Several thresholds based on the number of medications were mentioned. The majority of the publications  used a minimal threshold of five medications. Heterogeneous qualitative definitions were identified, mostly stating that polypharmacy is “more drugs than needed”. There was no significant divergence between domains as to the type of definitions used, although qualitative definitions were more common in clinical practice. Nearly half  of the publications provided no justification for the polypharmacy definition used. The wide variety of definitions for polypharmacy precludes comparisons, appropriate identification and management of polypharmacy in multimorbid older adults. Standardized definitions would allow more coherent judgments regarding the individual and collective stakes of polypharmacy.There are numerous definitions of polypharmacy to describe the use of many medications among older adults, but there is a need to clarify if they are purposive and meaningful. By means of a systematic review, we identified definitions of polypharmacy used in multimorbid older adults (≥65 years). We evaluated if the definitions align among the domains of research, clinical practice, and public health and appraised whether concepts of polypharmacy are based on strong foundations. More than 46 definitions of polypharmacy were retrieved from 348 publications (research:  As chronic diseases tend to accumulate over a lifetime, the ageing of the population has caused multimorbidity to become a significant condition . Caring Nonetheless, portraying the frequency and consequences of polypharmacy in multimorbid older adults remains challenging since there is no consensus definition for this term. Such a definition would be useful to understand the association between using many medications and health outcomes and to develop interventions designed to tackle inappropriate medication use. It would also facilitate the comparison of studies on polypharmacy in multimorbid older adults and hence help determine, for instance, how significant polypharmacy is as a public health issue. However, a universal and unique definition of polypharmacy may not be possible as it may depend on the aim it is intended to achieve .Indeed, the domains of research, clinical practice and public health, all of which must address the issues of polypharmacy in multimorbid older adults, have different needs and visions that could lead to different definitions of polypharmacy. Research activities focus notably on establishing etiological links between multiple medication use and health events. Therefore, the definitions of polypharmacy must be precise and allow for reproducibility. Clinical practice is based on research findings to establish standards of practice for the care of older adults. However, in this case, polypharmacy should probably be defined less comprehensively than for research, to allow a simple application in the clinical reality. Finally, for public health to be able to conduct its activities such as describing population health status in multimorbid older adults and assisting in resource planning, the definitions of polypharmacy should provide a clear picture of the associations between polypharmacy and health outcomes and use of health services. Thus, a certain variety of definitions of polypharmacy is expected. Although some publications have inventoried the definitions of polypharmacy in older adults ,9,10,11,Therefore, the objectives of this review were: (1) To identify the definitions of polypharmacy that are used in the context of multimorbidity among older adults (≥65 years) in the domains of research, clinical practice and public health; (2) to establish on what basis  these definitions were constructed or chosen. The final results of this work allowed us to highlight the knowledge gaps, the problems and the possible strengths of the identified definitions and to suggest which avenues should be privileged to advance towards consensus definitions for polypharmacy in multimorbid older adults.The complete protocol for our review has been registered in Prospero (CRD42014014989) and previously published . The sysWe considered all publications that targeted people aged 65 years and older with at least two concurrent chronic diseases (multimorbidity). We included publications if (1) at least 80% of participants were aged 65 years and older, (2) the data from people aged 65 years and older could be extracted, or (3) the focus of the publication was older adults (for reviews or other type of publications that did not recruit patients). We included all settings  and types of health care .A systematic search strategy was developed by the authors  in collaboration with an experienced librarian (V. Tessier). A second librarian specialized in health sciences revised the proposed strategy (S. Vachon). A first search was performed in December 2014 and was updated in May 2016 using the ovidSP  and EbscoHost  platforms. We also searched Google Scholar and Google to identify grey literature such as governmental reports.Four concepts were considered in our search strategy: Polypharmacy, older adults, multimorbidity and positive/negative outcomes . The search strategy was adapted to the syntax requirements of each database  . We inclNo restrictions were placed on the types of publications. Original studies, reviews, commentaries, editorials, practice guidelines, reports, theses and governmental publications were used to identify polypharmacy definitions and were searched for the primary references they cited. In cases of missing information, we contacted the authors to complete the required information if the publication corresponded to the inclusion criteria.® to group the results and exclude duplicated publications. Two independent reviewers examined the titles and screened the abstracts . Full-text publications were retrieved for all publications not excluded during the previous two steps. Full texts were reviewed by two independent reviewers (CS and NSD/AZ). All exclusion criteria were recorded at each step to generate flow charts according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) requirements.We used Endnote(1)The publication did not provide any operational definition of polypharmacy. For example, defining polypharmacy only as “large number of medications” did not qualify. To be considered operational, the definition could involve a specific number of medications  or indicate a specific condition .(2)The targeted population did not include people 65 years and over.(3)The publication referred to polypharmacy used for the treatment of a single medical condition in the absence of multimorbidity.Publications were excluded based on the following criteria:Publications were also excluded if there was missing information ; the document was published before 2004; the document was published in a language in which our team was not fluent  and we did not find adequate translation resources; or it was a duplicate publication .MD (https://www.filemaker.com). This allowed all researchers to access the data in real time.Two independent reviewers  conducted a full-paper evaluation and data extraction. In the event of disagreement that could not be resolved by discussion, a third reviewer was invited to make a final decision. Established data extraction forms adapted for the objectives of the review were created using FileMakerFor each publication, we recorded bibliographic details, type of publication, context, participants and the definition of polypharmacy. We then extracted information about the definition of polypharmacy, the types of medications included , the counting method and qualitative criteria  used in order to label a medication regimen as polypharmacy. We compared the definitions used in the publications according to whether the publication pertained to domains of clinical practice , research  or public health and surveillance . We also evaluated whether definitions (1) relied on a theoretical framework  or resulted from a methodological assessment ; (2) referred to other published material; or (3) were reported without specific foundation provided for their choice. We grouped definitions according to their nature .Our bibliographic search resulted in 2624 publications, of which 231 were included. Reference searching and evaluation of grey literature provided a further 117 publications . In totan = 202; 58%) and 10 or more medications  were the most popular. The definition cut-offs differed according to setting. While the minimal threshold of five medications was clearly the most frequent for research done in community (70%) and hospital (68%), research conducted in nursing homes used varied minimal cut-offs such as five (38%), six (33%) and nine medications (21%). Qualitative definitions were mentioned in 58 publications (16.7%), either independently or in addition to a quantitative element. A total of 13 (5.3%) research, 42 (47.7%) clinical practice (mostly reviews) and three (17.6%) public health publications contained such definitions  were the most frequently documented . They apinitions . We idenSeveral pieces of information relevant to the conceptualization of polypharmacy were missing from the quantitative definitions found . In the p = 0.168). In order to restrict results more closely to publications where polypharmacy was the main subject, we analyzed the 199 publications where the term polypharmacy appeared in the title: 73 of them (36.7%) did not provide information to justify their choice of a definition.From the 329 publications that stated a specific definition, 192 (58.4%) either stated a foundation or underpinning basis to their definition . ExampleOur systematic review demonstrates the manifold definitions of polypharmacy used in populations of older adults with multimorbidity, frequently without a strong basis or conceptual foundation. Given such arbitrary formulations, there is very little convergence between publications.Others have identified a wide variety of definitions of polypharmacy among older adults ,9,10,11.Several factors could explain the wide variety of definitions presented in the literature. First, as previously stated, the threshold for polypharmacy has evolved over time along with the concomitant increase in the number of medications used ,23. CurrMany essential elements for a proper, clear and operational definition of polypharmacy were missing in the majority of the publications retrieved, which is particularly concerning for research. The number of medications involved, the type of medications , the exposure length, or the distinction between acute and chronic polypharmacy, for example, were not often specified in the publications consulted. While it may be reasonable to omit such information in certain types of material , the degree of definitional rigor should be higher in research. However, the definition of polypharmacy remained vague in more than half of the research publications. One striking example is a study recruiting older people with polypharmacy without explicitly defining what polypharmacy entails . Other aThe lack of a theoretical basis or conceptual model is a major quality gap for most definitions of polypharmacy in multimorbid older adults. Only 28 (11.5%) research publications provided such information. Even after restricting analysis to publications mentioning polypharmacy in their titles, the percentage providing such information remained low (17.3%). Moreover, the diversity and wide array of references that were used to refer to specific definitions of polypharmacy is revealing. This corroborates the notion that there is neither consensus nor a clear path to a standard definition. Interestingly, the references which Hoffmann et al.  quote toThe quantitative definitions  and qualitative definitions  of polypharmacy are regularly opposed. We observed that publications from the clinical practice domain usually mention both positions. However, qualitative definitions remain rare in research publications. Considering the very strong association between the number of medications and the presence of inappropriate medications, concepts of polypharmacy and potentially inappropriate medications are entangled. Nonetheless, our review does not support the views of Hudhra et al.  who statSimilarly, many authors state that it is problematic to define polypharmacy with a threshold because some older adults will need a large number of medications to be adequately treated ,21,33. IOur review reveals that there are many knowledge gaps to fill, starting with the need for definitions allowing researchers to discern which combinations of treatments will result in adverse or beneficial effects in older adults with multiple chronic diseases. In fact, it remains a matter of concern that the definitions used in research suffer from several shortcomings. If clinical practice publications cannot be expected to provide important insights into such concepts as the type of drugs included or the route of administration, it would be crucial to document those elements in research, to ensure adequate integration of results in clinical practice and public health.We suggest that future definitions of polypharmacy integrate the following basic elements, especially when the definition is used for research and public health interventions: The number and type of medications involved , the exposure length, and the distinction between acute and chronic use of medications. Definitions should be based on a solid foundation and should serve the intended purpose. For example, a definition could be chosen on the basis that the medication threshold has been associated with an increased risk of adverse events in previous research, or that the use of health services increases significantly from this threshold. Definitions used in clinical practice should also be evidence-based to ensure credibility of the term “polypharmacy”. In fact, it is important for research to determine which health outcomes are associated with which type of polypharmacy. By ensuring reproducibility of results in different countries, settings and populations, it will be possible to develop standard definitions of polypharmacy that will ensure a better understanding of the issues at stake with polypharmacy. Such standard definitions could also provide other opportunities. For example, some authors have suggested that polypharmacy should be considered a disease in itself . If polyThis study has some limitations. Our definitions of clinical practice, research and public health may be arguable, and some publications could fall into more than one category. Although we strived to include all publications mentioning polypharmacy, our search strategy may have omitted some relevant ones. Nonetheless, we did retrieve a very large number of publications, including documents published in different languages, and we do not expect that results might have changed if further publications were included. We did not update our literature review to include recently published documents, as the number of publications was already significant; but from our recent work on the topic of polypharmacy , there aPolypharmacy in multimorbid older adults must be better defined according to its field of application. The panoply of definitions and means by which polypharmacy is conceptualized in research leads to much confusion surrounding the term, which hampers research from informing clinical practice and public health stakeholders adequately. There is an urgent need to better understand the basic pharmacological implications of using many medications in multimorbid older adults in order to evaluate the potential pitfalls (and benefits) of polypharmacy. At present, considering the variety of definitions used, it remains difficult to clarify what should be the best definitions of polypharmacy for multimorbid older adults. Most likely, several definitions will need to be employed to respond to the various imperatives of different domains, populations and settings.


