RankComp algorithm to identify DE genes for each of LARC sample compared with its own normal state. Then, we identified genes with significantly different deregulation frequencies between the non-responders and responders, defined as cancer-related pCRT-response genes. Pathway enrichment and protein-protein interaction analyses showed that these genes specifically and intensively interacted with currently known effective genes of pCRT, involving in DNA replication, cell cycle and DNA repair. In contrast, after excluding the cancer-related pCRT-response genes, the other DE genes between non-responders and responders were enriched in many pathways of drug and protein metabolisms and transports, and interacted with both the known effective genes and pharmacokinetic genes. Hence, these two types of DE genes should be distinguished for investigating mechanisms of pCRT response in LARCs.For patients with locally advanced rectal cancer (LARC) treated with preoperation chemoradiation (pCRT), identifying differentially expressed (DE) genes between non-responders and responders is a common approach for investigating mechanisms of chemoradiation resistance. However, some of such DE genes might be irrelevant to cancer itself but simply reflect the pharmacokinetic differences of the normal tissues. In this study, we adopted the  Currently, neoadjuvant preoperation chemoradiation (pCRT) with 5-fluorouracil (5-FU)-based regimens followed by surgical resection is extensively employed for the locally advanced rectal cancers (LARCs)5678910111213141516RankComp was proposed to detect the deregulated genes in a disease sample through comparing the REOs in this sample with the stable REOs in normal human tissuesHowever, it is difficult to identify DE genes in responders and/or non-responders compared with normal controls because currently few data for studying LARCs response to chemoradiation include normal rectal samples. Although there are some available data of normal rectal samples generated by different laboratories, they cannot be compared directly due to experimental batch effects18RankComp to identify DE genes for each of 38 non-responders and 34 responders of LARCs. Subsequently, we identified 186 genes that had significantly different deregulation frequencies between the non-responders and responders, defined as cancer-related pCRT-response genes. Among these genes, 57 genes were shared with DE genes between the non-responders and responders identified with RankProductIn this study, using data of 34 normal rectal tissue samples from three datasets, we extracted gene pairs with identical REOs in all the samples as the landscape of normal rectal tissue. Then, we used p-value = 1.0E-100), suggesting that the REOs of these gene pairs are highly stable in normal rectal tissues measured by the three different platform. Thus, taking the 35,127,930 gene pairs with identical REOs in the 34 normal rectal tissue samples measured by the three platforms as the normal REOs landscape of rectal tissues, we could apply RankComp to detect DE genes in rectal cancer samples measured by any of these platformsBecause only a limited number of normal rectal samples measured by a particular gene expression profiling platform could be obtained, we collected 34 normal rectal samples from three datasets measured by the Affymetrix, Illumina and Agilent platforms (see Methods), respectively, to construct cross-platform stable relative expression orderings (REOs) of gene pairs in normal rectal tissues. As reported in our recent paperRankComp through comparing with the stable REO normal landscape of rectal tissueThen, we identified DE genes in each of the 72 samples of rectal cancer collected from two datasets (GSE35452 and GSE53781) with p-value = 1.3E-10), indicating that at least the overlaps of the two lists of DE genes between the non-responders and responders were significantly reproducible in the two datasets. Notably, the percentage of overlapping genes between the two lists of DE genes was apparently low, which indicated that only a small percentage of DE genes could be found in each of the two datasets due to insufficient statistical power27Firstly, we identified 1288 and 805 DE genes between the non-responders and responders in the GSE35452 and GSE53781 datasets, respectively . The two lists of DE genes shared 101 genes, of which 80% showed consistent deregulation directions (up- or down-regulations) in the non-responders compared with responders across the two datasets. This consistency score was unlikely to be observed by random chance.Then, through the human protein-protein interaction (PPI) networkIn this work, we proposed to distinguish cancer-related and cancer-unrelated pCRT-response genes for genes differentially expressed between non-responders and responders of LARCs. We showed that these two types of genes affect LARCs response to pCRT in totally different ways. Notably, according to the framework of cancer hallmarks network3030In contrast, the cancer-unrelated pCRT-response genes were significantly enriched in typical metabolism pathways related to drug metabolisms such as cytochrome P450 which contributes to multidrug resistance in tumor30In summary, the two types of pCRT-response genes should be distinguished in studying mechanisms of LARCs response to pCRT.http://www.ncbi.nlm.nih.gov/geo/), as described in detail in Five microarray datasets for rectal normal samples and LARCs were downloaded from Gene Expression Omnibusin vivo7For the data of LARCs extracted from GSE35452 and GSE53781, there were some subtle differences in the protocol of chemoradiation and tumor response assessment. Firstly, the 5-fluorouracil-based regimens of chemotherapy in the two studies were ftorafur-uracil and capecitabine, respectively, both of which are metabolized to 5-FU The threshold value of frequency for stable relative ordering of gene pair in normal rectal samples was defined as 100%.X is differentially expressed in a given disease sample, Fisher’s exact test is used to test the null hypothesis that the frequency of reversal gene pairs supporting its upregulation and the frequency of reversal gene pairs supporting its downregulation are not significantly different. If the expression level of gene X is consistently lower (or higher) than another gene in normal samples but reversal in a disease sample, then this reversal gene pair is considered to support upregulation (or downregulation) of gene X in this disease sample. Finally, a filtering process is utilized to retain only those DE genes which are still significant after excluding their coupled reversal gene pairs including any other DE genes. The detail of this method is discribed in our previous workp-values were adjusted using the Benjamini and Hochberg procedure to control the False Discovery RateThen, we applied the RankComp algorithmp-values were adjusted using the Benjamini and Hochberg procedure to control the False Discovery Rate.For a specific gene, the Fisher’s exact test was used to test whether its deregulation frequency in the non-responders is significantly different from that in the responders. The p-values were adjusted using the Benjamini and Hochberg procedureWe used the RankProd algorithmk DE genes, of which s genes had the same up- or down-regulation directions, the concordance score was calculated as s/k. This score was used to evaluate the consistence of DE genes extracted from independent datasets. The probability of observing a concordance score(s/k.) by chance can be evaluated using the cumulative binomial distribution model as follows:For DE genes from two independent datasets sharing pe is the probability of a gene having the same dysregulation direction in two gene lists by random chance . The concordance score is considered significantly if the p-value < 0.05.In p-values were adjusted using the Benjamini and Hochberg procedureData of 223 pathways covering 6290 unique genes were extracted from the Kyoto Encyclopedia of Genes and Genomes (KEGG)The PPI data integrating eight databases were composed of 142, 583 distinct interactions involving 13693 human proteins, which were discribed in our previous workWilcoxon’s rank-sum test was used to test whether the number of interaction links between the known pCRT-response genes and a gene set were significantly different from the number of interaction links between the known pCRT-response genes and another gene set.http://www.r-project.org/).All statistical analyses were performed using the R 3.12 .Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


N,N’-methylenebisacrylamide was used as cross-linker and ammonium persulphate as initiator. As a consequence of the inclusion of clay into the polymer matrix and the intercalation of PMAA between the layers as well as the presence of hydrophobic interactions occurred between partners, the final hydrogel nanocomposites possessed greater swelling degrees, slower de-swelling process and enhanced mechanical properties depending on the clay type in comparison with pure hydrogel. In vitro MTS -5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt]) colorimetric assay showed that direct exposure with PMMA-clay-based constructs did not affect cell viability and proliferation in time (24 and 48 h) on either normal or adenocarcinoma cell lines.Present study refers to the synthesis of new advanced materials based on poly(methacrylic acid) (PMAA) with previously reported own advanced modified clays by edge covalent bonding. This will create the premises to obtain nanocomposite hydrogels with combined hydrophilic-hydrophobic behavior absolutely necessary for co-delivery of polar/nonpolar substances. For the synthesis,  Clay fu−] sheet . Highly −] sheet ,21,22,23N-isopropylacrylamide by introducing Laponite XLG or XLS. Fukasawa et al. . Am. Am60]. SEM analyses of the freeze-dried nanocomposite hydrogels were performed to observe the morphology of the hydrogels prepared with various types of clay . The micThe fineness of the structure, expressed by the mean of pore diameter and uniformity, can be visualized comparatively for the four samples, in TEM analyses were used to observe the intercalated or exfoliated structure of clay inside poly(methacrylic) hydrogels. TEM images of PMAA-Cl 93A-C18 nanocomposites  revealedCytotoxicity of biopolymer modified clays on different cell lines was assessed using a MTS method. Cell viability of normal Madin-Darby bovine kidney (MDBK) cells and HT-29 colorectal adenocarcinoma cells, two in vitro model cellular lines, was measured following treatment with different disk composite for 24 and 48 h. Metabolic activity of normal and tumoral cells after the two time points of direct treatment with biopolymer-modified clays was not affected . Thus, nThe morphology of the cell after biopolymer-modified clay disks treatment was analyzed using a brightfield microscopy system which scanned the entire probes. As seen in ® 93A; 90 meg/100 g)). The synthesis of in house modified clay with octyl dimethylmethoxysilane (Cl 93A-C8) or octadecyl dimethylmethoxysilane (Cl 93A-C18) was described elsewhere )colorimetric method  was used [One day before treatment with the disks cells were allowed to adhere at a density of 5 × 10was used . Briefly●  Microscopy methodThe morphology of HT-29 and MDBK cells after incubation with the disks for 24 and 48 h were monitored using an automated platform for live cell imaging. The probes were scanned using TissueFAXSiPlus brightfileld imaging system  with a 20× objective of a Zeiss AxioImager.Z1 inverted microscope and fields of view were generated.Newly synthesized hydrogel nanocomposites based on organically (unmodified and modified) montmorillonite were prepared and their properties were tested. The experiments derived from this study prove for the first time the capacity of hydrophobic modified commercial clay to slow down the release process of water from hydrogel network and moreover, the enhanced mechanical behavior of the final materials. These results are strongly related to the organomodified structure of clay leading to the intercalated structure of the final nanocomposites. In vitro studies revealed a good biocompatibility of PMMA-clay based novel structures. Altogether, the newly synthesized nanocomposite hydrogels could be recommended for situations where controlled release of the drug is required and hydrogel based material should reach to a specific target totally unaltered. Future perspectives include the synthesis of interpenetrated hydrogel matrices using a natural polysaccharide. The method will connect the biopolymer with pH responsive PMAA to design new semi-interpenetrated networks with encapsulated amphiphilic nanoclays to render tunable and efficient advanced materials exhibiting enhanced mechanical strength, improved stability and adjustable responsive properties, for co-delivery of anti-cancer drugs in gastrointestinal tract. The pH change from the stomach to the intestine could be used as a trigger for releasing encapsulated therapeutic agents from pH-responsive semi interpenetrated hydrogel networks-newly functionalized clay nanocomposites. In order to evaluate the influence of the novel functionalized clay system on the sustained release of the anticancer drugs, the final complex systems are foreseen to be investigated in vitro and in vivo. Biodistribution profiles, pharmacokinetic profile and antitumor activity of the drug loaded clay nanocomposite vehicle will be followed in comparison with the neat hydrogel network to prove its enhanced efficacy.


