The results provide a comprehensive catalog of the analyzed pathways across bacteria. The analysis indicates an enrichment of tryptophan metabolism pathways in five gut-associated phyla, namely, Actinobacteria, Firmicutes, Bacteroidetes, Proteobacteria, and Fusobacteria. Further, five genera, namely, Clostridium, Burkholderia, Streptomyces, Pseudomonas, and Bacillus have been predicted to be enriched in terms of number of the analyzed tryptophan metabolism pathways, suggesting a higher potential of these bacterial groups to metabolize tryptophan in gut. Analysis of available microbiome data corresponding to gut samples from patients of neurological diseases and healthy individuals suggests probable association of different sets of tryptophan metabolizing bacterial pathways with the etiology of different diseases. The insights obtained from the present study are expected to provide directions toward designing of microbiome based diagnostic and therapeutic approaches for neurological diseases/disorders.The link between gut microbiome and brain is being slowly acknowledged due to the speculated role of resident gut microbial community in altering the functions of gut-brain axis (GBA). Recently, a number of microbial metabolites (referred to as neuro-active metabolites) produced through tryptophan metabolism have been suggested to influence the GBA. In view of this, the current study focuses on microbial tryptophan metabolism pathways which produce neuro-active metabolites. An  Trillions of microbes residing on and within the human body  have been shown to play roles in health and disease . In partThe GBA comprises of the central nervous system (CNS) including the brain and spinal cord, the autonomic nervous system, the enteric nervous system, and the hypothalmic-pituitary-adrenal (HPA) axis . The comCertain metabolites produced by gut microbiome have been shown to influence the GBA. For example, short chain fatty acids (SCFAs) like butyrate synthesized by gut microbiota are reported to stimulate memory and synaptic plasticity by inhibiting histone deacetylases . ButyratLactobacillus and Bifidobacterium can produce neurotransmitters like acetylcholine and gamma-amino butyrate (GABA)  . Streptonephrine . Howevernephrine .Enterococcus, Pseudomonas, etc., to produce one of the important neurotransmitters, serotonin, in tryptophan-rich media  tryptophan metabolism pathways. Evaluation of gut microbiome’s role in brain diseases and disorders was also carried out using publicly available data. The insights obtained from the current study will be helpful in comprehending the understanding of host–microbiota interaction in context of gut-brain axis.One of the primary objectives of the present study pertains to the identification of bacterial tryptophan metabolizing pathways which leads to the production of metabolites that have been reported to influence the GBA. These metabolites include kynurenine, quinolinate, indole, IAA, IPA, and tryptamine . Furtherin silico identification of the below mentioned three categories of pathways for production of “TRYP-6” in bacteria.Prediction of pathways is widely performed based on the presence of genes/proteins homologous to the experimentally identified genes/proteins involved in the particular pathway. However, this approach is limited in cases where one or more of the participating enzymes perform generic functions  and thus are involved in multiple pathways. In order to address this limitation, the current study utilizes enzyme homology along with genomic proximity of the constituent genes for accurate prediction of a pathway. In addition, the pathways involving specific enzymes were also considered in the current analysis. Consequently, the present study includes (i)Pathways experimentally characterized to have constituent genes in proximity on the genome;(ii)Pathways comprised of enzymes specific to the corresponding reactions;(iii)Pathways involving enzymes having specific functional domains.The experimentally characterized pathways for each of the analyzed neuro-active tryptophan metabolites of microbial origin are described below and have been schematically shown in (a)L-tryptophan to N-formyl-L-kynurenine (kynA) and kynurenine formamidase (kynB) were reported to be present as a gene cluster in Ralstonia metallidurans . Thus, in the current analysis, the pathway was predicted based on the presence of this functional domain across bacterial genomes.Kynurenine: The kynurenine pathway is a well-characterized pathway leading to production of nicotinamide adenine dinucleotide (NAD) in mammals . The enznurenine  Cytophaga hutchinsonii, P. fluorescens, etc.) revealed that the genes were not always located as a cluster in the genomes. Hence, prediction of the pathway in bacteria was performed based on the presence of the specific functional domain 3-HAO as well as the other two domains FAD_binding_3 (PF01494.18) and Aminotran_5 (PF00266.18), corresponding to the enzymes KMO and KYN, respectively.Quinolinate: Quinolinate, in addition to kynurenine, is also produced through the kynurenine pathway where kynurenine is further converted to quinolinate . The con(c)1 and the target dataset contained the protein sequences of completely sequenced bacterial genomes obtained from NCBI2 and HMP3.Indole: The pathway for biosynthesis of indole from tryptophan is reported to be catalyzed by a single-step reaction involving a very specific enzyme tryptophanase (EC 4.1.99.1) . However(d)Pseudomonas syringae has been shown to be facilitated by tryptophan-2-monooxygenase (EC: 1.13.12.3)  . An alte and gut . The key and gut  with an (e)In vivo studies have reported that IPA can be produced by Clostridium sporogenes present in the gastrointestinal tract of humans C. sporogenes and Ruminococcus gnavus (4 and HMP5.Tryptamine: The pathway for the production of tryptamine has been suggested to involve a single-step decarboxylation reaction facilitated by the enzyme tryptophan decarboxylase (EC 4.1.1.28). It is noteworthy that this enzyme is a generic aromatic amino acid decarboxylase and is also involved in production of dopamine from tyrosine . Furthers gnavus , their hs gnavus  was perf7 was generated to represent the proportion of strains of a gut-associated phyla predicted to harbor pathways for “TRYP-6” metabolites.Heat-map using “R” package “gplots”tnaA; catalyzes the conversion of tryptophan to indole) in Escherichia coli  to sequester tryptophan from host. The most widely studied tryptophan specific transporter is TnaB, which is encoded by a gene operonic to the tryptophanase gene  was required. This evaluation was made for all bacterial groups analyzed at genera level. For each of the bacteria identified to have the selected tryptophan metabolizing pathway, a score “SCORBPEO”  was assigned. The “SCORBPEO,” for each pathway was determined by taking into account the proportion of bacterial strains predicted to have the pathway, the confidence value of the corresponding bacterial group, and a “gut-weightage” factor. The confidence value was included to allow a higher score for the bacterial taxa which had relatively higher amount of representative strains in the database. For the calculation of the confidence value, the number of strains present under a particular bacterial group was first noted. Further, based on this count, a percentile value was assigned and consequently a rank (between 1 and 10) was assigned to the corresponding bacterial group. Another factor called the “gut-weightage” was incorporated in “SCORBPEO” to assign a higher score to the bacterial genera having higher number of gut-associated strains with a predicted pathway. Therefore, a higher “gut-weightage” (for a genus) would indicate an enrichment of a pathway in the strains found in gut comparative to the strains found in other environments. Thus, the score “SCORBPEO” for a particular neuro-active compound “i” corresponding to a bacterial genus “j,” was computed using the following equation:P represents the proportion of strains predicted with the compound “i” producing capability for the particular bacterial genus “j” , α denotes the confidence value of the corresponding bacterial group , and “β” corresponds to a “gut weightage” which represents an enrichment value of a particular pathway in the gut environment . Thus, the values of the computed “SCORBPEO” scores ranged between 0 and 50. The values were further rescaled to “0–10.” For a particular pathway, a bacterial genus having a higher “SCORBPEO” would indicate a greater capability of production of a particular compound as opposed to a genus with a lower “SCORBPEO.”where 10 was generated to represent the normalized “SCORBPEO” scores of gut-associated genera predicted to harbor pathways for “TRYP-6” metabolites.Heat-map using “R” package “gplots”In order to study any probable correlation of bacterial tryptophan metabolizing potential with the etiology of neurological diseases/disorders, gut microbiome data (16S rRNA) available from seven published studies were investigated. In particular, the gut microbiome data corresponding to three studies related to autism, two studies related to Parkinson’s disease, and one study on schizophrenia