The notion of whether people focus on the past, present or future, and how it shapes their behavior is known as Time Perspective. Fundamental to the work of two of its earliest proponents, Zimbardo and Boyd , was the Past-Positive—a tendency to reflect on past events with a sentimental attitude; Past-Negative—a focus on the recollection of negative experiences, including the negative reconstruction of mundane events; Present-Hedonism—attraction toward immediate pleasure, including risk-taking and sensation seeking, with little concern for future consequences; Present-Fatalism—a belief that the future is predestined and humans have no influence on future outcomes; and, Future—forward thinking, and goal- setting, while often neglecting present enjoyment. Zimbardo and Boyd , moderately high level of past-positive (3.67) and future orientations (3.69), and low levels of past-negative (2.1) and present-fatalistic (1.67) scores. Zimbardo and Boyd  was originally proposed as a theoretical concept  and assessed the three operationalizations previously used to measure the construct. Of the three methods tested, they reported the best predictor of subjective well-being was the DBTP. That is, rather than measure balance—which categorizes individuals—their study demonstrated that measuring deviation from BTP was (a) the best predictor of subjective well-being, and (b) made more sense to measure how ill-balanced one was rather than how balanced.To date, different methods for measuring balance have been applied .In light of the reported evidence linking temporal balance with well-being indicators, the current study aimed to progress our understanding of BTP in older individuals by: (a) testing the relationship between DBTP and planning behavior, and (b) exploring the association between DBTP and well-being. Consistent with Drake et al.  and Bonin = 85). Fully retired individuals made up 62.2% (n = 79), and 37.8% (n = 48) were partially retired. Relationship status was reported as 65.4% (n = 83) married, 14.2% (n = 18) de facto relationship, 5.5% (n = 7) widowed, 11.8% (n = 15) divorced or separated, and 3.1% (n = 4) were single. In terms of educational achievement, 11.8% (n = 15) reported they had not completed high school, 6.3% (n = 8) completed high school, 7.9% (n = 10) graduate diploma, 10.2% (n = 13) advanced diploma, 25.2% (n = 32) Bachelor's degree, 37% (n = 47) post-graduate degree, and 1.6% (n = 2) reported “other.”One hundred and twenty-seven self-nominated retirees were recruited, primarily through My Longevity, a web-based provider of information about age-related risk factors and tips on boosting life expectancy. Participants were also sought from the general public through invitation pamphlets distributed at various council community centers. The age range of participants was 52–91 years. Men represented 66.9% of the sample ; Past-Positive (α = 0.80); Present-Hedonistic (α = 0.79); Present-Fatalistic (α = 0.74); and Future (α = 0.77). Each item is on a five-point Likert scale ranging from 1 (very uncharacteristic) to 5 (very characteristic). Higher scores reflecting a stronger orientation toward that particular item's TP.The 56-item ZTPI is a self-report measure consisting of five subscales designed to identify an individual's TP: illbalanced one is. This involved subtracting each empirical (e) TP from the ideal (i) and squaring it, thus eliminating negative values. The derived values for each of the five TPs were then summed, and the square root was taken (see Equation 1). This method calculated the total distance a participant deviated from zero—the balanced ideal. Calculations were performed using Microsoft Excel (2013).The method for calculating DBTP in this study was that proposed by Zhang et al. . DeviatiThe 28-item RPQII was used to measure the amount of effort participants invested in retirement planning. The measure was developed to assess a range of behaviors relevant to retirement including accessing services offered through government agencies, financial, health, social and other non-financial planning. Internal reliability in original study was α = 0.86. Participants were assessed on their effort contributed toward retirement planning on a scale of 1  to 5 (very large amount of effort). Scores for each participant were obtained by summing the ratings on all items and averaging; higher scores indicated greater effort invested on retirement planning.Least Applicable to (5) Most Applicable. Examples of items include “Happy,” “Excited,” and “Anxious.” Scores were calculated separately for positive mood and negative mood, ranging from 5 to 25. Higher scores reflected stronger positive or negative mood. The original study had good internal reliability for both positive (α = 0.70) and negative (α = 0.81) measures  s Field, .did not apply to me at all” to (3) “applied to me very much, or most of the time.” Ratings within each subscale were summed, and then multiplied by 2, in order to obtain scores for each subscale. Possible scores range from 0 to 34, depending on the number of items each subscale has. Higher scores indicated greater level of stress, anxiety or depression.The DASS-21 is a psychometric tool that has been widely administered, especially in clinical settings. It is a shorter version of the original DASS, and its psychometric properties were established in a study by Antony et al. . ParticiThe data collected were screened for assumptions of normality, and missing data, before conducting any analysis.We report the results of sub-scale TP scores from our sample to compare with the normative sample from Zimbardo and Boyd  on whichThe sample means from this study were; past-negative 2.44, past-positive 3.89, present-fatalism 2.15, present hedonism 3.11, and future 3.76. Normative sample means reported by Zimbardo and Boyd  were pasOur focus was on the balanced profile from which the TP subscale scores were based; we used only DBTP scores. As reported by Zhang et al. , DBTP waSD below the mean; that is, a low level of DBTP  was indicated by a score of ≤ 1.23. In accord with previous studies reporting 5–23% of participants were balanced , thus confirming our first hypothesis.In the current study, for the purpose of comparing our sample with other samples in the literature, well-balanced individuals were identified as having a DBTP score ≥ 1 n = 79) were fully retired, a large proportion of the sample were married or in a de facto relationship , and a large proportion were educated at tertiary level . All statistical analyses were conducted using the software program, IBM Corp , and correlation coefficients for all investigated variables, are presented in Table IBM Corp . As showF = 21.26, p < 0.001, and positive affect, F = 40.35, p < 0.001 (H2). Also, as anticipated, DBTP was positively related with depression, F = 46.10, p < 0.001, anxiety, F = 19.74, p < 0.001, stress F = 13.69, p < 0.001, and negative affect F = 31.65, p < 0.001 (H3).Therefore, we tested whether DBTP accounted for additional unique variance in retirement planning, depression, anxiety, stress, positive mood, and negative mood by conducting a series of multiple regression analyses. Demographic variables, age, gender, and relationship status (single or in a relationship) were controlled for in all the models by entering them in Step 1. DBTP scores were entered in step 2. As reported in Table Other than a small demographic contribution to the variance in the anxiety and positive mood models, there was no demographic contribution to retirement planning, depression, stress, or negative mood. DBTP scores contributed to a significant portion of additional variance in all of the outcome variables. Therefore, our hypothesis regarding additional unique variance in retirement planning, DASS subscales, and positive and negative mood was supported (H4). The evidence strengthens the validity of DBTP as a predictor of planning and subjective well-being.The aim of the present study was to: (a) investigate consistency in the prevalence of BTP, as defined by Zimbardo and Boyd , and (b)Our hypotheses regarding the relationship between DBTP and all of the outcome variables were confirmed. That is, DBTP explained additional unique variance in retirement planning, depression, anxiety, stress, positive and negative mood, beyond demographic characteristics. As a result, DBTP was significantly related to planning and well-being. Similar to the prevalence of balanced individuals reported in previous studies, 14.2% met the balanced profile criteria.actioned in terms of planning behavior, rather than what they plan to do. Thus, we could reliably conclude that having a healthy time balance allows one the ability to alternate between different temporal views of their life, as necessary, in order to put measures in place to ensure they promote or maintain their well-being during their retirement ; Stolarski et al.  excessive deviations from negative orientations are assessed the same way as excessive deviations from positive orientations, despite affective differences; and (2) the contribution of time perspectives is not even in this model, with only one future time perspective compared to two past, and two present perspectives. As a way to further validate DBTP, future research should consider investigating the negative effects of scoring extremely high on positive dimensions, or extremely low on negative dimensions. Despite the positive face value of these extremes, it would be worth investigating whether deviations in these directions hold true to the assumptions of poor functioning associated with ill-balance.As mentioned, previous studies have advanced the theoretical concept of BTP and operationalized it in ways that categorize individuals as either “balanced” or “not balanced” due to their methodological approaches. A different methodological perspective, deviation from the balanced ideal  - application number 2487.AM completed the study as part of an Honours in Psychology submission. JE supervised this project and secured funding. CM assisted with the development of the formula and application of the deviation from balance time perspective to create the datafile. HB helped secure original funding and reviewed manuscripts.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.


