Yiqi Huayu Jiedu (YQHYJD) is a traditional Chinese medicine decoction made up of eight traditional Chinese medicines. Although YQHYJD is effectively used to prevent and treat ARDS/acute lung injury (ALI) in rats, the molecular mechanisms supporting its clinical application remain elusive. The purpose of the current study was to understand its lung protective effects at the molecular level using network pharmacology approach.  In an ARDS animal model, the beneficial pharmacological activities of YQHYJD were confirmed by reduced lung tissue damage levels observed on drug treated rats versus control group. We then proposed a network analysis to discover the key nodes based on drugs and disease network. Subsequently, we analyzed interaction networks and screened key targets. Using Western blot to detect the expression level of key targets, the intervention effect of changes in expression level of key targets on ARDS was evaluated.  Pathway enrichment analysis of highly ranked genes showed that ErbB pathways were highly related to ARDS. Finally, western blot results showed decreased level of the AKT1 and KRAS/NRAS/HRAS protein in the lung after treatment which confirmed the hypothesis.  In conclusion, our results suggest that YQHYJD can exert lung tissue protective effect against the severe injury through multiple pathways, including the endothelial cells permeability improvement, inflammatory reaction inhibition, edema, and lung tissue hemorrhage reduction. Acute respiratory distress syndrome (ARDS) is a common acute critical illness. The main pathophysiological changes are reduced lung volume, reduced lung compliance, and severe ventilation/blood flow imbalance, with a mortality rate of up to 40% , 2, and As an important intervention due to its significant preventive and therapeutic effects, TCM treatment included novel coronavirus pneumonia diagnosis and treatment plan in China's fight against the epidemic of COVID-19 . Novel cRadix Astragali  Bunge, also named as Huangqi in Chinese), Panax notoginseng (the dried roots of Panax notoginseng (Burk.) F. H. Chen, also named as Sanqi in Chinese), Flos Lonicerae , Radix Scutellariae , Radix Paeoniae Rubra , Fructus Aurantii Immaturus , Cortex Mori , Semen Descurainiae  Webb. ex Prantl, also named as Tinglizi in Chinese).YQHYJD is the clinical experience decoction of Professor Du Huaitang from Dongzhimen Hospital of Beijing University of Chinese medicine. It is composed of 8 traditional Chinese medicines, which are commonly used in the clinical practice of traditional Chinese medicine, namely, Astragaloside IV in Radix Astragali which can alleviate LPS-induced acute lung injury by inhibiting the activation of NF-κB pathway and the expression of inflammatory genes [Astragalus polysaccharide inhibits the expression of ICAM-1 and VCAM-1 in TNF-α treated human vascular endothelial cells by blocking NF-κB activation [Panax notoginseng saponins) is very effective in promoting blood circulation to dissipate blood stasis and in improving blood circulation [Panax notoginseng can reduce the expression of ICAM-1 and ICAM-1, improve the connection between pulmonary microvascular endothelial cells, and reduce lung injury [Radix Scutellariae, can prevent LPS-induced acute lung injury in rats [κB signaling pathway. Therefore, baicalein reduced the formation and dysfunction of SOD-1 ROS induced by NADPH oxidase [Flos Lonicerae's active ingredient quercetin negatively regulates TLR4 signaling induced by lipopolysaccharide through Tollip expression and quercetin and quercitrin protect against cytokine induced injuries in RINm5F β-cells via the mitochondrial pathway and NF-κB signaling; these active compounds of Flos Lonicerae have been shown to exhibit a variety of modulatory effects on the inflammation process by reducing the induction of proinflammatory cytokines [Radix Paeoniae Rubra is able to reduce heat and cool blood, disperse stasis, and relieve pain. It was observed that total paeony glycoside (effective component of Radix Paeoniae Rubra) can inhibit apoptosis and oxidative stress by significantly reducing the phosphorylation level of PI3K and Akt and then regulate hemodynamics [Fructus Aurantii Immaturus; they have been proved to have the properties of antioxidant [cortex mori) treatment led to decreased levels of proinflammatory cytokines such as interleukin, IL-6, and IL-1β, and TNF-α and increased level of anti-inflammatory IL-10 in mice lung tissue. Morusin relieves mycoplasma pneumonia via the inhibition of the activation of Wnt/β-catenin and NF-κB pathways [Semen Descurainiae extract administration significantly reduced mucus production, inflammatory cell infiltration into airways, and eosinophil activation by reducing the expression of type 2 cytokines and inhibiting differentiation and activation of Th2 cells [Eight traditional Chinese medicines in YQHYJD are used with reference to the traditional efficacy of TCM in the “Pharmacopoeia of the People's Republic of China” . Pharmacry genes . Astragativation . PNS , interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), and other proinflammatory factors, reduce NF-κB p50, p65, mitogen-activated protein kinase p38, and other inflammatory proteins expressions, and increase anti-inflammatory factors IκBα, interleukin-4 (IL-4), and interleukin-10 (IL-10) expression. At the same time, the group confirmed the intervention effect of the compound on ARDS at the level of proteomics, but the mechanism of action was not clear.According to the clinical observation for many years, we found that the series prescription of YQHYJD had a significant effect on the treatment of critical respiratory system diseases , and ourThe concept of network pharmacology was firstly systematically elaborated by the British pharmacologist Andrew L. Hopkins in 2007. The “disease-gene-target-drug” interaction network is the research object, and a systematic analysis of the interaction relationship between drugs and the body is conducted to discover new drugs and explore the research methods of drug efficacy and mechanism of action . Using oIn this work, based on the network pharmacology research strategy, we integrate bioinformatics methods such as network construction and analysis, pathways, and target prediction to study the relevant molecular networks of YQHYJD in ARDS rats. Combined with animal experiments, the prediction results are validated and the mechanism of the compound intervention in ARDS is explored. The work in this paper can provide valuable references for the further quality control, product development, and clinical application of the compound. Escherichia coli and PBS solutions were purchased from Sigma Company in the United States; chloral hydrate, paraformaldehyde, absolute ethanol, xylene, methanol, and so on were purchased from Beijing Chemical Industry Group Co., Ltd. Hematoxylin and eosin dyes were purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd. Liquid paraffin, RIPA lysate, 30% polyacrylamide solution-bisacrylamide, 4 × separation gel buffer, 4 × concentrated gel buffer, 10 × electrophoresis solution, 10 × electrophoresis solution, and so on were purchased from Beijing Solibao Technology Co., Ltd. BCA protein quantitative analysis kit, protease inhibitor (100x), 10% ammonium persulfate solution, TEMED, skimmed milk powder, and TBST were purchased from Beijing Pulailai Gene Technology Co., Ltd. Anti-AKT1 antibody and anti-KRAS/HRAS/NRAS antibody were purchased from Abbot (Shanghai) Trading Co., Ltd. Primary antibody dilutions were purchased from Shanghai Biyuntian Biotechnology Co., Ltd. Goat anti-rabbit IgG/HRP was purchased from Beijing Baierdi Biotechnology Co., Ltd. Chemiluminescence liquid was purchased from GE Company in the United States.LPS A total of 30 clean male SD rats were selected, aging 8 weeks old and weighing 190–210 g . They were fed in the Barrier Laboratory of Experimental Animal Center, Dongzhimen Hospital, Beijing University of Chinese Medicine . The experiment was approved by the Experimental Animal Welfare and Ethics Committee of Dongzhimen Hospital of Beijing University of Chinese Medicine. The international experimental pharmacological principles were strict when handling animals.Radix Astragali 30 g, Panax Notoginseng 10 g, Flos Lonicerae 15 g, Radix Scutellariae 10 g, fried Fructus Aurantii Immaturus 12 g, Radix Paeoniae Rubra 15 g, Semen Descurainiae 15 g, and Cortex Mori 15 g; the medicinal materials used in the research are from Bozhou Yonggang Decoction Pieces Factory Co., Ltd. and the formulation was a TCM extract. Use 10 times the quality of water to decoct the traditional Chinese medicine for 2 times, each time for 30 minutes. The decoction obtained from the two decoctions was mixed, centrifuged, concentrated to 1.22g crude drug/ml, and finally stored at 4°C before use, which was produced and provided by Institute of Chinese Materia Medica, Chinese Academy of Chinese Medical Sciences (Batch No.:20170320).The composition of YQHYJD used for research was Chinese Pharmacopoeia (2015 edition), which contains more than 400 commonly used Chinese herbal medicines. In the Chinese medicine information setting interface, the 8 Chinese herbs contained in YQHYJD were searched and collected separately. For all chemical components, all drug targets with a similarity score ≥0.8 were selected as potential targets for YQHYJD at the Chinese medicine target prediction interface.TCMIP (V1.0)  databaseTCMIP disease/symptom target database integrates biological database information such as Drugbank , KEGG 4, OMIM 4, TTD 4242, and HBy integrating the above potential drug targets and ARDS disease targets and inputting into the STRING database (V11.0) , the PPIThe PPI network data obtained in the above steps were input into Cytoscape software (V 3.5.1)  for netwThe key nodes of the above network were input into the DAVID database (V6.8) , and relThe speculated mechanism of YQHYJD in the treatment of ARDS by a network pharmacological method was then verified by polypharmacology using an ARDS model.A total of 30 SD rats were randomly divided into 5 groups of a normal control group (NC), a model control group (MC), a low-dose group (LDG), a medium-dose group (MDG), and a high-dose group (HDG), with 6 rats in each group. The rats were weighed and recorded, with adaptive feeding for 1 week. Weighing and recording before each experiment. The dosage was calculated according to the adult's weight of 60 kg. The dose calculated by the rats and adults based on a factor of 6.25 times was the Chinese medicine dose. The low was 0.5 times as dose of the medium, and the high was 2 times as dose of the medium. Chinese medicine extract was prepared into 6.1 g/kg/d, 12.2 g/kg/d, and 24.4 g/kg/d (refer to the weight of the crude medicine) Chinese medicine decoction, which were equivalent to clinical equivalent doses of 0.5, 1, and 2 times, respectively. After the end of the adaptive feeding, Chinese medicine was administered to prevent the stomach. The normal group and the model group were given 1 mL/100 g/d of distilled water for 7 consecutive days. Each group had normal drinking water. The Chinese medicine solution was prepared with distilled water, and the medicinal solution and distilled water were stored in a refrigerator at 4°C and heated in a warm water bath before the operation of gastric administration.Inflammatory ARDS rat model was established by tail vein injection of lipopolysaccharides (LPS). Previous research of the research group proved that the modeling method was stable and effective , 47–49. The pathological changes of the lungs, including color, degree of edema, and degree of congestion, were observed with naked eye and recorded with a digital camera. The right lung tissue specimen was taken and paraffin sections were made through fixing, washing, dehydrating, clearing, immersing in wax, and embedding. After staining with hematoxylin and eosin (HE), it was observed under an optical microscope.Western blot method was used to detect the expression level of potential target protein. Lung tissue was subjected to protein extraction and protein concentration was determined using BCA method. The target protein expression level was determined by SDS-PAGE electrophoresis, membrane transfer, protein-antibody reaction, chemiluminescence, and development.P < 0.05 was the standard with significant difference. Graphpad Prism 7 software and