C. elegans mechanosensory neuron axotomy model. We identify an unexpected role of the nicotinamide adenine dinucleotide (NAD+) synthesizing enzyme, NMAT-2/NMNAT, in axon regeneration. NMAT-2 inhibits axon regrowth via cell-autonomous and non-autonomous mechanisms. NMAT-2 enzymatic activity is required to repress regrowth. Further, we find differential requirements for proteins in membrane contact site, components and regulators of the extracellular matrix, membrane trafficking, microtubule and actin cytoskeleton, the conserved Kelch-domain protein IVNS-1, and the orphan transporter MFSD-6 in axon regrowth. Identification of these new pathways expands our understanding of the molecular basis of axonal injury response and regeneration.The mechanisms underlying axon regeneration in mature neurons are relevant to the understanding of normal nervous system maintenance and for developing therapeutic strategies for injury. Here, we report novel pathways in axon regeneration, identified by extending our previous function-based screen using the  Axon regeneration after injury is an important and conserved biological process in many animals, involving a large number of genes and pathways . Upon axCaenorhabditis elegans  proteins, a Rab GTPase RAB-8, and the membrane transporter MFSD-6. We show that the endoplasmic reticulum (ER)-PM contact site protein Extended Synaptotagmin (ESYT-2) is sensitive to axonal injury, and that Junctophilin (JPH-1) inhibits axon-axon fusion. Our studies of genes encoding lipid or phospholipid metabolic enzymes indicate extensive functional redundancy. This expanded screen reinforces several themes from the previous study, such as the inhibitory role of ECM components and the permissive role of MT stabilization  (nmat-2 null (0) mutants, tm2905 and ju1512 (nmat-2(0) adult animals are sterile, while nmat-1(0) are fertile, indicating that these two NMNATs may have distinct tissue- or cell-type-specific roles.Among genes with significant inhibitory effects on axon regrowth, we identified NMAT-2, a member of the nicotinamide mononucleotide adenylyltransferase (NMNAT) enzyme family . NMNAT eand mice . In mammand mice . In C. eMEC-4(d) , but doeMEC-4(d) . We found ju1512 . A null d ju1512 . nmat-2(nmat-2(0) are related to NAD+ synthesis, we examined loss-of-function mutants of other enzymes in the invertebrate NAD+ salvage synthesis pathway  NMRK-1, nicotinate phosphoribosyltransferase (NAPRT) NPRT-1, nicotinamidase PNC-1 and PNC-2 (qns-1(0) mutants showed marginally increased axon regrowth , qns-1(0) mutants are sterile  and found that neither wild type or nmat-1(0) grown in FUdR showed increased PLM regrowth  under its endogenous promoter (juSi347). This transgene fully rescued the sterility of nmat-2(0) and restored the increased axon regrowth in nmat-2(0) mutants to wild-type levels (nmat-2(0) to normal  displayed sterility and enhanced regrowth of PLM neurons (nmat-2(0) mutants. Therefore, the role of NMAT-2 in axon regeneration likely requires its enzymatic activity. Here, we infer that the enhanced axon regeneration in nmat-2(0) reflects sustained low levels of NAD+.In addition to their enzymatic roles, several NMNAT proteins function as molecular chaperones, including n NMNAT3 . We thern NMNAT3   are regions where membranes from two organelles or an organelle and the PM are held together by protein tethers, most of which are conserved from yeast to mammals . MCSs caneration . Additioneration . We therC. elegans (jph-1(ok2823) mutants, likely null, exhibited a significantly increased rate of reconnection or fusion between the regrowing axon and distal fragment  are a family of proteins containing multiple C2 domains  that havd E-Syt3 . We founs system . In the s system  at the ps system . In unins system , we spec calcium . We gene editing . These m editing . Thus, wC. elegans has two orthologs, ANOH-1 and ANOH-2. ANOH-1 is expressed in mechanosensory neurons and acts together with the apoptotic factor CED-7 to promote phosphatidylserine exposure in the removal of necrotic cells (ced-7(0) reduces PLM axon regrowth  family of MCS-localized lipid transfer proteins includes multiple members. ORP5/8 act as tethers at ER-PM MCSs where they mediate PI4P/Phosphatidylserine counter-transport, while OSBP and the other ORPs function at different MCSs . We testregrowth . While tregrowth .Altogether, the above analysis echoes a recent study in yeast where elimination of multiple MCS components did not impair ER-PM sterol exchange , highligC. elegans, the majority of triglyceride is obtained from the diet, and lipogenesis accounts for less than 10% of stored body fat (C. elegans encodes a single ATGL (ATGL-1), three CGI-58/ABHD5 , a single HSL (HOSL-1), but lacks MGL by sequence homology , autophagy proteins (LGG-1 and LGG-2), and transcription factors (HLH-30/TFEB and MXL-3/MXL) act in lipophagy , preventing further analysis. Definitive conclusions will require tissue-specific and temporal manipulation of this pathway. Overall, our analysis suggests that the Kennedy pathway may affect axon regeneration.The Kennedy pathway synthesizes the most abundant phospholipids in eukaryotic membranes, phosphatidylcholine (PC) and phosphatidylethanolamine (PE) , and invregrowth . In testC. elegans IVNS-1 has the same overall domain organization as NS1A-BP  as an inhibitor of axon regrowth. BTB/POZ  domain and Kelch repeats function in a wide variety of biological processes including gene expression, protein ubiquitination, and cytoskeleton binding . Human N NS1A-BP .ivns-1 mutants (gk252 and ok3171) and observed increased axon regrowth, which was restored to control levels following transgenic expression of ivns-1 driven by its own promoter  (gm121) of EPI-1/Laminin α and a gain-of-function mutant (tk75) of EMB-9/Type IV collagen (ECM plays diverse roles in axon regeneration . In C. eane (BM) , which iane (BM) . We prevane (BM) . We furtcollagen  both shocollagen , supportC. elegans, multiple chondroitin proteoglycans are expressed, but are not sulfated  or membrane trafficking (rsef-1/RASEF) are required for axon regeneration   ; and NEX2/−3/–4) . We foun2/−3/–4) . These rmec-17; atat-2 double mutants showed reduced axon regrowth resembling the mec-17 single mutant  mutants are resistant to inhibitors of cholinesterase  are defective in axon regrowth  and have also specifically targeted several pathways not addressed in our earlier screen, such as NAD+ biosynthesis, MCS components, lipid metabolism, and actin regulators. Interestingly, both MCS components and lipid metabolism tested display a high degree of genetic redundancy, such that single mutants only occasionally display regeneration defects, and compound mutant strains are required to assess functional requirements. Nevertheless, our findings suggest that ER-PM contact sites may be regulated by axon injury and that phospholipid synthesis may be critical for axon regeneration. Further work will be required to define whether these pathways play a role in lipid addition to the regrowing axon membrane or a more general signaling role.Functional screening for axon regeneration phenotypes is a powerful approach to identify novel regulators of axon regrowth after injury. C. elegans and the results may be compared to assess reproducibility and generalizability of the results. The present work and our prior screen (nex-1 in PLM and motor neurons) may reflect cell-type-specific roles of the regulators in axon regeneration. A recent genome-wide in vitro axon regeneration screen in mouse cortical neurons revealed significant overlap with orthologous genes identified from C. elegans screen despite differences in neuron types, species, and experimental methods  also have functionally important roles in regrowth. One trend is that fewer mutants with dramatically reduced regrowth  were identified, and thus the number of genes essential for initiation of regrowth may be limited. On the other hand, the present screen identified new mutants with drastically enhanced regrowth . Interestingly, a recent genome-wide screen for enhanced regrowth in mouse cortical neurons reported a positive hit rate of 3%  plates seeded with E. coli OP50 bacteria unless mentioned. Wild type was the N2 Bristol strain  with Leica DMi8 microscope, laser axotomy was performed on the PLM axon ~45 μm away from the cell body. Images were taken immediately before and immediately after axotomy (0.81 s) with iXon ultra 888 EMCCD camera.L4 stage animals were immobilized using 2.5 mM levamisole in M9 buffer on 5% agar pads. PLM mechanosensory neuron cell bodies were imaged using a Zeiss LSM800 equipped with Airyscan. Z-stack planes were taken at 0.2 μm intervals in both mKate2 and GFP channels using Airyscan.nmat-2(ju1512), nmat-1(ju1564), qns-1(ju1563), and esyt-2(ju1409) deletion alleles using co-CRISPR (nmat-2(ju1514) point mutation allele using the homology-directed genome editing and single-strand oligodeoxynucleotide repair method (We generated the o-CRISPR . We gener method .We transferred worms onto plates containing 50 µg/ml 5-fluoro-2-deoxy uridine (FUdR) immediately after axotomy. No offspring were observed after 2 days, confirming FUDR-induced sterility.t-tests for two samples, one-way ANOVA followed by multiple comparison tests for multiple samples. p<0.05 (*) was considered statistically significant. *p<0.05; **p<0.01; ***p<0.001. Data are shown as mean ± SEM. ‘n’ represents the number of animals and is shown in graphs.Statistical analysis was performed using GraphPad Prism 5. Significance was determined using unpaired  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.C. elegans identifies new regulators and an inhibitory role for NAD+ in axon regeneration" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Eve Marder as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Marc Hammarlund (Reviewer #2); Benjamin Podbilewicz (Reviewer #3).Thank you for submitting your article "Expanded genetic screening in The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:C. elegans identifies new regulators and an