Helicobacter pylori, the effect of eradication in Helicobacter pylori-negative cases is unclear. In this case report, we describe a surgical option for a case of Barrett’s esophageal cancer with concurrent gastric mucosa-associated lymphoid tissue lymphoma.Although the first-line therapy for early-stage gastric mucosa-associated lymphoid tissue lymphoma is the eradication of A 79-year-old man was admitted to our hospital with Barrett’s esophageal cancer and gastric mucosa-associated lymphoid tissue lymphoma. Initially, we performed endoscopic submucosal dissection for Barrett’s esophageal cancer. Since residual tumor was observed after the endoscopic submucosal dissection, we performed an esophagectomy with two-field lymph node dissection, which was followed by placement of a gastric conduit via the posterior mediastinal route. He was discharged 14 days after surgery. Although no additional treatment exists for mucosa-associated lymphoid tissue lymphoma, no recurrent disease has been detected to date.An option to use a portion of the stomach with low-grade malignant mucosa-associated lymphoid tissue lymphoma as a conduit after esophagectomy was suggested. Helicobacter pylori (Hp) infection. Gastric MALT lymphoma usually manifests as a low-grade lymphoma, and in a minority of cases, low-grade disease transitions into a high-grade malignancy. The eradication of Hp infection with standard therapy leads to complete remission of the lymphoma in approximately 80% of cases [Barrett’s esophagus is present in 10–20% of patients with gastroesophageal reflux disease and 2–7% of the general population, with an incidence between 23.1 and 32.7 per 100,000 individuals [Subtotal esophagectomy followed by reconstruction using a gastric conduit is a standard procedure in patients with esophageal cancer . AnotherA 79-year-old Japanese man was referred to our hospital for esophageal cancer. Gastrointestinal endoscopy revealed a 37-cm tumor starting at the incisors with Barrett’s esophagus Fig. a and mulEsophagectomy with two-field lymph node dissection and gastric conduit reconstruction via the posterior mediastinal route were performed. Since a large lymph node was located in the mesentery of the small intestine, both the lymph node and the small intestine were resected together. The operation time was 6 h and 18 min, with an estimated blood loss of 80 mL.The resected tumor measured 30 × 20 mm in short-segment Barrett’s esophagus and contained a post-ESD scar Fig. a. PatholThe patient’s postoperative course was uneventful. The patient resumed eating on postoperative day 7 and was discharged from the hospital on postoperative day 14. No additional treatment was administered for MALT. Thus far, with respect to the MALT lymphoma, no recurrent disease except that in the gastric tube has been observed.According to the European Society of Medical Oncology guidelines for gastric MALT lymphoma , the patAlthough FDG uptake during PET-CT is frequently observed in many MALT lymphomas with high-grade malignant potential , this caInitially, we decided to perform ESD because Yu et al. reported that elderly patients who underwent esophagectomy had significantly lower cancer-related and 5-year overall survival (OS) rates than relatively younger patients . HoweverWe believe that chemoradiation therapy may likely worsen the stenosis. Therefore, we considered surgery to be the best option in this case. We elected to perform radical esophagectomy as an additional treatment. For this elderly patient, it was important to determine which organ should be used for reconstruction after esophagectomy. Reconstruction using the colon or jejunum should be performed after total gastrectomy . ActuallInitially, we considered the MALT lymphoma to be a localized lesion in the stomach, but a pathologic analysis revealed that the lymphoma in the mesentery of the small intestine was actually metastases of the MALT lymphoma. In many cases, the MALT lymphoma is less likely to have a worse prognosis even if residual lymphoma cells are present . RadiatiThieblemont et al. published data on a new prognostic factor for MALT lymphoma. Three individual factors with the greatest prognostic significance were age > 70 years, Ann Arbor stage III or IV, and an elevated lactate dehydrogenase (LDH) level. These factors allowed the three groups to be classified as follows: low, intermediate, and high risk . The 5-year OS rates in the low-, intermediate-, and high-risk groups were 98.7%, 93.1%, and 64.3%, respectively . Since tesophageal cancer, gastric MALT lymphoma, and surgery, this is the first report of esophageal cancer with gastric MALT lymphoma. We believe that it is possible to use the stomach despite the presence of low-grade MALT lymphoma as a reconstructed esophagus in elderly patients. If the stomach with MALT lymphoma can be used as a reconstructed organ, the choice of surgery may be expanded, and postoperative complications can be reduced.To our knowledge, based on a search for literature published between January 1983 and March 2020 in the Japan Medical Abstracts Society and PubMed databases using the key words Here, we present a surgical option for an elderly patient with Barrett’s esophageal cancer combined with gastric MALT lymphoma. Considering the balance of surgical risk and malignant potential, we used a gastric conduit containing MALT lymphoma.


The present study made use of a network pharmacological approach to evaluate the mechanisms and potential targets of the active ingredients of Epimedium for alleviating mild cognitive impairment (MCI) and treating Alzheimer's disease (AD).  The active ingredients of Epimedium were acquired from the Traditional Chinese Medicine System Pharmacology database, and potential targets were predicted using the TCMSP target module, SwissTargetPrediction, and PharmMapper database. Target proteins correlating with MCI and AD were downloaded from the GeneCards, DisGeNet, and OMIM databases. The common targets of Epimedium, MCI, and AD were identified using the Jvenn online tool, and a protein-protein interaction (PPI) network was constructed using the String database and Cytoscape. Finally, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the common targets was performed using DAVID, and molecular docking between active ingredients and target genes was modeled using AutoDock Vina.  A total of 20 active ingredients were analyzed, and 337 compound-related targets were identified for Epimedium. Out of 236 proteins associated with MCI and AD, 54 overlapped with the targets of Epimedium. The top 30 interacting proteins in this set were ranked by topological analysis. GO and KEGG enrichment analysis suggested that the common targets participated in diverse biological processes and pathways, including cell proliferation and apoptosis, inflammatory response, signal transduction, and protein phosphorylation through cancer pathway, MAPK signaling pathway, PI3K-Akt signaling pathway, HIF-1 signaling pathway, sphingolipid signaling pathway, FoxO signaling pathway, and TNF signaling pathway. Molecular docking analysis suggested that the 20 active ingredients could bind to the top 5 protein targets.  The present study provides theoretical evidence for in-depth analysis of the mechanisms and molecular targets by which Epimedium protects against MCI, AD, and other neurodegenerative diseases and lays the foundation for pragmatic clinical applications and potential new drug development. The incidence of Alzheimer's disease (AD) is increasing significantly with the globally increasing average population age . AD is aMCI is a clinically recognized neurodegenerative disease that is defined by cognitive changes, and represents a transition stage between healthy aging and dementia . This trModern pharmacological studies have demonstrated that Epimedium is a flavonoid compound with estrogen-like effects and simultaneously possesses anti-aging, antioxidant, anti-apoptotic, and anti-inflammatory properties , 16. Estβ production and amyloid plaque deposition, as well as enhancing learning and memory capacity [in vitro [Epimedium, also known as Horny Goat Weed, is a perennial herbaceous plant that is widely applied in Asian countries, such as Japan, South Korea, and China , 19. Epicapacity . Icariinin vitro . IcariinTraditional Chinese Medicine (TCM) has the peculiarities of multiple ingredients, multiple targets, and multiple pathways, and using experimental methods to explain the potential mechanisms of a TCM can therefore be difficult. Network pharmacology is a new methodological system based on pharmacology and pharmacodynamics that has the potential to enable exploration of the efficacy of TCMs . By analIn the present study, we made use of network pharmacology to investigate the active ingredients, drug targets, and key pathways of Epimedium against MCI and AD. The prediction of interactions between proteins implicated in MCI and AD and the active ingredients of Epimedium will highlight potential targets for further molecular studies, which may uncover the beneficial components of Epimedium and lead to the development of an effective treatment for neurodegenerative diseases. The experimental workflow is shown in http://lsp.nwu.edu.cn/browse.php) database. The TCMSP contains comprehensive information about traditional Chinese herbs, including the chemical structure, oral bioavailability, drug-likeness, half-life, Caco-2 intestinal epithelial permeability, blood-brain barrier, and associated drug-target-disease network. We used pharmacokinetic properties, including absorption, distribution, metabolism and excretion (ADME), oral bioavailability (OB) ≥ 30%, and drug-likeness (DL) ≥ 0. 18%, to screen the active ingredients of Epimedium. Compounds with OB ≥ 30% have good absorption, and a DL ≥ 0. 