The multi-phase epoxy syntactic foam (ESF) was manufactured with the epoxy-hardener system, HGMS (EP-hardener-HGMS), and AR-HEMS by “the compression modeling method.” In this experiment, in order to enhance the strength of the ESF, some different kinds of the carbon fiber (CF) were added into the EP-hardener-HGMS system (CFR-EP). The influence of the volume stacking fraction, inner diameter, and layer of the AR-HEMS and the content and type of the CF in the EP-HGMS (CFR-EP) system on the compressive strength of the ESF were studied. Weighing the two factors of the density and compressive strength, the ESF reinforced by 1.5 wt% CF with 90% AR-HEMS has the better performance. This kind of the ESF has 0.428 g/cm Because of their low weight, aerogel materials are favored in the fields of aerospace and deep-sea exploration [As a white powder material with extremely high porosity, aerogel is widely used in the fields of building insulation materials and military bulletproof materials due to its excellent thermal insulation resistance and thermal energy storage . As one loration . In receloration . Deep-seloration . Carbon loration ,7,8,9. Tloration ,11. The loration ,13,14. Iloration ,16. ThisIn addition, a lot of research has been done on the reinforcement materials of the hollow macro-spheres ,17, and 3 and 20.76 Mpa, which could be applied in 2076 m deep sea.In this experiment, the micron and millimeter level CF were used to study the influence of the length and content of CF on the compressive strength of the ESF. The influence of the volume stacking fraction, inner diameter, and layer of the AR-HEMS on the compressive strength of the ESF were also studied. From the data in the experiment, we found that, with the reinforced CF in the EP-hardener-HGMS, the density of the ESF is not changed clearly, but the compressive strength of the ESF is enhanced. Considering the two factors of the density and compressive strength, the ESF reinforced by 1.5 wt% CF with 90% AR-HEMS has better performance in the experiment. This kind of the ESF has 0.428 g/cm3 at 25 °C, respectively. The color of the curing agent used for the experiment is dark amber. Both of the resin and the curing agent are manufactured by AXSON Technologies Co., Ltd., Shanghai, China.Epoxy resin Araldite LY 1564 is a white transparent liquid with a viscosity of about 40 mPa·s. Polyamine curing agent Aradur 3486 is a light-yellow liquid. The Brockfield viscosity and density of the resin are 2800 mPa·s and 1.17 g/cm3 and 37.90 Mpa. This kind of the HGMS is manufactured by 3M company, MN, USA.The style of the hollow glass microspheres (HGMS) is S38HS, which is white. The density and compressive strength of the HGMS are 0.38 g/cm2. The specific surface area and bulk density of the aerogel are about 600 m2/g and 80 kg/m3. The porosity of the aerogel is about 90% to about 95%. This kind of the aerogel is manufactured by Zhongning Technology Co., Ltd., Shenzhen, China.Aerogel is a nano-porous solid material formed by the sol-gel method, using a certain drying method to replace the liquid phase in the gel with gas. The main components of aerogel is SiO3.The EPS beads selected for the experiment are produced by Hangchao Packaging Materials Co., Ltd., Hangzhou, China. The density of the EPS beads is about 10 kg/m3 and the wire diameter is about 7 μm.The CF used for preparing the CF reinforced EP-HGMS (CFR-EP) and added into the EP-hardener-HGMS system is manufactured by Weida Composite Materials Co., Ltd., Nanjing, China. The density of the CF is about 1.76 g/cmFirst of all, three parts of epoxy resin and one part of curing agent were mixed, and the mixed solution was fully mixed under magnetic stirring. Next, a certain volume fraction of EPS beads was measured into a plastic container, and the prepared epoxy-hardener was poured slowly and evenly into the plastic container. The mixture was stirred by the glass rod so that the epoxy curing system can be completely applied to the surface of the EPS beads. The EPS beads covered by the epoxy-hardener system were poured into the running ball machine and enough aerogel powder was sprayed into the ball machine. When the aerogel powder was completely wrapped on the EPS beads, the excess aerogel powder on the surface of the beads was filtered and poured into the tray. After the epoxy resin on the surface of the EPS beads was completely cured, one layer of AR-HEMS has been prepared. By repeating the above steps once or twice, two-layer and three-layer AR-HEMS could be prepared. The schematic diagram of the preparation of the AR-HEMS is shown in Multi-phase ESF mainly includes AR-HEMS and CFR-EP system. First, three parts of epoxy resin and one part of curing agent were measured, respectively. The solution is fully mixed under the magnetic stirring, and then the HGMS with 150% of the epoxy-hardener volume fraction and CF were added into the epoxy-hardener system. At this time, the viscosity of the mixed slurry gradually increased, which has a certain degree of plasticity. After the HGMS, CF and the epoxy-hardener were completely mixed, an amount of AR-HEMS was added into the slurry, and the macro-spheres were evenly distributed in the CFR-EP system. The mixed slurry was slowly poured into the circular mold. The mold filled with the slurry was placed in a hot press and kept at 100 °C for 1 h, and then placed in a cold press for about 15 min. After the epoxy resin in the system was completely cured, the multi-phase ESF had been prepared. The schematic diagram of the preparation of the ESF is shown in The influence of AR-HEMS with different stacking volume fractions on the compressive strength of ESF was studied in this group of experiments. Assuming that the volume fraction of the full mold is 100%, AR-HEMS with 20%, 40%, 60%, 80%, and 90% stacking volume fraction was added in the CFR-EP system with and without CF in this experiment. The influence of AR-HEMS with different layers on the compressive strength of ESF was studied in this group of experiments. AR-HEMS with no layer, 1 layer, 2 layers, and 3 layers were added into the CFR-EP system with and without CF in this experiment. The influence of AR-HEMS with different inner diameters on the compressive strength of ESF was studied in this group of experiments. AR-HEMS with an inner diameter of 7–8 mm, 9–10 mm, and 11–12 mm were added into the CFR-EP system. The influence of the CF content added into the EP-hardener-HGMS system on the compressive strength of ESF was studied in this group of experiments. 0%, 0.5%, 1%, 1.5%, 2%, and 2.5% CF were added into the EP-hardener-HGMS system in this experiment. The influence of the CF types added into the EP-hardener-HGMS system on the compressive strength of ESF was studied in this group of experiments. In total, 0.05 mm, 0.15 mm, 5 mm, and 10 mm of CF were added into the EP-HGMS system in this experiment. 3.In order to measure the diameter and mass of the AR-HEMS, 50 samples of the AR-HEMS were chosen for measurement, which is beneficial to avoid the accidental data. A digital vernier caliper  (Accuracy of 0.01 mm) and a digital analytical balance  (Accuracy of 0.001 g) were used to measure the diameters and the mass of the AR-HEMS. The specification of the mould used to prepare the ESF is Ø70 mm × 65 mm, H60 mm, and the volumes of the samples are all 199 cmThe Compressive Properties of the ESF were tested by an electronic universal testing machine . The GB/T16491-2008 is the standard in this experiment.Scanning electron microscopy (SEM)  was used to observe the EP-HGMS and the AR-HEMS system. The observed sample should be thin enough and be sprayed by the gold.It can be seen in This is the first example of an equation:3, 0.143 g/cm3, and 0.198 g/cm3. Although the strength of the sphere increases with the rise in the layers, the growth in the density of AR-HEMS will also lead to an increase in the density of the ESF material. In the deep-sea field, the increase in the quality will inevitably lead to the need for more power to drive the equipment to work. The great mass also increases the equipment load and reduces the service life to a certain extent. Therefore, when preparing ESF, it is necessary to select the appropriate layer of AR-HEMS, according to the specific working environment.As can be seen from 3, 0.143 g/cm3, and 0.142 g/cm3, respectively. When the layer is the same, the larger the inner diameter of AR-HEMS is, the smaller the strength of the sphere is, but its density also decreases. Therefore, density and strength are two contradictory materials. It is difficult to achieve both two good performances for the ESF materials. The key to preparing buoyant materials is to balance the density and compressive strength of ESF materials.In order to study the influence of AR-HEMS with different stacking volume fractions on the compressive strength of ESF, 20%, 40%, 60%, 80%, and 90% AR-HEMS were added into the matrix in this group. As shown in 3, 0.60 g/cm3, 0.53 g/cm3, 0.49 g/cm3, 0.45 g/cm3, and 0.423 g/cm3. When foam buoyancy materials are used in subsea equipment, ESF must have higher compressive strength to resist deep sea pressure, but lightweight materials can greatly reduce the energy consumption and provide more buoyancy. Therefore, strength and density are both important factors when preparing deep-sea buoyancy materials. In this group of experiments, when the stacking volume fraction is 90%, the ESF has the lower density and better compressive strength relatively, which can be used for subsea equipment working at a depth of about 1900 m.Although the compression strength of ESF decreases with the increase of the stacking volume fraction, the density of the composite material decreases. As shown in 3), it destroys the integrity of the ESF material. Therefore, the 0-layer AR-HEMS is not suitable for the preparation of the ESF materials. As shown in 3), but it is limited by the lower compressive strength, which is about 14.26 Mpa. In contrast, when the AR-HEMS is three layers, the ESF material has the higher density (0.45g/cm3), but this type of ESF has a wider range of applications and can be used for subsea equipment in the 2315-m deep sea. Therefore, the selection of materials during manufacturing must be based on the working environment and work requirements and the two performances of the density and compressive strength need to be weighed.In order to study the effect of the layers of AR-HEMS on the compression strength of ESF, 0-layer, 1-layer, 2-layer, and 3-layer AR-HEMS were added to the matrix with the stacking volume fraction of 90% in this experiment. As shown in 3. The ESF prepared by AR-HEMS with an