Acute rhinosinusitis (ARS) is a respiratory disease commonly caused by viral infections. Physicians regularly prescribe antibiotics despite bacterial etiologies being uncommon. This is of concern, as this use adds to the selection pressure for resistance. Here we present the descriptive epidemiology of acute rhinosinusitis and corresponding antibiotic prescribing practices by Canadian outpatient physicians from 2007–2013.Diagnosis and antibiotic prescription data for ARS were extracted from the Canadian Disease and Therapeutic Index for 2007 to 2013, and population data were acquired from Statistics Canada. ARS diagnosis and antibiotic prescription rates and frequencies of antibiotic classes were calculated.Eighty-eight percent of patients diagnosed with ARS in 2013 were adults, with a greater rate of antibiotic prescriptions observed among the adults relative to the pediatric patients . Between 2007 and 2013, the ARS diagnosis rate decreased from 596 to 464 diagnoses per 10,000 inhabitants, while the percentage of diagnoses with antibiotic prescriptions at the national level remained stable (87% to 84%). From 2007 to 2013, prescription rates for macrolides decreased from 203.5 to 105.4 prescriptions per 10,000 inhabitants. In 2013, penicillins with extended spectrum were more commonly prescribed compared to macrolides among adult patients .This study is the first to describe physician antibiotic prescribing practices for treatment of ARS in Canada. Results show that antibiotic treatment for ARS represents an area for implementing antimicrobial stewardship, and through it, managing antibiotic resistance. Further work is required to better understand diagnosing practices and treatment criteria for ARS, and use this information to further assist physicians to limit unnecessary antibiotic prescribing practices. There is global consensus that to address the problem of antibiotic resistance prudent management of antibiotics across all sectors is required. Since 2013, Canadian hospitals seeking accreditation have begun to develop antimicrobial stewardship programs, where antimicrobial therapy is monitored and regulated by a stewardship team .4 colony forming units per mL from bacterial cultures of sinus aspirates is a laboratory criteria for ARS; however, sinus aspirates are an invasive procedure and not routinely recommended in primary care [Acute rhinosinusitis (ARS) is a common respiratory condition seen by primary care health providers, with an incidence ranging from 15 to 40 episodes per 1000 patients per year . Howeverary care ,8. Antibary care ,7. HowevStudies from the United States indicate that there is a high utilization of antibiotics for treating sinus infections with over 80% of ARS patients having been prescribed an antibiotic, despite increasing evidence of their limited benefits –11. PresThe Canadian Disease and Therapeutic Index (CDTI) dataset  provides information about the patterns and treatments of disease encountered by office-based physicians across the country. Office-based physicians include general practice, internal medicine, obstetrics/gynecology, otolaryngology, ophthalmology, psychiatry, neurology, surgery, pediatrics and dermatology. For four consecutive quarters, 652 office-based physicians maintain a practice diary describing information on every patient visit during a randomly selected 48-hour period within that quarter. The participating physicians are provided a stipend for their participation and between 85% to 91% of them participate across all quarters . A two-sIMS Health Canada, Inc. assigns International Classification of Diseases—Ninth Revision System (ICD-9) codes to the diagnostic information provided by physicians. In turn, the Public Health Agency of Canada further classifies the ICD-9 codes into disease-specific classes for further analysis . For exaThe CDTI data were age stratified by IMS Health Canada, Inc. and aggregated for the following age-groups, 0–2 years, 3–9 years, 10–19 years, 20–39 years, 40–59 years, 60–64 years, and 65 years and older. For assessing regional differences, provinces were grouped into three geographical regions. The Eastern region included the provinces of New Brunswick, Newfoundland and Labrador, Nova Scotia, and Prince Edward Island. The Central region had the provinces of Ontario and Québec. British Columbia, Alberta, Saskatchewan and Manitoba represented the Western region.Population size information was acquired from Statistics Canada consisting of the 2011 Canadian census estimates . Both thChanges in the yearly percentage of patients recommended an antibiotic were used to estimate changes in physician behaviour over time. Due to the complexity of the data collection consisting of double-stratification and projection estimate methods, in addition to the small sample size of physicians included in the CDTI dataset, statistical comparisons between regions and over time are inappropriate and not possible; therefore, descriptive data alone is presented in this publication.All data analysed in this study were anonymous. Data processing and visualization were performed using R .Overall a total of 13 million diagnoses of ARS were provided by Canadian physicians between 2007 and 2013. During this time period, the diagnosis rates for ARS decreased from 596 diagnoses per 10,000 inhabitants in 2007 to 464 ARS diagnoses per 10,000 inhabitants in 2013 . Adults During the study period, an overall reduction of 25.0% in antibiotic prescription rates was observed with prescription rates decreasing from 517 antibiotic prescriptions per 10,000 inhabitants to 388 antibiotic prescriptions per 10,000 inhabitants . The perIn 2013, at the national level, the most frequently prescribed antibiotic class for treating ARS switched from macrolides to penicillins with extended spectrum . This swThe antibiotics prescribed for the treatment of ARS displayed regional variation in 2013 . PhysiciTo our knowledge this is the first study that describes the levels of ARS diagnosis provided by office-based physicians in Canada and their associated antibiotic prescription practices for treatment of these conditions. The ability to compare the diagnosis rates and antibiotic prescribing rates allowed us to evaluate changes in prescribing practices by physicians while assessing trends of disease over time. An overall decrease in the diagnosis rates were observed during the study period, with the prescribing rates remaining the same over time. However, the impact of new treatment recommendation and guidelines were observed with the change in the antibiotic of preference provided to patients for treatment.et. al reported that ARS visit rates among American adults between 2000 and 2009 did not change [Streptococcus pneumonia serogroups, which could lead to serogroup replacement. The serogroup replacement effect combined with the predominant viral etiology for ARS points to the small role that pneumococcal vaccination programs might have had on the observed ARS diagnosis rate in our study[While our results demonstrated a decrease of the ARS diagnosis rate between 2007 and 2013 in Canada, Fairlie t change ,17, chilt change ,19. HoweThe decrease in the number of patients presenting with ARS likely contributed to the simultaneous reduction of antibiotic prescriptions. This decline in antibiotic prescriptions for ARS follows the trend of an overall antibiotic use reduction observed in Canada from 1995 to 2010, while remaining relatively constant between 2010 and 2014s ,22. WhilA considerable variation in antibiotic prescribing practices was observed between the Canadian regions. In the Western Region, both the provinces of British Columbia and Alberta implemented in 2005 a community-based antibiotic stewardship program named “Do Bugs Need Drugs?” . An evalFor most of the study period, macrolide drugs were the most commonly recommended group of antibiotics for ARS patients. However, as of 2011, new national recommendations and guidelines for the treatment of ARS were released ,26,27 reOne of the main concerns regarding the inappropriate use of antibiotics is its role in increasing selection pressure for antibiotic resistant pathogens . In lighCertain limitations present in this data need to be considered interpreting the results. First, there was no clinical information available about the presentations categorized as ARS, leaving room for misclassification of the disease. Furthermore, the ICD-9 label for ARS does not differentiate between ABRS and AVRS; as such we were unable to determine which cases were consistent with ABRS presentations where antibiotics would be appropriate and which were likely AVRS. The high prescribing rate observed might suggest physicians believed cases to be ABRS, however, this data set does not allow for this determination. In addition, information on comorbidities or allergies was not available, reducing our ability to determine if the antibiotic selection was driven by other symptoms or diseases present at the time of diagnosis. Second, the prescribing practices of the small sample of physicians may not be representative of all physicians in Canada. However, the sampling strategy used by IMS Health Canada, Inc. in projecting to national levels has been identified to be fairly accurate when compared with other drug expenditures and health care services.As the physicians recorded diagnoses and prescriptions in hard copy, there is the potential that errors occurred during transcription to electronic formats. For example, there is the potential that amoxicillin-clavulanic acid was transcribed inaccurately as amoxicillin alone for some records and vice versa, which could have influenced the proportion of prescriptions reported here for both of these products.This study found that Canadian physicians commonly recommended antibiotics to ARS patients. This suggests antibiotic treatment of ARS continues to represent a target for improving antimicrobial stewardship and managing antibiotic resistance. Further work should seek to clarify the proportion of ARS patients who have appropriate criteria for antibiotics (ABRS) and which are likely viral presentations (AVRS), where antibiotics could be safely withheld. Such information could help physicians limit unnecessary antibiotic use in ARS treatment and decrease the selection pressure for antibiotic resistance.


The most common cause of perihilar obstruction is cholangiocarcinoma, especially in Thailand. Benign perihilar stricture represents less than 20% of cases. IgG4-related disease and IgG4-related sclerosing cholangitis, however, have been receiving increased recognition. Isolated IgG4-related sclerosing cholangitis is less common. The preoperative diagnosis of IgG4-related sclerosing cholangitis without pancreatic involvement is very difficult because the clinical presentation and preoperative evaluation are extremely difficult to distinguish from perihilar cholangiocarcinoma.We report the case of a 56-year-old man who presented with obstructive jaundice with preoperative imaging showing proximal common bile duct obstruction. He underwent right lobe liver hepatectomy with extrahepatic bile duct resection and regional lymph node dissection due to high suspicion of malignancy. The pathological report showed severe acute and chronic inflammation of the bile duct with morphology and immunohistochemistry suggestive of IgG4-related sclerosing cholangitis.IgG4-related sclerosing cholangitis with perihilar obstruction should be considered even in areas where cholangiocarcinoma is endemic. Thailand has the highest incidence of cholangiocarcinoma (CCA) worldwide, especially in the northeast area . MalignaA 56-year-old Asian male patient from northeast, Thailand with an unremarkable past-medical history was referred from a primary care center with painless obstructive jaundice for two weeks. He was previously admitted to a private hospital, and abdominal computed tomography (CT) was performed. He underwent endoscopic retrograde cholangiopancreatography (ERCP) and trans-ampullary biopsy and brush cytology. A plastic biliary stent was inserted across the obstruction point. At the time of the initial visit to the out-patient clinic, physical examination revealed a mildly obese patient, generally co-operative, with marked icteric sclera and pruritus. No acute distress was noted. There were no clinical signs of an abdominal mass or palpable supraclavicular lymph node.At the time of referral, laboratory analysis showed the following liver function test results: total bilirubin 16.0 mg/dL, direct bilirubin 11.7 mg/dL, alkaline phosphatase (ALP) 319 U/L , gamma glutamyl transferase (GGT) 33 U/L , aspartate aminotransferase (AST) 59 U/L , alanine aminotransferase (ALT) 55 U/L , white blood cell count 12,040/cumm, platelet count 464,000/cumm, hemoglobin 11.5 g/L, CEA 1.9 ng/ml (0–4.6 ng/mL), CA 19–9 19.8 U/mL , normal serum electrolytes, normal renal function. HBsAg, anti-HCV and anti-HAV were all negative.Transabdominal ultrasonography was performed at a private hospital and showed dilatation of both intrahepatic bile ducts. The common bile duct was not dilated. No gallstone was found. Abdominal CT showed a focal short segment, sheet-like, symmetric thickening of the common hepatic duct causing nearly complete obstruction just below the bifurcation of the right and left intrahepatic bile ducts and dilatation of the left intrahepatic duct Fig. . At privAt the time of referral, chest and mediastinum CT were performed, and the results showed no pulmonary metastasis or new lesions, along with a decreased degree of intrahepatic bile duct dilatation. Magnatic resonance imaging (MRI) and magnetic resonance cholangiopancreatography was not done in this patient. Ultimately, surgical resection was planned after adequate biliary drainage due to high suspicion of CCA.At surgery, the abdomen was opened by inverted J skin incision. There was no metastatic disease. There was a hard palpable lesion at the common bile duct along the common hepatic duct Fig. . ExtendeThe pathological examination of the surgical specimen revealed severe acute and chronic inflammation with dense lymphoplasmacytic infiltration, storiform fibrosis and obliterative phlebitis of the common hepatic duct and right and left hepatic ducts, with all margins inflamed  as shown in Fig. Postoperatively, the patient suffered from intra-abdominal infection from hepatico-jejunostomy anastomosis leakage that required re-laparotomy and repair of the anastomosis and abdominal toilet. After the re-laparotomy, he recovered uneventfully. The serum IgG4 level obtained after surgery was above 7230 mg/dL. At 6 months follow-up, he had recovered well and maintained normal activity with no recurrence of the disease. His laboratory analysis results were normal, and abdominal CT showed no other biliary stricture. There is no further treatment after discussing with gastroenterologist.th edition, the extrahepatic bile duct is divided into the perihilar  and distal common bile duct [The most common cause of perihilar biliary obstruction is perihilar CCA (Klatskin tumors) , especiaile duct . The incile duct –13. One ile duct . IgG4-SCile duct . IgG4-SCile duct . Zen et ile duct .The diagnosis of IgG4-SC is crucial. Because IgG4-SC lacks any highly specific diagnostic features, a high index of suspicion in all patients with unexplained biliary strictures can lead to the most accurate diagnosis. Some authors have proposed criteria for the diagnosis of IgG4-SC. Firstly, Ghazale et al. from the Mayo group proposed the worldwide acceptance of the HISORt criteria for IgG4-SC, adopted from previously reported criteria for AIP including histology of the bile duct, imaging of the bile duct, serology, other organ involvement and response to steroid therapy , 18. RecSerum IgG4 level is an important part of the diagnosis of IgG4-SC. However, an increased serum IgG4 level alone should not be used to diagnose of IgG4-SC. Ghazale et al. report the sensitivity of serum IgG4 to be 74% . Oseini Cross-sectional study, CT and MRI have proven difficult to use for differentiation from CCA –27. IgG4Recently, Chen L et al. have reported the accuracy rate, sensitivity, specificity, PPV and NPV of intraductal ultrasound (IDUS) with tissue sampling in proximal bile duct obstruction to be above 90% . The chaIsolated IgG4-SC without AIP is uncommon. Despite aggressive preoperative evaluation, especially in the center that have limitation of resource, it is very difficult to differentiate IgG4-SC without AIP from CCA. Preoperative histology in biliary stricture remains controversial. Davidson et al. concluded that there is currently no evidence to support routine histological examination for the diagnosis of CCA . But hisIn conclusion, surgeons should be aware of IgG4-RD as one of the causes of benign hilar and proximal bile duct stricture and consider this possibility preoperatively. The serum IgG4 level and associated other organ involvement should be investigated. However, the clinical presentation and imaging study are extremely difficult to distinguish from malignancy. A high index of suspicion and consideration of diagnostic criteria are important. The accurate diagnosis of IgG4-SC is important to avoid major surgical resection in patients with perihilar biliary obstruction. More invasive studies such as ERCP with IDUS and EUS show promising results in preoperative diagnosis.


