Although the respective potentials of magnifying endoscopy with narrow-band imaging (ME-NBI) and confocal laser endomicroscopy (CLE) in predicting gastric cancer has been well documented, there is a lack of studies in comparing the value and diagnostic strategy of these 2 modalities. Our primary aim is to investigate whether CLE is superior to ME-NBI for differentiation between gastric cancerous and noncancerous lesions. A secondary aim is to propose an applicable clinical strategy.We conducted a diagnostic accuracy study involving patients with suspected gastric superficial cancerous lesions. White light endoscopy, ME-NBI, and CLE were performed diagnostic accuracy, sensitivity, specificity, positive predictive value, and negative predictive value between ME-NBI and CLE were assessed, as well as agreements between ME-NBI/CLE and histopathology.P > 0.05). For undifferentiated/differentiated adenocarcinoma, CLE had a numerically but not statistically significantly higher accuracy than ME-NBI . Agreements between ME-NBI/CLE and histopathology were near perfect .This study involved 86 gastric lesions in 82 consecutive patients who underwent white light endoscopy, ME-NBI, and CLE before biopsy. The accuracy, sensitivity, and specificity for ME-NBI were 93.75%, 91.67%, and 95.45%, compared with 91.86%, 90%, and 93.48%, respectively, for CLE, for discrimination cancerous/noncancerous lesion (all CLE is not superior to ME-NBI for discriminating gastric cancerous from noncancerous lesions. Endoscopist could make an optimal choice according to the specific indication and advantages of ME-NBI and CLE in daily practices. However, routine biopsy sampling or endoscopic resection may increase the associated risks of bleeding and perforation. As for noncancerous lesions, multiple biopsies can be time-consuming and costly. In addition, local scars caused by previous biopsies make it difficult to perform subsequent examination or endoscopic resection.3 Thus in recent years there have been substantial interest in exploring new ways to diagnose gastrointestinal diseases real-time without biopsy.Annually there are over 900,000 new cases of gastric cancer worldwide, representing a major burden on health services (gastric cancer remains the second leading cause of cancer deaths across the world).6 CLE is the latest advanced endoscopic imaging technological modality. The first CLE platform was developed in 2004 which combined the application of confocal scanning microscope with conventional flexible endoscope.7 Subsequently, another system called probe-based CLE (pCLE), which conveys light with a fiber-optic probe bundled from a confocal microscope through the accessory channel of virtually any endoscope, was applied in clinical practice.8These so-called “red-flag” techniques or new powerful digestive endoscopies offer an absolutely new visualization for gastrointestinal tract, including high-resolution and magnification endoscopy, computed virtual chromoendoscopy, confocal laser endomicroscopy (CLE), and endocytoscopy. ME-NBI (magnifying endoscopy with narrow-band imaging) is confirmed to have achieved excellent diagnostic accuracy in differentiating between noncancerous and cancerous lesions.9 Kakeji et al10 conducted an ex-vivo and in-vivo study on diagnosing gastric cancer using CLE, and concluded that CLE images correspond well with histopathological images. Zhang et al11 and Li et al12 then developed and refined CLE diagnostic criteria for gastric lesions especially in differentiating between normal and cancerous tissues, thus making it easy to apply in clinical practice with high validity and reliability. However, there are few studies in the diagnostic accuracy of ME-NBI compared to CLE for diagnosis of gastric cancerous lesions.Our center has demonstrated that ME-NBI could successfully distinguish between cancerous and noncancerous lesions and between undifferentiated and differentiated gastric adenocarcinomas, with both accuracies approximating 90%.Consequently, we prospectively conducted this study to assess and compare the real-time diagnostic accuracy for gastric cancerous lesions between ME-NBI and CLE, and propose a practical clinical strategy, using histopathology as the “gold standard.”13 Exclusion criteria were as follows: patients with obvious advanced gastric cancer or previous gastrectomy; the patients’ condition was inadequate for receiving ME-NBI or CLE examination, such as those suffering liver cirrhosis, kidney dysfunction, acute gastrointestinal bleeding, esophageal and gastric varices, coagulopathy, or with known allergy to fluorescein sodium, etc.; patients with pregnancy, breastfeeding, younger than 18 year-old, or older than 80 years old; and those who were unable to sign informed consent.Participants were consecutively enrolled with suspected gastric superficial cancerous lesions previously diagnosed by conventional white light endoscopy (WLE) in either primary or secondary hospital, namely endoscopic findings corresponded with Paris endoscopic classification of superficial neoplastic lesions  of the gastrointestinal tract.All participants received informed consent before the study. The data collection was planned before endoscopic and histopathological examination at Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University. Local ethics committee approved the study protocol. The research was carried out in accordance with the Helsinki Declaration.15 We performed a randomization between ME-NBI and CLE endoscopic procedures to estimate the individual diagnostic value of each modality. Participants in group A had WLE followed by ME-NBI and CLE sequentially, while group B subjects had WLE, CLE, and ME-NBI in that sequence. All of these endoscopic procedures were performed by a single endoscopist (Han-Bing Xue) who was blind to patient's previous histopathological findings to avoid influence of varying experience of different endoscopists and previous diagnosis. We numbered patients according to the sequence of their examination date and carried out randomization via table of random number.This study was conducted in accordance with Standards for Reporting of Diagnostic Accuracy initiative (STARD).Before the procedure, all patients have received deep sedation with intravenous propofol in combination with fentanyl, which was performed under continuous monitoring of vital signs with supplemental oxygen. All procedures were first performed by WLE involving the use of Olympus GIF-H260Z endoscope. During the examination, 30 mL of water with 20,000 units of pronase  were used to wash the mucus and foam. When gastric lesions, especially superficial elevated, depressed and uneven lesions were discovered, ME-NBI or CLE should be performed to make a further examination.ME-NBI was performed by using a magnifying endoscope . A transparent hood was attached to the tip of the endoscope to maintain the appropriate distance during the procedure. The CLE used in this study was an EG-3870CIK . A contrast agent was injected intravenously . The sequence of these 2 equipments was randomized. All of above-mentioned procedures were performed by a single endoscopist (HBX) specialized in ME-NBI and CLE, and a real-time diagnosis of these 2 modalities were recorded by a research nurse. After examination of ME-NBI and CLE, forceps biopsy was obtained from the examined sites.16 Although there have been few studies on CLE diagnosing gastric cancer, the valuable results thereof showed high reliability and validity .17 We assumed that the sensitivity and specificity of ME-NBI versus CLE for detecting gastric cancerous lesions was 95% and 92%, 90% and 97%, respectively. The significance level of α was set at 0.05, and the allowable error of δ was set at 0.1, using the power of 80%. According to the sample size formula, the minimum sample size of cases required was 34 gastric cancerous lesions, and noncancerous cases should be at least 41.ME-NBI had been reported to have a sensitivity of 92.9% to 97.3% for distinguishing gastric cancer from benign lesions, while the specificity was 84.4% to 96.8%.13 During this current study, we chose vessel plus surface (VS) classification as our diagnostic criteria of ME-NBI.18 In addition, if the lesions were diagnosed as cancerous by ”VS“ classification, then we would further divide them into differentiated and undifferentiated type according to the appearance of the fine network pattern and corkscrew pattern.19The morphological class of lesions was recorded in accordance with the Paris Workshop guidelines.12 The simplified 2-tiered classification divides gastric lesions into noncancerous and cancer/high grade intraepithelial neoplasia (HGIN) lesions. The typical signs for cancer/HGIN lesions were as follows: irregular surface patterns, atypical glands, or disorganized patterns; irregular in shape and size and disordered dark cells; irregular, twisted, or unusual shaped microvessels. As for gastric cancerous lesions, type G1  and type G2  were responsible for undifferentiated and differentiated type, respectively. According to modified Vienna classification, high grade dysplasia (C 4.1) is regarded as mucosal high grade neoplasia and has a high risk of developing into intramucosal carcinoma or submucosal invasion; therefore, it should be given endoscopic or surgical local resection.20 In our study, we defined “cancerous lesion” as cancer or HGIN.As for CLE, we chose the gastric pit patterns classification and a simplified 2-tiered classification as diagnostic criteria.23 If the lesion was diagnosed as adenocarcinoma, then it should be further divided into D-type (well or moderately differentiated adenocarcinoma or papillary adenocarcinoma) and UD-type . The histological analysis after surgical or endoscopic resection was accepted as final.All biopsy specimens obtained from examined sites were immediately fixed in 10% formalin, sectioned into 4 mm thick samples, and stained with hematoxylin-eosin (H&E) for routine histopathological analysis. Two experienced GI pathologists (XYC and YC) who were ignorant of the patients’ clinical history or previous endoscopic diagnosis reviewed all of the specimens independently. The histopathological criteria were based on the Updated Sydney System, the World Health Organization classification of digestive tumors and Vienna classification.2 test was used to compare rate and ratio. A 2-tailed P value <0.05 was considered statistically significant. Cohen kappa (κ) was used to represent agreement between ME-NBI/CLE and histopathology, with value of 0.01 to 0.20 indicating poor agreement, 0.21 to 0.40 fair, 0.41 to 0.60 moderate, 0.61 to 0.80 substantial, and 0.81 to 1.00 almost perfect.All statistical analyses were performed using SPSS 13.0 software