Immunoglobulin G4-related sclerosing cholangitis (IgG4-SC) is a novel entity that belongs to the immune-mediated fibroinflammatory class of IgG4-related diseases (IgG4-RD). IgG4-SC is noted to be one of the most frequent manifestations of extra-pancreatic disease among IgG4-RD, which is significantly different from primary SC (PSC) and cholangiocarcinoma (CC) as is evident in the varied approaches to treatment. IgG4-RD includes IgG4-SC and autoimmune pancreatitis (AIP).Herein, we presented a case of IgG4-SC in a patient with obstructive jaundice secondary to AIP. We have also discussed the current recommendations for diagnostic and treatment modalities, with an emphasis on the issues that arise in obtaining a definitive classification of disease. IgG4-related sclerosing cholangitis (IgG4-SC) is an uncommon condition recently recognized as an independent disease entity in hope to better specify future diagnosis modalities and treatment. Its exact pathophysiologic mechanism is yet to be identified, but evidence of submucosal fibrosis and inflammatory cells infiltrating biliary ducts has been noted on biopsies -2. HereiThe patient is a 54-year-old male with no known past medical history who presented to the hospital with sudden onset of sharp, epigastric abdominal pain, weight loss, and nausea. Physical examination was remarkable for epigastric tenderness, scleral icterus, and painless jaundice. He was admitted to the hospital after being stabilized with intravenous saline, antiemetic medication, and analgesics. Liver function tests were elevated, Carcinoembryonic antigen (CEA) level was 652.9 ng/ml, and IgG4 was 464mg/dL. Computed tomography (CT) scan of the abdomen and pelvis revealed diffuse parenchymal enlargement, with surrounding inflammatory changes. Magnetic resonance imaging (MRI) of the abdomen revealed heterogeneous enhancement in the head of the pancreas along with a short segmental stricture of the common bile duct with extrahepatic biliary dilatation Figures -2. Liver biopsy was performed with hematoxylin and eosin stain showing cuff-like periductal lymphoplasmacytic infiltration and normal surrounding pancreatic parenchyma. Plasma cell-rich mixed infiltrate around bile ducts and periductal fibrosis were noted as well. These biopsy findings along with serum IgG4 levels were consistent with IgG4 AIP. The patient responded well to steroids, and the CEA levels dropped. He followed up in office and steroids were tapered with plans to start azathioprine. Ten months later, he presented to the hospital with obstructive jaundice and right upper quadrant pain. IgG4 at that time was >700 ng/ml. MRI of the abdomen and pelvis showed relapse of AIP with cystic changes at the level of the pancreatic neck, as well as a 1-cm long stricture of the proximal intrapancreatic portion of the common bile duct, wall thickening of the common hepatic duct, and the common bile duct. These findings were indicative of sclerosing cholangitis or IgG4-SC. The patient was again started on steroids and is currently doing well.Immunoglobulin G4-related diseases (IgG4-RD) consist of a spectrum of diseases including IgG4-SC and AIP -2. The cAIP is one of the most common extra-biliary etiologies of IgG4-RD. There are two distinct subtypes classified as type I and type 2 AIP -11. TypeIgG4-SC is a novel entity that has been documented to be associated with conditions such as autoimmune pancreatitis, primary SC (PSC), and CC -2. ThereIgG4-SC is a novel entity that has been documented to be associated with conditions such as AIP, PSC, and CC. Due to the overlapping clinical presentation of IgG4-SC with other disease processes, prompt diagnosis, classification, and appropriate treatment modalities are difficult to establish. To date, several diagnostic criteria have been proposed as a means to help identify IgG4-SC and other related diseases. However, further comparative diagnostic and treatment studies are needed with treatment remaining inconsistent and a lack of formally established diagnostic criteria.