inhibitory role for NAD+ in axon regeneration" Kim and colleagues expand on a previous screen also from the Jin lab and examine the role of 611 genes, selected for by genetic homology to humans and mutant availability, in axon regeneration using a single axon laser ablation model. The authors report that 82 of these knockouts significantly altered axon regeneration, 49 of which were important for normal regeneration, and 33 that would normally inhibit regeneration. The identified genes were distributed across a broad set of pathways and molecular functions and the authors do a nice job of covering the relevant background for such a wide set topics.In their manuscript entitled "Expanded genetic screening in Their new findings consist of:+ salvage pathway (via NAD+ itself) in axon regeneration;- An unexpected inhibitory role for the NAD- A role for junctophilin 1 in axon fusion;- Relocalization of extended synaptotagmin-1 to ER-PM contact sites after axotomy;- Strong functional redundancy in lipid metabolic pathways that can mask regenerative/degenerative effects;- Inhibition of regeneration by influenza virus NS1A protein largely due to faster initiation of regrowth independent of growth cone formation.emb-9, epi-1, adt-1, adt-3, mig-17), trafficking (rab-8), plasma membrane and resealing , microtubule acetylation  and actin . They also report a growth-potentiating role for the orphan receptor mfsd-6.In addition they build on themes from their previous screen by adding candidate regeneration modulators related to the ECM and basement membrane  Subsection 2 The conserved enzyme NMNAT and its product NAD+ salvage pathway reported here for PLM axon regeneration and its action in slowing Wallerian degeneration. If NAD+ rundown acts as an injury signal, then a lowered concentration could elicit a faster response to injury that is blocked by NAD+ supplementation. It would be very helpful if the authors could address the role of NAD+ levels in triggering the injury signal, perhaps by a delayed addition of NAD+ to the samples instead of incubating it from before the time of injury. If depletion is only needed to trigger the regeneration response, then later supplementation would not affect further growth. A dosage dependent response might also explain this. The reviewers felt that this could be addressed by varying the dosage of NAD+ or time of addition. We will leave it to you to decide if this experiment is doable within the time limit of submission, as the reviewers feel it will add, but it is not critical.2) Another experiment that two of the reviewers pointed out is the apparent conflict between the role of the NAD Essential revisions:+ inhibit axon regeneration”, first and second paragraphs: nmat-2 mutants are sterile, and have an enhanced PLM regrowth, while nmat-1 are not fertile and have no changes in regrowth. Is it possible that sterility, or genes affecting fertility affect PLM regrowth? To test this you can add FUDR to plates or use glp mutants. The reviewers felt that this is an experiment that you should be able to carry out.1) Subsection 2 The conserved enzyme NMNAT and its product NADnmat-1 cultured on FUDR and such treatment caused sterility, but no significant differences in PLM regrowth. The data is presented in revised Figure 3—figure supplement 1. Furthermore, we have previously shown that ablating germline by laser in L1 does not affect PLM axon regeneration in the wild type backgrounds . We conclude that germline sterility per se is unlikely to influence PLM regrowth.We tested the axon regeneration effects of wild type and nmat-1(0) grown in FUdR showed increased PLM regrowth . Additionally, we have previously reported that sterile animals following germline ablation do not affect PLM regrowth . Thus, we conclude that NMAT-2’s role in axon regrowth is independent of animal fertility.’Accordingly, we have revised the text:: ‘To address whether sterility of the animals might contribute to the observed effects on axon regrowth, we cultured animals on 5’fluoro-2’ deoxyuridine (FUdR) and found that neither wild type and + salvage pathway reported here for PLM axon regeneration and its action in slowing Wallerian degeneration. If NAD+ rundown acts as an injury signal, then a lowered concentration could elicit a faster response to injury that is blocked by NAD+ supplementation. It would be very helpful if the authors could address the role of NAD+ levels in triggering the injury signal, perhaps by a delayed addition of NAD+ to the samples instead of incubating it from before the time of injury. If depletion is only needed to trigger the regeneration response, then later supplementation would not affect further growth. A dosage dependent response might also explain this. The reviewers felt that this could be addressed by varying the dosage of NAD+ or time of addition. We will leave it to you to decide if this experiment is doable within the time limit of submission, as the reviewers feel it will add, but it is not critical.2) Another experiment that two of the reviewers pointed out is the apparent conflict between the role of the NADDrosophila and mammalian neurons that NAD+ effects in axon regeneration or degeneration are highly dose-dependent. In the course of revision, we tested acute treatment with NAD+ , and observed no major effects on PLM regrowth. In the course of this experiment, however, we observed that NAD+ supplementation is extremely sensitive to minor variations in how the NAD+ is prepared. We suspect that this might reflect the labile nature of NAD+ and the variability in delivery via the food supply C. elegans. In view of these inconsistencies in the effects of supplementation, which will take additional time to resolve, we have removed the NAD+ supplementation data.We agree with the reviewers’ opinions, as well as observations from other investigators on


In the present paper, experimental and numerical investigations were conducted on concrete bridge barriers utilizing glass fiber reinforced polymer (GFRP) bars with a hook at their ends. Implementation of these hooked bars instead of the bent bars or headed-end bars in the bridge barriers presented in the Canadian Highway Bridge Design Code (CHBDC) was investigated on American Association for State Highway and Transportation Officials (AASHTO) test level 5 (TL-5) concrete bridge barriers. This research aimed to reach a cost effective and safe anchorage method for GFRP bars at the barrier–deck junction, compared to the conventional bend bars or headed-end bars. Therefore, an experimental program was developed and performed to qualify the use of the recently-developed, small radius hooked bars at the barrier–deck junction. The experimental findings were compared with the design factored applied transverse load specified in CHBDC for the design of the barrier–deck junction as well as factored applied bending moment obtained at the barrier–deck junction using a recently-conducted finite-element modeling. Satisfactory behavior for the developed hooked GFRP bars as well as their anchorage resistance was established and a reasonable factor of safety in design of barrier–deck joint was achieved. Bending of glass fiber reinforced polymer (GFRP) bars at construction sites is not possible, and bent bars do not have the same strength as straight bars. This problem results in an increase in the number of GFRP bars used in construction. Alternatively, headed-end GFRP bars can address this problem. A vehicle crash test was conducted using a AASHTO test level 5 (TL-5) bridge barrier reinforced with headed-end, sand-coated GFRP bars, and resulted in a new structural design for bridge barriers . AnotherA design for precast ultra-high performance fiber-reinforced concrete (UHPFRC) TL-4 barriers, including investigation into their mechanical behavior under quasi-static transverse loading up to failure, was done both experimentally and using finite element modeling . ExperimTo qualify the revised barrier design in Five TL-5 barrier specimens of 900 mm length were constructed and tested by the authors to collapse at Ryerson University’s Structures laboratory to determine their failure patterns and load carrying capacities. Based on tested concrete cylinders on the day of casting, the average compressive strengths of concrete were 43, 40, 39, 49 and 52 MPa for Specimens # 1 to # 5, respectively, while the characteristic concrete strengths were 37, 35, 38, 45 and 44 MPa, respectively [In The photos of crack patterns at failure and test setups for Specimens # 1 to # 5 are shown in The load–displacement relationship for Specimen # 2 shown in  is depicThe load–displacement relationship for Specimen # 3 shown in  is depicThe load–displacement relationship for Specimen # 4 shown in , with a The load–displacement relationship for Specimen # 5 shown in  is depicAzimi et al.  conducteWhen an errant vehicle collides with a bridge barrier, the lateral impact force is distributed in the barrier wall and the deck slab with dispersal angles, leading to the design forces at the barrier–deck junction specified in CHBDC commentaries . Figure s, cantilever overhang length, Lc, and the barrier length, Lb, were considered in the parametric study by Azimi et al. [s; 0, 0.5, 1.0, 1.5, and 2.0 m for Lc; and 3, 4, 6, 8, 10, and 12 m for Lb. Linear elastic FEA was performed on the TL-5 barrier of c, of 0 represented a fixed base of the barrier wall, for a non-deformable deck slab. Barrier lengths were considered between 3 and 12 m, as this study showed that greater length does not have considerable effect on the results. The reason to choose these length is that the minimum barrier length, Lb, is usually considered to be 3 m in practice, and the maximum barrier length of 12 m was taken into consideration in this study because analysis showed that greater lengths would have insignificant effect on the distribution of forces in the inner portions of the barriers, and no effect at the end portions of the barrier. Transverse loads were applied at the middle of the barrier for interior locations and at the