Accelerated diagnostic pathways (ADP) have been designed to identify low-risk chest pain patients in the emergency department. This review article discusses the Asia-Pacific Evaluation of Chest Pain Trial (ASPECT) score, the Accelerated Diagnostic Protocol for Chest Pain Trial (ADAPT) score, the Emergency Department Assessment of Chest Pain Score (EDACS), the HEARTScore and the HEART pathway. These ADPs have been validated in various studies and aid the emergency provider with identifying the low-risk chest pain patient who is appropriate for discharge home, while at the same time highlighting those patients who would benefit from further in-patient work up. These approaches should be paired with patient input and shared decision-making strategies. Chest pain is one of the most frequent complaints of patients presenting to the emergency department (ED). Approximately 10–20% of patients who present to the ED with chest pain are suffering from an acute coronary syndrome (ACS), requiring early intervention and treatment.The American College of Cardiology and American Heart Association (ACC/AHA) have developed guidelines in an attempt to standardize the approach to patients with chest pain. The 2010 and 2014 guidelines recommend the use of the Global Registry of Acute Coronary Events (GRACE) score or thrombolysis in myocardial infarction (TIMI) risk score as part of the initial evaluation for possible ACS. However, neither TIMI nor GRACE was designed for ED chest pain risk stratification. The TIMI score was designed to be applied to patients with unstable angina or non ST-elevation myocardial infection (NSTEMI) to determine their risk for 14-day mortality, new or recurrent acute myocardial infarct (AMI) or severe recurrent ischemia requiring urgent revascularization.5Accelerated diagnostic pathways (ADP) were developed to provide guidance to emergency clinicians to determine the level of risk for patients with possible ACS and support appropriate testing for each patient with chest pain. The goal of ADPs is to identify the very low-risk patients for early discharge, while at the same time identifying those patients more likely to benefit from stress testing and angiography. ADPs are starting to appear in ACC/AHA guideline updates. A more detailed description of the most commonly used ADPs is found below.The ASia-Pacific Evaluation of Chest Pain Trial (ASPECT) aimed to prospectively validate the safety of a predefined two-hour ADP to identify ACS patients. The study was conducted in 14 EDs in nine countries in the Asia-Pacific region.6Those with a TIMI risk score of 0, no new ischemic changes on the electrocardiogram (ECG) and a negative 0- and 2-hour point-of-care biomarker results were deemed low risk and eligible for early discharge. Primary endpoint was major adverse cardiac events (MACE) within 30 days. MACE was defined as death (not clearly non-cardiac), cardiac arrest, an emergency revascularization procedure, cardiogenic shock, ventricular arrhythmia needing intervention, high-degree atrioventricular block needing intervention and acute myocardial infarction. A total of 3,582 patients were enrolled, of which 352 (10%) were considered low risk. Within the low-risk group there were three (0.85%) MACE. The ASPECT ADP had a 99.3%  sensitivity with a negative predictive value of 99.1% (95% CI 98–100) for MACE.The Accelerated Diagnostic Protocol for Chest Pain (ADAPT) trial was a prospective observational study from the same investigators as the ASPECT trial.In the Advantageous Predictors of Acute Coronary Syndrome Evaluation (APACE) validation study, patients were classified as low risk if they had a TIMI score of 0 or 1 , a non-ischemic ECG and normal 0- and 2-hour high sensitivity troponin measures .8 Of theThe Emergency Department Assessment of Chest Pain Score (EDACS) is the first emergency medicine-based risk score derived from clinical data and was developed as a chest pain score to identify patients safe for early discharge. The derivation cohort consisted of patients enrolled in the ADAPT study.The EDACS score was incorporated into an ADP, where low risk was identified as an EDACS score < 16, no new ischemia on ECG and a negative 0- and 2-hour troponin. In this observational cohort patients who met these criteria were identified who would have been safe for discharge home without further workup. The derivation cohort  and the validation cohort (608 patients) identified 40–50% of patients as low risk. The sensitivity was 99% (95% CI [96.9–99.7]) for MACE. EDACS has been validated in a randomized trial comparing EDACS to ADAPT.10The HEARTScore was developed to score predictors of primary end points based on clinical experience and previous medical literature.In the first retrospective validation study 122 patients presented to the ED with chest pain. Results are presented in Sixteen patients died (0.7%), 13 of whom died of a cardiac cause. One of these patients was in the low-risk HeartScore group, five were in the intermediate-risk group and seven in the high-risk HeartScore group. The C-statistics of the HeartScore when compared to TIMI and GRACE were as follows: HEART 0.83, TIMI 0.75, GRACE 0.70 (p<0.0001). This study provided additional support for use of the HeartScore as an ADP for low risk chest pain patients.th percentile upper-reference limit.While the HeartScore is predictive of MACE, many clinicians consider the 1.7% risk of MACE in a patient identified as low risk by the HeartScore to be too high. Furthermore, with the HeartScore it is possible to have a patient with a low-risk HeartScore, despite a positive troponin. The Heart pathway was designed to lower the missed MACE rate of the HeartScore below 1%, by separating the troponin results from the remaining “Hear” score and using two troponin measures (at 0 and 3 hours) instead of one. To be considered low-risk using the HeartScore pathway you must have a Hear(t) score of 0–3 and have both serial troponin measures less than the 99The first study to validate the HeartScore in the U.S. enrolled 1,070 chest pain patients in an observation unit and revealed that five patients with an NSTEMI had low-risk HeartScore.In recent years there has been growing attention to shared decision-making. Shared decision-making involves educating patients on their health risks, as well as the risks of testing, and discussing their treatment options. This is often done using a pictogram developed at the Mayo Clinic called the Chest Pain Choice.The low-risk patient with chest pain can be a high-risk scenario for the emergency physician. Accelerated decision protocols have been designed to aid the emergency physician in decision-making with regards to assessment of these patients. The use of these ADPs can reduce cost, length of stay and risk of unnecessary testing in chest pain patients. It is important for all emergency physicians to be familiar with different ADPs, and to know their benefits and limitations. All of the above-described ADPs are validated choices for risk assessment of low-risk chest pain patients in the ED. UsTe of any of these ADPs should be considered within standard of care. The choice to select a specific ADP for use in the ED can be done on an institutional level or can be the choice of the individual practitioner. Within the authors’  institution, the Heart pathway was implemented alongside a shared decision model for its high sensitivity, negative predictive value and ease of use. Shared decision-making tools may assist patients with acute chest pain and their providers to navigate difficult disposition decisions.


The mutualistic interaction between the gut microbiota (GM) and its host profoundly shapes many aspects of our physiology. The composition and activity of the gut microbiota is modulated by environmental factors such as dietary habits and antibiotic treatments. In rodents, studies demonstrate that the GM is a crucial regulator of bone metabolism and that modulation of the GM composition by probiotic interventions can prevent castration-induced bone loss. Short-term colonization of germ-free mice with GM results in an activation of CD4+T cells, resulting in increased levels of pro-inflammatory cytokines in bone and thereby activation of osteoclastic bone resorption. Besides these immune-mediated effects on bone mass, the GM is involved in nutritional uptake and may, thereby, regulate overall body growth and bone sizes possibly mediated via altered IGF-I levels. We recently introduced a new term “osteomicrobiology” for the rapidly emerging research field of the role of the microbiota in bone health. This research field is aimed to bridge the gaps between bone physiology, gastroenterology, immunology, and microbiology. Future studies will determine if the GM is a novel therapeutic target for osteoporosis and if the GM composition might be used as a biomarker for fracture prediction. Osteoporosis-related fractures are a huge economic burden on health care systems and fractures are associated with significant mortality and morbidity. The lifetime risk of any osteoporotic fracture is high in the western world and about 50% of women and 20% of men older than 50 years will have a fragility fracture in their remaining lifetime . OsteopoThe skeleton is remodeled by bone-forming osteoblasts (OBs) and bone-resorbing osteoclasts (OCLs) . The skeOsteoimmunology is a field describing the interactions between the immune and the skeletal systems . The assIn recent years, the development of effective sequencing technologies has made it possible to study the composition of the GM and its importance for both health and disease. The GM constitutes trillions of microorganisms, mostly anaerobic bacteria, which collectively contain 150-fold more genes than our human genome. It is acquired at birth and can be considered a multicellular organ that communicates with and affects its host in numerous ways . The comThe use of gnotobiotic animals has made it possible to characterize the effects of the GM. Germ-free (GF) animals have immature mucosal immune systems with poorly developed gut-associated lymphoid tissue (GALT). Furthermore, GF mice have reduced number of CD4+T cells in the spleen and fewer and smaller germinal centers within the spleen, suggesting that the GM is capable of shaping systemic immunity . BesidesOur group was the first to show that the absence of GM in GF mice leads to increased bone mass, compared to conventionally raised (CONV-R) mice . We foun−/− mice, cortical bone thickness in mice lacking Nod1 or Nod2 was not increased under GF conditions. The expression of TNFα and the osteoclastogenic factor RANKL in bone was reduced in GF compared to CONV-R WT mice but not in Nod1−/− or Nod2−/− mice indicating that the effect of the GM to increase TNFα and RANKL in bone and to reduce bone mass is dependent on both NOD1 and NOD2 signaling. It was recently demonstrated that muramyl dipeptide (MDP), known as a shared structural unit of peptidoglycans from both gram-positive and gram-negative bacteria and a ligand to NOD2, regulates bone mass indicating that the NOD2 signaling pathway is involved in GM effects on bone  and a mixture  of probiotic strains [L. para and the L. mix treatments protected the mice from ovx-induced bone loss , a commensal bacterium that also secretes beneficial immunomodulatory factors, on ovx-induced bone loss. Treatment with L. reuteri altered the GM composition and prevented ovx-induced trabecular bone loss and bone resorption [Bifidobacterium longum protected rats from ovx-induced bone loss [As several studies indicate that alteration of the GM by antibiotic treatment modulates the magnitude of bone loss in sex steroid-deficient female mice, we hypothesized that treatment with probiotics might protect mice from ovariectomy (ovx)-induced bone loss , 39. Bas strains , and thesorption . In addione loss . These fBased on this study, Li et al. proposed that GM products act as antigens passing through the intestinal cells and interact with antigen-presenting cells Fig. . This rewww.clinicaltrials.gov; Sept 2, 2017), there are currently three ongoing double-blind, placebo-controlled, randomized clinical studies of the effect of 12-month treatment with probiotics on bone mineral density in postmenopausal women or in osteopenic elderly women. The bacterial strains used in these three clinical studies were very recently all shown to protect mice from ovx-induced osteoporosis in three independent animal studies [The first small (21 subjects in each group) short-term (6 months) placebo-controlled clinical study on the effects of a probiotic supplement on bone health in osteopenic postmenopausal women was recently published . Consump studies , 41, 42.Animal studies demonstrate that the GM is a crucial regulator of bone metabolism and that specific modulation of the GM composition by probiotic interventions can prevent ovx-induced bone loss. The overall immune-related effect of short-term colonization of germ-free mice with GM is an activation of CD4+T cells, resulting in increased levels of pro-inflammatory cytokines in bone and thereby activation of osteoclastic bone resorption. Besides these immune-mediated effects on bone mass, the GM is involved in nutritional uptake and may, thereby, regulate overall body growth and bone sizes, possibly mediated via altered IGF-I levels.We recently introduced a new term “osteomicrobiology” for the rapidly emerging research field of the role of the microbiota in bone health and disease. This research field is aimed to bridge the gaps between bone physiology, gastroenterology, immunology, and microbiology. There has been a rapid translation from experimental animal studies to ongoing clinical trials of the effect of certain probiotic treatments on bone health in postmenopausal women and the results from these studies will be informative to determine if the GM might be a novel therapeutic target for osteoporosis. In addition, several large-scale cohort studies are initiated to determine if the GM composition might be used as a biomarker for fracture prediction.