were analyzed. A summary on the analyzed datasets has been provided in p-value threshold of ≤ 0.01. Subsequently, a comparative analysis was performed between disease and healthy datasets with respect to—(a) abundances of bacterial genera predicted with the selected tryptophan metabolism pathways and (b) distribution of the pathways.In order to identify differentially abundant genera in healthy and diseased  samples, “Wilcoxon rank-sum” test  was perf11 was utilized to generate the same.The association between differentially enriched genera and pathways for “TRYP-6” metabolites was represented using “Sankey” diagrams. Python library “plotly”12 and HMP13) were analyzed using various computational approaches . The results of the analyses indicated the presence of one or more tryptophan catabolism pathways in 19 phyla  and role of this pathway in biofilm formation  were identified to be comparatively enriched in terms of proportion of tryptophan metabolizing strains . The resormation . Three gormation . InteresA schematic diagram has been depicted in Going deeper at genera level, a clear occurrence of high proportions of tryptophan utilizing bacterial strains in gut environment was observed. In order to understand potential of utilization of tryptophan by various genera, each genus belonging to the gut-associated phyla  having tryptophan metabolizing capability was assigned a score “SCORBPEO” . A higher “SCORBPEO” value for a given bacterial genus would indicate a greater probability of production of the particular metabolite in comparison to a genus with lower “SCORBPEO” value. An in-depth investigation of the selected pathways in the bacterial genera previously reported to be associated with gut indicated 52 gut genera  to have at least one of the six analyzed pathways . For eacClostridium and Burkholderia contained five out of the six analyzed pathways for production of neuro-active metabolites. While kynurenine production pathway was found to be absent in Clostridium, Burkholderia did not possess required genes for IPA production. Further, out of six analyzed pathways, Streptomyces, Pseudomonas, and Bacillus genus were observed to have only four pathways . Also, the observed high “SCORBPEO” value (10) of Pseudomonas for kynurenine pathway indicates a higher probability of tryptophan metabolism via this pathway in the gut environment colonized by Pseudomonas strains. This is in line with the experimentally characterized enzymes of this pathway in Pseudomonas aeruginosa  in regulation of intestinal immunity  may also contribute to virulence through facilitating pathogenic colonization in cases of bacterial infection.The pathway distribution profile indicated that, among the six analyzed pathways, IAA and indole production pathways occurred in highest number of gut genera  . Bacilluimmunity . IAA hasimmunity . Severaltabolism . The preShigella . PreviouShigella . Apart fShigella . Thus, tPseudomonas, Bacillus, Burkholderia, and Streptomyces had higher “SCORBPEO” values (>5) for kynurenine production pathway, Klebsiella, Bacillus, and Burkholderia were predicted to have higher “SCORBPEO” value (≥2) for quinolinate production pathway  may indicate involvement of kynurenine as a major  inter-cellular signaling molecule in gut.Among the remaining four tryptophan metabolizing pathways (under study), those involved in production of tryptamine, kynurenine, and quinolinate were predicted in 15, 11, and 10 gut genera, respectively. While  pathway . Earlierthomonas . Kynurenthomonas . On the thomonas . Thus, aHoldemania, Tyzzerella, Desulfovibrio, and Yersinia  included Yersinia . Bacillu. gnavus . The curClostridium, Escherichia, and Proteus), indicating that synthesis of IPA in the gut is performed by limited gut bacterial groups. IPA production by certain species of Clostridium (such as C. sporogenes) in the gut has also been reported in literature , namely, production of IPA from tryptophan, was observed to be present in only three gut genera . The gene tnaA catalyzes the conversion of tryptophan to indole . In addiCitrobacter, Escherichia, Edwardsiella, and Providencia, all belonging to phylum Proteobacteria. On the other hand, only three strains  were predicted to utilize the low affinity/non-specific AroP transporter , 41 (37.2%) organisms utilize one or more tryptophan transporters for sequestering tryptophan from the host . A list nsporter . While tsporters , suggestA schematic representation has been shown in Since studies have indicated link between tryptophan metabolism by gut microbiome and neurological disorders like autism, Parkinson’s disease, depression, schizophrenia, etc. , availabBurkholderia was found to be significantly abundant genus  with five out of the six analyzed tryptophan pathways in autistic children  children were found to possess the analyzed tryptophan metabolizing pathways  were seen to be differentially abundant in diseased condition  in the disease etiology through production of indole and IAA, and thereby, altering tryptophan homeostasis in gut.Analysis of Parkinson’s disease microbiome included two publicly available datasets  correspoondition . One of  of pain . Thus, tStaphylococcus, obtained as differentially abundant in one of the analyzed Parkinson’s dataset, was predicted to have low probability of utilization of bacterial kynurenine and quinolinate pathways . Thus, the present analysis highlights significance of bacterial utilization of tryptophan through different sets of metabolic routes in different neurological disorders.Further, unlike in autism, the analysis indicated kynurenine and quinolinate pathways not to be distinctive in the analyzed datasets of Parkinson’s disease. Fusobacterium, one of the differentially abundant genera in Schizophrenia, was predicted to have indole production pathway with a relatively higher “SCORBPEO” value of 4.42 , having IAA production pathway with a higher “SCORBPEO” value of 7.48. Thus, correlation of the pathways under study with the disease etiology of schizophrenia, if any, is not very clear from the current analysis. Only the pathway for tryptamine production which was not enriched in disease microbiome was predicted in a relatively abundant genus Blautia in healthy microbiome  of bacterial tryptophan metabolism pathways for production of the neuro-active “TRYP-6” compounds . Results of the analysis revealed a relative enrichment of these pathways in bacterial phyla commonly found in the gut, such as Actinobacteria, Bacteroides, Firmicutes, Proteobacteria, and Fusobacteria, suggesting probable role of these bacteria in the functioning of GBA. Further, the “SCORBPEO” values assigned to each of the gut-associated bacterial genus provided an estimate of these bacteria’s potential for production of “TRYP-6” compounds. Such estimate is specifically helpful in inferring functional potential of a given microbiome (inhabiting a particular niche like human gut) in cases where taxonomic composition can be resolved only up to genera level due to technological limitations. Analysis of the “SCORBPEO” score suggested higher potential of tryptophan metabolism for some of the genera  belonging to the phylum Proteobacteria. Increase in the abundance of Proteobacteria has also been reported to be correlated with brain diseases and disorders  kynurenine were absent in this strain. This probably suggest a shared metabolism for quinolinate pathway where a group of organisms like Clostridium utilize kynurenine as a substrate which is produced  by a second group of bacteria like Bacillus, Burkholderia, Pseudomonas, etc. Further, the current results suggest probable presence of all the analyzed pathways except the one for the production of IPA in Burkholderia. Previous studies have reported production of kynurenine and quinolinate in various strains of Burkholderia  across bacterial groups. This knowledgebase is expected to be useful resource for future studies on GBA. The results further indicate a relative enrichment of tryptophan metabolizing capabilities in bacterial groups commonly associated with gut environment. The analyses of microbiome data indicate association of distinct modules of bacterial tryptophan metabolism with the etiology of different neurological diseases such as, autism, Parkinson’s, and schizophrenia. These insights are expected to aid future experiments which can enhance our understanding of microbiome-GBA and may also help in designing microbe-based diagnostic/therapeutic approaches for neurological diseases.In the present work, we have attempted to understand the communication between gut bacteria, tryptophan metabolism, and neurological pathologies. A comprehensive PRJEB27306, SRP093968, PRJEB15418, PRJEB15420, PRJEB4927, PRJEB14674, and CRA000653.Publicly available datasets were analyzed in this study. This data can be found using the following accession numbers: HK and CB conceptualized the work and designed the experiments. HK performed the computational analysis with the assistance from CB. HK, CB, and SM analyzed the results and prepared the manuscript. All authors reviewed the results and approved the final manuscript.HK, CB, and SM were employed by the company Tata Consultancy Services.