inner diameter of 11–12 mm has a lower density, which is about 0.41 g/cm3, but its compressive strength is not high. In this group of experiments, ESF material prepared by the AR-HEMS with the inner diameter of 9–10 mm has relatively high compressive strength and can also provide more buoyancy for deep-sea equipment. Therefore, AR-HEMS with an inner diameter of 9–10 mm is the best choice for preparing buoyant materials in this experiment.In order to study the influence of a different inner diameter of AR-HEMS on the compressive strength of ESF, in this group of experiment, AR-HEMS with 7–8 mm, 9–10 mm, and 11–12 mm inner diameter were selected as fillers, and added to the matrix. As shown in 3 to 0.43 g/cm3. It can be seen from the data that the compressive strength of ESF increases with the increase of CF content. When the CF content is 1.5%, the CF is evenly distributed in the matrix, so the compressive strength of ESF material reaches a maximum value. It can be seen in the SEM of the matrix that the fiber and resin are tightly combined. When a load is applied to the ESF material from the outside, the CF will enhance the bonding force of the resin matrix due to its good tensile strength, and enhance the compressive strength of the composite material.In order to study the influence of different content of CF added into the matrix on the compressive strength of the ESF, 5-mm CF was selected in this group of experiments. As shown in As shown in 3. As shown in In order to study the effect of different lengths of CFs on the compression strength of ESF, 0.05 mm, 0.15 mm, 2 mm, and 5 mm of CFs were chosen in this experiment. 1.5% mass fraction of CF were added into the matrix. As shown in As a lightweight material with extremely strong tensile strength, CF is always added to the polymer to improve the overall performance of the composite material, which has become the mainstream of making high-strength and low-density materials. In this experiment, CF is mixed with resin matrix and HGMS. From the SEM picture of the composite material, it can be seen that the CF is distributed evenly in the matrix, and there is no CF agglomeration. The CF reinforced resin material, which is uniform, is extremely conducive to the conduction of the load inside the composite material. The CF and the resin matrix are tightly combined without clear material defects. The addition of CF has no major effect on the density of the composite material. However, the compressive strength of the composite material has been significantly improved with the addition of CF. It could be found that the compressive strength of the ESF prepared by the AR-HEMS with different stacking volumes without reinforeced CF in the EP-hardener-HGMS system was showed in The addition of CF increases the viscosity of the resin system, which prevents the floating of the EPS beads to a certain extent. When adding unreinforced EPS beads to the resin matrix, if CF is not added to the resin matrix, the ESF material will not be able to form. A large number of EPS beads was poured into the upper half from the lower half during the forming process. Thin shell-like materials are easily damaged during demolding, and, finally, appear as a composite material collapsed in the upper half, as shown in In all, six compared groups were carried out in this experiment. The ESF prepared in this experiment includes two parts of AR-HEMS and CFR-EP system. From the test of the performance and appearance of the ESF, some conclusions could be made.(a) The AR-HEMS prepared in this experiment is almost round, which indicates that the ‘rolling ball method’ is beneficial for the preparation of the AR-HEMS. The regular sphere added into the matrix is conducive to combine with the matrix, which enhances the compressive strength of the ESF. From the data of the AR-HEMS, we could see that the density of the AR-HEMS increases with the layer increasing and the inner diameter decreasing.3) and better compressive strength (19.46 Mpa) relatively. The compressive strength and density are both important factors when preparing deep-sea buoyancy materials.(b) The compressive strength of ESF decreases as the stacking volume fraction increases, and its decrease tends to be gentle. However, the density of the composite material decreases. When the stacking volume fraction is 90%, the ESF has the lower density (0.423 g/cm(c) The compressive strength of ESF increases as the layer grows, but its density gets larger. Therefore, the selection of the layer during manufacturing must be based on the working environment and work requirements of different equipment.(d) For equipment and materials used in the deep-sea field, compressive strength and density are always a contradiction. The compressive strength of ESF increases as the inner diameter decreases, but its density gets larger. The ESF prepared with AR-HEMS with the inner diameter of 9-10 mm has relatively better performance.(e) When the CF content is from 0%~2%, the compressive strength of ESF increases with the increase of CF content. However, when the CF content is more, the compressive strength of the ESF material changes and shows a downward trend because of the material defects.(f) 1.5% CF can be evenly distributed in the resin matrix, and longer CFs can be more closely combined with the resin matrix, which further improves the integrity of the composite material.(g) When the AR-HEMS accumulation ratio is 20%, the compressive strength of the ESF reinforced by CF material has the biggest change, which is increased by about 28% compared with the ESF without CF. The addition of the CF could increase the viscosity of the matrix, which prevents the float of the AR-HEMS. The CF as the reinforced material in the matrix could also improve the strength of the EP-hardener-HGMS system.


Reflex atrioventricular block is well-recorded although it is considered rare. Recent data suggests that it is less rare than has been supposed. It has been shown to occur in both vasovagal and carotid sinus reflexes. It has to be distinguished from paroxysmal atrioventricular block due to ventricular conduction tissue disease. Low chronic adenosine levels combined with adenosine release may mimic reflex atrioventricular block. Explanations of the mechanism of these phenomena have been lacking until the recent past. The relevance of reflex atrioventricular block to clinical decision-making is as a possible indication for pacing the heart with consideration given to the vasodepressor component of the reflex. Atrioventricular block (AVB) is a well-recognized condition due in most cases either to idiopathic disease of ventricular conduction tissue, the His-Purkinje system, or to ischemic damage to this tissue. Amongst the less common causes are invasion of calcification from calcific aortic valve stenosis and congenital. As AVB became better understood, particularly by employing increasingly sophisticated electrocardiographic monitoring, it became evident that lesser examples of the disease, such as bundle branch block, tended to progress over months or years. Progression often presented evidence of paroxysmal atrioventricular block heralding permanent block. Evidence also emerged in 1980s and 1990s that paroxysmal AVB could be part of carotid sinus or vasovagal reflexes  Figure .Reflex AVB, a form of paroxysmal AVB, was less well accepted –5 as it Conservative thinking concerning reflex AVB has been very strong with a tendency to deny the existence of two of these three mechanisms, namely reflex and hypoadenosinemic types, despite presentation of 4 clear cases of reflex AVB in the Lancet in 1988  and giveThis matter of cause of AVB was particularly illustrated in the opposition of reviewers to publication of the ISSUE  3 study  on the bAnalysis of the ILR recordings during syncope in the ISSUE 2 study  surpriseThe description type 1c raised a question of ventricular conduction tissue disease in the minds of the classification's proposers. Thus, this finding in ~20% of the clinically determined reflex  was unexpected raising the possibility of a different mechanism, namely intrinsic disease of the His-Purkinje system as observed in Stokes-Adams attacks. At this time, even the ISSUE 2 investigators did not completely appreciate that such findings could be compatible with reflex AVB. When the ILR recordings were available from the ISSUE 3 study  a very sFollowing the results of the ISSUE 2 study, the literature again contained definite examples of reflex AVB  and evidThus, paroxysmal AVB can be considered to be of three types as put forward in the review by Aste and Brignole , Table 2In making a differential diagnosis, these aspects require consideration:- other associated reflex features e.g., nausea; coincident vasodepression;- evidence of already existing ventricular conduction tissue disease e.g., bundle branch block;- plasma adenosine level- lack of prodrome.Vasodepression as an integral part of reflex syncope must always be considered because it is always present . Not onlUnfortunately, adenosine has been a difficult to acquire measurement in routine clinical practice. However, shortly, a simple easily performed test will become available. It has been shown that there are clear differences between vasovagal patients  and those with low adenosine. Carotid sinus syndrome (CSS) patients show similar low adenosine levels to those presenting hypoadenosemic AVB . This maHow do these findings influence a decision to pace the heart? Complete AVB with or without asystole is generally considered an indication for permanent pacing whatever its mechanism. In the case of progression of ventricular conduction system disease the indication to pace is clear when complete AVB or asystole occurs. The relatively new findings reviewed here, reflex and hypoadenosinemic AVB prompt a reassessment of the indications for pacing.In vasovagal syncope even more than in CSS consideration needs to be given to the timing of bradycardia and timing of loss of consciousness, as vasodepression starts many minutes before cardioinhibition, which might prevent or markedly reduce pacing benefit –19. ThesIn hypoadenosinemia, the experience is, so far, small but pacing seems to be very effective  althoughIn summary, reflex atrioventricular block is more common than previously thought as evidenced by the ISSUE 2 and 3 studies , 14. It The author confirms being the sole contributor of this work and has approved it for publication.RS is a consultant to Medtronic Inc., a member of the speakers bureau of Abbott Laboratories Inc.  and stock holder in Edwards LifeSciences Corp., Boston Scientific Inc. and AstraZeneca PLC.