18% is chemically conducive to drug development [http://pubchem.ncbi.nlm.nih.gov/) for ulterior target prediction [We searched for the active ingredients of Epimedium in the Traditional Chinese Medicine System Pharmacology , and PharmMapper (http://www.lilab-ecust.cn/pharmmapper/) databases to predict the potential targets of Epimedium active ingredients. The TCMSP database provides the chemical composition of Chinese herbs, as well as all-sided drug targets of active ingredients, and their relation to diseases [http://www.UniProt.org/) [We used the TCMSP target module, SwissTargetPrediction (diseases . SwissTadiseases . PharmMadiseases . The resot.org/) .https://www.genecards.org/), DisGeNet (http://www.disgenet.org/), and OMIM (https://omim.org/) databases are regularly updated online tools containing information about human genes and genetic diseases. GeneCards includes proteomic, genetic, genomic, transcriptomic, clinical, and other information, which can be applied to predict human gene functions [Homo sapiens.”GeneCards (unctions . DisGeNeunctions . OMIM isunctions . In the https://string-db.org/) to generate a protein-protein interaction (PPT) network [We used the Jvenn online tool to generate a Venn diagram and identify common targets for Epimedium, MCI, and AD. Common targets were submitted to String ( network . The obthttps://david.ncifcrf.gov/), which is an online biological knowledge base and analysis tool, to identify enriched cellular components (CC), biological processes (BP), molecular function (MF), and KEGG pathways [GO is a constantly updated and internationally applicable gene function classification system that can be used to analyze functional enrichment of genes and proteins. KEGG pathway enrichment analysis provided a deeper explanation of different gene and protein functions by identifying enriched biochemical pathways in datasets. To gain insight into the gene functions and signaling pathways of the Epimedium targets, we used DAVID (pathways .https://pubchem.ncbi.nlm.nih.gov/) and used Open Babel 2. 3. 2 software to convert SDFs into PDB files [http://www.rcsb.org/) to obtain the structural data of binding sites on AKT1 (PDB ID: 4EKL), ALB (PDB ID: 6JE7), CASP3 (PDB ID: 2J32), IL6 (PDB ID: 1ALU), and TNF (PDB ID: 5MU8) [Molecular docking is a burgeoning approach to explore drug mechanisms, elevate the pharmacological effect of drugs to the molecular level, and provide a theoretical basis for treating diseases. In the present study, the top five protein targets of Epimedium were selected based on PPI network analysis and included serum albumin (ALB), RAC-alpha serine/threonine-protein kinase (AKT1), interleukin-6 (IL6), caspase-3 (CASP3), and tumor necrosis factor (TNF), to perform molecular docking with the 20 active ingredients. We retrieved the SDFs of the 20 compounds from Pubchem  . We usedA total of 130 chemical ingredients of Epimedium were obtained from the TCMSP database  indicating stronger interactions  is a fundamental treatment principle of TCM, which has accumulated vast experience in treating dementia over hundreds of years of clinical practice. Epimedium is one of the cross-sectional herbs that is prescribed as part of Chinese medicine, owing to its anti-apoptotic and anti-oxidative properties, and it is frequently applied to cure central nervous system diseases like forgetfulness, cognitive impairment, and dementia. The mechanism by which Epimedium exerts its therapeutic function is still poorly defined due to the complexity of its constituents. To enable better understanding of the mechanisms behind the protective effects of Epimedium, in the present study we made use of network pharmacology to explore multiple molecular perspectives, and to provide a theoretical basis for the treatment of neurodegenerative diseases.β1-42 and prevented the expression of amyloid precursor protein and β-site APP cleaving enzyme 1 in AD animal models [β polymerization, APP, and BACE1 expression and reduces oxidative stress [We identified 20 chemical compounds as the potential active ingredients of Epimedium, many of which have been widely reported to possess inhibitory effects on neurological aging. For instance, icariin was used in Tg2576 mice (containing the Swedish AD mutant gene), with a standard dose of 60 mg/kg. After three months of administration, the hippocampal nerve regeneration and working memory ability of the mice had improved . Icariinl models . Anotherl models . Quercetl models . It alsoe stress . Our molκB-dependent gene transcription. CREB1 phosphorylation induces the binding of auxiliary proteins that are also necessary for the transcription of pro-survival genes, such as BCL2 and MCL1 [In the present study, KEGG pathway and GO enrichment analysis was carried out for 54 common targets of Epimedium, MCI, and AD. Based on these analyses, we identified a subset of proteins, AKT1, MAPK1, CASP3, IL6, TNF, BCL2, TP53 and NOS3, which exert their effects via multiple different biochemical pathways. Such proteins are likely to be “master regulators” and could be major mediators of neurodegenerative diseases. For example, AKT1 regulates cell survival, proliferation, and growth and metabolism mediated by serine and threonine phosphorylation of downstream substrates , 42. AKTand MCL1 . The MAPand MCL1 .β. IL6 exacerbates the expression of inflammatory factor IL10 in downstream signaling pathways and promotes the oxidative stress response, leading to apoptosis or atrophy of neuronal cells. Moreover, IL6 promotes β-amyloid deposition and affects the integrity of neuronal axons or dendritic membranes [BCL2 is a pivotal apoptosis regulator in the apoptotic signaling pathway. BCL2 inhibits apoptosis in the outer mitochondrial membrane and inhibits the reduction of glutathione and redox state, controls membrane potential, and prevents the release of pro-apoptotic proteins such as cytochrome C and apoptosis-inducing factor AIF to suppress apoptosis , 46. Casembranes , 50.β-amyloid deposition and hyperphosphorylation of tau protein bring about cell autophagy, function loss, and apoptosis, and these pathological reactions are compactly associated with MAPK activation and signal conduction [in vivo and in vitro, preventing P38MAPK and MK2 activation and downregulating the NF-κB and SAPK/JNK-c-Jun signaling pathways to reduce cerebral cortex neurodegeneration and ameliorate cognitive function [The MAPK signaling pathway is one of the essential signaling systems in living organisms and has been broadly studied in plants, insects, and mammals. MAPK signaling participates in a wide variety of physiopathological processes, such as cell growth, development, differentiation, and apoptosis, and is a regulator of inflammation and the stress response . Neuroinnduction . Pinocemfunction , 54. DruκB, CREB, mTOR, GSK-3β, caspase, and the BCL2 family [β and ROS in in vitro AD models and APP/PS1 transgenic mouse models. Polyphenolic anthocyanins reduce oxidative stress to exert their neuroprotective effects, improve memory dysfunction, and delay neurodegenerative diseases through the regulation of the PI3K/Akt/Nrf2 pathway [The PI3K-Akt signaling pathway participates in the processes of cell reproduction, differentiation, and apoptosis and has many effectors downstream. Akt is the central link in the PI3K-Akt signaling pathway, and it translocates from the cell membrane to the nucleus, where it affects many transcriptional regulators and factors after activation, such as NF-2 family . The PI3 pathway .β and increasing the risk of AD [α upregulates BACE1 expression and catalyzes β-cleavage of APP, while BACE1 expression in the hippocampus and cerebral cortex of mice is reduced by the lack of HIF-1α. The above examples demonstrate that the HIF-1 signaling pathway is one of the most important pathways for regulating APP amyloidosis [The HIF-1 signaling pathway is a physiological response to hypoxia that is mediated by hypoxia-inducible factors (HIFs) and activated or initiated in mammals to adapt to hypoxic environments, also allowing for the accommodation of hypoxia and increased cellular energy metabolism . HIFs arsk of AD . Animal loidosis .β25-35 was applied to SH-SY5Y neuronal cells to create an AD model to induce SPHK1 inhibition and neuroamide accumulation, but the upregulation of sphingosine kinase prevented cell death and exerted a protective function [The sphingolipid signaling pathway plays crucial roles in aging, neurodegenerative diseases, and immune regulation . Sphingofunction . Sphingofunction .Forkhead box class O (FoxO) is a mammalian forkhead transcription factor that interferes with differentiated cell survival and stem cell proliferation and is a potential target for the control of neurological diseases . The Foxα, inducing destructive or toxic biological effects when it binds to specific receptors [α, such as NF-κB, P38 MAPK, and TNF receptor antagonist inhibitors, may be potential candidates for the treatment of cognitive impairment, AD, and other neurodegenerative diseases.Tumor necrosis factor (TNF) is a cytokine ligand that participates in systemic inflammation. Both TNF and its type 1 receptor (TNFRI) are involved in AD-associated cerebral neuroinflammation and regulate amyloid formation through secreted enzymes . Aging, eceptors . DevelopAs mentioned above, the targets identified in our study are consistent with previous attempts. Epimedium active ingredients have confirmed interacting partners with proteins that are important regulators of health and disease; these interactions could point future research towards novel therapeutic strategies for treating neurodegenerative diseases.In summary, we made use of network pharmacology to reveal the interactions between the possible active ingredients of Epimedium and known therapeutic genes associated with MCI and AD. Through further screening, we verified the potential targets of Epimedium at the molecular level and predicted possible major regulatory pathways that are involved in its neuroprotective effects. The pathogenesis of MCI and AD is complicated, forming a multi-path and multi-target action mode with interlaced signal pathways. The active ingredients of Epimedium against MCI and AD may function by modulating these pathways, demonstrating the multi-component and multi-target capabilities of Chinese herbs. Our findings provide a hypothetical basis for further practical molecular studies of the interactions between Epimedium and these proteins. The combination of Chinese medicine and modern analytical methods will allow in-depth studies of Epimedium's efficacy at treating neurodegenerative diseases. Our findings could lay the foundation for pragmatic clinical application of Epimedium, and new drug development.