end of barrier for the exterior locations, and were distributed over specified lengths. An equation for factored design moments at the barrier–deck interface was developed based on the statistical package of curve fitting (Slab thickness, ti et al. . The ass fitting , to deteComparisons between experimental and numerical results for the interior and end locations are shown in It was observed that using the proposed reinforcement arrangement presented in Regarding embedment length for the vertical bars, it was observed that 185 mm vertical embedment length for the GFRP bars in the deck slab was sufficient and satisfied the required barrier–deck anchorage capacity. The same GFRP bar arrangement can be used in solid slab and voided-slab bridge cross-sections with a minimum deck slab thickness of 500 mm.The barrier–deck anchorage capacity increased by 20% only when the 180° hooks were oriented towards the outer face of the barrier, compared to the arrangement in which these hooks faced toward the traffic side of the barrier wall.Tensile strains in the diagonal GFRP bars at the front face in the hooked bars and the adjacent middle bars at failure were far below the ultimate strain of the GFRP bars. Additionally, the concrete compressive strain at failure in the outer face of the barrier was also far below the ultimate concrete strain at failure. Moreover, the tensile strain at failure in the steel bars at the fixed end of the cantilever deck slab was below the steel yield strain. These observations support the observed failure mode at the barrier–deck region.Failure of the barrier specimen with deck slab cantilever was due to diagonal tension cracking in the deck slab cantilever just under the barrier wall. On the other hand, failure in the barrier specimen supported over the 500 mm thick deck slab was due to sudden concrete breakout at the location of the embedded GFRP bars in the deck.Based on the experimental findings in this research, the following conclusions can be made:


Diet plays a key role in the complex etiology and treatment of inflammatory bowel disease (IBD). Most existing nutritional assessment tools neglect intake of important foods consumed or omitted specifically by IBD patients or incorporate non-Western dietary habits, making the development of appropriate dietary guidelines for (Western) IBD patients difficult. Hence, we developed a food frequency questionnaire (FFQ), the Groningen IBD Nutritional Questionnaires (GINQ-FFQ); suitable to assess dietary intake in IBD patients. To develop the GINQ-FFQ, multiple steps were taken, including: identification of IBD specific foods, a literature search, and evaluation of current dietary assessment methods. Expert views were collected and in collaboration with Wageningen University, division of Human Nutrition and Health, this semi-quantitative FFQ was developed using standard methods to obtain a valid questionnaire. Next, the GINQ-FFQ was digitized into a secure web-based environment which also embeds additional nutritional and IBD related questions. The GINQ-FFQ is an online self-administered FFQ evaluating dietary intake, taking the previous month as a reference period. It consists of 121 questions on 218 food items. This paper describes the design process of the GINQ-FFQ which assesses dietary intake especially (but not exclusively) in IBD patients. Validation of the GINQ-FFQ is needed and planned in the near future. Diet is known to play a key role in the complex etiology of inflammatory bowel disease (IBD); comprising Crohn’s Disease (CD) and Ulcerative colitis (UC) . PatientLack of consensus about beneficial and harmful nutrition in patients with IBD is partly due to studies being affected by methodological issues such as information bias, recall bias, lack of proper measurement instruments or statistical incompliance . AdditioTo achieve qualitative dietary guidance, we took the first steps in a multistep approach. We composed a new food frequency questionnaire (FFQ), called the Groningen IBD Nutritional Questionnaires (GINQ-FFQ), dedicated (but not limited) to assessing dietary intake in IBD patients. Development of this new nutritional assessment tool could potentially catalyze the development of evidence-based nutritional therapeutic strategies since this tool may identify nutritional gaps. The aim of this present paper is to describe how this dietary assessment tool was developed.To develop the GINQ-FFQ, several steps were taken . First, Our goal is to design an FFQ specific for IBD patients. Therefore, as initial step, the literature was searched to identify IBD specific foods, meaning foods that have been reported to be important in the development or treatment of IBD or are preferably consumed or intentionally avoided by IBD patients as a coping strategy for their disease. This procedure has led us to form a comprehensive list of food items which build the backbone of the GINQ-FFQ.Secondly, we conducted a literature search to identify what FFQs are currently used to assess dietary intake of IBD patients worldwide. The following search terms were used to search the PubMed database: “Food frequency questionnaire” AND “Inflammatory Bowel Disease (IBD)”. We compared the received questionnaires reciprocally and distilled important (IBD-) foods and added these to the list of our previously identified foods (step 1).™ for realizing and validating web-based food frequency questionnaires (FFQ). This FFQ Tool™ converts frequency of food items into nutritional intake using the Dutch food composition database (NEVO table/“Nederlands Voedingsstoffenbestand”)  wa wa™. AddThe GINQ-FFQ is based on standard methods of WUR and therewith the content validity and face validity are assured. Construct validity, precision, and reproducibility still need to be checked; this will be done in a follow-up study. To validate the GINQ-FFQ, we plan to compare it to mean dietary intake deducted from 24-hour dietary recalls. Therefore, we will administer the GINQ-FFQ and additional three times 24h dietary recall to approximately 300 IBD patients within our outpatient IBD population.The first step in the process of developing the GINQ-FFQ was to compile a comprehensive list of food items based on the literature and information provided by patients, dieticians, and physicians. This list is presented in Secondly, we reached out to IBD-researchers who used an FFQ in their studies. As shown in the flowchart , 42 artiThirdly, based on expert views , several items from the extensive list composed in step 1 and step 2 were excluded from being incorporated in the GINQ-FFQ. Reasons for exclusion were: (1) on several items, no food composition data was available in the Dutch food composition database (NEVO table )  and subsequently these items were not incorporated. After expert views and technical implementation, 218 items ended up in the FFQ. To get some insight in the level of fresh food vs. ultra-processed foods consumed by patients, fast food products were incorporated in additional questions. The time needed to fill out the questionnaire was reported by participants during the readability process. It took them approximately 45 min to fill out the GINQ-FFQ. Additional nutritional and IBD related items were digitized as well. The result was a patient friendly online GINQ which contains, in total, 204 questions (web-version); 121 food frequency questions on 218 food items and embeds 83 questions on additional nutritional and IBD related topics.In step 4 we digitized the questionnaire. Due to some technicalities with the FFQ ToolA new IBD-specific FFQ—The GINQ-FFQ—was developed (and digitized) to assess dietary intake of IBD patients, using the preceding month as a reference period. The result is an FFQ with 121 questions on 218 food items embedded in an online web environment together with 83 additional questions on nutrition and IBD related topics. It takes approximately 45 min to fill out the GINQ-FFQ. Where other FFQs neglect the intake/avoidance of IBD specific products and Western dietary habits, the GINQ-FFQ includes as many as possible of these foods. The GINQ-FFQ assesses dietary intakes of IBD patients in an extensive way, which is urgently needed for the development of proper dietary guidelines and dietary advice for patients.Food-related coping strategies are common among IBD patients; Vagianos et al.  found thTo obtain long-term dietary intake, using an FFQ is the method of choice and is recommended by nutritional epidemiologists . Using aThe strength of the GINQ-FFQ is that the predefined list on which the FFQ-part is based, is extensive and includes items that are specific for the IBD population. Therefore, it allows us to get a good overview of the intake of IBD patients, even if they have these particular unguided habits. Since the GINQ-FFQ covers a broad range of food items and was composed in collaboration with WUR using their standard methods to create a valid questionnaire, the use of this FFQ is designed for IBD patients but is not limited to this specific population. This newly developed questionnaire may also be used in populations with immune-mediated inflammatory diseases (IMIDs)  or in he™, and to assure the questionnaire is not too extensive. Since content and face validity are already assured due to the standard methods of WUR, only the construct validity, precision, and reproducibility of the GINQ-FFQ need to be checked and this will be done in a follow-up study.Specific limitations of the GINQ-FFQ are that certain items of interest specific for IBD could not be included due to lack of information on the food composition, technicalities with the FFQ ToolThe GINQ is potentially an appropriate dietary assessment tool for future epidemiological studies in Western IBD populations. Although validity and reliability of this newly developed questionnaire is planned to be studied in the near future, development of this GINQ-FFQ is a first necessary step in increasing our knowledge on the role of diet in IBD and to guide personalized anti-inflammatory dietary advice in the future.