Here, we reveal that the transcription factor Y-box binding protein 1 (YB-1) interacts with β2-adrenergic receptor (β2-AR) following stimulation with the agonist isoproterenol (ISO). Clinicopathological analysis demonstrated that β2-AR is significantly correlated with YB-1, which favours the progression of HCC. The binding of YB-1 with β2-AR resulted in YB-1 phosphorylation at serine 102 (S102) via the β-arrestin-1-dependent activation of the PI3K/AKT pathway, followed by the translocation of YB-1 to the nucleus to carry out its tumour-related function. β2-AR-mediated activation of YB-1 facilitated epithelial-to-mesenchymal transition (EMT) and HCC metastasis. The interference of YB-1 expression significantly attenuated liver tumour metastasis induced by chronic stress. Analysis of the transcriptional profile and chromatin immunoprecipitation (ChIP) identified β-catenin as a crucial target of YB-1. Our results unveiled a novel β2-AR-mediated regulatory axis in HCC metastasis that might be helpful for the development of HCC therapeutics. Hepatocellular carcinoma (HCC) is the most common primary liver cancer2. Despite an increasing number of treatment options for HCC, the overall prognosis of this disease remains unsatisfactory due to tumour metastasis3. HCC metastasis is attributed to two models: a genetic model based on genomic mutations and an epigenetic model defined mainly as modifications in gene expression that do not change the DNA nucleotide sequence1. Both the genetic and epigenetic models of HCC metastasis complement each other, as genetic alterations result in the formation of a metastatic cell phenotype early in tumorigenesis, while epigenetic modifications induced by external signals such as chemokines and neurotransmitters facilitate the manifestation of such a phenotype4. A broader interpretation of this epigenetic model of metastasis results from the regulation of the migratory activity of tumour cells by G protein-coupled receptors (GPCRs). Once GPCRs bind with potential ligands, they directly or indirectly activate intracellular protein kinases, subsequently triggering a complex series of invasion/metastasis-related cascades5. Stress-induced neurotransmitters released from the neuroendocrine system are prominent GPCRs ligands. They not only induce the dissemination of metastatic cells from the primary tumour but also guide these cells in a selective and organ-specific way4. These neurotransmitters have been found to participate in the metastasis of multiple types of tumours, including breast, colon and small-cell lung carcinoma (SCLC), suggesting that psychosocial factors favour the progression of cancer11. However, the crosstalk between these factors and the initiation and progression of HCC metastasis remain unclear.Liver cancer, which has an incidence of ~850,000 new cases every year, is the second leading cause of cancer-related death worldwide13. Abundant β2-AR expression is closely correlated with poor clinicopathological features, tumour recurrence, metastasis, and reduced survival17. Therefore, β2-AR is a cancer-relevant biomarker in carcinogenic processes. Due to the clinical significance of β2-AR expression in tumour patients, different antagonists of β2-AR, such as propranolol, atenolol, and ICI118,551, began to be gradually tried for cancer treatment18. However, the inhibitory effects of these β-AR blockers vary dramatically18. Elucidation of the β2-AR functional domain and the mechanism of its signal transduction would be beneficial for the development of pharmaceutical tumour therapies.The catecholamines epinephrine and norepinephrine, which are classic stress hormones, bind with β-adrenergic receptors (β-ARs), members of a family of seven transmembrane GPCRs. β-ARs, especially β2-adrenergic receptor (β2-AR), are aberrantly expressed in a variety of tumour tissues, where they mediate malignant cancer cell behaviours19. Additionally, this receptor can induce epidermal growth factor receptor (EGFR) dimerization, tyrosine autophosphorylation and EGFR internalisation, a mechanism of ERK1/2 transactivation that stimulates its phosphorylation20. Several signal transducing adaptor proteins, such as β-arrestin, are recruited to activate assorted intracellular proteins that carry out a broad range of functions21. In the context of psychological stress, the β2-AR-dependent interaction between β-arrestin-1 and p53 leads to the Mdm2-mediated degradation of p53 and the accumulation of DNA damage22. Chronic stress also promotes HIF-1α expression through a β2-AR-dependent pathway to drive cancer progression. However, the regulatory mechanisms by which β2-AR promotes HCC progression have not been fully clarified.As a transmembrane receptor, β2-AR initiates intracellular signalling cascades, including the cAMP/PKA, MAPK/ERK1/2, p38/MAPK, PI3K/AKT, VEGF and Src/STAT pathways, through an allosteric mechanism24. In the present study, stimulation of β2-AR with isoproterenol (ISO) induced the phosphorylation of YB-1 at S102 and the subsequent β-arrestin-dependent nuclear translocation of YB-1 into HCC cells. YB-1 drove the progression of HCC cells through the transcriptional activation of β-catenin. These findings delineate a novel mechanistic link between chronic psychological stress and tumour progression and open up new avenues for the development of therapies to treat tumour metastasis.To search for the crucial factor(s) involved in β2-AR-regulated HCC metastasis, we performed immunoprecipitation-mass spectrometry (IP-MS) analysis of HCC tissues. A vital oncoprotein, Y-box binding protein 1 (YB-1), was shown to interact with β2-AR in HCC cells. The pro-metastatic effect of YB-1 on HCC cells was regulated by β2-AR via the phosphorylation of serine 102 (S102) in YB-1, which facilitates YB-1 nuclear localization and activityn = 200) and the Affiliated Hospital of Nantong University  between January 2004 and December 2009 were included in this study. The study was approved by the Research Ethics Committee of the two institutes in accordance with the Helsinki Declaration, and written informed consent was obtained from every patient. Pre-operative clinical diagnoses of HCC met the diagnostic criteria of the American Association for the Study of Liver Diseases. These inclusion criteria were (a) a distinctive pathologic diagnosis, (b) no pre-operative anticancer treatment or distant metastases, (c) curative liver resection, and (d) the availability of complete clinical-pathologic and follow-up data. The pathological tumour-node-metastasis (pTNM) classification of HCC was based on the staging system from The American Joint Committee on Cancer/International Union against Cancer . Differentiation statuses were graded according to the method of Edmondson and Steine. The main clinical and pathological characteristics of both cohorts are shown in Supplementary Table A total of 300 adult patients with HCC who underwent curative hepatectomy at the Affiliated Cancer Hospital of Nantong University  staining intensities were graded from 0 to 2 . The staining extent was graded from 0 to 4 based on the percentage of immunoreactive tumour cells . The final scores for the specimens calculated from the staining intensity score and the staining extent score ranged from 0 to 8. Based on the final scores, the IHC results were classified as indicating either low expression (<4) or high expression (≥4). The above procedure of evaluation was performed by three independent pathologists with a multihead microscope blindly, and a consensus was achieved among the three pathologists.2 at 37 °C. The source of cell lines were identified and they were recently authenticated by STR profiling and tested for mycoplasma contamination. A 10 μM ISO solution was prepared fresh for every experiment by dissolving ISO bitartrate salt (Sigma) immediately before stimulation. ICI118,551 , 5-(2-benzothiazolyl)-3-ethyl-2-(2-(methylphenylamino)ethenyl)-1-phenyl-1H-benzimidazolium iodide  or LY294002  were added to the culture medium for 30 min before stimulation with ISO.Human HCC cell lines , a normal liver cell line (LO2), and HEK293 cells were obtained from the Institute of Cell Biology and cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% foetal bovine serum , 100 U/ml penicillin, and 100 μg/ml streptomycin in an incubator with 5% CO6 cells in 50 μL of PBS were injected into the left hepatic lobes of the nude mice. The restraint-stress procedure refers to a physical restraint system for 2 h each day described in previous studies16. To track tumours in vivo, the transplanted cells were infected with firefly luciferase-expressing lentiviral particles (Research Science). Tumour formation and metastases were imaged by bioluminescence using an IVIS 100 Imaging System (Xenogen). Luminescent images were captured using the desired exposure times (1–60 s). The resulting greyscale photographic and pseudo-coloured luminescent images were automatically superimposed using IVIS Living Image (Xenogen) software. Four weeks following cell transplantation, the mice were sacrificed, and their organs were dissected and subjected to standard histological examination. The procedure were performed blindly.Forty 12-week-old athymic female BALB/C nude mice were housed under standard conditions and cared for according to institutional guidelines for animal care. All animal experiments were approved by the Committee on the Use of Live Animals in Teaching and Research, Nantong University. The mice were randomly divided into four groups. A total of 1 × 1025. The antibodies used are listed in Supplementary Table 26.The experimental procedures were performed as previously describedChromatin immunoprecipitation (ChIP) assays were performed using a Pierce Agarose ChIP kit  in accordance with the manufacturer’s protocol. The primers used for the ChIP assay are listed in Supplementary Table t-test, and qualitative variables were compared using the Pearson χ2 test or Fisher’s exact test. Kaplan–Meier analysis was used for survival analysis, and the log-rank test was used to compare differences. The Cox proportional hazards model was used to determine independent factors that influenced survival and recurrence based on variables identified as significant by univariate analysis. All analyses were performed using SPSS® software . P values < 0.05 indicated statistical significance in all analyses. All the experiments were carried out on at least three separate occasions.Quantitative variables were compared using Student’s Many candidate β2-AR-interacting proteins , among which YB-1 exhibited the greatest abundance and correlation with oncogenesis, were identified by using mass spectrometry. To understand the clinical relevance of YB-1 and β2-AR, we used two sets of HCC patient cohorts including 83 and 300 paired HCC tissues respectively. At first, we examined the mRNA level of β2-AR and YB-1 in 83 paired HCC samples and normal liver tissues Fig. . CompareNext, we did immunohistochemistry and further analyzed the protein expression of β2-AR and YB-1 in 300 paired HCC tissues. In HCC tissues, β2-AR primarily localized in the cytoplasm, while YB-1 primarily localized in the nuclei Fig. . In addiTo gain better insight into the interaction between β2-AR and YB-1, co-immunoprecipitation (Co-IP) was performed in HCC tissue specimens and SMMC-7721 cells. As expected, YB-1 was pulled down in metastatic HCC specimens and SMMC-7721 cell lysates pre-treated with 10 μM ISO with an anti-β2-AR antibody, and β2-AR was pulled down with an anti-YB-1 antibody Fig. . These rVectors containing the sequences of full-length or truncated HA-tagged β2-AR and Myc-tagged YB-1 were subsequently constructed to identify the precise domain(s) responsible for the interaction between β2-AR and YB-1. HEK293T cells were transfected with these constructs, and Co-IP was performed to detect the potential interaction. Myc-tagged YB-1 immunoprecipitated with anti-HA antibody Fig. . Three dTo determine the underlying mechanism by which β2-AR regulates YB-1, the β-adrenoceptor agonist ISO was used to stimulate HCC cell lines. The addition of 10 μM ISO rapidly induced the phosphorylation of YB-1 (P-YB-1) at S102 in SMMC-7721 cells whereas β2-AR knockdown  and HCC cell lines  were determined. The expression of β2-AR and YB-1 at the transcriptional and translational levels was remarkably elevated, along with an increase in metastatic potential in HCC cell lines compared to that in normal hepatocytes , a key step in the metastasis of epithelial tumours, including HCC Fig. . ImmunofTrim28, rbpj, lims1, hnrnpab, snail, wnt5a, fam101b, ctnnb1, and gsk3b) responded to stimulation with ISO and YB-1 depletion, suggesting that they are target(s) of the YB-1 protein  of nuclear YB-1, SK-Hep1 cells and SK-Hep1 cells that had been transfected with YB-1 siRNA for 24 h were stimulated with 10 μM ISO for 6 h. Microarray analysis was performed to compare the transcriptional profiles of SK-Hep1 cells subjected to YB-1 knockdown. A total of 18,893 differentially expressed genes (DEGs) in the three groups  were identified. Gene ontology (GO) analysis revealed 34 DEGs related to EMT, the expression levels of which were dynamic, as shown by hierarchical cluster analysis Fig. . Among tein Fig. . Specifiant Fig. . ConsideThe putative Y-box elements in the β-catenin promoter region were cloned into pRL-CMV vectors, which were used to transfect SK-Hep1 cells. The cells were stimulated with ISO for 48 h. A luciferase reporter assay demonstrated a time-dependent increase in β-catenin promoter activity, which peaked at 6 h, following ISO treatment Figs. c and 6d.In the context of chronic stress, catecholamines are released by the neuroendocrine system to modulate carcinogenesis. To determine whether HCC metastasis is modulated by chronic stress via β2-AR/YB-1 signalling, a restraint-stress model utilizing a physical restraint system for 2 h each day was designed. Orthotopic xenografts