MET-amplified Hs746T cells was reduced by means of siRNA transfection. The effects of MET knockdown on signal transduction, cell proliferation and motility were examined. In addition to the manual assessment of cell motility, a computational motility analysis involving parameters such as (approximate) average speed, displacement entropy or radial effectiveness was realized. Moreover, the impact of afatinib was compared between MET knockdown cells and control cells. MET knockdown in Hs746T cells resulted in impaired signal transduction and reduced cell proliferation and motility. Moreover, the afatinib resistance of Hs746T cells was reversed after MET knockdown. Therefore, the amplification of MET is confirmed as a resistance factor in gastric cancer cells. Whether MET is a useful resistance marker for afatinib therapy or other HER-targeting drugs in patients should be investigated in clinical trials.The therapeutic options for advanced gastric cancer are still limited. Several drugs targeting the epidermal growth factor receptor family have been developed. So far, the HER2 antibody trastuzumab is the only drug targeting the HER-family that is available to gastric cancer patients. The pan-HER inhibitor afatinib is currently investigated in clinical trials and shows promising results in cell culture experiments and patient-derived xenograft (PDX) models. However, some cell lines do not respond to afatinib treatment. The determination of resistance factors in these cell lines can help to find the best treatment option for gastric cancer patients. In this study, we analyzed the role of MET as a resistance factor for afatinib therapy in a gastric cancer cell line. MET expression in afatinib-resistant  Gastric cancer, an important malignancy worldwide, is the fifth most frequently diagnosed cancer and the third leading cause of cancer death . AlthougTo date, the HER2 antibody trastuzumab is the only anti-HER therapeutic which is available to patients with advanced gastric cancer. Since trastuzumab is only approved for HER2-positive gastric cancers (6–30%) and approximately 50% of HER2-positive cancers are resistant to trastuzumab treatment, there is an urgent need for alternative therapies (reviewed by ). The efMET-amplified cell line Hs746T, we suggested MET as resistance factor for afatinib therapy [We previously compared the effects of trastuzumab and afatinib on kinase activity in gastric cancer cell lines. Besides inhibiting the phosphorylation of HER2, EGFR and HER3, the tyrosine kinase inhibitor afatinib also had strong effects on downstream kinases MAPK1/2, AKT 1/2/3, PRAS40 and WNK1 in NCI-N87 cells. Moreover, cell proliferation was markedly reduced after afatinib treatment. By showing afatinib resistance in the  therapy .EGFR amplification or HER2 amplification, respectively [The effects of afatinib were also investigated in mouse xenograft and patient-derived xenograft (PDX) models. Afatinib treatment resulted in a dramatic regression of tumor volume in a NCI-N87 mouse xenograft model , 7. In pTaken together, data from cell culture and xenograft models reveal afatinib as a promising candidate for gastric cancer therapy. However, the influence of response and resistance factors on therapy outcome needs further evaluation and should be considered carefully.MET-amplified advanced solid tumors. A partial response was demonstrated in 3 out of 5 patients with MET-amplified tumors , whereas the 36 patients with tumors without MET amplification did not respond [MET-amplified gastric, esophageal or GEJ cancer responded to tyrosine kinase inhibitor AMG-337 [MET-amplified gastric/esophageal carcinoma or other solid tumors was terminated (NCT02016534). However, another phase II study currently investigates AMG-337 in advanced solid tumors that overexpress MET or bear MET exon 14 skipping (NCT03147976).The hepatocyte growth factor receptor (MET) pathway plays an important role in the regulation of growth, survival and invasiveness of gastric cancer , 10. Abe respond . In a ph AMG-337 . The phaIn this study, we investigated the role of MET as a resistance factor for afatinib therapy in the gastric cancer cell line Hs746T by means of MET knockdown. The effects of MET knockdown on signal transduction and its phenotypic effects on cell proliferation and cell motility were considered. We were able to show at the molecular and phenotypic level that it is possible to restore a therapeutic response to afatinib therapy by downregulation of MET.2. Cells were grown in Dulbecco’s Modified Eagle Medium with GlutaMAX (Thermo Fisher Scientific) with 0.5% penicillin/streptomycin (Thermo Fisher Scientific) and 10% Sera Plus (Pan Biotech). The absence of mycoplasma was tested as described elsewhere [The gastric cancer cell line Hs746T (ATCC) was cultured at 37°C in humidified atmosphere with 5% CO4 cells/cm2. Two hours before transfection, the medium was replaced by antibiotic free medium. Cells were transfected with a pool of 4 siRNA oligomers (5.7 pmol/cm2) against MET  and 0.57 μl/cm2 Lipofectamin®2000 (Thermo Fisher Scientific) according to the manufacturer’s instruction. As negative control, cells were transfected with equal amounts of All Star Negative Control siRNA (Qiagen). All Star Negative Control siRNA AF488 (Qiagen) was used to determine the transfection efficiency. The transfection was stopped by medium replacement after 24 h. Cells were then plated for proliferation assay, motility analysis and generation of protein lysates.Hs746T cells were plated one day before transfection with a density of 1.7 x 10Western blot analyses were performed according to a standard protocol described earlier , 17. One3 cells/cm2 and were treated with 0.5 μM afatinib or 0.05% DMSO (solvent control) for 72 h.Cell proliferation was analyzed using WST-1 cell proliferation assay (Roche Diagnostics) as described earlier . Hs746T 5 cells/dish were plated onto the coated plate. In order to determine the percentage of motile cells and the average speed, time-lapse microscopy was carried out as described earlier [Glass-bottom culture dishes (MatTek Corporation) were coated with 100 μg/ml collagen type I  for 30 minutes at 37°C. One day after transfection, 1.5 x 10 earlier , 19.For each image frame of a video and for each cell having its nucleus entirely present on the frame a point in the center of the nucleus is placed and its position  is recorded together with the number t of the frame on which the point is placed, leading to a value  for each frame and cell.This procedure is carried out based on an algorithm -sequences –have been selected which did not undergo division and for which the -values are available for each image frame t. For a given -sequence in a first step a sequence of displacement vectors is computed  =  where  and  correspond to the cell's position in two successive frames and the displacement length is min (5.0 times the pixel length are chosen), sub-sequences of displacements , , … , relating the points , , …, , are replaced by the displacement  with length dapprox for the first k for which dapprox ≥ dmin  < dmin).The approximate average speed is defined as the approximate trajectory length divided by the total elapsed time of the video sequence. To compute the approximate trajectory length for a given minimum displacement length di of displacements dt (in pixel length) with i ≤ dt < i + 1 divided by the total number N-1 of displacements of the considered trajectory, hence hi = ni / (N-1). The entropy value is then calculated as the sum of all terms -hi*log(hi) for all histogram bins i. The natural logarithm log = ln to the base e (Euler's number) is taken. In the same way the orientation entropy is defined by replacing the displacement lengths by the angles of the displacement vectors with respect to the x axis. The parameters displacement entropy and orientation entropy are illustrated in The displacement entropy consists in a magnitude and in an orientation entropy where the former one is the entropy value computed for the histogram of displacement lengths dt . One pixmax between any two points of the trajectory we obtain the radial effectiveness (= distmax / L). The radial effectiveness is illustrated in The displacement effectiveness is defined as the ratio of the direct distance between the first and last point of the trajectory divided by the total trajectory length L  . If the At least three biological experiments were analyzed. The data are presented as mean +/- standard deviation. One-sample or two-sample t-test was used for pairwise comparisons (IBM SPSS Statistics). The significant differences are indicated by *p<0.05, **p<0.01 or ***p<0.001.We analyzed protein lysates after MET knockdown and afatinib treatment by Western blot to assess the influence on MET and EGFR signaling. The afatinib response was compared between MET knockdown (MET KD), control (Ctr) and non-transfected (NT) cells. DMSO was used as solvent control.After MET KD, MET expression was reduced to 14% compared to NT cells. Afatinib treatment did not influence MET expression in any of the three groups . DMSO treatment reduced MET expression to 78% in non-transfected cells, but not in Ctr cells or MET KD cells . The actEGFR expression was not reduced after MET knockdown. Neither afatinib nor DMSO treatment showed any effect on EGFR expression . MET knoAfter MET knockdown, activation of downstream kinases MAPK and AKT was reduced to 28% and 26% respectively, compared to NT cells . To illuAfatinib reduced activation of MAPK and AKT, but not EGFR in MET knockdown cells. One would expect that reduced EGFR activation results in reduced MAPK and AKT activation. However, small, not detectable changes in EGFR activation could be enough to result in changed MAPK and AKT activation. Within these experiments, only the pEGFR Y1068 phosphorylation site was analyzed. In earlier experiments using the proteome profiler, we compared effects of afatinib on EGFR phosphorylation using site-specific and pan EGFR antibodies. EGFR Y1086 phosphorylation was not reduced after afatinib treatment in NCI-N87 cells, but phosphorylations of AKT and MAPK were reduced. The treatment effect of afatinib was more pronounced with the use of a pan EGFR antibody, which detects multiple EGFR phosphorylation sites .Taken together, the results indicate that knockdown of MET in Hs746T cells