Adobe Photoshop CS6 version software were used to make data chart.In this study, SPSS 21.0 software was used for statistical analysis. Each group of data was expressed as mean ± standard deviation Radix Astragali, Panax Notoginseng, Flos Lonicerae, Radix Scutellariae, Radix Paeoniae Rubra, Fructus Aurantii Immaturus, Cortex Mori, and Semen Descurainiae were collected with the number of 29, 95, 47, 54, 20, 49, 54, and 21  and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. It was found that the drug targets involved in biological functions mainly include oxidation-reduction process, mitochondrial electron transport, mitochondrial respiratory chain complex I assembly, membrane depolarization during action potential, aldehyde dehydrogenase (NAD) activity, voltage-gated sodium channel activity, NF-kappaB signaling, and response to oxidative stress . Key target functions mainly involve ErbB signaling pathway, VEGF signaling pathway, focal adhesion, FoxO signaling pathway, influenza A, MAPK signaling pathway, regulation of actin cytoskeleton, Toll-like receptor signaling pathway, insulin signaling pathway, adherens junction, mTOR signaling pathway, gap junction, Ras protein signal transduction, positive regulation of ERK1 and ERK2 cascade, and so on. The correlation between ErbB signaling pathway with the highest P value and key targets was analyzed, and 14 key targets were found to be enriched in ErbB signaling pathway, as shown in The key targets of the above network in the DAVID database were analyzed to obtain the related pathways and biological function information  and sortBy analyzing the upstream and downstream of the ErbB signaling pathway, it was found that key targets were mainly located on the downstream PI3K-AKT signal axis and MAPK signal axis. Combined with our previous research results, it was believed that YQHYJD might play an interventional role in ARDS by regulating these 2 signal axes. In order to verify the network analysis and prediction results, animal experiments were designed for verification.During the onset of ARDS, the occurrence of explosive inflammatory reactions led to pathological changes in lung tissue damage. Therefore, it is of great significance to reduce inflammation damage and protect lung tissue function in terms of treatment. As shown in P < 0.01), while there was a dose-dependent inhibition of AKT1 overexpression; each dose group of the compound significantly reduced the expression of KRAS/HRAS/NRAS (P < 0.001) (Each dose group can reduce the expression levels of AKT1 and KRAS/HRAS/NRAS in lung tissue of ARDS rats to varying degrees. The compound high-dose group significantly reduced the expression of AKT1 (< 0.001) .in vivo experiments, to provide a reliable basis for the further research of the compound.ARDS is a clinically critical illness characterized by progressive respiratory distress, refractory hypoxemia, and nonuniform exudation of pulmonary imaging . At presκB pathway and MAPK pathway and improve LPS-induced ARDS/ALI [α-ENaC, β-ENaC, and γ-ENaC on the surface of alveolar cells [According to the enrichment analysis of the core target pathways of the ARDS network that YQHYJD interferes, the core target pathways include ErbB signaling pathway, VEGF signaling pathway, focal adhesion, FoxO signaling pathway, influenza A, MAPK signaling pathway, Toll-like receptor signaling pathway, insulin signaling pathway, adherens junction, mTOR signaling pathway, gap junction, Ras protein signal transduction, and positive regulation of ERK1 and ERK2 cascade . These iARDS/ALI . In ARDSARDS/ALI . Insulinar cells . The mTOar cells . Gap junar cells . Blockinar cells . ERK1/2 ar cells . Accordiin vivo studies. As shown in Based on the above analysis, YQHYJD can interfere with ARDS by targeting different genes through multiple signaling pathways. Therefore, we evaluated the predictions through in vivo and in vitro experiments [Staphylococcus aureus-induced lung injury, the increase in p-AKT expression in the PI3K/AKT cascade was positively correlated with the degree of cell damage [The full name of AKT1 is RAC-alpha serine/threonine-protein kinase, which is involved in regulating cell metabolism, proliferation, cell survival, growth, and angiogenesis through serine and/or threonine phosphorylation of a series of downstream substrates. It is an important downstream signal protein of the PI3K-AKT pathway . AKT1 aneriments . In the l damage . Ligustrl damage . As showκB inflammatory signaling pathway and blocking the expression of p-p38MAPK, NF-κB65, HIF-1α, p-IκB-α, and TGF-β1 ALI [κBp50, MAPK P38, p65, and the expression of TNF-α, IL-1β, IL-6, IL-8, and other proinflammatory factors, which plays a protective role in the lung tissue of ARDS rats [κB signaling pathway activation, thereby reducing the release of inflammatory proteins and inflammatory factors, which is in line with the results of our previous research. Therefore, we believe that YQHYJD might block the MAPK signaling pathway by inhibiting the expression of KRAS/HRAS/NRAS, thereby reducing lung tissue inflammation.KRAS, HRAS, and NRAS are different structural monomers of GTPase Ras protein, and they are activation proteins of MAPK transduction pathway. Studies have found that inhibiting the MAPK cascade can reduce lung tissue damage, and its mechanism may be related to inhibiting oxidative stress and apoptosis and blocking inflammatory responses –71. BlocF-β1 ALI . At the F-β1 ALI . We alsoRDS rats –33. As sIn this study, each dose group of YQHYJD downregulated the expressions of PI3K-AKT pathway downstream protein AKT1 and MAPK pathway activating protein KRAS/NRAS/HRAS to different extents, which played a role in the intervention of ARDS. The predictions are consistent.in vivo experiments. The experimental results are consistent with those predicted by network analysis. This study not only evaluated the anti-inflammatory effects of YQHYJD in intervening ARDS, but also proposed that YQHYJD regulates vascular endothelial function and anticoagulant function, which deserves further study.In short, a systematic study of YQHYJD was conducted through predictive analysis and


Traditional Chinese medicine (TCM) believes that the pathogenic factors of colorectal cancer (CRC) are “deficiency, dampness, stasis, and toxin,” and Fuzheng–Jiedu Decoction (FJD) can resist these factors. In this study, we want to find out the potential targets and pathways of FJD in the treatment of CRC and also explain from a scientific point of view that FJD multidrug combination can resist “deficiency, dampness, stasis, and toxin.”  We get the composition of FJD from the TCMSP database and get its potential target. We also get the potential target of colorectal cancer according to the OMIM Database, TTD Database, GeneCards Database, CTD Database, DrugBank Database, and DisGeNET Database. Subsequently, PPI analysis, KEGG pathways analysis, and GO biological processes analysis were carried out for the target of FJD in the therapy of colorectal cancer. In addition, we have also built a relevant network diagram.  In this study, we identified four core compounds of FJD in the therapy of colorectal cancer, including quercetin, kaempferol, beta-sitosterol, and stigmasterol. At the same time, we also obtained 30 core targets, including STAT3, INS, TP53, VEGFA, AKT1, TNF, IL6, JUN, EGF, CASP3, MAPK3, MAPK1, MAPK8, SRC, IGF1, CCND1, ESR1, EGFR, PTEN, MTOR, FOS, PTGS2, CXCL8, HRAS, CDH1, BCL2L1, FN1, MMP9, ERBB2, and JAK2. FJD treatment of colorectal cancer mainly involves 112 KEGG pathways, including FoxO (hsa04068) signaling pathway, PI3K-Akt (hsa04151) signaling pathway, HIF-1 (hsa04066) signaling pathway, T cell receptor (hsa04660) signaling pathway, and ErbB (hsa04012) signaling pathway. At the same time, 330 GO biological processes were summarized, including cell proliferation, cell apoptosis, angiogenesis, inflammation, and immune.  In this study, we found that FJD can regulate cell proliferation, apoptosis, inflammation and immunity, and angiogenesis through PI3K-Akt signaling pathway to play an anti-CRC effect. Colorectal cancer (CRC), as the third most common cancer in the world, accounts for the fourth highest cancer mortality rate in the world . It is aHerbal medicine and traditional medical practice have been reported as a world upsurge by the World Health Organization's global Atlas of traditional, complementary, and alternative medicine . TraditiPanax ginseng C.A. Mey , Pinellia ternata , and Smilacis Glabrae Rhizoma . Not only does the drug combination have a good antitumor effect, but also it can improve the symptoms of CRC such as asthenia and abdominal distension [There are four main factors in TCM Syndromes of colorectal cancer: “deficiency, dampness, stasis, and toxin” . Accordistension . Based ostension . BX can stension . Variousstension . AlthougNetwork pharmacology is an effective new strategy integrating bioinformatics, chemical informatics, network biology, traditional pharmacology, and network analysis . NetworkThe compounds of RS, BX, and TFL in FJD were extracted from the TCM systems pharmacology (TCMSP) database. TCMSP database is one of the most comprehensive databases of Chinese herbal medicine, which is used to find the relationship among herbs, natural compounds, target protein, and diseases .Although FJD is composed of many natural compounds, only a few of them have pharmacological effects. Therefore, it is necessary to screen the pharmacokinetics of each compound of Chinese herbal medicine . Based oAll the active ingredients were input into the TCM systems pharmacology (TCMSP) database to get the known targets. The 2D structure of the compounds obtained from PubChem was imported into the SwissTargetPrediction database, and a threshold (probability >0.6) was set to obtain more credible targets for each compound.We collected the CRC targets from six resources: (1) Online Mendelian Inheritance in Man database (OMIM); (2) Therapeutic Target Database (TTD); (3) GeneCards Human Gene database (score ≥ 30); (4) Comparative Toxicogenomics Database (CTD); (5) DrugBank Database; and (6) DisGeNET Database (Score_gda ≥0.3). The official names of the genes (FJD and CRC) were obtained as UniProt, and the species was selected as “Homo sapiens.” Then, the names of the targets were uniformly converted into gene and UniProt ID . FinallyThe target gene of FJD for CRC was input into the STRING database, and its amplification and prediction were carried out. The network diagram of the target protein interaction in vivo was obtained. We first amplified the target protein of FJD against CRC with the STRING database and then input it into Cytoscape software. We use the “analysis network” tool in Cytoscape to get the protein interaction network to obtain the relevant parameters. Based on the four parameters of “Degree,” “BetweennessCentrality,” “ClosenessCentrality,” and “Stress,” we do topology analysis on the PPI network to get hub nodes .GO biological process analysis and KEGG pathway analysis are effective methods to explain the role of target genes . We inpuOn the basis of the compounds, targets, and pathways of FJD in the treatment of colorectal cancer, combined with the functions of Cytoscape 3.7.1 software, a network model of herbs, ingredients, targets, and diseases was constructed. According to this network model, we made a scientific analysis of the relationship between herbs, ingredients, targets, and diseases. We constructed (1) “compound-target” network diagram; (2) “herbal-compound-target” network diagram; (3) PPI network diagram of FJD potential targets for CRC; and (4) “pathway-target” network diagram. Nodes in the network diagram are linked by edges, and these nodes can represent herbs, compounds, targets, and KEGG pathways.According to the TCMSP database, there are 202, 123, and 78 compounds in FJD, respectively. These compounds were screened by OB greater than or equal to 30 percent and DL greater than or equal to 0.18. After filtering and deduplicating values, 46 potential compounds were included. There are 22, 13, and 15 compounds in RS, BX, and TFL, respectively. The composition screening diagram is shown in In this process, we screened 46 active compounds according to chemical similarity and got 212 related targets, including RS 128, BX 91, and TFL 188. As shown in Through the method of multisource database integration, the CRC target data in OMIM, TTD, GeneCards, CTD, DrugBank, and DisGeNET databases are integrated. A total of 1018 CRC targets were obtained according to the screening and removal of duplicate values. The intersection of FJD and CRC related targets was collected and 58 common targets were obtained as the related targets of FJD in CRC.We input 58 target genes of FJD for colorectal cancer into the STRING database and got the PPI network diagram of 468 nodes. The obtained targets are divided into two parts, including 468 targets (58 public targets and 410 other human protein targets), which may represent FJD in in vivo response to CRC. There are nodes 468 and 12359 edges in total. Then, the topological characteristics of PPI are analyzed. We use the “analysis network” tool in Cytoscape to get the protein interaction network to obtain the relevant parameters, and based on the four parameters of “Degree,” “BetweennessCentrality,” “ClosenessCentrality,” and “Stress,” the index above the median value is selected as the key index  Figure  and the P < 0.01.After enrichment analysis of 85 hub targets by DAVID v6.8, we got 112 KEGG pathways and 330 GO biological processes according to P value, we selected the first 20 KEGG  can control the progression of cancer by inducing apoptosis, upregulating DNA repair protein, and maintaining genome stability , 47. At α also has an inhibitory effect [The disorder of apoptosis is directly or indirectly related to the occurrence of tumors . In FJD,y effect . Diosgeny effect .