were produced by injecting HCC cells into mouse livers to establish mouse liver tumour models. Mice subjected to chronic restraint stress for 4 weeks exhibited rapid tumour growth and metastasis Fig. . However28. This led to the consensus that tumour cell migration and invasion are not only genetically determined but also distinctly environmentally regulated29. Long-term anxiety in patients with HCC triggers adrenergic signalling, which worsens prognosis30. As an important receptor of stress-induced catecholamines, β2-AR is implicated in tumour development, growth, and progression, including migration. Currently, the exact mechanism linking β2-AR activation to malignant progression is unclear. Here, we identified a novel β2-AR/YB-1/β-catenin axis that participated in the regulation of HCC metastasis in response to chronic stress. Our results provide a new avenue for the development of β-blocker-based therapy for HCC metastasis.Since 1926, psychosocial stress has been closely associated with carcinogenesis1A, α1B, α1D, α2A, α2B, α2C, β1, β2 and β3) that transduce the effects of (nor)epinephrine in a variety of cell types31. Signalling from β2-AR is first initiated by either a G protein or β-arrestin. β2-AR agonist triggers the coupling of β2-AR to Gs, activating either cAMP-dependent protein kinase A (PKA) or guanine exchange proteins directly activated by cAMP (EPACs). PKA subsequently either activates cytoplasmic kinases, including mitogen-activated protein kinase (MAPK)32, or enters the nucleus to phosphorylate the CREB transcription factor11. PKA can also mediate phosphorylation of β2-AR, causing it to couple with Gi. This G protein switching is associated with the Gbγ-mediated activation of alternative signal pathways, such as the PI3K/AKT pathway33. EPACs further transduce signals through the Rap family of small Ras-like GTPases34. Alternatively, β2-AR can induce cellular responses that involve members of the β-arrestin protein family. β-Arrestins not only participate in the attenuation of β2-AR signalling and receptor internalisation but also play roles in signal transduction by their connection to multiple signalling pathways, such as the p38, ERK1/2 and NF-κB signalling pathways35. In the present study, we have shown that the β2-AR agonist ISO can facilitate the phosphorylation of YB-1 in an AKT activity-dependent manner in HCC cells. Meanwhile, β-arrestin was shown to be an indispensable signal transducer rather than a signalling inhibitor. These findings suggest that the synergistic effects of β2-AR-mediated pathways are involved in the progression of HCC.β2-AR is one of nine ARs  of YB-1. The critical and multi-functional protein β-catenin, in addition to other EMT-promoting factors including Snail and Twist, was shown to be regulated by YB-1 in response to the stimulation of β2-AR in HCC cells. It is reported that knockdown of YB-1 impairs Wnt/β-catenin signalling pathway and reduces the numbers of HCC initiating cells, whereas YB-1 also mediates breast cancer invasion and metastasis via regulation of MMP1 and β-catenin41. Such findings might be helpful to address the complex cellular events during HCC metastasis and provide a potential target for pharmaceutical development.YB-1 is implicated in the malignant phenotypes of various carcinomas. Cytoplasmic YB-1 facilitates the translational activation of Snail, HIF1α and Twist, which induces EMT in cancer cellsIn the context of chronic stress, β2-AR signalling induces the phosphorylation of YB-1 at S102 via β-arrestin-1-dependent activation of the PI3K/AKT pathway. Activation of YB-1 promotes the expression of β-catenin, which mediates the progression of HCC.Supplementary TablesSupplementary Figure 1Supplementary Figure 2Supplementary Figure legends


In 2007, and in a series of later papers, Joy Christian claimed to refute Bell’s theorem, presenting an alleged local realistic model of the singlet correlations using techniques from geometric algebra (GA). Several authors published papers refuting his claims, and Christian’s ideas did not gain acceptance. However, he recently succeeded in publishing yet more ambitious and complex versions of his theory in fairly mainstream journals. How could this be? The mathematics and logic of Bell’s theorem is simple and transparent and has been intensely studied and debated for over 50 years. Christian claims to have a mathematical counterexample to a purely mathematical theorem. Each new version of Christian’s model used new devices to circumvent Bell’s theorem or depended on a new way to misunderstand Bell’s work. These devices and misinterpretations are in common use by other Bell critics, so it useful to identify and name them. I hope that this paper can serve as a useful resource to those who need to evaluate new “disproofs of Bell’s theorem”. Christian’s fundamental idea is simple and quite original: he gives a probabilistic interpretation of the fundamental GA equation  In this introductory Recall that Bohm and Aharonov (1957) , in ordeOf course, this author’s opinion is that Christian’s approach is fundamentally flawed, and throws no light whatsoever on the foundations of quantum mechanics. Moreover, Christian’s “model” went through a number of major transformations, and one can better talk about Christian 1.0, Christian 2.0, and Christian 3.0. With each new edition, more complexity was added, the mistakes of the previous edition were hidden more deeply, new errors were made, new misconceptions were exposed. Since numerous authors criticise Bell’s theorem on the grounds of the same misconceptions, and more generally, follow similar tactics in their attempt to rescue Einstein from Bohr’s victory, I think it is useful to look at this whole process. The work does throw light on how Bell is repeatedly misunderstood.a and b are ordinary 3D real vectors, with scalar product To warm up, I will recall in The GA equation is actually the core of Christian’s opus, a book  and verya and b on two entangled spin-half particles in the singlet state, is just the negative cosine of the angle between the two directions, i.e., Note that the famous negative cosine curve of the singlet correlations, i.e., the correlations between spin measurements in directions  theorem  on the iBefore going into Christian’s works in more detail, I will summarise, in As another brief interlude, in In I will conclude the paper in By the way, I personally have benefitted enormously from interaction with Bell-deniers, and in particular from a ten year interaction with Joy Christian himself. We have in common a tenacity and conviction that we are right which is probably not healthy. Several Bell-deniers, whom I enormously respect, actually laid their finger on very sore points concerning past experiments, and on very sore points in the common ways that experimental data were analysed. In fact, up till 2015, no experiment had really proved anything. They had, of course, always confirmed quantum mechanics, but that could hardly be a surprise. They had not disproved local realism. It was the unrelenting criticism of a small number of intelligent Bell deniers, in the face of ostracism from the physics establishment, with all the serious effects which this has on their academic careers or scientific reputation, which did actually force the experimental refinements which led up to the first “loophole-free” experiments. The success of these experiments is vital to the reliability of quantum technology which is already being marketed successfully.The two problems I mentioned are historical problems, since science is evolving, and new generations see scientific hierarchies and divisions differently from old generations. Hopefully these problems will go away, but I fear that new sorts of problems will arise. New dogmas and misconceptions and fashions will take over, for instance, concerning the power of AI and the power of analysis of unscientifically collected “big data”.Of course there remains the big problem of interpretation of quantum theory, itself connected to the much older problem of the interpretation of probability. These problems are not addressed in this paper, even though Christian believed that he was making a revolutionary contribution to the foundations of quantum theory.An algebra over a field is a vector space over that field endowed with a bilinear product.Our primary example is the set of Every We can also multiply matrices. The matrix product is bilinear, that is, it is linear in each argument. Matrix multiplication is associative. We already mentioned the zero matrix; there is also a multiplicative unit, the For now a side remark, it must be mentioned that it is sometimes useful to discuss more general abstract algebras: algebras which are not necessarily associative and not necessarily unital. In particular, we will later briefly meet the octonions, which, oh horror, are not associative. Together with the real numbers, the complex numbers, and the quaternions, these four spaces are sometimes called number systems and denoted Let me return to the example of our 8-dimensional real algebra My description was not quite the “official” definition of a Clifford algebra, but is sufficient for our purposes. We are mainly interested in the 8-dimensional algebra p and q be nonnegative integers. The Clifford algebra p square to q to A bit more generally, let Each Clifford algebra has an even sub-algebra, which by definition is the smallest algebra containing all multiples of an even number of the generating elements Thus within our favourite algebra We already mentioned that its even subagebra can be identified with the quaternions. This is the real linear algebra of linear combinations of the four elements It must be emphasised that switching some signs or permuting the order does not change the algebra generated by M commutes with everything, and obviously p and q are quaternions, M commutes with the quaternions, and M is itself another square root of minus one.Obviously, Notice that a, b of The real reason for calling this algebra a geometric algebra comes from locating real 3D space within it, and also recognising geometric operations and further geometric structures with the algebra. To start with, look at the linear span of In the latter incarnation  , has twoThe reader is referred to Chapters 8 and 9 of that work whose endnotes contain many, many further references. What is important to notice for the main theme of the present paper, is that prior to Christian’s work, there already was a large literature devoted to expressing quantum theory in terms of geometric algebra. Though it seems that this literature did not gain a place in mainstream quantum theory, one of Christian’s aims had already been fulfilled: geometric algebra had already been put forward as a vehicle for re-writing quantum theory with geometry instead of Hilbert space at the forefront. I will later try to explain why, I believe, Doran and Lasenby’s project  to gBefore continuing, I would like to make some remarks on computation. In order to test formulas, or in order to simulate models, it is convenient to have access to computer languages which can “do” computer algebra.First of all, the fact that For those who like to programme in the statistical language R it is good to know that there is an R package called “onion” which implements both the quaternions and the octonions.GAViewer which accompanies the book Dorst, Fontijne and Mann (2007)  mBut anyway, within Christian’s “model” as we have it so far, we can just as well define So now we have the modelThis is where things go badly wrong. On the one hand, the model is not yet specified, if Christian does not tell us how, exactly, he means to map the set of two possible values Let us take a look at each mis-step separately. Regarding the first (incomplete specification), there are actually only two options (without introducing completely new elements into the story), since Christian already essentially told us that the two values Regarding the second mis-step, Christian proceeds to compute the geometric product I next would like to take the reader to Christian’s “one page paper” Christian (2011) , simultaIt seems clear to me that by 2011, Christian himself has realised that his “model” of 2007 was incomplete: there was no explicit definition of the measurement functions. So now he does come up with a model, and the model is astoundingly simple … it is identical to my second model:A and B by dividing the mean value of the product by the non random “scale quantities” Now the cunning device of representing these two random variables as products of fixed bivectors and random bivectors allows Christian to propose a generalised Pearson bivector correlation between Since the geometric product is non commutative, one must be careful about the order of these operations (and whether we divide on the left or the right), but I will follow Christian in taking what seems natural choices: “a” always on the left, “b” on the right.Unfortunately, since l (2012)  I have aChristian’s computer programming supporters discovered themselves that his formulas did not give the right answer, and discovered the “fix”. His GA model is an amusing way to compute a cosine function by a Monte-Carlo simulation, in which the cosine itself is already built in: GA knows the cosine function since it already knows how to compute I will now go on to discuss subsequent versions of Christian’s model. He has elaborated more or less the same “theory” with the same repertoire of conceptual and algebraic errors in various papers with increasing levels of complexity. This work shows a remarkable degree of dedication and persistence, and erudition too, as more and more complex mathematical constructions are brought into play. However the flaws in the foundation are unchanged and one of them (as has been said by many before) is unredeemable: Bell’s theorem is about probabilities of different