EIN3/EIL1 transcription factors are the key regulators of ethylene signaling that sustain a variety of plant responses to ethylene. Since ethylene regulates multiple aspects of plant development and stress responses, its signaling outcome needs proper modulation depending on the spatiotemporal and environmental conditions. In this review, we summarize recent advances on the molecular mechanisms that underlie EIN3/EIL1-directed ethylene signaling in Arabidopsis. We focus on the role of EIN3/EIL1 in tuning transcriptional regulation of ethylene response in time and space. Besides, we consider the role of EIN3/EIL1-independent regulation of ethylene signaling. L-methionine, which is consequently converted to S-adenosyl-L-methionine (by SAM-synthetases), 1-aminocyclopropane-1-carboxylic acid (ACC) (by ACC synthases), and ethylene (by ACC oxidases) , as well as diverse biotic and abiotic stress responses . EthylenArabidopsis thaliana genome   . They ha EIL1-5) . EIN3, E EIL1-5) . The mosxpressed . In Figd pollen .via i) translational repression of EBF1/2 mRNA in the cytosol promoted by EIN2-C  and EBF2 3A Gagn. Stabiliy EIN2-C , and ii)y EIN2-C   (Climacteric fleshy fruits (the ones that demonstrate a respiratory burst at the beginning of ripening) use ethylene as a ripening signal . Mature nd ACOs) . To prevRIN in tomato, NAC in peach and banana)—are associated with the repressive histone mark H3K27me3 in leaf and immature fruit. They become demethylated and therefore accessible only in the ripening fruit tissues. Presumably, this epigenetic mechanism prevents autocatalytic ethylene production in vegetative and immature fruit tissues.Epigenetic modifications often promote spatiotemporal regulation of plant hormone responses  play essential roles in these processes. They contribute in chlorosis and increased apical hook curvature of buried seedlings, induce shortening and thickening of hypocotyl to enhance lifting capacity of the seedling, and finally promote seedlings greening upon light irradiation , a central repressor of light signaling, directly targets EBF1/2 for ubiquitination and degradation . As seedWhen the seedling reaches the soil surface, light triggers a dramatic developmental transition known as de-etiolation that leads to immediate termination of ethylene responses. Light-activated photoreceptor phytochrome B (phyB) directly interacts with both EIN3 and EBF1/2 proteins, thereby stimulating robust EIN3 degradation, rapidly turning off ethylene signaling .ERF1 and ORA59, directly regulated by EIN3/EIL1, as well as their downstream target PDF1.2, are upregulated in response to JA.In this section, we consider the cross-talk of ethylene signaling pathway with jasmonic acid (JA) and gibberellins (GA) based on an inhibition of EIN3/EIL1 transcriptional activity due to their physical interactions with repressor proteins Table 1.ERF1, ORA59, and PDF1.2 genes are upregulated in myc2 mutants. This inhibitory mechanism underlies ethylene-JA antagonism. Particularly, JA-directed abolishment of ethylene-promoted apical hook formation proceeds via MYC2-mediated attenuation of HOOKLESS1 (HLS1) expression, which is the key regulator of hook development and a direct EIN3/EIL1 target  genes in a cooperative manner to synergistically suppress their expression. Upon light exposure, the levels of EIN3 and PIF3 decrease, and activation of LHC expression triggers chloroplast differentiation.EIN3/EIL1’s capability to function cooperatively with the transcriptional regulators of the other signaling pathways provides another possibility to shape spatiotemporal patterns of ethylene response. This cooperation implies the cross-talk of TFs bound to DNA that goes along with the physical interaction of these TFs Table 1.HLS1, the key regulator of apical hook development  gene  gene . Besides as well . Both EIem alone . The rol as well . Similarripening . Both TFvia physical interactions with other TFs and modulation of their activity (via upregulation of TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1 (TAA1) plays a pivotal role in ethylene-induced inhibition of root growth (TAA1 promoter through a “piggyback” interaction with RESPONSE REGULATOR 1 (ARR1), a transcriptional regulator of cytokinin signaling, thereby enhancing ARR1 transcriptional activity  and a sustained phase II . Both phesponses . Moreoveesponses . These oesponses . EER5 re1 signal .via PPIs) that facilitate the “shaping” of ethylene response according to spatiotemporal and environmental conditions. At the same time, these findings open up new perspectives for further research. Growing evidence of the important role that epigenetic landscape plays in EIN3/EIL1 functioning requires its more detailed characterization. Particularly, the contribution of distinct epigenetic modifications as well as ENAP1 patterns in modulation of EIN3/EIL1 function is of interest. In view of interdependent cooperation of EIN3/EIL1 with some TFs described recently, the detailed analysis of nucleotide context surrounding EIN3 binding sites requires more attention, and genome-wide research appears helpful both to generalize resent findings and to predict new connections. Moreover, it would be interesting to clarify the role of epigenetic regulation and PPIs in suppression of gene expression upon ethylene treatment.Ethylene response is a target for a complex regulation, in which EIN3/EIL1 TFs play a crucial role. Recent studies shed light on multiple layers of complexity in tuning EIN3/EIL1 function (including epigenetic gene silencing and modulation of EIN3/EIL1 stability and activity Yet, despite their essential role, EIN3/EIL1 are not indispensable regulators of ethylene response. To couple the molecular events and phenotypic responses more precisely, EIL2 function in ethylene signaling and EIN3/EIL independent pathways are to be elucidated.VAD and EMP performed the literature search and drafted the paper. VAD performed the analysis of the transcriptome datasets. EVZ revised and edited the manuscript. All authors read and approved the final manuscript.The work was funded by the Russian Foundation for Basic Research through grant № 18-29-13040. Meta-analysis of the transcriptome datasets was done in the frame of the project supported by the Russian Foundation for Basic Research and the government of Novosibirsk region through grant № 18-44-540039.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.


Aims: 25-Hydroxyvitamin D [25(OH)VD] deficiency/inadequacy is a major public health issue affecting more than 1 billion people worldwide. A convincing association exists between low levels of circulating 25(OH)VD and the poor health outcomes associated with chronic diseases. However, high supraphysiological doses of VD are needed to achieve the required 25(OH)VD levels in the blood, because many subjects respond poorly to supplementation.Results: This study reports a link between 25(OH)VD deficiency and a reduction in glutathione (GSH) in obese adolescents. The improvement in GSH status that results from cosupplementation with VD and l-cysteine  significantly reduced oxidative stress in a mouse model of 25(OH)VD deficiency. It also positively upregulated VD regulatory genes (VDBP/VD-25-hydroxylase/VDR) in the liver and glucose metabolism genes (PGC-1α/VDR/GLUT-4) in muscle, boosted 25(OH)VD, and reduced inflammation and insulin resistance (IR) levels in the blood compared with supplementation with VD alone. In vitro GSH deficiency caused increased oxidative stress and downregulation of VDBP/VD-25-hydroxylase/VDR and upregulation of CYP24a1 in hepatocytes and downregulation of PGC-1α/VDR/GLUT-4 in myotubes. This study demonstrates that improvement in the GSH status exerts beneficial effects on the blood levels of 25(OH)VD, as well as on the inflammation and IR in a VD-deficient mouse model. Thus, the VD supplements widely consumed by the public are unlikely to be successful unless the GSH status is also corrected.Innovation: These studies demonstrate a previously undiscovered mechanism by which GSH status positively upregulates the bioavailability of 25(OH)VD.Conclusion: Supplementation with a combination of VD and LC or GSH precursor, rather than supplementation with VD alone, is beneficial and helps achieve more successful VD supplementation. Epidemiological studies provide conclusive evidence that lower circulating levels of 25(OH)VD are associated with the poor outcomes frequently associated with several chronic metabolic diseases VD inadequacy/deficiency found in populations worldwide.The status of 25-hydroxyvitamin D [25(OH)VD] plays a crucial role in preventing disease and maintaining optimal health. These studies demonstrate a previously undiscovered mechanism by which increasing glutathione (GSH) status positively influences the bioavailability of 25(OH)VD. The improvement