package . Accuracy, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for ME-NBI and CLE were estimated with McNemar test, respectively, along with exact binomial 95% confidence intervals, with histopathology considered as the “gold standard”. χThe study was performed from January 2013 to January 2014. A total of 86 consecutive patients were prospectively enrolled for study analysis. Four patients were withdrawn from the study before the randomization. Two patients were unable to receive deep sedation and the other 2 refused to have an injection of fluorescein sodium. Ultimately, 82 patients with 86 gastric lesions were analyzed. A total of 39 and 43 patients were assigned to group A and group B, respectively. There were 58 males and 24 females with a mean age of 59.3 ± 8.5 years (range from 29 to 79 years). The median size, macroscopic type, and histopathology were all summarized in Table It took nearly 1 week to get histopathological diagnoses after examination of ME-NBI and CLE. No special treatment was given to participants except drug therapy for someone during this period and no adverse event was found. Figure As showed in Table The agreements between new imaging modality and histopathology were all almost perfect with κ value of 0.87 for ME-NBI and 0.84 for CLE. Agreement remained perfect when combined the use of ME-NBI and CLE with a κ value of 0.85. The data also showed perfect agreement between ME-NBI and CLE, with a κ value of 0.82.Our study demonstrated CLE was not superior to ME-NBI for discriminating between gastric cancerous and noncancerous lesions with the sensitivity, specificity, and accuracy of 90%, 93.48%, and 91.86%, respectively. In the current study, the sensitivity was improved to 94.44% when ME-NBI combined with CLE, and the overall accuracy reached 92.5%. On the other hand, PPV of ME-NBI + CLE is worse than that of ME-NBI or CLE alone. That means, ME-NBI + CLE is helpful not to overlook cancerous lesion, but they will increase unnecessary therapy and take longer time. Although our primary single center study demonstrated the high validity and reliability of ME-NBI and CLE for gastric cancerous lesions, the high diagnostic accuracy and observer agreement were reported from limited experienced institute and lack of external validation from Western countries. It might be concerned with the different research areas between Eastern and Western countries. Owing to relatively different cancer prevalence, scholars from Western countries paid more attention to colorectal and esophageal cancers.18 Their research group proposed the initial VS classification system. Recently, they conducted a multicenter prospective study to validate the VS classification system for screening EGC involving large samples (1092 patients). The accuracy, sensitivity, and specificity of high confidence ME-NBI diagnosis were 98.1%, 85.7%, and 99.4%, respectively.24 According to our experience, the observation of MS and MV patterns would be obstructed in lesions covered by dirty ulcerative things or fragile mucous membrane with a bleeding tendency. With regard to the current study, 6 of 86 lesions were indeterminate owing to invisible MS and MV patterns in ME-NBI group.Unlike esophageal or colorectal cancers, no consensus on the criteria for the diagnosis of gastric cancer based on ME-NBI features has been established to date. In 2009, Yao et al reported an irregular MV pattern and/or an irregular MS pattern together with a clear demarcation line were the hallmarks of EGC.25 Nakayoshi et al19 have described 2 characteristic patterns, one of which was called fine network pattern with an irregular MV network and the other was corkscrew pattern with isolated corkscrew-like vessels. They were confirmed to correlate with D-type and UD-type carcinoma, respectively. Another characteristic pattern proposed by Yokoyama for differentiated carcinoma was intra-lobular loop: irregular papillary MS, irregular MV pattern located in the gland duct.26 Some scholars believe that the MS structure of undifferentiated gastric cancer not invading the full layer of mucosa could still be mild dysplasia sometimes.27 For poorly differentiated adenocarcinoma and signet-ring cell carcinoma, mucosal surface may be covered by nonneoplastic foveolar epithelium, which shows the same color as the background mucosa at the early stage. It may be responsible for the low sensitivity in diagnosis of UD-type carcinoma using ME-NBI in our study.In our subgroup analysis, we paid attention to the differentiation of gastric cancer. According to Japanese gastric cancer treatment guidelines, D-type carcinoma has a lower rate of lymph node metastasis and a better prognosis than UD-type, thus resulting in different therapeutic strategy.28 In the present study, some endomicroscopic aspects for differential diagnosis between UD-type and D-type carcinoma were observed and summarized. D-type carcinoma appears to be hypervascular with vessels that were tortuous and dilated as well as irregular in shape and size, and the glands are atypical with irregular black cells . Multicenter studies with larger sample sizes are crucial for confirming this trend in the future.CLE is the latest advanced imaging device providing endoscopist a real-time in vivo histopathological images or optical biopsies of living cells and tissues during endoscopy. Asia, as a region of relatively higher gastric cancer prevalence than in the West, reports the most literatures on the utility of CLE in stomach. Nevertheless, the features of gastric cancer under CLE have not been standardized due to the difficulty in reading images which are totally different from either routine endoscopy or histopathology. Recent studies in China have described characteristic of gastric intraepithelial neoplasia and cancerous lesions. The sensitivity, specificity, and accuracy of real-time CLE for diagnosis of gastric superficial cancer or HGIN could be up to 88.9%, 99.3%, and 98.8%, respectively.s Figure . Our res31 Our study showed CLE was not superior to ME-NBI for differentiation between gastric cancerous and noncancerous lesions. The reasons for this outcome might be that our center had richer experience on ME-NBI than CLE, and we withdraw 6 indeterminate cases for final statistical analysis, thus increasing diagnostic validity in ME-NBI group. The high PPV (94.29% and 92.31%), NPV (93.33% and 91.49%), and agreement with histopathology (0.87 and 0.84) of ME-NBI and CLE confirmed the enormous significance of these 2 modalities in clinical practice. Clinician could make an appropriate and quick judgment based on either single or combined use of them. If the lesion is suspected to be cancerous, then a target or multiple biopsies should be performed to confirm the histopathology. On the other hand, when the lesion is considered noncancerous by experienced endoscopist using ME-NBI or CLE, a negative biopsy could be avoided.Some researchers suggest CLE was superior to NBI on gastric intestinal metaplasia and intraepithelial neoplasia.32 There has been no consensus published on diagnosis of gastric cancer by CLE. In our opinion, CLE is not suitable for screening EGC due to the difficulty in surveying the whole gastric lumen with limited field of vision. We recommend conventional WLE in screening endoscopic examination, as soon as gastric superficial lesions have been identified. ME-NBI or CLE could be applied in further examination according to endoscopist's experience. CLE is preferred if: surface of lesion is covered by thick and dirty ulcerative things, and MS and MV patterns could not be observed; fragile mucous membrane with a bleeding tendency; fibrosis after biopsy or endoscopic surgery; and suspicious undifferentiated gastric carcinoma. If the patient is insufficient to receive deep intravenous sedation or allergic to fluorescein sodium, ME-NBI would be an appropriate choice. Histopathological examination remained “gold standard” for final diagnosis. A clinical strategy for diagnosis of gastric cancerous lesions can now be proposed based on advantages of each modality mentioned above (Figure The secondary aim of the study was to propose an applicable clinical strategy for diagnosing gastric cancerous lesions. The current Asia-Pacific consensus does not recommend NBI as the initial modality for the detection of EGC during screening endoscopy. However, they suggest that NBI can be used in differentiation of neoplastic from nonneoplastic lesions, as well as distinguishing tumor margins from nonneoplastic surrounding mucosa.e Figure .The current study has several limitations that should be noted. First, we only enrolled patients suspected with gastric cancerous lesions that have been diagnosed previously, thus raising the proportion of cancerous lesions. In addition, patients with obviously advanced gastric cancer were excluded. Second, the subjective experience of the endoscopists in ME-NBI and CLE is varied. Namely, the bias of endoscopist herself could affect the diagnostic accuracy. The standardized diagnosis criteria and learning curve of each modality are crucial. To avoid the influence of varying experience of different endoscopists, we chose a single endoscopist to perform examinations. However, since the single endoscopist was unable to be blind to previous findings among ME-NBI and CLE observation, it might improve diagnostic accuracy of the later one. So, we made a randomization between the order of 2 devices, this intervention could balance the validity of each modality, nevertheless, enhanced the real diagnostic accuracy of ME-NBI and CLE. As is known to all, NBI was perhaps hampered by mucous and blood due to endoscope contact on the mucosa during NBI or CLE. The fact demonstrated an advantage of CLE, but also a possibility that the tandem randomized comparison was unfair to compare the 2 modalities. Third, the number of gastric cancerous lesions is relatively small, though it meets the required sample size, especially when comparing the diagnostic yield between UD-type and D-type gastric adenocarcinoma. Finally, this was a single-center study where cost-effectiveness of ME-NBI and CLE is not evaluated. Multicenter randomized controlled prospective studies with larger sample sizes are necessary.In conclusion, gastric cancerous lesions could be detected by ME-NBI or CLE with high validity and reliability. CLE is not superior to ME-NBI for discriminating gastric cancerous from noncancerous lesions. Endoscopist could make an optimal choice according to the specific indication and advantages of ME-NBI and CLE in daily practices. The unified diagnostic strategy and consensus on the criteria of ME-NBI and CLE for the diagnosis of gastric cancerous lesions are expected to be proposed in the future.