combinations of pairs of binary outcomes. A local hidden variable theory has to explain the correlations between binary outcomes which are predicted by quantum mechanics, following standard, minimal, interpretations: quantum mechanics predicts the probability distribution of measurement outcomes. Experimenters (in this field) count discrete events and estimate probabilities with relative frequencies, and then compute correlations as defined to be the probability of equal outcomes minus the probability of opposite outcomes. A statistician would call them unnormalised and uncentered (or “raw”) empirical product-moments. In the case of the singlet correlations, the expectations are zero, hence the standard deviations are one and the means are zero; the physicist’s theoretical correlations are actually also the statistician’s theoretical correlations.IJTP. I wrote to the editors, saying that the paper should be retracted because of its obvious errors, but the editors preferred to print my critique—for a fee. So this became a short paper Gill (2016) . The publisher apologizes for any inconvenience this may cause.Through an administrative error, the author had not been informed.Christian (2015)  is entitAmusingly, if there were a local realistic model of the singlet correlations, it would have been easy to experimentally violate Bell inequalities. The editors clearly have no clue whatsoever as to the logic of Bell’s theorem. The experiment would have proven that the “theoretical” result of Bell was actually wrong, just as Christian claims.Quite amazingly, this paper included a computer simulation, allegedly of the author’s model, but actually a simulation of Pearle’s famous detection loophole model of 1970 . The autIncidentally, the Pearle detection loophole model, and the earlier discussed GA cancellation tricks, have nothing whatsoever to do with one another. Of course, the author does not even mention the mismatch. One marvels that referees can recommend publication of a series of papers with such evident self-contradictions, and that editors do not have a clue. I am getting old. O tempora, o mores!Royal Society Open Science, entitled "Quantum correlations are weaved by the spinors of the Euclidean primitives". Here he presents a (for him) new argument why Bell’s theorem is wrong. He claims that the use of expressions involving simultaneously measurement functions evaluated at several different measurement settings cannot possibly have any physical meaning and hence that proofs which depend on the evaluation of such expressions are physically meaningless and hence invalid.At last Christian managed to publish a really major paper  in the jBut under local realism, the local hidden variable Note that when I use the word “exists”, I speak as a mathematician, and I am discussing mathematical models. For me, local realism means the existence of a mathematical model which reproduces the predictions of quantum mechanics (or approximately does so). It is up to physicists and to philosophers to thrash out what they mean when they use words like local, real, or exist. The mathematics which they do should “stand alone”, the words used to describe various objects and to describe the relations between them are irrelevant.Christian is not the first physicist to get confused on this issue. It keeps popping up. It has kept popping up for 50 years. Yes, the proof of the CHSH inequality involves combining measurement functions with different settings inserted but depending on the same value of the hidden value. Yes, quantum physics says that one cannot measure spin in different directions at the same time. So what? Who says we are doing that?a, b, can only be Apart from that, the paper yet again proposes a local hidden variables model; but a new one this time. The hidden variable The proof that the singlet correlations are produced by this model is more sophisticated than ever; and the paper actually claims to deal with all quantum correlations, not just the EPR-B correlations. The usual errors can be located in the proofs, together with a new one: a limit is computed as There is also a proof of an astonishing pure mathematical result in this paper, which has been separated off and submitted as a separate publication to a pure mathematics journal. I will deal with that at the end of this section. That paper has not yet appeared in print.The referee reports are published online. Three referees wrote short reports saying that the paper was pure nonsense, giving some documentation and/or argumentation. Several earlier papers have been written and published in decent journals explaining why Christian’s model is no good, and the old criticism applies to the new paper, too. Two referees wrote very, very short reports saying no more and no less than that the paper was brilliant. The author has proudly remarked elsewhere that the journal kindly waived his publication fee, so at least that is something.Again several folk wrote to the journal complaining about the paper and arguing that it should be withdrawn. I wrote complaining that my computer code had been stolen. A committee is evaluating the paper and an “expression of concern” has been posted. I have the impression that the journal is just waiting for the problem to go away.IEEE journal, entitled “Bell’s Theorem Versus Local Realism in a Quaternionic Model of Physical Space” [Annals of Physics paper “Local Causality in a Friedmann–Robertson–Walker Spacetime” which I discussed earlier. It combines elements from all previous works!Again, a big success came with a publication in an l Space” . This isI complained of plagiarism to the journal, and I am told that an investigation is underway.In this latest paper "Eight-Dimensional Octonion-Like but Associative Normed Division Algebra"  which ChHe writes “the corresponding algebraic representation space … is nothing but the eight-dimensional even sub-algebra of the M squares to This space is well known to be isomorphic to v. It satisfies Take any unit bivector Bulletin of the American Mathematical Society reproduced on his web pages http://math.ucr.edu/home/baez/octonions/. Full proofs are given but already the key definitions and main theorems are clearly stated in the first couple of pages.Recall that a normed division algebra is an algebra that is also a normed vector space and such that the norm of a product is the product of the norms; a division algebra is an algebra such that if a product of two elements equals zero, then at least one of the two elements concerned must be zero. A splendid resource on the Hurwitz theorem and the octonions is John Baez’s celebrated 2002 article in the GAviewer, in order to verify for themselves whether or not the even sub-algebra of To the computer-savvy person who is familiar with the basic concepts of geometric algebra, I recommend use of Dorst et al.’s  already The isomorphism can be verified by first drawing a correspondence between n-sphere. That is, a sphere from the point of view of all its topological properties, but carrying a smooth structure that is not the familiar one. They were first discovered by Milnor in 1956 for the 7-sphere, and have been further classified by him and many other researchers. It is unknown if exotic 4-spheres exist. There are no other exotic spheres in dimensions 1 up to 6; from then on, they only exist in some dimensions, and much is still unknown about them.There do exist some exotic objects called “exotic spheres”: differentiable manifolds that are homeomorphic but not diffeomorphic to the standard Euclidean Christian claims that his model depends on the Hopf fibration, a wonderful way to look at the 3-sphere. He claims that a uniform distribution on The huge number of attempts  to refute Bell’s theorem has to do, in my somewhat biased opinion , with two problems in physics. One is that physicists use mathematics as a language in which they speak about the physical world. This makes it harder for them to distinguish between models and reality. Mathematicians do not only calculate; they also study mathematical models, thought of as stand-alone abstract worlds. The common distinction between theoretical physics (part of physics) and mathematical physics (part of mathematics) corresponds, in my mind, to this same difference between on the one hand talking with mathematics about the real world and on the other hand talking with logic about imaginary or toy mathematical worlds. The second problem is the lack of appreciation of many old school physicists for modern probability theory, and lack of knowledge about modern statistics. Both of these problems are historical problems, science is evolving, and new generations see scientific hierarchies and divisions differently from old generations. Incidentally, Bell, who was a physicist, had a consummate understanding of statistical issues, though he was hampered by the language he was obliged to use in order to communicate with the physicists of his day.Christian’s main problem is that he does not understand Bell’s theorem as a mathematical theorem, and that he does not have the mathematical discipline to be sufficiently careful with notation. On the other hand he is so confident of his physical insight that he is not going to let any mathematical difficulties get in his way. The ultimate authority for him is nature, not logic. However he does not provide any new understanding of nature.His original errors were to ignore the fact that measurement outcomes in Bell’s theorem, and also in experimental tests of Bell’s theorem, are binary. The statistics which experimentalists calculate are called correlations, but they are just simple functions of counts of various kinds of combined events. Changing the co-domain of the measurement functions, by which Christian means that he embeds the numbers Foundations of Physics). Clearly, Christian realised that he had made a mistake, though he never admitted to one. In his next try he did define measurement functions which took as outcomes the values In his very first paper, Christian took as measurement outcomes the bivectors of the standard Clifford algebra of real 3D space. Plenty of authorities pointed out that this does not disprove Bell’s theorem—it results in some mathematics which is irrelevant to Bell’s theorem . The Hurwitz theorem is a good deal harder to prove, and a good deal deeper. Fortunately, there is no need to go into the mathematical details of Christian’s work to see that his conclusions must be wrong – the conceptual misunderstandings of Bell’s theorem are immediately obvious. His counter-examples must be wrong. His arguments that Bell made some mistakes are completely misguided.Many attempts to disprove Bell’s theorem use very sophisticated mathematics to come up with a counter-example, the mathematical details of which are beyond most people’s capacity to check. In fact, the authors’ enthusiasm with their vindication of Einstein and their proof that Bell and Bohr were wrong also clouds their vision and the errors are hidden in little notational slip-ups at the end of a huge analysis, when they feel they are “on the home stretch”, and discipline is relaxed. In fact this is much like accidents in mountain climbing.There is no need to analyse the proof, since one usually can easily see by the general introductory remarks in such papers that the authors do not understand the point or the logic of Bell’s theorem. I hope that readers of this cautionary tale will learn not to be impressed by apparently learned use of abstract mathematics, and understand how simple Bell’s theorem is. Extraordinary claims require an extraordinary degree of support. Usually it is not necessary to work through obscure mathematical details to find an error. One can see in the introductory words that the author did not understand Bell’s theorem or its proof in the first place, hence his or her counter-example or meta-physical arguments can reasonably be taken with a grain of salt. As a last resort, ask the author to establish their claim by publishing computer programs which can be run on a network of ordinary home computers and which violate Bell inequalities by a substantial margin (see Gill (2014)  for a diIJTP and Annals of Physics used to have good reputations. In their defence, it must be said that Christian himself deliberately formulated his work as an exercise in relativity theory in order to avoid close inspection by experts in quantum information. The journals RSOS and IEEE Access have a world expert on geometric algebra on their editorial boards. Of course, such a person would believe that GA should de-mystify Bell’s theorem and possibly then even solve the problems of interpretation of quantum mechanics, just as David Hestenes had intimated. Unfortunately such an expert is typically not an expert on Bell’s theorem, quantum information, and the interpretation of quantum mechanics.We also learn that the peer-reviewed literature is full of pure nonsense. Even established and reputable publishing houses now offer their own “tame quality predatory journals” in order to satisfy the huge world-wide economic/social demand for peer-reviewed publishing venues. After all, how else can university managers decide who should get promotion or tenure? At least, these more established and more professional publishing houses can offer a bigger guarantee that their publications will still be accessible in, say, twenty years, and maybe even in a century . More than ever, young researchers have to learn not to trust anything, even if that is going to impede their own academic progress. The journals A bit more amateur psychology: we all too easily see what we want to see. So, dear reader, do not trust me either. Check and find out for yourself whether or not I am right. You might even like to learn a bit of geometric algebra, like I had to. Maybe it will come in useful for you. I look forward to hearing from you exactly what I have got wrong.