The identification of four Consensus Molecular Subtypes (CMS1–4) of colorectal cancer forms a new paradigm for the design and evaluation of subtype-directed therapeutic strategies. The most aggressive subtype - CMS4 - has the highest chance of disease recurrence. Novel adjuvant therapies for patients with CMS4 tumours are therefore urgently needed. CMS4 tumours are characterized by expression of mesenchymal and stem-like genes. Previous pre-clinical work has shown that targeting Platelet-Derived Growth Factor Receptors (PDGFRs) and the related KIT receptor with imatinib is potentially effective against mesenchymal-type colon cancer. In the present study we aim to provide proof for the concept that imatinib can reduce the aggressive phenotype of primary CMS4 colon cancer.n = 27) will receive treatment with imatinib (400 mg per day) starting two weeks prior to planned tumour resection. To assess treatment-induced changes in the aggressive CMS4 phenotype, RNA sequencing will be performed on pre- and post-treatment tissue samples.Tumour biopsies from patients with newly diagnosed stage I-III colon cancer will be analysed with a novel RT-qPCR test to pre-select patients with CMS4 tumours. Selected patients  distant recurrence are almost always first tested in patients with heavily pre-treated metastatic disease. Second, measuring on-target drug effects and biological consequences in tumour tissue is not commonly a part of the study design. Third, due to the lack of patient selection tools, clinical trials in the adjuvant setting require large patient populations. Finally, the evaluation of recurrence-prevention requires a long-term follow-up. In the ImPACCT trial these issues are addressed by including newly diagnosed pre-selected patients with CMS4 tumours prior to primary tumour resection, rather than non-selected patients with late-stage disease. By making use of the pre-operative window period, the biological effect of imatinib treatment on CMS4 tumours can be rapidly assessed. Delivering proof-of-concept for drug action in early stage disease should form the basis for the design of future trials with subtype-targeted therapies in colon cancer patients.NCT02685046. Registration date: February 9, 2016.ClinicalTrials.gov:  Mortality from colon cancer is almost invariably due to the development of distant metastases. In current practice, pathological (TNM stage) and clinical characteristics  mainly determine the choice of adjuvant chemotherapy. However, these features have limited value in predicting which patients are at risk of developing metastases. In clinical trials, the five-year recurrence rate in stage III colon cancer patients is approximately 50% without adjuvant chemotherapy. With adjuvant treatment this is reduced to ~35%, implying that such treatment is only effective in a subgroup of patients . ConsequNovel adjuvant therapies should be based on an understanding of the pathways that drive metastasis formation. Recent studies on molecular classification of colon cancer have provided insight into these pathways. Several research groups have independently developed classification systems for primary colon cancer based on gene expression profiles –8. CrossThe pro-metastatic pathways that are upregulated in CMS4 provide opportunities for subtype-targeted therapy. CMS4 tumours are characterised by high expression of stem cell and mesenchymal genes, and a high stromal content . We haveImatinib as Pre-operative Anti-Colon Cancer Targeted therapy). In ImPACCT, patients with CMS4 colon cancer are identified with a recently developed 4-gene RT-qPCR test that measures PDGFRA, PDGFRB, PDGFC and KIT expression levels in diagnostic tumour biopsies [To test this hypothesis in a proof-of-concept study, we designed the ImPACCT trial  to systemic imatinib concentrations; to relate the level of inhibition of PDGFR/KIT signalling and the extent of changes in gene expression to the systemic and intra-tumoural PK of imatinib and its active metabolite CGP74588; to assess changes in tumour markers during treatment by measuring the concentrations of plasma-CEA and circulating tumour DNA (ctDNA) and to study the effects of imatinib on organoid-forming potential. Finally, the effect of short-term exposure to imatinib immediately followed by bowel surgery on the complication rate will be monitored.PDGFRA, PDGFRB, PDGFC and KIT [All patients who are scheduled for a diagnostic colonoscopy on account of clinical suspicion of colon carcinoma will be approached for permission to obtain extra biopsies for this study in case a tumour is found in the colon. These biopsies are pre-screened with the new RT-qPCR test that predicts the chance of a tumour being CMS4, based on the combined expression of imatinib targets  and KIT . If the Patients pre-selected with the RT-qPCR test will be screened by a medical oncologist for inclusion in the second part of this study. Included patients will receive treatment with imatinib starting two weeks prior to planned tumour resection. Imatinib will be administered orally at a daily dosage of 400 mg for two weeks, with the last dose given within 12 h before surgery. Patients are requested to register drug intake and any adverse events in a patient diary. Before the start of imatinib therapy and at the end of the treatment period, blood samples will be obtained to measure ctDNA and plasma-CEA. Plasma imatinib trough levels will be determined on the day of surgery. Immediately following tumour resection, biopsies will be taken from the surgical specimen (post-treatment biopsies). Gene and protein expression of the pre-treatment biopsies (from colonoscopy) and post-treatments biopsies will be compared to assess the effects of imatinib therapy on PDGFR- and KIT-signalling and on the mesenchymal gene expression profile. After surgery, patients will be monitored according to standard of care. Any postoperative adverse events up until 14 days after discontinuation of study medication (end of study) will be documented.This study is designed as a proof-of-concept study with multiple outcomes of interest. We expect the effect size of imatinib treatment on the various parameters to be very high, since we specifically select patients who express high levels of the drug targets. However, we are aware of possible factors that may reduce the observed effect size. This includes intra-tumour heterogeneity in target expression – causing potential misclassification – and variation in drug distribution throughout the tumour and between patients. Therefore, we anticipate a medium to high Cohen effect size for the primary endpoint (i.e. Cohen’s effect size of 0.65). To demonstrate such an effect, we need to include 27  patients, based on a two-sided paired-samples t-test with a significance level α of 0.05 and power 1-β of 90%. We specifically chose this high power in order not to dismiss effects that are potentially relevant for further development of imatinib therapy in colorectal cancer patients. Given that approximately 25% of colon cancers are CMS4, at least 4*27 = 108 eligible patients with newly diagnosed colon cancer will need to be pre-screened with the RT-qPCR test. Since <10% of the patients undergoing colonoscopy are diagnosed with colon cancer, at least 1.100 patients will have to be approached for pre-screening informed consent.Imatinib-induced changes in the expression of specific gene signatures associated with CMS4 will be assessed by RNA-sequencing analysis. The signatures will be obtained from published literature and our own data. In addition, imatinib-induced gene expression changes will be analysed in a non-biased way by gene ontology and gene enrichment analyses. Using the appropriate statistical tests depending on distribution of the data  we will test whether there is a significant change in gene signature expression. Phosphorylation of PDGFRα/β and KIT will be quantitatively assessed as a fraction of the total amount of PDGFRα/β and KIT present in a sample. Post-treatment imatinib-induced inhibition of PDGFR phosphorylation will be compared relative to the pre-treatment sample  using the appropriate statistical tests. Correlations between plasma trough levels and intratumoural concentration of imatinib, and between the extent of PDGFR and KIT inhibition and systemic/intratumoural imatinib concentrations will be evaluated using Pearson correlation coefficient. The Wilcoxon matched pairs signed rank test will be used to compare serial ctDNA and CEA levels. Organoid-forming potential of the tumours after imatinib therapy will be compared with the general success rate of organoid-establishment from colon cancers, using a Fisher exact test. In case of missing data, samples will be excluded pair-wise.Targeted anti-cancer therapies that seem promising in pre-clinical and early-phase clinical trials often fail to show benefit in phase III randomized controlled trials. Up to 60 % of phase III trials have negative outcomes , 15. ThiProspective stratification and/or inclusion based on predictive molecular biomarkers will presumably improve trial results by enrichment for responsive patients . HoweverImPACCT is conducted with treatment-naïve patients during the pre-operative window period, which allows us to obtain high quality tissue material before and after imatinib therapy without additional interventions: pre-treatment samples will be obtained during diagnostic colonoscopy and post-treatment biopsies are collected from the resection specimen; both procedures are part of standard of care. By comparison of pre- and post-treatment tissue samples, the effects of imatinib on CMS4 tumours can be evaluated at a cellular level. The treatment period of 14 days is within the normal time frame from diagnosis to surgery (the pre-operative window). We expect that two weeks of treatment will be sufficient to induce changes in gene expression: given that steady state plasma concentrations will be attained within four days , patienExperimental therapy during the pre-operative window period could potentially lead to an increase in postoperative complications. However, based on two phase II trials that evaluated the use of neoadjuvant imatinib therapy for otherwise irresectable or metastatic GIST, we believe it is safe to administer the last dose of imatinib within 12 h before surgery. In these trials imatinib was administered for several months and stopped one day prior to surgery. Both studies concluded that this approach was feasible, and the reported postoperative complications were acceptable and not out of the ordinary considering the extensive abdominal surgery , 26.Instead of assessing a clinical endpoint , this proof-of-concept trial investigates a surrogate endpoint (gene expression changes) to demonstrate a ‘biological’ treatment effect. This endpoint requires a relatively limited sample size and can be determined after only two weeks of therapy, which limits the burden placed on participants. If treatment effects are indeed demonstrated, knowledge of the size of the effect on tumour biology can be used to drive the design of a larger randomised phase II trial.The design of the ImPACCT trial presented here allows rapid evaluation of the mechanism of action of a targeted therapy in a subtype-stratified patient population by analysis of paired pre- and post-treatment biopsies, without interfering with or delaying standard-of-care, by making use of the pre-operative window period. This design can serve as a blueprint for subtype-directed proof-of-concept trials in colon cancer, with the ultimate goal of designing effective adjuvant therapy that eradicates occult metastases.