reduced the activation of MET and EGFR as well as the activation of downstream kinases MAPK and AKT. Moreover, only the MET KD cells responded to afatinib treatment as shown by decreased MAPK and AKT activation.In order to assess the success of MET knockdown during phenotypic assays, protein lysates were generated in parallel. The expression of MET was analyzed by Western blot.During cell proliferation assay, MET expression was reduced to 13% compared to NT cells on day 1 and 12% on day 5 . At the Thus, we demonstrated successful MET knockdown during the duration of proliferation and motility assays.In order to investigate the effects of MET knockdown and afatinib treatment on proliferation, we performed the WST-1 cell proliferation assay. The afatinib response on proliferation (measured as metabolic activity) was compared between MET KD, Ctr and NT cells. DMSO was used as solvent control.The metabolic activity of MET knockdown cells was reduced to 30% in comparison to NT cells . AfatiniWe showed reduced proliferation after MET KD in Hs746T cells. Furthermore, only the MET KD cells were sensitive to afatinib treatment.The effects of MET knockdown on cellular motility were assessed by time-lapse microscopy. MET KD, Ctr and NT cells were filmed for 7 hours. Different motility parameters were assessed by manual and computer-driven analysis.The manual analysis of motility parameters allowed the assessment of motile cell number and approximate average speed. A cell was defined as motile when it completely left the field previously occupied by the cell itself. The movement of each cell was tracked while the film was running and the approximate average speed was calculated. To determine the approximate average speed manually the cell was not tracked at each point in time but its path is marked on the screen while the video is running . In addiAfter MET knockdown 42% of cells were classified as motile, whereas 79% of cells were classified as motile after control transfection. The percentage of motile cells in non-transfected cells was 61%. The motility reduction in MET knockdown cells compared to control cells was significant, while the decrease was not statistically significant when compared to non-transfected cells . The appTo sum up, the percentage of motile cells as well as the motility parameters (approximate) average speed, displacement entropy and radial effectiveness were reduced after MET KD in Hs746T cells. A high proportion of MET KD cells demonstrated an altered, tangle-like movement behavior.MET amplification and MET exon 14 skipping due to a splice-site mutation. The binding site of CBL (Y1003), which mediates MET degradation, is missing due to exon 14 skipping. Consequently, MET oncogenic signaling is sustained [To investigate the importance of the receptor tyrosine kinase MET as resistance factor we used the gastric cancer cell line Hs746T, which bears a ustained , 24. We ustained . In the MET is currently under scrutiny for its role as resistance factor for anti-HER therapies in different entities. Experiments in lung cancer cell lines  revealed an effect of MET inhibition with su11274 and afatinib treatment on cell growth and apoptosis. Treatment of lung cancer cells with a combination of MET siRNA and EGFR siRNA enhanced the growth inhibiting effect of EGFR siRNA alone .In vivo generated afatinib-resistant clones of H1975 lung cancer cells showed decreased expression of EGFR, HER2, HER3 and HER4 and increased expression of MET, c-KIT and PDGFRβ. The combined but not the single or double knockdown of HER3, c-KIT and MET resulted in cell death [ll death . This inIn a 3D culture system of colorectal cancer cells, the phosphorylation of MET and RON was higher in cells with weaker response to EGFR-directed antibody cetuximab. Furthermore, this resistance was reversed by treatment with MET/RON TKI crizotinib .HER2-amplified gastric cancer cell lines NCI-N87 and SNU-216 were sensitive to EGFR tyrosine kinase inhibitor lapatinib. In presence of MET ligand HGF, these cells were resistant to lapatinib. MET knockdown reversed the lapatinib resistance after HGF stimulation. Moreover, the resistance was also reversed by treatment with a MET inhibitor. The authors conclude that MET is an important resistance factor in these cell lines [MET, MET knockdown improved sensitivity to lapatinib.The two ll lines . AlthougMET-amplified cell lines MKN45, Hs746T and SNU-5 were amongst the 4 cell lines with the highest IC50 value [Data from our group in combination with GDSC (Genomics of Drug Sensitivity in Cancer) dataset show a correlation of afatinib sensitivity and MET Y1234/1235 activation. Gastric cancer cell lines with higher IC50 value for afatinib sensitivity showed stronger MET Y1234/1235 activation. Moreover, the three 50 value .MET amplification was not found in any of the patients. However, the analysis of post-afatinib progression sites of one patient revealed a MET amplification which was not present in the analyzed tumor before treatment. The response of one post-afatinib progression site to a combination treatment of afatinib and AMG 337 (MET inhibitor) was demonstrated in a xenograft model [Afatinib as well as the combination of trastuzumab and afatinib were tested in a cohort of 32 patients with disease progression on trastuzumab. ft model .MET-amplified gastric cancer cell line can be reversed by MET knockdown is in line with data from studies using different entities or anti-HER therapeutics. Taken together, MET is a resistance factor for anti-HER therapies in different cancer entities, including gastric cancer. Studies with cell lines without MET amplification indicate that it is the high MET expression and not necessarily the amplification that may lead to resistance to anti-HER therapies. One might argue that using one cell line is not sufficient to show the relevance of MET as resistance factor. However, as indicated in this paragraph, the relevance of MET as resistance factor to EGFR-targeted therapies was already shown in various cell lines, including gastric cancer cell lines, and in clinical samples.Our observation that afatinib resistance in a MET knockdown resulted in reduced signal transduction, cell proliferation and motility. We conclude that Hs746T cells are addicted to MET, according to the concept of oncogene addiction . AlthougMET-amplified lung cancer cell lines  resulted in growth inhibition. This growth inhibiting effect was not observed in cell lines without MET amplification, with lower MET expression and no basal MET activation [MET amplification in these cell lines could explain the minor response to MET inhibition.MET knockdown in two tivation . In H358tivation . The lacMET-amplified gastric cancer cell lines  was reduced after treatment with MET inhibitors PHA-665752 or crizotinib [MET amplification predicts the sensitivity to MET inhibitors. Six MET-amplified gastric cancer cell lines, amongst them Hs746T, were sensitive to both MET antibodies and MET TKIs in cell viability assays [et al. who showed sensitivity of MET-amplified gastric cancer cells lines to the MET inhibitor PHA-665752. They further showed decreased viability after MET knockdown in MET-amplified cell line SNU-5 [MET inhibitors were also tested in gastric cancer cell lines. The selective MET inhibitor KRC-00715 suppressed tumor growth in a Hs746T mouse xenograft model . Anchoraizotinib . The anay assays . This stne SNU-5 . Effectsne SNU-5 .MET amplification [MET amplification, also MET exon 14 skipping is being discussed as a possible marker for anti-MET therapies. In lung cancer, patients with MET exon 14 skipping responded to MET inhibitors [To date, no drug targeting the HGF/MET pathway has been approved for gastric cancer treatment, but antibodies or tyrosine kinase inhibitors against MET are currently being investigated in clinical trials see . Both MEfication , 15. Beshibitors , 38. In in vitro, xenograft and clinical studies came to the same conclusion that MET-amplification is an indicator for oncogene addiction to MET and consequently for the efficacy of MET inhibitors. Our own MET knockdown data in MET-amplified cells support these observations.Different It has been shown that targeting tumor cell motility may be a strategy against invasion and metastasis . In the Activation of MET signaling cascade by its ligand HGF results in colony dispersal, epithelial-mesenchymal transition (EMT) and increased motility . The comOur data confirm the important role of MET as well as MAPK and PI3K/AKT pathways, as regulators of cell motility and consequently of invasion and metastasis.In our experiments, cell proliferation and downstream signaling were at the lowest following combined MET knockdown and afatinib treatment. Thus, the combined inhibition of EGFR, HER2, HER3 and MET is most effective with regard to inhibition of proliferation.MET amplified progression site from one gastric cancer patient [The expression of MET, EGFR and HER2 in 293 patients with unresectable or recurrent gastric or gastroesophageal junction cancer was examined by immunohistochemistry. The combined overexpression of MET, HER2 and EGFR was observed in 3% of gastric tumors. 5% of tumors were MET- and HER2-positive, but EGFR-negative. The co-overexpression of MET and EGFR without HER2 overexpression was present in 12% of tumors. In total, 20% of gastric cancers were either MET-, EGFR- and HER2- positive or MET- and EGFR- or HER2-positive . For the patient .In malignant pleural mesothelioma, the combination of afatinib and MET/ALK/RON/ROS inhibitor crizotinib has been investigated in cell culture and mouse xenograft models. In both systems, the combination was more effective than each drug alone in suppressing cell proliferation . The comin vitro studies, pan HER tyrosine kinase inhibitor afatinib is a promising therapy option for advanced gastric cancer. However, the presence of resistance factors should be considered before therapy. In the present work, we identified MET amplification as a resistance factor for afatinib therapy in gastric cancer cell line Hs746T. Moreover, we identified MET as an important regulator of cell motility. Future clinical trials should investigate the importance of MET as a resistance factor for afatinib therapy and other HER-targeting therapies.Based on S1 Raw Images(PDF)Click here for additional data file.