IMPALA (Inferred Modularization of PAthway LAndscapes) integrates information from high throughput gene expression experiments and genome-scale knowledge databases to identify aberrant pathway modules, thereby providing a powerful sampling strategy to reconstruct and explore pathway landscapes. Here IMPALA identifies pathway modules associated with breast cancer recurrence and Tamoxifen resistance. Focusing on estrogen-receptor (ER) signaling, IMPALA identifies alternative pathways from gene expression data of Tamoxifen treated ER positive breast cancer patient samples. These pathways were often interconnected through cytoplasmic genes such as IRS1/2, JAK1, YWHAZ, CSNK2A1, MAPK1 and HSP90AA1 and significantly enriched with ErbB, MAPK, and JAK-STAT signaling components. Characterization of the pathway landscape revealed key modules associated with ER signaling and with cell cycle and apoptosis signaling. We validated IMPALA-identified pathway modules using data from four different breast cancer cell lines including sensitive and resistant models to Tamoxifen. Results showed that a majority of genes in cell cycle/apoptosis modules that were up-regulated in breast cancer patients with short survivals (< 5 years) were also over-expressed in drug resistant cell lines, whereas the transcription factors JUN, FOS, and STAT3 were down-regulated in both patient and drug resistant cell lines. Hence, IMPALA identified pathways were associated with Tamoxifen resistance and an increased risk of breast cancer recurrence. The IMPALA package is available at  For example, BIRC5 intersects multiple pathways essential for cell proliferation, survival, and resistance to growth inhibition3. The goal is to identify anticancer drugs that interfere with multiple molecular targets in different subcellular compartments while minimizing damage to normal cells5. However, to be effective, such combinatorial drug design must address the complexity and heterogeneity inherent in most cancers, which, in turn, requires the development of systems biology tools to characterize multiple cancer-specific pathways and signaling networks6. Although there are computational methods for deciphering complex signal transduction pathways by integrating multi-platform genomic data with biological knowledge like GESA7 and PARADIGM8, their ability to discover novel pathway interactions is limited.A new direction9 provides an alternative source of information for signaling pathway identification, which typically has been formulated as a mathematical problem of reconstructing paths between source and target genes10. The main challenge for such methods—which include, for example, Netsearch11, random color coding12, integer linear programming (ILP)10 and ResponseNet14—is inferring signaling directions between genes given non-directed PPI network information. Gitter et al. proposed to use maximum edge orientation (EO) on a PPI network to determine the most likely signaling directions that fulfil global optimality15. However, EO relies heavily on the assumption that most biological pathways are short (length < 5) in order to accommodate the requirement of exhaustive enumeration of possible pathways and fails to utilize important biological knowledge such as subcellular information. Hence, assigned signaling directions are usually difficult to interpret in a biological meaningful way. Furthermore, EO fails to jointly analyze individual pathways for structural or functional similarities, which are important for studying pathway crosstalk.The current abundance of genome-wide protein–protein interaction (PPIs) dataIMPALA (Inferring Modularization of PAthway LAndscape) integrates gene expression data and biological knowledge within a Bayesian framework to reconstruct aberrant pathway modules. IMPALA defines three potential functions representing gene expression, gene co-expression and prior network interactions. These functions, which jointly measure the aberrancy of individual pathways, are converted to probability distributions for pathway sampling. IMPALA estimates edge directions by aggregating pathway samples. To study crosstalk between multiple pathways, sampled pathways are clustered into interconnected modules based on structural similarities.Here we use IMPALA to identify and explore estrogen-receptor (ER) signaling associated with Tamoxifen resistance in breast cancer and to build an aberrant pathway network connecting ER to transcription factors involved in cell proliferation and apoptosis. The identified pathway network was significantly enriched in ErbB, MAPK and JAK-STAT signaling components. Pathway clustering by IMPALA identified key functionally associated ER signaling, cell cycle and apoptosis modules with crosstalk. We validated the expression of module genes using breast cancer cell line models. Hence, IMPALA provides a novel and effective approach to investigate alternative pathways and pathway crosstalk in cancer cells.16 and identified aberrant signal pathway transduction associated with Tamoxifen resistance. We normalized the data using PLIER (http://www.affymetrix.com), and then corrected the batch effects using ComBat17. A 5-year cut-off on distant-metastasis-free-survival (DMFS) was used to divide Loi samples into ‘early recurrence’ (DMFS ≤ 5 years) and ‘late recurrence’ (DMFS > 5 years) groups, yielding 88 and 92 samples, respectively.IMPALA is a Bayesian approach to infer signaling pathway modules from gene expression data Fig. . We appl18 a gene network was extracted from protein–protein interaction data to predict genes and interactions likely associated with ER signaling. Candidate pathways were constructed starting from the estrogen receptor ESR1 gene and targeting breast cancer-associated transcription factors, such as JUN, FOS, STAT1, STAT3, STAT5A, ELK1, and ETS1  Gibbs sampling to Infer Signal Transduction (GIST) and (2) Structural Organization to Uncover pathway Landscape (SOUL) , ErbB , MAPK , and JAK-STAT  signaling pathways, each of which plays a key role in breast cancer21. We further examined the association of the pathway network with Tamoxifen recurrence by using the network to predict the survival of breast cancer patients based on a similar, but independently generated gene expression dataset (termed Symmans)22. Specifically, using the above ER signaling pathway network and the Loi gene expression data, we trained a NetSVM classifier23 to group samples as early or late. Threefold cross-validation using Loi data returned the area under ROC curve (AUC) as 0.8. Applying the classifier to the Symmans dataset, which includes 103 patient samples, we obtained a prediction AUC of 0.79. Kaplan Meier analysis of Symmans data returned a hazard ratio of 3.26  include signal transduction genes like YWHAQ, YWHAZ and PTPN11, the chaperone HSP90AA1, and STMN1, which functions in cytoskeletal rearrangements. HSP90AA1 is an intracellular gene that is actively expressed in breast cancer cells—high levels of which correlate with a low chance of survival31. TSC2 is a negative regulator of mTOR, which in turn inhibits autophagy. Although cellular stress from therapeutic drugs can induce cell death via autophagy, lysosomal degradation or prolonged stress32 can sustain long-term survival or dormancy by enabling autophagy of some tumor cells33.We found ESR1 and IGF1R to be overexpressed in the ‘late recurrence’ group  dataset; consisting of 47 ‘early recurrence’ and 56 ‘late recurrence’ samples based on a 5-year DMFS cutoff). Source receptor genes were the same as selected for the Loi data analysis, while target transcription factors were identified using GibbsOS for ER signaling, cell cycle, and apoptosis  shown in Fig. 34. MCF7RR-STR and LCC2 are Tamoxifen resistant, whereas MCF7-STR and LCC1 are sensitive. As shown in Fig. We used in vitro breast cancer cell line models to validate the expression of genes in aberrant pathway modules identified by IMPALA. Four MCF7 derived cell models were included in the analysis: MCF7-STR, MCF7RR-STR, LCC1, and LCC2IMPALA characterizes intracellular signal transduction pathways by integrating multi-platform data and by identifying crosstalk among pathways. Using this approach, we identified breast cancer-associated aberrant pathways by integrating breast cancer gene expression data with protein-DNA and protein–protein interaction data, and with published information regarding signaling pathways.IMPALA has several notable advantages over existing methods. First, GIST allows users to incorporate the subcellular location of genes in order to focus on signal transduction components in the nucleus, the cytoplasm, or the plasma membrane. Second, most existing methods either fail to assign signaling directions between genes or else infer signaling direction in an ad hoc manner. GIST assigns a posterior probability for each signaling direction, thereby estimating a degree of confidence. Third, SOUL models network components as structurally related modules to better identify local modules within a large-scale pathway landscape. This identifies overlap between modules, which corresponds to crosstalk between pathways.35. Here, IMPALA revealed that breast cancer-associated pathway modules are structurally interconnected with crosstalk between ER signaling, cell cycle and apoptosis pathways, thereby imparting tamoxifen resistance. And, by characterizing the pathway landscape, IMPALA systematically categorized complex pathway interactions into within-module and between-module interactions. This echoes the increasing emphasis among researchers on networks, rather than pathways, as a reflection of the complex and integrated nature of molecular signaling.Unravelling signaling pathways from complex molecular networks in cancer cells is challengingIMPALA applies GIST to identify signaling pathways by integrating gene expression data with protein–protein interactions (PPIs), and SOUL to explore the pathway landscape for pathway module and crosstalk identification.36 to the Loi and Symmans datasets, respectively, and selected transcription factors significantly associated with the survival difference, as listed in Supplementary Tables To build the candidate pathway landscape, we pre-selected the source and target genes for each signaling pathway. Specifically, we selected ESR1 for ER signaling, membrane receptors and the growth factors EGFR, TGFB1, IGF1R, INSR, FGFR1 for cell cycle, and canonical death receptors IL1R1, FAS, and TNFRSF1A for apoptosis. Based on literatures, we selected transcription factors associating to breast cancer recurrence as pathway targets. Categorized transcription factors selected for ER signaling, cell cycle, and apoptosis are listed in Supplementary Table 18 to pre-screen human PPIs for an ER-related, Tamoxifen resistant sub-network. An ESR1-centered PPI subnetwork including 2326 genes  was selected.To build a candidate pathway landscape, we used MRWOGTo infer signal directions between genes, GIST constructs a flow network of a given pathway length between source and target genes. To weight the flow network, node (gene), edge (interaction) and flow (network) potentials are defined for individual pathways. GIST converts the three potentials into a joint probability distribution so that samples of candidate pathways can be drawn probabilistically. Signaling pathway directions were inferred by aggregating the pathways samples and then selecting the interconnected linear pathways with the largest potentials.L genes, where ith gene in the pathway. n genes and m samples with two conditions , we derive gene potential 37; edge potential 38; and flow potential 39. Derivations of the three potentials are provided in the Supplementary Methods.We define a vector GIST integrates the three potentials into a pathway energy function as follows:T is the "temperature" that controls the shape of the distribution. GIST samples pathway genes iteratively from a conditional distribution as Due to the large number of genes and their interactions, finding the optimal solution of Eq.  is a NP i to gene j and its reverse direction are both estimated  could bias the sample distribution. Instead of directly ranking pathways based on their GIST sampling frequency, SOUL first clusters pathway samples based on their structural similarities using hierarchical clustering, resulting in a re-organized pathway topological pattern visualized as a pathway structural heatmap  and to simulated pathway networks .We evaluated the performance of GIST for pathway identification on simulated datasets generated by two different pathway structures: type I, corresponding to alternative pathways between a single source gene and a single target gene; and type II, corresponding to multiple pathways with crosstalk among multiple sources and targets 10. Note that we only applied ILP to pathway gene identification because ILP does not infer signaling directions. IMPALA consistently obtained comparable or better performance in all cases. When the level of noise was set to 0.2 , IMPALA gained about a 16% increase in precision for type I pathway gene identification, and an even larger improvement of 24% for edge identification. Similarly, for type II GIST achieved about a 15% increase in average precision for gene identification, and a 17% increase for edge identification.Supplementary Figures Supplementary Information.