α [α plays a biological role in tumor cells through the STAT3 signaling pathway, so the STAT3 signaling pathway is a bridge between inflammation and tumor [Patients with inflammatory bowel disease (IBD) including ulcerative colitis have a higher probability of developing colitis-related CRC . During α , and forα , activatα . At the nd tumor . PTGS2 end tumor , which hnd tumor . T cell nd tumor . IL-6 prnd tumor .α, by preventing the phosphorylation and degradation of IκB in the process of inflammation [α and IL-6 in the serum of colon cancer mice, relieves tumor-related immunosuppression, and prolongs the survival of colon cancer mice [Chronic inflammation is an important factor leading to CRC. TNF gene is regulated by quercetin, kaempferol, astilbin, and ginsenoside rh2 in FJD and IL6 gene is regulated by quercetin. Quercetin has a strong anti-inflammatory effect. It can reduce some special inflammatory factors including TNF-ammation . Studiescer mice .α (HIF-1 α), which can lead to the enhancement of tumor cell invasion and metastasis [α played a “bridge” role in the process from hypoxia to VEGF increased expression [α can also combine with the hypoxia response element (HRE) of hypoxia response gene, promote the transcription of the downstream target gene to enter the nucleus, promote Akt activation, activate PI3K/Akt signal transduction pathway, inhibit endothelial cell apoptosis, and promote angiogenesis [VEGFA can induce angiogenesis by promoting the abnormal proliferation and migration of vascular endothelial cells, which is conducive to the growth, invasion, and metastasis of tumor . Hypoxiatastasis . Under hpression . Under togenesis .α and VEGFR2 in endothelial cells through PI3K/Akt/mTOR signals, thus showing its antiangiogenesis ability [As we all know, angiogenesis is a necessary condition and a key step for tumor growth and metastasis. Therefore, the inhibition of tumor angiogenesis can block the occurrence, development, invasion, and metastasis of tumors to a certain extent . In FJD, ability .Traditional Chinese medicine has been passed down for thousands of years in China. The idea of Chinese herbal medicine treating tumors is the combination of tonifying deficiency and resisting evil. Based on the combination of modern medicine, a lot of experience in treating malignant tumors has been summarized. In this study, we first recognized from the perspective of network pharmacology that FJD regulates cell proliferation, apoptosis, inflammation, immunity, and angiogenesis through the PI3K-Akt signaling pathway. In addition, we also provide a scientific explanation for FJD in treating “deficiency, dampness, stasis, and toxin,” and we hope to open a window for understanding the complex mechanism of action of Chinese herbal medicine and the essence of more concepts of traditional Chinese medicine.


Hiccups are involuntary contractions of the diaphragm and intercostal muscles, which lead to sudden contractions of the glottis. CAM, such as acupuncture is commonly used and stimulation of the vagus nerve and interference with phrenic nerve conduction are also used to treat hiccups. However, there is little evidence on the effectiveness of acupuncture for cancer-related hiccups. We will plan to conduct a systematic review and meta-analysis of RCTs to evaluate the current evidence on the effects of acupuncture for cancer-related hiccups.The following databases will be searched: PubMed, the Cochrane Library, Wanfang Data, China National Knowledge Infrastructure (CNKI), SinoMed, VIP, Medline, Embase, and EI. Randomized controlled trials will be included to evaluate the effect and safety of acupuncture on cancer patients with hiccups. We will set standards for the curative effect on the basis of the standard of cure and improvement for clinical disease diagnoses. The risk of bias will be assessed by the Cochrane risk of bias tool. We will conduct a meta-analysis and sensitivity analysis, as well as a subgroup analysis if high heterogeneity is present, using Revman 5.3. We will use funnel plots to identify potential reporting biases. We will test asymmetry using Egger test. The Grading of Recommendations Assessment, Development, and Evaluation (GRADE) will be used to evaluate the quality of evidence.This study will be to assess the effect and safety of acupuncture for cancer-related hiccups.This study will assess the effect of acupuncture for cancer-related hiccups and provide reliable evidence for the choice of treatments. Intractable hiccups last longer than 48 hours. Cancer, radiotherapy, chemotherapy, and other cancer-related problems often cause hiccups.Hiccups are involuntary contractions of the diaphragm and intercostal muscles, which lead to sudden contractions of the glottis. Hiccups caused by a central lesion involve the compression of hiccup reflex centers by intracranial and cervical tumors. Hiccups caused by peripheral lesions involve a tumor that metastasizes to the upper diaphragm or diaphragm  or subdiaphragmatic or mediastinal carcinomas, such as gastric carcinoma, lung carcinoma, or alimentary canal carcinoma. Second, cancer-related treatments, such as chemotherapy and radiotherapy, lead to hiccups. Third, electrolyte disturbances and acid-base imbalances cause hiccups in patients with tumors.The main reason for cancer-related hiccups is an increase in excitability of the phrenic nerve and vagus nerve. First and foremost, there are organic lesions, which are characterized as either central lesions or peripheral lesions.; GABA-B receptor agonists; antipsychotics, such as chlorpromazine and haloperidol; antiepileptic drugs, such as sodium valorous, carpentry, and carbamazepine; calcium antagonists, such as nifedipine and nimodipine; acid suppressants, such as omeprazole; central excitatory drugs, such as methylphenidate; and narcotic drugs, such as lidocaine and propofol. Surgical treatments, including unilateral and bilateral phrenic nerve block, are often used. CAM, such as acupuncture and moxibustion therapy, which includes milliacupuncture, a combination of scalp acupuncture and body acupuncture, electroacupuncture, moxibustion, and ear acupuncture, is used. Last, stimulation of the vagus nerve and interference with phrenic nerve conduction are also used to treat hiccups.Currently, treatment options for cancer-related hiccups include medications, surgical treatment, complementary, and alternative medicine (CAM), stimulation of the vagus nerve and interference with phrenic nerve conduction. The medications that are often used include dopamine receptor antagonists, such as metoclopramide1.2 Acupuncture is defined by the insertion of filiform needles into the skin at precise locations (acupuncture points) or other specific areas and the use of twiddle and lifting techniques to treat diseases. Acupressure is known as the noninvasive form of acupuncture. Acupuncture is simple, convenient, effective, and inexpensive, and it has few side effects; it is gaining attention as a treatment for hiccups. Acupuncture is one of the major treatments used in traditional Chinese medicine and is widely used for the supportive and palliative care of patients with cancer.Acupuncture has been used for over 2000 years to treat various diseases in China. It was first described in the Yellow Emperors Classic of Internal Medicine. There is little evidence on the effectiveness of acupuncture for cancer-related hiccups, which was summarized in this systematic review. The total number of RCTs included in the analysis was low, and the risk of bias for the RCTs was high. Therefore, it is of great importance to perform systematic reviews and meta-analyses of the randomized controlled trials (RCTs) on the effects of acupuncture in treating cancer-related hiccups. In this study, we plan to conduct a systematic review and meta-analysis of RCTs to evaluate the current evidence on the effects of acupuncture for cancer-related hiccups.In 2012, systematic reviews (SRs) were conducted to summarize the evidence on the use of acupuncture for the management of cancer-related hiccups.22.12.1.1Randomized controlled trials will be included to evaluate the effect and safety of acupuncture in cancer patients with hiccups.2.1.2Participants diagnosed with acute, persistent, or intractable hiccups for cancer will be included. The participants will include patients who have hiccups as a result of various cancers, including central lesion cancer and peripheral lesion cancer, and cancer-related treatments, including chemotherapy, radiotherapy, other medicines administered for cancer, and electrolyte disturbance and acid-base imbalance treatments. Patients with series of hiccups caused by an issue unrelated to cancer will be excluded.2.1.3The only type of intervention included for the treatment of hiccups in cancer patients is acupuncture. Acupuncture combined with other therapeutic methods to treat hiccups related to cancer will be excluded. All randomized controlled trials (RCTs) using either traditional or contemporary acupuncture will be included. Traditional acupuncture is defined by the insertion of needles in classic meridian points. Regardless of the source of electrical stimulation or fine needle, contemporary acupuncture is defined by the insertion of needles in nonmeridian or trigger points. Treatment without needling will be excluded, such as laser acupuncture, acupressure, auricular acupuncture with a specific pressure device, pharmacoacupuncture, tap-pricking, point injection, or moxibustion. There will be no restrictions on the duration and duration of treatment.2.1.4Types of comparisons will include medication, no treatment, placebo acupuncture, and sham acupuncture. For example, chlorpromazine, sodium valorous, carbamazepine, nifedipine, omeprazole, lidocaine, and other medicinal treatments for hiccups related to cancer will be included. Valsalva movement, drinking a large amount of water, and other physiotherapeutic treatments for hiccups related to cancer will be included.2.1.5We set standards for the curative effect on the basis of the standard of cure and improvement for clinical disease diagnoses. When the hiccups stop after treatment and their associated symptoms resolve, the hiccups are considered to have been cured. The relief of, shortened duration of, or increased intervals between hiccups after treatments indicate improvement in patients. When the hiccup duration and hiccup attacks do not exhibit obvious changes, ineffectiveness of the treatment is noted.2.1.6The secondary outcomes include adverse events related to the interventions.2.2The following databases will be searched: PubMed, the Cochrane Library, Wanfang Data, China National Knowledge Infrastructure (CNKI), SinoMed, Chinese Science and Technology Periodical Database (VIP), Medline, Embase and EI. The search will be conducted in Chinese and English with the following terms:  AND (hiccup OR hiccough OR hiccups OR hiccupping OR singultus OR intractable hiccup OR intractable hiccups) AND (cancer OR tumor OR carcinoma). Table 2.32.3.1The titles and abstracts will be selected and identified by 2 independent reviewers (MR and LY). The full text will be assessed. If there are any disagreements, they will be discussed and resolved by an experienced reviewer (L-SJ). All articles will be stored in a separate database. The 2 reviewers will independently assess all eligible studies according to the inclusion criteria. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) flow chart will be used to report the reasons for excluding and including eligible studies. The study selection procedure will be shown in a PRISMA flow chart Fig. .2.3.2All data from the studies will be independently extracted by 2 reviewers (MR and LY) according to the criteria previously described. We will create a standardized data extraction form to record the extracted data. The data extraction form will include the following items: general information, author, trial characteristics (RCT), year of publication, participants, inclusion and exclusion criteria, acupuncture intervention, control intervention, and outcomes measures. If there are any disagreements, we will discuss them with a third experienced reviewer (L-SJ) to resolve them. When data is unavailable, the corresponding authors will be contacted to obtain the missing data.2.3.3The risk of bias for all included studies will be assessed by 2 independent authors (MR and LY) according to the Cochrane Handbook for Systematics Reviews of Interventions. Any disagreements will be settled by a third experienced reviewer (L-SJ). The following aspects will be assessed for risk of bias: selection, performance, detection, attrition, reporting, and other factors. All included studies will be classified into 3 categories: high risk, low risk, and unclear level of risk.2.3.4For dichotomous outcomes, we will calculate risk ratios (RRs) with 95% CIs. We will calculate mean differences (MDs) with 95% CIs for the continuous outcomes. If different scales are used to measure outcomes, we will use standard mean differences (SMDs) with 95% CIs.2.3.5The unit of analysis will be all individuals participating in the trials.2.3.6When there are missing data, we will call or email the original authors of the articles to obtain the missing data. However, if missing data cannot be obtained, we will only use the data available.2.3.7χ2 tests (P = .1). We will check the I2 statistic to quantify the level of inconsistency. When I2 < 50%, the trials can be considered to represent low heterogeneity. When P < .1 or I2 > 50%, the trials can be considered to represent high heterogeneity. We will perform subgroup analyses if high heterogeneity exists.We will assess the statistical heterogeneity using 2.3.8We will use funnel plots to identify potential reporting biases. We will test asymmetry using Egger test.2.3.9Data synthesis will be performed by Review Manager 5.3. We will use a fixed effects model if there is no statistical heterogeneity. We will use a random effects model if high heterogeneity is found.2.3.10We will perform subgroup analyses if there is high heterogeneity in the included studies. Subgroup analyses will be conducted according to the age, sex, cause of hiccups, type of hiccups, type of acupuncture , type of control group , different acupoints of the electroacupuncture or course of treatment.2.3.11To evaluate the robustness of the pooled results, we will perform a sensitivity analysis to assess the impact of the trials with a high risk of bias. We will compare the findings to determine whether lower quality studies should be excluded based on the size of the sample, the strength of the evidence and its impact on the overall scale of effectiveness.2.3.12The Grading of Recommendations Assessment, Development and Evaluation (GRADE) will be used to evaluate the quality of evidence. The studies will be assigned 1 of 4 possible ratings: very low, low, moderate, or high.3The effective treatment of acupuncture on cancer-related hiccups is significant. We will summarize the available evidence for acupuncture on cancer-related hiccups, and evaluate the effectiveness and the adverse effects of these treatments on cancer-related hiccups. Our findings may assist clinicians make clinical decisions and promising way for treatment of cancer-related hiccups.4The review will not need ethical approval because no issues of participant privacy exist. The results of this systematic review will provide evidence about the effect and safety of acupuncture for hiccups. The results will be disseminated through peer review.Conceptualization: Rui Ma, Yan Li, Songjiang Liu, Wenhui Zhao.Data curation: Rui Ma, Yan Li.Funding acquisition: Yan Li.Methodology: Rui Ma.Software: Rui Ma, Yan Li.Supervision: Songjiang Liu, Wenhui Zhao.Writing – original draft: Rui Ma, Yan Li.Writing – review & editing: Songjiang Liu, Wenhui Zhao.


The program, source code and data can be downloaded from https://github.com/hyr0771/BPFinder.Finding non-standard or new metabolic pathways has important applications in metabolic engineering, synthetic biology and the analysis and reconstruction of metabolic networks. Branched metabolic pathways dominate in metabolic networks and depict a more comprehensive picture of metabolism compared to linear pathways. Although progress has been developed to find branched metabolic pathways, few efforts have been made in identifying branched metabolic pathways via atom group tracking. In this paper, we present a pathfinding method called BPFinder for finding branched metabolic pathways by atom group tracking, which aims to guide the synthetic design of metabolic pathways. BPFinder enumerates linear metabolic pathways by tracking the movements of atom groups in metabolic network and merges the linear atom group conserving pathways into branched pathways. Two merging rules based on the structure of conserved atom groups are proposed to accurately merge the branched compounds of linear pathways to identify branched pathways. Furthermore, the integrated information of compound similarity, thermodynamic feasibility and conserved atom groups is also used to rank the pathfinding results for feasible branched pathways. Experimental results show that BPFinder is more capable of recovering known branched metabolic pathways as compared to other existing methods, and is able to return biologically relevant branched pathways and discover alternative branched pathways of biochemical interest. The online server of BPFinder is available at  Computational search of branched metabolic pathways is a fundamental problem in metabolic engineering and metabolic network analysis, which provides a systematic way of understanding the metabolism and discovering alternative pathways for synthesis of useful biomolecules. We propose BPFinder, a novel computational approach to identify branched metabolic pathways via atom group tracking. Different from other pathfinding methods using atom tracking, BPFinder tracks the movement of atom groups in metabolic network to find linear atom group conserving pathways, and merge the found linear pathways by the selected branched compounds to generate branched pathways. Based on the structure of conserved atom groups in branched compounds, we design two merging rules for branched compounds: overlapping rule and non-overlapping rule. The user can flexibly adopt these rules to accurately find the branched pathways that contain overlapping/non-overlapping conserved atom groups. BPFinder also enables the user to combine the information of compound similarity, Gibbs free energy of reactions, and conserved atom groups to sort resulting pathways. Compared with other existing methods, BPFinder can more accurately recover the known branched pathways. The alternative branched pathways returned by BPFinder reveal that the user can flexibly utilize our proposed merging rules to discover biochemically meaningful pathways of interest. In the past decades, the quantity of metabolic data in the metabolic databases, such as KEGG (Kyoto Encyclopedia of Genes and Genomes) –3 and MeNevertheless, a graph-based pathfinding strategy –21 sometHowever, these approaches require defining the atoms to be tracked. This may result in missing of the pathways that do not conserve the tracked atoms. For solving this problem, based on the KEGG RPAIR database ,30, we pOn the other side, branched metabolic pathways consist of multiple pathways that biochemically interact in metabolic networks. Comparing with linear metabolic pathways, the branched metabolic pathways are dominant in metabolic networks and describe a more comprehensive picture of metabolism ,33. IdenA number of pathfinding methods have been proposed for finding branched metabolic pathways. For example, Gerard.et al  developeHomo sapiens from L-Glutamate to L-Ornithine in KEGG database. This branched pathway consists of the linear pathways 1 and 2. As shown in Branched compound is the compound that acts as branch point to connect multiple linear pathways in metabolic network. It is known that, in many known branched metabolic pathways, linear pathways contain overlapping conserved atom (groups) in branched compounds. For example, Quercus suber from D-Ribose 5-phosphate to beta-D-Fructose 1,6-bisphosphate in KEGG. This branched pathway consists of linear pathways 3 and 4. As shown in On the other hand, some linear pathways in the known branched pathways contain non-overlapping conserved atom (groups) in branched compounds. For example, Branched pathway is a universal characteristic of metabolism. From thBPAT-M is an effective method for finding branched pathways containing non-overlapping conserved atoms in branched compound . For exaHowever, BPAT-M only merges the linear pathways that do not contain overlapping conserved atoms in the branched and target compounds to produce branched pathways. This may result in failing to predict the branched pathways that contain overlapping conserved atoms in the branched and target compounds.de novo synthetic pathway [y] = 0 when x is not mapped to y. Here, the node (atom) mappings in M between GA and GB can be obtained from the atom mapping information between substrate S and product O in the reaction R of the KEGG RPAIR. We can compute the mapped sub-graphs between GA and GB based on the following formulas:The matrices MB of graph GB and the node(atom) mapping matrix M of graphs GA and GB, we can use formula  between nodes u, v of GA is potentially mapped to GB, and MC[u][v] = 0 when the edge between nodes u,v of GA is not mapped to GB. Next, we can use formula  of GA is correctly mapped to the edge of GB when MA[u][v] = 1 and MC[u][v] = 1 and M[u][u’] = 1 and M[v][v’] = 1, and this edge  is called the correct mapped edge for GA and GB. We can use such correct mapped edges to construct the sub-graphs mapped from GA to GB. And these mapped sub-graphs of graphs GA and GB are the atom groups transferred from substrate S to product O in the reaction R.First, based on the adjacency matrix  formula  to compu formula  to check formula , the edgk shortest pathways between source and target compound in the metabolic network constructed by the reactions and compounds of the KEGG RPAIR. Then BPFinder utilizes the Ullmann algorithm to compute the atom groups transferred from source to target compound for each shortest pathway, and selects the linear pathways whose target compound contains the atom groups transferred from the source compound as the linear atom group conserving pathways. Note that, different from our previous linear pathfinding method AGPathfinder  and brings the values of fe(ri) and sim into alignment . αs, αsf and αt are weight parameters, which are used to adjust the relative weights of compound similarity, Gibbs free energy of reaction and conserved atom groups in formula  by the following formula:We can obtain the score of the linear pathway that consists of branched pathway by formula . Combininb is the number of the branched compounds in the branched pathway pb. lb is the number of linear pathways in pb. pi is the ith linear pathway that consists of pb. Score(pi) is the score of pi computed by formula Click here for additional data file.S2 Text(DOCX)Click here for additional data file.S3 Text(DOCX)Click here for additional data file.S4 Text(XLSX)Click here for additional data file.