IgG4-related disease (IgG4-RD) is a nosological entity defined as a chronic immune-mediated fibro-inflammatory condition characterized by a tendency to form tumefactive, tissue-destructive lesions or by organ failure. Urologic involvement in IgG4-RD has been described in some short series of patients and in isolated case reports, most often involving the kidneys in so-called IgG4-related kidney disease (IgG4-RKD). The disease can occasionally mimic malignancies and is at risk of being misdiagnosed due to its rarity. We report the case of a 56-year-old man presenting with a right renal mass suspected of being malignant. Laboratory tests showed normal creatinine levels, a high erythrocyte sedimentation rate, and high levels of C-reactive protein and microalbuminuria. The patient underwent radical right nephroureterectomy and histopathologic examination revealed features proving IgG4-RKD. He was therefore referred to immunologists. Typical clinical presentation of IgG4-RKD includes altered renal function with inconstant or no radiologic findings. Conversely, in the case we presented, a single nodule was detected upon imaging evaluation, thus mimicking malignancy. This raises the issue of a proper differential diagnosis. A multidisciplinary approach can be useful, although in clinical practice the selection of patients suspected of having IgG4-RKD is critical in the cases presenting with a renal mass that mimics malignancy. IgG4-related disease (IgG4-RD) is a recent nosological entity defined as a chronic immune-mediated fibro-inflammatory condition  characteIgG4-related kidney disease (IgG4-RKD) is the most common among urologic manifestations of IgG4-RD, usually presenting in the form of tubulointerstitial nephritis (TIN) –9, althoWe report the case of a 56-year-old Caucasian man presenting with a right renal mass (12 × 9 × 8 cm) revealed by CT  and MRI Laboratory tests performed within four months prior to urologic evaluation showed creatinine level of 1.10 mg/dL, a erythrocyte sedimentation rate up to 120 mm/h, C-reactive protein levels ranging from 65 to 70 mg/L, and microalbuminuria levels of 82.5 mg/g. These high levels drove the decision for abdominal imaging. Comorbidities included mellitus diabetes type 2. No further relevant aspects were recorded in the patient's previous history.Due to the suspicion of malignancy, the patient underwent open-surgery radical right nephroureterectomy, which was complicated by firm adhesions between the kidney, its fascia, and surrounding structures so that the posterior renal fascia could not be excised. The postoperative period was uneventful.Histopathologic examination showed storiform fibrosis with lymphoplasmacytic infiltrate made up of B and T cells , as well as focal obliterative phlebitis. The IgG4+/IgG ratio was 70% . Given tThe patient was prescribed 5 mg prednisone daily and 50 mg azathioprine twice daily for the first five months after surgery and experienced dramatic pain improvement. Afterwards, a drug maintenance regimen of 50 mg azathioprine twice daily was prescribed by immunologists. Currently, at 18 months after surgery, the patient is healthy. His creatinine level is 1.8 mg/dL, his erythrocyte sedimentation rate is 17 mm/h, and his C-reactive protein level is 7.71 mg/L.IgG4-related disease (IgG4-RD) is a recent nosological entity defined as a chronic immune-mediated fibro-inflammatory condition  characteIn 1995, the concept of autoimmune pancreatitis (AIP) was proposed by Yoshida et al. ; in 2001As different terms have been proposed, the two independent teams headed by Umehara and Okazaki agreed to define the disease as IgG4-RD . The disMany papers have investigated urologic involvement , with thOur patient presented with normal IgG4 serum levels, sampled one month after surgery.However, his IgG4/IgG ratio upon histopathologic examination was > 40%, which is considered a diagnostic criterion of IgG4-RKD .IgG4-RKD is the most common among urologic manifestations of IgG4-RD, usually presenting in the form of TIN –9, althoIn 2011, Kawano et al. described a series of 41 patients identified between 2004 and 2011 in Japanese hospitals presenting with histopathologic findings consistent with IgG4-RKD . CT was It has been reported that conventional imaging—including ultrasound scans, CT, and MRI—has proven to be of limited usefulness in determining IgG4-RD .Over the past decades, the management of overall renal masses has evolved from radical surgery to minimally invasive approaches, or even active surveillance in selected cases. Thus, the use of renal biopsy has shown an increase, although with a lack of standardization. Consequently, the identification of patients who may benefit from renal biopsy still remains an individualized clinical decision . NeverthIn our case, prior knowledge of the proper diagnosis could have directed the management towards medical therapy, thereby avoiding surgery at least as the first option . That said, a smaller renal mass would definitely have been easier to manage for both diagnosis and treatment, because retrospective analysis could not establish whether our patient might have been an adequate responder to medical therapy due to the mass volume.Concerning urologic sites, kidney is the organ most frequently involved in IgG4-RD.However, the disease is rare and can be difficult to diagnose. Its typical clinical presentation includes altered renal function, with inconstant radiologic findings. In the case presented here, a single solid nodule was detected upon imaging evaluation, thus mimicking malignancy. This raises the issue of a proper preliminary differential diagnosis, which is not always easy. Conventional imaging—including ultrasound scans, CT, and MRI—has proven to be of limited usefulness in determining IgG4-RD. A multidisciplinary approach can be useful, although in clinical practice the selection of patients suspected of having IgG4-RKD is critical in cases presenting as a renal mass, thus mimicking malignancy.