S-adenosylmethionine–mediated methylation of proteins, RNA, and DNA. The de novo biosynthetic pathway of Met is well conserved across prokaryotes but absent from vertebrates, making it a plausible antimicrobial target. Using a systematic approach, we examined the essentiality of de novo methionine biosynthesis in Salmonella enterica serovar Typhimurium, a bacterial pathogen causing significant gastrointestinal and systemic diseases in humans and agricultural animals. Our data demonstrate that Met biosynthesis is essential for S. Typhimurium to grow in synthetic medium and within cultured epithelial cells where Met is depleted in the environment. During systemic infection of mice, the virulence of S. Typhimurium was not affected when either de novo Met biosynthesis or high-affinity Met transport was disrupted alone, but combined disruption in both led to severe in vivo growth attenuation, demonstrating a functional redundancy between de novo biosynthesis and acquisition as a mechanism of sourcing Met to support growth and virulence for S. Typhimurium during infection. In addition, our LC-MS analysis revealed global changes in the metabolome of S. Typhimurium mutants lacking Met biosynthesis and also uncovered unexpected interactions between Met and peptidoglycan biosynthesis. Together, this study highlights the complexity of the interactions between a single amino acid, Met, and other bacterial processes leading to virulence in the host and indicates that disrupting the de novo biosynthetic pathway alone is likely to be ineffective as an antimicrobial therapy against S. Typhimurium.Methionine (Met) is an amino acid essential for many important cellular and biosynthetic functions, including the initiation of protein synthesis and  S-adenosylmethionine (SAM),de novo pathway for Met biosynthesis is present in the vast majority of prokaryotes, albeit with variations in the enzymes that drive the biosynthetic cascade  is a sulfur-containing proteinogenic amino acid that is required for the initiation of protein synthesis . S-adeno cascade , 4. In cut flora , 6. In rut flora . The appSalmonella enterica is a Gram-negative, facultative intracellular bacterium that causes gastrointestinal and systemic diseases in animals and humans. There are ∼2500 serovars in this species, which include the human-adapted enteric fever pathogens S. enterica var. Typhi and Paratyphi, alongside non-typhoidal Salmonella (NTS) serovars that cause debilitating gastroenteritis, including S. enterica var. Typhimurium   , 12. Thed mapped . It is lde novo biosynthetic pathway for Met has been previously reported in S. enterica  transporter composed of three subunits: the ATPase (MetN), a transmembrane permease (MetI), and a periplasmic Met-binding protein (MetQ). Biochemical analysis has shown that MetNIQ transports both the d- and l-enantiomers of Met  for MetP remains unidentified to date.The enterica . This paenterica . With itept metH . In contand metH . In addis of Met , 18. In d “MetP” –21. In cS. enterica may encounter markedly different nutrient levels during local (e.g. the gut) and systemic (e.g. the spleen and liver) infections, which, in turn, may vary the bacterium's dependence on de novo biosynthesis and nutrient import pathways for growth in different tissue niches. The enzymes required for Met biosynthesis and the Met transporter have been implicated in the virulence of S. enterica and other bacteria  are expressed under a variety of growth conditions  genes were expressed or when the bacteria were inside macrophages  were generated in S. Typhimurium SL1344, sequenced, and tested for their ability to grow in M9 minimal medium, with or without added l-Met, and in nutrient-rich Luria–Bertani (LB) medium. All mutants grew in LB medium at a rate comparable with WT SL1344 (data not shown). In M9 minimal medium supplemented with Met, Met mutants grew as efficiently as SL1344 over a 24-h period (A), suggesting that the absence of the de novo biosynthetic pathway was compensated by the transport of exogenously available Met. As expected, the ΔmetA, ΔmetB, ΔmetC, ΔmetE, and ΔmetF mutants exhibited Met auxotrophy in M9 medium, whereas ΔmetH showed substantial growth over the 24-h period (A), confirming that MetH is not essential for de novo biosynthesis of Met under aerobic conditions , as ΔmetEΔmetH failed to grow in M9 medium in the absence of exogenous Met (A). It has been suggested that the functionality of MetH in E. coli is vitamin B12–dependent . The ΔmetE mutant, which depended on MetH to synthesize Met, grew only when vitamin B12 was added (B), confirming that MetH activity in S. Typhimurium is vitamin B12–dependent. As S. Typhimurium synthesizes vitamin B12 only under anaerobic conditions . These observations are consistent with an understanding that S. Typhimurium synthesizes vitamin B12 under anaerobic conditions and support a level of functional redundancy between MetE and MetH during de novo biosynthesis of Met in S. Typhimurium.Defined mutants in the Met biosynthetic pathway . The ΔmetE mutant only showed obvious growth after 5 h. In contrast, the ΔmetH mutant showed intracellular growth comparable with SL1344, suggesting that a functional MetE is sufficient for supporting intracellular replication in the SCV (D). Importantly, when HeLa cells were cultivated in DMEM containing physiological levels of l-Met, none of the mutants showed any significant defect in intracellular growth , demonstrating that exogenous Met can rescue Met auxotrophy, presumably reflecting direct or indirect transport of Met from the culture medium to the lumen of the bacteria-occupied SCV.To assess the requirement for  control D. The Δm the SCV D. ImportS. Typhimurium in mice or chickens  and spleen (F) was determined by viable count. The number of bacteria recovered from mice infected with the mutants was comparable with those from mice infected with SL1344. Hence, none of the mutants showed virulence defects in mice following intravenous infection. Another group of mice were infected orally with ΔmetB, ΔmetE, ΔmetH, or ΔmetEΔmetH. The bacterial load in the spleen and liver of mice infected with the mutant strains was comparable with those from mice infected with the WT S. Typhimurium . These data showed that de novo Met biosynthesis is not essential for the growth of S. Typhimurium in C57BL/6 mice, regardless of the route of infection.Previous reports have proposed that Met biosynthesis might be required for virulence of chickens , 34. To nfection , 36. Gronfection E and spld spleen F was detS. Typhimurium mutants were able to grow intracellularly and in vivo without a functional de novo Met biosynthetic pathway suggested that Met acquisition via the transporters may play an important role in bacterial virulence. To test this hypothesis, a high-affinity transporter mutant of S. Typhimurium, ΔmetNIQ, was constructed. As expected, the ΔmetNIQ mutant grew in M9 minimal medium in the absence of Met supplementation (A), suggesting that the high-affinity Met transporter is not essential for growth in M9 medium provided that de novo synthesis is intact.Our observation that entation A, suggesde novo biosynthesis and the high-affinity transporter of Met affected the growth of S. Typhimurium in vitro and in vivo, ΔmetNIQΔmetEH and ΔmetNIQΔmetB mutants were generated. As expected, the ΔmetNIQΔmetEH and ΔmetNIQΔmetB mutants were unable to grow in M9 minimal medium without added Met (B); this Met auxotrophy confirms that a combined deficiency in both biosynthesis and high-affinity transport is inhibitive for growth. Interestingly, the addition of l-Met into M9 medium restored the growth of all of these mutant strains, consistent with the presence and activity of the putative and functional low-affinity transporter MetP . The presence of l-Met in DMEM fully restored the growth of the mutant strains in HeLa cells , suggesting that the activity of the cryptic transporter MetP was sufficient to support intracellular growth in HeLa cells.To determine whether the observed de novo biosynthesis and the high-affinity transporter of Met reduced S. Typhimurium virulence in vivo, C57BL/6 mice were intravenously infected with ΔmetNIQ, ΔmetNIQΔmetB, and ΔmetNIQΔmetEH mutant strains or WT. At day 5 post-infection, mice infected with ΔmetNIQ had a similarly high bacterial load in the liver (D) and spleen (E), comparable with that of SL1344-infected mice, indicating that loss of the high-affinity Met transporter alone does not reduce S. Typhimurium virulence in vivo. In contrast, infection with ΔmetNIQΔmetB and ΔmetNIQΔmetEH mutants resulted in a significantly reduced bacterial load in the liver (D) and spleen (E), and this attenuation was reversed when the ΔmetNIQΔmetB mutant was complemented by metB (D and E). Similar results were obtained when the ΔmetNIQΔmetB and ΔmetNIQΔmetEH mutants were used to infect mice via the oral route , confirming that ΔmetNIQΔmetB and ΔmetNIQΔmetEH are attenuated in mice regardless of the route of infection. Taken together, these results demonstrate that restriction of Met availability has a strong impact on the virulence of S. Typhimurium in vivo, and the presence of the putative cryptic transporter (MetP) alone is insufficient to sustain maximal bacterial growth in the permissive murine host.To determine whether dual mutations in he liver D and spld spleen E, comparhe liver D and spld spleen E, and th by metB , D and ES. Typhimurium WT and mutants lacking either or both of the de novo biosynthesis pathway and the high-affinity Met transporter were grown in LB medium, and polar metabolites were extracted and analyzed by LC-MS. The intracellular accumulation of 5-methyl-tetrahydropteroyltri-l-glutamate, a member of the polyglutamate forms of 5-methyltetrahydrofolate (metH (∼10-fold) and further increased in ΔmetNIQΔmetEH (∼25-fold), but not in ΔmetE, compared with SL1344  feature analysis of the mutant lines led to the identification of a number of unexpected metabolite changes. Pairwise comparisons were performed to identify differences that were statistically significant (p < 0.05) and to filter interesting m/z features. Deletion of metB led to significant increases in the intracellular levels of SAM (5.5-fold increase compared with WT), and a number of other metabolites, including glycerate, adenine, hexose 6-phosphate, 3-phosphoglycerate, and gluconate, were also increased compared with the WT , whereas only the m/z feature corresponding to 5-methyl-tetrahydropteroyltri-l-glutamate, a representative form of 5-methyltetrahydrofolate, was significantly different in the ΔmetH mutant .An