Circ-centro-some/spindle pole-associated protein (CSPP1) has been confirmed to be characterized in diverse human malignancies and its ectopic expression may regulate tumor progression and development. However, in hepatocellular carcinoma (HCC), its biological role, clinical significance and molecular mechanism are still unclear.Circ-CSPP1 expression and its prognostic values in HCC tissues were detected by qRT-PCR or in situ hybridization (ISH), and enriched by using Rnase R. The functional experiments (Circ-CSPP1 was overexpressed or knocked down) were performed in HCC cells. The HCC cell growth was analyzed by CCK-8 assay, transwell, wound healing and colony formation assays. The interation between circ-CSPP1 and miR-577/miR-577 and cyclin E2 (CCNE2) were determined by dual luciferase assay or RNA binding protein immunoprecipitation (RIP) assay. The RNA fluorescence in situ hybridization (FISH) assay was used to detect the subcellular distribution. Finally, an in vivo nude mouse tumor model was constructed.In HCC patients and cells, circ-CSPP1 was aberrantly expressed, and its upregulation predicted poor prognosis, and closely correlated with tumor size and TNM stage. Circ-CSPP1 resisted RnaseR digestion, indicating it is a circular RNA structure. Moreover, overexpression of circ-CSPP1 promoted HCC cell viability, colony formation, migration, and invasion in vitro. Knockdown of circ-CSPP1 showed contrary results. Circ-CSPP1 acts as a miR-577 sponge and positively regulated the target of miR-577, CCNE2. Besides, miR-577 inhibitor rescued the suppressive effects of circ-CSPP1 knockdown on HCC cell growth, whereas was completely reversed by silencing of CCNE2. Finally, the in vivo experiments confirmed that circ-CSPP1 knockdown regulated xenograft tumor volume and downregulated CCNE2, p-Rb, E2F1 and c-myc expression.These findings revealed that circ-CSPP1 contributed to HCC progression by positively regulating CCNE2 via miR-577, thus established its potential as new a prognostic and therapeutic marker for HCC patients. Hepatocellular carcinoma (HCC) ranks the fifth leading and third-most lethal cancer globally , with a Circular RNAs (circRNAs), represented in all types of organisms, are a naturally occurring family of noncoding RNAs formed by two mechanisms, ‘exon skipping’ and ‘direct back-splicing’. In mammalian cells, circRNAs has been widely and successfully identified due to the achievements in the bioinformatics analysis, technique of high-throughput sequencing and novel computational methods. CircRNAs were firstly found highly in the eukaryotic transcriptome and located in cytoplasm, which act as pivotal regulators in a variety of cell lines and across different species via diverse biological processes , such asMicroRNAs (miRNAs) are endogenous, small RNAs of approximately 20–24 nucleotides in length that functions through interacting with the 3′-UTR of mRNAs. It is recognized widely that miRNAs are important for tumor biology. The upregulation in oncogenic miRNAs can result in the reduction of tumor suppressor genes, whereas the downregulation in tumor suppressor miRNAs can upregulate oncogenes in cancer. Therefore, the upstream regulators of miRNAs need deep investigations. It is hypothesized that the communications and regulatory effects are exist among lncRNAs, mRNAs and pseudogenes by competitively interacting with the microRNA response elements (MREs), providing new mechanisms for gene regulatory system . It is aHere, this is the first evidence to determine the expression profile of circ-CSPP1 in HCC patients and cell lines. By using a series of cell and mouse experiments, the novel biological roles of circ-CSPP1 was identified and the molecular mechanism in HCC biology was confirmed. These observations may have implications to provide a possible novel therapeutic strategy involving circ-CSPP1 for HCC patients.http://www.ncbi.nlm.nih.gov/geo) from the platform GPL19978 and was analyzed using Agilent-069978 Arraystar Human CircRNA microarray V1 . By using an online tool GEO2R (http://www.ncbi.nlm.nih.gov/geo/geo2r/), the differentially expressed circRNAs (DECs) was analyzed. By Venn analysis (http://bioinformatics.psb.ugent.be/webtools/Venn/), the intersections of differentially expressed circRNAs among the three groups  were determined. By fold changes (fold change > 1.5) and P values (P < 0.05.), the DECs between tumor and adjacent normal tissues were determined.The gene expression profile GSE97332, GSE78520 and GSE94508 of HCC samples were downloaded from the Gene Expression Omnibus database . All enrolled patients had not accepted chemotherapy, radiation therapy, immunotherapy and so on. The clinical information of HCC patients were summarized in Table 2.HCC cell lines  and human liver cell line LO2 were from BeNa Culture Collection . Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with fetal bovine serum , 100 U/ml penicillin, and 100 µg/ml streptomycin in a condition of 37 °C and 5% CO#1, GATCCGGTGTCTCCCAGTGCTCCAGACAATTCAAGAGATTGTCTGGAGCACTGGGAGACACCTTTTTTC.Sh-circ-CSPP1#2,CCGGCGGAAAGGACTAGACATTGATCTCGAGATCAATGTCTAGTCCTTTCCGTTTTTTG. MiR-577 mimics, UAGAUAAAAUAUUGGUACCUG.Circ-CSPP1 plasmid, circ-CSPP1 short hairpin RNA (shRNA) plasmid, miR-577 mimics, miR-577 inhibitor, CCNE2 short hairpin RNA (shRNA) plasmid and the corresponding controls were provided by GenePharma Co., Ltd. . The sequences of sh-circ-CSPP1 were as follows: Sh-circ-CSPP1MiR-577 inhibitor, AUACAUAUACUUCUUUACAUUCCA. Sh-CCNE2, CCGGGCTCTTAAAGATGCTCCTAAACTCGAGTTTAGGAGCATCTTTAAGAGCTTTTTG. Lipofectamine 2000 (Invitrogen) was used for cell transfection.Circ-CSPP1 expression in HCC tissues was detected by ISH assay. The paraffin-embedded HCC samples were dewaxed by xylene and rehydrated with gradient alcohol, hybridized with specific digoxin-labeled probe , and followed by incubated with anti-Digoxin-AP  at 4 °C overnight. The tissues were finally stained and quantified.RNase R linear RNA digestion experiment was used to examine the circ-CSPPA resistance to digestion of RNase R. In brief, total RNA (5 μg) was incubated with a 20-μl reaction including RNase R  for 15 min at 37 °C, and subsequently purified using an RNeasy MinElute cleaning Kit .−△△CT method. GAPDH or U6 was used as internal references  and Qiangen miRNeasy Mini kit , respectively. By using PrimeScript RT Reagent Kit , total RNAs were reversely transcribed into cDNA. By using a TransGen One-Step qRT-PCR SuperMix kit , qRT-PCR assays were used to detect messenger RNA or lncRNA expression, with the following primers. Data was analyzed using the 2The experiment was performed according to kits from Life Technologics . In brief, the cell lysis buffer was used to collect total RNA from cells, and treated with separation buffer, the supernatant is used to extract cytoplasmic RNA, and the precipitate is used to extract nuclear RNA:Cells after transfection and tissues were lysed with RIPA lysis buffer. The samples with equal amounts of 10 µg of total proteins were fractionated on 10% SDS-PAGE. Then proteins were transferred to polyvinylidene fluoride membrane . After blocking, the membrane were incubated with the following primary rabbit antibodies at 4 °C overnight.: anti-CCNE2 , anti- twinfinlin-1 (TWF1) , anti-Rb , anti-p-Rb, anti-E2F1, anti-c-Myc , anti-E-cadherin , anti-N-cadherin  and anti-GAPDH. Membranes were then incubated with secondary antibody . The bands were visualized using enhanced chemiluminescent substrates  and protein expression was quantified with Image J software  .3 cells/well) and cultured for 24 h, and transfected with Circ-CSPP1 plasmid, circ-CSPP1 short hairpin RNA (shRNA) plasmid, miR-577 mimics, miR-577 inhibitor or the corresponding controls. 10 μl of CCK-8 solution  was then added per well and cultured for indicated times . Then, absorbance was measured at 450 nm.Hep3B and SK-HEP-1 cells were seeded into 96-well plates (4 × 103 cells/well) and maintained in RPMI-1640 medium for 2 weeks (the medium was replaced every 3 days). The visible colonies were fixed, stained and photographed under light microscope  and counted using Image J software .48 h after transfection with Circ-CSPP1 plasmid, circ-CSPP1 short hairpin RNA (shRNA) plasmid, miR-577 mimics, miR-577 inhibitor or the corresponding controls, Hep3B and SK-HEP-1 cells were seeded into 6-well plates  plasmid, miR-577 mimics, miR-577 inhibitor or the corresponding controls were resuspended in 6-well plates and scratched with a 200-μl pipette tip in the middle of the wells at 24 h posttransfection, and cultured in serum-free medium. Then, the cell debris was washed away and the rest of the cells continued to be cultured. The cell images were captured using a microscope  at 0 h and 24 h.2 × 104) were inoculated into the upper chambers of transwell plates  pre-coated with Matrigel . Serum-free culture medium was added into the upper chamber and 400 μl cultured medium containing FBS (10%) was added into the bottom chambers. After 24 h, cells on the bottom chamber were fixed, stained and counted under a microscope from five randomly selected fields.Hep3B and SK-HEP-1 cells  and propidium iodide  in the dark. Finally, the apoptotic cells were analyzed by Calibur flow cytometry equipped with CellQuest software (BD Biosciences).To observe the location of circ-CSPP1 and miR-577 in Hep3B and SK-HEP-1 cells, FISH assay was performed. In brief, frozen sections and cell climbing piece were prehybridized and hybridized with specific Cy3-labeled circ-CSPP1 probes/FITC-labeled miR-577 probes at 37° Covernight, and dyed with DAPl. Slides were photographed with a fluoresaence microscope .After culture for 24 h, Hep3B and SK-HEP-1 cells were transfected with the wild type clone containing the binding site sequence between circ-CSPP1 and miR-577 (circ-CSPP1-WT)/miR-577 and CCNE2(CCNE2-WT), as well as the corresponding mutant control clones (circ-CSPP1-MUT and CCNE2-MUT). Meanwhile, cells were co-transfected with miR-577 mimic or NC mimics into psiCHECK2 vector expressing Renilla luciferase. 