Yixin Ningshen tablet is a CFDA-approved TCM formula for treating coronary heart disease (CHD) clinically. However, its active compounds and mechanism of action in treating CHD are unknown. In this study, a novel strategy with the combination of network pharmacology and proteomics was proposed to identify the active components of Yixin Ningshen tablet and the mechanism by which they treat CHD. With the application of network pharmacology, 62 active compounds in Yixin Ningshen tablet were screened out by text mining, and their 313 potential target proteins were identified by a tool in SwissTargetPrediction. These data were integrated with known CHD-related proteomics results to predict the most possible targets, which reduced the 313 potential target proteins to 218. The STRING database was retrieved to find the enriched pathways and related diseases of these target proteins, which indicated that the Calcium, MAPK, PI3K-Akt, cAMP, Rap1, AGE-RAGE, Relaxin, HIF-1, Prolactin, Sphingolipid, Estrogen, IL-17, Jak-STAT signaling pathway, necroptosis, arachidonic acid metabolism, insulin resistance, endocrine resistance, and steroid hormone biosynthesis might be the main pathways regulated by Yixin Ningshen tablet for the treatment of CHD. Through further enrichment analysis and literature study, EGFR, ERBB2, VGFR2, FGF1, ESR1, LOX15, PGH2, HMDH, ADRB1, and ADRB2 were selected and then validated to be the target proteins of Yixin Ningshen tablet by molecular docking, which indicated that Yixin Ningshen tablet might treat CHD mainly through promoting heart regeneration, new vessels' formation, and the blood supply of the myocardial region and reducing cardiac output, oxygen demand, and inflammation as well as arteriosclerosis . This study is expected to benefit the clinical application of Yixin Ningshen tablet for the treatment of CHD. CHD, with clinical condition presented as angina, myocardial infarction, sudden death, and consequent chronic heart failure, is the leading cause of death in noncommunicable diseases and accounted for about 1/3 of all deaths worldwide (17.5 million) in 2012 . For exaSchisandra chinensis (Turcz.) Baill. (Wu Wei Zi), Panax ginseng C. A. Mey (Ren Shen), Albizzia julibrissin Durazz (He Huan Hua), and Ganoderma Lucidum Karst (Ling Zhi). In China, its clinical indications include CHD, palpitations, anxiety, depression, neurasthenia, and memory loss. However, until now, no research studies had been conducted to identify its active compounds and potential mechanism of action in treating CHD, which urgently remains to be investigated.Traditional Chinese Medicine (TCM) formulae with low toxicity and high efficiency had been long applied clinically for several thousand years. The mode of action with multicomponent and multitarget is the key feature of TCM formulae. Yixin Ningshen tablet, a CFDA-approved cardiac protective formula, consists of four different herbs: The advent of network pharmacology, whose “multicomponent-multitarget network” theory coincides well with the integrity and systemic nature of TCM formulae, provided us a novel approach for mechanism investigation of TCM formulae . Many prIn this study, network pharmacology and proteomics were combined to identify the active compounds in Yixin Ningshen tablet and the action mechanisms by which they relieved CHD. The flowchart of this study is shown in http://tcm.cmu.edu.tw) [http://www.megabionet.org/tcmid) [http://www.organchem.csdb.cn). The active compounds of Wu Wei Zi, Ren Shen, He Huan Hua, and Ling Zhi were identified by text mining from the National Center of Biotechnology Information PubMed database (http://www.ncbi.nlm.nih.gov/pubmed/). The search bar was composed of a compound name in Yixin Ningshen Tablet and heart or cardiac or myocardial. Compounds which showed cardioprotective effect were regarded as potential active compounds for the treatment of CHD.The compound information  present in Yixin Ningshen tablet was obtained from the TCM Database @ Taiwan (.edu.tw) , Traditig/tcmid) , and CheThe potential targets of active compounds in Yixin Ningshen tablet were predicted by SwissTargetPrediction , which iThe CHD-related differential proteins were obtained by text mining from the National Center of Biotechnology Information PubMed database with a search bar (coronary heart disease AND proteomics). Literatures reporting the differential proteins regulated by CHD were downloaded –19, and https://string-db.org/). The interaction score was set at 0.9 to obtain a high confidence. Cytoscape 3.6.0 was applied to visualize the PPI network. Afterwards, PPI combined with CTPI was used to build a new compound-target protein-differential protein network (CTPP). This network analysis approach can identify target proteins which associate compounds in Yixin Ningshen tablet with differential proteins.A protein-protein interaction (PPI) network was constructed by uploading the gene names of the potential target proteins and differential proteins regulated by CHD to the public database STRING  and imported into discovery studio 2.5. Water molecules were then removed from the structures, and the hydrogens were added. The ligand in the cocrystallographic structure was selected and defined as the binding site. At last, the active compounds were imported into discovery studio 2.5 and docked with related proteins using the standard docking protocol.CDOCKER was applied to validate the interation between the active compounds and target proteins. The protein structures were downloaded from RCSB Protein Data Bank  was established through the integration of CTPI and PPI to improve the target prediction accuracy of SwissTargetPrediction . This nep value <0.05 and observed gene count ≥3. Generally speaking, pathways with more target proteins involved in are more meaningful than pathways containing fewer target proteins. Thus, a target protein pathway network , and β2-adrenergic receptor (ADRB2), as clinically validated target proteins of drugs for CHD, were also selected to validate their interaction with active compounds in Yixin Ningshen tablet. Of note, available protein structures and active pockets were the prerequisites for molecular docking experiments; thus, STAT3, VEGFA, STAT1, FGF2, CP1A2, and CDK4 without known active pockets were excluded from docking validation. A precise docking program called CDOCKER was applied for docking, and the CDOCKER energies were calculated to reflect the binding affinity of compounds with proteins. The binding energies (CDOCKER energies) of compounds with target proteins were listed in Supplementary As SwissTargetPrediction could not provide the interaction details between target proteins and active compounds, here, we applied an accurate docking program called CDOCKER to investigate the binding modes between compounds and target proteins. Aforementioned enrichment analysis indicated the importance of 29 processes, including the calcium signaling pathway, MAPK signaling pathway, PI3K-Akt signaling pathway, cAMP signaling pathway, Rap1 signaling pathway, steroid hormone biosynthesis, AGE-RAGE signaling pathway in diabetic complications, insulin resistance, relaxin signaling pathway, endocrine resistance, HIF-1 signaling pathway, necroptosis, arachidonic acid metabolism, prolactin signaling pathway, sphingolipid signaling pathway, estrogen signaling pathway, IL-17 signaling pathway, Jak-STAT signaling pathway, Th17 cell differentiation, VEGF signaling pathway, Wnt signaling pathway, folate biosynthesis, tryptophan metabolism, adipocytokine signaling pathway, toll-like receptor signaling pathway, choline metabolism, platelet activation, linoleic acid metabolism, and fluid shear stress and atherosclerosis. We selected target proteins related with more than three aforementioned processes and more than three active compounds in Yixin Ningshen tablet, including the epidermal growth factor receptor (EGFR), signal transducer and activator of transcription 3 (STAT3), vascular endothelial growth factor A (VEGFA), signal transducer and activator of transcription 1-alpha/beta (STAT1), receptor tyrosine-protein kinase erbB-2 (ERBB2), vascular endothelial growth factor receptor 2 (VGFR2), muscarinic acetylcholine receptor M1 (ACM1), muscarinic acetylcholine receptor M2 (ACM2), fibroblast growth factor 2 (FGF2), fibroblast growth factor 1 (FGF1), cytochrome P450 1A2 (CP1A2), estrogen receptor (ESR1), arachidonate 15-lipoxygenase (LOX15), cyclin-dependent kinase 4 (CDK4), and prostaglandin G/H synthase 2 (PGH2), as examples to validate. In addition, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMDH), π cation interaction with Lys745. π sigma interaction with Val734. π-π interactions (Phe916 and Phe1045), and a π sigma interaction (Leu838). Trehalose could form three hydrogen bonds with Asp105, Ser109, and Tyr404 in ACM1 (π-π interaction (His366). π sigma interactions with Phe306. The interaction of gomisin A and ADRB2 is presented in The interplay between quercetin and EGFR is shown in  in ACM1 . Trehalo in ACM1 . As to F in ACM1 . As showß-caryophyllene, protopanaxatriol, kaempferol , citric acid, ginsenoside-Rb1, ginsenoside-Rb3, ginsenoside-Rd, ginsenoside-Re, ginsenoside-Rg1, ginsenoside-Rg3, ginsenoside-Rk3, pseudoginsenoside f11, spathulenol, and ß-sitosterol  from Ren Shen as a principal herb could bind with 2, 2, 2, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, and 1 target proteins, respectively. In addition, ß-D-Glucan, trehalose, and ergosterol from Ling Zhi as a principal herb could target 3, 3, and 1 proteins, respectively. Specially, ergosterol was validated to bind with HMDH, a confirmed CHD target protein. Quercetin, formononetin, apigenin, chrysoeriol, isoliquiritigenin, isorhamnetin, linoleic acid, luteolin, rhamnetin, and taxifolin from He Huan Hua could bind with 3, 3, 2, 2, 2, 1, 1, 1, 1, and 1 target proteins, respectively. Linalool , α-terpineol, gomisin A, schisandrin B, deoxyschizandrin, and schisanhenol from Wu Wei Zi could bind with 3, 3, 3, 2, 1 and 1 target proteins, respectively. Aforementioned molecular docking results also validate the interactions obtained from SwissTargetPrediction.Among the aforementioned 12 target proteins, statistical results  in Wu Wei Zi and apigenin, chrysoeriol, and formononetin in He Huan Hua could treat CHD through targeting ESR1 inhibitor in LDL receptor-deficient mice was reported to show a protective role in early atherosclerosis . In addiß-sitosterol  and citric acid in Ren Shen and ergosterol in Ling Zhi treated CHD by reducing low-density lipoprotein cholesterol through inhibiting HDMH  for the treatment of CHD. Our study provides a new strategy for the study of TCM formulae, and the findings are also expected to provide a basis and reference for the further invesitgation of action mechanism of Yixin Ningshen tablet.