untargeted mass/charge . This, in turn, suggested an association between Met biosynthesis and peptidoglycan biosynthesis. Disruption of metB led to a decrease in intracellular concentrations of UDP-N-acetylmuraminate (∼0.4-fold) and a large increase of UDP-N-acetylmuramoyl-l-alanine (12-fold), UDP-N-acetylmuramoyl-l-alanyl-d-glutamate (1400-fold), and UDP-N-acetylmuramoyl-l-alanyl-d-γ-glutamyl-meso-2,6-diaminopimelate (4-fold) (D). A decrease in UDP-N-acetylmuraminate and an increase in UDP-N-acetylmuramoyl-l-alanine (9-fold), UDP-N-acetylmuramoyl-l-alanyl-d-glutamate (1000-fold), and UDP-N-acetylmuramoyl-l-alanyl-d-γ-glutamyl-meso-2,6-diaminopimelate (5-fold) was also observed in the double mutant ΔmetNIQΔmetB (D).Interestingly, several lutamate D. This, (4-fold) D. A decrNIQΔmetB D.Ex vivo, the ΔmetB mutant had a reduced growth rate compared with the WT in bile salts. To study the impact of the altered levels of peptidoglycan-related metabolites, the ΔmetB mutant and WT were tested in LB broth in the presence of EDTA, SDS, lysozyme, and fasted state–simulated intestinal fluid, but no significant differences in growth were found (Table S1). A small increase (∼2 mm on 15 mm) in β-lactam antibiotic sensitivity, using a validated disc sensitivity method, was observed for the ΔmetB mutant (data not shown). In addition, when the ΔmetB and WT were tested in MacConkey broth supplemented with 0.6% bile salt, a small but consistent delay in the growth of ΔmetB was observed .S. enterica enzymes are dispensable and that essential pathways are often protected against random mutation by redundancy, reflecting the selective pressure placed on metabolism as a key virulence trait , suggesting that transport systems can compensate for the loss of de novo biosynthesis. Met biosynthetic mutants require the presence of Met in the culture medium in order to grow inside of HeLa cells , suggesting that extracellular Met becomes available to bacteria in the SCV within a few hours of infection. It is likely that Met is relatively abundant in the SCV during in vivo infection, as all of the Met biosynthetic mutants were as virulent as WT S. Typhimurium in mice (E and F), presumably because the Met acquisition systems have access to a sufficient source of Met for supporting bacterial growth in the infected animal.We showed that de novo biosynthesis and high-affinity transport of Met are both disrupted that we observed severe growth attenuation in HeLa cells (C) and, importantly, in infected mice (D and E). This result strongly suggests functional redundancy between transport and biosynthesis as a source of Met for S. Typhimurium inside of the host. Whether Met is acquired (e.g. ΔmetB) or synthesized (e.g. ΔmetNIQ and ΔmetNIQΔmetB complemented with metB), the growth in vivo appears to be comparable, suggesting that either de novo biosynthesis or the high-affinity transport system of Met alone can provide Met in excess to what is required for S. Typhimurium to grow at maximal capacity in the host. Hence, at least in the case of S. Typhimurium, it is probably quite difficult to inhibit growth by targeting the Met biosynthetic pathway. The observation that ΔmetNIQΔmetB and ΔmetNIQΔmetEH were still able to grow in mice is suggestive of a Met acquisition system independent of MetNIQ, which is consistent with the presence of a low-affinity transporter MetP , which may put more pressure on the mutant to grow in the competitive environment. It is not uncommon to have divergent results from single infections and competitive infections . This analysis revalidated the pathway model for Met biosynthesis shown in S. enterica until now.To generate novel insights about the Met biosynthesis pathway in by LC-MS  and 5. Mi.e. ΔmetNIQΔmetEH) leads to a profound difference in 5-methyltetrahydrofolate concentration, a metabolite in the one-carbon cycle .The observed accumulation of substrates provides interesting insights into the kinetics of enzyme activities. The deletion of both Met synthases . Enterobacteriaceae peptidoglycan usually consists of alternating molecules of GlcNAc and N-acetylmuramic acid that are linked by a tetrapeptide of l-alanine, d-glutamate, diaminopimelic acid (DAP), and d-alanine. Because Met biosynthesis and m-DAP biosynthesis are linked through aspartate metabolism (l-cystathionine can substitute for DAP (e.g. β-lactam antibiotics) that act through peptidoglycan synthesis or directly on peptidoglycan with the exception of growth in bile. Bile is known to remodel S. enterica peptidoglycan  overnight, and 100 μl of the overnight culture was subcultured into 10 ml of fresh LB broth and grown with shaking (180 rpm) at 37 °C for 3–4 h until the optical density reading at 600 nm reached 0.6–0.8. Growth phenotypes were characterized in LB broth or M9 minimal medium . Met or vitamin B12 was supplemented at 100 μm. For assessing growth under anaerobic conditions, cultures were grown shaking in air-tight jars with AnaeroGen (ThermoFisher) for depletion of oxygen.The bacterial strains and plasmids used in this study are listed in eviously ; its virmet biosynthesis genes metA, metB, metC, metE, metF, and metH and Met high-affinity transporter metNIQ were generated in S. Typhimurium SL1344. The biosynthetic mutant and high-affinity transporter mutant were combined together to generate the double mutant ΔmetNIQΔmetB and the triple mutant ΔmetNIQΔmetEH. The double mutant ΔmetNIQΔmetB was complemented by introducing the de novo biosynthetic metB gene into ΔmetNIQΔmetB strain in trans. This strain is named ΔmetNIQΔmetB pACYC184metB. Gene deletions and concomitant insertions of an antibiotic resistance cassette were constructed using a Lambda Red-mediated “gene gorging” method .Defined deletions of sequence encoding the ” method . All con” method , 56. Priml-GlutaMAX (Life Technologies), in a humidified 37 °C, 5% CO2 incubator. One day before infection, HeLa cells were seeded in 24-well plates at 2 × 105 cells/well. S. Typhimurium strains were grown to mid-exponential phase before and were frozen in LB with 10% glycerol at −80 °C. Bacteria were thawed immediately before infection, washed in antibiotic-free tissue culture medium, and diluted in DMEM with or without l-Met and then added to HeLa cell monolayers at a multiplicity of infection (MOI) of 5–10. The cfu in the inoculum was estimated by plating on LB agar plates. Infected HeLa cells were centrifuged at 600 × g for 5 min immediately after the addition of bacteria and then incubated for 1 h at 37 °C. After 1 h, the tissue culture medium was replaced with DMEM with or without Met and containing 100 μg/ml gentamicin to kill extracellular bacteria. The concentration of gentamicin was reduced to 10 μg/ml at 2 h post-infection and maintained for the remainder of the experiment. To enumerate intracellular bacteria, cells were washed twice with PBS and lysed with 1% Triton X-100 (Sigma) for 15 min to release the intracellular bacteria. The bacteria were enumerated by plating appropriate dilutions on LB agar plates.Infection of HeLa cells (sourced from American Type Culture Collection (ATCC)) was conducted using established gentamicin protection assay protocols , 58. BriAll animal research conducted in this study was approved by the Animal Ethics Committee  at the University of Melbourne, under project number 1413141. All experiments were conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes, 8th edition, 2013.7 cfu of S. Typhimurium strains. To prepare the inoculum, all strains of S. Typhimurium were grown shaking at 180 rpm in M9 minimal medium supplemented with 100 μm Met, at 37 °C for 24 h, and stored in 10% glycerol at −80 °C until use. Immediately before infection, the stored aliquots were thawed and diluted in PBS to the required concentration. At designated time points post-infection, the spleen and liver were removed aseptically, homogenated using the Stomacher 80 Biomaster paddle blender (Seward), and serial dilutions were plated on LB agar plates with streptomycin to determine the bacterial load.Age- and sex-matched C57BL/6 mice were used between 6 and 8 weeks of age to assess the virulence of mutant strains, using either the intravenous or oral route of infection. For intravenous infections, 200 cfu of each bacterial strain was prepared in 200 μl of PBS and injected into the lateral tail vein. For oral infections, mice were orally gavaged with 100 μl of 10% sodium bicarbonate immediately before oral gavage of 200 μl containing ∼5 × 1013C15N-Aspartate (Cambridge Bioscience) was used as an internal standard at 1 μm in Milli-Q water. All stock solutions were stored at −20 °C.Stock solutions of the related underivatized metabolites were prepared at 1.0 mg/ml in an appropriate solvent. Before using, the solutions were combined and diluted with water to give an appropriate standard metabolite mix solution. S. Typhimurium strain was grown at 37 °C to mid-exponential phase in 10 ml of LB, with shaking at 180 rpm. The culture was diluted into 30 ml of PBS and chilled in an ice/water slurry for 5 min and subsequently centrifuged . The pellet was then washed in 1 ml of PBS and centrifuged . This washing step was repeated before removing the PBS and storing the cell pellet at −80 °C until metabolite extraction was performed.Each g, 5 min, 1 °C). The metabolic extract was transferred to a new tube and stored at −80 °C until analysis.Cell pellets were resuspended with 400 μl of 75% ethanol solution containing 1:1000 of internal standard. Cell lysis was ensured by repeated cycles of freeze-thaw, for a total of 10 times, while cooling to −80 °C. Cellular debris was pelleted by centrifugation  coupled to a 1260 series HPLC system (Agilent) was used to separate metabolites. The method used was described previously . Data were analyzed using MAVEN in parallel to validate the LC-MS results (p value < 0.05 with Benjamini correction.LC-MS.d files were converted to .mzXML files using MS convert and analyzed using the LCMS R package , 61. Fol results . FollowiA. U. H. and R. A. S. conceptualization; A. U. H., N. W., S. A. C., H. J. N., J. J. T., M. J. M., and R. A. S. formal analysis; A. U. H., N. W., S. A. C., D. M. H., J. J. W., T. A. S., M. R. D., and J. C. H. investigation; A. U. H., N. W., S. A. C., H. J. N., D. M. H., J. J. W., T. A. S., M. R. D., J. C. H., M. J. M., and R. A. S. methodology; A. U. H. and S. A. C. writing-original draft; N. W., H. J. N., M. J. M., and R. A. S. supervision; N. W. and J. J. T. visualization; N. W., H. J. N., T. L., M. J. M., and R. A. S. writing-review and editing; T. L., M. J. M., and R. A. S. funding acquisition.


