DPP4), opioid receptor gene d1 (OPRD1), and opioid receptor m1 (OPRM1) are key targets for the Yiqi Qingre Ziyin method. Therefore, this study proposed a potential mechanism for the treatment of AR by affecting the expression of neuropeptide-related genes , which may potentially improve the immune microenvironment of the nasal mucosa.Atrophic rhinitis (AR) is a chronic disease that causes severe structural changes to the nasal mucosa leading to squamous epithelial metaplasia. However, treatment regarding AR remains a major challenge. We used network pharmacology and molecular docking methods to explore the potential mechanisms of the Yiqi Qingre Ziyin method to modulate neuropeptides in the treatment of AR. The active ingredients of the Yiqi Qingre Ziyin method and their targets of action were obtained from the Traditional Chinese Medicine Systematic Pharmacology Database Analysis Platform (TCMSP). Disease targets for AR were obtained from four databases: GeneCards, PharmGKB, DrugBank, and Online Mendelian Inheritance in Man (OMIM). A total of 59 active ingredients, 39 potential targets, and 76 relevant neuropeptides were obtained after deduplication. We constructed target interaction networks with the STRING database. Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed on the 14 potential target proteins. We used Cytoscape software to construct the “drug-active ingredient-potential target” and “ingredient-target-pathway” networks of the Yiqi Qingre Ziyin method for treating AR. Molecular docking results suggest that dipeptidyl peptidase 4 ( Atrophic rhinitis (AR) is a chronic disease that causes severe structural changes to the nasal mucosa leading to squamous epithelial metaplasia, loss of cupped cells, and occlusion of the vascular cavity , 2. ChroIn Chinese medicine, AR, also known as “Bigao,” is commonly associated with the deficiency of the “lung and spleen.” The identification and typing of diseases is the core of diagnosis in the theory of Chinese medicine. The treatment should benefit the Qi, clear heat, and nourish Yin. However, that theory of Chinese medicine was performed at the subjective level, lacking conclusive evidence. In previous studies, scholars found that neurogenic inflammation tests are more specific than traditional eosinophil counts or sIgE tests. And there is a stronger correspondence between these tests and TCM evidence –9. With The sympathetic and parasympathetic nervous systems regulate the complex structure of the nasal mucosa. However, it is also controlled by the nonadrenergic noncholinergic nervous system (NANC) –12. NANCThis study explored the mechanism of the Yiqi Qingre Ziyin method for the treatment of AR through network pharmacology and molecular docking.radix astragali (20 g), Poria (15 g), Atractylodes macrocephala Koidz (AMK) (12 g), angelica (12 g), scrophulariae (12 g), Ligustrum lucidum Ait (LIA) (15 g), kudzu (12 g), and red peony root (RPR) (9 g). Codonopsis pilosula (20 g) was added to the prescription when patients presented with significant “qi” deficiency syndrome. In previous research, ingredients and targets of these herbs were downloaded from the traditional Chinese medicine systems pharmacology database [Based on the literature review, an herbal prescription was summarized . The predatabase –17. An ihttps://www.uniprot.org/) database. Proteins related to AR were gathered from four datasets, including GeneCards (https://www.genecards.org/), OMIM (https://omim.org/), PharmGKB (https://www.pharmgkb.org), and DrugBank (https://www.drugbank.com). The aggregation and repetition cut-out work was finished using R software [Drug targets were converted to gene names using the UniProt  as previous researches [Homo sapiens. Protein interaction relationships were obtained using a filter with a combined score ≥ 0.7 and removing free nodes. Download the result file in tsv format, and import it into Cytoscape 3.7.1. Use the network analyzer in this software to perform network topology analysis and build a PPI network [Potential targets were imported into the STRING 11.0 database  and Gene Ontology (GO). The top-ranked genes and those with searches –26.http://www.rcsb.org/) and the ZINC database (http://zinc.docking.org/) were used to obtain the structures of target proteins and ligands. Docking results are presented in the form of binding energy, smaller scores indicating more stable binding [The molecular docking software AUTODOCK 4 was used to validate the molecular docking of the target with the highest correspondence value for each herbal medicine in the PPI network . The RCS binding , 29.The complex system of compound and target protein obtained by molecular docking was used as the initial structure for all-atom molecular dynamics simulations (MDS). Create small molecule force fields using ACPYPE Server , 31. TheGROMACS software was used in this study to perform the system simulations , 34. EneCodonopsis