cellular milieu in the cancer tissues, due to the lack of ER targeting chemical moieties to recognize the ER markers. To address this, in the last decade, numerous biomaterials were explored to selectively impair and image ER in cancer cells to induce ER stress. This review outlines those biomaterials which consists of carbon and silicon materials, lipid nanoparticles (liposomes and micelles), supramolecular self-assembled nanostructures, cell membrane-coated nanoparticles and metallic nanoparticles. Moreover, we also discuss the challenges and possible solutions of this promising field to usher the readers towards next-generation ER targeted cancer therapy.

Isolation of Food Grade Dye from Flower Petals of Butea monosperma and Determination of Marker Compounds for Its Quantitative Analysis.

Health concerns associated with synthetic dyes/colorants have fostered the use of natural coloring materials for food applications. This study has been carried out to extract a natural dye from the flower petals of Butea monosperma (family Fabaceae) under an eco-friendly and organic solvent-free approach. Hot aqueous extraction of dry B. monosperma flowers followed by lyophilization of the resulting extract furnished an orange-colored dye in ∼35% yield. Silica gel column chromatography of dye powder resulted in the isolation of three marker compounds, viz. iso-coreopsin (1), butrin (2), iso-butrin (3) which were characterized by spectral methods, e.g., ultra violet, Fourier-transform infrared spectroscopy, nuclear magnetic resonance, and high-resolution mass spectrometry. The XRD analysis of isolated compounds established an amorphous nature for compounds 1 and 2 while compound 3 showed good crystallinity. The stability of dye powder and the isolated compounds 1-3 was determined by thermogravimetric analysis which showed excellent stability up to 200 °C. In trace metal analysis, the product B. monosperma dye powder exhibited low relative abundance <4% for Hg along with negligible concentrations of Pb, As, Cd, and Na. The detection and quantification of marker compounds 1-3 in the B. monosperma flower extracted dye powder were carried out by a highly selective UPLC/PDA method of analysis.

Transcriptional advantage influence odorant receptor gene choice.

Odorant receptors (ORs) obey mutual exclusivity and monoallelic mode of expression. Efforts are ongoing to decipher the molecular mechanism that drives the 'one-neuron-one-receptor' rule of olfaction. Recently, single-cell profiling of olfactory sensory neurons (OSNs) revealed the expression of multiple ORs in the immature neurons, suggesting that the OR gene choice mechanism is much more complex than previously described by the silence-all-and-activate-one model. These results also led to the genesis of two possible mechanistic models i.e. winner-takes-all and stochastic selection. We developed Reverse Cell Tracking (RCT), a novel computational framework that facilitates OR-guided cellular backtracking by leveraging Uniform Manifold Approximation and Projection embeddings from RNA Velocity Workflow. RCT-based trajectory backtracking, coupled with statistical analysis, revealed the OR gene choice bias for the transcriptionally advanced (highest expressed) OR during neuronal differentiation. Interestingly, the observed selection bias was uniform for all ORs across different spatial zones or their relative expression within the olfactory organ. We validated these findings on independent datasets and further confirmed that the OR gene selection may be regulated by Upf3b. Lastly, our RNA dynamics-based tracking of the differentiation cascade revealed a transition cell state that harbors mixed molecular identities of immature and mature OSNs, and their relative abundance is regulated by Upf3b.

Long-term non-phosphorus application increased paddy methane emission by promoting organic acid and methanogen abundance in Tai Lake region, China.

Rice paddy is a significant source of atmospheric methane (CH4), a major global warming source. CH4 emission from paddy fields is greatly influenced by phosphorus (P) management, especially the long-term non-P application on CH4 emission is largely unexplored. In the present study, long-term non-P application (NK) and P application (NPK) treatments of two paddy fields in Suzhou (from 1980) and Yixing (from 2009), Tai Lake region was done. The effect of P application on CH4 emissions and related microorganisms (i.e., methanogens and methanotrophs) from 2019 to 2020 was analyzed. Results revealed that long-term NK treatment didn't alter the seasonal trend of CH4 flux, but significantly promoted CH4 emissions at the tillering stage. The non-P application for >12 years caused the cumulative CH4 emissions of NK treatment in the whole rice season significantly increased by 41.9-221 % in two fields compared to NPK treatment in 2019 and 2020. NK treatment increased the abundance and diversity of methanogens, while reducing the abundance and diversity of methanotrophs. Community composition of soil pmoA gene differed in two experiment sites. Correlation analysis revealed that the CH4 emission was significant and positively related to soil mcrA gene and C/P while negatively related to soil pmoA gene and P. Structure equation model analysis show the low soil available P content was the dominant driving factor for the high CH4 emission under long-term non-P application through its direct impact on soil mcrA and pmoA genes. The increased soil organic acid content was another driver which was positively related to soil mcrA gene and negatively to soil pmoA gene. Our findings demonstrate the important role of soil P in regulating CH4 emissions from paddy fields in the Tai Lake region, China, and suitable P application is necessary for ensuring the yield while reducing CH4 emission.