48 h later, the luciferase activity was detected and normalized to the ratio of firefly to Renilla luciferase signal.HCC cells were lysed in RIP lysis buffer. Anti-AGO2  or anti-IgG  conjugated with RNA magnetic beads were subjected to qRT-PCR to determine levels of circ-CSPP1 and miRNA-577.6) stably transfected with shNC or sh-circ-CSPP1 were subcutaneously injected into the either side of flanks of nude mice (n = 6 in each group). Tumor volumes were measured every 3 days and calculated by the formula: length × width2 × 0.5. At 21th days after inoculation, the body weight of mice was measured and the tumor tissues were taken out for subsequent IHC assay. Animal experiments were performed by the guideline for the care and use of laboratory animals with the approval of The First Affiliated Hospital of Zhengzhou University.Twelve female athymic BALB/c mice (5–6 weeks old) were purchased from the Shanghai Experimental Animal Center (Chinese Academy of Sciences). Hep3B cells  and CCNE2 , p-Rb , E2F1  and c-myc . Slides were dried, dewaxed, rehydrated, added with DAB substrate chromogen solution, and counterstained with haematoxylin.t test or one-way ANOVA. P < 0.05 was considered as statistically significant.All results were shown as mean ± SD and analyzed by SPSS (version20.0) and GraphPad Prism 7. To analyze overall survival, survival curves plotted via Kaplan–Meier method and log-rank test were used. The comparisons between two groups or among multi-groups were analyzed using Student’s Three microarray datasets  were from the platform of Agilent-069978 Arraystar Human CircRNA microarray V1. A total of 115 DECs were found in gene chip GSE78520; 13 DECs were determined in gene chip GSE94508; 233 DECs were identified in gene chip GSE97332  and advanced TNM stage (P = 0.0005). ISH assay showed the similar results with qRT-PCR . These data suggested the abnormal expression of circ-CSPP1 in HCC patients.To confirm the differential expression of circ-CSPP1 in 72 human HCC tissues (HCC), qRT-PCR analysis was performed. Results showed that circ-CSPP1 was remarkedly higher in HCC tissues (Tumor) than that in the adjacent normal tissues   than human liver cell line LO2 (P < 0.01 or P < 0.001). Lower expression level of circ-CSPP1 was found in Sh#1 group than that in Sh#2 group, which was used for the following analysis (P < 0.001) and named as sh-circ-CSPP1. Importantly, overexpression or knockdown of circ-CSPP1 could not affect the mRNA expression of linear CSPP1 .The schematic illustration of circ-CSPP1 expression vector and shRNAs were shown in Fig. P < 0.01 or P < 0.001). Besides, down-regulation of circ-CSPP1 markedly increased the cell number in G1 phase and decreased the cell number in S phase in Hep3B and SK-HEP-1 cells .Growth curves and colony formation assay demonstrated that circ-CSPP1 overexpression enhanced the proliferative viability and colony formation of Hep3B and SK-HEP-1 cells observably, whereas silencing of circ-CSPP1 showed the completely contrary results , whereas was decreased after circ-CSPP1 was knocked down . Transwell assay showed that overexpression of circ-CSPP1 caused the significant increased numbers of invasive cells, but circ-CSPP1 knockdown significantly reduced numbers of invasive cells . Comprehensively, circ-CSPP1 might participate in the progression of HCC by promoting cell growth and decreasing cell apoptosis.The migratory ability of Hep3B and SK-HEP-1 cells was increased evidently when circ-CSPP1 was overexpressed , were clustered by venn analysis. The results showed that hsa-miR-520 h, hsa-miR-577, hsa-miR-193a-5p, hsa-miR-1182, hsa-miR-1299, hsa-miR-1276 and hsa-miR-324-5p may be target genes of circ-CSPP1. The binding sequences between miR-577 and circ-CSPP1 were shown in Fig. P < 0.001). RIP assay showed that the enrichments of circ-CSPP1 and miR-577 in Ago2-containing miRNAs than that in anti-IgG control group . It is well-accepted that the function of circRNA could be determined by its subcellular localization. FISH analysis revealed that about 60–70% of circ-CSPP1 and miR-577 was distributed in the cytoplasm of Hep3B and SK-HEP-1 cells , while circ-CSPP1 overexpression had the opposite result in Hep3B and SK-HEP-1 cells . Besides, miR-577 was down-regulated in HCC tissues compared to the Normal tissues  revealed that CCNE2 and TWF1were targets of miR-577 , while circ-CSPP1 overexpression showed the opposite result in Hep3B and SK-HEP-1 cells . Interestingly, silencing or overexpression of circ-CSPP1 had no obvious effects on TWF1 expression . The binding sequences between miR-577 and CCNE2 were shown in Fig. P < 0.001). Besides, miR-577 mimic or inhibitor was transfected into Hep3B and SK-HEP-1cells, miR-577 mimic caused decreased mRNA and protein expression level of CCNE2, while miR-577 inhibitor had the opposite result . Besides, in HCC tissues, CCNE2 was up-regulated as compared to Normal tissues  from the platform of Agilent-069978 Arraystar Human CircRNA microarray V1 were used, which were also used in other studies . Venn anCircRNAs have a covalently closed loop structure without 5′–3′polarity and polyadenylation tails, making they are more stable than linear RNA and less susceptible to RNA exonuclease or RNase R. Here, RNA samples obtained from HCC cell lines were digested by RNase R and the linear mRNA and circRNA of CSPP1 before and after treatment were detected. The results showed that linear mRNA was easily degraded by ribonuclease, while the circ-CSPP1 was stable and not easily degraded. In other words, circ-CSPP1 could resist RNaseR digestion both in Hep3B and SK-HEP-1 cells, indicating it is a circular RNA structure. Besides, FISH analysis revealed that about 60–70% of circ-CSPP1 was distributed in the cytoplasm of Hep3B and SK-HEP-1 cells, indicating it mainly play the corresponding biological roles in the cytoplasm.Moreover, functional experiments revealed that overexpression of circ-CSPP1 promoted cell viability, colony formation, migration, and invasion in vitro, whereas knockdown of circ-CSPP1 showed contrary results. Besides, the in vivo experiments showed that circ-CSPP1 knockdown reduced tumor volume. Consistently, the DECs between HCC and paired normal tissues were determined by Qiu et al. , and hsaCircRNAs have been discovered to affect HCC progression due to their regulatory function in various biological processes including regulation of cell growth via directly targeting miRNAs and proteins. For instance, circMTO1 act as a sponge of miR-9 and suppressed HCC cell proliferative ability and thus positively regulated p21, a cell cycle inhibitory protein; hsa_circ_100338 promoted HCC cell migratory and invasive abilities through directly regulating miR-141; ciRS-7 facilitated HCC cell proliferative and invasive abilities by targeting hsa-miR-7-5p (miR-7) and thus upregulating CCNE1, EGFR and PIK3CD , 35–38. To sum up, this is the first study demonstrating the biologic significance of circ-CSPP1 in HCC progress and diaplayed a relationship between circ-CSPP1, CCNE2 and miR-577, established its potential as new prognostic and therapeutic marker for HCC patients.


AR) gene alterations. Three clinically relevant AR variants related to response/resistance to standard-of-care treatments  were evaluated by digital PCR in 15 samples from patients affected by Castration-Resistant Prostate Cancer (CRPC). Plasma was processed to obtain circulating RNA and DNA using protocols based on tumor EVs enrichment through immuno-affinity and peptide-affinity compared to generic extraction kits. Our results showed that immuno-affinity enrichment prior to RNA extraction clearly outperforms the generic isolation method in the detection of AR-V7, also allowing for a distinction between responder (R) and non-responder (NR) patients. The T878A mutation was detected, overall, in nine out of 15 samples and no approach alone was able to reveal mutations in all harboring samples, showing that the employed methods complement each other. AR amplification was detected in the majority of CRPC samples analysed using either cell-free DNA (cfDNA) or exosome isolation kits (80%). We demonstrated that selective isolation of a subset of circulating exosomes enriched for tumor origin, rather than analysis of total plasma exosomes, or total plasma nucleic acids, increases sensitivity and specificity for the detection of specific alterations.We evaluated the advantages and the reliability of novel protocols for the enrichment of tumor extracellular vesicles (EVs), enabling a blood-based test for the noninvasive parallel profiling of multiple androgen receptor ( In recent years, the therapeutic landscape of Prostate Cancer (PCa), in particular for patients with advanced and Castration-Resistant Prostate Cancer (CRPC), has rapidly expanded with the introduction of several new approved drugs that represent the second generation of anti-androgens promising remarkable survival benefits. Regrettably, the blind and unoptimized use of anti-androgen therapies, in an inappropriate patient selection scenario had led to the poor overall efficiency of treatment, elevated costs, a loss of time and reduced patient survival and quality of life .AR) signaling in Prostate Cancer (PCa) oncogenesis and disease progression. AR gene aberrations are rare in primary tumors before exposure to hormone therapy, but occur in over 60% of patients with metastatic disease [AR gene [AR splice variants that display ligand-independent activity [AR mutations with high specificity and sensitivity emerges as crucial for a dynamical and readily monitoring of the therapeutic resistance and progression of the disease.Many preclinical and epidemiologic data corroborate the central role of androgen receptor , would positively affect the management of PCa patients. However, there still exist important challenges that affect the application of liquid biopsies in clinical practice.Circulating tumor DNA (ctDNA) is a very small fraction of cell-free DNA (cfDNA) that cannot be selectively isolated and distinguished from a large background of non-tumor derived circulating DNA. Moreover, it is confirmed that the major sources of circulating DNA fragments, either complexed with nucleo- or lipoproteins or naked, are apoptosis or necrosis, making them less representative of living and proliferating cells . The avaCirculating extracellular vesicles (EVs) offer a new liquid biopsy approach that could overcome drawbacks related to the use of CTCs or circulating nucleic acids (CNA), mostly ctDNA. These vesicles, which are exceptionally stable, cluster preserved and functionally relevant tissue and disease markers (proteins and nucleic acids) and are accessible in biofluids with minimally or noninvasive procedures. Exosomes are a nanosized subclass of EVs, originating from the endosomal cell compartment, that are found to be abundant in biofluids such as blood and urine, and have been extensively studied as biomarker reservoirs. Despite promising features , exosomeA common feature of recently reported studies exploiting plasma-derived exosomes is that the biomarkers are isolated from the bulk of circulating exosomes and not from a specific subpopulation of cancer cell-derived vesicles, thus limiting the effectiveness and advantage of the exosome-based approach .AR alterations in relation to response/resistance to standard-of-care treatments for CRPC. In a pilot study on a small cohort of 15 patients with CRPC, we concomitantly assessed three AR gene variants falling into different biomarker categories, namely a somatic point mutation T878A, a copy number variation in the AR gene as an indication of gene amplification, and levels of expression of full-length AR and AR-V7 splice variants.With this study, we investigated the feasibility of novel protocols for the enrichment of tumor EVs that employ specific affinity-mediated selection platforms. The objective is to develop a robust and clinically feasible blood-based test which enables the noninvasive parallel profiling of multiple The aim of the present study was to explore the feasibility and the advantage of a tumor exosome-based approach to support the accurate and timely stratification of CRPC patients in the context of their response to antiandrogen therapy. The results were evaluated based on the patients’ classification according to conventional clinicopathological criteria and on the determination of the same genomic variations in free CNA extracted from the same blood samples.The study sample was 13 patients with metastatic CRPC ; all werg for 10 min at room temperature and then stored at −80 °C until analysis. RNA from plasma samples was extracted using four different protocols: (1) a SoRTEV™ RNA Enrichment Kit , providing tumor EVs enrichment by proprietary immuno-affinity (IA) selection (hereafter IA-RNA protocol). The antibody employed within the SoRTEV kit is a part of a proprietary library of antibodies (Exosomics S.p.A) raised against exosomes (nanosized EVs) derived from primary tumor cell lines. Although its target is undisclosed, this antibody has been selected during the extensive screening on tumor and non-tumor models and samples in which this SoRTEV Ab displayed preferential binding to EVs, in particular exosome-like vesicles (in terms of size and molecular content), from tumor cell lines grown in hypoxic conditions, and to exosomes from