It is based on a recent large-scale dataset of ~20,000 drug-induced expression profiles in multiple cancer cell lines, which provides i) a global impact of transcriptional perturbation of both known targets and unknown off-targets, and ii) rich information on drug’s mode-of-action. First, the drug-induced expression profile is shown more effective than other information, such as the drug structure or known target, using multiple HTS datasets as unbiased benchmarks. Particularly, the utility of our method was robustly demonstrated in identifying novel DR candidates. Second, we predicted 14 high-scoring DR candidates solely based on expression signatures. Eight of the fourteen drugs showed significant anti-proliferative activity against glioblastoma; i.e., ivermectin, trifluridine, astemizole, amlodipine, maprotiline, apomorphine, mometasone, and nortriptyline. Our DR score strongly correlated with that of cell-based experimental results; the top seven DR candidates were positive, corresponding to an approximately 20-fold enrichment compared with conventional HTS. Despite diverse original indications and known targets, the perturbed pathways of active DR candidates show five distinct patterns that form tight clusters together with one or more known cancer drugs, suggesting common transcriptome-level mechanisms of anti-proliferative activity.An  In silico DR is considered an alternative and efficient route to establish novel connections between diseases and existing drugs [in silico DR [et al. identified simvastatin and ketoconazole as potent anti-proliferative agents in breast cancer by analysing a drug-target network [Drug repositioning (DR) refers to the identification of novel indications for existing drugs  and is cng drugs ,4. Advanilico DR ,6. A broilico DR ,8, drug-ilico DR , and pheilico DR –14. For  network . Particu network , have prin silico DR methods were developed using this dataset either alone or in combination with other information [et al. further revealed the modes of action for multiple drugs using drug-induced expression modules conserved between humans and rats. Recently, a similar but highly expanded version of a chemical genomics dataset was publicly released by the NIH LINCS program (Library of Integrated Network-based Cellular Signatures). This dataset consists of gene expression signatures and protein binding, cellular phenotypic, and phosphoproteomic profiles due to chemical or genetic perturbation. Specifically, it produced the gene expression profiles of ~1,000 landmark genes (L1000) in response to >20,000 chemical perturbations across many cell lines. Additionally, they inferred transcriptome-level expression profiles of ~20,000 genes computationally using the 1,000 landmark genes [The latest version of CMAP consists of gene expression profiles of five cell lines treated with ~1,300 compounds, from which many ormation –25. The ormation ,20. Iskark genes .in silico DR using the expression signature (E) derived from the recent large-scale, chemical genomics dataset (LINCS) as well as chemical structure (S) and target signatures (T). Next, we applied our method to infer DR candidate anti-cancer drugs for glioblastoma, lung cancer, and breast cancer. We focused on the ability to identify novel DR candidates that are not structurally related to known anti-cancer drugs because structural analogues may be inferred easily by other structure-based methods [In this study, we adopted an integrative approach for  methods ,28. The gold standard) was extracted from several public databases of DrugBank [e.g., Ki < 10 nM). Throughout the analysis, compounds and protein targets were mapped to NCBI PubChem Compound Identifiers (CID) and Entrez Gene ID as standard identifiers, respectively.The known drug set  or NCBI GEO [The expression profiles for the target diseases were downloaded from either the TCGA data portal (NCBI GEO  that incNCBI GEO . The DEGwww.lincsproject.org) that consisted of the 100 most up- and down-regulated genes in response to each compound.The LINCS dataset included an extensive catalog of gene-expression profiles generated by the Library of Integrated Network-based Cellular Signatures (LINCS) project from 59 human cancer cells in response to ~20,000 chemical perturbations. The LINCS team has produced the expression profiles of 1,000 landmark genes using a high-throughput Luminex-based assay . The whoThe compounds used for the cell viability assay were purchased from Sigma-Aldrich and Enzo Life Science. Both the glioblastoma cell lines  and primary cells (GBL cells) were cultured in DMEM high-glucose cell culture medium  supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin solution, and 30 μg/ml Plasmocure  reagent to prevent mycoplasma contamination.To measure the cytotoxic activity of the candidate drugs, the metabolic activity of the viable cells was measured using WST reagent  in 96-well plates. The number of seeded cells was adjusted according to the growth rate of each cell type (100–500 cells/well). Twenty-four hours after seeding, the cells were treated with drugs at a concentration of 10 μM and further cultured for 72 h at 37°C. One-tenth of the medium volume of WST reagent was added to the cells, and the absorbance was measured at 450 nm after 2 h using a SpectraMax 190 microplate reader (Molecular Devices). The experiments were repeated five times.http://www.broadinstitute.org/gsea/msigdb), which consist of 186 gene sets representing various biological processes. Since many disease-related pathways are redundant with other signalling pathways , we excluded such 28 pathways and the remaining 158 pathways were used. The significance of enrichment was calculated using the hypergeometric test. The LINCS dataset contains multiple signatures for the same drug from different cells and conditions, where the harmonic mean of the corresponding p-values was taken as its representative p-value. The adjusted q-value was then calculated using the Benjamini-Hochberg method. The distances between drugs were calculated as the cosine distance of–log p-value.KEGG pathways were downloaded from the MSigDB database  as the benchmark for the three types of cancer  from four public databases as well as by manually curating 243 publications . We consCPD) or known drugs (SKD). The target information was collected from 10 public databases comprising 342,311 compound-target interactions among 205,570 unique compounds . The expression signature  was downloaded from LINCS and consisted of the differentially expressed genes (DEGs) by chemical perturbation[core set of 2,250 compounds for which all three types of signatures  were available. The intersection of the core set and CD set was 304 drugs  for glioblastoma (4 sets), lung cancer (11 sets), and breast cancer (16 sets) from TCGA [Next, the structural signature was extracted as 1,024 bits of FP2 chemical fingerprints implemented in Open Babel  for compturbation. Multiplrom TCGA  or publiWe developed a series of classifiers to predict DR candidate drugs for the treatment of glioblastoma, lung cancer, and breast cancer. Our method utilizes three types of signatures that are derived from chemical structure (S), drug-target relation (T), and gene expression data (E). DR candidates were predicted based on the similarity of these signatures between the compounds and disease (or its known drugs). The prediction performance was thoroughly inspected in an unbiased manner using i) a conventional cross-validation scheme that utilizes known drugs (KD set) as a benchmark, ii) the 29 anti-cancer HTS datasets for 11,000–41,000 compounds, and iii) assays based on glioblastoma cancer cell lines and patient-derived primary cells.CPD-SKD). The expression (E) and target (T) signatures essentially are a list of genes that could be associated by any method for gene set enrichment analysis. Therefore, we could generate six additional types of associations between the compounds  and disease . We used the Tanimoto coefficient as a measure of signature similarity because all signatures can be represented as a binary vector of 0s and 1s—i.e., the presence or absence of genes or structural fingerprints. Because multiple signatures are allowed for a single compound and disease, we calculated the mean of the Tanimoto coefficient values for a given compound-disease pair.The work described herein consisted of three stages: 1) building association signatures, 2) constructing a series of classifiers, and 3) evaluating the prediction performance. The aim of the first stage was to associate compounds and a target disease (or its known anti-cancer drugs) based on the similarity of the three signature types . In totagold standard, ii) the 29 large-scale HTS datasets for anti-proliferative activity, and iii) assays that use both established glioblastoma cell lines and patient-derived glioblastoma primary cells. We further interpreted the potential modes of action of drugs using pathway enrichment analysis followed by comparisons with other cancer drugs.The second stage involved the construction of a series of classifiers to predict DR candidates . To compTo ensure unbiased evaluation, we limited our analysis to the core set of 2,250 compounds so that different classifiers can be compared using exactly the same benchmark. The core set included 79, 100, and 132 known anti-cancer drugs (KD set) as benchmarks for glioblastoma, lung cancer, and breast cancer, respectively. The negative set  was prepared by excluding the CD set from the core set.DR score—scaled from zero to one. The target-based classifiers (T) perform better than the other classifiers  in identifying known drugs (KD core set) in all three types of cancer  or combination of multiple signatures . Their relative performances were evaluated using 100 rounds of three-fold cross-validation schemes. The classifiers were based on a logistic regression that automatically weighs the component features to yield a unified prediction score—the f cancer . This tre.g., only a 20% hit overlap was observed between the two assays for GBM   best predicted anti-cancer hits in all three cancers . By contGlioblastoma multiforme (GBM) is the most common and aggressive form of brain cancer showing a highly poor prognosis despite concurrent or sequential chemo-radiotherapy . Temozoli.e. the percentile rank of the predictions by the same classifier (E) as described in the previous section. Next, we applied several filters: a) FDA-approved; b) DR score >0.9; and c) indication to pass brain-blood barrier (BBB) either by literature or by in silico prediction at http://www.cbligand.org/BBB. One high-scoring candidate  was also included despite being indicated not to pass BBB. The final 14 DR candidates were selected, and their anti-tumor activities were tested using four GBM cell lines and eight patient-derived primary cells. Our DR scores correlated well with the anti-tumor activities among the cell lines tested in terms of both anti-proliferative activity and the fraction of significant growth inhibition  strongly increased lipid metabolism . Accordingly, amlodipine, a calcium channel blocker, was previously reported to induce steroidogenesis . AmlodipWe also performed a cluster analysis of the eight DR candidates together with the 69 cancer drugs included in the LINCS dataset using the enrichment pattern of the same 32 up- and 17 down-regulated pathways . These cAlthough most drug development projects have been aimed to design a specific modulator for a target, polypharmacology—multiple targets for a single drug—may be more prevalent than expected. According to a recent report by Ciceri and colleagues, an unbiased screening of 628 kinase inhibitors identified 20 hits (3.2% hit rate) that also strongly inhibited BRD4 (>90% inhibition at 50 μM) . Two of in silico DR methods were developed, their prediction performances are not thoroughly assessed in most cases. Until recently, an in silico DR method was usually evaluated based on the AUC (area under the curve) of its receiver operating characteristic (ROC) curve using a collection of known or gold standard drugs. Experimental validation was performed in only a small number of the top-scoring DR candidates. Caution is required because the gold standard set is almost always incomplete—i.e., it includes a significant fraction of unknown false negatives or hidden hits. In fact, the very purpose of any in silico DR is to identify such hidden hits rather than to rediscover known drugs. Often, the ability to identify such novel DR candidates has not been thoroughly investigated or estimated using indirect information . As we demonstrated, the benchmark dataset e.g., known drugs vs large-scale HTS dataset, can dramatically influence the performance evaluationThe efficient and systematic identification of clinically relevant off-targets and novel indications is a challenge. Although many in silico DR methods that employ chemical genomic approaches, the success of these methods was mostly anecdotal due to the limited number of transcriptome-profiled compounds. The recent LINCS dataset includes the transcriptional profiles of ~10,000 unique compounds from multiple cell lines. It not only increased the compound coverage by eight fold compared with CMAP, but allowed quantitative and unbiased evaluations via cross-comparisons with multiple benchmarks, such as known cancer drugs, public HTS dataset, and wet experiments. We also managed to compare the performance of the three most frequently used data types  for in silico DR, an analysis that was previously unfeasible due to the small size of the CMAP dataset. Although our analysis is limited to a simple logistic regression classifier, it strongly suggests that the expression signature was the most predictive, particularly in identifying novel DR candidates. Next, we identified eight DR candidates for glioblastoma based solely on expression signatures. Our DR score positively correlated with the anti-proliferative activities in cancer cell lines and primary cells, showing an approximately 20-fold enrichment of active hits. Notably, none of the expression signatures from LINCS were generated using glioblastoma cell lines, which demonstrated that our in silico DR method remains valid across different cancer types and potentially for other diseases. We reasoned that the consensus of multiple signatures from diverse cell lines provided sufficient information to overcome the heterogeneity and noise from individual signatures. Finally, we exploited large-scale, drug-induced transcriptional profiling to interpret the modes of action of our DR candidates. These candidates showed unique patterns of pathway perturbation that were shared with multiple other cancer drugs. Common patterns were observed not only among the drugs of the same target but also among drugs of different classes, which strongly indicated many unknown off-targets or cross-talk between different targets. In the post-chemical genomic era, we may expand the scope of drug repositioning from merely seeking polypharmacology (i.e. one drug for multiple targets) to broadly considering omics-scale effects by both known targets and unknown off-targets, such as the entire transcriptome, proteome, and metabolome.The CMAP dataset is the first large-scale transcriptional profile and includes >1,300 compounds. Although it triggered the development of many S1 File(DOCX)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.