Gallus gallus)-specific immunometabolic kinome peptide array. Using this tool to examine the active enzymes responsible for phosphorylation events (kinases) provides important information on host and cellular functions. The objective of this project was to determine if feeding a microencapsulated product comprised of a blend of organic acids and botanicals (AviPlus®P) impacts the intestinal kinome of broiler chickens . Day-of-hatch chicks were provided 0 or 500g/MT of the additive and jejunal and ileal segments collected for kinome analysis to determine the mode-of-action of the additive. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was performed by uploading the statistically significant peptides to the Search Tool for the Retrieval of Interacting Genes database. As a whole, GO and KEGG analysis showed similar activities in the ileum and jejunum. However, there were a small number of KEGG pathways that were only activated in either the ileum or jejunum, but not both. Analysis of the adipocytokine and PI3K-AKT signaling pathways showed differences between ileal and jejunal activity that were controlled, in part, by AKT3. Additionally, cytokine/chemokine evaluation showed the ileum had higher IL1β, IL6, IL10, TNFα, IFNγ, CXCL8, and CCL4 mRNA expression levels (P<0.05). As a whole, the data showed the addition of microencapsulated organic acids and botanicals to a broiler diet activated many of the same signaling pathways in the ileum and jejunum; however, distinctions were observed. Taken together, the findings of this study begin to define the mode-of-action that microencapsulated organic acids and botanicals have on two important intestinal segments responsible for nutrient digestion and absorption in chickens.The use of natural products as feed additives in the poultry industry is increasing; however, most studies focus on performance and growth with little regard for determining mechanism. Our laboratory designed a chicken ( In the past 10–15 years, the poultry industry has curtailed the use of antibiotic growth promoters (AGP) in poultry production . Some exAnalysis of the host kinome with a peptide array provides site-specific protein details, has analogous biochemical properties to full-length protein, and provides a detailed picture of phosphorylation-mediated events  making iCommercial meat-type chickens consume large amounts of feed and the distinct sections of the digestive tract including the crop, proventriculus, gizzard, small intestine and the ceca all carry out specific functions  as the fad libitum access to food supplied in hanging feeders and fresh water through nipple drinkers. Chickens were provided 24 hr of continual light at placement to ensure sufficient water and food intake, then transitioned to 18 hr of light and 6 hr of darkness for the remainder of the study. The temperature of the pens was maintained at 35°C for day 1 to 3, 32 to 34°C for day 4 to 7, and 29 to 31°C for day 8 to 15. Chickens were monitored each morning (08:00) for mortality, behavioral changes, litter quality, and feed and waterers were checked to ensure they were in proper working order. No mortality, behavioral changes, or other animal welfare concerns were observed during the study. The chicks were not treated with any medications or other therapeutic interventions during the study. The experiments were conducted on two separate occasions with chicks from a different hatch. Chicks were randomly assigned to either a control or supplement-fed pen (n = 20). Those assigned to the control pen were allowed ad libitum access to a balanced, unmedicated corn and soybean meal-based starter diet that met or exceeded the established nutrient requirements [®P, Vetagro S.p.A., Reggio Emilia, Italy). The experiments were conducted in accordance with the recommended code of practice for the care and handling of poultry and followed the ethical principles according to the Guide for the Care and Use of Laboratory Animals [Day-of-hatch by-product breeder chicks were obtained from a commercial hatchery . The chicks were transported in standard chick boxes and placed in a BL2 building in floor pens (3 m × 3 m) containing wood shavings and provided supplemental heat and  Animals . All birlater , and stored at -20°C for quantitative real-time RT-PCR (qRT-PCR).For both experimental replicates, the chickens on the control (n = 10) and supplemented diet (n = 10) were euthanized by cervical dislocation and necropsied at 15-days-of-age. Sampling time was established based on previous work , 21 and 3VO4, 1 mM NaF, 1 μg/mL leupeptin, 1 g/mL aprotinin and 1 mM Phenylmethylsulphonyl fluoride). All chemicals were purchased from Sigma-Aldrich, Co.  unless specified otherwise. Arrays were then imaged using a Tecan PowerScanner microarray scanner  at 532–560 nm with a 580 nm filter to detect dye fluorescence.Peptide array protocol was carried out as previously  describelater and transferred to prefilled microtubes containing zirconium beads . Lysis buffer  was added, and the tissues homogenized for two minutes at the maximum speed. Each tissue was handled separately. Following homogenization, total RNA was isolated according to the instructions, eluted with 50 μL RNase-free water, and stored at -80°C.The tissue samples were homogenized with a BeadBug homogenizer . Briefly, a piece of ileum and jejunum (30–40 mg) was removed from RNAT 1 Step Kit . Sample standardization was done using 28S RNA. Results were calculated as 40-cycle threshold (CT) for each tissue sample from control- and supplement-fed chickens and the data are presented as the fold-change from controls. Fold change was calculated as 2^(supplement-fed corrected mean–control-fed corrected mean) for each tissue.Ileum and jejunum segments from control- and supplement-fed chickens were subjected to qRT-PCR to determine mRNA expression of select cytokines and chemokines as described previously  using puArray images were then gridded using GenePix Pro software , and the spot intensity signal was collected, thus ensuring peptide spots were correctly associated with their phosphorylation sites. Greater intensity fluorescence correlates to greater phosphorylation at the target site. Fluorescent intensities for treatments were then compared with controls using a data normalization program—Platform for Intelligent, Integrated Kinome Analysis, version 2 (PIIKA2) . The resP-value. The P value is calculated by conducting a one-sided paired t-test between treatment and control values for a given peptide. This consistent analysis method facilitates a more direct comparison between the two distinct array datasets and allows for a statistically robust analysis of the phosphorylation events being measured. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was performed by uploading the statistically significant peptide lists to the Search Tool for the Retrieval of Interacting Genes database as previously described [Normalization and analysis of the data was performed as previously described . Images escribed .P ≤ 2 × 10−8).The GO Consortium recognizes three areas relating to gene function and include cellular component, molecular function, and biological process (BP). The BP terms generally focus on the “larger processes or biological programs” that result from numerous molecular activities. Using STRING functionality, GO BP terms were generated for each tissue. The total number of BP terms associated with the ileum and jejunum were 309 and 355, respectively. Fifteen of the most significant BP terms (based on the observed gene count) for the ileum and jejunum are shown in Tables KEGG pathways for each tissue were generated using the STRING database. For the ileum and jejunum tissues, the results were corrected using the corresponding tissue from birds on the control diet to confirm the phosphorylation changes were the result of dietary supplementation with the microencapsulated blend of organic acids and botanicals. In total, 148 and 144 KEGG pathways were significantly different in the ileum and jejunum, respectively.−17). The common pathways include: B cell receptor signaling pathway, Chemokine signaling pathway, ErbB signaling pathway, Fc epsilon RI signaling pathway, Focal adhesion, FoxO signaling pathway, HIF-1 signaling pathway, Insulin signaling pathway, MAPK signaling pathway, mTOR signaling pathway, Neurotrophin signaling pathway, and the PI3K-Akt signaling pathway and are all shown in bold font. To be included, a pathway had to be significant for each tissue over both replicate studies (n = 20 chicks/group). The pathways specific to cancer and viral infections were excluded from the analyses.As seen with the GO BP terms, most of the top 15 KEGG pathways were similar between the ileum  and jejuP < 0.04) different in the respective tissue from supplement-fed chickens compared to the comparable tissue from those on the control diet.There were several pathways that were completely unique to each tissue and are shown in The adipocytokine signaling pathway was one of the top 15 pathways in the ileum  but was −34) in both tissues, but not all of the same proteins were represented in the two tissues  signaling pathway was highly significant (FDR < 6 × 10 tissues . The ileP ≤ 0.05) different proteins in the ileum and jejunum were subjected to the Markov Cluster (MCL) algorithm. Using the STRING database MCL clustering tool, the clusters were generated and are shown for the ileum and jejunum in Figs The protein-protein interactions of all statistically (P < 0.05) compared to expression in the tissue from the control-fed chickens whereas the expression levels in the jejunum only changed from 0.15 to 1.4 -fold above the controls. These data showed the differences observed in the array were supported by differential activity in the form of cytokine and chemokine mRNA expression between the ileum and jejunum in supplement-fed chickens compared to controls.The chemokine signaling pathway was significantly different in both tissues Tables  and 4. A® P; identification number 4d3) is recognized by the European Union Commission and European Food Safety Authority (EFSA) for its ability to enhance growth and feed efficiency in healthy chickens. However, the precise mechanism(s) driving the improved feed efficiency and growth rate has not been fully characterized. The study described herein was designed to address this gap in knowledge and function by evaluating the kinome profile of ileal and jejunal segments isolated from broiler chickens fed a diet supplemented with a microencapsulated blend of organic (citric and sorbic) acids and botanicals (thymol and vanillin) compared to segments from control-fed birds.The blend of organic acids and botanicals used in the current study has been shown to enhanced gut immune and barrier function in the ileum and jejunum of weaned pigs ; howeverUse of microencapsulation allows for the slow release of active compounds, such as organic acids and botanicals, into the intestinal tract instead of disappearing after exiting the stomach . AdditioThe avian digestive tract includes the crop, proventriculus, gizzard, small intestine, and ceca with much focus on the crop and ceca; however, less is known about a properly functioning small intestine. Collectively, digestion and nutrient absorption occurs in the small intestine which is comprised of three segments including the duodenal loop, jejunum, and ileum. As might be expected, based on their close proximity, there are some overlapping functions between the jejunum and ileum. However, each segment does function independently with nutrient digestion and absorption mainly occurring in the jejunum with water and mineral adsorption primarily taking place in the ileum as well as starch digestion and absorption in commercial fast-growing chickens , 30.Numerous studies have evaluated cytokine or chemokine expression in various tissues following dietary supplementation with a feed additive –33 or ev® P; EFSA identification number 4d3) is recognized by the EU as an approved feed additive that improves feed efficiency and growth in healthy broilers. It is possible the observed differences in intestinal kinome activity contribute to the enhanced performance, but additional studies are required to draw this correlation.Clearly, cytokine and chemokine responses are observed in chicken intestinal segments following supplementation with various natural compounds that are intended to replace antibiotics and/or act as growth and performance enhancers. A deeper evaluation of the kinome data showed the PI3K-AKT and adipocytokine signaling pathways were important in the tissues from the supplement-fed chickens compared to those on the control diet Tables . SpecifiIn addition to AKT3, downstream phosphorylation of the transcription factor CREB1 (CAMP responsive element binding protein 1) was also unique to the ileum  which coWe have shown giving broilers a diet supplemented with the microencapsulated blend of organic acids and botanicals results in a distinct kinome profile in the ileum compared to the jejunum, and future studies will further explore these specific immune and metabolic pathways. From a purely scientific standpoint, understanding mechanism(s) is important, but even more so, this understanding will be vital for the poultry industry moving forward so they can make decisions based on sound science.S1 Data(XLSX)Click here for additional data file.S2 Data(XLSX)Click here for additional data file.