It uses Oryza sativa (rice) as a reference species for manual curation of pathways and extends pathway knowledge to another 82 plant species via gene-orthology projection using the Reactome data model and framework. It currently hosts 298 reference pathways, including metabolic and transport pathways, transcriptional networks, hormone signaling pathways, and plant developmental processes. In addition to browsing plant pathways, users can upload and analyze their omics data, such as the gene-expression data, and overlay curated or experimental gene-gene interaction data to extend pathway knowledge. The curation team actively engages researchers and students on gene and pathway curation by offering workshops and online tutorials. The Plant Reactome supports, implements and collaborates with the wider community to make data and tools related to genes, genomes, and pathways Findable, Accessible, Interoperable and Re-usable (FAIR).Plant Reactome ( From thierarchy .The Plant Reactome extensively collaborates with various plant genomics projects. The basic platform and data model was adopted and remodeled in collaboration with the Reactome ,2. The iIn the following sections, we summarize updates to the Plant Reactome since our last publication  coveringhttps://plantreactome.gramene.org) is the primary entry point to Gramene's pathway portal. It has an entirely new, fully-responsive front-end design and includes the content revision as well as full implementation of Secure Sockets Layer (SSL). Our new homepage (https://swagger.io). Users can navigate this website to search or browse pathways using a hierarchical schema, access and download data in standardized formats, visualize curated baseline expression of pathway-associated genes fetched from EMBL-EBI Expression Atlas  database using an automated script. In addition, our curators utilize GO annotations for assigning molecular function, subcellular location, and biological process to the reference entities, add cross-references to the ChEBI , PubChemAt present, we host 1824 gene products mapped to 1723 reactions associated with 298 manually curated reference rice pathways. It reflects an increase of 76 reference pathways, 698 reactions and 651 reference gene products since our last update . We havePlant growth and developmental pathways are grouped into vegetative organ development (i.e. leaf and root formation) and reproductive organ development . The plant developmental processes were curated by combining information related to genes, transcripts, and regulatory factors  to provide a better understanding of the biological events occurring in different cellular compartments. For example, the ‘Reproductive meristem phase change in rice’ pathway shows interactions of miRNAs , transcription factors , their target genes and associated protein complexes. Similarly, transcriptional events associated with early embryogenesis and their connections to various hormone signaling pathways are shown in Pathways representing a plant's response to various stimuli or stressors are arranged under two categories: (i) response to abiotic stresses, and (ii) response to biotic stresses. The response to abiotic stress category includes pathways for the response to temperature (heat or cold), nutrient deficiency (phosphate), submergence, drought, salinity and heavy metal  stress. We have recently begun to curate pathways involving plant-pathogen interactions, for example, ‘Recognition of fungal and bacterial pathogens and immunity response’ Figure .We continue to utilize a combinatorial approach involving manual curation of reference rice pathways and automated gene orthology-based projection to scale up pathway knowledge to other species rapidly. Such projections provide the opportunity to expand the reach of the original curation to more plant species and allow users to upload experimental data for analysis from a broader spectrum of plant life. This list of species has now grown from 62 to 82 diverse members of the plant kingdom Figure  with a phttps://github.com/PlantReactome/external-data-analysis/tree/master/orthology_stats), to generate heatmap clustering, as shown in Figure To assess the scope and the quality of comparative pathway projections, we used the Plant Reactome API to grab all terminal pathways from the event hierarchy for all available species and then used two Python scripts, ‘orthology_data_grabber.py’ and ‘orthology_rebuilder.py’  thehttps://www.ebi.ac.uk/gxa)  . From th.uk/gxa) , transcrThe built-in analysis tools are accessible from the Plant Reactome homepage by clicking on the ‘Analyze Data’ icon or from the icon in the header of the Pathway Browser. As described earlier , users cAnother analysis tool, ‘Analyze your data’ , allows https://www.ebi.ac.uk/intact)  , or our /intact) ,29. The /intact)  in one o/intact)  to accesd-xylulose 5-phosphate reductoisomerase (DXR) has 130 interactors; AT2G02500 encoding 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (MCT) has 67 interactors; AT2G26930 encoding 4-(cytidine 5′-diphospho)-2-C-methyl-d-erythritol kinase (CMK) has 43 interactors; AT5G60600 encoding 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (HDS) has 134 interactors; and AT4G34350 encoding 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR) has 68 interactors , ClpC2 (AT3G48870) and ClpD (AT5G51070), and two plant-specific accessory proteins ClpT1 (AT4G12060) and ClpT2 (AT4G25370). The Clp protease complex is composed of a catalytically active proteolytic core formed by ClpP1, ClpP3-P6 and inactive ClpR1-R4 subunits, and is stabilized by two other subunits ClpT1-T2 . The Clpet al. . Previouet al. , includiet al. .Similarly, users of the Plant Reactome can get information about the gene interactors that play an important regulatory role at the transcriptional, translational, or post-translational level using interactome overlay analysis. Furthermore, the data provided on gene-gene interaction are generated using diverse evidence consisting of a mix of heterogeneous methods, including published experiments and predictive computational methods. Thus, unlike expression analysis or metabolite concentration data, interaction data contain an associated confidence score, which the user can apply as a filter using a slider feature. Interactors will appear and disappear as the confidence threshold is crossed on the slider. Each displayed interactor provides a link to the appropriate Ensembl Gramene gene page, where available.When the human Reactome and the Plant Reactome were first envisioned, a relational database model was the predominant choice for complex and rigorous biological data modeling. Thus, MySQL has long served as a solid foundation for describing, storing, and providing access to the biological models depicting metabolic and signaling pathways. Moving forward, as the size and scope of both the human Reactome and Plant Reactome knowledgebases have grown, performance and software design considerations have necessitated a re-evaluation of the most efficient and flexible data engine for modeling pathway space. Graph databases, born of network theory and experimental data retrieval systems, have emerged as a viable alternative to the classical relational database .https://neo4j.com) an exceedingly popular graph database, was selected to drive internal data delivery on the Reactome platform (https://mariadb.org) is still retained as the versioned, reference data source and the foundation for curating with the Reactome Curator Tool used by our biocurators, but Neo4j has become the basis for most of the web-based data retrieval and services within the Plant Reactome. An example of an internal Neo4j server graph is shown in Graph theory helped to ground major advances in omics assembly algorithms ,42, inteplatform . MySQL oUsers, depending on their comfort level and informatics skills, can access publicly-available Plant Reactome data via the pathway browser described earlier, the Content Service API, and single-file and bulk downloads of data and diagrams.https://plantreactome.gramene.org/ContentService) is a RESTful web interface (https://www.ics.uci.edu/∼fielding/pubs/dissertation/rest_arch_style.htm) designed for both internal uses in the Plant Reactome knowledgebase and by external users and resources importing our data. The majority of data access within the Plant Reactome application arrives via responses from requests to the Content Service. Plant Reactome data can be requested remotely via this service. For functional examples of how to query the service, Plant Reactome has implemented a Swagger interface to document and give access to the underlying data calls (https://plantreactome.gramene.org/ContentService/data/species/main’ -H ‘accept: application/json’). All queries are transported via http/https protocol, and results returned from the Content Service consist of responses formatted as JSON . Other examples of service queries include requesting a hierarchical listing of all pathways and reactions for a given species (curl -X GET ‘https://plantreactome.gramene.org/ContentService/data/eventsHierarchy/Populus%20trichocarpa’ -H ‘accept: application/json’), or requesting all participating physical entities present in a pathway or reaction . Projects using the Content Service to access Plant Reactome data include our partners within Gramene and at Ensembl Plants.The Content Service . Gramene's integrated search feature has indexed Plant Reactome gene product listings and uses the Diagram Widget to display gene to pathway association in its gene search results. Using the same association mapping, Ensembl Plants embeds available Plant Reactome pathway diagrams on its individual gene pages  (PubChem (nih.gov)  is an ophttps://plantreactome.gramene.org/download/current/biopax3.zip) and the associated SVG format pathway diagram images (https://plantreactome.gramene.org/download/current/diagrams.svg.tgz) from the Plant Reactome. Subsequently, a BioPAX3 parser based on the raptor2 C library (http://librdf.org) was developed to identify small molecules, proteins and genes, and reactions from each pathway. For small molecules, they used ChEBI cross-references to PubChem compounds and proteins/genes were mapped to NCBI protein/gene identifiers via UniProt cross-references.In order to import and integrate pathway data from the Plant Reactome, PubChem developers first downloaded pathways in the BioPAX3 data description format (An example of the Plant Reactome ‘Thiamin biosynthesis’ pathway display from PubChem page  shows lihttps://github.com/ebi-gene-expression-group/atlas-heatmap) as described previously (https://www.ebi.ac.uk/gxa/home), thus providing two-way connections between the two platforms. The Expression Atlas data can be searched with Plant Reactome stable identifiers for pathways and reactions, For example, if users type a pathway description and species, such as ‘circadian rhythm’ and ‘Triticum aestivum’, the search results will provide two tabs, (i) a list of experiments with baseline expression of genes associated with the query parameters and (ii) heatmap with differential expression data correlated to all annotations for that pathway from the Plant Reactome, UniProt and Ensembl Biomart .To assist Plant Reactome users and the broader community of plant genomics researchers, we offer onsite workshops and outreach booths at the annual Plant Biology meeting (organized by the American Society of Plant Biologists), Plant and Animal Genome conference, Maize Genetics conference’, etc. We routinely disseminate information on the project's developments via scientific journals, webinars, social media, and blog posts. We also produce recorded video-tutorials and conduct online live webinars using case studies from a variety of plant species to demonstrate the utility of the available resources, data, and tools for comparative genomics and pathway analysis ; Undergraduate Learning Experiences in working with Big Data in Agriculture; and the Biological Data Science Capstone course for graduate students at Oregon State University. In each case, students accessed Plant Reactome data via the Content Service, downloadable mapping files, and/or custom data extractions for hands-on exercises, analysis and software development, and customized the content according to the needs of the students and the participating program.Oryza sativa, to an additional 82 species. These functionalities include pathway browsing, analysis and visualization of omics and gene-gene interaction data in the context of plant pathways. In addition, the species comparison tool provides a phylogenetic framework to understand functional conservation and diversification of various reactions and pathways across a wide spectrum of photoautotrophs ranging from cyanobacteria to higher plant clades.As an open-access knowledgebase and resource for plant pathways, the Plant Reactome provides a foundational resource and an environment for learning and discovery that is accessible to plant researchers, educators, and the general public. This integrated resource provides a framework for scaling up functionalities from the reference species, At present, the curation of reference pathways is ongoing, and our focus is on the curation of complex biological processes and their projection onto other species using the gene-orthology framework. The Plant Reactome is updated 2–3 times per year.Plant Reactome and Gramene's commitment to free, open and FAIR access to data, open-source code, and software interoperability has led to an improved cyberinfrastructure for genomic and functional annotation, data visualization and analysis. Our streamlined user interfaces and back-end functions for the integrated search interface demonstrate these efficiencies. Plant Reactome has contributed to the open data initiative by making all its resources freely accessible in standard human and machine-readable formats. We are working with communities to frame recommended best practices to support annotations of genes and pathways, and standard workflows for functional annotation . We conthttps://plantreactome.cyverse.org) to facilitate training, education and integration with the CyVerse platform and user community . We also look forward to extending collaborations with the Plant Metabolic Network , MapMan ommunity .In summary, our scientific, cyber-infrastructure, and community-building efforts and contributions have had a profound impact on the plant research community. We continue to seek feedback from users and consult the experts in reviewing curated pathways. We also invite the community to expand and enhance the model for exploring gene interactions by contributing gene-gene interaction data across the scope of the plant species we currently support.https://plantreactome.gramene.org).Plant Reactome knowledgebase webiste (https://plantreactome.cyverse.org).Plant Reactome Mirros site Powered-By-CyVerse (https://goo.gl/qQ2Pjn).Training and Tutorial videos (gkz996_Supplemental_FileClick here for additional data file.