Caenorhabditis elegans. While RNA-sequencing (RNA-seq) data contain information on such events, properly documented methods to extract them are lacking.The spliceosomal transfer of a short spliced leader (SL) RNA to an independent pre-mRNA molecule is called SL trans-splicing and is widespread in the nematode SL-quant, a fast and flexible pipeline that adapts to paired-end and single-end RNA-seq data and accurately quantifies SL trans-splicing events. It is designed to work downstream of read mapping and uses the reads left unmapped as primary input. Briefly, the SL sequences are identified with high specificity and are trimmed from the input reads, which are then remapped on the reference genome and quantified at the nucleotide position level (SL trans-splice sites) or at the gene level.To address this, we developed SL-quant completes within 10 minutes on a basic desktop computer for typical C. elegans RNA-seq datasets and can be applied to other species as well. Validating the method, the SL trans-splice sites identified display the expected consensus sequence, and the results of the gene-level quantification are predictive of the gene position within operons. We also compared SL-quant to a recently published SL-containing read identification strategy that was found to be more sensitive but less specific than SL-quant. Both methods are implemented as a bash script available under the MIT license [ Caenorhabditis elegans nematode where there are two classes of SL, SL1 and SL2, which trans-splice about 70% of the mRNA transcripts. Strikingly, the SL2 trans-splicing is highly specific for genes in position two and over within operons that range from two to eight genes expressed from a single promoter  option with the following enhancement:The input reads, if strand specific, are aligned to the SL sequences only (not their reverse complement).With paired-end data in single-end mode, only the left-most unmapped reads are considered as input.With paired-end data in paired-end mode, only the left-most unmapped reads whose mates are mapped are considered as input.The method developed in  has a hiSL-quant), with almost no compromise on sensitivity regarding SL trans-splice site detection  from the SL-quant output as a predictor of gene positions in operons, receiver operating characteristic curve analysis reveals a high TP rate (>90%) at a 5% false discovery rate threshold, regardless of SL-quant options  on the modENCODE_4594, modENCODE_4705, modENCODE_4206 [C. elegans, C. briggsae, C. brenneri, and C. remanei reference genome and annotation (WS262) were downloaded from wormbase [T. brucei reference genome and annotation (Apr_2005 version) were downloaded from Ensembl [SL-quant and at the end of the pipeline were performed using HISAT2 [trimmomatic [Samtools [picard [bedtools [cutadapt (v 1.14) [FeatureCounts [Bedtools [C. elegans genome for 50-nt segments. Sequence logo were made with weblogo [We ran ODE_4206 , SRR2832ODE_4206 , SRR4404ODE_4206 , SRR0387ODE_4206 , and SRRODE_4206  poly-A +wormbase . The T. g HISAT2  (v 2.0.5mmomatic  v 0.36)Samtools  (v 1.5), [picard  (v 2.9),bedtools  was usedbedtools , 34 with(v 1.14)  was usedreCounts  was used weblogo . FinallySamtools Project name: SL-quanthttps://github.com/cyaguesa/SL-quantProject home page: Operating system(s): UNIX-based systems Programming language: Shell, ROther requirements: The BLAST+ suite (2.6.0 or higher), samtools (1.5 or higher), picard-tools (2.9.0 or higher), featureCounts from the subread package. (1.5.0 or higher), bedtools (2.26.0 or higher), cutadapt (1.14 or higher), hisat2 (2.0.5 or higher). Installation instruction for those requirements is provided online.License: MITRRID:SCR_016205GIGA-D-18-00139_.pdfClick here for additional data file.GIGA-D-18-00139_Revision_1.pdfClick here for additional data file.Response_to_Reviewer_Comments_Original_Submission.pdfClick here for additional data file.Reviewer_1_Report_ -- Wenfeng Qian, PhD5/20/2018 ReviewedClick here for additional data file.Reviewer_1_Report_(Revision_1) -- Wenfeng Qian, PhD6/8/2018 ReviewedClick here for additional data file.Reviewer_2_Report_ -- Serghei Mangul05/22/2018 ReviewedClick here for additional data file.Reviewer_2_Report_(Revision_1) -- Serghei Mangul6/16/2018 ReviewedClick here for additional data file.Reviewer_3_Report_ -- Ben Langmead22 May 2018 ReviewedClick here for additional data file.Reviewer_3_Report_(Revision_1) -- Ben Langmead6/12/2018 ReviewedClick here for additional data file.Supplemental FigureClick here for additional data file.


The combination therapy would be ideal for heroin abuse treatment as the effects of two therapeutic agents targeting two independent mechanisms are cooperative.Heroin is a growing national crisis in America. There is an increasing frequency of heroin overdoses. All of the currently used therapeutic approaches to treatment of heroin abuse and other opioid drugs of abuse focus on antagonizing a brain receptor (particularly µ-opiate receptors). However, it has been known that the therapeutic use of certain µ-opiate receptor antagonist may actually increase heroin overdose. Once overdosed, heroin addicts may continue to get overdosed again and again until fatal. Here we report our design and validation of a novel therapeutic strategy targeting heroin activation based on our analysis of the chemical transformation and functional change of heroin in the body. An effective blocker of heroin activation, such as ethopropazine tested in this study, may be used as a standalone therapy or in combination with a currently available, traditional medications targeting µ-opiate receptors ( The connection between heroin abuse and prescription opioid abuse is related to the actual availability, in addition to the common brain protein target (µ-opiate receptors), of these opioid drugs. In fact, “80% of recent heroin initiates reported that they began their opioid use through the nonmedical use of prescription opioid medications”2. The prevalence of prescription opioid abuse is similar among men and women. Those who abuse the prescription drugs most often obtain them from friends and family either through sharing or theft. When they are no longer able to get prescription opioid drugs, they start to use illegal opioid heroin, because heroin is easy to obtain on the street and even online. In addition, heroin has become much cheaper than any other drug of abuse, e.g. $10–$20 for a typical single dose (0.1 g) of heroin purchased on the street2. Due to the connection between heroin abuse and prescription opioid abuse, further enhancing law enforcement through improving existing prescription drug monitoring programs3 might effectively reduce the prescription opioid abuse. On the flip side, the reduced prescription opioid abuse could lead to increase of the heroin abuse. As a result, the overall opioid overdose deaths might not really decrease. For example, study showed prescription drug abuse declining in Kentucky4, due to the strict Kentucky All Schedule Prescription Electronic Reporting Program (KASPER). Unfortunately, the heroin overdose has dramatically increased4, and the total number of drug overdose deaths actually went up in Kentucky5. So, further enhancing law enforcement through improving prescription drug monitoring programs should be associated with development of a more effective therapeutic strategy for heroin abuse.Heroin is known as a growing national crisis in America, due to the rapidly increasing overdose deaths. From 2001 to 2014, heroin overdose deaths nationwide increased 594 percent and continued to dramatically increase since 2014, according to the Centers for Disease Control  in the brain and, thus, block the physiological effects of heroin or another opioid used. The overdose treatment with naloxone appears to be effective, but the precondition is that the naloxone treatment can begin soon enough after a heroin overdose. For the problem, once overdosed, heroin addicts may continue to get overdosed again and again until fatal. Some heroin addicts survived from one overdose with treatment in hospital, and then died of another overdose the next day2. Even worse, the use of naltrexone or its extended-release formulation Vivitrol actually increased heroin overdose10. A truly effective heroin treatment should account for not only rescuing heroin addicts who have already been overdosed, but also preventing the addicts from overdose again.Currently used therapeutic agents for treatment of heroin abuse and other opioid drugs of abuse include naloxone for overdose treatment and buprenorphine, methadone, and naltrexone for addiction treatment. These therapeutic agents may be used in various formulations/devices, such as nasal spray device for naloxonee.g. naltrexone or its extended-release formulation Vivitrol). The combination therapy would be ideal for heroin abuse treatment as the effects of two therapeutic agents targeting two independent mechanisms are perfectly cooperative.Here we report our mechanism-based rational design and validation of a novel therapeutic strategy targeting heroin activation to complement the currently available antagonist approach. Our strategy is based on the fact that heroin itself is actually a prodrug which must be activated to be effective, and our further analysis of the chemical transformation and functional change of heroin in the body. An effective blocker of heroin activation may be used as a standalone therapy or in combination with one of the currently available traditional medications targeting µ-opiate receptors  first, and then to morphine  which has the highest binding affinity with the µ-opiate receptors. 