Rhodospirillum rubrum S1H is an α-proteobacterium that is capable of photoheterotrophic assimilation of volatile fatty acids (VFAs). Butyrate is one of the most abundant VFAs produced during fermentative biodegradation of crude organic wastes in various applications. While there is a growing understanding of the photoassimilation of acetate, another abundantly produced VFA, the mechanisms involved in the photoheterotrophic metabolism of butyrate remain poorly studied.The great metabolic versatility of the purple non-sulfur bacteria is of particular interest in green technology. In this work, we used proteomic and functional genomic analyses to determine potential metabolic pathways involved in the photoassimilation of butyrate. We propose that a fraction of butyrate is converted to acetyl-CoA, a reaction shared with polyhydroxybutyrate metabolism, while the other fraction supplies the ethylmalonyl-CoA (EMC) pathway used as an anaplerotic pathway to replenish the TCA cycle. Surprisingly, we also highlighted a potential assimilation pathway, through isoleucine synthesis and degradation, allowing the conversion of acetyl-CoA to propionyl-CoA. We tentatively named this pathway the methylbutanoyl-CoA pathway (MBC). An increase in isoleucine abundance was observed during the early growth phase under butyrate condition. Nevertheless, while the EMC and MBC pathways appeared to be concomitantly used, a genome-wide mutant fitness assay highlighted the EMC pathway as the only pathway strictly required for the assimilation of butyrate.Rs. rubrum with butyrate as sole carbon source requires a functional EMC pathway. In addition, a new assimilation pathway involving isoleucine synthesis and degradation, named the methylbutanoyl-CoA (MBC) pathway, could also be involved in the assimilation of this volatile fatty acid by Rs. rubrum.Photoheterotrophic growth of  Rhodospirillum rubrum S1H (Rs. rubrum) is an α-proteobacterium which has been chosen by the European Space Agency (ESA) to recycle the volatile fatty acids (VFAs) produced in the MELiSSA  loop, envisaged to autonomously sustain the metabolic needs of a crew for a long-term space mission [Rs. rubrum is known to grow photoheterotrophically, using light as an energy source and VFAs as carbon and electron sources. VFAs are common byproducts from fermentative processes. They are produced in the first compartment of the MELiSSA loop where raw biomass is liquefied and subjected to fermentation. Similar to acetate, butyrate is one of the most abundant VFAs, and is usually produced during acidogenic digestion [Purple non-sulfur bacteria (PNSB) are known for their remarkable metabolic versatility. They represent a promising resource for biotechnology such as biofuel and biodegradable plastic production, wastewater treatment, or biofertilization.  mission . In anoxigestion .Desulfosarcina cetonica, butyrate can be used as carbon and energy sources. Butyrate is completely oxidized into CO2 through the formation of acetyl-CoA which is further oxidized through the acetyl-CoA/carbon monoxide dehydrogenase pathway [Rs. rubrum lack the key enzyme of this pathway, the isocitrate lyase (Icl). Among the alternative pathways recently proposed for Icl-negative organisms, the ethylmalonyl-CoA pathway was demonstrated to be a key acetate assimilation route in Rhodobacter sphaeroides [Methylobacterium extorquens [Rs. rubrum [Despite a growing interest in biotechnology, the assimilation metabolism of butyrate under photoheterotrophic conditions remains relatively poorly described. Under catabolic conditions in  pathway . In phot pathway . Neverthaeroides , Methylotorquens –9 and Rs. rubrum .Rs. rubrum under photoheterotrophic conditions, using growth analysis, proteomics, targeted mutagenesis, and genome-wide mutant fitness profiling. We highlighted two main assimilation pathways for butyrate. First, the expected conversion into acetyl-CoA, and the anaplerotic ethylmalonyl-CoA pathway was detected. Second, we also highlighted a potential and unexpected new assimilation pathway involving enzymes related to the biosynthesis and degradation of branched-chain amino acids.This study aimed to offer a better understanding of the photoassimilation of butyrate in Rs. rubrum S1H in photoheterotrophic conditions with butyrate or succinate as the carbon sources. Compared to succinate, we observed a drastically lower biomass production in the presence of butyrate and 3 mM bicarbonate with stationary phase appearing at an OD680nm of 1.3 while the OD680nm reached almost 5 in the presence of succinate . Five biological replicates  were considered. With 1752 proteins identified  and quantified, these proteomic data covered approximately half of the theoretical proteome of Rs. rubrum S1 . The complete dataset is available in the supporting material (Table S1) and the data discussed below are listed in the Table P-value< 0.05; fold change > 1.5 or < 0.66), 162 and 191 proteins were quantified with higher or lower abundances, respectively, under butyrate growth condition compared to succinate growth condition. Our proteomic data lead us to propose that multiple metabolic pathways are involved in the photoassimilation of butyrate. These pathways are depicted in Fig. To gain better understanding of the metabolic pathways responsible for butyrate photoassimilation, we compared the proteome of the biomass obtained under butyrate and succinate growth conditions. To avoid bias related to different illumination perceived at the cellular level, the biomass was harvested at equivalent cell densities . As a role in redox balancing is more likely to be observed at the beginning of the growth curve [680nm ± 0.7). If no difference was observed between succinate and butyrate condition regarding valine content, we observed a large increase in isoleucine relative abundance under butyrate condition . In medium supplemented with succinate or acetate only, the culture reached an optical density of 4.5, which corresponded to the exhaustion of the carbon source  that permits the parallel assessment of gene fitness using a quantitative DNA barcode sequencing strategy [ccr, a key gene of this pathway, was not essential. The detailed interpretation of the results is given in the discussion section.To further explore the requirement of the EMC and MBC pathways for optimal growth under butyrate condition, we used genome-wide mutant fitness assays to probe the importance of most strategy  and has strategy . Of the ccr::KmR) mutant under butyrate condition. This mutant was recently shown to be unable to grow under photoheterotrophic conditions with acetate as the sole carbon source [wt and mutant strain while growing in medium supplemented with succinate or butyrate . The results of this targeted analysis revealed that our mutation had no effect on the abundance of Rru_A3062 . Alternatively, Rru_A1927, annotated as an acetyl-CoA hydrolase, was strongly upregulated  under butyrate growth condition and could also be involved in butyrate activation. According to the InterPro database [The first step involved in the assimilation of butyrate is its activation in butyryl-CoA Fig. . One of cetonica . Howeverdatabase , Rru_A19P-value, 5.6e-6) and is annotated as a butyryl-CoA dehydrogenase. Rru_A1835 is also the best homologue, in the Rs. rubrum genome, of the butyryl-CoA dehydrogenase reviewed in Clostridium acetobutylicum [Megasphaera elsdenii [The butyryl-CoA is then oxidized to crotonyl-CoA through a butyryl-CoA dehydrogenase activity. Rru_A1835 is highly upregulated in the presence of butyrate  under butyrate growth conditions. A 3-hydroxybutyryl-CoA dehydrogenase  catalyzes the oxidation of (S)-3-hydroxybutyryl-CoA to acetoacetyl-CoA which finally undergoes a thioclastic cleavage, releasing two molecules of acetyl-CoA. The acetoacetyl-CoA thiolase  is probably the best candidate to catalyze this last step. Other acetyl-CoA C-acetyltransferases  were identified in our dataset but were downregulated or not significantly upregulated under butyrate growth condition.As already described in cetonica  in the cP-value, 4.8e-1), this protein is probably involved in the maintenance of this route. Conversely, an (R)-specific acetoacetyl-CoA reductase (Rru_A0273) was strongly downregulated under butyrate growth conditions . Beyond its (R)-isomeric specificity, this enzyme was also associated with acetate photoassimilation, catalyzing the reaction in the reverse direction [The conversion of crotonyl-CoA to (R)-3-hydroxybutyryl-CoA is known to be the entry point for the poly (3-hydroxybutyrate) (PHB) biosynthesis pathway , 22. Eveirection . The speP-value, 1.1e-4). Phasin (Rru_A2817), constituting the protein layer on the surface of PHB granules was also strongly upregulated . Higher production of PHA granules under butyrate conditions was beside confirmed by transmission electron microscopy.The production of (R)-3-hydroxybutyryl-CoA was therefore maintained under butyrate growth condition and sustained PHB biosynthesis Fig. . The polP-value, 6.9e-3). PhaZ1  and its phasin-like activator [P-value, 6.0e-6) were also more abundant under butyrate growth condition. PhaZ1 was described as being highly specific to native PHB granules and short chain-length PHAs while being periplasmic and connected with the extracellular medium [Ralstonia eutropha [Rs. rubrum when butyrate is used as the sole carbon source. In the context of acetate assimilation, the biosynthesis of PHB from acetyl-CoA has been proposed to have a redox balancing role through the reduced equivalent-consuming conversion of acetoacetyl-CoA to 3-hydroxybutyryl-CoA [Surprisingly, enzymes responsible for the mobilization of PHB were also significantly upregulated Fig. . The intctivator , 24 ApdAr medium . Its celeutropha –28. Thisyryl-CoA , 29. In Under photoheterotrophic conditions, the conversion of butyrate into acetyl-CoA necessarily implies the reassimilation of the latter through an anaplerotic pathway to maintain the pool of TCA cycle intermediates required as biosynthetic precursors. Interestingly, the first intermediate of butyrate photoassimilation, crotonyl-CoA, is also the first intermediate of the ethylmalonyl-CoA pathway, a C2 compound anaplerotic pathway –8, 10. BIn the second part of the EMC pathway, the enzymes of the methylmalonyl-CoA pathway are responsible for the conversion of propionyl-CoA to succinyl-CoA. These enzymes  were also expected to be upregulated, as recently observed with acetate as a carbon source . HoweverRs. rubrum genome, some of which were downregulated (Rru_A0508), while others were more abundant under butyrate conditions (Rru_A2223). The poor annotation of these enzymes and the lack of information regarding their specificity make interpretation of these results complicated. Interestingly, a recent study by McCully et al. [Rs. rubrum as an electron balancing mechanism during malate and fumarate assimilation. In our case, starting from butyrate, isoleucine synthesis could be considered reductive  biosynthesis pathway in acetate photoassimilation . The acey et al.  concludeons Rru_A223. The The results of the monitoring of the relative abundance of isoleucine in the biomass during the early phase of growth under butyrate condition also claim for the involvement of isoleucine production in the assimilation of butyrate, at least during the early growth phase. If this production of isoleucine effectively plays a role in terms of redox balancing will require furthers investigations.Rs. rubrum and requires some clarifications. To detect genes belonging to