in GSH caused by supplementation with vitamin D (VD) combined with Risk factors for 25(OH)VD deficiencies include race (darker pigmented skin tones), higher body mass index (BMI), winter season, higher geographic latitudes, and diet. Circulating 25(OH)VD is considered to be a comprehensive and stable metabolite, levels of which can be used to diagnose 25(OH)VD deficiency and monitor VD consumption . The metCYP2R1, CYP27A1) (VDBP) and transported into the circulation. VDBP is primarily synthesized and secreted by the liver (CYP27B1), which converts 25(OH)VD to its active metabolite , is present in both renal (major site) and nonrenal tissues (CYP27B1) in nonrenal cells and tissues, indicating localized 1,25(OH)2VD formation and its tissue-specific paracrine function in different tissues  , 45, 56.he liver , 55. (CYCYP24A1) is involved in the catabolic inactivation of 1,25(OH)2D3 and its inhibition is thought to limit 1,25(OH)2D3 signaling (CYP2R1) are known to influence 25(OH)VD blood levels in response to VD supplementation (VDR) (2VD are directly related to the VDR content of target tissues. Muscle is a major site of glucose metabolism and maintenance of glucose homeostasis (VDBP/VD-25-hydroxylase) in the liver, while the downstream actions of the active 1,25(OH)2VD in muscle are mediated by glucose metabolism genes . . The bioeostasis . Therefol-cysteine  has been successfully used to improve the GSH status in blood and tissues while lowering inflammation and IR in humans and animals (Glutathione (GSH) is a major antioxidant and its depletion increases oxidative stress and extensive carbonylation of proteins , 16–18.  animals , 58, 61.This article discusses our investigation of the dual roles of GSH in increasing circulating 25(OH)VD and augmenting the actions of active VD metabolites in one of the target tissues , which is a major site for glucose metabolism.TNF-α) levels and homeostatic model assessment (HOMA)-IR were significantly higher in obese compared with lean or overweight subjects .Blood levels of GSH and 25(OH)VD were significantly lower in obese compared with lean or overweight adolescents . VDBP lesubjects . Figure www.liebertpub.com/ars) shows that 25(OH)VD has a negative association with IR ; IR also showed a negative correlation with GSH  and a positive association with TNF-α . BMI shows negative association with 25(OH)VD (r = −0.25) and positive association with TNF-α (r = 0.39). TNF-α showed positive association with BMI (r = 0.39), protein carbonyl (r = 0.37), and IR (r = 0.29). Protein carbonyl association with GSH was not significant. Ages of subjects in each group were similar, while BMI levels were significantly different in each group. Subject enrollment detail is given previously (VDBP or in VDBP-knockdown (KD) mouse models VD levels in obese adolescents. A positive association exists between blood levels of GSH and those of 25(OH)VD and has been previously shown in adult diabetic patients , food intake, parathyroid hormone (PTH), calcium, and blood count levels in the blood of mice fed normal diet and HFD mice groups were similar  were purchased from The Jackson Laboratory . The animals were fed either a standard chow diet  or a high-fat diet (HFD) for 16 weeks . Composi similar .GCLC /GCLM (glutamate-cysteine ligase regulatory subunit)/GSS/NRF2 (nuclear factor erythroid-2-related factor) involved in GSH synthesis VD, were significantly downregulated (CYP24A1 that degrade 25(OH)VD are upregulated in the liver of mice fed HFD in comparison with mice fed normal diet.ynthesis , and genegulated  in liverGCLC/GCLM/NRF2) and VD regulatory genes (VDBP/CYP2R1/CYP27B1) are downregulated significantly (VDR/PGC-1α/GLUT-4) is significantly downregulated and TNF-α increased in the muscle of mice fed HFD compared with normal diet-fed mice group.ficantly  in HFD-fThere was a significant increase in lipid peroxidation and protein oxidation with decreased GSH levels in skeletal muscle  of HFD-fOverall, HFD feeding resulted in a significant downregulation of genes that synthesize GSH and lower levels of GSH in blood, liver, and muscle. Similarly, there was a significant downregulation of VD regulatory genes in the liver and muscle of mice fed HFD. In addition, there was a significant downregulation of glucose metabolism genes in muscle of mice fed HFD in comparison with normal diet-fed mice group. A decrease in blood and tissue GSH reflects exhaustion or impaired antioxidant pathways and increased oxidative stress in tissues in mice consuming HFD.ad libitum for 16 weeks +LC significantly corrected the GSH status and showed a significant decrease in TNF-α, IR, and HbA1c in the blood compared with levels seen in the mice supplemented with OO (vehicle)-control group. In addition, supplementation with combined VD +LC showed a greater increase in 25(OH)VD and decrease in protein carbonylation levels in the blood compared with the OO-supplemented control mice. There were no changes in food intake, RBC indices, or calcium among these groups. There was also no change in blood counts, which indicates that the decrease in HbA1c values seen in the VD+LC group was not due to any effect on cell viability VD, VDBP, iability .GCLC/GSS/NRF2), GSH status, and VD regulatory genes (CYP2R1/CYP27A1/VDBP/VDR) in the liver VD to 1,25(OH)2VD. VDR-KD was induced using siRNA and was able to achieve nearly 80% VDR-KD in hepatocytes. VDR-KD caused a simultaneous decrease in VD metabolism genes CYP27A1, VDBP, and VDR, while upregulation of genes by VD+LC was abolished in VDR-KD hepatocytes  caused a simultaneous increase in TNF-α and decrease in VDR/PGC-1α/GLUT-4 mRNA expression in GCLC-KD C2C12 myotubes 2VD in muscle. Deficient GSH levels can result in inflammation, which can be reversed by improving GSH status. Further studies examined the effect of combined LC and 1,25(OH)2VD and of PGC-1α KD on GLUT-4 in myotubes.myotubes . Stimulamyotubes . Supplemmyotubes . This su2VD (active vitamin D) resulted in significantly greater upregulation of PGC-1α/NRF2/GLUT-4 2VD was used to understand its efficacy on glucose metabolism genes in muscle cells, whereas cholecalciferol was used with hepatocyte studies to understand 25(OH)VD biosynthesis from cholecalciferol in liver.Cotreatment with LC and 1,25(OH)2/GLUT-4  gene expin vivo defense against oxidative stress VD and GSH status in adolescents. This led us to examine whether GSH regulates VD regulatory genes and 25(OH)VD status in the blood. Using the mouse model of 25(OH)VD deficiency, we show a significantly greater increase in blood 25(OH)VD levels after cosupplementation with VD +LC (a GSH precursor) compared with results in a group supplemented with VD alone. In addition, supplementation with VD alone does not affect GSH, in vitro and in vivo studies demonstrate the dual roles of GSH in increasing circulating 25(OH)VD and augmenting the actions of active VD metabolites in one of the target tissues , which is a major site for glucose metabolism.Improvement in GSH status by LC prevented the downregulation of VD regulatory genes in hepatocytes and glucose metabolism genes in myotubes. These VDBP/VD-25-hydroxylase/VDR, thereby helping protect the status of 25(OH)VD levels. VDBP is required for efficient transport and VD-25-hydroxylase is needed for the hydroxylation of cholecalciferol.2VD complex to the nucleus for activation of the VDR/PGC-1α/GLUT-4 pathway responsible for metabolic actions of 1,25(OH)2VD. The PGC-1α is an inducible transcriptional coactivator that upregulates expression of GLUT-4 in skeletal muscle and is a coactivator of the retinoic X receptor (RXRα) r (RXRα) . PGC-1α s of GSH . TherefoVDR, both of which are needed to increase the bioavailability and blood levels of 25(OH)VD and reduce inflammation levels. GSH provides not only upregulation of VD regulatory genes but also adds to GLUT-4 activation. Thus, increasing GSH status by combined supplementation with LC or a GSH precursor along with VD provides a novel approach to treat widespread 25(OH)VD deficiency/inadequacy in populations worldwide.This study provides evidence for a previously undiscovered mechanism that describes how 25(OH)VD deficiency/inadequacy is linked to lower GSH levels and that boosting GSH status beneficially upregulates the genes of VD metabolism and VDBP/VD-25-hydroxylase/VDR as well as PGC-1α/GLUT-4 genes. Blood levels of 25(OH)VD are lower in humans with a genetic mutation for VDBP or in VDBP-KD mouse models (glutamate-cysteine ligase (GCL) and GSH synthetase VD levels in the blood . In factdiabetes . ObservaThese findings focus attention on the fact that the VD supplements widely consumed by the public are unlikely to be efficacious unless the status of the VD metabolism genes is improved by first correcting the status of GSH. This suggests that combined consumption of GSH precursors and VD, rather than solely using high-dose VD, is both novel and a better strategy with which to achieve a more efficacious bioavailability in response to cholecalciferol consumption and to increase blood levels of 25(OH)VD.