Klotho gene. In mice, defects in Klotho expression lead to complex phenotypes resembling those observed in dialysis patients. However, the relationship between the level of serum soluble Klotho and overall survival in hemodialysis patients, who exhibit a state of Klotho deficiency, remains to be delineated. Here we prospectively followed a cohort of 63 patients with a mean duration of chronic hemodialysis of 6.7 ± 5.4 years for a median of 65 months. Serum soluble Klotho was detectable in all patients . Patients with serum soluble Klotho levels below the lower quartile (<309 pg/mL) had significantly higher cardiovascular and all-cause mortality rates. Furthermore, the higher all-cause mortality persisted even after adjustment for confounders . We conclude that there may be a threshold for the serum soluble Klotho level associated with a higher risk of mortality.Klotho is a single-pass transmembrane protein predominantly expressed in the kidney. The extracellular domain of Klotho is subject to ectodomain shedding and is released into the circulation as a soluble form. Soluble Klotho is also generated from alternative splicing of the  Klotho gene extends longevity, while defective Klotho proteins are associated with premature death . The normalized protein catabolism rate (nPCR) and the Kt/V urea index, which integrates the efficiency of solute removal as urea clearance (K), treatment duration (T), and the patient's size as urea distribution volume (V), were determined using previously described formulas [2D levels were performed by a commercially based clinical diagnostic testing service .Demographic and medical data, including age, gender, smoking history, and comorbid conditions, were obtained from the subjects' medical records in addition to standardized interviews. The body mass index (BMI) was calculated from the weight and height measurements as the weight (kg) divided by the square of the height  ; congestp values of less than 0.05 were considered to be statistically significant. The statistical analyses were performed using the JMP software package version 9 , unless otherwise specified.The data are expressed as either the number of participants or the percentage of the study population. The remaining data are expressed as the mean ± standard deviation (SD), or median and interquartile range (IR) for variables with a skewed distribution. The groups were compared using a one-way analysis of variance for normal distributions and the Kruskal-Wallis rank test for skewed distributions. The chi-square test was used to evaluate the proportional differences in categorical variables. Prior to conducting the survival analysis, the 25th, 50th, and 75th percentiles of serum sKlotho and serum FGF23 levels in the current study population were determined. The subjects were categorized as follows: low-Klotho group (low-KL), subjects with a serum sKlotho level under the 25th percentile; middle-Klotho group (middle-KL), subjects with a serum sKlotho level equal or over the 25th percentile and under the 75th percentile; high-Klotho group (high-KL), subjects with a serum sKlotho level of equal to or over the 75th percentile; low-FGF23 group (low-FGF23), subjects with a serum FGF23 level under the 25th percentile; middle-FGF23 group (middle-FGF23), subjects with a serum FGF23 level equal to or over the 25th percentile and under the 75th percentile; high-FGF23 group (high-FGF23), subjects with a serum FGF23 level of equal to or over the 75th percentile. We then assessed the impact of the serum sKlotho and FGF23 levels on the cumulative patient and cardiovascular event-free survival rates according to the Kaplan-Meier method combined with the log-rank test. Cox regression models were used to analyze the relationships between the primary outcomes and the serum sKlotho levels. Multivariate models were also applied to adjust for age, gender, diabetes mellitus, sPi, sAlb, and Hb, which are known to be associated with the risk of mortality in patients under chronic HD treatment . The resSoluble Klotho was detectable in the serum of all 63 chronic HD patients, with serum concentrations of sKlotho ranging from 167 to 720 pg/mL . No significant difference in serum sKlotho levels was observed between males and females. The baseline clinical and demographic profiles of the subjects with the three varied degrees of serum sKlotho levels are summarized in p = 0.018). This trend remained significant even after adjusting for age, gender, diabetes mellitus, sPi, sAlb, Hb, 1,25(OH)2D, and FGF23 . The cardiovascular event-free survival, cardiovascular mortality, and all-cause mortality rates did not differ between the low-FGF23 (FGF23 < 1184 pg/mL), middle-FGF23 (1184 ≤ FGF23 < 15134 pg/mL), and high-FGF23 (FGF23 ≥ 15134 pg/mL) groups (Twelve patients (19%) died within the median follow-up period of 65 months (IR 63–67). Four patients in the low-KL group, three in the middle-KL group, and two in the high-KL group died of cardiovascular events, while one patient in the low-KL group died of infectious disease. Only two patients in the low-KL group died of carcinoma, versus one patient in the middle-KL group. The serum Klotho levels of the nonsurvivors at study entry were numerically lower  than those of the survivors . The Kaplan-Meier analysis also showed that the cardiovascular event-free survival and mortality rates did not differ significantly between the three groups, whereas the cumulative survival rate was significantly lower in the low-KL group . The car) groups .In the current study, we demonstrated that chronic HD subjects have worse cardiovascular and all-cause mortality rates with low serum sKlotho levels than those without low serum sKlotho levels. Moreover, our results showed that a reduced level of serum sKlotho is associated with all-cause mortality, even after adjusting for confounding variables, implying that the circulating sKlotho is a potential predictive indicator of overall mortality among patients with end-stage renal disease (ESRD). Previous findings have demonstrated that low serum sKlotho is associated with adverse kidney disease outcomes and/or arterial stiffness in some subsets of CKD patients and is related to all-cause mortality in older community-dwelling adults , 25, 26.Information regarding the kinetics of circulating sKlotho has been poorly understood , 27, 29.The soluble form of Klotho may target multiple remote tissues and organs, comprising a wide range of biological activities, including antiaging properties , 42. It Several studies in CKD patients have shown that circulating FGF23, a hormone involved in phosphorous and vitamin D homeostasis, predicts adverse outcome , 50, 51.In this study, we demonstrate for the first time that chronic hemodialysis subjects with low serum soluble Klotho levels have worse cardiovascular and all-cause mortality rates than those without low soluble Klotho levels. Moreover, we found that reduced serum soluble Klotho is associated with all-cause mortality, even after adjusting for confounding variables, implying that the level of serum soluble Klotho plays a role as a predictive indicator of overall mortality in patients with ESRD.