To be able to visualize the abundance and spatiotemporal features of RNAs in bacterial cells would permit obtaining a pivotal understanding of many mechanisms underlying bacterial cell biology. The first methods that allowed observing single mRNA molecules in individual cells were introduced by  The rapid development and implementation of fluorescence microscopy techniques has led to the realization that, even in tiny bacterial cells, key molecular processes can be highly orchestrated and organized to temporarily or statically occur at specific subcellular sites . Most maInitially, gene expression, like many other cellular processes, could only be studied in cultures of cells and, thus, in an ensemble fashion. In the latter approach, total RNA is usually extracted from tissue samples or cell cultures, after which it is further analyzed by Northern blotting, quantitative reverse transcriptase (qRT)-PCR, DNA microarrays or RNA-sequencing. This type of data yields important information but typically represents averaged and normalized values that cannot be regarded as quantitative or absolute. In addition, ensemble RNA measurements do not provide information on cell-to-cell variations in RNA content and intracellular spatiotemporal distributions of transcripts. Biological processes such as transcription are inherently stochastic and can show great differences within clonal populations, such as bacterial cultures . TissuesEscherichia coli) and therefore require a potentially bias-introducing pre-amplification step to prepare cDNA libraries  recognizes distinct RNA sequences with high affinity. One of the first high-specificity RNA-RBP pairs that was characterized originates from the S2 phage . The MS2S2 phage . RepressS2 phage . RealiziS2 phage . By intrst cells . These sst cells . More re embryos . The MS2 embryos . In 2016peptides . A summaAlthough protein-based RNA labeling is now widely employed in eukaryotic organisms, several caveats have thwarted the extensive use of MS2-based RNA labeling and similar systems in bacteria. The main problem is that most employ intact autofluorescent proteins, causing substantial background noise that obscures the proper visualization of single transcripts. The background noise is effectively reduced in eukaryotic cells by tagging unbound MS2-GFP proteins with a nuclear localization signal (NLS) sequence, which directs the surplus of NLS-MS2-GFP proteins to the nucleus . Such coE. coli cells to study transcription kinetics (ms2) transcriptionally fused to the transcript under study resulted in sufficient fluorescent signal to visualize and follow single mRNA molecules over time in individual E. coli cells. In comparison, the incorporation of 24 binding sites typically suffices to monitor single transcripts in eukaryotic cells. It was recognized that extensive tethering of MS2-GFP reduces the diffusion rate of the transcript under study and renders the molecule perpetual . This can be a laborious task and, as discussed above, may influence the endogenous properties of the transcript under study. The RNA-labeling system employing PumHD and homologous proteins does not suffer from the latter drawback . It remains to be elucidated whether this system is functional in bacteria to track individual transcripts due to a potentially insufficient signal amplification.The RNA-guided Cas9 protein of the bacterial CRISPR-Cas9 immune system is a DNA endonuclease that was long thought to solely recognize DNA targets. The site where Cas9 interaction with DNA occurs is dictated by the combination of the protospacer adjacent motif (PAM) domain in the target DNA and the Cas9-bound single-guide (sg) RNA. Recently, Cas9 was found to also recognize single-stranded RNA after slight modifications of the CRISPR/Cas9 system : The sgRrinciple . It was lization . As a prlization . SubsequTo reduce signal-to-noise ratios, care needs to be taken in expressing the proper number of RBP-GFP molecules in relation to the estimated number of available RNA target binding sites. Even low expression of MS2-GFP may lead to a surplus of unbound MS2-GFP proteins in the cytoplasm, which would thwart capturing single mRNA molecules using fluorescence microscopy. This is true for all RBP-FPs expressed in bacterial cells in which RBP-FPs cannot be contained within a specific compartment. Even though Pumby and Cas9 have the advantage of targeting endogenous mRNAs, the background noise issue remains. It is essential to carefully consider which RBP is the right choice for RNA visualization in bacteria. For instance, a detailed analysis of the binding kinetics of MS2 and PP7 to their respective aptamers revealed that PP7 binds with a higher affinity than MS2 because PP7 occupies more aptamer repeats. Importantly, the construction of a tandem dimer of both MS2 and PP7 increased protein-RNA interactions in various eukaryotic cell lines, effectively improving signal-to-noise ratios . BacteriFigure 1C; Figure 1C; Figure 1C; Figure 1C; Protein complementation employs split fluorescent proteins that reconstitute into a functional fluorescing protein upon reunion via the interaction of their respective fusion partners . The impE. coli are degraded within 3–10 min post-synthesis , followed by cyclization and a final oxidation step in order for the active site to fluoresce. In addition to folding, the last step can take a relatively long time and greatly depends on the presence of sufficient molecular oxygen. Maturation of full-length super-folder GFP is estimated to take around 11 min under optimal conditions . A commoynthesis . Thus, tynthesis . Both shFigure 1D). Rather, RNA binding mediated FRET (RB-FRET) was developed to detect the interaction between eukaryotic RBPs and their cognate RNA (Förster Resonance Energy Transfer fluorescence microscopy makes use of a donor fluorophore that dissipates energy to and thereby excites a nearby (<10 nm) acceptor fluorophore upon excitation. Donor-acceptor pairs can exist of fluorophores and/or fluorescent proteins. Live single-cell and single-molecule FRET studies have been executed and well-performing FRET pairs consisting of fluorescent proteins have been carefully selected for various bacterial species . A combinate RNA . FRET isFigure 1E). The MS2 protein can be fused to, for instance, a trimethoprim (TMP)-tag  or a SNAP-tag   . The TMPsferase) ]. The STembranes . Many meFigure 1F). Hence, self-labeling protein tags hold great promise for both the visualization of RNA molecules and proteins, also because the diversity of commercially available tags and fluorophores is rapidly growing.Although the genetically encoded self-labeling protein tags can be relatively bulky and may require longer incubation times depending on the tag and cell type, they allow detecting mRNAs in live cells with photostable fluorophores. The latter is key to achieving single-molecule tracking and super-resolution microscopy . FluoropFigure 2A; in vitro  is a combinatorial method for the generation of nucleic acids binding to virtually any molecule, including proteins and small molecules such as organic dyes and metabolites  have been designed to visualize RNA through fluorophore-based FRET , of which the Spinach RNA-fluorophore pair was reported first . Spinach2 shows improved folding kinetics through the presence of stabilizing RNA hairpins but still requires a tRNA scaffold for optimal functionality (E. coli using fluorescence-activated cell sorting (FACS). In this way, the 49-nt RNA molecule Broccoli was developed , it would be highly desirable to be able to detect these molecules through RNA labeling. A recent study shows that arranging multiple Spinach aptamers in a tandem array increases fluorescence  is a triphenylmethane dye with low quantum yield in solution. The fact that the autofluorescence of MG increases in cold or viscous environments led Babendure and colleagues to develop a 38-nt-long MG-binding aptamer (MGA) that resulted in an over 2000-fold increase and red-shift in MG fluorescence  binds derivatives of thiazole orange (TO) at high binding affinities in the presence of potassium , resulting in a 100-fold increase in SB fluorescence  quenching is most effective. Static quenching is completely abolished when the quencher-fluorophore complex becomes dismantled . Quencherescence . SRB-2-tFigure 2B). Importantly, non-conditional fluorophores with different spectral properties, such as rhodamine green, tetramethylrhodamine or sulforhodamine can be used in combination with the same DNB aptamer  aptamer was introduced . Binding aptamer . To thisin vivo imaging of RNAs in bacterial cells or whether further advances of current or new aptamers are needed to obtain systems that accurately function in an intracellular environment.Future studies will have to show if recent improvements of existing aptamers are applicable and truly robust enough for in situ hybridizations of oligonucleotide probes to complementary nucleic acid sequences (in situ hybridization (FISH) has remained the predominant tool for the examination of cell-to-cell variations in transcript numbers. The technique itself was initially developed to visualize DNA but adapted to allow quantification and localization of mRNA in single cells using fluorescence microscopy. Contemporary RNA FISH protocols use oligonucleotides of 17–22 nucleotides that are complementary to the RNA under study (in vivo hybridization (FIVH) for