® tube. Ethylenediaminetetraacetic acid (EDTA) blood tubes were brought to the research laboratory. Isolated plasma was stored in different aliquots and frozen immediately at −80°C for analyses of the biochemical parameters.This study was carried out after informed written consent was obtained from all subjects according to the protocol approved by the Louisiana State University Health Sciences Center (LSUHSC) Institutional Review Board (IRB). This study enrolled adolescent boys and girls ages 14–17 years, in good health (other than being overweight or IR), who provided written informed assent and parental consent. Inclusion criteria were children who are not smoking or taking any medicines, food supplements, or antihistamines. All subjects who gave written informed consent were invited to return to have blood drawn after fasting overnight (8 h). The exclusion criterion was that subjects were excluded if pregnant or suspected to be pregnant, applied only to female subjects. Urine was collected from the female subjects for urine pregnancy testing. Subjects were allowed to sit quietly for 10 min after which blood was collected from an easily accessible forearm vein using a butterfly needle and Vacutainerl-cysteine (LC+VD)  were purchased from The Jackson Laboratory and acclimatized in the institutional animal house for 1 week. Mice were divided into various groups by computer-generated randomization and then housed and labeled in individual cages. They were fasted overnight and then weighed. Blood glucose was assessed by tail prick using an Accu-Chek glucometer . BW and blood glucose were monitored weekly. Control animals were fed a normal diet (lower in fat), while animals in the HFD group were fed HFD for 16 weeks. Flow diagram showing details of diet feeding of mice is given in  (LC+VD) .via heart puncture with a 191/2-gauge needle into heparinized Vacutainer tubes. Plasma was isolated after centrifuging the blood in a 4°C centrifuge at 3000 rpm for 10 min. The livers were perfused with cold saline to free them of residual blood. Liver and gastrocnemius muscle were collected immediately, weighed, quickly diced, and frozen in liquid nitrogen at −80°C.In addition, two groups of mice maintained on the VD-deficient HFD were also simultaneously supplemented by oral gavage with either water or the same dose of the vehicle used for dissolving cholecalciferol (OO) . The aniLC can be taken as a supplement, it is also formed in the body from methionine. An adult ingests about 500 mg LC from dietary sources, assuming that an average protein intake is 90 g/day and that LC is about 0.6% of total protein  were cultured and maintained in F-12K complete medium. Mouse C2C12 myoblasts were cultured at 37°C in an atmosphere of 5% CO2 in growth medium (GM) consisting of Dulbecco's modified Eagle's medium (DMEM) supplemented with heat-inactivated 10% fetal bovine serum and antibiotics (penicillin and streptomycin). Differentiation of myoblasts into myotubes was induced when the cells had achieved 90–95% confluence by switching the medium from GM to differentiation medium consisting of DMEM supplemented with 2% horse serum (5 days), then treated as described in the figures. siRNAs were purchased from Santa Cruz Biotechnology, Inc. , catalog numbers sc-41979 (GCLC), sc-36811 (VDR), and sc-38885 (PGC-1α). The control siRNA, a scrambled nonspecific RNA duplex that shares no sequence homology with any of the genes, was used as a negative control.FL83B mouse hepatocytes  following the method described earlier (M) for 6 h for LC-alone experiments. Pretreatment of the cells, maintained at a concentration of 1 × 106/mL media, was done for 2 h with LC (0–300 μM), followed by treatment for 22 h with cholecalciferol or 25(OH)VD (10 nM). TNF-α (0–250 pg/mL) was exposed for 6 h to differentiated myotubes.Cells were transiently transfected with 0–100 n earlier , 43. The3) to 25-hydroxy-vitamin D. FL83B mouse hepatocytes express VDBP, CYP27A1, CYP27B1, CYP24A1, and VDR, but expression of CYP2R1 is very low and could not be accurately quantitated. However, we got reproducible gene analysis results for expression of VDBP, CYP27A1, CYP27B1, CYP24A1, and VDR genes using FL83B mouse hepatocytes. Liver contains both CYP27A1 and CYP2R1 and participates in the conversion of cholecalciferol (vitamin D3) to 25-hydroxy-vitamin D. The population studies have also shown a link between CYP2R1, GC (VDBP), CYP24A1, and VDR with that of circulating 25(OH)VD concentrations. Recent studies have shown a regulatory role of CYP27A1 gene expression on the blood concentrations of 25(OH)VD VD , 62, 63.Thus, mouse hepatocytes used in this study have much strength and can be used for investigating the link between GSH-deficiency, oxidative stress, and VD regulatory genes. Muscle is a major site for glucose metabolism. Differentiation of myoblasts into myotubes is very reproducible and these cells express the glucose metabolism genes such as PGC-1α, RXRα, VDR, and GLUT-4. GLUT-4 is a master regulator for the maintenance of glucose metabolism. Thus, mouse C2C12 myoblasts have much strength to investigate the role of GSH deficiency in regulation of glucose metabolism pathways .Total RNA was extracted from cells or tissue using the TRIzol reagent (Life Technologies) following the manufacturer's instructions. The quality and quantity of the extracted RNA were determined on a NanoDrop spectrophotometer (Thermo Scientific). First-strand complementary DNA (cDNA) synthesis was performed using a commercially available High Capacity RNA-To-cDNA kit (Life Technologies) in a final reaction volume of 20 μL. Amplification of cDNA was performed on a 7900HT Real Time polymerase chain reaction (PCR) system (Applied Biosystems). PCR conditions were 2 min at 50°C, 10 min at 95°C, 40 cycles of 95°C for 15 s, and then 60°C for 60 s. Details of the TaqMan-FAM-labeled primer/probe used are given in To exclude nonspecific amplification and/or the formation of primer dimers, control reactions were performed in the absence of target cDNA. All of the experiments were run in triplicate. The relative amounts of mRNAs were calculated using the relative quantification (ΔΔCT) method. M Tris pH 8, 150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid, and 0.1% SDS) was supplemented with protease and phosphatase inhibitors . Lysates were then centrifuged for 10 min at 10,000 g at 4°C. Supernatants were collected and the protein concentrations were determined using a BCA assay kit  for Western blot analysis and high performance liquid chromatography (HPLC) assay. Equal amounts (20 μg) of proteins were separated on 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinyl difluoride membrane. Membranes were blocked at room temperature for 2 h in a blocking buffer containing 1% bovine serum albumin to prevent nonspecific binding and then incubated with an appropriate primary antibody at 4°C overnight  for 30 min and incubated with an appropriate Horseradish peroxidase-conjugated secondary antibody (1:5000 dilution) for 2 h at room temperature. The protein bands were detected using ECL detection reagents (Thermo Scientific) and exposed on blue X-ray film . The technical replicates (n = 2) and biological replicates (n = 4) were done in all our immunoblot experiments. Western blot scans were analyzed using ImageJ software . Plasma VDBP quantification was carried out using a kit purchased from ALPCO Diagnostics . TNF-α was measured using an ELISA kit from R&D Systems . PTH (1–84) and insulin were determined using ELISA kits from ALPCO Diagnostics, and the HOMA-IR index was calculated (Plasma levels of 25(OH) vitamin D were determined using an ELISA kit  and 1,25(OH)lculated . VDBP wa1c, Complete Blood Count, glucose, and calcium were done at the clinical chemistry laboratories of LSUHSC-Shreveport. Due to limited amount of blood collected from each mouse, we borrowed diluents from the clinical laboratory and diluted the blood before taking it to the clinical laboratory, which reduced the amount of blood required for clinical tests. All chemicals were purchased from Sigma Chemical Co.  unless otherwise mentioned.Levels of GSH in plasma, tissues, and cultured cells were determined using HPLC . This asp-value of ≤0.05 for a statistical test was considered significant.Data from clinical, cell culture, and mouse studies were analyzed using regression analyses and ANOVA with Sigma Stat software . A The data supporting the findings are available within the article and the associated Supplementary Data of this study. Any other data are available from the corresponding author on reasonable request.