The Wei Pi Xiao (WPX) decoction, based on the theory of traditional Chinese medicine, has been widely used for the treatment of gastric precancerous lesions (GPL). Although WPX is known to be effective for the treatment of GPL, its active ingredients, cellular targets, and the precise molecular mechanism of action are not known. This study aimed to identify the multiple mechanisms of action of the WPX decoction in the treatment of GPL. The active compounds, drug targets, and the key pathways involved in the therapeutic effect of WPX in the treatment of GPL were analyzed by an integrative analysis pipeline. The information pertaining to the compounds present in WPX and their disease targets was obtained from TCMSP and GeneCards, respectively. The mechanisms underlying the therapeutic effect of WPX were investigated with gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. A total of 82 bioactive compounds and 146 related targets were identified in this study. Following target analyses, the targets were further mapped to 26 key biological processes and 21 related pathways to construct a target-pathway network and an integrated GPL pathway. The study demonstrated that the WPX formula primarily treats the dysfunctions of GPL arising from cell proliferation, apoptosis, and mucosal inflammation, which offered a novel insight into the pathogenesis of GPL and revealed the molecular mechanism underlying the therapeutic effects of the WPX formula in GPL. This study offers a novel approach for the systematic investigation of the mechanisms of action of herbal medicines, which will provide an impetus to the GPL drug development pipeline. Gastric cancer is the fifth common malignancy in the world and continues to be the most prevalent cancer in Eastern Asia, especially China . Althoug Hedysarum multijugum Maxim , Pseudostellaria heterophylla (Miq.) Pax , Atractylodes macrocephala Koidz , Salvia miltiorrhiza Bunge , Curcuma zedoaria (Christm.), Roscoe , and Hedyotis diffusa Willd . HMM, PHP, and AMK, widely used for the treatment of general weakness, have anti-inflammatory and anticarcinogenic activities [ RMB and CZR are known to promote blood flow and remove blood stasis according to the theory of TCM, and studies have proven their anti-inflammatory and anticarcinogenic activities [ HDW is frequently used for the treatment of cancers such as gastric cancer, colorectal cancer, and breast cancer by mediating tumor angiogenesis, proliferation, and apoptosis [β-catenin pathway [In China, traditional Chinese medicine (TCM) has been used for more than 4000 years for the treatment of various diseases . Typicaltivities –10 and ptivities , 12. RMBtivities , 14. As poptosis –18. A pr pathway . AlthougA new advanced analytical technique called systems pharmacology has been used in TCM research , which hThis study attempted to investigate the mechanism of action underlying the therapeutic effect of WPX by employing a systems pharmacology approach. The protocol of the integrated systems pharmacology approach used herein is depicted in http://lsp.nwu.edu.cn/tcmsp.php), which is a database of Chinese herbal medicines providing information on the relationships between drugs, targets, and diseases [ HMM, 25 in PHP, 55 in AMK, 202 in RMB, 81 in CZR, and 37 in HDW. In order to identify the potential active compounds present in WPX, the compounds were screened on the basis of OB, DL, and Caco-2 cell permeability.The compounds present in WPX were identified from the Traditional Chinese Medicines for Systems Pharmacology Database and Analysis Platform , which were calculated on the basis of Dragon soft descriptors [DL is a comparative measure of the functional or physical properties of compounds with those of the majority of known drugs . DL is ecriptors . The comCaco-2 cell permeability is another vital parameter frequently used as a model for studying the passive diffusion of drugs across the intestinal epithelial barrier . In thishttps://pubchem.ncbi.nlm.nih.gov/), which is an open chemistry database containing information on the chemical structures, identifiers, chemical and physical properties, biological activities, and other molecular properties of compounds. The compounds were subsequently submitted to the Swiss Target Prediction database for identifying the potential targets, and the confidence score of the prediction probability of a target protein was more than 40%.Owing to the diversity of the compounds present in the herbal constituents of WPX, the formulation is capable of targeting multiple proteins, thus making target identification a crucial step in understanding the molecular mechanism underlying the therapeutic properties of WPX. In this study, target prediction was achieved using TCMSP, which uses the SysDT model and the Herb Ingredients' Targets (HIT) database . The Syswww.uniprot.org/) was used. The identified candidate targets were treated as the query, and the hits categorized under ‘Homo sapiens' were selected from UniProtKB. The molecular targets of the compounds of WPX, along with their gene symbols, were retrieved from the database.Owing to the noncanonical description of the targets thus identified, the UniProt Knowledgebase , which is a searchable, integrative, user-friendly database that provides comprehensive information on all predicted and annotated human genes, proteins, and diseases [The genes associated with GPL were identified from the Human Gene Database, known as GeneCards (diseases . The Gendiseases . In ordehttp://david.abcc.ncifcrf.gov). The enriched GO terms and pathways having a false discovery rate (FDR) of less than 0.01 according to Fisher's exact test were selected and subjected to further analyses.The GO defines concepts related to gene function and the interrelationships among the functions of different genes. It describes the functions of herbal components in terms of the molecular function(s), the cellular component(s) involved, and the biological process affected . In thisIn order to elucidate the molecular mechanisms underlying the complex therapeutic property of WPX in the treatment of GPL, a compound-target network (C-T network) and a T-P network were generated for studying the relationships among the candidate compounds and the potential disease targets. (1) C-T network: The compound-target interactions were visualized by the C-T network, in which all the active ingredients were connected to their corresponding targets. (2) T-P network: The potential targets identified by preliminary analyses were mapped to the DAVID database for conducting pathway enrichment. The relationships between these potential pathways and GPL were subsequently elucidated by literature mining.The bipartite graphs were visualized and analyzed by Cytoscape version 3.2.1 , which iFor a better understanding of the mechanisms underlying the therapeutic effects of WPX against GPL, an integrated “GPL pathway” was manually constructed based on the T-P network previously generated. The pathways in the T-P network which had no direct and close connections with the disease were removed. HMM, 7 from PHP, 7 from AMK, 54 from RMB, 3 from CZR, and 7 from HDW. Since it was possible that the compounds which did not satisfy the screening criteria could also have therapeutic effects in humans, certain compounds were retained as active components on the basis of available information pertaining to their pharmaceutical activities, even if they did not match the screening criteria. For instance, although astragaloside IV  has poor OB, DL, and Caco-2 permeability, it was retained as an active compound since it is the major constituent of HMM [α and IL-1β [ RMB, known as danshensu  is a potential antithrombotic and antiplatelet agent, owing to its highly selective inhibition of COX-2 [In order to identify the active ingredients in the WPX decoction, three classical ADME parameters, namely OB, DL, and Caco-2 cell permeability, were employed for screening the compounds. Following screening, 88 potential active compounds, representing 20.37% of the total number of compounds present in WPX, were identified, which included 16 compounds fromt of HMM . Studiesnd IL-1β , and resnd IL-1β . Additioof COX-2 . AtractyBased on the aforementioned target fishing approach, a total of 306 targets were predicted to interact with the 93 compounds identified from the 6 herbs in the WPX decoction. However, the targets specific to GPL as well as their mechanisms of interaction were not known. In order to validate the relevance of these proteins in the development of GPL, the GeneCards database was employed for identifying the disease-related genes. Since GeneCards is a gene-centric database, the disease-associated genes are presented in an integrated web card, representing nearly 90% of the human protein-coding genes . A total p-values, FDR, and counts are provided in In order to validate whether the 146 targets are associated with GPL, a GO analysis was performed for elucidating the concerned biological processes. In order to understand the relationships among the herbal constituents in the WPX decoction, the compound targets, and the GPL targets, a C-T network was constructed. The active compounds, targets, and the interactions among them are represented in In this study, 146 targets were mapped to 115 pathways following KEGG pathway enrichment. After combining the pathological data obtained from literature mining with the FDR scores (FDR ≤ 0.01), the pathways that had no relationships with GPL were discarded. Finally, 21 remarkably enriched pathways that were likely to be the major pathways in the treatment of GPL were selected refer . The T-PAs demonstrated in Based on the aforementioned results, an integrated “GPL pathway” was constructed by integrating the key pathways, which included the pathways involved in cancer (hsa05200), the PI3K-Akt signaling pathway (hsa04151), the MAPK signaling pathway (hsa04010), the Ras signaling pathway (hsa04014), the FoxO signaling pathway (hsa04068), the HIF-1 signaling pathway (hsa04066), the TNF signaling pathway (hsa04668), the p53 signaling pathway (hsa04115), and the NF-kappa B signaling pathway (hsa04064). As shown in  HMM and HDW and luteolin (MOL000006) from PHP and RMB, have been shown to be effective in downregulating the phosphorylation of Akt, which leads to the inhibition of cell proliferation [The disruption in the balance between the proliferation and apoptosis of gastric epithelial cells is linked to the progression from chronic gastritis, to atrophy, intestinal metaplasia, dysplasia, to ultimately cancer . As demoferation , 54. Addferation –57. All  PHP, oleanolic acid (MOL000263) from RMB and HDW, and kaempferol (MOL000422) from HMM. In the Ras and FoxO pathways, the Fas ligand (FasL) that belongs to the TNF family leads to apoptosis upon binding to its receptor and significantly influences the progression of cancer [Apoptosis is a crucial mechanism leading to cell death, and the failure to inhibit apoptosis induces the formation of certain gastrointestinal malignancies . It has f cancer . The afo Helicobacter pylori is recognized as a risk factor for atrophic gastritis, intestinal metaplasia, and adenocarcinoma [ RMB, has COX-2-dependent anticancer properties [Chronic mucosal inflammation is associated with a high risk of progression from chronic gastritis to gastric cancer . Chronicarcinoma . As indiarcinoma . A studyoperties . These dGastric cancer is one of the most common types of cancer, and the mortality rate has markedly improved , 68. Ear HMM and HDW exhibited the highest degree number of interactions with various protein targets. Being the main dietary flavonoid, quercetin not only functions