The number of refugees arriving in Europe increased dramatically in 2015, challenging the German health system. Amongst others, the treatment of infectious diseases is an important topic in refugee healthcare. A high prevalence of multi-drug-resistant organisms has been identified among the refugee population. Still, little is known about the prescription of antibiotic medication for refugees. We conducted a descriptive analysis of all antibiotics prescribed to newly arrived refugees who were treated as outpatients between 10/01/2014 and 09/30/2015 in Erlangen, an average sized German town. The City’s invoicing documents were used to collect data on prescriptions written for refugees. Basic penicillins, aminopenicillins with beta-lactamase inhibitor and cephalosporins constituted the largest proportion of antibiotics prescribed in the adult refugee group. Of these, both aminopenicillins with beta-lactamase-inhibitor as well as basic penicillins were prescribed significantly more often compared to non-refugees. We conclude that the high percentage of prescriptions of aminopenicillins with beta-lactamase inhibitor is striking and should be further investigated. Staphylococcus aureus (MRSA) [Since the beginning of the refugee crisis in 2015, medical care for refugees has posed a challenge to European healthcare systems, including the German health system. Amongst many other health problems these patients present with, infectious diseases are salient [s (MRSA) –6. Littls (MRSA) , but theFor a better comparison with literature reporting on antibiotic prescriptions in Germany, we calculated the defined daily dose (DDD)  of each We analysed the antibiotic prescribing pattern of two groups which, due to their size, allowed a meaningful analysis: Persons from 0 to 14 years, in the following referred to as “children group”, and persons from 20 to 39 years, in the following referred to as “adult group”.While we did not find major differences in the children group (0–14 years) compared to children insured by the German statutory health insurance , as our results , cephalosporins (13%) and macrolides (10%) (see Table 1%, cephaIn comparison to GKV-insured patients, refugees received broad spectrum antibiotics and aminopenicillins with BLI more often. Bätzing-Feigenbaum et al.  found a Unfortunately, we can only report on a small number of antibiotic prescriptions. It was also not possible to gain information about the diagnoses leading to the prescriptions. However, we offer the first insight into the antibiotic prescription pattern of refugees in Germany.Due to the fact, that the prevalence for MRSA in refugees is not only higher compared to the German population , but exc


PIK3CD, FGFR3, TSC2 and RASGRP2 contribute to greater oncogenic potential compared with corresponding European American (EA)-expressing variants. Ectopic overexpression of the newly cloned AA-enriched variant, PIK3CD-S, in EA PCa cell lines enhances AKT/mTOR signalling and increases proliferative and invasive capacity in vitro and confers resistance to selective PI3Kδ inhibitor, CAL-101 , in mouse xenograft models. High PIK3CD-S expression in PCa specimens associates with poor survival. These results highlight the potential of RNA splice variants to serve as novel biomarkers and molecular targets for developmental therapeutics in aggressive PCa.Clinical challenges exist in reducing prostate cancer (PCa) disparities. The RNA splicing landscape of PCa across racial populations has not been fully explored as a potential molecular mechanism contributing to race-related tumour aggressiveness. Here, we identify novel genome-wide, race-specific RNA splicing events as critical drivers of PCa aggressiveness and therapeutic resistance in African American (AA) men. AA-enriched splice variants of  In prostate cancer tumour aggressiveness can be related to race. Here, the authors identify an alternative RNA splice variant of PIK3CD as a potential mechanism to explain racial disparities in the incidence and aggressiveness of prostate cancer between African American and European American men. Prostate cancer (PCa) is the most commonly diagnosed cancer and the second leading cause of cancer death among American men236789BCL2L1) pre-mRNA is alternatively spliced into two variants, Bcl-xS and Bcl-xL, encoding protein isoforms with opposite biological effectsFGFR2) pre-mRNA also undergoes AS, where FGFR2-IIIb is predominately expressed in epithelial cells and FGFR2-IIIc is primarily associated with epithelial-to-mesenchymal transition of PCa cellsTMPRSS2-ERG gene fusion commonly found in PCa and associated with poor clinical outcome21TMPRSS2-ERG variants ectopically overexpressed in prostatic epithelial cells, variants containing a 72 base exon (+72) mediate increased cell proliferation and invasionAR-V7 is overexpressed in hormone-refractory PCa, being correlated with poor patient survival and higher recurrence rates24Alternative splicing (AS) is a post-transcriptional process allowing for the generation of alternative mRNA transcripts that encode structurally and functionally disparate protein isoforms. Next-generation sequencing suggests that >90% of human genes undergo AS121314FGFR2-IIIc, TMPRSS2-ERG+72, AR-V7 and/or other as yet undiscovered variants associated with more aggressive PCa might be predominantly or selectively expressed in AA PCa, thus contributing to PCa disparities. In addition, it is unknown whether differences in mRNA splicing along racial/population lines occur in only a limited number of genes or more globally across the transcriptome. If the latter, it will be important to ascertain whether these genome-wide, differential splicing (DS) events are overrepresented within specific gene ontologies . Lastly, assessment of the functional consequences of any race-specific (or enriched) splicing events will provide critical further insight into the genetic/molecular mechanisms underlying PCa disparities. To this end, we have applied a functional genomics approach to address these questions. Our results underscore the leveraging of population differences in tumour biology to discover novel splice variants that will likely serve as novel biomarkers and/or molecular targets for developmental therapeutics against aggressive AA PCa, identify previously hidden splice variants encoding oncogenic signalling proteins resistant to small-molecule inhibitors (SMIs), and assimilate splice variant information for prognostication of cancer aggressiveness and/or therapeutic responsiveness.Despite the significance of alternative/aberrant splicing in PCa progression irrespective of race, the occurrence of race-specific/-enriched PCa splicing events and a causal relationship between these events and observed PCa disparities remains unexplored. For example, it is unclear whether P>0.05, Fisher’s exact test). In AA PCa versus EA PCa and AA NP versus EA NP, the significant differentially expressed exons  and 35 patient-matched normal prostate (NP) specimens (20 AA/15 EA) derived from chemo-/hormone-/radiation-naive patients were interrogated using the Affymetrix Human Exon 1.0 ST GeneChip to assess DS events. Gleason scores of PCa specimens (range 6–8) and patient ages range 49–81 years) were not significantly different between AA and EA cohorts , vascular endothelial growth factor (VEGF), phosphatase and tensin homolog (PTEN), phosphatidylinositol-3-kinase (PI3K)/AKT, extracellular signal–regulated kinase/mitogen-activated protein kinase (ERK/MAPK) and nuclear factor-κB (NF-κB) signalling  and short  variants, whereas EA PCa samples predominately expressed PIK3CD-L. Hence, the RT-PCR results were in agreement with the exon array data, indicating the presence of an AA-enriched PIK3CD-S variant. Analogous findings were obtained where either a short or long variant of each gene was confirmed by RT-PCR to be enriched or uniquely expressed in AA  or EA PCa  (n=22–25 AA and n=21–24 EA PCa specimens). Exon array data also revealed two alternative RASGRP2 transcripts with apparent mutually exclusive exon skipping events (RASGRP2-b variant (exon 11 excluded) was exclusively expressed in AA PCa, while a RASGRP2-a variant (exon 12 excluded) was enriched in EA PCa  identified by exon arrays as not undergoing DS were confirmed by RT-PCR. In summary, there was strong agreement  between exon array and RT-PCR results, thus providing an internal quality metric to our global DS analysis of AA and EA PCa .We proceeded to validate a subset of both proto-oncogenes and tumour suppressor genes with DS events in our composite cancer signalling pathway, including  RASGRP2 . Real-ti RASGRP2 . As showg events . RT-PCR n EA PCa . We werePIK3CD variants was particularly interesting owing to recent findings implicating PI3Kδ (p110δ) kinase activity in haematologic malignancies as well as other cancer types303132P>0.05), quantitative RT-PCR validation was performed reaffirming significantly higher levels of PIK3CD-S relative to PIK3CD-L in AA versus EA PCa specimens  (genome.ucsc.edu) or Ensembl Genome Browser (www.ensembl.org). Consequently, we cloned the full-length versions of PIK3CD-S from AA PCa cell line MDA PCa 2b, and PIK3CD-L from MDA PCa 2b as well as EA PCa cell lines VCaP and LNCaP using standard molecular approaches (5′- and 3′-RACE (rapid amplification of cDNA ends)PIK3CD-S and PIK3CD-L variants from PCa patient specimens. PIK3CD-L  along with three different AA PIK3CD-S variants  and one AA large deletion variant of the PIK3CD gene. Interestingly, exclusion of exon 8 eliminates a 30-amino acid segment situated between the Ras-binding and C2 domains, while exclusion of exon 20 deletes a 56-amino acid segment located in the catalytic domain of PI3Kδ. In subsequent functional studies involving ectopic overexpression of the short variant (see below), we concentrated our efforts on the variant missing exon 20 given the possibility that kinase activity may be affected.The AA-enriched PIK3CD-L and PIK3CD-S, respectively, in EA and AA PCa cell lines and examined the functional consequences of these knockdowns on cell proliferation and invasion. A similar strategy was applied to investigate the biological significance of the variants of FGFR3, TSC2 and RASGRP2. VCaP and MDA PCa 2b cells were used as population-specific PCa models, as these two cell lines represent bone metastases derived from castration-resistant EA and AA PCa patients, respectively3520) successfully knocked down PIK3CD-L expression by >8-fold compared with nonsense siRNA (PIK3CD-L increased the ratio of PIK3CD-S/PIK3CD-L expression by nearly twofold (S/L ratio for nonsense versus 3.46 S/L ratio for siP20-transfected cells), and this ‘enrichment’ of AA-enriched PIK3CD-S subsequently enhanced proliferation and invasion