pilosula. A total of 843 disease-associated genes were screened , were found to be associated with neuropeptide-related genes  were used for enrichment analysis. The enriched biological processes are responses to lipopolysaccharide, molecules of bacterial origin, chemical stress (cellular response), oxygen levels, hypoxia, metal ion, nutrient levels, and oxidative stress (cellular response). The enriched cellular components are membrane raft, membrane microdomain, membrane region, nuclear chromatin, transcription regulator complex, RNA polymerase II, caveola, plasma membrane raft, ficolin-1-rich granule, and ficolin-1-rich granule lumen. The enriched molecular functions are nuclear receptor activity, ligand-activated transcription factor activity, RNA polymerase II general transcription initiation factor binding, steroid hormone receptor activity, heme binding, tetrapyrrole binding, Hsp90 protein binding, general transcription initiation factor binding, RNA polymerase II-specific DNA-binding transcription factor binding, and DNA-binding transcription factor binding. The above results suggest that the Yiqi Qingre Ziyin method presents a multitarget and multipathway action for the treatment of AR  from DS bound to DPP4 with binding energies of -7.6 kcal/mol, -8.6 kcal/mol, -7.8 kcal/mol, and -8.9 kcal/mol, respectively. 7-Methoxy-2-methylisoflavone, 3-beta-hydroxymethyllenetanshiquinone, and 11-hydroxyrankinidine bound to OPRM1 with binding energies of -8.9 kcal/mol, -8.9 kcal/mol, and -7.4 kcal/mol, respectively. 3-beta-Hydroxymethyllenetanshiquinone also binds to OPRD1 with a binding energy of -8.3 kcal/mol. It is generally accepted that a docking fraction below -4.5 kcal/mol is a binding possibility, and a docking fraction below -6 kcal/mol is strong binding power. The binding docking fractions in this analysis were all below -6 kcal/mol, indicating stable and strong binding. This suggests that the effective components in Codonopsis pilosula have a high binding capacity towards neuropeptide-related proteins DPP4, OPRD1, and OPRM1.pilosula , DPP4, Os for AR . Luteoli Figures   β, and granulocyte-macrophage colony-stimulating factor. It also increases the levels of IL-10, an anti-inflammatory cytokine. 7-Methoxy-2-methylisoflavone significantly reduces depression in patients, and this antidepressant affects both menopausal women and men [DPP4 in Th17 cells, thereby alleviating the symptoms of AR.Luteolin, frutinone A, 7-methoxy-2-methylisoflavone, 3- effects –41. Lute and men –44. GSEADPP4 was found to be associated with AR. It is also a target gene of Codonopsis pilosula. Indeed, as a dipeptidyl peptidase, DPP4 could cleave dipeptides from the n-terminus of dipeptides, including enterotrophic hormone, neuropeptides, and some chemokines. DPP4 was found abundantly expressed in IL-17-producing CD4 T cells (Th17 cells) [DPP4 is an enzymatically active molecule expressed by Th17 cells and has a biologically active enzymatic role that may contribute to the regulation of inflammatory T cell chemotaxis [DPP4-mediated modifications of enzymatic chemokines may regulate T cell migration. Therefore, upregulation of DPP4 is also associated with disease activity in human autoimmune diseases, such as rheumatoid arthritis and multiple sclerosis [beta-hydroxymethyllenetanshiquinone stably binds to DPP4 protein in a computer-simulated system by MDS technique. This suggests that 3-beta-hydroxymethyllenetanshiquinone, the active ingredient in Yiqi Qingre Ziyin method, may be able to inhibit the progression of AR by targeting binding to DPP4.In this study, 7 cells) , 46. DPPclerosis –50. In tOPRM1) gene and is the primary site of action for the most used opioids. The delta-opioid receptor (DOR) is encoded by the opioid receptor gene (OPRD1). Opioid receptors are the targets of a large number of drugs, including heroin and many analgesics. Genetic diversity and DOR expression levels of OPRD1 are associated with a variety of diseases, including substance abuse and addiction, anorexia nervosa (AN), obesity, and Alzheimer's disease (AD) [OPRD1 and OPRM1 in AR are needed, and this study found that they may serve as potential targets for the Yiqi Qingre Ziyin method in the treatment of AR.The m-opioid receptor is encoded by the opioid receptor m1 (ase (AD) –53. Undease (AD) –56. HoweDPP4, OPRD1, and OPRM1, were found to be potentially associated with the development of AR. Nonetheless, the particular mechanism of these proteins' action on AR has to be explored further. The active ingredients luteolin, frutinone A, 7-methoxy-2-methylisoflavone, 3-beta-hydroxymethyllenetanshiquinone, and 11-hydroxylancidin from Codonopsis pilosula bind with DPP4, OPRM1, and OPRD1. These could be key targets for the Yiqi Qingre Ziyin method. The results of this study suggest that the Yiqi Qingre Ziyin method has good potential efficacy against AR. In this study, the pathogenesis of AR was found to be associated with OPRD1 and OPRM1. However, the exact mechanism remains to be further investigated.Network pharmacology and molecular docking were used to explore the potential mechanisms of Yiqi Qingre Ziyin method-mediated neurogenic peptides for the treatment of AR. Three neuropeptide-related proteins, DPP4, OPRD1, and OPRM1), which provides a theoretical basis for further elucidating the molecular mechanisms of this method in the treatment of AR.This study revealed that the Yiqi Qingre Ziyin method may improve the immune microenvironment of the nasal mucosa by affecting the expression of neuropeptide-related genes (including