An increased risk of mortality in patients with hyperuricemia has been reported. We examined (1) the risk of all-cause and cardiovascular disease (CVD) mortality in untreated hyperuricemic patients who did not receive urate-lowering therapy (ULT), and (2) the impact of ULT on mortality risk in patients with hyperuricemia.In this retrospective case-matched cohort study during a mean follow-up of 6.4 years, 40,118 Taiwanese individuals aged ≥17 years who had never used ULT and who had never had gout were examined. The mortality rate was compared between 3,088 hyperuricemic patients who did not receive ULT and reference subjects  matched for age and sex (1:3 hyperuricemic patients/reference subjects), and between 1,024 hyperuricemic patients who received ULT and 1,024 hyperuricemic patients who did not receive ULT (matched 1:1 based on their propensity score and the index date of ULT prescription). Cox proportional hazard modeling was used to estimate the respective risk of all-cause and CVD (ICD-9 code 390–459) mortality.After adjustment, hyperuricemic patients who did not receive ULT had increased risks of all-cause  and CVD  mortality relative to the matched reference subjects. Hyperuricemic patients treated with ULT had a lower risk of all-cause death  relative to hyperuricemic patients who did not receive ULT.Under-treatment of hyperuricemia has serious negative consequences. Hyperuricemic patients who received ULT had potentially better survival than patients who did not. Hyperuricemia has recently attracted world-wide attention . Its preNon-pharmacotherapy with moderately intense physical activity is recommended to reduce the mortality risk that is increased with high serum uric acid (sUA) . HoweverThe survival benefit of allopurinol for all-cause death was reported in an observational study of hyperuricemic veterans in the US . A recenIn our previous demonstration of a beneficial effect of physical activity in patients with hyperuricemia , a lowerThis retrospective case-matched cohort study used clinical data collected from the nationwide MJ Health Screening Centers, which enroll participants from all regions of Taiwan . All memThe MJ dataset is linked to the National Health Insurance Research Database (NHIRD), which records all medical claims for drugs dispensed to Taiwanese patients who are covered by National Health Insurance (NHI), with minimal under-reporting and misclassification . The MJ Among participants in this MJ cohort, 10,103 subjects with sUA > 7 mg/dL were defined as patients with hyperuricemia. The definition of gout was a patient with a diagnosis code 274.X  and with concomitant use of a non-steroidal anti-inflammatory drug or colchicine between January 1, 1997 and December 31, 2002 . AccordiAccording to the Taiwanese guideline, asymptomatic hyperuricemic patients with sUA > 9 mg/dL along with comorbidity or patients with sUA > 10 mg/dL are suggested to consult doctors to decide if they should receive ULT . MeanwhiThe cohort of hyperuricemic patients who did not have gout and were not treated with a ULT (hyperuricemia [HUA] /No gout /No ULT) , were matched with reference subjects (No HUA /No gout /No ULT) at a 1:3 ratio according to age (within a 5-year age span) and sex. The observation time was calculated from the entry date of the health examination in 1996 to the censored date, which was either the time of death or the end of follow-up .The primary outcome was all-cause mortality, and the secondary outcome was CVD (ICD-9 390–459) mortality. Kaplan-Meier survival curves were used to plot the survival probability of all-cause and CVD mortality for the 30,475 reference subjects (No HUA /No gout /No ULT), the 7,522 hyperuricemic patients who were not treated with ULT (HUA /No gout /No ULT), and the 1,032 hyperuricemic patients who were treated with ULT (HUA /No gout /ULT), all of which  were tesA propensity score (0 to 1) was used to determine the likelihood of a hyperuricemic patient being prescribed a ULT by a physician given his or her individual characteristics . This ant-test for continuous data and the chi-square test for categorical data. The mortality events resulting from all causes and CVD were compared (1) between hyperuricemia patients who did not receive ULT and the 1:3 matched reference subjects (No HUA /No gout /No ULT), and (2) between the 1:1 matched pairs of hyperuricemic patients with and without ULT. The Cox proportional hazard model was used to estimate the adjusted hazard ratio (HR) of all-cause and CVD mortality.Baseline demographic data were compared between hyperuricemic patients and their respective reference groups using the Student’s The concomitant use of anti-hypertensive , anti-dip = 0.76; In total, 39,029 subjects satisfied the inclusion criteria and were followed up for a mean of 6.4 years. There were 3,088 hyperuricemic patients who did not receive ULT (HUA /No gout /No ULT) were matched with 9,264 reference subjects (No HUA /No gout /No ULT) . p < 0.001) (p < 0.05) (< 0.001) , and amo < 0.05) .Matching was performed to improve the similarity of confounding variables across comparison groups. Comparison of hyperuricemic patients who did not use ULT (HUA /No gout /No ULT), to the matched reference group (No HUA /No gout /No ULT) showed a marginally significant effect on all-cause mortality  0.97–1.59) due to attrition of case number after matching procedure, and a significant risk of hyperuricemia for CVD mortality  .In the 1:1 matched groups of hyperuricemic patients who did and did not receive ULT, the all-cause and CVD mortality rates of ULT users were lower than the rates in non-ULT users . HyperurA potential effect of the type of ULT on mortality outcomes was examined. We compared all-cause and CVD mortalities in hyperuricemic patients who used allopurinol (a xanthine oxidase inhibitor) or benzbromarone (a uricosuric agent) with those who did not use any ULT . AlthougIn each subgroup stratified according to concomitant medication, which consisted of anti-hypertensive, anti-diabetic, and lipid-lowering agents, fewer mortality events were generally seen in hyperuricemic patients who used ULT than in the 1:1 matched non-users. However, only subgroups of patients with hyperuricemia in which we considered concomitant use of anti-hypertensive medication (721 pairs) and no lipid-lowering medication (860 pairs) had sufficient power for a risk estimate of all-cause mortality . We obseAmong the hyperuricemic patients who received ULT, 48.5% of these patients used ULT cumulatively for <60 days . We obsePrevious studies indicated an independent association between hyperuricemia and increased risk of premature all-cause and CVD death , 8. Our Throughout the follow-up period, compared with the matched non-ULT users, ULT users had lower mortality rates, and a significant survival benefit of ULT use was demonstrated after >2 years of use. The observation for each matched pair with variably cumulative ULT use intervals started from the same index date. This finding, based on the comparison between ULT users and their 1:1 matched non-ULT users, is consistent with a recent general population study indicating an association between reduced risk of death in patients with hyperuricemia and initiation of treatment with allopurinol .Our present study did not, however, show a beneficial effect of allopurinol on premature mortality that was due to hyperuricemia. This negative finding is consistent with a recent Taiwanese report using the database from NHIRD . One conAlthough uric acid is regarded as an anti-oxidant, it can act as a damage-associated molecular pattern that contributes to gout and its comorbidities of atherosclerosis and metabolic syndrome . The rolOur study has several limitations. First, this was not a randomized clinical trial, so we cannot attribute all the observed mortality risk or benefit exclusively to ULT. Nonetheless, in the model we used, we adjusted for several potential variables that may affect mortality. A balanced pattern of known comorbidities  between ULT users and non-users in hyperuricemic patients was established by the greedy matching technique. ULT appeared to have a direct and independent effect on all-cause mortality in Taiwanese patients with hyperuricemia who did not have gout. Second, our information on ULT prescriptions came from the NHI database, a record of dispensed drug claims. Compliance with ULT may have been poor in many hyperuricemic patients , making Our study also has several strengths. A potential bias, which can result from different baseline risks between hyperuricemic patients who were or were not treated with ULT was attenuated by propensity score matching. The baseline characteristics between ULT users and non-users were balanced and showed no significant difference. In addition, a potential bias caused by the immortal time effect was also reduced, because if non-ULT hyperuricemic users died before the date of initial ULT prescription to their matched ULT users, we reselected subjects by matching the index date of ULT prescription. In addition, the current study relied on the large amount of recorded information that is available in the national database to examine the potential benefit of ULT in hyperuricemic patients. The use of a large number of measured covariates  may overcome the limitation of using propensity scores in 1:1 matched case-cohort analyses . The modWe recently reported serious negative consequences on mortality in under-treated gout patients, and gout patients who received ULT had significantly better survival than those who did not . This cuS1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.


Macaca mulatta) using recombinant soluble WNV envelope (E) ectodomain adjuvanted with Matrix-M, either with or without DNA priming. The DNA priming immunization was performed with WNV-DermaVir nanoparticles. Both vaccination strategies successfully induced humoral and cellular immune responses that completely protected the macaques against the development of viremia. In addition, the vaccine was well tolerated by all animals. Overall, The WNV E protein adjuvanted with Matrix-M is a promising vaccine candidate for a non-infectious WNV vaccine for use in humans, including at-risk populations.The mosquito-borne West Nile virus (WNV) causes human and animal disease with outbreaks in several parts of the world including North America, the Mediterranean countries, Central and East Europe, the Middle East, and Africa. Particularly in elderly people and individuals with an impaired immune system, infection with WNV can progress into a serious neuroinvasive disease. Currently, no treatment or vaccine is available to protect humans against infection or disease. The goal of this study was to develop a WNV-vaccine that is safe to use in these high-risk human target populations. We performed a vaccine efficacy study in non-human primates using the contemporary, pathogenic European WNV genotype 1a challenge strain, WNV-Ita09. Two vaccine strategies were evaluated in rhesus macaques ( West Nile virus (WNV) is a mosquito-borne flavivirus that is maintained in an enzootic transmission cycle between avian hosts and mosquito vectors, but WNV can also be transmitted to humans and other mammals In recent years, WNV infection has become a public health concern in Europe because of the increasing number of human outbreaks with severe neurological consequences and mortality Seven different phylogenetic lineages of WNV have been described so far The increasing number of outbreaks, as well as emergence of novel strains belonging to both major lineages, emphasizes the necessity to develop a WNV vaccine Because of their genetic relatedness to humans, and their relative susceptibility to WNV infection, rhesus macaques may provide a better animal model for the evaluation of the immunogenicity and efficacy of prototype human WNV vaccines. Candidate WNV vaccines that have been tested in non-human primates include recombinant chimeric yellow fever virus or dengue virus as backbone expressing WNV structural genes We recently performed an experimental infection study in rhesus macaques and common marmosets using the European WNV genotype 1a strain, WNV-Ita09 E. coliNeutralizing antibodies are associated with protection against WNV infection Quillaja saponaria Molina, phosphatidyl choline and cholesterol, and has been shown to increase the migration of the antigen towards the draining lymph nodes To increase vaccine induced T-cell responses we formulated the E protein in Matrix-M (Novavax AB). The adjuvant Matrix-M