plasma samples obtained from cancer patients  moreover, the exoRNeasy serum/plasma kit , a more generic extraction protocol that takes advantage of membrane-affinity columns (exoEasy columns) to bind exosomes and other EVs, was used. They are afterwards lysed by Qiazol and spin column-based RNA purification is performed; (4) finally, the miRNeasy serum/plasma kit  was used for a subset of samples  from which leftover plasma was available; the cell-free total RNA from plasma was purified following the manufacturer’s instructions. Briefly, plasma is lysed with Qiazol and, after chloroform addition, the upper phase is purified through a silica membrane column.Peripheral blood was collected in 4.9 mL K2EDTA tubes and processed within 30 min after drawing. Plasma was obtained by centrifuging at 2000× patients . This spThe DNA from plasma was extracted by employing three methods: (1) EVs enrichment with proprietary IA selection (using the same isolation agent as the SoRTEV RNA Enrichment Kit—see above) followed by column-based DNA purification (hereafter IA-DNA protocol); (2) PA isolation of EVs with the ME™ Kit  followed by column-based DNA purification (hereafter PA-DNA protocol); (3) a QIAamp Circulating Nucleic Acid kit , which purifies generic free-circulating nucleic acids from body fluids in four steps: sample lysis with the subsequent release of nucleic acids from vesicles, lipids and proteins, binding to QIAamp Mini column, washing steps, elution. For each nucleic acid extraction, 0.5 mL plasma was employed (0.2 mL for the miRNeasy serum/plasma kit); the elution volume was 15 µL for RNA and 50 µL for DNA.AR T878A variant , AR gene copy numbers with RNaseP as the internal reference standard . A volume of 15 μL of the reaction mix was loaded on a QuantStudio 3D Digital PCR 20K Chip  and thermocycling ran at 95 °C for 8 min, 40 cycles (37 cycles for the AR-V7 assay) at 95 °C for 15 s and at 60 °C for 1 min, with a final extension step at 60 °C for 2 min. Copies/µL reaction were retrieved with QuantStudio 3D AnalysisSuite Cloud Software and copies/mL plasma were calculated by multiplying this value by the dilution factors applied during the process.For each RNA sample, 12 µL of the template were retrotranscribed to cDNA using a QuantiTect Reverse Transcription Kit  for a final volume of 20 µL. The assessment of AR-V7, AR-FL and RNY4 copies was performed on cDNA using custom assays (primers described elsewhere) . DNA wasp-value was less than 0.05.The obtained results refer to the absolute target concentration expressed as copies per milliliter, as determined by dPCR QuantStudio 3D AnalysisSuite Cloud Software . As our study is the first one with the simultaneous assessment of all fractions in a single sample, the number of technical replicates was limited and the analysis was done by sample set comparisons. Briefly, the one-way analysis of variance (ANOVA) and Bonferroni post-hoc tests were used to determine the statistical significance of the observed differences for group comparison. The statistical analysis was performed using Prism . The difference between groups was considered statistically significant if the AR alterations in CRPC.The aim of this study was to take advantage of cutting-edge protocols that enrich tumor-derived exosomes both by IA or PA methods in comparison to benchmark or more generic kits, in order to explore the feasibility and reliability of exosome-based tests to allow for the detection of AR variants, known to be associated with patients’ response to therapy.Nucleic acids were isolated from 15 CRPC plasma samples and from a pool of healthy donors, using different sample preparation methods for the collection of either total cfDNA, total circulating EVs or the enrichment of tumor originated EVs, and afterwards tested by digital PCR for the presence of AR-V7 splicing variant, AR-full length (FL) (the full-length version of the transcript) and the small noncoding RNY4 as a control gene transcript known to be abundant in EVs and human plasma [RNA was extracted from plasma samples using different sample preparation kits. dPCR assay was performed to detect the n plasma .AR-V7 transcript (AR-V7+ ones) and in 7/15 (46.6%) samples purified with the PA-RNA protocol .The anscript A was detAR-V7+ (average of 60.4 copies/mL plasma) while among the five R samples, two were positive and three were AR-V7− (average of 10.1 copies/mL plasma).The IA-RNA protocol distinguished NRs from Rs better than the generic extraction method. With the IA-RNA protocol, all 10 NR samples were A parallel analysis with generic extraction detected the transcript in 4/10 NR samples and in 4/5 R samples . The splicing variant was detected in 5/10 NR samples and in 2/5 R samples  with the PA-RNA protocol, showing an inverse correlation with patient classification in comparison to the IA-RNA method.AR-V7 was detected in 3/6 (50%) samples, of which two were NR (average of 60.5 copies/mL) and one was R (32 copies/mL)  D.AR-V7 compared to the more generic method for EV RNA extraction. When the AR-V7 copies were normalized to Y4 copies, the normalized values were much higher in the samples processed with the IA protocol  compared to the benchmark generic kit .DNA was extracted from patient plasma using three sample preparation kits; AR T878A+   using the PA-DNA protocol and in 6/15 samples (40%) after generic extraction (18.7–294.2 copies/mL) A. All poctively) B.AR gene amplification, RNaseP, a gene that is not known to be overexpressed in CRPC, was employed as an internal reference standard to normalize AR gene copies. The ratio between AR and RNaseP copies was calculated for each sample. The ratio of each disease sample was then normalized to the ratio of a healthy pool of male individuals. A cutoff of >1.5 was considered for gene amplification, as previously described [To analyze escribed .AR amplification was detectable with the PA-DNA protocol and the QIAamp Circulating Nucleic Acid extraction kit in 12/15 samples (80%). The PA-DNA protocol returned borderline values (1.49 and 1.48) for two of the three negative samples (samples 3 and 15); the IA-DNA protocol returned positive results in 8/11 (72.7%) samples  .The detection of tumor markers in both research and diagnostic settings has historically relied on the direct analysis of tumor tissue samples. However, tumor tissue biopsies collected at diagnosis or local surgery do not capture tumor clonal evolution; in addition, biopsies are associated with substantial intra-patient heterogeneity and discomfort, risks and costs. On the other hand, liquid biopsy, using the tumor material shed in circulation in the form of free CNAs, or encapsulated within CTCs or EVs, promises to provide a minimally invasive platform for the routine sampling and monitoring of advanced cancer patients.EVs, exosomes in particular, carry tumor-derived nucleic acids, proteins, metabolites and lipids in the peripheral circulation.AR gene alterations.In this study, we show the advantage of novel methods for enriching tumor-derived exosomes from plasma, over conventional and more generic methods for the recovery of overall EVs or overall circulating cfDNA. We have applied these preanalytical methods to process 15 plasma samples from CRPC patients who received anti-androgen therapy prior to the detection of three clinically relevant AR splice variants, the most common of which is AR-V7, are associated with a resistance to AR-targeted therapies, as well as to common taxane-based treatments [AR-V7 variant in CRPC plasma samples. As expected, the IA-RNA protocol isolated much less RNA than the generic method, as shown by a 20-fold decrease in the number of copies of the reference gene RNY4 (AR-V7 detected in 12/15 samples) vs. 53% (8/15 samples), respectively. The AR-V7 detection rate after preparation with the generic RNA extraction protocol is consistent with studies that used the same method for AR-V7 detection [AR-V7 is appreciated when normalized values (AR-V7 vs. RNY4) or AR-V7 allelic frequency are compared ). A recent meta-analysis reported that relative changes in PSA rather than absolute levels during treatment are an important independent prognostic factor [AR-V7 copies from the total plasma exosomes or the tumor-enriched exosomes, however, the samples processed with the IA-RNA protocol better matched the PSA RR than those processed with the generic isolation protocol (73% vs. 40%), , .AR-V7 is a prostate-specific transcript, its detection in whole blood is affected by the contaminating RNA from blood cells that constitutes most of the RNA recovered. Besides directly comparing exosome isolation kits, we also investigated the detection of AR-V7 in whole plasma which, unlike whole blood, lacks leucocyte-derived RNA. We detected AR-V7 in 50% of samples, while reference gene RNY4 and the full-length AR transcript were successfully detected in all of them  by which diverse exosome (sub)populations are harvested along with other plasma components. In this view, the hypothesis is that the IA enrichment protocol preferentially isolates intact exosomes released from living tumor cells, while the peptide-based isolation agent also captures RNA fractions associated to DNA released by dead cells. Therefore, we can expect to observe the increased detection of tumor markers with the IA-RNA protocol in the NR group, and with the PA-RNA protocol in the R group. This exciting hypothesis is indeed supported by our initial data  [AR T878A mutation occurs in the ligand binding domain of the androgen receptor and alters the steroid binding properties of the mutated receptor [The prevalence of atients) . The AR receptor . Its impreceptor ,28. In oAR copy number gain has been associated with disease progression and with a decreased response to abiraterone and enzalutamide [AR amplification can be detected in cfDNA at the same rate as that obtained from metastatic tissue [AR amplification is a non-binary variable; therefore, the elevated sensitivity not only in detection, but also in the quantification of the AR gain, is likely to be required for a meaningful association to patient outcomes. If we consider a proposed criterion [AR amplification in most of the CRPC samples analysed using either cfDNA or exosome isolation kits (80%)  . AlthougAR alterations that were previously reported to be associated with a differential response to AR inhibitors as well as to generic treatments, can be detected with a different robustness and sensitivity to different fractions of plasma samples. Moreover, in “qualitative” and unique tumor marks such as AR-V7 isoforms , the enAR alterations, including amplification and mutations, was higher in this study than previously reported [Interestingly, the number of patients with concomitant reported .AR) and, ultimately, multiple fractions of the sample that likely reflect the cell or tissue origin of the measured marker. We hereby assessed the feasibility of such an approach and highlighted the advantage of accurate sample fractionation for the detection of shared and unique PCa alterations. Therefore, opting for plasma fractions that are restricted but enriched in tumor exosomes/markers can increase the sensitivity and specificity of downstream analytical methods such as dPCR (used in the current study). The validation of this approach in a larger cohort of both CRPC patients and PCa patients in relation to biochemical progression after primary treatment is warranted to obtain robust evidence of its clinical utility.This study supports the idea of a multiparametric approach in liquid biopsy as a next step that would integrate multiple blood-based phenotypes to yield a maximally informative disease profile . The uni