The effectiveness of radar interferometric techniques in non-urban areas can often be compromised due to the lack of stable natural targets. This drawback can be partially compensated through the installation of reference targets, characterized by a bright and stable radar response. The installation of passive corner reflectors (PCR) often represents a valid aid, but these objects are usually cumbersome, and suffer from severe weather conditions; furthermore, the installation of a PCR can be difficult and costly, especially in places with hard accessibility. Active reflectors (AR) represent a less cumbersome alternative to PCRs, while still providing a stable phase response. This paper describes the design, implementation, and test of an AR prototype, designed to operate with the Sentinel-1 synthetic aperture radar (SAR), aimed at providing a fair performance/cost benefit. These characteristics, obtained through a tradeoff between the use of off-the-shelf components and a simple architecture, can make the setup of a dense network  in the monitored areas feasible. The paper reports the design, implementation, and the analysis of different tests carried out in a laboratory, and in a real condition in the field, to illustrate AR reliability and estimate its phase stability. The use of Sentinel-1 SAR images for interferometric synthetic aperture radar (InSAR) in non-urban areas is often challenging because most of these areas are covered by vegetation or heterogenous surfaces, which are geometrically and dielectrically instable, and hence have a low coherence. The deployment of stable artificial targets in these areas can remarkably improve the situation. Strong reflectors, with a stable amplitude and phase response, can be classified as persistent scatterers ; thus, pThe installation of a PCR can be difficult when accessibility to the installation is hard, and weather conditions can jeopardize their performance. An alternative to PCR is represented by the installation of active reflectors (ARs). These systems have been introduced since the 1990s for SAR system calibration ,14,15,16This study does not take into consideration complex transponders developed to assist—during and after the commissioning phase—the calibration and test of space sensors, representing, in terms of cost, performance, and use, very different systems. Nowadays ARs designed for field installations are available from the market at a moderate cost. These systems are compact enough, and they provide users an advanced interface to allow the control of some acquisition parameters. An outstanding example is the MetaSensing Electronic Corner Reflector at C-band (ECR-C) . The perThe AR was designed with a simple rationale, which basically consists of two compact microstrip antennas (size 14 cm × 14 cm), and a radio frequency (RF) section to provide the requested signal gain. One of the design requirements is to maintain the current absorption very low, to allow the use of a power system with a small capacity 12 V battery (7 Ah) and a small (25 cm × 24 cm) photovoltaic panel (5 W and 25 × 24 cm size), and provide long autonomy in case of a lack of buffering power. The size of the case of the prototype here discussed is 52 cm × 32 cm × 7 cm. Phase stability has been achieved with a tradeoff among low-cost, simple functioning, and easy and rugged hardware, using off-the-shelf components. This choice allows for minimizing the cost of most of the components. In addition, the antennas, designed and developed at Centre Tecnològic de Telecomunicacions de Catalunya (CTTC), have also an implemented a cost, lower than 10% of the cost of the entire system. A rough estimate of the total cost of the current version of the AR discussed here is less than 1000 euros. These goals have been achieved with a simple radio frequency (RF) architecture, based on linearity and neatness, and taking care of all the aspects of the overall design. In order to evaluate its performance, the implemented device was tested at different levels and conditions. First, the system without antennas has been tested in a laboratory to check the performances of the RF amplifying section. Then, the performance of the entire apparatus was measured through a field experiment carried out