Astragalus membranaceus (AM) has been widely used for DN in China while the pharmacological mechanisms are still unclear. This work is aimed at undertaking a network pharmacology analysis to reveal the mechanism of the effects of AM in DN. Materials and Methods. In this study, chemical constituents of AM were obtained via Traditional Chinese Medicine Systems Pharmacology Database (TCMSP), and the potential targets of AM were identified using the Therapeutic Target Database (TTD). DisGeNET and GeneCards databases were used to collect DN-related target genes. DN-AM common target protein interaction network was established by using the STRING database. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were carried out to further explore the DN mechanism and therapeutic effect of AM. The network diagrams of the active component-action target and protein-protein interaction (PPI) networks were constructed using Cytoscape software.  Diabetic nephropathy (DN), characterized by hyperglycemia, hypertension, proteinuria, and edema, is a unique microvascular complication of diabetes. Traditional Chinese medicine (TCM) β, JUN, and EGFR. GO and KEGG analyses revealed that these targets were associated with inflammatory response, angiogenesis, oxidative stress reaction, rheumatoid arthritis, and other biological process.  A total of 16 active ingredients contained and 78 putative identified target genes were screened from AM, of which 42 overlapped with the targets of DN and were considered potential therapeutic targets. The analysis of the network results showed that the AM activity of component quercetin, formononetin, calycosin, 7-O-methylisomucronulatol, and quercetin have a good binding activity with top ten screened targets, such as VEGFA, TNF, IL-6, MAPK, CCL3, NOS3, PTGS2, IL-1 This study demonstrated the multicomponent, multitarget, and multichannel characteristics of AM, which provided a novel approach for further research of the mechanism of AM in the treatment of DN. The etiology and pathogenesis of diabetic nephropathy (DN) are complex and diverse . There aAstragalus membranaceus (AM) has a longstanding history and has gained widespread clinical applications in China, with the functions of lowering blood sugar, decreasing blood lipid, eliminating edema, antioxidative stress, and so on [Traditional Chinese medicine (TCM) has been playing an important role in the improvement of life quality and curing disease. In recent years, much attention has been focused on the Chinese herb medicine for the treatment of diabetes and metabolic syndrome. nd so on , 6. In and so on , 8.It is well known that TCM is guided by the theory of TCM and characteristics of being multicomponent, multitarget, and multipathway in the treatment of diseases. AM therapeutic effects on DN by regulating Nrf2/HO-1 signaling pathway, antioxidant stress, nuclear factor-kappa B, and immunoregulation were reported , 10. HowNetwork pharmacology is an interactive network based on the drug-target-gene-disease, including chemoinformatics, bioinformatics, network biology, and pharmacology , 12. It http://lsp.nwu.edu.cn/tcmsp.php) was used to collect the chemical constituents of AM; it was a unique system pharmacology platform that captures the relationships between herbal ingredients, targets, and diseases [Traditional Chinese Medicine System Pharmacology Database , a comprehensive multifunctional data platform that contains integrated diseases, genes, and experimental research [https://www.genecards.org/) as a comprehensive database of functions including genomics, proteomics, and transcriptomics [http://db.idrblab.org/ttd/). It provides information regarding the known and explored therapeutic protein targets, the targeted disease, and the corresponding ID at each of the targets.Information on DN-associated target genes were gathered from the following database. DisGeNET (research . The Geniptomics . Therefohttp://www.ehbio.com/ImageGP/index.php) platform for analysis, and the common targets of compound-disease were obtained as the potential targets for further analysis [The screened chemical targets and disease targets database were imported into the ImageGP , which covered almost all functional interactions between the expressed proteins [https://www.cytoscape.org/) software where the interaction network was drawn and analyzed. The node size was used to reflect the number of combined targets (degree), and the degree greater than twice the median degree of all nodes was selected as a hub according to our previous studies.Protein-protein interaction (PPI) network was derived based on STRING database  pathway enrichment analysis were using the DAVID system (https://david.ncifcrf.gov/) [P value less than 0.05 was employed for further analysis.The biological process (BP), molecular function (MF), cell component (CC), and Kyoto Encyclopedia of Genes and Genomes (KEGG) database (rf.gov/) , 24. In The active compound targets of AM were searched via the TCMSP databases for each chemical component. Eighty-seven compounds were collected from the TCMSP, with the values of OB ≥ 30% and DL ≥ 0.18 properties, applied to screen the active compounds from AM. At last, 16 candidate ingredients were selected out from AM compounds . In totaTo further understand the interaction relationship between the compounds and their corresponding targets, we constructed a compound-target network, as shown in Through the DisGeNET and GeneCards databases, the retrieved results were integrated to obtain the DN-related disease protein targets. As shown in β, NOS3, JUN, and EGFR have higher degree in this process, which explained their significance in the network.A total of 48 potential genes associated with DN were uploaded to the STRING database for analysis. The systematically selected protein targets with a medium confidence score of 0.400 were plotted as an interaction network. The network of protein-protein interactions (PPI)  was estaWe imported the selected potential 48 target genes into the DAVID system for GO analysis. The results revealed that the functions of these potential targets are related to many biological processes, molecular function, and cellular components. It may be important for the occurrence and development of DN. A total of 139 biological processes were enrichment, and the top 20 remarkably enriched BP terms were selected for analysis, such as positive regulation of angiogenesis, inflammatory response, and removal of superoxide radicals . The resP < 0.05. These targets of molecular function mainly involved identical protein binding, cytokine activity, MAP kinase activity, heme binding, peroxidase activity, growth factor activity, and many genes related to the molecular functions described above.A total of 21 molecular function  GO termsP < 0.05. The targets are closely related in extracellular space, membrane raft, cytosol, extracellular matrix, caveola, Golgi apparatus, mitochondrion, and extracellular exosome, and many targets were ranked highly as potential related genes.In all, 16 cell component  GO termsP < 0.01  is one of the common chronic microvascular complications in diabetes mellitus. At present, the incidence of DN is increasing year by year, which seriously threatens the prognosis and quality of life in diabetic patients . The occEvidence showed that some compounds found in this study not only have anti-immune and antistress effects but also have the effect of alleviating endocrine and metabolic disorders. It was reported that quercetin could inhibit glomerular mesangial cell proliferation in high glucose-treated DN mouse and via reactivation of the Hippo pathway . MeanwhiAs we can see from the compound-target network, many targets can be adjusted by multiple compounds, such as NOS3, PTGS2, JUN, MAPK1, and CCL2. Meanwhile, MAPK1, SOD1, TGFB1, and others can be only regulated by quercetin. It indicated that AM has the biological characteristics of multicomponent and multitarget in treating DN. Common target PPI network showed that the targets are also controlled by AM and DN, which revealed that AM could regulate the expression of DN-regulated targets and alleviate DN symptoms. VEGFA (degree = 41), TNF (degree = 40), IL-6 (degree = 39), MAPK1 (degree = 37), and CCL2 (degree = 35) might be the hub target of this network.P < 0.05) were chosen from the 138 enrichment results, which indicated that the major hubs were significantly in multiple biological processes, including positive regulation of angiogenesis, lipopolysaccharide-mediated signaling pathway, angiogenesis, positive regulation of endothelial cell proliferation, and positive regulation of peptidyl-serine phosphorylation, as shown in α play a key regulatory role in this pathogenic process. Among them, DPP4 target inhibitors have been widely used in the treatment of diabetes mellitus, which has an important impact on reducing blood sugar in patients [In order to predict the mechanism of AM in the treatment of DN, we analyzed the candidate targets by performing GO enrichment results, such as biological processes, molecular function, and cellular components. The top 20 GO terms (patients .α, and TGF-β1 mRNA expression was downregulated in DN mice [α, and VEGFA play a crucial role in angiogenesis and endothelial cell proliferation. A further investigation showed that VEGF increased hTERT expression in a mechanism that implicates the PI3K/AKT/mTOR pathway and HIF-1 signaling pathway.HIF-1 signaling pathway activates the expression of VEGFA in inflammatory response to counteract hypoxia and prevent kidney tissue damage in conjunction . VEGFA c DN mice . Meanwhiα, one of the main regulatory factors, which can induce the production of IL-1β, IL-6, and other cytokines, participates in the process of oxidative stress and inflammation and aggravation of renal tissue damage [β, and TNF-α into the serum [α, IL-1β, and ICAM-1 expression was all markedly blocked in DN mice [In the pathogenesis of DN, inflammatory cells will proliferate and differentiate, increase the infiltration of renal tissue cells, and secrete a large number of cytokines to mediate renal tissue damage. In addition, these inflammatory cells and cytokines secreted by them can stimulate and activate kidney cells, regulating the expression of chemokines, cytokines, adhesion molecules, and extracellular matrix components, and aggravate the pathological changes of kidney tissue. TNF-e damage . IL-6, ae damage . Howeverhe serum . What is DN mice . Collectα, and IL-1β entry into glomerular cells by activating nod-like receptor signaling pathway and increase cell damage. CCL2 is a secretory protein that mediates the phosphorylation of C-terminal serine/threonine of receptor protein by binding to the N-terminal of CCR2 on target cell membrane [α, which can repair infection or tissue damage [MAPK1 is an important transmitter of signals from the cell surface to the nucleus in NOD-like receptor signaling pathway. It can activate the release of various inflammatory factors, chemokines, and adhesion factors mediated by NF-KB signaling pathways and aggravate the pathological changes of DN . When inmembrane . It parte damage . TherefoIn summary, AM has significant advantages in the treatment of DN, which is consistent with previous studies. At the same time, the biological functions of active ingredients and their corresponding targets of AM were analyzed by network pharmacological method, which further revealed the molecular biological mechanism of AM in treating DN. It has a significant value to provide theoretical basis for clinical treatment of DN.