as a radical-scavenging antioxidant, but also suppresses inflammation for inhibiting carcinogenesis [α (HIF-1α) [The results show that 82 active compounds were obtained from WPX, and 146 potential targets were found to be linked to multiple compounds from different herbs. These indicate that WPX exerts therapeutic effects on GPL through multiple compounds and targets. Among the active compounds linked to the network, quercetin , luteolin , kaempferol , isorhamnetin , acacetin , and astragaloside IV  are well-known bioactive compounds in the treatment of gastric cancer –76. For ogenesis . Accordiogenesis . Further(HIF-1α) .On the other hand, numerous targets were found to be linked to multiple compounds from different herbs, which indicated the synergistic property of the compounds in the WPX decoction in the treatment of GPL. The target PTGS2 (Prostaglandin-Endoperoxide Synthase 2) for instance, also known as COX-2, was connected to 64 active compounds of the WPX formula. COX-2 plays an important role in the progression of GPL , and a rAlthough numerous researches have demonstrated that TCM formulations are effective and safe in treating GPL , the mecA total of 82 bioactive compounds were identified from WPX, which provided potential clues for investigating the molecular mechanism underlying the therapeutic effect of WPX.A total of 146 targets were predicted by comparing the targets of the WPX compounds with the GPL targets, which further demonstrated the characteristic multitargeting property of WPX. The results of GO enrichment and C-T network analysis revealed that WPX exerts a therapeutic effect by interfering with apoptosis and cell proliferation.The results of GO enrichment and the integrated GPL pathways revealed that the WPX decoction targets 26 key biological processes and 21 pathways involved in the pathogenesis of GPL, which further demonstrated the three dysfunctions of GPL, namely, cell proliferation, apoptosis, and mucosal inflammation.On the whole, this study investigated the mechanisms of action of WPX in the treatment of GPL and provides a novel approach for exploring TCM formulations. The results will advance the cognitive understanding of TCM formulations and promote the therapeutic application of traditional medicines for the treatment of diseases in the present day scenario.GPL is related to the development of gastric cancer that progressively advances through the formation of sequential lesions, and the treatment of precancerous lesions offers an effective measure for decreasing morbidity in the initial phase of gastric carcinogenesis. In this study, we employed a systems pharmacology approach by integrating active compound screening, target prediction, network analysis, and pathway analysis to explore the molecular mechanisms underlying the therapeutic effect of the WPX decoction in the treatment of GPL. The key results of this study are as follows:


Porphyromonas gingivalis is a pivotal periodontal pathogen, and the epithelial cells serve as the first physical barrier to defend the host from bacterial attack. Within this host-bacteria interaction, P. gingivalis can modify the host immune reaction and adjust the gene expression, which is associated with periodontitis pathogenesis and developing strategies. Herein, a meta-analysis was made to get the differential gene expression profiles in epithelial cells with or without P. gingivalis infection. The network-based meta-analysis program for gene expression profiling was used. Both the gene ontology analysis and the pathway enrichment analysis of the differentially expressed genes were conducted. Our results determined that 290 genes were consistently up-regulated in P. gingivalis infected epithelial cells. 229 gene ontology biological process terms of up-regulated genes were discovered, including “negative regulation of apoptotic process” and “positive regulation of cell proliferation/migration/angiogenesis”. In addition to the well-known inflammatory signaling pathways, the pathway associated with a transcriptional misregulation in cancer has also been increased. Our findings indicated that P. gingivalis benefited from the survival of epithelial cells, and got its success as a colonizer in oral epithelium. The results also suggested that infection of P. gingivalis might contribute to oral cancer through chronic inflammation. Negative regulation of the apoptotic process and transcriptional misregulation in cancer pathway are important contributors to the cellular physiology changes during infection development, which have particular relevance to the pathogenesis and progressions of periodontitis, even to the occurrence of oral cancer. Porphyromonas gingivalis is an opportunistic pathogen and mainly colonized in periodontal tissues P. gingivalis may modify gene expression, even host immune response by degrading host cell surface proteins or receptors et al. have employed microarray analyses to speculate the gene expression signatures in periodontitis Periodontitis is a ubiquitous inflammatory disease and the primary cause of tooth extraction in adults resulted from infection of periodontopathic bacteria. The host manifests an inflammatory immune response to this bacterial infection. P. gingivalis infection. The present study gives us a synthesized assessment of the gene expression signatures in the epithelial cells with P. gingivalis infection. The differentially expressed genes (DEGs), the gene ontology (GO) terms and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways concerned in the transcription signatures were identified A meta-analysis deals with the transcriptome data by combining the individual microarray study, and counters the above-mentioned disadvantage Porphyromonas gingivalis” or “gingival epithelial cells” or “oral epithelial cells” were downloaded. Epithelial cells infected with P.gingivalis were considered as “case group”, while non-infected epithelial cells as “control group”. Sample-sourced datasets from epithelial cells infected with other bacteria were excluded. Three separate microarray datasets were enrolled with raw data. The details of these datasets were listed in Table An electronic search was performed in Gene Expression Omnibus  database. Microarray data of the gene expression with the keywords “P.gingivalis or non-infected control. Microarray data for epithelial cells with P.gingivalis infection (n = 11) and non-infected control (n = 11) from GEO transcriptome database (http://www.ncbi. nlm.nih.gov/geo/) were compared for their transcriptome profiles.The basic data were obtained from the 3 individual studies, including GEO accession; sample source; platform; numbers of cases or controls in the array data. Two of the datasets were performed in Affymetrix Human Genome U133 Array. The third dataset was conducted in Affymetrix Human OElncRNAs520855F Array. This analysis contains totally 22 GEO transcriptome datasets for epithelial cells infected with The research was authorized by the Ethics Committee of School of Stomatology, China Medical University (Shenyang). They determined the current analysis of the public data set did not require the consents of the patients.P.gingivalis infection or not, the datasets extracted from the individual microarray assays were submitted to the network-based meta-analysis program for gene expression profiling (http://www.inmex.ca/INMEX/), starting with multiple gene expression tables for meta-analysis 2 transformed and normalized to zero mean and unit variance. While performing differential expression analysis on individual data set, the False Discovery Rate (FDR) of Benjamini-Hochberg's was set 0.05 in order to adjust the cut-off P-value. Third, data integrity was assessed as described previously P values were detected according to Fisher's method. A P value of less than 0.05 was considered as the criterion of statistically significant in our study. The genes selected were classified based on the grade products of the combined P-value. The statistics were downloaded and visual exploration was performed. A Venn diagram was made to compare the difference between the findings of the meta-analysis and the individual study (http://bioinformatics.psb.ugent.be/webtools/Venn/).DEGs were identified in epithelial cells with P-value < 0.05 were utilized to determine a critical analysis. Our figures listed the typical GO biological process terms, which were chosen from the functional annotation charts that were significantly enriched at the top. Representative KEGG pathways chosen from the most significantly enriched charts were shown in our figures.Subsequently, we exploited a web-based tool  to conduct the GO Biological Process (GO_BP) terms and KEGG pathway analysis PLK2, CYP24A1, TNFAIP3, PTGS2, EDN1, IL6, GADD45A, IL1B, etc. The top 50 up-regulated DEGs were shown in the heatmap  signaling pathway, tumor necrosis factor (TNF) signaling pathway, mitogen-activated protein kinase (MAPK) signaling pathway, we found that the pathway associated with a transcriptional misregulation in cancer, Janus kinases/signal transducer and activator of transcription (JAK/Stat) pathway, and transforming growth factor-β (TGF-β) signaling pathway, had also been increased. Representatively, the involved DEGs of the KEGG pathways of transcriptional misregulation in cancer were listed in Table P. gingivalis is one of the most prominent pathogens in the etiology of chronic periodontitis. Epithelial cells are now considered to be the first immune barrier in the pathogenesis of periodontal disease confronted by the microorganism that colonizes or even intrudes mucosal surfaces. The intercommunication of P. gingivalis with epithelial cells involves several intricate signaling pathways P. gingivalis can not only invade the local periodontal tissue but also persist in other systems of the human being , we should be more cautious in the active performance of P. gingivalis with epithelial cells.As a Gram-negative anaerobic bacterium, P. gingivalis-infected epithelial cells based on comparisons to the GO Now we have obtained the transcriptomic profile with our meta-analysis using several public microarray data. Taking advantage of this enhanced statistical power, our results were more reliable BCL2A1, BCL2, BARD1, CFLAR, were significantly up-regulated in epithelial cells infected with P. gingivalis compared to non-infected ones. It is conceived that P. gingivalis improves the survival of host cells by directly inhibiting different pro-apoptotic pathways P. gingivalis in these cells. It is reported that this resistance to apoptosis may be related to the BCL2 protein family, including BCL-2 and BAX P. gingivalis infection was phosphatidylinositol 3 kinase/Protein Kinase B (PI3K/Akt) -dependent, furthermore, the PI3K signal pathway could regulate transcription or post-translational modification of certain BCL2 family member In our meta-analysis we showed several genes about negative regulation of the apoptotic process, including P. gingivalis infection has numerous anti-apoptotic effects on epithelial cells . The diversity of these multiple genes and pathways demonstrates that P. gingivalis may affect apoptotic pathways at different levels. Some of the up-regulated genes and pathways surround the mitochondria, which is a critical component of the intrinsic apoptosis process. The analysis of our study, along with others reports, showed that P. gingivalis suppressed the apoptosis of epithelial cell partially through the JAK/Stat signal pathway, which regulates the inherent mitochondria cell death mediated by the caspase-dependent apoptotic pathway.Our results verified that P. gingivalis participates in the regulation