live-cell mRNA imaging in mammalian cells (discussed below) and Turbo RNA FISH in which the hybridization time is increased by changing to alcohol-based fixation and increasing probe concentration  in individual cells . Both strategies have been successfully applied to quantify endogenous transcripts in E. coli  hybridization (BDH) , multiplFigure 3C). Locked nucleic acid (LNA) probes are more rigid because the 2′ oxygen and the 4′ carbon in the RNA nucleotide are linked, leading to highly specific Watson-Crick base pairing . Importantly, this technique has been successfully applied in single E. coli cells, in which it has led to the acknowledgment that mRNAs that include genes encoding membrane proteins are generally localized in proximity of the cytoplasmic membrane, whereas mRNA species that only contain open reading frames for soluble, secretory or outer membrane proteins are scattered throughout the cytoplasm (Multiplexed error-robust FISH was introduced by the group of Zhuang in 2015 and is a strategy based on multiplexed fluorescence formance . Elaboraytoplasm .in vivo hybridization (FIVH) can be achieved but requires alternative probe delivery and circumvention of at least the degradation of probes by cellular nucleases. Synthetic probes, such as LNA and PNA probes, but also 2′-O-methylated oligonucleotides  display improved nuclease stability, which make them especially suited for in vivo imaging of RNA . One end of the probe contains an organic fluorophore, while the other is labeled with a quencher that keeps the fluorophore in a dark state as long as the termini of the probe are in close proximity. Quenching is alleviated when the molecular beacon adopts a linear structure upon interaction with target RNA, yielding fluorescence. mRNAs have been visualized in vivo via microinjection of 2OMe-based molecular beacons in a variety of mammalian cells  and Exciton-controlled hybridization-sensitive fluorescent oligonucleotide (ECHO) probes consist of PNA or 2OMe-based backbones that carry a fluorogenic base . The incic cells . Interes oocytes .Figure 3D; The so-called black hole quencher (BHQ) aptamer initially showed insufficient functionality as mRNA appendix and was modified into a probe that anneals to target RNA  were used to detect 16S rRNA sequences in living E. coli cells with high specificity  and sodium dodecyl sulfate (SDS) surfactant  probes  can therefore be used in mammalian 293T cells to specifically incorporate an azide-carrying agmatine in 5S RNA carrying a genetically encoded archaeal tRNAIle2-tail, after which the tRNAIle2-5S fusion RNA can be fluorescently labeled with Sulfo-Cy5-azide via AAC , which requires the incorporation of toxic Cu(II) molecules. Therefore, the studies presented above utilized cell fixation prior to CuAAC. However, copper-free AAc reactions, like strain-promoted AAc (SPAAC), could be employed to realize site-specific and covalent RNA labeling in living cells.Last but not least, efforts are undertaken to covalently label RNAs in cells with reporter molecules via so-called bio-orthogonal “click” chemistry, which often makes use of one-pot azide-alkyne cycloaddition (AAC) to achieve bioconjugation. The RNA of interest can be post-synthetically and chemo-enzymatically modified by transferases to contain click-compatible moieties that are then joined to other substrates through biocompatible AAC  aptamer is a bright light in bacterial mRNA detection as the system combines the advantages of targeting native transcripts with high specificity and bright and stable fluorophores.RNA-binding protein-based techniques, such as the MS2, PP7 and eIF4A systems have been adapted for use in bacterial cells more than a decade ago but have, unfortunately, met with limited success. Although MS2 can be used to follow transcript production in time, one has to keep in mind that the diffusion and localization of MS2-labeled transcripts are severely estranged from the natural situation due to the addition of bulky complexes. The number of MS2-binding appendices can be reduced when studying high-abundant RNAs and/or by careful fine-tuning of the ratio between target RNAs and RBP levels. However, this would still not allow quantifying low-abundant or single RNA molecules to reflect native cell genetics. New RBP-based methods have recently emerged that make use of Cas9 or Pumby modules . Their uin vivo imaging of bacteria. Rather, it conveys static data of - in fact – highly dynamic processes. On top of that, cell fixation required for FISH could also influence the localization of the studied RNA by itself.To date, smFISH is the only available technique to reliably count the number of mRNA molecules in bacterial cells while at the same time acquiring data with high spatial resolution . Recent E. coli while at the same time keeping cells in a viable state  compatible with the small-sized and cell wall-enclosed bacteria. Electroporation can be used to internalize short DNA fragments or proteins up to 100 kDa in le state . It woulle state . More rele state . Alternain vivo RNA studies. Moreover, the recently developed aptamers and their organic dye ligands are, in theory, compatible with other aptamer-dye pairs by taking advantage of the variation in spectral characteristics of the fluorophores. Especially those techniques that would allow detecting single RNA molecules for prolonged periods of time would boost the knowledge on bacterial cell biology. So far, all fluorogen-binding aptamer methods that have been used to detect transcripts in bacteria have employed multicopy plasmids to express the mRNA. Also, all of the studies have so far been undertaken in E. coli only. These are clear indications of the fact that most techniques are still under development and none have yet yielded physiological information on endogenous bacterial transcripts, many of which are typically low-abundant and short-lived.The shortfall of FISH in imaging RNAs in living bacterial cells has led to the development of methodologically different RNA-labeling techniques. Approaches that have received the most attention in recent years make use of aptamer-binding fluorogenic dyes. Many small organic dyes are able to diffuse into cells and are good candidates for less intrusive RNA imaging strategies. A number of fluorogen-aptamer pairs have been reported for live-cell RNA imaging, of which most required considerable optimization after their first introduction. Next-generation aptamers are now coming of age. Additional technical improvements and development of new fluorogen-binding aptamers circumvent many of the problems that are encountered with protein-based RNA-labeling techniques and, thus, hold great promise for future E. coli at the single-cell level (Although monitoring of single mRNA molecules in bacteria has proven difficult, important information has been gained regarding the spatial behavior of RNA species inside bacterial cells. The techniques have allowed uncovering the transcription kinetics of promoter systems in ll level  and led ll level . The sysLvG has drafted the manuscript. JK and LvG have reviewed and edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The ability to predict the sensory feedback of self-generated stimuli against incoming sensory information is of importance to distinguish internal from external stimuli and is associated with sensory attenuation. Furthermore, it has been proposed that deficits in sensory attenuation could contribute to clinical symptoms of schizophrenia, including hallucinations and delusions, involving potential deficits in corollary discharge. The current study examined the hypothesis whether sensory attenuation is present in participants at clinical high–risk (CHR) for psychosis.Sixty-four CHR-participants and 32 healthy controls were presented with auditory stimuli during two experimental conditions: 1) In a passive condition, participants were presented with ripple sounds  and responded to flat sounds  with the right-index finger and 2) In an active condition, the ripple sounds were elicited by a button press with the right-index finger every 4s. MEG-data were acquired with a 248-magnetometers whole-head MEG system  at a sampling rate of 1017Hz. We focussed on the M100 response during the passive and active conditions at sensor- and source-level. A LCMV beamforming approach was employed for source reconstruction and virtual channels in primary auditory cortex and the left superior temporal cortex were used further analysis of sensory attenuation effects. Condition and group effects were tested with a cluster-based nonparametric test implemented in Fieldtrip with a window of interest for the M100 component between 120ms-150ms.There was a significant decrease (P =0.009) in the amplitude of M100 component in the active vs. passive conditions across groups at both sensor- and source-level. Interaction-effects revealed that that sensory attenuation was significantly reduced in auditory cortices in the CHR group vs controls (P=0.032).The current results highlight that sensory attenuation can be studied with ASSR-paradigms and that both primary auditory and superior temporal cortices underlie this effect. Moreover, our current findings suggest that sensory attenuation is impaired in CHR-participants, suggesting the possibility of impaired corollary discharge processes as a potential biomarker for the early diagnosis and detection of schizophrenia.