of the AA cell line . Transfection of siPj had no effect on VCaP proliferation and invasion, as expected since this EA line does not significantly express PIK3CD-S  to target se siRNA , left. Cked down , right, ked down , right. siFGFR3-ex14), exon 20 (siTSC2-ex20) and exon 11 (siRASGRP2-ex11) were used to selectively suppress expression of FGFR3-L, TSC2-L and RASGRP2-a variants (predominately expressed in EA), respectively. Upon siRNA-mediated knockdown of FGFR3-L, TSC2-L or RASGRP2-a  to activate downstream signalling components within this pathway. SiRNA (siP20)-mediated knockdown of PIK3K-L expression (confirmed by qRT-PCR) led to a drastic decrease in AKT phosphorylation at Thr308 and Ser473 while moderately decreasing (approximately twofold) phosphorylation of mTOR in EA VCaP cells (PIK3CD-L in AA MDA PCa 2b cells (resulting in an increased PIK3CD-S/-L ratio confirmed by qRT-PCR) led to a sizable increase (two- to threefold) in phosphorylation of AKT, mTOR and ribosomal protein S6 (S6)  signalling pathway, we examined the ability of different PI3Kδ isoforms (encoded by j)-mediated knockdown of PIK3K-S (confirmed by qRT-PCR) in MDA PCa 2b cells resulted in a drastic decrease in the phosphorylation status of AKT, mTOR and S6  induced a significant reduction in basal AKT and S6 phosphorylation (−1) had negligible effects on inhibiting basal AKT signalling in EA PCa cell lines overexpressing PI3Kδ-S, as phosphorylation states of AKT and S6 were comparable to vehicle-treated cells  isoform into the left hind flank of nonobese diabetic–severe combined immunodeficient (NOD-SCID) mice. Mice harbouring PI3Kδ-L-overexpressing or PI3Kδ-S-overexpressing PC-3 cell xenografts were administrated either vehicle  or CAL-101 (50 mg kg−1) by daily intraperitoneal (i.p.) injection. CAL-101 treatment for 30 days significantly reduced the growth of PI3Kδ-L-expressing xenografts compared with the vehicle treatment  via i.p. injection (3 times per week). After 8 weeks, vehicle-treated mice carrying PI3Kδ-L-overexpressing cells developed prominent tumour metastases in the lungs (P<0.05) of metastases (P>0.05). Taken together, the in vitro and in vivo functional studies suggest that SMIs such as CAL-101  in the catalytic domain of the PI3Kδ-S isoform (The consequence of excluding exon 20 (168 bp) in the  isoform . To gainNext, PI3Kδ isoforms were purified from the lysates of PC-3 cells overexpressing either His-tagged PI3Kδ-S or PI3Kδ-L using Ni-NTA resin columns. As shown in 4243in vitro and in vivo results (Purified PI3Kδ isoforms (minus p85α) were incubated with vehicle, nonselective PI3K inhibitor wortmannin (100 nM) results  and 6.AR-V7 splice variant in PCa disparities has been suggested in a recent study. Selective downregulation of miR-212 observed in AA PCa is correlated with upregulation of splicing factor hnRNP-H1, upregulation of AR-V7 and antiandrogen resistance in PCa cell lines2728The phenomenon of DS, much less global DS events, has not been adequately explored as a possible mechanism underlying PCa disparities. Potential involvement of the constitutively active PIK3CD-S, FGFR2-S, FGFR3-S, TSC2-S, RASGRP2-b, ATM-S and GSK3-S  in AA PCa compared with EA PCa9PIK3CD-L, FGFR3-L and TSC2-L) detected in EA PCa, each conferred a less aggressive oncogenic phenotype compared with the corresponding in-frame variants detected in AA PCa .Studies on AS indicate that approximately half of such events occurring in the coding sequence are in-frame, while the remaining events are frameshifts leading to truncated or extended C-terminal proteins48d GSK3-S . In compd GSK3-S , while tresolved . Presumade novo events (occurring as NP evolved into PCa). Accordingly, the AA-enriched/-specific variants already present in NP specimens have the potential to serve as inherent ‘at-risk alleles’ for poor PCa prognosis in AAs. In comparison, the de novo appearance of tumour-specific variants may drive poorer outcomes. PIK3CD-S would be an example of a potential AA ‘at-risk allele’ contributing to increased PCa aggressiveness upon disease presentation. Indeed, ectopic overexpression of the AA-enriched PIK3CD-S in PCa cell lines was demonstrated to enhance oncogenic potential  compared with the corresponding EA-enriched PIK3CD-L. Moreover, genetic manipulation of AA MDA PCa 2b cells to favour expression of the -S variant over the -L variant likewise increased oncogenic behaviour. Conversely, genetic manipulation in the opposite direction decreased oncogenic behaviour. Interestingly, survival plots generated from The Cancer Genome Atlas (TCGA) RNA-sequencing data demonstrate that a high S/L ratio is associated with significantly worse survival for PCa and trending for worse survival in both breast and colon cancer  were identified that may be associated with poor PCa prognosis in AAs. Further experimentation is needed to investigate whether these variants can serve as novel biomarkers to address PCa disparities. In contrast to the ‘at-risk alleles’, AA-enriched variants FGFR-S and TSC2-S were detected in AA PCa, but not in patient-matched NP specimens. The appearance of these de novo variants during PCa formation may contribute to driving the more aggressive PCa phenotype observed in the AA population, since in vitro genetic manipulation favoring expression of these -S variants over the -L variants promoted oncogenesis in MDA PCa 2b cells. It is noteworthy that three PCa-associated splice variants identified in previous studies, Bcl-xL, FGFR2-IIIc and TMPRSS2-ERG+72  confers resistance to enzalutamide and abiraterone acetate in castration-resistant PCa patientsBRAF splice variants encoding protein isoforms that are missing the RAS-binding domain exhibited resistance to the RAF inhibitor vemurafenibPIK3CD-S imparts PCa cell lines with significant resistance to SMIs targeting PI3Kδ, as demonstrated in both in vitro assays and preclinical mouse models of PCa. This short variant is missing exon 20, encoding a 56-amino acid segment that is present in PIK3CD-L. Amino acids residing in the exon 20-encoded cassette appear critical for the docking of CAL-101 and wortmannin. Indeed, molecular modelling studies predict that Glu826 and Val828 (missing in PI3Kδ-S) undergo hydrogen bonding with CAL-101  have been identified that abrogate the inhibitory action of p85α on PI3Kα in cancers64P85 regulatory subunits are known binding partners of class I PI3Ks, resulting in protein stabilization and suppression of basal kinase activity62PIK3CD-S. The identification of novel splice variants as biomarkers and/or development of therapeutics targeting protein isoforms have the potential to reduce cancer disparities.The identification and functional validation of global AS in cancer pathogenesis remains challenging and largely unexplored. We have undertaken such an analysis in the context of race-related aggressive PCa and identified a large number of DS events in cancer-associated pathways in EA and AA PCa, with a subset of these events also being detected in patient-matched NP specimens. These events will have both biological and clinical consequences, case in point 20, 5′-CCAACAUCCAACUCAACAA-3′; siPj, 5′UGAGGGAGGCCCUGGAUCGA-3′; siF, 5′-CUCGACUACUACAAGAAGA-3′; siTSC2-ex20, CUGCGCUAUAAAGUGCUCA-3′; siRASGRP2-ex11, 5′-CCACAUCUCACAGGAAGAA-3′. siPIK3R1 Smart Pool .EA PCa cell lines LNCaP (CRL-1740), VCaP (CRL-2876) and PC-3 (CRL-1435), and AA PCa cell line MDA PCa 2b (CRL-2422) were obtained from the American Type Tissue Collection , authenticated at the ATCC by short tandem repeat profiling of multiple unique genetic loci, and tested negative for mycoplasma. Primer sequences for RT-PCR are provided in t-test, P>0.05) between the two racial groups with respect to age  and Gleason score . No distant metastasis was detected in the enrolled patients.Prostate biopsy samples were collected at the George Washington University Medical Faculty Associates according to an institutional review board-approved protocol (IRB no. 020867). Informed consent was obtained from all study participants. High-quality PCa and patient-matched normal prostate (NP) biopsy cores from each of 20 AA and 15 EA primary PCa patients were collected and processed for the exon array analysis. PCa cores were determined by a pathologist to have Gleason scores of 6–7 (17 AA and 13 EA) or 8–9 (3 AA and 2 EA), while NP cores were diagnosed negative for cancer. There were no significant differences . Detection of differential expression at the gene level  was performed in Partek using the One-Step Tukey’s Biweight algorithm for detection of outlier probe-sets. Statistical analysis of exon expression data was based on ANOVA with multiple-correction testing using 10% false discovery rate (FDR)Total RNA was purified from PCa and patient-matched NP biopsy cores using the RNeasy micro kit as per manufacturer’s protocol . Briefly, total RNA samples were extracted using Trizol reagent, then treated with DNase I and further purified by RNeasy MinElute spin column. High-quality RNA isolation was confirmed by using the Agilent Bioanalyzer as per the manufacturer’s protocol . For exon array analysis, 1 μg of purified RNA sample from each biopsy core was interrogated with the Affymetrix Human Exon 1.0 ST GeneChip . Exon microarray data can be assessed at GEO (Gene Expression Omnibus) using accession number GSE64331. The exon array raw data were subjected to quantile normalization, GC-content adjustment, RMA background correction and logEIF1AX and PPA1, using the ΔΔCt approach9QRT-PCR was performed using the 7300 Real-Time PCR System  to validate and quantify AS events. Primers were designed to amplify the flanking regions of skipped exons or the junctions across catenated exons of variant mRNAs . AmplifiPIK3CD-L and PIK3CD-S transcript variants from purified RNA of PC-3, VCaP and MDA PCa 2b cells . PC-3 and VCaP cells were maintained in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum (FBS), while MDA PCa 2b cells were grown in BRFF-HPC-1 medium  supplemented with 20% FBS. All the cell lines were grown at 37 °C and 5% CO2. Primers were designed according to the National Center for Biotechnology Information (NCBI) reference sequences of PIK3CD mRNA (NM_005026.3). The forward primer contained the start codon (bold) (5′-ATGCCCCCTGGGGTGGACT-3′) and the reverse primer was upstream of the stop codon (5′-CTGCCTGTTGTCTTTGGACA-3′). Full-length PCR products were ligated into pcDNA3.1/V5-His TOPO vector  using the manufacturer’s protocol. A total of 8–10 independent clones were selected for each of the amplified PIK3CD-L and PIK3CD-S variants and sequence verified. The consensus sequences of PIK3CD-S and PIK3CD-L mRNAs were deposited to GeneBank (accession number KU612116 and KU612117). The plasmids pcDNA3.1-PIK3CD-L/V5-His