of the extrinsic apoptotic pathway. Our KEGG pathway analysis demonstrated NF-κB signal pathway was up-regulated. It is reported that NF-κB, together with TNF and FasL, takes its role in the composition of the extrinsic apoptotic process P. gingivalis to benefit the survival of epithelial cells may assist its successful colonization in the oral mucosal epithelium.At the meantime, CXCL8), CCAAT/enhancer binding protein epsilon (CEBPB), Meis homeobox 1 (MEIS1), interleukin 6 (IL-6), myeloperoxidase (MPO) and plasminogen activator urokinase (PLAU) were increased significantly.Besides, the current analysis found that the transcriptional misregulation in cancer, another KEGG pathway, was up-regulated. Related genes expression, including C-X-C motif chemokine ligand 8  et al. revealed that both CEBPB and IL6 were up-regulated in late OSCC and CEBPB was activated as an upstream regulator of IL6. Moreover, CEBPB was recently identified as one of the master regulators in cancer biology CXCL8 and its mRNA in P. gingivalis-infected epithelial cells or oral squamous cells P. gingivalis stimulated the tumorigenic features and invasiveness of oral squamous cells, which was correlated with enhanced expression of CXCL8Nowadays, more researches have reported a significant increase of In addition, over-expression of MEIS1 in acute leukemia has been observed P. gingivalis in contribution to oral cancer. Previous research by our group showed P. gingivalis enhanced the cell proliferation, migration, and invasive ability of human oral epithelial cells. Thus it promoted the cells tumorigenic properties et al. reported that chronic inflammation induced by bacterial infection drove carcinogenesis, involving induction, progression, invasion, and metastasis So far, several studies have attempted to elucidate the role of P. gingivalis played a role in promoting tumor transformation in some inflammatory conditions. Here pathways enrichment investigation showed that several pathways were enhanced significantly, including the NF-κB pathway, TNF pathway, TGF-beta pathway, MAPK signal pathway, and cytokine-cytokine receptor interaction. These inflammation-related signaling pathways were listed as the top 5 of our KEGG pathway analysis results. Here we believe that this chronic bacteria infection should be identified as probable effector which may result in oral cancer.Our KEGG pathway analysis further confirmed P.gingivalis. The bioinformatics analyses of the up-regulated genes showed us that the GO terms of the negative regulation of apoptotic process, several inflammation-related signaling pathways, and the transcriptional misregulation in cancer were significantly enriched. It provided us some new sights into the alteration in cytopathogenicity that took place in the process of infection, which had particular relevance to periodontitis pathogenesis and developing strategies. Meanwhile, it provided more evidence to support the view that P.gingivalis might be related to the occurrence and development of OSCC.Generally speaking, the current meta-analysis using microarray datasets offered us an outlook about the gene expression characteristics in epithelial cells infected with Supplementary table: All the up-regulated genes were listed in supplementary table S1; All the down-regulated genes were listed in supplementary table S2; GO Biological Process terms of up-regulated genes were listed in supplementary table S3; KEGG pathway of up-regulated genes were listed in supplementary table S4.Click here for additional data file.


N1PAS quantifies the degree of alternative splicing via Hellinger distances followed by two-stage clustering to determine pathway enrichment. We provide a clinically relevant “odds ratio” along with statistical significance to quantify pathway enrichment. We validate our method in clinical samples and find that our method selects relevant pathways (p < 0.05 in 4/6 data sets). Extensive Monte Carlo studies show N1PAS powerfully detects pathway enrichment of ASGs while adequately controlling false discovery rates. Importantly, our studies also unveil highly heterogeneous single-subject alternative splicing patterns that cohort-based approaches overlook. Finally, we apply our patient-specific results to predict cancer survival (FDR < 20%) while providing diagnostics in pursuit of translating transcriptome data into clinically actionable information. Software available at https://github.com/grizant/n1pas/tree/master.RNA-Sequencing data offers an opportunity to enable precision medicine, but most methods rely on gene expression alone. To date, no methodology exists to identify and interpret alternative splicing patterns within pathways for an individual patient. This study develops methodology and conducts computational experiments to test the hypothesis that pathway aggregation of subject-specific alternatively spliced genes (ASGs) can inform upon disease mechanisms and predict survival. We propose the N-of-1- Most RNA-Seq analytics ignore alternative splicing patterns despite recent evidence that alternative splicing is implicated in nearly a third of common diseases. In cancer, a tumor often displays dysregulation of the cellular machinery that controls alternative splicing (RNA-Sequencing (RNA-Seq) data offers an opportunity to enable precision medicine, but most methods rely on gene expression alone . RNA-Seqsplicing . Yet, thThis study develops methodology and conducts computational experiments to test the hypothesis that pathway aggregation of subject-specific alternatively spliced genes (ASGs) can inform upon disease mechanisms and predict survival, thereby providing clinical interpretation of alternative splicing patterns. By “alternative splicing” patterns, we mean that the distribution of isoforms of a certain gene differs between two samples. Specifically, in the context of cancer, our hypothesis is driven by the high likelihood that comparing non-cancer  tissue to cancer tissue will unveil cell-type specific expression in cell-type specific pathways. This, in turn, will affect the proportion of the ASGs in the overall comparison between tissues (where the cell-type elements have changed) and will distribute in pathways. Two facts taken together form this opinion: (1) cellular-specific splicing occurs and (2) if a differentially expressed gene (DEG) occurs between paired samples, it is in part due to the change in activated pathways within the concordant cell types that have become cancerous, and in part due to the change of cell-type proportions in the cancer tissue vs. normal tissue .pathways, to provide subject-specific interpretations of the transcriptome  method transforms a patient’s paired-sample RNA-Seq isoform expression data  into a pathway enrichment profile of ASGs. N1PAS quantifies the degree of alternative splicing using gene-wise Hellinger distances followed by two-stage clustering to determine pathway enrichment using a robust, existing procedure testing procedure – local false discovery rate (locFDR). The single-subject output provides an interpretable odds ratios describing the overrepresentation of ASGs along with uncertainty quantification through locFDR.In this study, we propose a novel methodology to improve the clinical interpretation of subject-specific alternative splicing patterns derived from paired RNA-Seq samples. The N-of-1-The article continues with some brief mathematical background and description of the proposed method. Then, several computational experiments explore and validate our proposed methods in clinical samples. In this proof of concept study, we demonstrate the potential for alternative splicing interpretation as one of the Omics signals which should be considered for predicting cancer survival. We also compare the proposed method with alternative approaches. Finally, we conduct extensive simulation studies to explore empirical operating characteristics of N1PAS. A discussion concludes the article.This section motivates the use of and describes two mathematical concepts employed in the proposed method.Hellinger distance. Such an approach has been shown to be useful in the quantification of alternative splicing in the context of clustering  by definition. To establish a convention, if a gene is not expressed in both samples, we choose to record a missing value for the distance.Simply stated, the p-values) derived from these measurements will also be correlated. So, we need a model that either specifically accounts for this co-expression or does not assume independence. Efron discusses the statistical issues in detail in Efron’s local false discovery rates provides a flexible and robust tool for multiple hypothesis testing under correlated test statistics or effect sizes , 2013. Ri be an observed test statistic from i = 1,…, N testing procedures. N must be large to ensure quality locFDR estimates, say at least in the hundreds. But the zi need not be independent. We assume that the N-values can be sorted into two classes (“null” and “non-null”), occurring with prior probabilities of p0 or p1 = 1 - p0:Local FDR results from modeling test statistics as arising from a two-component mixture density. Formally, let znull subdensity as:Define the mixture density:And the Then, define the local false discovery rate  as the Bayes posterior probability that a case is null given z:The definition provides a straightforward interpretation: It is the probability an observed value came from the null density. In practice, Efron indicates that a locFDR < 0.2 provides strong statistical evidence that the case is from the non-null distribution.k-means) clustering (pathway). That is, odds ratios  by fitting the two-component mixture model to the distribution of pathway odd ratios . This whThis section describes computational experiments to explore, validate, and apply N1PAS. We conduct these studies using RNA-Seq data derived from clinical samples housed in The Cancer Genome Atlas (TCGA). Pathway annotations are based on the Kyoto Encyclopedia Genes and Genomes KEGG; . Surviva1. The UCSC Table Browser (Data sets were selected based on the availability of: (1) paired normal-tumor isoform-level quantification from each cancer patient, (2) a KEGG pathway annotated to the same cancer, and (3) survival data. Six TCGA data sets were identified  meeting  Browser  was usedThe RNA-Seq data sets were filtered to include patients with paired normal-tumor data. Such patients were identified via the R library TCGA2STAT . FurtherGenes were annotated to KEGG gene sets (pathways) using the Bioconductor database KEGG.db version 2.3.5, downloaded 16 Sep 2009. In total, 230 gene sets were downloaded. To improve efficiency of the algorithms developed, the 73,599 isoforms measured were filtered to only those that mapped to a gene annotated to a KEGG pathway , resulting in 18,823 isoform-level quantities for the 5,757 genes measured in the TCGA data set. Following standard practice in alternative splicing analytics  only gentarget pathways. Our strategy here was inspired by the work of target pathway as a KEGG pathway whose description matches the disease associated with the RNA-Seq data set  × P (pathways). The assessment begins with the original matrix of alternatively splicing calls – 1 indicating that a pathway had both an odds ratio greater than 1 and locFDR was less than 0.20, and 0 otherwise. Then each patient’s values (rows) are shuffled. This procedure will preserve the number of 0’s and 1’s within each patient while disrupting the correspondence across individuals and pathways. Thus, each column forms a synthetic null pathway with values not tied to any particular pathway annotation while preserving the patient