is composed of a specific purified saponin fraction obtained from the tree Two different WNV vaccine strategies were evaluated for immunogenicity and efficacy against WNV-Ita09 challenge. The first strategy consisted of three immunizations with recombinant E protein adjuvanted with Matrix-M. The second strategy entailed a priming immunization with WNV-DermaVir, followed by two booster immunizations with recombinant E protein and Matrix-M. Nine weeks after the last immunization the animals were challenged with the European WNV-Ita09 strain. Both strategies had been evaluated previously in mice, and in that model induced neutralizing antibodies and WNV-specific cellular immune responses This protocol was approved by the Institutional Animal Care and Use Committee . The qualification of the members of this committee, including their independence from a research institute, is requested in the Dutch law on animal Experiments . At the BPRC, all animal handling is performed within the Department of Animal Science (ASD) according to Dutch law. A large experienced staff is available, including full-time veterinarians and a pathologist. ASD is regularly inspected by the responsible authority , and by an independent Animal Welfare Officer.The Council of the Association for Assessment and Accreditation of Laboratory Animal Care  has awarded full accreditation to the BPRC. The BPRC is fully compliant with international demands on animal studies and welfare as set out by the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes, Council of Europe (ETS 123 including the revised Appendix A), Dutch implementing legislation, and the Guide for Care and Use of Laboratory Animals.Macaca mulatta) used in this study were captive-bred for research purposes and housed socially at the Biomedical Primate Research Centre (BPRC) in Rijswijk, The Netherlands. BPRC facilities comply with Dutch law on animal experiments , the European Council Directive 86/609/EEC, as well as with the ‘Standard for humane care and use of Laboratory Animals by Foreign institutions’ identification number A5539-01, provided by the Department of Health and Human Services of the United States of America’s National Institutes of Health (NIH).The rhesus macaques  were performed under anesthesia using ketamine. Before euthanasia, animals were first sedated deeply with ketamine, and subsequently euthanized by intracardiac injection of an overdose of pentobarbital.Macaca mulatta) were used in this study. All monkeys were adult animals, ranging in age from 5 to 12 years. The animals were in good physical health with normal baseline biochemical and hematological values. At the start of the study, the animals tested negative for antibodies to WNV. To prevent sex, age and weight bias, the animals were assigned randomly to different treatment groups.Eighteen rhesus macaques (E. coli and purified as described previously The ectodomain of the E protein (amino acid residues 1 to 404) of WNV-NY99 was cloned into the bacterial expression plasmid the pET21a, expressed in 5 TCID50 of WNV lineage 1a strain Ita09 A schematic outline of the study is given in During the immunization period, blood was collected using standard aseptic methods from the femoral vein at the start of the study, two weeks after each immunization, 5 weeks after the last booster immunization, and at challenge (week 15) for determination of biochemical and hematological parameters, and for the analysis of vaccine-induced humoral and cellular immune responses. After challenge, 0.5 ml blood samples were collected on a daily basis until euthanasia for viral load determination using real-time RT-PCR. Additional, larger blood volumes were collected at days 3, 7, and 14 post challenge for hematological and biochemical analysis, and for the evaluation of WNV-specific humoral and cellular immune responses.A panel of hematological parameters, i.e. white blood cell count (WBC), red blood cell count (RBC), hemoglobin, hematocrit, mean cellular volume (MCV), mean corpuscular hemoglobin (MCH), platelets, neutrophils, lymphocytes, monocytes, eosinophils and basophils, was analyzed in peripheral blood using a Sysmex XT-2000iV Automated Hematology Analyzer . Biochemical analysis, i.e. creatinine, urea, bilirubin, gamma-glutamyltransferase (γGT), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase, lactate dehydrogenase (LDH), iron, albumin, total protein, cholesterol and glucose, was assessed using a COBAS Integra 400 plus system .2SO4 was added to stop the reaction and plates were measured at 450 nm and 520 nm (reference wavelength) in an ELISA Reader .WNV-specific antibodies in EDTA-plasma were detected by ELISA. Briefly, 96-well microtiter plates were coated overnight with 400 ng of the ectodomain of the WNV-NY99 E protein in vitro neutralizing capacity of sera from vaccinated macaques, plaque-reduction neutralization tests (PRNT50) were performed essentially as described in the Guidelines for plaque reduction neutralization testing of human antibodies to dengue virus . Heat-inactivated EDTA plasma samples taken at various time points were serially diluted and mixed with 25 TCID50 of WNV-Ita09 (lineage 1), or WNV-AUT08 (lineage 2), before addition to adherent Vero cells. Cytopathic effect (CPE) was visualized using a standard microscope, and the TCID50 was calculated using the Karber formula To determine the Cell-mediated immune responses were determined in peripheral blood mononuclear cells (PBMCs) isolated from EDTA-treated blood. PBMCs were tested for WNV-specific secretion using WNV-E protein in ELIspot assays according to the manufacturers’ guidelines . ELIspot assays were performed on freshly isolated cells at weeks 0, 2, 5, 8, and 11.To obtain more detailed information on the quality of WNV-specific responses, intracellular cytokine staining (ICS) was performed. Frozen PBMCs, isolated at weeks 0 and 15 were thawed, and intracellular staining was performed as described previously Viral loads were determined in EDTA-plasma by quantitative real-time RT-PCR as previously described Monkeys were euthanized by infusion of pentobarbital , and full necropsy was performed. Based on the dissemination data of WNV obtained from an earlier experimental infection study Data obtained with the two vaccine strategies were analyzed and compared using an unpaired t-test in GraphPad Prism version 6.0.Both vaccines were well tolerated by the animals and no local reactions were observed after immunization.Induction of WNV-specific IgG was measured two weeks after each immunization, and 4 weeks before WNV challenge Figure 1Vaccine-induced neutralizing antibodies (VN) against WNV were measured using a plaque reduction assay on Vero cells in plasma samples collected at week 0 and week 11. No VN were detectable at the start of the immunization period, but plasma samples collected 5 weeks after the third immunization (week 11) inhibited the infectivity WNV-Ita09, with individual VN titers ranging from 1/3,698 to 1/48,000  in group 1, and from 1/1,567 to 1/21,657  in group 2  . Next, CD4 T-cells and CD8 T-cells were analyzed for their intracellular expression of IFNγ. At week 15, the day of WNV challenge, IFNγ was produced by both CD4 , hematological and biochemical parameters were seen, suggesting that all animals remained clinically healthy during the 2-weeks post-challenge follow-up.At week 15, all of the animals were challenged intradermally with 2×10The protective capacity of both WNV vaccine strategies was assessed by measuring WNV RNA levels in plasma from the macaques by real-time RT-PCR, and in solid tissues by diagnostic PCR. All vaccinated macaques remained negative for WNV in plasma during the entire follow-up period. In contrast, one day after intradermal infection with WNV, 4 of 6 non-vaccinated controls had become positive for WNV (7.700 to 58.000 RNA copies/ml plasma) Table 1.In addition to quantifying WNV by real-time PCR, we tested the plasma taken at 1 to 5 days after WNV exposure for the presence of infectious WNV particles. Plasma of EDTA-treated blood samples was serially diluted, and cultured for 7 days on Vero cells. Only plasma from R03027 (a non-vaccinated control) collected 3 days after WNV exposure caused CPE in Vero cells. Based on a standard curve analysis, the virus titer in this sample was calculated 144 infectious particles per ml of plasma. Sequence analysis confirmed that the CPE in Vero cells was caused by WNV-Ita09 infection.In contrast to the control animals, the vaccinated animals did not show plasma viremia despite exposure to WNV. To assess if the vaccine strategies employed in this study conferred sterilizing immunity, we performed PCR analysis on solid tissues that were collected at euthanasia, 14 days post-challenge. The kidney and urinary system have been suggested as potential target organs for WNV infection in humans Since its introduction in 1999 into the USA, and the subsequent spread into the New World, WNV has emerged as a serious threat to public health. This opinion is confirmed by an increasing incidence of WNV infections in South-East Europe caused by lineage 1 and 2 WNV strains. Currently, no antiviral treatment or vaccine is available to protect humans from WNV infection. In our study, two WNV vaccine strategies (protein prime + protein boost and DNA prime + protein boost) fully protected monkeys against the development of viremia. Although sterilizing immunity was not achieved, only 3 of 12 vaccinated macaques were positive for WNV RNA in one or more solid tissues compared to 6 out of 6 challenge controls.This project aimed to develop a vaccine against WNV that is safe for use in the high-risk human target populations, elderly individuals and those having a compromised immune system. Our prototype WNV vaccines were composed of antigens that are safe to use, as they are well-characterized recombinant components  that lack the ability to replicate. In preparation of the monkey study, the individual antigens, in combination with DermaVir and Matrix-M, showed safety, immunogenicity, and efficacy in rodents et al. Protein-based vaccines can efficiently protect against a number of viruses by eliciting antibodies, while priming the immune system with a DNA vaccine has been shown to induce CD8 T-cell-mediated immunity in vivo in macaques, but in vitro assays for detection of virus neutralizing IgG showed cross-neutralization of the lineage 2 WNV-AUT08 strain. This is likely because of the conservation of dominant neutralizing epitopes in different regions of WNV E protein between lineage 1 and 2 strains We observed cross-protection between North American and European lineage 1 strains, as the vaccine components were based on WNV-NY99, and the challenge strain used in the monkeys was WNV-Ita09 (>99% sequence identity). In mice, E protein/Matrix-M immunizations also afforded protection against lethal challenge with a lineage 2 strain of WNV 4 and 105 PFU 5 TCID50 challenge dose may have been relatively high.At present, our results do not tell us which WNV vaccination strategy is best. Both induced complete protection against viremia, but failed to induce sterilizing immunity against intradermal challenge. Although it is speculative, the vaccine may have failed to achieve sterilizing immunity because of the relatively high challenge dose used. Depending on the mosquito species, the dose of WNV inoculated by one mosquito during blood feeding varies between 10in vitro with a WNV lineage 2 strain. It can therefore be concluded that the vaccines described here are promising candidates for the further development of WNV vaccines for at-risk human populations.Several WNV vaccines have been clinically evaluated Figure S1Gating strategy for intracellular IFNγ staining in CD4 and CD8 T-cells. Representative gating strategy to define intracellular IFNγ-staining in CD4 and CD8+ T-cells of vaccinated rhesus macaques. Cytokine-producing T-cells were defined as LIVE/DEAD negative and CD3 positive cells. Next, CD4 positive cells and CD8 positive T-cells were analyzed for IFNγ production.(TIF)Click here for additional data file.Figure S2Rectal body temperatures of rhesus monkeys during the immunization period and after WNV challenge. Rectal body temperature (°C) measured at indicated time points in animals from group 1 , group 2 , and group 3 . Median rectal body temperature (D) per group at indicated days after experimental WNV infection. Statistically significant differences were defined as p<0.05 and are indicated with arches in the figure.(TIF)Click here for additional data file.