Studies have shown that combination drug therapy which corresponding treatment involves multiple genes and targets is more effective against cerebral ischemia. To identify the synergistic mechanism of ursodeoxycholic acid and jasminoidin based on differential pathway network, which protect against brain ischemia-reperfusion injury. Totally 115 mice with focal cerebral ischemia-reperfusion injury were allocated into five groups: sham, vehicle, ursodeoxycholic acid (UA), jasminoidin (JA), and JA and UA combination group (JU). The differentially expressed genes identified by microarray which consisted of 11,644 complementary DNAs were loaded to the GeneGo MetaCore™ software to analyze the enriched pathways and processes among different groups. Of the top 10 pathways and process networks, 5, 6, and 3 overlapping pathways as well as 5, 3, and 4 overlapping process networks were observed between UA and JA, UA and JU, and JA and JU, respectively. Of these, three pathways and three process networks overlapped across the three groups. Interestingly, four representative pathways and six process networks were only noted in the JU group. Gene Ontology process analysis showed 2 processes were shared by all three treatment groups in the top 10 processes. The UA and JA combination resulted in synergistic effects through affecting multi-signal transduction pathways, different locations in the same pathway, and the new signaling pathway emerged in drug combination group, those together may enhance the treatment of cerebral ischemia-reperfusion injury through promoting neural cell apoptosis, decreasing calcium levels, inhibiting inflammation, and protecting neurons. Cerebral ischemia is one of the leading lethal disorders worldwide . Its patConcha margaritifera (CM) has been shown to display protective effects against focal cerebral ischemia and reperfusion injury , ursodeoxycholic acid (UA), jasminoidin (JA), and n injury . It has n injury , of whicn injury . Our pre™ is a pathway analysis tool based on a proprietary manually curated database of various high throughput data  were placed at a constant temperature of 25°C and were subjected to a 12-12 h light-dark cycle. All experiments complied with the Prevention of Cruelty to Animals Act (1986) and National Institutes of Health (NIH) guidelines for the care and use of laboratory animals. The experimental protocol gained approval from a local committee review.After anesthesia with 2% pentobarbital , surgical operation to induce middle cerebral artery occlusion (MCAO) was performed. Focal cerebral ischemia-reperfusion was induced by occluding the left middle cerebral artery for 90 minutes with an intraluminal filament followed by 24 h of reperfusion. Sham animals were treated in the same manner except that the filament was not inserted. Throughout the surgical procedure, the rectal temperature was kept at 37.0–37.5°C by use of a heating pad; brain temperature was monitored with a thermocouple implanted in the right striatum and was maintained at 36–37°C using a thermostatically controlled lamp. Besides, blood pressure, blood gas, glucose levels, and EEGs were also monitored during operation. Infarction volume and subsequent mice behavior for each mouse was used to assess the operation success according to the method described by Bederson et al. .via the tail vein at a dose of 2 ml/kg body weight.The experimental animals were divided into five groups (n = 23): sham, vehicle (0.9% NaCl), UA , JA , and JU (combination of ursodeoxycholic acid and JA at a ratio of 1:1) treatment groups. The herbal preparation was dissolved in 0.9% NaCl immediately before use. All preparations were injected intravenously ® Reagent . Total RNA was extracted using the RNeasy Micro Kit  following the protocol supplied by the manufacturer. RNA quality was evaluated by analyzing the 26S/18S ratio with Bioanalyzer microchip . Microarrays were created from a total of 374 cerebral ischemia-related complementary DNAs. The collection procedure of 11,644 genes, as well as the detailed protocols for RNA extraction and microarray sample preparation have been described in our prior study . Enrichment analysis is a useful tool that determines the functional distribution and the significantly enriched functional categories  of the genomic/proteomic expression profile. It would help to understand the functions of these differentially expressed genes and offer essential information for further bioinformatics analysis. The biological process, sub-cellular location, and functional distribution of the differentially expressed genes were computed using MetaCore™ based on Gene Ontology. The network construction and analysis were done by use of the analyze network algorithm provided in the software. And network distribution of the selected genes was also determined via MetaCore based on GeneGo network ontologies. The enrichment p-values in result tables were calculated based on EASE Score, which is a rigorously modified Fisher exact test p value. Statistical relevance of the identified ontology matches was calculated as p-value, or a probability of a match to occur by chance. The more genes/proteins belong to a process/pathway, the lower the p-value.All experimental data were uploaded to the ArrayTrack system . All microarray data obtained were normalized with locally weighted linear regression (Lowess) to reduce the variability in datasets . In ordeThe expression levels of JunB proto-oncogene (JUNB) were measured using Western blotting. The hippocampus tissue of 15 mice from five groups was excised from the brain at 24 h post-ischemia. Following cell lysis, 40 mg of protein were subjected to electrophoresis on 10% sodium dodecyl sulfate-polyacrylamide gels and then transferred to polyvinylidene difluoride membranes. Next, the primary and secondary antibodies rabbit anti-JUNB (CST3753) and mouse anti-b-actin  were applied. Optical density-calibrated images were captured with a video camera for analysis. All measurements were performed in triplicate. The JUNB protein expression levels were calculated by normalization to b-actin levels.It has been shown that the JA-UA combination had a pharmacodynamic synergistic effect in treating cerebral ischemia .Based on microarray data analysis, we found 414, 470, and 401 differential expression genes in JA, UA, and JU group, respectively. Compared to sham group, the enrichment pathways in vehicle and treatment groups were mostly focus on functions such as development, signal transduction, immune response, transcription, G-protein signaling, and apoptosis and survival, and the top 10 signaling pathways in each group were shown in via Janus kinase/signal transducers and activators of transcription (JAK/STAT) and mitogen-activated protein kinase (MAPK) cascades, and transcription-role of activator protein 1 (AP-1) in regulation of cellular metabolism. In addition, three overlapping pathways were found between UA and JU groups. Meanwhile, there were specific pathways in each treatment group, for example, there were four specific pathways in JU groups, including Mucin expression in CF via IL-6, IL-17 signaling pathways, transcription-role of vitamin D receptor (VDR) in regulation of genes involved in osteoporosis, regulation of cystic fibrosis transmembrane conductance regulator (CFTR) activity 9 (norm and CF), development-granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling  processes Table 1,The Western blot analysis indicated that, in comparison with the sham group, the expression level of JUNB was significantly up-regulated in the vehicle, JA and JU groups (P < 0.05)  downregulation. Excitatory amino acids, calcium overload, free radicals, and increased NO all involve the expression of c-Fos in the central nervous system during brain ischemia/reperfusion. c-Fos can combine with c-Jun to form the AP-1 complex, which affects the transcription of genes involved in brain ischemia/reperfusion injury, thus promoting neural cell apoptosis . In our Another example is the development-Hedgehog and parathyroid hormone (PTH) signaling pathways in bone and cartilage development pathway, which is a common pathway between UA and JU Figure 1via interleukin 6 (IL-6), IL-17 signaling pathways, and regulation of CFTR activity. The development-GM-CSF signaling pathway was specifically altered by JU. GM-CSF plays an important role, inhibiting inflammation and protecting neurons  and National Institutes of Health (NIH) guidelines for the care and use of laboratory animals for experimental procedures. The experimental protocol was approved by a local committee review.ZW and JW designed the study. YZ and HL wrote the manuscript; HG, BL and XZ performed the research; YZ and ZZ modified tables and figures; PWa, HW, JL ,YC and PWu analyzed the experimental data. All authors participated in the discussion and data analysis and reviewed the manuscript.This study was partially funded by the Basic Research Fund of Guest Professor Innovation Research Project in China Academy of Chinese Medical Sciences (No. ZZ070803), and partially by the Natural Science Foundation of China (NSFC) (No. 81303132).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.