Rosa damascena, prevented fibril formation since PEA-L was able to trap the oligomeric form of HEWL in contrast to PEA-S where protofibrils but not mature fibrils were formed; (2) Cin, previously shown to prevent fibril formation in the liquid form, was also shown to do so in the aroma form by producing protofibrils and not mature fibrils in both Cin- L and Cin-S aroma forms and (3) the aroma of TEMED-L was able to retain HEWL’s native structure completely and prevented both aggregation and fibril formation, while TEMED-S prevented HEWL fibril formation and instead directed the pathway towards amorphous aggregate formation. Furthermore, the ability to trap oligomeric species (by PEA-L aroma) is of great importance for further research as it provides routes for preventing the formation of toxic oligomeric intermediates along the fibrillation pathway. Last but not least, the novelty of this in vitro study on the effect of aroma at the molecular level with a unique experimental set-up using HEWL as a model protein in assessing amyloid fibril formation paves the way for more and detailed studies on the importance of aroma producing molecules and their effects.This study investigated for the first time the molecular effectiveness of 'aroma' from three small molecules including a phenol  and an aldehyde  both containing an aromatic ring, and a diamine  at two different amounts  in preventing hen egg white lysozyme (HEWL) amyloid fibril formation using Thioflavin T and Nile red fluorescence assays, circular dichroism spectroscopy, SDS-polyacrylamide gel electrophoresis, atomic force microscopy, dynamic light scattering and HEWL activity test. Interestingly, the results revealed that (1) the aroma of PEA, identified as an active constituent of  Amyloidcidic pH , leadingcidic pH  with 2–5cidic pH –8. In thcidic pH –8. ThereOn this basis, several therapeutic approaches have been suggested for the treatment of amyloidogenic diseases including (a) reduction in the production of the amyloidogenic form of proteins (by blocking step III); (b) increasing the clearance rate of misfolded or aggregated proteins; (c) increasing the stability of the native state of amyloidogenic proteins (by blocking step I); and (d) directly inhibiting the self-assembly process . AmongstThe use of aroma in diseases is being practiced in aromatherapy, which is a subdivision of phytotherapy making use of natural pure essential oils from aromatic plants  to help relieve health problems and improve the quality of life in general . AromathRosa Damascena [Cinnamomum zelanicum, which is an aldehyde giving cinnamon its flavour as well as odour [Rosa Damascena rose flower extract  and cinnamaldehyde both in solution have been shown in previous studies to inhibit fibril formation [water-white coloured liquid with a fishlike odour [In this study, three different compounds  were investigated for their effectiveness of aroma in preventing hen egg white lysozyme (HEWL) amyloid fibril formation in two different amounts . The phenol used was phenyl ethyl alcohol (PEA) from amascena , which gas odour . The comormation –35. The ke odour . TEMED wke odour .in vitro to understand the mechanism(s) involved at the molecular level. HEWL amyloid formation was assessed using atomic force microscopy (AFM), Thioflavin T (ThT) fluorescence assay and dynamic light scattering (DLS). The secondary structure and hydrophobic patch analyses were performed by circular dichroism (CD) and Nile red fluorescence assay, respectively. The relative enzymatic activity of HEWL was also assessed to further our understanding of the effect of aroma on inhibition of amyloid formation.Therefore, in this study the effect of ‘aroma’ of the three different compounds were assessed on HEWL amyloid fibril formation Micrococcus lysodeikticus (ATCC No. 4698), glycine (CAS No. 56-40-6), potassium phosphate , sodium dodecyl sulphate , N,N,N,N'-Tetramethylethylenediamine  and Trans-cinnamaldehyde  were all purchased from Sigma-Aldrich. Excelband all blue regular range protein marker (PM1500) was purchased from SMOBIO. Mica for atomic force microscopy  was purchased from PELCO. Acrylamide (UN-NO 2074) and N, N’methylendiacrylamid (EC number 203-750-9) were purchased from Merck.Hen egg-white lysozyme , phenyl ethyl alcohol , Thioflavin T (ThT), Nile red, 3g−1cm−1at λ = 280 nm [UV–vis absorbance spectra were recorded using a UV-vis Spectrophotometer . The protein concentration was calculated using the extinction coefficient ε = 2.65 dm= 280 nm . Three sSample solutions of HEWL at 2 mg/ml (concentration determined by UV spectrophotometer at a wavelength of 280 nm) in 50 mM glycine-HCl (pH 2.2) was used in this study. PEA, Cin and TEMED were all used in their original purchased forms. The experimental set-up  was suchHEWL samples incubated with or without aroma were diluted 50-fold with ThT solution (at 25 μM) and fluorescence intensities were recorded at 484 nm after excitation at 440 nm. Excitation and emission slit widths were both set at 5 nm. Samples of not-heated and not-treated HEWL were also used and ThT recorded as positive and negative controls, respectively. The results were repeatable and standard deviation bars were calculated for the graph using multiple data.Nile red powder was initially dissolved in ethanol and then 5 μl of the Nile red solution (at 2 mM) was diluted in 990 μl acidic deionized water (pH = 1.6). 5 μl of HEWL sample post-incubation were added to the Nile red solution and incubated in a dark room for 20 min. The excitation wavelength was 553 nm and emission spectra were recorded between 600 and 700 nm. Excitation and emission slit widths were both set at 10 nm. The control samples were also recorded.2 dmol−1) versus wavelength (nm). The control samples were also measured using CD.Circular dichroism (CD) spectra of HEWL samples were recorded from 250 to 195 nm with an AVIV 215 spectrophotometer . The HEWL samples post incubation were diluted 10 times for CD. CD spectrum of glycine buffer (pH 2.2) was recorded and subtracted from the CD spectra of HEWL samples post-incubation. Three scans of each sample was measured and averaged. The control buffer scans were run in duplicates, averaged, and then subtracted from the sample spectra. The results were plotted as ellipticity . For preparation of AFM samples, 10 μl of each sample was loaded onto a mica surface and allowed to fix on mica by incubation for 30 minutes at room temperature. The mica surface was then washed once with 100 μl deionized water and left to dry at room temperature before being examined by the atomic force microscope .Dynamic light scattering measurements were performed using the Malvern Zeta sizer Nano ZS ZS . The apparatus was equipped with a laser with a wavelength of 633 nm. Samples were placed into disposable cuvettes with a 1 cm optical path. Scatter radiation was collected at an angle of 173° (backscatter mode). Each scan consisted of 70 runs and each run duration was 60 seconds. The temperature of the measurements was set at 25°C. The refractive index of the material (protein) and the absorption were set to 1.59 and 0.01, respectively. The following parameters were used: the dispersant viscosity was 0.8872 cP and the refractive index was 1.330.Micrococcus lysodeikticus (M. luteus) by HEWL was measured as reported [M. luteus in 0.1 mM of potassium phosphate buffer, 10 μl of the HEWL samples were added to a final concentration of 0.2 mg/ml. Changes in the turbidity at 450 nm was recorded per minute using a spectrophotometer. The two formulae [The rate of lysis of reported . The lytformulae  (see Eqsdf = dilution factor450 as per the Unit Definition0.001 = ΔA0.01 = Volume (in milliliters) of Enzyme SolutionThree compounds were tested for their ability to inhibit amyloid fibril formation of HEWL by ThT fluorescence assay. It is known that ThT interacts rapidly and specifically with amyloid fibrils , and an To assess the effect of different aroma on the exposure of hydrophobic surfaces and secondary structure of HEWL, Nile red fluorescence spectroscopy and circular dichroism (CD) measurements were carried out. Nile red is an uncharged phenoxazone dye which interacts with exposed hydrophobic sites of proteins resulting in the enhancement of the fluorescence intensity and blue shifting of the emission maxima. Proteins which are known to have hydrophobic binding domains such as k-casein, 8-lactoglobulin and serum albumin exhibit greater enhancement and greater shifts of fluorescence than proteins which do not have such domains, such as lysozyme and ovalbumin. Therefore, Nile red can be used as a fluorescent probe of hydrophobic domains of proteins in their native state as well as monitoring conformational changes resulting in the formation or destruction of such domains, including partial denaturation of proteins, aggregation and unfolding of proteins . Beta-shThe secondary structure changes of HEWL in this study were assessed using CD measurements in the far-UV region of the spectrum (CD-UV). Dynamic light scattering (DLS) results have shown that the diameter of not-heated HEWL was shifted from 3.36 nm to 193 nm in the heated HEWL in the absence of aroma (i.e. the not-treated sample) upon 24 hours of incubation under fibrillation conditions . The effAFM images confirm previous results since in the absence of aroma (in the not-treated HEWL sample) fibrils with width size of 8.54 nm were seen  but whenMicrococcus luetus as HEWL's substrate. The slope of the activity plot is an indicator of the enzyme activity  and protofibrils , while the diamine was capable of fully retaining HEWL's native structure (TEMED-L) or directing HEWL towards misfolded or amorphous aggregate formation (TEMED-S). Several different techniques had to be used to derive at these conclusions.The results seen for the aroma of the phenol and aldehyde with a single aromatic ring using different techniques were as follows: ThT results revealed that the aroma of all the compounds studied here were able to reduce mature fibril formation compared to the not-treated samples, however, the major discrepancy seen in PEA-L can be explained by the study of Hudson et al (2009) on the effect of presence of exogenous compounds on ThT fluorescence assay. They state that phenolic aromatic rings of polyphenols are often connected via conjugated systems, resulting in strong ח-ח* electronic transitions and making them chromophoric and even intrinsically fluorescent at the same wavelength of ThT fluorescence . Hence PHydrophobic patch analysis results show that the Nile red fluorescence intensity increases as the hydrophobic patches are exposed on the surface of HEWL. The not-treated HEWL had the most intense Nile red fluorescence compared to the not-heated sample since the beta sheet structure is more hydrophobic than the native structure. The aroma of PEA and Cin were able to reduce the exposure of hydrophobic patches compared to the not-treated sample, indicating their ability to prevent mature fibril formation.CD spectra showed that the secondary structure of HEWL in the presence of the aroma of these aromatic compounds consists of beta-sheet structure (same as 'not-treated' HEWL). Additionally, CD results revealed a surprisingly higher beta-sheet structure content in HEWL treated with PEA-L (consisting of oligomers) in comparison to the 'not-treated' HEWL sample (consisting of mature amyloid fibrils). This can be explained by the fact that precursors of soluble aggregates are highly structured whereas amyloid fibrils have minimum structured conformations ,45.AFM and DLS results confirmed the formation of a mixture of oligomeric and/or protofibrillar and mature fibril structures when HEWL was treated with aroma of these aromatic compounds. This was in line with other results seen. As for SDS-PAGE analysis of the samples, the analysis was based on earlier studies showing that protofibrils often have curvilinear shape and are shorter and narrower than mature fibrils  and hencAs for the diamine, all the techniques used here supported the finding that TEMED-L was able to retain the native form of HEWL and completely prevented fibril formation. TEMED-S aroma, however, directed HEWL away from the fibril formation pathway and formed amorphous aggregates, again supported by the techniques used in this study. As it has been shown in previous studies, multiple amine groups and short alkyl chains in diamines play critical roles in preventing thermal aggregation and inactivation of lysozyme . It was + ions and inhibiting degradation of HEWL as seen in SDS-PAGE for HEWL treated with TEMED  and shortening the water H-bonds leading to the promotion of tighter protein folding  [Moreover, we propose that even the aroma of TEMED (with TEMED in the liquid form having a pH of 10.36) can decrease the acidity of HEWL solution environment by interacting with Hability) . The latThis research has unraveled novel data showing the effects of aroma from three small molecules in inhibiting HEWL fibril formation. This is the first time, the effect of aroma has been studied at the molecular level in preventing amyloid fibril formation using HEWL as a model protein in studying protein stability, folding and aggregation.Rosa damascene in producing odour has been recognised to prevent mature fibril formation since the PEA-L aroma is able to trap the oligomeric form of HEWL in contrast to PEA-S aroma where protofibrils but not mature fibrils were formed. The possibility to trap oligomeric species along the fibrillation pathway by PEA-L aroma is of importance for further research as it provides routes for preventing the formation of toxic oligomeric and or other unwanted intermediates along the fibrillation pathway. Additionally, previous research have suggested the presence of at least two phenolic rings each with three hydroxyl groups as essential in preventing fibril formation but results here show that PEA with just one phenolic ring and a single hydroxyl group may be sufficient even in the aroma form.The data from this study suggest different pathways for preventing or inhibiting amyloid fibril formation by the aroma of three different small molecules used in this study . It can As for cinnamaldehyde, it was also recognised to prevent fibrillation in the aroma form where predominantly protofibrils and not mature fibrils were formed in both small and large amounts. It is plausible to say that aromatic groups of PEA and Cin may be involved in stacking between beta-sheet structures to prevent fibril formation ,13,18,19+ ions in acidic environments and shortening the water H-bonds leading to the promotion of tighter HEWL folding (seen in TEMED-L) similar to TMAO [TEMED on the other hand, was able to fully retain the native form in TEMED-L and completely stop the fibrillation process, while TEMED-S took the route of amorphous aggregate formation. The nitrogen atoms in TEMED are important and may act as hydrogen acceptors interacting with H to TMAO . FurtherIn conclusion, considering that amyloid fibrils are toxic, any inhibitor which can hamper amyloid fibril formation could potentially be considered as a way for delaying and/or inhibiting amyloid formation. Additionally, identification of these small aroma producing molecules and the results showing their effect in inhibiting the fibril formation pathway could help improve our knowledge about the very important neurodegenerative disease-related aggregation, misfolding and fibrillation pathways.S1 Fig(XLSX)Click here for additional data file.S2 Fig(XLSX)Click here for additional data file.S3 FigTurbidity seen in HEWL treated with TEMED-S after 24 hours incubation compared to the other samples.(TIF)Click here for additional data file.S4 Fig(XLSX)Click here for additional data file.S5 FigA mixture of protofibrils and mature fibrils are present in samples of HEWL treated with PEA-S (a), Cin-S (b) and Cin-L (c).(TIF)Click here for additional data file.S6 Fig(XLSX)Click here for additional data file.