and pcDNA3.1-PIK3CD-S/V5-His were individually transfected into the PCa cell lines (VCaP and PC-3) using the cationic lipid-mediated methodPIK3CD-L or PIK3CD-S.RT-PCR was performed to amplify PIK3CD, FGFR3, TSC2 or RASGRP2 using DharmaFECT4 transfection reagent (Dharmacon), according to the manufacturer’s protocol. The in vitro functional assays, including cell proliferation and invasion, were performed following siRNA transfections for 24 h. Cell proliferation and invasion assays were performed using 5-bromodeoxyuridine Cell Proliferation Assay kit  and the Matrigel Invasion Chambers , respectively, as per the manufacturers’ protocol9VCaP and MDA PCa 2b cells were grown in DMEM with 10% FBS for 24 h, and then were transfected for 24 h with siRNAs (50 nM) designed to target splice variants of Tyr308, pAKTSer473, AKT, pmTOR, mTOR, pS6 and S6 , rabbit polyclonal antibodies for His-tag , HA-tag, PI3Kδ, p85α and β-actin . Horseradish peroxidase-conjugated secondary antibodies for rabbit and mouse IgG were purchased from Southern Biotech .Antibodies used in western blot analysis were rabbit monoclonal antibodies for pAKT6 PC-3 cells stably overexpressing PIK3CD-L or PIK3CD-S were subcutaneously injected into the left flank of NOD-SCID mice. Tumour xenograft growth was measured with calipers and the volume was determined as 1/2 × length × width2. Mice were randomized into groups once the average tumour size reached ∼200 mm3 and treated with vehicle  or CAL-101 (50 mg kg−1) through daily i.p. injections. After 30 days, mice were euthanized and the dissected xenografts were photographed and weighed using a blinded design.All animal work was approved by the George Washington University institutional animal care and use committee (protocol A272). Male NOD-SCID mice, 4–6 weeks old, were purchased from the Jackson Laboratory . To establish a PCa xenograft model, 2 × 106 PC-3 cells stably overexpressing PIK3CD-L or PIK3CD-S were injected into the tail vein of NOD-SCID mice. The mice were then treated with vehicle or CAL-101 (50 mg kg−1) via i.p. injections, 3 times a week. After 8 weeks, lungs of mice were collected and stained with India ink and bleached with Fekete’s solution . India ink-stained lungs were photographed and lung metastases were quantified using the NIH ImageJ programTo establish the PCa metastasis model, 1 × 10PIK3CD-L or PIK3CD-S were maintained in DMEM (Life Technologies) supplemented with 10% FBS. After growing the cells for 24 h, cell extracts were prepared and His-tagged PI3Kδ protein was purified using a column HisPur Ni-NTA purification kit . Briefly, cell lysates were mixed with Ni-NTA resin and incubated at room temperature for 30 min. After incubation, the resin was washed with wash buffer  and applied to a HisPur Ni-NTA spin column, centrifuged and wash buffer eluate discarded after centrifugations. His-tagged proteins were eluted from the resin by adding one-resin-bed volume of elution buffer . The purified PI3Kδ-His protein was mixed with 2 × Laemmli sample buffer, boiled and analysed by immunoblotting.PC-3 cells stably overexpressing Plasmids pcDNA3.1-PIK3CD-S/V5-His (or pcDNA3.1-PIK3CD-L/V5-His) and pSV-p85α  were co-transfected into PC-3 cells. After growing the cells for 48 h, the co-transfected cells were collected and cells were lysed with RIPA lysis buffer (Santa Cruz Biotechnology). The cell lysates were then subjected to Co-IP assays with anti-His antibody  and immobilized on protein G-Sepharose beads . Cell lysates and precipitates were subjected to western blotting, and visualized by enhanced chemiluminescence system .−1) at room temperature for 1 h. After incubation, plates were washed three times and incubated with 100 μl of TMB  substrate solution at room temperature for 5–20 min. Reactions were stopped by adding 100 μl of stop solution and plates read at 450 nm. The colourimetric signal was inversely proportional to the amount of PIP3 produced by PI3K activity and the relative amount of PIP3 produced was determined with a standard curve.PI3Kδ activity was evaluated with a PI3K activity/inhibitor assay kit  according to the manufacturer’s instructions. Briefly, purified His-tagged PI3Kδ-L or PI3Kδ-S isoform was pretreated with the PI3Kδ inhibitor (100 nM of wortmannin or 100 nM of CAL-101) or vehicle in 96-well plates for 10 min and subjected to a competitive ELISA. PIP2 substrate and kinase reaction buffer were added to the pretreated His-tagged PI3Kδ-L or PI3Kδ-S isoform and incubated at room temperature for 1 h. After incubation, biotinylated PIP3 and GST-GRP1 working solutions were added to the wells and the reaction samples were further incubated at room temperature for 1 h. Plates were washed three times with 1 × Tris-buffered saline with Tween-20  and incubated with streptavidin-horseradish peroxidase conjugate .A total of 4,253 significant differentially expressed exons (2% FDR) were identified. Using a 1.5-fold cutoff, the number of differentially expressed exons  was narrowed down to 3,112 . The reference coordinates for the Affymetrix probe sets used to identify these skipped exons were cross-referenced with the Ensembl database, release GRCh37, resulting in final set of 2,517 well-curated, differentially expressed exons that corresponded to 1,484 DS genes . Based oValidation success rate. Eight of 9 genes (89%) identified by Exon GeneChip analysis to undergo differential RNA splicing between AA PCa and EA PCa were successfully validated by quantitative RT-PCR. The only gene that did not validate was EPHA1. In addition, 2 out of 2 genes (100%), defined not to exhibit differential RNA splicing (GSK3A and ATM), were successfully validated by quantitative RT-PCR. Aggregate success rate was 91% (10/11).P values. The P values ranged from 1 × 10−3 to 1 × 10−15 for the 9 genes identified by Exon GeneChip analysis to undergo differential RNA splicing between AA and EA PCa specimens (P=6 × 10−4 for ATM). In comparison, P values for all genes identified by Exon GeneChip analysis to exhibit differential RNA splicing ranged from 3 × 10−3 to 1 × 10−20  and a computed s.d.=0.4, our findings correspond to >85% power to distinguish 1.5-fold changes at P<0.01.Affymetrix Exon GeneChip analysis has revealed ∼2,500 differential splicing events between AA and EA PCa. While we can only draw firm conclusions on a subset of differential splicing patterns that were validated by a second approach , the following metrics have been provided to allow evaluation of the overall reliability of Exon Genechip results:PIK3CD was determined using the method IsoformExhttps://tcga-data.nci.nih.gov/tcga/, accessed 22 January 2016). The ratio of short to long isoforms was calculated for survival analysis.Expression of the short and long isoforms of https://tcga-data.nci.nih.gov/tcga/, accessed 22 January 2016). Patients who did not have a relapse event during the study were considered as censored. The expression values of the short and long isoforms as well as the interaction term were used as predictors to fit the Cox proportional hazards regression model under L2-regularization, where the disease-free survival is the response variable. For each patient, a prognosis index score was computed from the Cox proportional hazards modelP value was then calculated to assess the statistically significant difference between the Kaplan–Meier curves of the high- versus low-risk groups.Disease-free survival data for BRCA, COAD and PRAD were obtained from TCGA clinical data (PIK3CD-S and PIK3CD-L were deposited to GenBank (accession numbers KU612116 and KU612117).The authors declare that all data supporting the findings of this study are available within the article and its How to cite this article: Wang, B.-D. et al. Alternative splicing promotes tumour aggressiveness and drug resistance in African American prostate cancer. Nat. Commun.8, 15921 doi: 10.1038/ncomms15921 (2017).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Escherichia coli (EHEC) ever reported. Four years thereafter, we systematically searched for scientific publications in PubMed and MEDPILOT relating to this outbreak in order to assess the pattern of respective research activities and to assess the main findings and recommendations in the field of public health. Following PRISMA guidelines, we selected 133 publications, half of which were published within 17 months after outbreak onset. Clinical medicine was covered by 71, microbiology by 60, epidemiology by 46, outbreak reporting by 11, and food safety by 9 papers. Those on the last three topics drew conclusions on methods in surveillance, diagnosis, and outbreak investigation, on resources in public health, as well as on inter-agency collaboration, and public communication. Although the outbreak primarily affected Germany, most publications were conducted by multinational cooperations. Our findings document how soon and in which fields research was conducted with respect to this outbreak.In 2011, Germany experienced one of the largest outbreaks of entero-hemorrhagic  Escherichia coli (EHEC) ever reported, almost 3,000 people fell ill with acute gastroenteritis, 855 of them developed hemolytic uremic syndrome (HUS). In total, 55 people died due to the infection , the German federal public health institute, together with local and state health and food safety agencies, conducted a total of 13 epidemiological field investigations, using different study designs. Initial investigations pointed at lettuce, raw tomatoes, and cucumbers as potential sources of the infection. On June 10—3 weeks after the first notification of the outbreak—epidemiological evidence supported that fenugreek sprouts, produced in Germany from seeds imported from Egypt, were the vehicle causing the outbreak  guidelines , we searBy applying the search criteria, we found 346 publications in PubMed and 304 in MEDPILOT; 214 articles (32.9%) were duplicates, resulting in a total of 436 unique publications written in Chinese, Dutch, English, French, German, Polish, Russian, Spanish, or Swedish. Of these, 255 publications were excluded based on content criteria, 47 for being editorials, commentaries, replies, diaries, or meeting abstracts, and five because of being submitted or published before May 1, 2011, leaving 129 (29.6%) after full-text screening. We identified an additional 11 publications on scrutinizing reference lists of eligible reviews, of which four articles fulfilled our eligibility criteria. In total, 133 eligible publications  were included in the systematic review  contributed to the study design, interpretation of the data, and to the writing and revision of this paper. All authors agree to be accountable for the content of the work.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.