distributional characteristics. Once shuffled, a null pathway is randomly selected and the number of 1’s (significant pathways) are counted to calculate the null capture rate. The procedure is repeated 2000 times to form an approximate null distribution for the capture rate for a single pathway. An empirical p-value is the proportion of null pathways with a higher capture rate than the observed target capture rate for a given data set. To assess the capture rate for at least one of nine selected pathways (mimicking the cancer-annotated capture rate), the capture rate of at least one of nine pathways is computed by selecting nine columns at random, without replacement, and counting a “success” as at least one “1” in the vector of 9 values. This procedure is repeated 2000 to produce a null distribution corresponding to the chance of capturing at least 1 out of 9 cancer-annotated pathways. The empirical p-value for this assessment is proportion of times the null capture rate is larger than the observed cancer capture rate. The assessment is limited as it makes no adjustment for the fact that smaller pathways correspond to more variable odds ratios .The above-mentioned empirical significance assessment entails producing 2000 R (locfdr) contains default parameters that assume there are a large number of tests (at least 1000). Since there are approximately 200 pathways under consideration, custom configurations needed to be developed. Based on practical experience with the pathway odds ratio distributions, the model fits reasonably well using the following heuristic procedures: (1) filter outlier odds ratios and set FDRloc to 0 for those pathways, (2) the number of breaks in the histogram is set to 25, (3) nulltype is set to “Central Matching” to provide less conservative results , and (4) the mixture density estimate’s degrees of freedom is set to 4. In larger ontologies, program defaults should be adequate, but the model fit should always be inspected. It is important to note that the number of genes in each pathway does not play into any locFDR calculations. We chose to forgo any formal inferences at the gene level as it is rate prohibitive to manually inspect and adjust the fit at the first stage of clustering . This produces a new odds-ratio matrix (P minus the number of filtered pathways). Now, patients are clustered (unsupervised) into two groups  using only the odds ratios for a single pathway (a priori set to two. For each clustering (one per pathway), Kaplain–Meier estimates . Pathways withFDRBH < 20% are identified as survival-relevant pathways , all WI isoform measurements are included in the call to the PAM clustering algorithm. The second method uses both the tumor and normal isoform expression, but is only concerned with differential expression not alternative splicing patterns. The third method quantifies differential isoform usage, but does not aggregate these patterns at the pathway level (as in N1PAS). As we defined the Hellinger distance only when both samples expressed the gene, care was needed to form a complete 51 × 4133 matrix of Hellinger distances. Specifically, any missing gene-wise distances were imputed with a patient’s mean Hellinger distance across all genes. Each method produces two patient clusters as in empirical null of survival p-values, each patient’s WI isoforms or genes were shuffled to disrupt correspondence with the specified pathway’s true annotations (similar to the other permutated distributions above) and two clusters are determined. Relevant pathways are identified using the FDR-corrected empirical p-values as shown in To explore whether pairing and aggregating alternative spliced genes at the pathway level improves the detection of survival-relevant pathways, we modify the above pipeline by systematically modifying the input to the binary clustering in three ways: (1) use only the tumor isoform expression (as opposed to N1PAS odds ratios and locFDR), (2) use the difference in tumor isoform expression from the normal isoform expression, and (3) use the Hellinger distances corresponding to each of the 4133 KEGG-associated genes The first method differs from N1PAS in that for each pathway G, in the pathway and (2) proportion, π, of ASGs within the pathway over the background percentage of ASGs. We study the empirical false positive rates and  power to detect an enriched pathway based on permutations of patient-specific Hellinger distances for all 246 TCGA patients  number of expressed genes, patients  while vabackground level and denote this quantity asπall. Next, for each patient, we shuffle the gene labels to approximate a ‘null’ distribution of Hellinger distances as the values do not aggregate meaningfully into pathways. Then the number of expressed genes G (approximate gene set size) is selected from the set {15, 30, 50, 100} and an effect size π is selected from the set {0, 0.05, 0.10, 0.15, 0.20}. Next, we randomly select one of the 206 KEGG pathways that has at least G expressed genes  to label Hellinger distances with specific gene labels to induce the effect size π. Call this selected pathway the specified pathway. To induce the specified effect size, G∗ gene labels within the specified pathway were randomly chosen and assigned randomly sampled Hellinger distances from the ASG group. The remaining genes were randomly assigned values from the non-ASG group. Then N1PAS was run on the permuted, modified Hellinger distance data for all 206 KEGG pathways. This process was completed 100 times across the 4 × 5 simulation configurations for a total of 2000 runs per patient. This results in 246 patients × 2000 runs per patient for a total of 492,000 simulated N1PAS runs.To avoid over-simplistic parametric and statistical assumptions  and to anchor simulation results to our studied setting, we restricted our Monte Carlo experiments to permutated TCGA patient-specific Hellinger distances for genes annotated to KEGG pathways. First, Hellinger distances within each gene were computed for all 246 patients across the 6 TCGA data sets. This produces patient-specific distributions of distances that are then used to classify genes into two groups: ASG or not as in  for eachThis section describes observations from the target KEGG pathway validation study across six TCGA data sets , an applexpressed genes (in either sample from a patient). Of the 206 KEGG, the number of pathways scored across all data sets was fairly constant (with a median of approximately 185 pathways). The locFDR significance threshold appears somewhat severe with the median percent significantly enriched ranging from 3 to 7.8% across data sets. So, it is quite difficult in this small ontology to call a pathway as enriched with ASGs. One should keep in mind that pathway can have a high proportion of ASGs yet still not be significantly enriched, especially if alternative splicing is rampant through the entire transcriptome.The frequency of significantly enriched pathways of ASGs varies greatly from data set to data set and patient to patient. bladder cancer was the most often-captured target pathway, with 9 of the 19 (47%) Bladder Cancer (BLCA) patients significantly enriched. To put this in context, a random null pathway for the BLCA data was never captured at such a high rate in 2000 empirically-derived pathways (p < 0.001). In fact, the target capture rate is greater than expected for all data sets except for the two lung cancer data sets (LUSC and LUAD). Moreover, the rate of cancer pathway capture is higher than expected from four of the six data sets.The target pathway capture rate  is greater than expected for most data sets. The KEGG pathway While the capture rates on the surface appear lukewarm, we view the results as unveiling the need for subject-specific metrics. If one relied on cohort-based methods, the heterogeneity in splicing patterns would be missed. To explore this notion, we clustered patients into two groups using the odds ratios within target pathway. impoverishment of ASGs for some patients  and high cancer pathway capture rate (47%) for the LUSC data set . This will require care to uncouple for the patient at hand. For example, a patient may exhibit a worse prognosis in the staphylococcus aureus infection pathway, but a better prognosis in the cancer-annotated pathways. Hypothetically, this patient may then respond well to the standard treatment in combination with an innovative treatment to address the dysregulation in the staphylococcus pathway.One practical issue with subtyping a patient using multiple survival-relevant pathways is that different clusterings may disagree with prognosis . Better subtype . This is interesting as it may indicate that individual lung cancer patients display unique dysregulated pathways (motivating precise treatments). We also observe that subtyping based on the three cancer-associated pathways  generally agree well for the Better patients. But these Better subtypes from cancer pathways agree poorly with the staphylococcus aureus infection subtyping. The Worse subtypes generally agree less well than the Better subtypes, and with a similar trend in disagreement with the staphylococcus pathway. This suggests a distinct survival-related signal in this pathway from the cancer-annotated dysregulation. Of course, conflicting subtypes will complicate clinical application and slow any decision process, thus limiting our proposed approach.To gain insight into subtype overlap within our LUSC case study, we explore agreement across the top-four survival-relevant pathways . To quanAs detailed in section Alternative Approaches to Survival Subtyping, the input data to the clustering step of our proposed subtyping pipeline was modified using three straightforward alternative approaches for the 51 LUSC patients: (1) using tumor isoform expression, (2) using the difference in isoform expression between tumor and normal samples, and (3) Hellinger distance data for the 4133 genes annotated to KEGG pathways. non-small cell lung carcinoma target pathway is ranked relatively poorly  when compared to the N1PAS method reported in chronic myeloid leukemia, was significant at FDR < 20% – in contrast to the two other alternative methods.Finally, we use the Hellinger distances for each gene across the 51 LUSC patients to cluster patients. This signal is somewhat nearer to N1PAS and is concerned with alternative splicing patterns. N1PAS takes these distances one step further by aggregating the signal into pathway-level enrichment of ASGs . The average πall across the 246 patients is 0.1914, but vary from 0.1088 to 0.4417 with the middle 50% of the rates vary from 0.1613 to 0.2122.Our simulation results provide insight into N1PAS empirical operating characteristics, specifically simulated false-positive rates and statistical power. Among the 492,000 N1PAS runs, there were 625 algorithm failures (0.127%) due to misfit odds ratio mixture modeling. This may happen in practice, although rarely, and the parameters of locFDR will have to be manually adjusted. Here, we discarded these runs from the results. Each patient displays a different Hellinger distance distribution with their own background level of ASGs . ASGs can aggregate in pathways by chance in this setting. We calculated the simulated false-positive rate as the number of detected pathways among the 206 KEGG pathways under permuted patient-specific Hellinger distances. We performed 100 simulation replicates per patient and computed the patient-specific average false-positive rates. Further, we pooled these mean rates within each TCGA data set and computed the empirical standard error of the mean to assess variability across patients. Finally, we computed pointwise 95% confidence intervals for the data set mean false-positive rate using a normal approximation centered at the overall mean with the observed standard error. power the observed detection rate of a specified pathway, with an induced enrichment of ASGs. G). We increment the effect size π from 0.05 to 0.20 to calibrate how sensitively N1PAS can detect an enriched pathway. 