the ILV degradation pathway in Rs. rubrum, we searched in the genome of Rs. rubrum for the ILV box, the consensus DNA sequence that the regulator of the ILV degradation genes binds to [rru_A1994, annotated as a merR-like transcription factor. Using blast, we observed that rru_A1994 is the best homologous sequence for liuR of Sh. oneidensis (Table S2). The ILV degradation pathway has been well characterized in this species [Rs rubrum. Rru_A1994 was clearly upregulated when butyrate was used as sole the carbon source.Surprisingly, our proteomic data revealed a concomitant upregulation of some enzymes involved in the ILV degradation pathway. Although the existence of the ILV degradation pathway has been known for a long time , 32, thibinds to . The ILV species . Rru_A19rru_A1835, rru_A1834 and rru_A1833 which, based on sequence comparison with Sh. oneidensis data, correspond to ivdC, ivdE and ivdF respectively. The ivd gene cluster encodes proteins specifically responsible for L-isoleucine and L-valine degradation. Rru_A1835, Rru_A1834 and Rru_A1833 were all clearly upregulated in cultures grown in the presence of butyrate  which was observed in higher abundance in the butyrate assimilating cultures. The thiolase coded by rru_A1946 links L-isoleucine degradation pathway to the methylmalonyl-CoA pathway.The L-valine/L-isoleucine degradation pathway usually leads to the production of propionyl-CoA, which is then assimilated through the methylmalonyl-CoA pathway. In the case of L-valine degradation, the last intermediate is the methylmalonate-semialdehyde, which requires dehydrogenase to be converted to propionyl-CoA. Rru_A1542 and Rru_A2071 were identified as the best candidates to catalyze this step in Rs. rubrum. The ILV box could also be detected downstream of the leucine dehydrogenase-encoding gene Rru_A1040, which was highly downregulated under butyrate conditions, also suggesting the involvement of a global regulatory mechanism. The organization of the liu and ivd operons seems to be largely different in Rs. rubrum than in other proteobacteria. In particular, the genes of the ivd cluster are present in the liu operon, and some genes, such as liuFG and ivdB and ivdD, are not dependent on the ILV box. It is currently unknown whether this organization is specific to Rs. rubrum or generalized to α-proteobacteria.Altogether, these data suggest that the ILV degradation regulon, regulated by LiuR via the ILV box, is involved in butyrate assimilation in Rs. rubrum. Our proteomic data show that all the proteins involved in the L-isoleucine degradation pathway were present in higher abundance when butyrate was used as the sole carbon source, suggesting the involvement of the isoleucine biosynthesis and degradation in butyrate assimilation. It is important to mention that the role of ILV synthesis and degradation is not fully understood in purple bacteria. A recent study in Rh. capsulatus highlighted that genes of the ILV degradation such as mmsA,B; isobutyryl-CoA dehydrogenase; 3-hydroxyisobutyryl-CoA hydrolase; mccA,B, hmglL or menB were downregulated in a mutant lacking AerR, which acts as an indirect photosynthesis activator. This observation already linked ILV degradation metabolism with photosynthetic metabolism in another purple bacteria [Based on these results, we could thus reconstruct the complete L-isoleucine degradation pathway Fig.  of Rs. rbacteria .It can also not be ruled out that the production of branched chain amino acids also plays a role in signaling as valine and leucine were reported to be involved in alarmones regulation and in particular in development of the stringent response .P-value, 1.0e-11). This result is paradoxical in the context of a concomitant upregulation of the biosynthesis and the degradation pathways of ILV. A decoupling of these two pathways was considered and a hypothetical isobutyryl-CoA mutase (ICM) able to directly convert butyryl-CoA to isobutyryl-CoA as described in Streptomyces cinnamonensis [Rs. rubrum genome. Sequence comparison of the coenzyme B12-dependent isobutyryl-CoA mutase of S. cinnamonensis showed 43% identity  with Rru_A2479, a methylmalonyl-CoA mutase that apparently catalyzes the conversion of (R)-methylmalonyl-CoA to succinyl-CoA in Rs. rubrum. Nevertheless, Rru_A2479 showed 79% identity  with the closely-related methylmalonyl-CoA mutase of S. cinnamonensis, which was reported to exhibit no isobutyryl-CoA mutase activity [Rs. rubrum genome is therefore unlikely and the upregulation of the branched-chain-amino-acid aminotransferase Rru_A2223 remains puzzling.Our proteomic data suggest that the L-isoleucine biosynthesis and degradation pathways are upregulated when butyrate is used as a unique carbon source. Surprisingly, while the class IV aminotransferase Rru_A0508 and the L-leucine dehydrogenase Rru_A1040, both of which are involved in the final transamination of the ILV biosynthesis pathway, were clearly downregulated under butyrate growth condition, a third enzyme, the branched-chain-amino-acid aminotransferase Rru_A2223, was conversely upregulated  pyruvate oxidoreductases alpha (Rru_A1978) and beta (Rru_A1977) subunits might be involved in the conversion of 3-methyl-2-oxopentanoate directly into 2-methylbutanoyl-CoA as the first step of the isoleucine degradation pathway; however, this requires further investigations that fall out of the scope of this paper.On the other hand, 3-methyl-2-oxopentanoate is also the first intermediate in the catabolism of L-isoleucine, a 3-methyl-2-oxopentanoate dehydrogenase could be the connecting enzyme between the L-isoleucine biosynthesis and degradation pathways  pathway.2 + 3H2O + 1ATP + 5/3ETF(ox) + 2/3NADPH + 2/3H+ + 2/3FAD + 1/3 NAD+ = 4/3malate + 1ADP + Pi + 5/3ETF(red) + 2/3NADP+ + 2/3 FADH2 + 1/2NADH + 1/3H+. The carbon/redox balance of the MBC pathway is as follows: 1butyrate + 1 ATP + 5H2O + 2ETF(ox) + 2Fd(red) + 2NAD+ + 1FAD = 1malate + 1ADP + Pi + 2ETF(red) + 2NADH+ 2H+ + 1FADH2 + 2 Fd (ox). The MBC pathway produces twice as much reduced cofactor per mole of assimilated butyrate which could seem completely unfavorable under photoheterotrophic conditions where redox balance is a major issue [The two pathways (EMC and MBC) suggested to be involved in the photoassimilation of butyrate differ in their global balance. Starting from butyrate, the balance of the EMC pathway to produce malate corresponds to 1butyrate + 4/3COor issue . On the P-value, 3.0e-8). On the other hand, the expected butyryl-CoA:acetate CoA transferase, Rru_A1471, which had a lower abundance in the first proteomic analysis was not identified here. These results tend to show that the activation of butyrate is probably entirely mediated by Rru_A1927.We performed a kinetic proteomic analysis to try to determine if these two pathways could be sequentially used for butyrate assimilation. This kinetic proteomic analysis first allowed us to better understand the activation of butyrate. Indeed, our data showed a clear correlation between Rru_A1927, annotated as an acetyl-CoA hydrolase, and the presence of butyrate. The enzyme remained at the same level of abundance and dropped only when butyrate was totally consumed . Interestingly, at this moment of growth, the intracellular PHB depolymerase PhaZ2 was strongly downregulated, while the periplasmic depolymerase PhaZ1 was upregulated. On the other hand, phasin (Rru_A2817) was also strongly downregulated between T3 and T4, the abundance of which dropped to minimal levels. Assuming that the intracellular PHB granules were completely consumed between T3 and T4 (resulting in the lowered abundances in phasins and PhaZ2), PhaZ1 is probably involved in the depolymerization of native PHB granules released in the culture medium through cell lysis as already hypothesized by Sznajder et al. .P-value: 4e-8). The other transaminase, Rru_A0508, showed the opposite trend and was upregulated during the stationary phase. Nevertheless, the abundance remained far lower than that of Rru_A2223.No significant variations were observed in the abundance of the enzymes involved in the ILV biosynthesis pathway. A very slight and continuous downward trend was observed between T0 and T4, but the fold changes were not biologically relevant (fold changes higher than 0.8). Interestingly, the upregulated transaminase Rru_A2223 exhibited a similar profile with a more significant decrease during the exponential phase  and Rru_A1944 (unknown function) presented an increased abundance when stationary phase started. In contrast, the two enzymes expected to connect the ILV biosynthesis and L-isoleucine degradation pathways, Rru_A1977 and Rru_A1978, were observed with a clear upregulation exclusively during the butyrate assimilation phase (T1 to T3) followed by a strong downregulation once butyrate is consumed, reinforcing the likelihood of their involvement in butyrate assimilation.Concerning the L-isoleucine degradation pathway, the enzymes of the Although our kinetic proteomic approach allowed to highlight a few trends, we could not observe a clear sequential expression of the enzymes of the EMC and MBC pathways that seem therefore to be simultaneously used at least during the analyzed growth period.Rs. rubrum when butyrate is the carbon source -3-hydroxybutyryl-CoA, appeared to be beneficial in butyrate growth conditions, meaning that this gene has a deleterious effect on the growth of Rs. rubrum on butyrate , the key enzyme of this pathway, does not appear to be essential in the butyrate assimilation. In the study of the assimilation of acetate, the RB-TnSeq library clearly demonstrated that ccr was also essential for growth in medium supplemented with acetate. Data obtained with our (∆ccr::KmR) mutant confirmed the mutant library observation that ccr was not essential for growth in butyrate conditions. This observation, which contrasted with the essentiality of the other members of the EMC pathway, may suggest the use of an alternative route from butyryl-CoA to ethymalonyl-CoA bypassing the crotonyl-CoA carboxylase/reductase. In 1968, Olsen and Merrick demonstrated that propionyl-CoA carboxylase, identified here as Rru_A0052 and Rru_A0053, also catalyzed the carboxylation of butyryl-CoA, but produced ethylmalonyl-CoA at much lower rate (358 μmole/h.mg proteins vs. 32.2 μmole/h.mg proteins for propionyl-CoA and butyryl-CoA respectively). The ubiquitous activity of propionyl-CoA carboxylase against butyryl-CoA could explain why the EMC pathway is essential for butyrate assimilation, while its key enzyme, CCR, is not.On the other hand, almost all the enzymes of the EMC pathway as well as enzymes involved in the conversion of propionyl-CoA to succinyl-CoA are strongly required for the optimal growth of ate Fig. . The fitThe ILV biosynthesis pathway or at least the conversion of pyruvate to 3-methyl-2-oxopentanoate appears to be essential Fig. . This reivd and liu pathways were not detected as being essential for growth in butyrate conditions  was used in this study using medium and culture conditions already described [3 filtered solution . For comparison of PHA production through electron microscopy, cells were also obtained from acetate-supplemented medium . The cultivation experiments performed with mutant strains under photoheterotrophic conditions were performed with 3 mM bicarbonate in the culture medium.escribed . This stescribed . The cul680nm between 0.5 and 0.7, light was supplied by halogen lamps  at an intensity of 50 μmole.m− 2.sec− 1. Under dark aerobic conditions, cultures were inoculated at a starting OD680nm of 