Hepatocyte growth factor receptor  is an essential member of the receptor tyrosine kinase (RTK) family that is often dysregulated during tumor progression, driving a malignant phenotypic state and modulating important cellular functions including tumor growth, invasion, metastasis, and angiogenesis, providing a strong rationale for targeting HGF/c-Met signaling axis in cancer therapy. Based on its protumorigenic potentials, we developed IRCR201, a potent antagonistic antibody targeting the plexin-semaphorin-integrin (PSI) domain of c-Met, using synthetic human antibody phage libraries. We characterized and evaluated the biochemical properties and tumor inhibitory effect of IRCR201 in vitro and in vivo. IRCR201 is a novel fully-human bivalent therapeutic antibody that exhibits cross-reactivity against both human and mouse c-Met proteins with high affinity and specificity. IRCR201 displayed low agonist activity and rapidly depleted total c-Met protein via the lysosomal degradation pathway, inhibiting c-Met-dependent downstream activation and attenuating cellular proliferation in various c-Met-expressing cancer cells. In vivo tumor xenograft models also demonstrated the superior tumor inhibitory responsiveness of IRCR201. Taken together, IRCR201 provides a promising therapeutic agent for c-Met-positive cancer patients through suppressing the c-Met signaling pathway and tumor growth. Aberrant activation of c-Met, a receptor tyrosine kinase, is frequently observed in various cancer types via a hepatocyte growth factor (HGF)-dependent or -independent manner ,2,3,4. cHGF/c-Met axis targeting therapeutic antibodies have been developed to prevent molecular interaction between c-Met and its ligand HGF or to inactivate c-Met via protein degradation or shedding (release from the cell surface) ,14. RiloHeterotopic or orthotopic tumor mouse models have been used extensively for the pre-clinical evaluation of human c-Met-specific therapeutic antibodies ,22,23,24In an effort to overcome this potential problem, we developed IRCR201—a novel human and mouse cross-reactive c-Met-targeting antibody. IRCR201 is a bivalent fully-human antibody that binds to the plexin-semaphorin-integrin (PSI) domain of c-Met and exhibits a tumor inhibitory effect by regulating tumor cellular growth, proving to have ideal effects in cancer treatment. IRCR201 also demonstrates low agonistic activity and disrupts c-Met signaling pathway activation induced by both HGF-dependent and independent mechanisms in various cancer types, eliciting distinct molecular traits compared to other c-Met-targeting antibodies. In addition, IRCR201 provides exceptional anti-tumor activity in both gastric cancer and NSCLC xenograft models. Collectively, our results demonstrate that targeting the PSI domain of c-Met provides a promising therapeutic approach, and its human and mouse cross-reactivity allows successful preclinical studies to assess precise therapeutic efficacy, pharmacokinetics (PK), and toxicity profiles, resulting in the further development of IRCR201 as an ideal cancer-targeted therapeutic agent.D of 0.7207 nM, and mouse c-Met ECD-Fc with a KD of 0.8448 nM (IRCR201 is a cross-reactive c-Met-targeting human immunoglobulin G1 (IgG1), and was developed through panning techniques using synthetic human antibody phage libraries. To evaluate whether IRCR201 specifically binds to the c-Met extracellular domain (ECD)-fragment crystallizable region (Fc), we performed enzyme-linked immunosorbent assay (ELISA) using recombinant human c-Met and recepteur d’origine nantais (RON) proteins. The RON receptor tyrosine kinase is a member of the c-Met family, and has similar biochemical and structural properties to c-Met ,31,32. W.8448 nM . However.8448 nM d. Additi.8448 nM b–e. To c.8448 nM f. To ass.8448 nM g.To determine the binding epitope of IRCR201 against c-Met, we performed epitope mapping using the IRCR201 single-chain variable fragment (scFv) format . The binTo expand the structural understanding of the docking of IRCR201 onto c-Met, computational modeling was conducted using a published c-Met structure (PDB accession: 1SHY) . A threeIn the development of c-Met inhibitory antibodies, the bivalent nature of the c-Met antibodies enabled the antibodies to mimic the role of HGF and thus activate the c-Met signaling pathway . BecauseIn vitro functional inhibition activity of IRCR201 was analyzed in various cancer types. IRCR201 did not alter cancer cell growth of the low c-Met-expressing cell line MCF7 a. In U87To elucidate the cellular growth inhibitory mechanism of IRCR201 in c-Met-expressing cancer cell lines, we measured the change in total c-Met protein levels according to antibody treatment time in various cancer cell lines using ELISA. In multiple cancer cell lines, the total c-Met protein level was rapidly attenuated after 15 min from the initial treatment with IRCR201 a–c. FourImmunoblot analysis of HGF-dependent (U87MG) and HGF-independent c-Met-amplified (MKN45) cell lines were conducted to analyze the inhibitory effect of IRCR201 on the c-Met signaling pathway. In both models, IRCR201 efficiently depleted total c-Met levels, and inhibited c-Met phosphorylation and its downstream signaling pathway e. In addIn vivo anti-tumor activity of IRCR201 was observed in an A549 NSCLC subcutaneous model. Our results showed that 3 mg/kg IRCR201 treatment (48.4% tumor inhibition) could potently inhibit tumor growth, while 3 mg/kg huOA5D5.v2 treatment (13.3% tumor inhibition) showed a similar tumor growth rate compared to the PBS-treated group a. In addBased on the inhibitory effects of IRCR201 in vitro and in vivo, the cellular mitotic index was further evaluated through immunohistochemical analysis of Ki-67-positive nuclei in the paraffin sections of MKN45 xenograft models that had been treated with either PBS (vehicle) or 10 mg/kg IRCR201 and harvested after 48 h. Immunohistochemistry (IHC) staining showed that Ki-67 positive cells were significantly reduced in the IRCR201-treated group compared to the PBS-treated group a. AdditiAberrant activation of the HGF/c-Met signaling pathway has been reported in numerous human cancers ,2. In c-IRCR201 does not mimic the role of HGF and exhibits lower agonist activity compared to huOA5D5.v2 as it binds to the SAPPFVQ amino acid sequence of the PSI domain, as distinct from the Sema domain, which is an HGF binding site of c-Met. The PSI domain containing the SAPPFVQ sequence—the IRCR201 epitope—acts as a domain that gives flexibility to c-Met, promoting molecular interaction between c-Met and HGF ,42. AlthIRCR201 possesses a different binding epitope from previously developed c-Met inhibitory antibodies, and rapidly promoted c-Met depletion primarily through the lysosomal degradation pathway, thereby abrogating downstream signaling cascades in U87MG, A549, and MKN45. To further ascertain the effect of IRCR201 at a cell surface receptor level, a flow cytometry assay was configured to detect cell surface c-Met of U87MG, A549, and MKN45 following the antibody treatment. Similar results were observed throughout various cell lines, as the rapid degradation of cell surface c-Met was observed at the 15-min mark. Total receptor expression levels in the membrane were decreased by approximately 40% post IRCR201 treatment. Furthermore, we found that IRCR201 resulted in c-Met co-localization within the lysosome, suggesting an underlying mechanism behind IRCR201-induced c-Met degradation. The c-Met depletion activity of IRCR201 was inhibited by concanamycin A, supporting the notion that IRCR201 deteriorates c-Met through the lysosomal degradation pathway. These findings demonstrate that IRCR201 induces receptor-mediated internalization and lysosomal degradation, providing a potential underlying mechanism behind the loss of total c-Met protein.IRCR201 does not inhibit the interaction between HGF and c-Met, but it exhibited excellent growth inhibition capacity in various types of c-Met-expressing cancer cell lines compared to huOA5D5.v2 in vitro. IRCR201-induced c-Met degradation demonstrated a therapeutic effect mediated by the downregulation of tyrosine kinase activity and the subsequent inhibition of cellular proliferation in U87MG, A549, and MKN45. IRCR201 also showed significant antitumor effect in an A549 NSCLC xenograft tumor model. Since c-Met amplification leads to shorter survival in patients with gastric cancer and NSCLC , we asseThe HGF/c-Met signaling pathway is a pivotal component of tumor-associated angiogenesis that leads to tumor progression and metastasis via a sufficient supply of oxygen and nutrients through blood vessels ,2. ImmunPrevious efforts to develop bivalent c-Met antagonistic antibodies were unsuccessful, as the antibodies incited agonistic effects . ThroughBy impairing both HGF-dependent and c-Met-amplified downstream activation in various cancer types, IRCR201 is differentiated from other therapeutic antibodies by securing a wide range of drug response groups. The anti-cancer activity of IRCR201 may be further enhanced by antibody-mediated cell cytotoxicity (ADCC), as the antibody’s isotype is human IgG1, which binds to the Fc receptors on immune cells. IRCR201 is also applicable to antibody–drug conjugate (ADC) platforms for toxin delivery, as it demonstrated the lysosomal degrading traits of c-Met.This study currently has an accompanying research program to investigate the specific degradation pathway responsible for IRCR201-induced c-Met depletion. IRCR201 is also being used in toxicological studies using mouse models to confirm that its mouse cross-reactivity has an adverse effect on mouse health.In summary, the newly identified traits of IRCR201 allow the evaluation of precise therapeutic efficacy in mouse models. Our comprehensive results show that IRCR201 is capable of inhibiting a variety of cancer types with HGF-dependent c-Met activation or gene amplification-driven constitutive c-Met activation. Taken together, IRCR201 represents a promising therapeutic antibody to treat cancer patients suffering from dysregulation of the HGF/c-Met signaling pathway.Biopanning was performed using phage displayed synthetic human scFv libraries according to basic panning protocol . Brieflyv/v) fetal bovine serum  and 1% (v/v) penicillin/streptomycin . MCF7, MKN45, A549, and Caki1 were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium  containing 10% (v/v) FBS  and 1% (v/v) penicillin/streptomycin . All cells were cultured according to the manufacturer's manual and had been passaged for fewer than three months after thawing. Cell counting was performed with the Scepter™ 2.0 automated cell counter  and c-Met expression was confirmed by Western blot using cell lysates.The MCF7 , U87MG , A549 , and Caki-1  cell lines were obtained from American Type Culture Collection , and the MKN45  cell line was purchased from the Japanese Collection of Research Bioresources Cell Bank . All cell lines were tested for mycoplasma, cross-contamination, and genetic fingerprints. U87MG were cultured in Eagle’s Minimum Essential Medium  containing 10%  were used for the in vivo studies. All animals were obtained from Orient Bio Inc. , and were maintained under specific pathogen-free conditions in facilities approved by the Association for Assessment and Accreditation of Laboratory Animal (AAALAC) International in accordance with the current regulations and guidelines of the Laboratory Animal Research Center (LARC) at the Samsung Medical Center (Project identification code: NTX1161851-20160324002). Mice were acclimated for at least a week before they entered the studies.w/v) skim milk  for 1 h at room temperature. The wells of the plates were washed three times with 1× PBST and then incubated with 100 µL of IRCR201 diluted to the appropriate concentration for 1 h at room temperature. After three washes with 1× PBST, the wells of the plates were incubated with 100 µL of 1:10,000 goat anti-human IgG F(ab’)2 conjugated horseradish peroxidase (HRP)  for 1 h at room temperature, and then again washed three times with 1× PBST. 