95% confidence intervals for mean power were constructed at each pair (π G) within each data set as above in the false-positive rate study.Empirical power estimates, as detection rates, from our simulations are graphed in G for immediate values of π .Patterns in In general, these results suggest that N1PAS procedure exhibits good false-positive error control and excellent power, at least under the settings chosen for these simulations.This study creates a first look at personalized alternatively splicing patterns within pathways. As such, these patterns are likely complementary to other ‘Omics measures, and thus the proposed N1PAS strategy could become an additional tool to bridge the gap between RNA-Seq data and clinical translation. For example, one could investigate whether splicing events occur in a coordinated way in response to a stimulus or perturbation like a medication. A practical limitation is the relative rarity of paired RNA-Seq data (compared to single-sample expression data). It’s true that paired unaffected and cancer tissues are currently uncommon. Yet novel experimental designs and corresponding analyses will drive data collection protocols. Indeed, as the National Institute of Health and National Cancer Institute announced , there iBetter or Worse survival groups. This informs on both prognosis and on patient-specific disease mechanisms. Our proposed method, however, only considers one pathway at a time and therefore only partially explains survival, compared to using all the odds ratios. Future studies could improve survival prediction through aggregation of pathway metrics and potentially have greater clinical utility in terms of prognosis in addition to combining multiple ‘Omics measurements.To make clear of a potential clinical use of N1PAS within the survival subtyping pipeline of Omics signals, imagine the following scenario: a patient suffering from non-small cell lung cancer consents to paired tumor-normal RNA-sequencing. Patient-specific N1PAS odds ratios are first computed in the five survival-relevant pathways. Each survival-relevant pathway has an associated odds ratio threshold  as well as exhibit differential isoform usage. As such, the signal obtained by N1PAS may not be purely alternative splicing as traditional DEGs may still contribute. Another issue is that different isoforms may not always be indicative of differential protein structure or activity and the biological impact may be minimal in these situations. The model does not currently account for any noise that may be present in RNA-Seq measurements, an important consideration in the N-of-1 setting. Along those lines, the estimated proportions pgA in Equation (1) depend on the initial accuracy of the isoform abundances. This accuracy depends on several aspects such as read depth, number of expressed isoforms, and the specific method that has been used for estimating the transcript abundance. Therefore, the estimated proportions may have different variances and other statistical properties. Our method does not explicitly account for these differences. An important extension of our model could include this uncertainty in estimating the proportions for a more holistic and realistic formulation.There are, of course, limitations and caveats to the methodology. The proportion clustering approach seeks to quantify differential relative isoform usage and not differential p-value aggregation methods (such as Fisher’s method) could be explored. Care must be taken, however, as these methods often assume independent measurements. Future studies could develop more sophisticated and novel p-value aggregation approaches in this setting. Additionally, it would be interesting to vary the pathway definitions across several databases in future studies. Lastly, this proof of concept study was designed to demonstrate the utility of alternative splicing signals in absence of other signal to avoid confounders. Therefore, in future studies, this method should be opportunistically combined with any ‘Omics as well as clinical signals in order to translate clinically useful predictions with high accuracy.We acknowledge some limitations to the computational experiments in the study. The significance of the survival curves has been assessed on a single retrospective dataset and could be over-optimistic. Future studies should include prospective independent datasets. Our choice of pathway database is outdated and more current databases may provide more informed discoveries. One could imagine a variety of alternative methods to form comparisons against N1PAS. For example, pathways Alternatively Spliced, to quantify differential mRNA isoform usage within biological pathways from paired-sample RNA-Seq data. A target pathway validation study on paired normal-tumor samples from TCGA reveals that in most data sets the identified pathways generally concur with the annotated disease (bladder cancer pathway was more likely to be enriched with ASGs). More than just providing validation, this study also highlights the vast patient-to-patient heterogeneity in alternative splicing patterns. This heterogeneity actually motivates our N-of-1 approach: despite having the same disease, patients vary greatly in their splicing dysregulation. Our identification of subject-specific splicing dysregulation offers targets for personalized interventions and monitoring plans. Next, we applied our N1PAS single-subject metrics to predict survival within a novel subtyping pipeline. The output of this pipeline contains easy-to-interpret diagnostics to enable precision medicine from transcriptome data. Finally, we showed adequate statistical power and false-positive rates in simulation studies.We proposed a single-subject methodology, N-of-1-AS and YA convinced the foundational concepts, designed experiments, and explored the results. AS developed the methodology and drafted the manuscript. AS and DA conducted the computational experiments. YA contributed sections. AS and CK developed the figures. All authors contributed to manuscript revision, read and approved the submitted version.https://github.com/grizant/nof1-splice. An R package to conduct N1PAS, including vignettes, is freely available at https://github.com/grizant/n1pas/tree/master.All code used to conduct this study are freely available at The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


For years, microbes have been widely applied as chassis in the construction of synthetic metabolic pathways. However, the lack of in vivo enzyme clustering of heterologous metabolic pathways in these organisms often results in low local concentrations of enzymes and substrates, leading to a low productive efficacy. In recent years, multiple methods have been applied to the construction of small metabolic clusters by spatial organization of heterologous metabolic enzymes. These methods mainly focused on using engineered molecules to bring the enzymes into close proximity via different interaction mechanisms among proteins and nucleotides and have been applied in various heterologous pathways with different degrees of success while facing numerous challenges. In this paper, we mainly reviewed some of those notable advances in designing and creating approaches to achieve spatial organization using different intermolecular interactions. Current challenges and future aspects in the further application of such approaches are also discussed in this paper. Corynebacterium glutamicum was used in the biosynthesis of medical materials such as l-amino acids  can precisely meet these requirements and provide clues for new spatial organization systems. To precisely edit genome, genome editing tools often rely on highly effective and sequence-specific DNA-binding. For example, zinc finger nuclease contains a engineerable zinc finger domain, which consists of three to six replaceable individual zinc finger repeats to bind desired DNA sequence . SimilarE. coli was also reported [Accordingly, recent studies have developed multiple enzyme clustering approaches with these DNA-binding proteins considering their potential for scalability and robustness. Currently, approaches inspired by ZFN and TALEN have already been used for the spatial organization of exogenous enzymes. In 2012, Conrado et al.  reportedreported . Comparereported . Using tMore interestingly, a recent study applied the CRISPER–Cas9 technique as a novel system to concentrate specific enzymes for metaplastic functions . In thisAs a new and rapidly advancing strategy for the spatial organization of pathway enzymes, the DNA-binding mechanism in genome editing tools provides a promising way to establish highly stable, extendable, and low-cost systems for such applications. On the other hand, the development of such methods also provides a new vision on how techniques such as genome editing could be used besides its current application in genome editing. In the meanwhile, it should be noted that, with further study on the structural basis and mechanisms of protein-nucleotide interactions, current DNA-binding domains can be further engineered to generate more robust DNA-binding proteins for new spatial organization systems in the near future.Since the metabolic pathways are basically a set of enzymes with different dynamics, finding ways for balancing the metabolic flow and reducing negative impact to the chassis is now an important field for synthetic biologists. Despite the strategies employed, the purpose of the spatial control and organization of pathway enzymes remains the same. As an important complementary and parallel approach along with traditional ways, such as rational design of expression systems and optimization of enzyme expression levels, the spatial organization of pathway enzymes aims to provide a highly predictable and stable control of the heterogeneous metabolic pathways and reduce the effect on the survival and growth of the chassis.However, since the detailed reason why spatial organization of pathway enzymes can enhance the heterologous pathways is yet to be understood, improving the predictability and stability of such scaffolds is still a challenge. Supported by both computational modeling and wet-lab experiments , the metIn addition, according to either numerical experiments or wet-lab results currently reported, the benefits of the spatial organization of pathway enzymes are affected by multiple factors such as spatial orientation of enzymes, distance between enzymes, and copy number of scaffold molecules. With multiple, convoluted unknown variables, the rational design and functional examination of scaffold systems are difficult. Hence, further study on structural biology, especially on the detailed mechanisms of the pathway-enhancing effect of spatial organization, is needed. An in-depth investigation on the substrate channeling process on natural existing enzyme clusters, such as the tryptophan synthesis complex, would be a great start. Knowledge on the influencing mechanisms of these factors will provide a sound foundation for the design and creation of new spatial organization methods, especially for highly tunable protein-nucleotide interaction strategies. Moreover, the rapid development and uncovering of new genome editing techniques such as the CRISPR-C2c2  and CRIS