0.1, wrapped in aluminum foil and incubated at 30 °C with shaking at 150 rpm. Three monoclonal biological replicates were performed for each culture condition, except for the cultivation experiments with mutant strains that were performed on five monoclonal biological replicates. The growth was monitored by measuring the optical density at 680 nm using a 1-cm-path-length cuvette and a Thermo Scientific Helios Zeta spectrophotometer. When the OD was higher than 1.0, the samples were diluted, and the measured OD values were corrected for the dilution.Anaerobic phototrophic conditions were obtained by purging upper gas phase with pure nitrogen before hermetically sealing the flask. Cultures were inoculated at a starting ODRs. rubrum S1H cultivated in medium supplemented with butyrate or succinate. For each experiment, cells from 5 biological replicates were used to extract proteins and obtain tryptic peptides following already described procedure [Rs. rubrum S1H grown on acetate, succinate and butyrate containing medium using the Rs. rubrum ATCC11170 UniProt entries  as reference sequence database.Quantitative proteomic analyses were performed on protein extracts of rocedure . A SWATHrocedure  with twot-test with a statistical threshold set at 5% .SWATH wiff files were processed using the AB Sciex PeakView 2.1 software and the SWATH™ Acquisition MicroApp considering up to 6 peptides (with at least 99% confidence) and 6 transitions per peptide. The XIC peak area was extracted and exported in AB Sciex MarkerView™ 1.2 software for normalization using Total Area Sums. A principal component analysis (PCA) was routinely performed on normalized data to eventually highlight trends across groups of samples. The significance of the relative abundance  observed between our experimental conditions was determined through Student’s 2SO4 as the mobile phase (flow rate: 1 ml/min.). The detection was performed by refractometry at 210 nm. The amounts of succinate, butyrate and acetate were determined by integrating their specific peaks  and compared to standard curves.VFA consumption was monitored through culture supernatants obtained by centrifugation at 16,000 g for 10 min at 4 °C and stored at − 20 °C before analysis. One hundred microliters of culture supernatant were analyzed by HPLC-refractometry . The separation was realized in isocratic mode using a Shodex Sugar SH1011 column (300 mm × 8 mm) with 5 mM H2O were added. After centrifugation , the aqueous upper phase was collected and submitted to SpeedVac. Dried samples were solubilized in loading buffer (acetonitrile 3% (v/v), formic acid 1% (v/v)). The metabolites were analyzed using an eksigent LC 425 system coupled to a Q-TRAP6500+ (Sciex) used in multiple reaction monitoring (MRM) mode. Peptides were separated on a C18 YMC-Triat 0.3x150mm column operated at a flow rate of 5 μl/min in isocratic mode (3% acetonitrile (v/v), formic acid 1% (v/v)) for 5 min followed by an acetonitrile gradient from 3 to 55% in 3 min. Following transitions were used to quantify the following amino acids: Lysine 147/130; Valine 118/72 Arginine 175/116 and isoleucine 132/69. In order to avoid extraction bias between the samples, isoleucine abundance was expressed as the ratio of the area under the curve for its specific transition on the area under the curve of the specific transition of each of the other amino acids.The pellets issued from the centrifugation of 500 μL of culture of Rs. rubrum in succinate or butyrate condition were snap frozen using liquid nitrogen, freeze-dried and stored at − 20 °C until the extraction. Briefly, the pellets were resuspended using 1.5 mL of a methanol:chloroform solution . Five freeze/thaw cycles were applied to the solution and 400 μL of distilled H680nm around 1), harvested by centrifugation at 16,000 g for 10 min at 4 °C, washed with a cacodylate buffer  ruthenium red, pH 7.8) and immediately fixed with 3% (v/v) glutaraldehyde in cacodylate buffer for 2 h at 4 °C. The samples were rinsed with cacodylate buffer (2 × 10 min.), postfixed for 2 h with 1% (w/v) OsO4 cacodylate buffer at room temperature and washed again. Subsequent dehydration steps were conducted in a graded ethanol series up to 100%. Dehydrated samples were finally embedded in Spurr resin . Ultrathin slices (70–90 μm) were obtained with a Leica Ultracut UCT ultramicrotome and stained with uranyl acetate and lead citrate. Prepared slices were observed with a Zeiss LEO 906E transmission electron microscope operated at 60 kV, and TEM images were acquired with analySIS  software.Cells were grown to the mid-exponential growth phase  , which it-test with a statistical threshold set at 5%) were further considered as essential to adapt specifically to butyrate condition. Further details about mutant fitness experiments are given in Supporting Information.The mutant fitness assays were performed as described previously  using thAdditional file 1:Table S1. Differential protein abundance in butyrate- vs succinate-grown Rhodospirillum rubrum S1H (Complete Dataset). Table S2. Sequence comparison for ILV degradation pathway in Rs. rubrum, based on Shewanella oneidensis ivd operon. Table S3. Differential protein abundance in butyrate-grown Rhodospirillum rubrum S1H during different growth phases (Complete Dataset). Table S4. Fitness profiling of and succinate- and butyrate-grown Rhodospirillum rubrum S1H (Complete Dataset).Additional file 2: Fig. S1. Targeted mutagenesis of the crotonyl-CoA carboxylase/reductase (ccr - Rru_A3063). Scaled diagram of the ccr gene region both in the wild type and ∆ccr::KmR strain. “H.R.1” and “H.R.2” are the homologous regions used for the final double homologous recombination between the genomic DNA and cloning vector, and “KmR Cass.” is the kanamycin antibiotic resistance cassette. Fig. S2. MRM based quantification of abundance of Rru_A3062 (A), Rru_A3063 (B) and Rru_A3064 (C) wt and ∆ccr::KmR strain. We use 5, 3 and 7 peptides to quantify Rru_A3062, Rru_A3063 and Rru_A3064, respectively, based on 6 transitions for each peptide. Five biological replicate cultures of wt (orange bar) and ∆ccr::KmR strains (blue) were grown in butyrate containing medium and harvested in mid-exponential phase. Fig. S3. Growth of Rs. rubrum S1H in medium supplemented with succinate or acetate and a mixture of ILV. Rs. rubrum was cultivate under anaerobic photoheterotrophic conditions in medium containing 31 mM of succinate (A) or acetate (B) and 3 mM of bicarbonate (dashed lines). The medium was also eventually supplemented with an equimolar (10 mM each) mixture of ILV (solid lines). The growth was monitored by measuring OD680nm. n = 5. Table S5. Oligonucleotides used for targeted mutagenesis. Table S6. MRM transition used to monitor abundance of protein expressed by gene close to Rru_A3063.


Dysidea species in 1981 and have been under continuous research to the present day. This article summarizes the two research aspects, (i) the marine compound chemistry research dealing with naturally produced PBDEs and (ii) the environmental toxicology research dealing with synthetically-produced brominated flame-retardant PBDEs. The different bioactivity patterns are set in relation to the structural similarities and dissimilarities between both groups. In addition, this article gives a first structure–activity relationship analysis comparing both groups of PBDEs. Moreover, we provide novel data of a promising anticancer therapeutic PBDE phenol; termed P01F08). It has been known since 1995 that P01F08 exhibits anticancer activity, but the detailed mechanism remains poorly understood. Only recently, Mayer and colleagues identified a therapeutic window for P01F08, specifically targeting primary malignant cells in a low µM range. To elucidate the mechanistic pathway of cell death induction, we verified and compared its cytotoxicity and apoptosis induction capacity in Ramos and Jurkat lymphoma cells. Moreover, using Jurkat cells overexpressing antiapoptotic Bcl-2, we were able to show that P01F08 induces apoptosis mainly through the intrinsic mitochondrial pathway.Polybrominated diphenyl ethers (PBDEs) are a group of molecules with an ambiguous background in literature. PBDEs were first isolated from marine sponges of  The search for new bioactive substances, which can overcome intrinsic or acquired resistance, are core topics of pharmaceutical research. Thus, there is a constant need for new types of resistance-breaking drugs due to the spread of multidrug-resistant microorganisms and tumors. Ecological niches under high evolutionary pressure often yield bioactive compounds with high antibacterial or antineoplastic capacity . These compounds and their analogs from stress-exposed marine organisms or fungal endophytes could serve as a pool for new, potentially active compounds to elucidate the modes of action and overcome resistance at the molecular level. The global pharmaceutical market amounts to 1.1 trillion US dollars . NaturalMarine organisms are an especially diverse and rich source of natural products, with manifold bioactivities ranging from the inhibition of growth to the induction of apoptosis ,5. A hug1), which showed promising antineoplastic capacity, will be presented, followed by a structure activity relationship (SAR) analysis.This article aims to summarize data about the bioavailability and diversity of polybrominated diphenyl ethers (PBDEs), a class of marine natural products, mainly extracted from marine sponges. Additionally, the newest data of a unique PBDE named P01F08 (1) DysideidaIt was assumed in several sources that not the sponges themselves are the producers of polyhalogenated (either polybrominated or polychlorinated) compounds ,42,52,53Dysidea herbacea with the cellular source of filamentous cyanobacteria pyrrole (17). The formation of 2,3,4,5-tetrabromopyrrole (18) was also observed during the heterodimerization of 2,3,4-tribromopyrrole.The biosynthetic pathway described in the following is derived from Agarwal and Moore and will be explained in brief  Figure A. As an ogenase) . CYP450 Proposed steps for radical generation, rearrangement (i–iv), and coupling of 2,4-dibromophenol by Bmp7 to generate biphenyls and OH-BDEs are shown in bmp pathway can synthesize dibenzo-p-dioxins by employing different phenolic initiator molecules. They further diversified the structural classes of diphenyl ethers accessed by the pathway . I. I10) orhylation . Agarwal50 of >5 g/kg but upon chronic exposure, liver, kidney, and thyroid gland were the organs to suffer most from toxicity . . DysideaDysidea sp., termed P01F08 (1) and P01F03 (37) (1) showed a 3.2-fold lower IC50 value in primary leukemic cells compared to the PBMNCs of healthy donors. Thus, these data demonstrate the therapeutic relevance of polybrominated diphenyl ethers for patients with a cancerous disease such as AML i at high concentrations (5–20 µM) in chromaffin and pheochromocytoma (PC12) cells along with the inhibition of depolarization-evoked [Ca2+]i [2+]i by Ca2+ release from intracellular stores by (27) than to those due to influx of extracellular Ca2+ by (19) or (42) i . This in or (42) .27), had fewer effects than OH-PBDEs that shielded only at one side ((19) and (42))  for 120 min at 4 °C. After lysis, the PI fluorescence was measured via flow cytometry. PI binds stoichiometrically to DNA, thus, PI fluorescence mirrors DNA content of the prepared nuclei [Cells were seeded at a density of 1 × 10Experiments were replicated at least three times, and representative data are shown. Error bars indicate standard deviation.