Manufacturers of surgical instrumentation have increasingly sought to decrease the size of ophthalmic surgical instruments. We have used finite element modeling to model the stress and strain present in a fragmatome as a function of driving frequency and fragmatome dimensions.Finite element calculations using the COMSOL Multiphysics system v3.5 were used to elucidate the influence of wall thickness, length, and excitation frequency on a titanium fragmatome tube with outer diameters of 20, 23, 25, and 27 gauge.By coupling structural mechanics, fluid mechanics, and acoustical physics, we were able to determine the eigenfrequencies (resonant frequencies) as well as parameters in which the von Mises stress in a fragmatome tube exceeds the yield strength, leading to destruction of the instrument.Solid fragmatomes have far fewer possible failure modes than fragmatomes with a standard wall thickness. Eigenfrequency analysis and finite element calculations can be critical in predicting potentially catastrophic designs in modern surgical instruments.Instruments developed for microsurgical applications cannot always simply be scaled down versions of conventional instruments. Such an approach can lead to potentially dangerous intraoperative failures, such as a fragmatome shattering inside the eye. Modern engineering techniques are increasingly necessary to investigate potential instrument failure mechanisms and to optimize device performance in a design in silico before in vivo testing. Decrease in the size of an incision in surgery is associated with decreased pain,14In vitreoretinal surgery, with a decrease in incision size, the instrument gauge also must increase . In the case of retained lens material, a complication of cataract surgery, the retained lens fragments often are removed from the vitreous cavity by a fragmatome, a long tube that can apply ultrasonic energy to a lens fragment to emulsify it as well as to aspirate the resulting small lens particles. While modern vitrectors often can be used to remove soft nuclei and lens cortex, a fragmatome continues to be helpful in the management of hard nuclei. The advantages of a thinner fragmatome, such as a 23- and 25-gauge versus conventional 20-gauge fragmatome, have been well documented in terms of surgical efficiency,11Finite element calculations using the COMSOL Multiphysics system v3.5  were used to elucidate the influence of wall thickness, tube length, and excitation frequency on a titanium alloy fragmatome tube with outer diameters of 20, 23, 25, and 27 gauge. By coupling a linear elastic model of structural mechanics, fluid mechanics, and acoustical physics, we were able to determine the eigenfrequencies as well as parameters in which the internal von Mises stress (force/area) in the fragmatome exceeds the yield strength and, thus, according to the von Mises yield criterion,Typical driving frequencies for phacoemulsification systems and fragmatomes vary from 35 to 55 kHz and, for some of our calculations, we chose a “typical” ultrasonic frequency of 45 kHz. For comparison with the calculations included in this study, the fragmatome used with Alcon's Constellation system operates at a frequency of 39.0 ± 1.9 kHz and has a length of 30.5 mm. Relevant engineering terms used in this study, with their definition, can be found in R2 of 0.9996  and the gauge (20-gauge) of the fragmatome constant, the wall thickness can influence the maximum displacement of the tip of the fragmatome . The disTo predict potential failure, the von Mises stress was calculated for 20-, 23-, 25-, and 27-gauge hollow fragmatomes at all predicted eigenfrequencies and at 45 kHz at a series of lengths . Figure At the tip of a 26 mm long, 20-gauge hollow fragmatome, driven at a frequency of 44.8 kHz, the volumetric strain calculation shows greatest compression at the tip and greatest extension at the base of the fragmatome . DrivingAt the tip of a 14 mm long, 23-gauge hollow fragmatome, driven at a frequency of 45 kHz, the volumetric strain calculation shows greatest stretching at the tip of the fragmatome . DrivingAt the distal shaft of a 30 mm long, 23-gauge hollow fragmatome, driven at a frequency of 45 kHz, the volumetric strain calculation shows greatest stretching at the distal shaft of the fragmatome . As mentAt the distal shaft and tip of a 20 mm long, 27-gauge hollow fragmatome at a frequency of 45 kHz, the volumetric strain calculation shows greatest compression at the distal shaft and greatest stretching at the tip of the fragmatome .To predict potential failure, von Mises Stress was calculated for 20-, 23-, 25-, and 27-gauge solid fragmatomes at all predicted eigenfrequencies and at 45 kHz at a series of lengths . Figure Larger von Mises stress was found only in a 25-gauge, 24 mm long fragmatome, near 45 kHz . This frWe find that the resonant frequencies at which a given fragmatome can vibrate vary strongly with the length of the fragmatome, and do not vary significantly with thickness or gauge. On the other hand, the thickness of the wall and the gauge strongly influence the internal stress and strain that develops in the device. Only by performing detailed computational modeling is it possible to determine sharp resonances in the response of a given fragmatome to a given ultrasonic frequency. It should be mentioned that simulations at different fragmatome length should be performed because, if the fragmatome is held firmly by the trocar in the sclera, the effective length of the fragmatome will be shortened and the eigenfrequencies of that shortened fragmatome are critical . In addition, if the driving force is not at an eigenfrequency, vibrations at an eigenfrequency will be induced as there is a coupling between the driving frequency and eigenfrequencies of the system.There is a significant risk of intraoperative instrumentation failure, including having the end of the instrument move in an uncontrolled and large-amplitude manner or having a piece of a given fragmatome break off. These failure modes can be predicted using advanced computational modeling. We find, in our simulations, that these risks may be minimized by reducing the size of the fragmatome lumen.Note that this analysis does not discuss failure due to fatigue, where the fragmatome is subject to repeated cyclical loading. Fatigue often results in the maximal stress that results in failure being much lower than the yield stress, considered here. In this way, our results are an upper limit on the stress needed to cause failure in a new fragmatome and the actual risk of failure is is often computed using statistical considerations.Eigenfrequency analysis and finite element calculations can be critical in optimal instrument design and in predicting potentially catastrophic designs in modern surgical instruments. For example, the calculated stress and strain for a 30 mm long, 23-gauge hollow fragmatome at a frequency of 45 kHz demonstrates a maximal von Mises stress that exceeds tensile strength of titanium, (880 MPa) at the distal shaft of the fragmatome. Our calculations, thus, predict the fracture of such a fragmatome, as has been noted in a published report of fracture of a 23-gauge fragmatome.It is possible to use finite element calculations to predict the complex motion of a surgical device, such as a fragmatome, as the thickness of the wall of the fragmatome tube or the length of the fragmatome tube is changed. We found that changing the thickness of the wall of the fragmatome did not change the primary frequency that the device vibrated at and the length of the fragmatome changed the primary frequency in a predictable manner. In addition, there were very narrow ranges of frequencies over which the vibration of the device suddenly became large (a resonance), sometimes large enough to break apart a titanium device. In fact, we were able to predict the failure of a 30 mm fragmatome, which occurred clinically and was previously reported. Such resonances require a calculation and cannot be easily predicted. Thus, instruments developed for microsurgical applications cannot always be simply scaled down versions of conventional instruments. Such an approach can lead to potentially dangerous intraoperative failures, such as a fragmatome shattering inside the eye. Modern engineering techniques are increasingly necessary to investigate potential instrument failure mechanisms and to optimize device performance in a design in silico before in vivo testing.