Data is collected from multiple providers in standard formats, including the Biological Pathway Exchange (BioPAX) language and the Proteomics Standards Initiative Molecular Interactions format, and then integrated. Pathway Commons provides biologists with (i) tools to search this comprehensive resource, (ii) a download site offering integrated bulk sets of pathway data (e.g. tables of interactions and gene sets), (iii) reusable software libraries for working with pathway information in several programming languages  and (iv) a web service for programmatically querying the entire dataset. Visualization of pathways is supported using the Systems Biological Graphical Notation (SBGN). Pathway Commons currently contains data from 22 databases with 4794 detailed human biochemical processes (i.e. pathways) and ∼2.3 million interactions. To enhance the usability of this large resource for end-users, we develop and maintain interactive web applications and training materials that enable pathway exploration and advanced analysis.Pathway Commons ( Pathway information that describes interactions between molecules in biological processes can help in solving research problems, such as the interpretation of genomics data , generatwww.pathguide.org), increasing the need for integration. Unfortunately, making this knowledge available to the research community has been hindered by fragmentation from the use of diverse data representation schemes and software, making pathway information from multiple sources difficult to combine and use.The number of available pathway and interaction resources has nearly tripled over the last decade, from 190 in 2006 to 702 in 2018 (Pathway Commons (PC) is a resource that aggregates data from publicly available biological pathway and molecular interaction databases and provides it from a single access point on the web . In thisPC has been used to analyze transcriptomics, proteomics and metabolomics data in a large number of projects across diseases to further our understanding of human biology in health and disease ,10–17. SPC currently integrates data from 22 public databases, up from the 9 in our initial report. This has more than tripled the number of pathways (from 1477 to 4794) and interactions (from 687 883 to over 2.3 million) Figure . PC focuThe core software tools driving PC are cPath2 and Paxtools. cPath2 is an open-source database and web application for collecting, storing and querying biological pathway data, and has been completely rewritten based on cPath . cPath2 pathwaycommons.org/pc2/formats; pathwaycommons.org/archives).Users can freely access PC data by either downloading data files , through a web service , or via a series of interactive web-based search tools. Pathway information downloads are made available in BioPAX format, Gene Matrix Transposed (GMT) format, which is used in gene set enrichment analyses ,30, SimpWe have developed a number of tools using the core cPath2 and Paxtools PC infrastructure, including programming libraries as well as desktop and web-based applications for use by a broad audience.apps.cytoscape.org/apps/cypath2), and the CellDesigner BioPAX export plugin allows export from Cytoscape in BioPAX format  and fCoSE graph layout algorithms that are capable of laying out SBGN-styled pathways and complexes (graphs including nesting); the CoSE algorithm has been implemented both in Java and JavaScript .newteditor.org), which is a fully-featured SBGN editor that can load data from PC and other sources.Together, these libraries provide the fundamental components needed to build rich applications to visualize pathways stored in PC and elsewhere. An example of a mature application using these components is Newt . The system recognizes specific search types (e.g. genes) that are typically part of user queries (e.g. ‘cell cycle arrest involving TP53 and CDKN1A’). In this case, the search results display additional information about each gene along with links to additional apps that use this gene-based information as input (below)  Figure . ClickinDepending on the nature of the input query, links to other apps will become available. For instance, if one or more genes are recognized in a search query, they are used to seed an interactive network visualization, called the Interactions app Figure . When thAn additional web-based network visualization app called PCViz, helps users in obtaining details about genes and their interactions from PC. When queried with one or more gene or protein identifiers, PCViz displays an interaction neighborhood both between and surrounding the query genes Figure . InteracAll network views in the above-described web apps are implemented using the Cytoscape.js graph visualization JavaScript library .pathwaycommons.org/guide) that aims to be an online textbook for pathway analysis approaches. A current focus is pathway enrichment analysis that translate observed differences at the gene-level due to state  or experimental testing (e.g. control versus treated samples) into higher-level changes at the pathway level. The Workflows section guides users through a step-by-step, example-driven tutorial to create Enrichment Map visualizations in Cytoscape from the analysis of RNA-seq data using Gene Set Enrichment Analysis (GSEA)  used in popular software packages and apps.A major goal of Pathway Commons is to support the analysis and interpretation of molecular and genomic profiling datasets. To support this, we developed PC Guide (s (GSEA) . The PriThe goal of Pathway Commons is to provide a comprehensive and user-friendly access point for researchers desiring pathway and molecular interaction information to support the analysis of biological data and the discovery of interesting relationships. Since our original report , the resWhile PC incorporates over 20 large pathway and molecular interaction resources and over 700 of these resources are known, the vast majority of pathway resources are unfortunately no longer active or available. Further, even for the 22 databases currently integrated, much effort was required to work with data providers to create or tune BioPAX output to enable integration of the available data. For this reason, even with 700 created pathway-related databases, few additional ones will be integrated. As new databases are created, they can now use PC software components, such as Paxtools, to make available standard BioPAX formatted output. Another major barrier to pathway data access is that only a small handful of pathway and molecular interaction resources that curate data from the literature remain actively funded and they are only able to cover a relatively small part of the rapidly growing literature. To address this, the PC team is advancing text-mining technology to extract pathway information directly from the existing literature ,65,66, agithub.com/PathwayCommons/cpath2/issues or github.com/BioPAX/Paxtools/issues).All software developed as part of Pathway Commons is freely available, open-source and hosted on GitHub repositories. Software for the Pathway Commons project is hosted at github.com/PathwayCommons and software related to the BioPAX initiative is hosted at github.com/BioPAX. Users of Pathway Commons are able to provide feedback and ask questions of the development team using a discussion group (groups.google.com/forum/#!forum/pathway-commons-help). Users can submit developer feedback, file bug reports, and request new features using project-specific issue trackers (e.g. gkz946_Supplemental_FileClick here for additional data file.