The clinical and cognitive responses to repetitive transcranial magnetic stimulation (rTMS) in bipolar II depressed patients remain unclear. In this study, thirty-eight bipolar II depressed patients were randomly assigned into three groups: (i) left high-frequency (n = 12), (ii) right low-frequency (n = 13), (iii) sham stimulation (n = 13), and underwent four-week rTMS with quetiapine concomitantly. Clinical efficacy was evaluated at baseline and weekly intervals using the 17-item Hamilton Depression Rating Scale (HDRS-17) and Montgomery-Asberg Depression Rating Scale (MADRS). Cognitive functioning was assessed before and after the study with the Wisconsin Card Sorting Test (WCST), Stroop Word-Color Interference Test (Stroop), and Trail Making Test (TMT). Thirty-five patients were included in the final analysis. Overall, the mean scores of both the HDRS-17 and the MADRS significantly decreased over the 4-week trial, which did not differ among the three groups. Exploratory analyses revealed no differences in factor scores of HDRS-17s, or in response or remission rates. Scores of WCST, Stroop, or TMT did not differ across the three groups. These findings indicated active rTMS combined with quetiapine was not superior to quetiapine monotherapy in improving depressive symptoms or cognitive performance in patients with bipolar II depression. Bipolar disorder (BpD) is a severe, recurrent, and chronic psychiatric disorder with an estimated population prevalence of 3%21667Repetitive transcranial magnetic stimulation (rTMS) has been employed in various neuropsychiatric illnesses, especially for major depressive disorder912131415The aim of this study was thus to explore the clinical efficacy and cognitive remediation of rTMS in treating patients with a depressive episode of bipolar II disorder, compare the advantages and disadvantages between left high-frequency and right low-frequency rTMS in reference to its common settings of stimulation parameters in unipolar depression. Given its antidepressant role and preventative effects in treatment-emergent mania or hypomania6F2,32). A hypomanic switch was detected in one patient in the left high-frequency group after 3 weeks of rTMS therapy (BRMS score of 11). This patient recovered spontaneously in several hours without any medical intervention and successfully completed the 4th week of stimulation. Flowchart of subjects in this study was shown in After randomization, 12, 13 and 13 participants entered the left high-frequency, right low-frequency and sham stimulation groups, respectively. The three groups reported withdrawal of one subject each due to intolerable rTMS-induced headache or noise.Thirty-five patients were included in the final analysis. No significant difference was found among the three groups in age, gender, years of education, marital status, onset or duration of illness, or total number of episodes . The aveF2,32) or remission rates  was detected across the three groups.No significant difference in response rates .Meanwhile, all factor scores of the HDRS-17 across the three groups, including anxiety/somatization, cognition, sleep, weight, and retardation, failed to demonstrate any significant difference at baseline and over the 4-week treatment  across the three groups before and after the rTMS treatment  nor cognitive functioning  differed significantly after treatment among the three groups. Similar results were found in exploratory analyses of factor analytic scores of HDRS-17 .rTMS is now widely used in clinical practice for the treatment of unipolar depression91012131419The rTMS parameters in our study were selected for consideration of both effectiveness and safety based on previously published guidelines22Most studies reported no significant differences in cognitive performance between active and sham rTMS25262728Our study had several limitations: (i) The relatively small sample size limited its statistical power to detect real effects, which also weakened the group difference between left high-frequency and right low-frequency stimulation; (ii) The blinding effectiveness of mood raters was not assessed, which may potentially influence the trial validity and the interpretation of our findings; (iii) Although the average quetiapine doses did not differ significantly across the treatment groups, the dose was not determined before initiation of the study, and the serum concentration of quetiapine was not monitored during the trial. Moreover, the antidepressant role of quetiapine might cover up the true effect of active rTMS on bipolar depression; (iv) We used a relatively low stimulation intensity of 80% of motor threshold and daily stimulation of 1200 pulses for safety reasons, which may have lowered the treatment efficacy. Meanwhile, 4-week daily active rTMS may not be sufficient to achieve apparent clinical outcomes, which possibly require longer stimulation sessions.In conclusion, as our data demonstrated, active rTMS (either left high-frequency or right low-frequency stimulation) in combination with quetiapine was not superior to quetiapine monotherapy with sham-stimulation in improving either depressive symptoms or executive functioning in patients with a depressive episode of bipolar II disorder. Further investigations with larger sample size are needed to determine the optimal stimulation parameters, including stimulation location and duration of rTMS of bipolar depression.This prospective, randomized, and controlled study was approved by the Ethics Committee in the First Affiliated Hospital of Zhejiang University, registered in the Chinese Clinical Trial Registration (ChiCTR-TRC-14004814) on 16/06/2014, an institution belong to the World Health Organization international clinical trial registry platform. The methods were carried out in accordance with the approved guidelines.Between July, 2014 and July, 2015, we recruited totally 40 patients in this study. Their diagnosis of depressive episodes in bipolar II disorder was ascertained by a senior clinician (N.W.) with the Structured Clinical Interview for DSM-IV axis I (SCID-I). All participants were drug-naïve or had withdrawn from psychoactive medications for at least 1 week prior to commencement of the study. Patients’ baseline total scores exceeded 20 on the 17-item HDRS and ≥4 on the Clinical Global Impression severity of illness scale (CGI-S). Physical examination and routine laboratory tests were performed to exclude the presence of severe medical illnesses or neurologic disorders. Other exclusion criteria included any comorbid psychiatric illness, any form of metal implants, or any history of suicide attempt, drug abuse, seizures, or medications known to lower seizure threshold. Two participants did not meet the inclusion criteria and the remaining 38 participants were randomly assigned in a 1:1:1 ratio to 1 of 3 groups: left high-frequency rTMS, right low-frequency rTMS, or sham stimulation. Prior to the trial, a sequence number was randomly generated for each participant by a computer and a randomization table was also generated. Randomization block, which was constituted by each 6 participants (two were randomly assigned to each group), was used to minimize the imbalance of group sizes. Written informed consent was provided by all participants before commencement of the study. Demographic and clinical profiles are listed in In addition to the assigned rTMS or sham therapy, all patients received quetiapine, which was previously reported to be effective in treating bipolar depressionAll rTMS procedures were conducted in a certificated laboratory located in the Department of Mental Health equipped for emergency care. Magnetic stimulation was conducted on a Magstim rapid stimulator . The resting motor threshold (MT) was determined by the minimum intensity of magnetic stimulation on the primary motor cortex to elicit five visible muscle contractions out of ten consecutive stimuli in the contralateral abductor pollicis brevis muscleIn the left high-frequency rTMS group, every treatment session consisted of 30 trains of 4 s stimulation at 10 Hz and 80% MT followed by 12 s off . In the right low-frequency rTMS group, treatment consisted of 120 trains of 10 s stimulation at 1 Hz and 80% MT followed by 2 s off . In the sham-stimulation group, all rTMS parameters were consistent with the left high-frequency rTMS group, except that the coil was positioned vertical to the scalp, thereby eliminating the influence of magnetic field on the cortexDepression severity was assessed using the Hamilton Depression Rating Scale (17-item HDRS) at baseline, with the Montgomery-Asberg Depression Rating Scale (MADRS) at weekly intervals throughout the study. A clinical response was defined as a decline in HDRS-17 score greater than 50%; remission was defined as an HDRS-17 score of ≤7. We also compared groups at each time point on the 5 factor scores derived from the HDRS-17: anxiety/somatization, cognition, sleep, weight, and retardation. We used the Beth-Rafaelsen Mania Scale (BRMS) to monitor for any rTMS-induced mania/hypomania symptoms. Two experienced clinicians (J.B.L. & M.L.H.), who were blinded to grouping and stimulation, rated the HDRS-17 and the MADRS, respectively.Cognitive functioning was assessed at the beginning and the endpoint of the study using the Chinese modified versions of the Wisconsin Card Sorting Test (WCST)3335p < 0.05 was considered statistically significant.Statistical analyses were conducted using SPSS version 16.0. One-way analysis of variance (ANOVA) was used to analyze measurement data involving sociodemographic and clinical profiles. An Intent-To-Treat method was used to analyze outcome measures  with repeated-measures ANOVA. Chi-square test was used to compare gender, marital status, response rate, and remission rate across the three treatment groups. A two-tailed How to cite this article: Hu, S.-H. et al. Efficacy of repetitive transcranial magnetic stimulation with quetiapine in treating bipolar II depression: a randomized, double-blinded, control study. Sci. Rep.6, 30537; doi: 10.1038/srep30537 (2016).