6-MAM  is mainly responsible for the acute toxicity17, whereas morphine  is mainly responsible for the long-term toxicity, of heroin. So, cholinesterases are crucial enzymes that control the metabolic profile, and thus affect the toxic and physiological effects, of heroin.It has been known that heroin  behaves as a prodrug and its effects are mediated by its metabolites 23, we performed the Michaelis-Menten kinetic analysis of the BChE-catalyzed hydrolysis of heroin at 37 °C. The kinetic data , which is qualitatively consistent with the conclusion obtained by Lockridge et al.13. According to reported pharmacokinetic studies17, heroin is converted to 6-MAM with a very high metabolic rate in blood (with a rate constant for heroin hydrolysis to 6-MAM as high as 12.092 min−1), and the estimated metabolic rate of heroin in brain was much lower. The very different metabolic rates for heroin in blood and brain calculated by the model were confirmed by in vitro experiments18, which also supports the notion that BChE (which is the dominant cholinesterase in serum) is the primary enzyme responsible for heroin activation. Further, heroin is either eliminated very rapidly from the body  or metabolized rapidly to 6-MAM in plasma, according to the well-established pharmacokinetic (PK) model (the rate constant is 13.757 min−1 for direct heroin elimination and 12.092 min−1 for heroin hydrolysis to 6-MAM)18. Therefore, we propose to target heroin activation by using a BChE-selective inhibitor in order to attenuate the toxic and physiological effects of heroin. When the metabolic pathway of heroin by BChE is blocked, heroin will mainly go through the direct elimination process to urine. Hence, blocking BChE-catalyzed hydrolysis of heroin should help to significantly attenuate the actual toxicity and physiological effects of heroin.There are two main cholinesterases, including acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Which cholinesterase (AChE or BChE) is mainly responsible for heroin hydrolysis has been controversial. It was once proposed that human erythrocyte AChE is responsible for hydrolyzing heroinata Fig.  reveal tetc. Most of these cholinesterase inhibitors are able to inhibit both AChE and BChE, without the desirable high selectivity for BChE over AChE. Nevertheless, ethopropazine (a BChE inhibitor approved by the FDA for treatment of Parkinson’s disease) is highly selective for BChE over AChE, with IC50 = 210–300 nM for BChE and IC50 = 210 μM for AChE (~1000-fold selectivity)24. So, ethopropazine was used to examine whether a BChE-selective inhibitor can really attenuate the toxic and physiological effects of heroin in mice. First, we determined the effects of ethopropazine on heroin-induced hyperactivity by performing locomotor activity tests in high density, non-porous plastic chambers measuring 50 cm (L) × 50 cm (W) × 38 cm (H) in a light- and sound-attenuating behavioral test enclosure . As seen in Fig. Notably, many cholinesterase inhibitors have been approved by the FDA as therapeutic agents for treatment of Alzheimer’s disease (AD), myasthenia gravis, glaucoma, and Parkinson’s disease 100) of heroin . At 10 min after IV administration of ethopropazine or saline (negative control), the mice were given a lethal dose (LD100) of heroin. The protection experiments were performed under various dose conditions (n = 6 for each scenario). Depicted in Fig. Ethopropazine was tested further for its effectiveness in protecting mice against heroin-induced acute toxicity. Heroin-induced acute toxicity was characterized by the lethality (occurrence of death). For the protection experiments, a single dose of ethopropazine was administrated (IV) prior to administration of a lethal dose  can also attenuate heroin-induced toxicity, and found that pre-treatment of mice with Galantamine at a dose of 10 or 5 mg/kg (IV or IP) before the heroin injection did not protect any mouse against the acute toxicity of a lethal dose of heroin , which is consistent with the concept that BChE, rather than AChE, is the primary heroin-activating enzyme.Additional data in Fig. All of the animal data obtained consistently support the concept of using a potent BChE-selective inhibitor, such as ethopropazine tested in this study, as a blocker of heroin activation to attenuate the toxicity and physiological effects of heroin. For this reason, ethopropazine may be repurposed for clinical use to attenuate the acute toxicity of heroin.26. The cooperative effects of the two therapeutic agents should be much more effective. For example, if a heroin activation blocker with a given concentration in plasma can block heroin activation by 90% and if the μ-opiate receptor antagonist with a given concentration in the brain can block the receptors binding with the available active heroin metabolites  also by 90%, then only 10% of the 10%  can bind with μ-opiate receptors. Thus, the two types of therapeutic agents together can block the toxicity and physiological effects of heroin by 99%, when each one can block the toxicity and physiological effects of heroin by 90%. For the same logics, the two types of therapeutic agents together can block the toxicity and physiological effects of heroin by 75%, when each one can block them by 50%; the two types of therapeutic agents together can block the toxicity and physiological effects of heroin by 99.99%, when each one can block them by 99%. So, the novel combination therapy is potentially more promising for heroin addiction treatment and for prevention of heroin overdose. In addition, the general concept of a combination therapy with two therapeutic agents  targeting two independent mechanisms may also be useful for therapeutic treatment of other drugs of abuse.Further, although a heroin activation blocker, such as ethopropazine which we have identified in this study for the first time, may be used as a standalone therapy for heroin addiction treatment, the combined use of a heroin activation blocker and a μ-opiate receptor antagonist should be more effective. This is because the two mechanisms are independent and, therefore, their effects can be perfectly cooperative (Bliss Independence model)27. Briefly, the BChE protein was expressed in CHO-S cells, and the secreted enzyme in the culture medium was purified by a two-step approach, including ion exchange chromatography using QFF anion exchanger and affinity chromatography using procainamide-sepharose. The purified protein was stored at −80 °C before the use. Heroin and its metabolite 6-MAM were provided by the National Institute on Drug Abuse (NIDA) Drug Supply Program . All other supplies  were purchased from Sigma-Aldrich .The purified recombinant human BChE protein was prepared in our previous studyoC. Experiments were performed in a same colony room in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health. The animal protocol was approved by the IACUC  at the University of Kentucky.Male CD-1 mice (28–32 g) were ordered from Harlan , and housed in cage. All mice were allowed ad libitum access to food and water and maintained on a 12 h light/12 h dark cycle, with the lights on at 8:00 a.m. at a room temperature of 21–22 g. The resulting supernatant was subjected to reverse-phase HPLC (RP-HPLC) on a 5 µm C18 110 Å column  and RP-HPLC was performed using the mobile phase consisting of 20% acetonitrile in 0.1% TFA. The remaining substrate (heroin) and the formed reaction product (6-MAM) were monitored by a fluorescence detector with an excitation wavelength of 230 nm and emission wavelength of 315 nm and by monitoring UV absorbance at 230 nm. The quantification was based on a standard curve prepared using an authentic standard compound. The measurement was performed in triplicate, and the catalytic parameters were determined by using the Michaelis-Menten kinetic equation.For kinetic analysis of the catalytic activity of BChE against heroin, the purified enzyme and heroin were incubated in 0.1 M phosphate buffer, pH 7.4, at 37 °C. The enzymatic reaction was terminated and the protein was precipitated by adding 100 µl of iced 50% acetonitrile/0.5 M hydrochloric acid, followed by 5 min centrifugation at 15,000 e.g., 2.5 mg/kg, IP). After the heroin administration, mice were immediately returned to the test chamber for a 60-minute session of activity monitoring.To determine the effects of ethopropazine on heroin-induced hyperactivity, locomotor activity tests were performed in high density, non-porous plastic chambers measuring 50 cm (L) × 50 cm (W) × 38 cm (H) in a light- and sound-attenuating behavioral test enclosure . This system available in our lab can test 8 mice at the same time. Cumulative distance traveled and speed were recorded by EthoVision XT video tracking system  to represent the locomotor activity. Mice were introduced to the test chambers for one habituation session (no injection). Before the test session, mice were allowed to acclimate to the test chambers for at least 60 minutes, and the total distance traveled during this period of time were determine the basal activity. Then, ethopropazine or saline (negative control) was administered through intravenous (IV) injection. At 10 min after the ethopropazine or saline injection, the mice were given a pharmacological dose of heroin  and/or naltrexone (0.3 mg/kg) 10 min before administration of heroin . Following the heroin administration, mice were immediately placed in containers for observation. The presence or absence of death was recorded for 6 hours following heroin administration.