Background: Pulmonary hypertension is the major cause of morbidity and mortality in congenital diaphragmatic hernia (CDH). Mutations in several genes that encode signaling molecules of the transforming growth factor β (TGFβ) and bone morphogenetic protein (BMP) pathways have previously been associated with CDH. Since studies on the activation of these pathways in CDH are scarce, and have yielded inconsistent conclusions, the downstream activity of both pathways was assessed in the nitrofen-CDH rat model.Methods and Results: Pregnant Sprague-Dawley rats were treated with nitrofen at embryonic day (E) 9.5 to induce CDH in offspring. At E21, lungs were screened for the expression of key factors of both signaling pathways, at both the mRNA transcript and protein levels. Subsequently, paying particular attention to the pulmonary vasculature, increased phosphorylation of SMAD2, and decreased phosphorylation of Smad5 was noted in the muscular walls of small pulmonary vessels, by immunohistochemistry. This was accompanied by increased proliferation of constituent cells of the smooth muscle layer of these vessels.Conclusions: Increased activation of the TGFβ pathway and decreased activation of the BMP pathway in the pulmonary vasculature of rats with experimentally-induced CDH, suggesting that the deregulated of these important signaling pathways may underlie the development of pulmonary hypertension in CDH. Congenital diaphragmatic hernia (CDH) is a severe developmental anomaly characterized by a diaphragmatic defect. The concomitant pulmonary hypertension (PH) that develops in affected lungs can cause severe problems in the newborn, and is responsible for the high morbidity and mortality in these patients. Although the muscularization of the pulmonary vessels has been demonstrated to be increased in CDH , the patTGFβ is a negative regulator of airway branching in early lung development. However, TGFβ signaling is also active in the vascular and airway smooth muscle and alveolar and airway epithelium during late lung development. Both up- and down-regulation of TGFβ signaling impairs the alveolarization process , 4, depeStudies on the TGFβ pathway in CDH have not yielded consistent conclusions. Decreased expression of TGFβ1 was found at the mRNA level in the hearts of the nitrofen-exposed rat pups with CDH , where iIn contrast to the TGFβ pathway, conclusions drawn in several reports on components of the BMP pathway in CDH are consistent. Reduced expression of BMPR2 , 12 and Investigations conducted to date have focused largely on the expression of receptors in both the TGFβ and BMP pathways, but little is known about the actual activation of these pathways. Therefore, we hypothesized that the analysis of downstream mediators would identify changes in TGFβ and BMP signaling pathways in the lungs of rats in which CDH was induced by nitrofen exposure.Pregnant Sprague-Dawley rats received either 100 mg nitrofen dissolved in 1 ml olive oil or just 1 ml olive oil by gavage on gestational age day E9.5. Nitrofen induces CDH in ~70% of the offspring, while all pups have pulmonary hypertension , 19. At RNA isolation, cDNA synthesis and subsequent qPCR analysis on right lung lobes was performed as previously . The genImmunohistochemistry (IHC) was performed on 5-μm paraffin sections of the left lobe according to standard protocols, using the Envision™ detection system  . PrimarySnap-frozen right lung lobes were homogenized on ice in Carin buffer , containing protease inhibitor Complete . Samples were centrifuged at 14,200 r.p.m. for 15 min and protein concentration in the supernatant was measured using the Bradford method. Subsequently 50 μg of protein per lane was loaded onto an SDS-PAGE and transferred to nitrocellulose membranes using wet blotting. Antigens were detected with TGFβ , pSMAD2 , SMAD2 , pSMAD5 , SMAD5 , and Zeb2 . Cofilit-tests for normally distributed variables. The analyses were performed using SPSS 21.0 for Windows . All statistical tests were two-sided and used a significance level of 0.05.Data are presented as percentages, means (SD) for normally distributed variables. Univariate analyses were performed using independent samples Tgfbr1 and Tgfbr2 receptors, but no difference in the abundance of the ligand Tgfb1 mRNA transcript was noted. The abundance of mRNA transcripts encoding both the receptor-activated SMADs, Smad2, and Smad3, as well as the co-SMAD, Smad4, which together form a signaling complex for translocation into the cell nucleus, was increased in CDH , another receptor in the BMP/TGFβ pathway which mediates the signal of Bmp9 and Bmp10, was slightly increased in CDH. Bmp4, one of the important ligands in this pathway, and the receptor-activated Smad1 and Smad5 showed an increase in CDH  , and incStudies available in the literature have reported conflicting trends in expression of different signaling factors in CDH, which might be explained by the differences in the gestation of the animals under study. In the present study, some variability between samples was also noted, suggesting that small differences in gestational age may have an appreciable impact on trends in the expression of signaling molecules under study.We initially analyzed the TGF-β and BMP pathways in whole lungs, and given our previous report on vascular abnormalities in CDH , we focuIn conclusion, increased phosphorylation of SMAD2 and decreased phosphorylation of SMAD5 was noted in the in the vessel walls of small pulmonary vessels of nitrofen-CDH pups. These data indicate increased activation of the TGFβ pathway and decreased activation of the BMP pathway in the pulmonary vasculature of these animals at day 21 of gestation, possibly leading to increased proliferation of the muscularized vessel wall. Since the different factors in these pathways are differently expressed during gestation and might differ from the human situation, further research must be conducted at different developmental stages, and most importantly, in material of human patients.The original contributions presented in the study are included in the article/The animal study was reviewed and approved by independent animal ethical committee (EMC).DT, RM, and RR: conceptualization and writing—original draft. DM, MB-vK, RM, and RR: data curation. DM, MB-vK, and RR: formal analysis and methodology. RM and RR: funding acquisition and supervision. DM and MB-vK: investigation and validation. DT and RW: resources. DM, MB-vK, RW, DT, RM, and RR: writing—review and editing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Hiccups are a common and poorly understood pathologic phenomenon. While hiccups often occur suddenly and episodically, they may persist for weeks and sometimes months. There is a paucity of data regarding the precise etiology and optimal treatment for persistent hiccups. Frequently considered a benign and frustrating condition, hiccups are sometimes a presenting symptom for pulmonary embolism and cardiac disease. We present a patient with gastroesophageal reflux disease who developed 11 days of recurrent hiccups following an orthopedic procedure. A healthy 68-year-old Caucasian male with a past medical history significant for gout, asthma, and gastroesophageal reflux disease presented for evaluation of subacute right posterior tibial tendon dysfunction. All vital signs were stable on arrival, and all labs were normal. His medications were famotidine 10 mg (recently changed from omeprazole 40 mg), allopurinol 100 mg, and rosuvastatin 10 mg. He drank one glass of wine every evening and had no tobacco or drug use. EKG showed normal sinus rhythm at a regular rate without any evidence of ischemia or infarction. Chest X-ray was without any active cardiopulmonary disease. The patient underwent general endotracheal anesthesia with propofol along with a right popliteal nerve block prior to tibial tendon transfer, medializing calcaneal osteotomy, and posterior tibial tendon reconstruction. He received standard of care intraoperative cephalosporin, and there were no surgical or airway complications. The patient was discharged home with a recommendation to avoid weight-bearing status and to take aspirin 162 mg daily.On postoperative day 1, the patient developed hiccups as described by a sudden diaphragmatic contraction coupled with vocalization. Following the initial vocalization, the patient progressed to silent diaphragmatic contractions up to 9-10 in a row that persisted for 10 seconds . This prThe following medications were trialed without successfully terminating contractions: metoclopramide 10 mg every 6 hours, chlorpromazine 25 mg three times daily, baclofen 10 mg twice daily, clonazepam 0.5 mg as needed, and gabapentin 300 mg three times daily. On day 8 of persistent hiccups, the patient went to the emergency department for evaluation. EKG and all labs, including troponin, were normal. A computed tomography of the chest was obtained and excluded a pulmonary embolism. He was given metoclopramide and aluminum/magnesium hydroxide suspension and discharged home without relief. The hiccups continued for a total of 9 days prompting pulmonary consultation. The patient was then started on gabapentin 600 mg three times daily and omeprazole 40 mg two times daily. One complete day on this regimen resulted in complete cessation of hiccups. He completed a slow taper of gabapentin and omeprazole and was continued on famotidine 10 mg daily monotherapy.Hiccups are referred to as both synchronous diaphragmatic flutter and singultus. They result from a sudden reflexive spasmodic contraction of the diaphragm that precedes sudden closure of the glottis with corresponding vocalization. The hiccup process occurs over 35 milliseconds . The mamAlthough there is no universally recognized etiology of hiccups, there are a variety of hypotheses related to their origin. From a Darwinian perspective, the burping reflex signifies a survival advantage as young mammals who depend on milk for their nutrition need to displace swallowed air in the abdomen from continuous suckling in order to make room for more milk . HiccupsWhile transient hiccups are commonly of unclear etiology, persistent hiccups often result from gastroesophageal dysfunction and disease . DespiteAs cardiac ischemia and pulmonary embolism are potentially life-threatening clinical conditions, persistent and intractable hiccups necessitate further investigation of a potentially lethal origin. Further investigation may include a thorough history, physical, labs, and imaging studies. Frequent lack of definitive etiological understanding in presentations of hiccups leads to considerable variation in treatment approaches. Although not validated in randomized controlled trials, physical maneuvers to terminate hiccups may be successful if the hiccups last for less than 48 hours [In addition to physical maneuvers, pharmacologic treatment, presented in order of escalatory therapy, is indicated if the hiccups persist for more than 48 hours . Most ofThere are limited data to support the use of second-line agents including anticonvulsants, antidepressants, antiarrhythmics, and central nervous system stimulants . If pharAlthough hiccups can be a transient and benign entity, the persistent duration of hiccups in this case for 11 days postoperatively is peculiar. Persistent hiccups in the postoperative setting are an underreported and important phenomenon to both recognize and investigate. This patient had risk factors for venous thromboembolic disease after recent orthopedic operation and subsequent immobility and endothelial injury. After pulmonary embolism and cardiac ischemia were excluded, other, less critical, triggers were explored. Development of recurrent hiccups in this current patient was likely multifactorial given the history of gastroesophageal reflux disease and recent discontinuation of omeprazole. Although the use of propofol carries a rate of hiccups <1% , it is pHiccups are a common and frequently transient nuisance. In rare cases, recurrent or intractable hiccups may signify a potentially life-threatening cardiopulmonary condition and require clinical evaluation. In addition to a thorough history and physical exam, labs, imaging, and further diagnostic tests may be indicated to investigate the origin. Vagal stimulation as well as therapies targeting dopaminergic and GABAergic pathways may provide relief from persistent hiccups. While hiccups can be a benign entity, persistent hiccups should prompt evaluation for serious, and potentially fatal, life-threatening conditions.


Our analysis of genomic neighbourhood of cad and motifs of the encoded polypeptide and its orthologs suggests a close functional association between cAD1 and ApbE protein (Q82Z24), a FMN insertion and trafficking facilitator. The cad and apbE orthologs were coupled in the genomes and ApbE-specific motifs for FMN covalent attachment were identified in cad-encoded protein sequence and its orthologs. These findings suggest a potential role of FMN-based reductase function of the cAD1 lipoprotein precursor in its processing and release of the active sex pheromone peptide. They may lead to a new approach in prevention of antibiotic resistance spread via targeting sex pheromone processing chaperones or by suppression of the FMN availability and covalent binding. This methods can be also applied to a controlled evolution of bacterial pathogenicity in microbial fuel cells, as the findings suggest the crosstalk between bacterial pathogenicity and bacterial electro-activity.Functional coupling and comparative genomics analysis have been applied to study functional associations of orthologs of enterococcal cAD1 sex pheromone (P13268) known to be responsible for biofilm formation, conjugative plasmid transfer and spreading of bacterial antibiotics resistance. cAD1 peptide pheromone is released from the membrane lipoprotein with the peptide precursor encoded by a gene  As other known bacterial sex pheromones, cAD1 is secreted by plasmid-free cells to be recognised by plasmid-baring cells and initiate cell clumping, conjugation and the virulence pAD1 plasmid transfer  A n A n8] A transfer , 11], ,  ar ar17] ar2cad homologs in different genomes suggests close functional association between cAD1 and ApbE protein (Q82Z24), known to be involved in thiamine metabolism in Enterobacteria . T. Tcad hohttp://pubseed.theseed.org) have been used in this study. The SEED provides precise genome annotations that are cross-validated and unified across the genomes. It is a platform for a comparative functional and phylogenetic analysis of multiple sets of genomes and the basis for RAST annotation service. , e, eapbE hcad-apbE gene cluster in a genome may be used as a predictive measure. There are still no reports for electroactivity of Oenococcus genera but, based our analysis, for instance, we can suggest that this genus also has electrogenic properties. We may even suggest, taking in mind pheromone properties of cAD1, that its processing can be one of regulators of relevant bacteria response to an applied electric potential and, for these bacteria groups, a trigger of the anodic biofilm formation in MFC ..cad-apbEcad gene functional coupling suggests that prenylation of the terminal cysteine , , , , , , . T. TEnteropathways , [8], Ho