Randomized trials have shown that voluntary medical male circumcision (VMMC) significantly reduces the risk of HIV acquisition in men. However, the rate of complications associated with the surgical procedure varies from 0.7% to 37.4% in real-world settings. We assessed the frequency, type and severity of adverse events following VMMC among 427 adult men surgically circumcised in southeastern Botswana; 97% completed ≥1 follow-up visit within seven days post-circumcision. Thirty moderate AEs were observed in 28 men resulting in an overall AE rate of 6.7%. Patient satisfaction was high: >95% were very or somewhat satisfied with the procedure and subsequent follow-up care. Three randomized controlled trials demonstrated that male circumcision, when performed in a clinical setting by a trained provider, safely and significantly reduces the risk of heterosexually-acquired HIV infection in men –3 leadinIn Botswana, similar to other VMMC programs in sub-Saharan Africa, monitoring and evaluation of VMMC-related AEs relies on a passive surveillance system. Although the system uses a standardized approach for assessing and recording complications, it is in place only in facilities that regularly provide circumcision services, and patients experiencing complications following VMMC may seek care at any healthcare facility. If the facility does not routinely provide circumcision services, clinic staff may lack sufficient training and/or clinical experience to appropriately identify and treat complications. Furthermore, complications treated at these facilities will not be captured in the surveillance system. Finally, patient completion of follow-up care during the wound-healing period can be very poor. Among more than 5,100 males circumcised in KwaZulu Natal, South Africa between 2011 and 2013, less than half were seen at the seven-day post-operative visit . This isWe sought to actively assess circumcision-associated AEs by prospectively following a cohort of men undergoing surgical VMMC at two government-run clinics in southeastern Botswana.This prospective cohort study was designed to assess (a) the frequency, type and severity of adverse events following VMMC; (b) prevalence and correlates of re-initiation of sexual activity and; (c) changes in risky sexual behavior following VMMC. The study was conducted by the International Training and Education Center for Health (I-TECH), a collaboration between the University of Washington and University of California, San Francisco. Ethical approvals were obtained by the Health Research and Development Committee at the Botswana Ministry of Health (MOH) and the University of Washington Institutional Review Board. Recruitment and enrollment of study participants occurred before undergoing VMMC but after individuals completed group education about the risks and benefits of VMMC, received individual counseling with clinic staff (including HIV testing) and provided written, informed consent for the procedure. Neither the pre-procedure activities described above nor the procedure itself were performed by study staff. A complete description of Botswana’s National Safe Male Circumcision program, including details on the procedure itself, can be found elsewhere .Adult men undergoing VMMC through the National Safe Male Circumcision program were enrolled between November 2013 and April 2015 at two government-run clinics providing free circumcision services in Gaborone, the capital city of Botswana. Participant eligibility criteria were age 18 to 49 years, residence within 25 km of Gaborone, ever had sexual intercourse and documented HIV-negative test result. All participants provided written informed consent for participation in the study in addition to the consent obtained by clinic staff for the circumcision procedure.After circumcision, study staff reviewed the registry for complications occurring during the surgery and performed a physical examination to assess post-procedure bleeding and other immediate adverse events. Post-procedure visits at two days, seven days, six weeks and three months were scheduled as outlined by the Botswana MOH guidelines for adult VMMC. A follow-up visit at 12 months was planned to coincide with annual HIV testing as per standard of care in Botswana. Each study participant was provided with a wallet-size reminder card noting the date of each follow-up visit. In advance of each visit, study staff telephoned participants to remind them of the upcoming visit. At each follow-up visit, study staff performed a physical examination, including inspecting the circumcision site and assessing for signs of STIs. Participants received BWP100 (approximately USD$8 at study initiation) at each post-operative visit as compensation for their time and travel costs. In the event of a missed visit, study staff made telephone calls to reschedule the appointment. If participants were unable to be reached by telephone after three attempts, study staff them at home.The current analysis is restricted to data collected at two and seven days post-circumcision because the majority of adverse events occur during or immediately following VMMC and to ensure comparability of our results to previously-published safety studies conducted in other settings –15. PartThe primary outcomes of the current analysis were (1) frequency, type and severity of short-term adverse events following VMMC; (2) participant satisfaction with the procedure and follow-up care by clinic staff and (3) time to resumption of normal activities. We defined the types and severity of adverse events potentially related to VMMC prior to study initiation based on WHO guidelines for the recognition and management of post-circumcision complications  and inclThe AE rate at two and seven days post-circumcision was calculated as the number of participants with ≥1 moderate or severe adverse events divided by the number of participants assessed at the respective visit. Pain-related events were not included in the non-type-specific AE rates because they often are associated with another complication . The oveBetween November 2013 and October 2015, research staff screened 577 men preparing to undergo VMMC for study participation .A total of 528 (92%) participants were determined to be eligible for participation and 523 (99%) subsequently enrolled. Reasons for ineligibility included not sexually active (4%), residence outside of the area (5%), HIV-infection (<1%) and age <18 years or >49 years (<1%). Four individuals who met study eligibility criteria and consented to study participation were not circumcised due to medical contraindications that were identified prior to the procedure. Ninety-two participants were non-surgically circumcised with the PrePex device. Of the 427 surgically circumcised participants, 415 (97%) completed one or both follow-up visits at two and seven days post-circumcision . Three hth, 75th percentile) age of study participants was 27 years . Most men reporting being in a dating relationship but not living with that partner (53%); only 31 men (7%) were currently married. A minority of participants completed less than secondary education (4%), and most reported being currently employed (70%).Median  of 6.7%. The most common AE was hematoma, followed by infection and bleeding. The overwhelming majority of participants were very or somewhat satisfied with the circumcision procedure and the follow-up care they received. More than three-quarters of participants resumed normal activities seven days post-operative.Although we found an overall AE rate that was approximately twice that observed in the trials, the rate was consistent with previously published reports with similarly high retention. A 2015 evaluation by Reed and colleagues of 1,699 surgically circumcised men in the Nyanza Province, Kenya within the public sector (of whom 94.2% were assessed for post-operative complications) reported an overall AE rate of 5.1% . StudiesA strength of this study was the high retention rate (97%), achieved by a combination of activities including providing participants with wallet-sized appointment reminder cards, telephone calls prior to each appointment, a financial incentive for each completed follow-up visit and in the event of a missed visit, up to three telephone calls and one home visit. Another strength was the data from a real-world setting, rather than a clinical trial setting, which likely closely represent the experience of mature, government-run urban VMMC clinic. While study staff worked closely with site clinicians to share all patient safety data in real time, they did not perform any circumcision procedures or directly provide post-procedure clinical care.all persons undergoing VMMC for a pre-specified list of complications potentially associated with VMMC.Our study is also subject to limitations. We restricted enrollment to men who resided within 25 km of our study sites . This inclusion criterion likely contributed to our very high retention rate (97%) but results may not generalize to men undergoing VMMC in rural or remote areas. We also provided reimbursement to participants, which may not be feasible in resource-constrained settings. Additionally, our findings do not apply to non-surgical circumcision with devices, such as the PrePex device. Unlike surgical techniques, devices do not require injectable anesthesia and can be applied by non-physician personnel in non-sterile settings. The Botswana Ministry of Health began offering the PrePex procedure to adult men in August 2014. However, VMMC programs must maintain a surgical option given that 5–7% of adult men and up to 53% of adolescent boys are ineligible for the PrePex procedure due to anatomical reasons –26. AddiIn summary, while patient satisfaction was overwhelmingly positive, we observed a higher overall AE rate compared to that reported in trial settings. VMMC programs must improve post-procedure retention to ensure accurate monitoring and evaluation of patient safety. Wallet-sized appointment reminder cards and active tracing of men who do not return for follow-up care used in the current study may be effective strategies. As national VMMC programs grow, safety of services must be prioritized to ensure the success of this research-tested, evidence-based HIV prevention intervention.S1 Appendix(PDF)Click here for additional data file.S2 Appendix(PDF)Click here for additional data file.S3 Appendix(PDF)Click here for additional data file.S4 Appendix(PDF)Click here for additional data file.