MECP2, especially on DNA maintenance, cell cycle, transcription, and translation. In conclusion, using pathway models for classical enrichment and more advanced network analysis enables a more comprehensive analysis of gene expression data and provides novel results.Pathway and network approaches are valuable tools in analysis and interpretation of large complex omics data. Even in the field of rare diseases, like Rett syndrome, omics data are available, and the maximum use of such data requires sophisticated tools for comprehensive analysis and visualization of the results. Pathway analysis with differential gene expression data has proven to be extremely successful in identifying affected processes in disease conditions. In this type of analysis, pathways from different databases like WikiPathways and Reactome are used as separate, independent entities. Here, we show for the first time how these pathway models can be used and integrated into one large network using the WikiPathways RDF containing all human WikiPathways and Reactome pathways, to perform network analysis on transcriptomics data. This network was imported into the network analysis tool Cytoscape to perform active submodule analysis. Using a publicly available Rett syndrome gene expression dataset from frontal and temporal cortex, classical enrichment analysis, including pathway and Gene Ontology analysis, revealed mainly immune response, neuron specific and extracellular matrix processes. Our active module analysis provided a valuable extension of the analysis prominently showing the regulatory mechanism of  In a diseased state, many molecular processes in the human body are affected and dysregulated. Performing pathway analysis on molecular data sets comparing healthy vs. diseased subjects is immensely effective in finding affected pathways and it enables researchers to study the underlying processes in detail, to reveal possible disease mechanisms. While standard enrichment methods have limitations and pathways are analyzed independently with their arbitrary process boundaries . The original study was published by Lin et al.  and three age-, gender-, and ethnicity-matched controls.The publicly available dataset studying the transcriptome in human brain tissue of Rett syndrome patients and healthy controls from the Gene Expression Omnibus (GEO) was used  and subjected to quality control, including signal distribution and sample grouping analyses, using plotting functions from ArrayAnalysis.org  with an absolute fold change cutoff of 1.5. Ensembl identifiers of all measured genes in the datasets were provided as the background list. Number of permutations was set to 2,000. Pruned GO-term results  were filtered based on Z-score (> 1.96), permuted p-value (< 0.05) and a minimum number of changed genes of five. Pathway analysis was performed on a combined human pathway collection from all curated WikiPathways pathways including the Reactome pathway set . Differential gene expression was mapped to genes on the pathway diagrams using the Hs_Derby_Ensembl_91.bridge identifier mapping database. Thereafter, pathway statistics was performed on differential gene expression for temporal and frontal cortex using the following criteria to select only significantly differentially expressed genes :For GO analysis with GO-Elite, the input gene lists for frontal and temporal cortex contained all significantly changed genes  AND .p-value (< 0.05), and minimum number of changes (positive) genes of five.The resulting ranked pathway list was filtered based on Z-score (> 1.96), permuted Biological pathway models are small sub-networks describing specific biological processes. Connecting and integrating pathway models in one large network enables us to use network biology tools and approaches to study and investigate the network.https://github.com/wikipathways/wprdf2cytoscape). Interactions with at least two annotated interaction participants  are included. Gene products have unified Ensembl  for both frontal and temporal cortex were added as node attributes to the network.The constructed network was loaded into Cytoscape (version 3.7.0), a network analysis and visualization tool , BridgeDbR package (version 1.16.0) with Hs_Derby_Ensembl_91.bridge database.Enrichment analysis: PathVisio (version 3.3.0) and GO-Elite (version 1.2).Pathway-based network construction: Script available on Github(https://github.com/wikipathways/wprdf2cytoscape)Active module analysis: Cytoscape (version 3.7.0), jActiveModules app (version 3.2.1)The total number of probes measured was 37,707 from which 29,024 could be linked to Ensembl identifiers. After merging multiple probe identifiers for the same Ensembl identifier, 19,023 unique gene identifiers remained. Differential gene expression analysis revealed 1,953 in the frontal cortex and 2,436 significantly changed genes in the temporal cortex samples of RETT syndrome patients vs. controls. Only 221 in frontal and 341 of the significantly changed genes in temporal cortex had a more than 1.5-fold increase or decrease in expression (|log2 fold change| > 0.58). In both brain regions, more genes were down-regulated in Rett syndrome patients than up-regulated, see Gene Ontology overrepresentation analysis identified 39 and 50 biological processes as altered in frontal and temporal cortex, respectively , 3. SummMicroglia Pathogen Phagocytosis Pathway , see From the 903 pathway models in the WikiPathways and Reactome collection, 860 pathways contained 27,410 unique interactions. On average, a pathway contained 35 interactions . Interestingly, 3,264 interactions occur multiple times but only 2,103 interactions are present in more than one pathway. As an example, one of the highest occurring interactions is the complex binding of the three subunits of the IκB kinase complex which plays an important role in the propagation of cellular response to inflammation  and 427 smaller components with each less than twenty nodes. The network contains 8,643 gene products, 2,704 metabolites and 9,882 complex / group nodes. Most common interaction types are directed interaction , complex / group participation , catalysis , inhibition , and conversions (896).Active modules were calculated using the jActiveModules app. The top five modules with the highest active paths scores were identified for both comparisons, frontal and temporal cortex. The modules for frontal cortex contained between 300–350 nodes and 560–1,020 edges. The top modules for temporal cortex tended to be smaller ranging from 230–290 nodes and 450–1,000 edges. p-value < 0.05). Twelve gene products have an absolute log2 fold change > 0.58. The subnetwork contains eight significantly down-regulated genes (blue rounded rectangles) including two F-Box genes, FBOX32 and FBXO9, involved in phosphorylation-dependent ubiquitination. The subnetwork contains five significantly up-regulated genes (red rounded rectangles) with diverse roles. The genes identified as hubs in the active module network of frontal cortex are two gene products which are not measured in the dataset, RPS27A and UBA52. Both are involved in protein degradation via 26S proteasome, ubiquitination, translation, and DNA excision repair. In the central part of the network, the ribosomal proteins including RPL14, RPL29, and RPL3 form a cluster. This cluster is connected via PPP2CA and PPP2R1A, two phosphatases involved in cell cycle, DNA replication and transcription, to a cluster of centrosomal proteins including CEP78, CEP57, and CEP131. The module combines interactions from 47 unique pathways (WP3577), non-sense-mediated decay (WP2710), cell-cycle related pathways , and eukaryotic translation elongation and initiation .The highest ranked active module for frontal cortex contains 303 nodes (79 interactions and 224 gene products) and 568 edges, see pathways  includinp-value < 0.05). Twenty-nine gene products have an absolute log2 fold change > 0.58. The module contains 24 significantly down-regulated genes (blue rounded rectangles) including several ubiquitin conjugating enzymes  and translation initiation factors . Only five significantly up-regulated genes are found in the subnetwork (red rounded rectangles) but the distance between them is large. This subnetwork contains similar hub nodes as in the frontal cortex subnetwork including RPS27A, UBA52, and PPP2R1A. Additionally, NCBP2 and NCBP1, proteins involved in RNA processing, play an important role in the subnetwork. The module combines interactions from 51 unique pathways , cell cycle , and immune response  related processes.The highest ranked active module for temporal cortex contains 238 nodes (84 interactions and 154 gene products) and 457 edges, see pathways  includinMECP2 is a multifunctional protein which is involved in several transcriptional inhibitory and activational processes. MECP2 was generally regarded as a repressor, however its role as genetic activator has also been confirmed  coverage is enabled. Identifying active modules from a large network has some major benefits, such as the easy applicability to any gene expression dataset, ignoring predefined boundaries used in traditional pathway diagrams, and incorporating the relations and overlap between the pathways. Additionally, this method does not require researchers to predefine a certain cutoff, since genes are ranked based on their significance.Some considerations arose when constructing and analyzing the network. For instance, some common metabolites like ATP, ADP, or NADH, while biologically necessary, were excluded from the network, since their involvement in a multitude of interactions created links between almost every node. Additionally, this approach is strongly depending on the a priori input of pathway data in terms of coverage and quality. Currently, human pathway databases contain a little over 50% of the protein coding genes  known, the resulting downstream consequences can be diverse and affect a variety of pathways. By using pathway models in an integrative network approach, further use of the invaluable resources present in pathway databases is enabled and subnetworks of interest can be retrieved based on the entire body of pathways available. Using Cytoscape allows using all available apps such as the jActiveModules app to analyse our network. Importantly, the complete interaction network of WikiPathways with 48,639 nodes and 106,137 edges can be opened and analyzed in Cytoscape, despite of the network to be too large to be visualized. The use of graph databases like Neo4j, which already have connections available to Cytoscape .The dataset analyzed for this study can be found in the Gene Expression Omnibus (RM and FE: data analysis, literature search; LE: data preprocessing and statistical analysis; DS: data analysis; LC: literature search; CE and EW: study design; MK: data analysis, study design, literature search. All authors contributed in writing and editing of the manuscript.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.