Young women are under-represented in cardiovascular disease research, with obesity and cardiometabolic risk factor interventions generally targeting older adults. Furthermore, appropriate study designs for young women remain uncertain. This study aimed to assess the impact of a 12 week multi-disciplinary lifestyle intervention on cardiometabolic risk factors in premenopausal women with abdominal obesity.Women aged 18–30 y with abdominal obesity [waist circumference (WC) ≥ 80 cm] were randomised to a 12 week lifestyle intervention (n = 26) of physical activity, nutrition education and cognitive behavioural therapy, or a wait-list control group (n = 17). Both groups completed anthropometric, biochemical, nutrition and fitness testing, at pre (0 weeks) and post (12 weeks), with intervention participants completed follow-up testing at 24 weeks.2max, and total energy intake. Most changes were maintained at 24 weeks post-intervention. Similar within-group improvements were observed in control participants in WC, WHR, WHtR, and systolic blood pressure but no changes were detected in physical activity and nutrition.Results from a linear mixed model showed no between-group differences, other than increased physical activity in the intervention group, at post. In the intervention group alone, positive within-group changes were observed in WC, waist-hip-ratio (WHR), waist-height-ratio (WHtR), resting heart rate, blood pressure, predicted VOCardiometabolic risk factors were decreased as a result of a lifestyle intervention in young women with abdominal obesity. It is difficult to describe observations in the control group without greater understanding of the behaviour of wait-list participants.ACTRN12612001017819Australian New Zealand Clinical Trials Registry  Cardiovascular disease (CVD) represents a major health threat to women worldwide , consequDespite global strategies for preventive health, there is poor understanding of early risk factors (cardiometabolic risk factors) in young women, and lifestyle interventions can improve health outcomes. Moreover, research assessing the effectiveness of weight management interventions specifically targeting young women is relatively recent . EffectiPoorer retention but greater success has been reported when results of an online team-based weight loss lifestyle intervention were compared in younger and older adults . HoweverTherefore, the primary aim of this study was to assess the effectiveness of a lifestyle intervention for reducing CVD risk in young women with abdominal obesity, using a randomised controlled trial (RCT) design. Specifically, the RCT involved a 12-week multi-disciplinary program  with Caucasian women aged 18 to 30 years, who shared the cardiometabolic risk factor of abdominal obesity [elevated waist circumference (WC) ≥ 80 cm]. A secondary aim was to examine the effectiveness of the intervention through an improved understanding of the sustainability of any changes. It was hypothesised that the lifestyle intervention would be effective (and sustainable) in reducing cardiometabolic risk in young women with abdominal obesity.th 2009  on December 18th 2009  Texts. Tth 2009 . Data weSixty-two female Caucasian tertiary students at risk of CVD volunteered for this study. Advertisements for recruitment specifically sought young women with abdominal obesity (WC ≥ 80 cm), who were also leading a sedentary lifestyle. Included were women aged 18 to 30 years; with a waist circumference ≥ 80 cm, and who were physically inactive . Exclusion criteria were being pregnant or breastfeeding; a history of bariatric surgery; and/or having a diagnosis of liver or kidney disease; heart arrhythmia; insulin dependent diabetes mellitus; polycystic ovarian syndrome; thyroid abnormalities. All participants were non-smokers.A power analyses estimated that 18 participants per group would provide the appropriate sample size to detect a large between-subject difference of 1.0 standard deviation  in waist circumference from pre-intervention to post-intervention. To allow for 20% attrition, there was an attempt to recruit an initial sample size of 44 participants (22 per group). For each testing period , participants attended the laboratory on two occasions. They were requested to refrain from strenuous physical activity in the 24 hours prior to all laboratory sessions. The first visit required the participant to arrive in a fasted state and clinical testing lasted 75 minutes. The second visit of 60 minutes required participants to abstain from caffeine and alcohol for 12 hours.For the intervention group, testing of cardiometabolic risk factors was performed at pre-intervention (0 weeks), post-intervention (12 weeks) and following a short-term sustainability phase (24 weeks). The control group underwent testing at pre-control and post-control (12 weeks) time periods only. For all testing, measurements were taken at the same time of day (±2 hours) and by the same researcher. For participants in the control group, monthly contact was made to remind them of the control criteria. For the sustainability phase between post-intervention (12 weeks) testing and the following 12 weeks, a sustainability strategy was delivered electronically to the intervention group. This involved a fortnightly newsletter on healthy living tips from evidence-based resources.Neither the investigators nor the participants were blinded to group allocation as this was considered impractical for the long-term investigation and limited members of the research team. However, to minimise contamination, participants in the intervention group were asked to refrain from disclosing their intervention experience to researchers assigned to data collection and/or analysis and to wait-list control participants. To further minimise bias, assessor blinding occurred within dietary measures and biochemical analyses. For the primary outcome variable of WC, measures were completed in duplicate with reported measures of reliability: coefficient of variation (CV), intraclass correlation coefficient (ICC) and measurement error (ME).A self-administered lifestyle survey was completed to provide data on (i) health status and medical conditions, (ii) nutrition , and (iii) current physical activity habits. The lifestyle survey  was deve-2 [Body composition was assessed via waist circumference (WC), hip circumference, body mass index (BMI), and body mass. WC was measured to the nearest 0.1 cm in the horizontal plane at the level of the midpoint between the iliac crest and lower costal margin . For WC,Metabolic syndrome markers and additional biochemical parameters: Metabolic syndrome was defined according to the most recent and unified criteria [criteria . Markers-1 was deemed high risk [Following an overnight fast, intravenous blood was collected Blood lipid profiling of serum concentrations of triglycerides, total cholesterol and high-density lipoprotein (HDL) cholesterol were measured using the Reflotron Plus desktop analyser . Fasting plasma glucose, insulin and hs-CRP concentrations were analysed by clinical pathology at a leading hospital. Insulin resistance was estimated by HOMA-IR using the equation . For hs-igh risk .After 10 minutes of rest in a quiet, temperature controlled room SBP and DBP were obtained in duplicate from the left arm with an automated digital sphygmomanometer  with the participant in the supine position.2max), where heart rate was extrapolated against work rate (W) using regression analysis. Physical activity behaviour  physical activity (2) nutrition education, and (3) cognitive behavioural therapy . In contParticipants undertaking the intervention completed two supervised exercise sessions (progressive circuit training) and one unsupervised, but were prescribed one home-based session  per week. All sessions were devised and administered by a qualified Exercise Scientist who has experience in exercise prescription for elite and healthy populations. The supervised sessions consisted of a general warm-up, a combination of aerobic activities, dynamic strength and/or resistance training, abdominal conditioning, and stretching. Session duration lasted approximately 60 min, with the intensity of the exercise increasing from 6.0 to 8.5 on the OMNI Picture System (ranging from 0 = extremely easy to 10 = extremely hard), by the Participants in the intervention group received weekly nutrition education sessions guided by a qualified dietician about healthy eating choices from the existing Australian Dietary Guidelines . This inWithin the framework of self-determination theory , weekly P ≤ 0.05. A linear mixed-model analysis was used to calculate the differences between groups and across time for the intervention and control groups. Hedge’s g effect size was used to assess the magnitude of effect. An effect size ≥ 0.2 was considered small, ≥ 0.5 medium, and ≥ 0.8 large [Data were analysed using IBM SPSS Statistics, Version 20 for Windows . Data were tested for normal distribution , with lo.8 large . Mean diA total of 39 participants were included in the linear mixed-model analysis. For reasons described , 27% of g = 0.79). With the exception of WC and weekly physical activity most cardiometabolic risk factors were within normal limits for the population at baseline testing (see footnotes g = 2.14). Similarly, both the absolute and percentage change in physical activity from pre to post testing were greater for the intervention group, as were the absolute and percentage changes in predicted VO2max when compared to controls.2max , physical activity  and total energy intake   changes pre-to post-intervention for WC , WHR , WHtR , SBP , DBP , resting heart rate , predicted VOj, -22%) . Absolut2max and physical activity reduced during the 12-week sustainability phase however, physical activity remained greater than at pre-intervention.Many of the improvements observed at post-intervention were maintained at sustainability (24 weeks) testing including, WC, WHR, WHtR, DBP and resting heart rate . The afo2max, resting heart rate and energy intake following the 12-week control period.Despite being requested to maintain normal lifestyle habits, the control group displayed several improvements from pre- to post-testing, including WC , WHR , WHtR  and SBP , while circulating triglycerides rose . In contThe effectiveness of the multi-disciplinary lifestyle intervention for reducing CVD risk in young women was highlighted by within-group improvements in a range of risk factors for the intervention group, with several of these improved markers retained 12 weeks after completion of the lifestyle intervention. Thus, these data suggest a relatively successful intervention for reducing CVD risk with promising sustainability. However, when between-group comparisons were made with the control group, the findings suggested a research design that was largely unsuccessful in identifying the effectiveness of the intervention phase. This raises several concerns associated with a wait-list control design when used with overweight/obese young women. Concerns from the current study support previous findings that describe more difficulties in retaining younger than older females to research trials .Positive changes within the intervention group were demonstrated with improvements in WC related measures, systolic and diastolic blood pressure, aerobic fitness and physical activity, and dietary energy intake. Collectively, these measures imply that the intervention produced cardiovascular, more so than metabolic, benefits for the intervention group. The use of investigative procedures such as non-invasive echocardiography or MRI may provide insight into the significance of these changes .Improvements in fitness and physical activity and reduced energy intake also suggest that the exercise and nutrition education components of the intervention, respectively, were effective. Moreover, there was strong evidence for sustainability of intervention-induced improvements at 24 weeks, suggesting that the CBT component produced positive behavioural change in participants. It has been shown that poor adherence to behavioural programs is a barrier to successful long-term weight maintenance beyond the completion of the intervention . In the However, similar post-intervention changes in our wait-list control group, made it difficult to detect any anthropometric, biochemical, fitness or dietary differences between groups following the 12-week intervention phase. The control group in this RCT also displayed a decrease in WC that paralleled the intervention group, despite undetectable changes in self-reported physical activity and nutrition. The decreased WC of the control group is particularly difficult to explain without further investigative procedures such as accelerometry for physical activity and more rigorous dietary monitoring, but does suggest that basic awareness of CVD risk might be enough to evoke change in targeted populations ,44.Randomised controlled trials provide the highest level of evidence for the effects of an intervention and are deemed to be scientifically rigorous , with coapps or support groups might improve commitment and maintain control group compliance. There were also some difficulties encountered with recruitment, with almost a quarter of interested and eligible participants failing to engage after initial commitment . Complexities associated with recruiting young women for weight management trials, especially from ‘Generation Y’, may result in smaller sample sizes and require shorter periods of engagement [Retention and compliance of wait-list participants is also an issue for consideration when planning control conditions essential for maintaining the rigour of a randomised control design. In this study, more than one-third of the control group failed to return for post-testing at 12 weeks despite researcher attempts to maintain contact. In contrast, once engaged in the intervention group, retention to lifestyle change was high for at least 24 weeks. It is postulated that assigning participants to the control group decreased their motivation to participate. Thus, the duration (12 weeks) and/or conditions (no changes to existing lifestyle) of the control group do not appear suitable for women of this age group and demographic. Alternative strategies for immediate engagement, perhaps via topics of interest using multimedia such as health-related gagement .Although not always the case , successThis study is not without limitations. Despite recruitment and retention strategies, the sample size, particularly in the control condition, was lower than anticipated at completion of the study, suggesting it was slightly under-powered. Additionally, the results are specific to Caucasian women at a tertiary institution. To capture any potential changes to control groups in a wait-list design, objective measures  might be useful. In addition, the use of more objective measures of physical activity and dietary compliance would strengthen evidence of change in this age group of women. In agreement with previous reports, not all food and activity diaries were completed with precision. Future researchers may benefit from the use of diet quality changes rather than diet intake.Nonetheless, the study contributes to a very limited number of healthy lifestyle interventions in young adult women (< 30 years of age) with cardiometabolic risk factors . The mulWithin-group analysis showed that the multi-disciplinary lifestyle intervention comprising physical activity, nutrition education and CBT was positive for the reduction of CVD risk factors both immediately after and beyond the completion of the program. However, we also observed positive, but unexpected and difficult to explain, changes in the wait-list control group. Therefore, comparative lifestyle benefits for the intervention group may have been masked by undetectable weight management behaviour in the control group. When considered alongside the difficulties faced with recruitment and retention, especially in the nature of the control group, these results provide a challenge for prospective study designs with young women with cardiometabolic risk factors. Traditional RCT designs may be problematic for healthy lifestyle interventions in young women.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S1 Text(DOC)Click here for additional data file.S2 Text(DOC)Click here for additional data file.S3 Text(DOC)Click here for additional data file.