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate solution  was added to each well, and then reactions were quenched by STOP solution .To analyze the specificity and selectivity of IRCR201, 0.1 µg human c-Met protein  or 0.1 µg human RON protein  was coated in 96-well EIA/RIA plates  at 4 °C overnight. Following three washes with 1× phosphate-buffered saline with Tween 20 , they were blocked with 3%  or 0.1 µg/well mouse c-Met ECD-Fc  was coated in 96-well EIA/RIA plates . The subsequent steps were performed as described above. Since 5D5 was a mouse antibody, goat anti-mouse Fab conjugated HRP  was used as a secondary antibody.To confirm the binding property of IRCR201 to human c-Met domain proteins, 96-well EIA/RIA plates  were immobilized with 100 µL/well of 0.1 µg recombinant human Sema-Fc (protein consists of amino acids 1 to 515 of human c-Met), 0.1 µg recombinant human Sema-PSI-Fc (protein consists of amino acids 1 to 562 of human c-Met), and 0.1 µg recombinant human whole c-Met ECD-Fc . Recombinant human c-Met domain proteins with an Fc tag (rhSema-Fc and rhSema-PSI-Fc) were expressed in an Expi293™ system  and purified from HiTrap™ Mabselect SuRe . Then, 100 µL of IRCR201 (1 µg/mL) was used as the primary antibody, and 100 µL of 1:10,000 goat anti-human IgG F(ab’)2 conjugated HRP  was used as the secondary antibody. The color of the plates was developed using 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution  and the reaction was terminated with STOP solution .® M200 pro .For the HGF/c-Met blocking assay (competitive ELISA), 96-well EIA/RIA plates  were coated with recombinant human c-Met  at 4 °C overnight. After washing the wells with 1× PBST, they were blocked with 3% skim milk for 1 h at room temperature. After pre-binding with/without 2.5 μg/mL HGF  at room temperature for 1 h, IRCR201 and huOA5D5.v2 were incubated at room temperature for 1 h. After three washes with 1× PBST, the wells were incubated with 100 μL of 1:10,000 goat anti-human IgG F(ab’)2 conjugated HRP  for 1 h and then washed three times with 1× PBST. TMB substrate solution  was added to each well and incubated at room temperature until color was developed. Afterwards, reactions were quenched by STOP solution . Optical density (OD) of the wells was measured at 450 nm with an InfiniteD values. To obtain the kinetic and affinity constants of IRCR201 to human and mouse c-Met, Biacore™ T100 evaluation software  was used.The binding affinities and kinetics of IRCR201 for human c-Met and mouse c-Met were measured using a Biacore™ T100 . Human c-Met, mouse c-Met, or BSA were immobilized on a CM5 sensor chip  using an Amine coupling kit . Different concentrations of purified IRCR201 were injected into immobilized human and mouse c-Met to determine KEscherichia coli (E. coli)  and purified from the bacterial lysates by immobilized metal affinity chromatography (IMAC) using nickel–nitrilotriacetic acid (Ni-NTA) agarose  according to the instructions of the manufacturer. The c-Met 15-mer peptides corresponding to 1-932 of the c-Met extracellular domain were synthesized at 5 nmol/spot and covalently bound to a Whatman® 50 cellulose support by the C-terminus . The membrane of c-Met 15-mer peptides was blocked with 5% skim milk for 3 h at room temperature with shaking. The membrane was incubated with the scFv of IRCR201 in 5% skim milk at 4 °C overnight. The scFv of IRCR201 bound to the c-Met 15-mer peptides was transferred to a polyvinylidene difluoride (PVDF) membrane  using the electroblotting method. The transferred PVDF membranes were washed in 1× TBST  twice for 10 min. The PVDF membrane was blocked with 5% skim milk for 3 h and incubated with anti-HA-peroxidase  for 2 h with agitation. The membrane was washed three times with 1× TBST for 5 min and incubated with enhanced chemiluminescence (ECL) detection reagents  for about 1 min. A sheet of film on the membrane was placed into a film cassette in a darkroom, according to the manufacturer’s instructions. The intensity of the bands was quantified by Multi Gauge software V3.0 .The epitope mapping of IRCR201 to c-Met was performed using the scFv format of IRCR201. The scFv of IRCR201 was produced using TOP10F' H and VL segment of IRCR201 were assembled in the scFv format by homology modeling using the Rosetta-based computational homology modeling technique [The sequences of the Vechnique . The IRCechnique . Structuv/v) FBS. After culture for 24 h, cells were starved in serum-free RPMI1640 medium for another 24 h. The cells were then cultured in the presence of anti-c-Met antibodies and HGF in serum-free medium for 30 min at 37 °C. Next, the medium was removed and the cells were washed once with 1× PBS (pH 7.4). Cells were lysed and p-Akt levels were quantified by PathScan® phospho-Akt1 (Ser473) chemiluminescent sandwich ELISA kit  according to the manufacturer’s protocol.Wells of 96-well tissue culture plates  were seeded with 5000 Caki1 cells in RPMI1640 medium supplemented with 10%  FBS, 0.03% (w/v) NaN3, and 25 mM HEPES in 1× PBS, pH 7.4). To confirm the expression of c-Met on the cell surface depending on IRCR201 treatment times, fixed cells were incubated with 1:50 Met (D1C2) XP® Rabbit mAb conjugated Alexa Fluor® 488 . Cells were run on a FACSAriaTM III  and analyzed using the CellQuest™ program .U87MG, A549, and MKN45 cells were treated with 100 nM of IRCR201 for 0 min, 15 min, 30 min, 60 min, 120 min, and 240 min, and then detached by mild trypsinization, fixed using 4% paraformaldehyde solution  at 4 °C, and resuspended at a concentration of 5 × 10® ELISA kit  according to the manufacturer’s protocol.To analyze the total c-Met degradation of c-Met-expressing cells by IRCR201, an ELISA-based quantification method was used. When U87MG, A549, and MKN45 cells in 96-well cell culture plates  reached approximately 70% confluency, cells were treated with 100 nM of IRCR201 for 0 min, 15 min, 30 min, 60 min, 90 min, and 120 min. After washing with 1× PBS (pH 7.4), cells were resuspended with lysis buffer  supplemented with protease inhibitor cocktail tablets  and phosphatase inhibitor . The changes in c-Met protein were analyzed in a timely manner by a human HGFR/c-MET DuoSet2 atmosphere. The number of viable cells was estimated by a CellTiter-Glo® luminescent cell viability assay kit . The luminescent signal intensity of the plates was read by an Infinite® m200 pro  and normalized to the untreated group.MCF7, U87MG, A549, and MKN45 cells were seeded into 96-well white plates  at a density of 3,000 cells/well and cultured for 72 h with 0–100 nM of IRCR201, huOA5D5.v2, or human IgG control at 37 °C in a humidified 5% CO® 3/7 Luciferase assay kit  in accordance with the manufacturer's recommendations. Cells in appropriate complete medium were incubated for 24 h with the untreated group, Human IgG, or IRCR201. After the addition of Caspase-Glo® 3/7 reagent, the luminescent signal intensity was measured by an Infinite® m200 pro  and normalized to the untreated group.The caspase 3/7 activity was detected using the Caspase-Glo2 atmosphere. In the case of A549, conditions for treatment with HGF (50 ng/mL) were added. Cells were then lysed with Lysis-M reagent  supplemented with protease inhibitor cocktail tablets  and phosphatase inhibitor . Samples were denatured with 4× lithium dodecyl sulfate (LDS) sample buffer  and 10× reducing reagent  at 70 °C for 10 min. For Western blotting, cell lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 4–20% Mini-PROTEAN® TGX™ gels  and transferred to nitrocellulose membranes  using a Trans-Blot® Turbo™ transfer system . Blots were blocked with 5% BSA solution for 1 h at room temperature and then incubated overnight with appropriate primary antibodies at 4 °C with agitation. Primary antibodies used include the Met (D1C2) XP® Rabbit mAb , Phospho-Met (Tyr1234/1235) (D26) XP® Rabbit mAb , Akt Rabbit mAb , Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb , p44/42 MAPK (Erk1/2) Rabbit mAb , Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) Rabbit mAb , and Actin (13E5) Rabbit mAb , all used according to the manufacturer’s recommendations. Following overnight incubation with primary antibodies, blots were washed three times with 1× PBST for 20 min, and then incubated with 1:2000 goat anti-rabbit IgG-HRP  as a secondary antibody for 1 h at room temperature. Blots were then washed three times with 1× PBST for 20 min, and immune-reactive bands were visualized using ECL Western blotting detection reagents  and scanned by ImageQuant™ LAS 4000 .When U87MG, A549, and MKN45 cells reached 70% confluency in six-well tissue culture plates , cells were incubated with IRCR201 (100 nM) or PBS in full-serum media for 24 h at 37 °C in a humidified 5% CO2 atmosphere. Cells were lysed, and the Western blotting steps were conducted as described above.To confirm the degradation mechanism of c-Met in A549, 100 nM concanamycin A  or 5 μM MG132 /5 μM lactacystin  mixture was pre-treated for 2 h, and IRCR201 or 5D5 was treated for 4 h at 37 °C in a humidified 5% CO2 atmosphere. After being washed with 1× PBS (pH 7.4), the cells were fixed by 4% paraformaldehyde , permeabilized with 0.1% Triton™ X-100 , and blocked by adding 1% BSA. IRCR201 binding to c-Met were stained by anti-Human IgG secondary antibody conjugated Alexa Fluor® 647 , and LAMP1 was detected by anti-LAMP1 antibody conjugated Alexa Fluor® 488 . MKN45 cells stained fluorescence dyes were mounted with VECTASHIELD® Mounting Medium with 4′,6-diamidino-2-phenylindole . All fluorescence images were obtained by LSM780  and analyzed by ZEN2012 .MKN45 cells were cultured on eight-well chamber 15 µ-slide . MKN45 cells were incubated with 10 µg/mL IRCR201 at 37 °C in a humidified 5% CO®  were subcutaneously injected into the right flank region of female BALB/c-nu mice (1 × 106 cells per mouse in 100 μL). When the mean tumor volume reached approximately 150 mm3 (day 0), animals were randomized according to tumor volume to minimize intragroup and intergroup variation. After regrouping, IRCR201, huOA5D5.v2, or vehicle was intravenously administered twice per week. Each treatment group consisted of six mice. Tumor volumes were measured using three-dimensional calipers. Body weights were measured as general confirmation of toxicity.The efficacy of IRCR201 was evaluated in various tumor xenograft models. A549 and MKN45 single cells suspended in Hank’s Balanced Salt Solution  with Matrigel® Rabbit mAb , Phospho-Met (Tyr1234/1235) (D26) XP® Rabbit mAb , Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb , Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) Rabbit mAb , and PECAM1 (D8V9E) XP® Rabbit mAb ; all were used according to the manufacturer’s recommendations. After slides were washed twice in 1× PBST, tumor sections were incubated with 1:200 biotinylated anti-rabbit IgG . Detection and visualization of the tumor sections were performed using the avidin/biotin/peroxidase complex  and 3,3′-diaminobenzidine . For TUNEL assay, the ApopTag® Peroxidase In Situ Apoptosis Detection Kit  was used according to the manufacturer’s protocol. The IHC images of the tumor sections were obtained using the Aperio imaging system .For the immunostaining of Ki-67, c-Met, and phospho-c-Met, phospho-Akt, phospho-Erk1/2, and PECAM1, antigen retrieval was processed in formalin-fixed and subsequently paraffin-embedded MKN45 tumor tissues. After blocking with 5% bovine serum albumin in 1× PBS (pH 7.4) for 1 h at room temperature, the sections were incubated overnight with primary antibodies at 4 °C. Primary antibodies used include Ki-67 (D2H10) Rabbit mAb , Met (D1C2) XP® V5.01  and expressed as the mean ± SEM unless otherwise noted. The statistical significance in tumor growth between different groups was analyzed by one-tailed two sample t-test.All data were analyzed with GraphPad PrismIRCR201 exhibits tumor growth inhibitory activity in various cancer types harboring HGF-dependent or HGF-independent/c-Met-amplified activation.