Even though the exact mechanism is largely unknown until now, statins are supposed to improve survival outcomes in various malignancies. For prostate cancer however, statins are known to compete with dehydroepiandrosterone (DHEAS) for the transport into the cytosol both using the cell by the Solute Carrier Transporter and thus diminish the cellular uptake of DHEAS as a precursor of androgens. Abiraterone inhibits CYP17A1 and thus effectively decreases the production of all relevant androgens including DHEAS. In this study we examined whether statins still affect survival outcome in patients with metastatic castration resistant prostate cancer (mCRPC) when treated with Abiraterone.108 men with mCRPC treated with Abiraterone from 02/2010 to 07/2015 with (n = 21) or without (n = 87) concomitant treatment with statins were investigated. Progression free survival (PFS) and overall survival (OS) were analyzed using Kaplan-Meier-estimates and univariate Cox-regression analysis. The influence on best clinical benefit under Abiraterone treatment was analyzed with bivariate and multivariate logistic regression analysis.PSA-decline ≥ 50% was not significantly different in both groups . The median PFS  and OS  did not differ significantly between those men treated with and without concomitant statin therapy, respectively. Accordingly, there was no improvement for best clinical benefit in patients using statins (odds ratio: 1.2 (CI: 0.4–4.2); p = 0.76).Use of statins as concomitant medication did not improve survival outcomes or best clinical benefit in men with mCRPC treated with Abiraterone. Statins, also known as 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, are originally used to treat hypercholesterolemia. However, next to inhibiting the synthesis of cholesterol, statins also lead to a decreased production of farnesyl pyrophosphate and geranyl pyrophosphate which both are essential for growth and proliferation of cells . This anin vitro-part of the study, the group demonstrated that statins block the uptake of DHEAS competitively by binding to SLCO2B1, thus effectively decreasing the available intratumoral androgen pool and improving and prolonging the effect of primary ADT [In prostate cancer another property of statins may add to their positive effect on survival. The solute carrier transporter (SLCO2B1) is an organic anionic transporter, which enables various anticancer compounds or hormones to enter cells . Next tomary ADT . IntriguAbiraterone is a potent 17α-hydroxylase / C17, 20-lyase (CYP17)-inhibitor that effectively blocks the synthesis of all relevant androgens, amongst others DHEAS, testosterone and DHT and has recently been approved for the treatment of metastatic castration resistant prostate cancer (mCRPC) in a setting prior to and after Docetaxel chemotherapy , 13. ThiWe reviewed all patients with mCRPC out of a prospectively maintained who were treated with Abiraterone at the Department of Urology, Muenster University Medical Center, between 02/2010-07/2015. Best clinical benefit, progression free survival (PFS) and overall survival (OS) was evaluated with regard to concomitant statin use. The study was approved by the Institutional Review Board of the University of Muenster Medical Center and was conducted according to the principles expressed in the Declaration of Helsinki. Written informed consent has been obtained from the participants.All patients presented with confirmed mCRPC as defined by prostate cancer working group 2 (PCWG2)-criteria for which metastatic PCa has to be progressing radiographically, clinically and/or biochemically with testosterone levels being in the castrate range . All patPatients presented the day before start of Abiraterone-therapy to have blood drawn for baseline analysis of prostate specific antigen (PSA), two and four weeks after initiation of therapy and four-weekly thereafter. PSA-progression was defined according to PCWG2-criteria .For baseline evaluation of soft tissue metastases CT-, or MRI-scans of thorax, abdomen and pelvis and for the acquisition of information on bone metastases bone scans were performed. Imaging was repeated during the course of the Abiraterone-therapy when clinically indicated but not in a routine fashion. PD was defined according to RECIST 1.1 criteria for cross sectional imaging  and by PThe current response status i.e. complete remission (CR), partial remission (PR), stable disease (SD) or PD was assessed at each visit. PD was declared upon deterioration of general condition or worsening of pain when unequivocally caused by prostate cancer, when PSA-progression according to PCWG2-criteria occurred, or when radiographic progression was seen.The status of patients being concomitantly treated with statins or not was clarified by interviews directly before initiation of Abiraterone therapy. The patients were subdivided according to statin use . All patients who received a statin used it beforehand to control dyslipidaemia and not for control of PCa.For descriptive statistics we report medians with 95% confidence intervals (CI) or interquartile range (IQR) for continuous variables and populations and frequencies for categorical variables. We determined the significance of the differences between categorical and continuous variables using the χ2-test, Fisher’s exact-test or Mann-Whitney U-test. For analysis of survival outcomes were applied Kaplan-Meier-estimates. We performed univariate and multivariate analysis with the use of binary logistic-regression for determining significance for best clinical benefit and of Cox-regression-models for survival outcomes. All reported p-values are two-sided and we assumed statistical significance when p was ≤0.05. We used SPSS-Statistics V.22  for statistical assessment.Descriptive characteristics of the cohort are given in 2-test)) and a smaller proportion of a Gleason score ≥8 ). All other baseline characteristics, including the use of bone protective medication (66.7 vs. 55.2%), did not differ significantly.Concomitant statin medication was present in 21 (19%) patients. Patients with statin medication had significantly higher baseline median ALP than patients without statin use ), a larger proportion of patients with LDH-levels above the upper limit of normal . A decline of ≥90% was found in 38.1 vs. 24.1% of the patients   .177Lutetium-PSMA-Ligand therapy (4%).In the follow-up period after progression on Abiraterone the following subsequent treatments were given to the patients: Enzalutamide (35%), Docetaxel (25% (only patients with Abiraterone prior to chemotherapy)), Cabazitaxel (4%), Radium-223 (7%) and In univariate analyses for best clinical benefit (defined as CR/PR/SD vs. PD) concomitant treatment with statins was not associated with an improved benefit in patients on Abiraterone. The odds ratio was 1.2 for patients with statin use regarding this endpoint : 0.4–4.1; p = 0.76). The status of metastases and the line of Abiraterone therapy did all not significantly influence best clinical benefit .Results of univariate analyses for PFS and OS are displayed in Concordant with the regression analyses, the Kaplan-Meier estimates showed no significant differences in median survival both for PFS and OS . For PFSAfter multivariate adjustment with clinically important covariates (status of metastases and line of therapy), the concomitant use of statins remained not associated with improved survival outcomes or best clinical benefit. Only use of Abiraterone post chemotherapy and presence of visceral metastases were independent prognosticators of worse progression free survival .in vitro that statins diminish DHEAS-stimulated proliferation of hormone sensitive PCa cells [Almost all PCa initially respond to ADT. However, in the end continuous ADT inevitably leads to development of CRPC. One reason for this development is that androgen receptor signaling can still be triggered by residual androgens even if serum testosterone levels are reduced below castration limits –18. DHEACa cells . In the These results are in line with epidemiological studies that mostly showed significant associations between the use of statins and lower PSA-levels, reduced incidence of clinically significant and advanced PCa, decreased recurrence rates after local treatment as well as better survival outcome , 19–21. in vitro study on different PCa cell lines including CRPC (castration resistant LNCaP subtype and VCaP) by Murtola et al. demonstrated that statins inhibited only hormone-sensitive but not the CRPC cell lines [However, an ll lines . Reasonsll lines –18. Anotll lines , 24. Spell lines . All of In clinical routine statins are effectively used to treat hypercholesterolemia by inhibiting the HMG-CoA-reductase. Androgens are steroid hormones and the basic component of all androgens is cholesterol. So by reducing the available cholesterol in PCa cells with accelerated androgen metabolism and therefore increased need for cholesterol, statins may preclude proliferative activity of PCa. The impact of statins on the SLCO2B1-transporter may add to this potential effect.Abiraterone is a very potent CYP-17-Inhibitor and subtotally reduces the levels of all relevant androgens, including DHEAS. Therefore, the effect of statins on the SLCO2B1-transporter and thus reducing the levels of DHEAS might not be of additional value in patients receiving Abiraterone .de novo diabetes mellitus, may even increase this risk. Furthermore, statins increase the risk of muscle-related side effects. These side effects occur in 5–10% of patients treated with statins [Statins have the reputation of being rather non-toxic. However, there are some important caveats. In susceptible patients with risk factors for developing diabetes mellitus, statins can hasten the progression to manifest diabetic disease . The use statins . HoweverAs the recent reports of the potentially PCa-protective effects of statins get to the public domain and due to the alleged favourable toxicity profile of statins, more and more men with PCa are seemingly treated with statins to improve their respective outcome of PCa even when no hypercholesterolemia is evident.In our study we found that in patients treated with Abiraterone the concomitant use of statin medication did not improve the best clinical benefit, progression-free survival or overall survival. Owing to the method of action of Abiraterone with subtotal depression of cholesterol dependent steroid-androgens, including DHEAS, our results support the hypothesis that the effect of statins to reduce the available cholesterol pool and of interfering with the SLCO2B1-transporter adds no further to the effect caused by Abiraterone alone.Our study is limited by the problems inherent to its retrospective approach even in the setting of a prospectively maintained database. We did not collect statin specific toxicity data. Statin users may represent a population that cares more about their individual health and therefore might bias our results. Finally, we did not stratify between different available statins with potentially different potency and pharmacokinetic impact that could have influenced the extent of our findings.Despite these limitations, our study is the first that relates to the impact of statins on survival outcomes in patients treated with next generation ADT in CRPC and suggests caution with respect to the application of statins in patients without coincidental hypercholesterolemia.In mCRPC patients treated with Abiraterone statins as concomitant medication do not seem to improve the best clinical benefit under Abiraterone and do not improve survival outcomes. Overdosing of statins or interaction with concomitant medication can lead to severe side effects. Therefore, the administration of statins should be critically reviewed in CRPC patients treated with Abiraterone in case of normocholesterolemia.S1 File(SAV)Click here for additional data file.


The physical data of the semiconductor materials used in the design of a CIGS absorber based thin film photovoltaic cell have been presented in this data article. Besides, the values of the contact parameter and operating conditions of the cell have been reported. Furthermore, by conducting the simulation with data corresponding to the device structure: soda-lime glass (SLG) substrate/Mo back-contact/CIGS absorber/CdS buffer/intrinsic ZnO/Al-doped ZnO window/Al-grid front-contact, the solar cell performance parameters such as open circuit voltage  Specifications TableValue of the data•The numerical data described in •Researchers could be able to use this dataset to design and analyze another theoretical model of a photovoltaic cell.•Analyzing these data, one can compare and ensure the validity of other simulation approaches and models.•The values of the performance parameters can be used to compare the simulation results of CIGS solar cell.1The physical data for input parameters of different materials used for designing a highly efficient CIGS solar cell have been presented in 22.12/CdS/i-ZnO/ZnO:Al/Al-grid was proposed for epitaxial growth on the substrate.The schematic design for CIGS absorber based solar cell has been depicted in 2.2ADEPT 2.1