in a controlled environment, with temperature variations between 10 °C and 35 °C, at short distances, and using a signal, simulating that of Sentinel-1. Finally, the AR was installed in an agricultural site, in real conditions, processing almost a one-year long slot of Sentinel-1 images, i.e., more than 50 Single Look Complex (SLC) images, in interferometric wide (IW) mode, descending orbit geometry. The stability of the AR amplitude and phase response has been analyzed, paying attention to the influence of the air temperature.This paper is organized in five sections: this introduction, followed by a brief recall of the working principle of an AR, including a description of some design details and of the applied methodology, as described in In this section, a short description of the working principle is provided. Then, a design rationale is discussed. Details about the antennas and the amplifying section—the most critical points of the device—are also commented on. The methodology used to test the AR is also described.2. It is worth noting that it corresponds to the RCS of a TT CR, whose size is 1.7 m. The main goal of this study is to report the feasibility of a low-cost, reliable and easy to use AR, able to assist InSAR use of Sentinel-1 SAR images. The simplest AR scheme consists of a receiving antenna, an amplifying section, and a second antenna. The signal received from the satellite is re-transmitted after an amplification towards the same satellite. The essential working principle is shown in GRF: gain of the RF amplifying section. Gtx, Grx: gain of the transmitting and receiving antennas, respectively. λ: wavelength of the received signal.The radar cross section of an AR can be determined by characterizing the gains of its components. We used the relationship obtained from the radar equation used in . AccordiGtx = Grx. Considering that the RCS of the AR must be > 40 dBm2, and that the measured gain of the patch array antenna specifically designed and implemented for the AR is greater than 17 dB , it is clear that the gain of the antennas is of major concern, because fixing the maximum RF gain, the higher the gain, the lower the 11, the parameter conventionally used to describe the matching performance of the antenna, was measured and simulated at the antenna connector. The measured value below −20 dB in the operating band assures good matching between the two antennas and the amplifying section; the measured S11 is compared to the simulated behavior. The antenna patterns, shown in A good standing wave ratio (SWR) is mandatory to reduce losses and reflections, and hence, noise, which can make the system more unstable and sensitive to temperature variations. SAccording to the RCS requirement, the antenna gain, and Equation (1), the required gain for the amplifying section must be greater than 40 dB and not exceed 50 dB. This value was obtained by designing this part of the system using components selected according to the requirements of a low power consumption. A band-pass filter is also necessary to avoid interferences from external sources, such as wireless or mobile networks. Different prototypes of the RF section were implemented and characterized in order to test different amplifiers and design schemes. The final architecture used off-the-shelf components with a negligible cost of the single RF components. The RF scheme is composed of a low noise amplifier (LNA) just after the receiving antenna, followed by a band-pass filter (5150–5990 MHz), and three amplifiers here indicated as Medium Power Amplifier (MPA), also with a low noise figure. The nominal gain of the amplifiers is about 15 dB each. Laboratory tests suggested adding an attenuator and an isolator to reduce the risk of saturation and optimize the matching among the different components. The implemented prototype is based on the use of evaluation boards of commercial low-cost components, but it is planned that the final version will be integrated in a single board. In The external size of the device used for this study, which is shown in To test the performance of the AR, three main steps were taken. First, an indoor laboratory experiment was carried out to analyze the response, amplitude, and phase of the RF section, without the antennas, at different temperatures. Then, a test of the entire sensor was carried out, arranging a setup to evaluate the thermal stability of the AR at a short range, and using as input a frequency modulated continuous wave (FMCW) signal generated by a vector network analyzer (VNA). Finally, the prototype was installed in real conditions, together with a PCR. The AR and the PCR were installed in an experimental agricultural field of the campus above a concrete base see . A firstIn this section, different tests carried out to evaluate the performance of the AR are reported. The first one consists of an indoor laboratory experiment aimed at a basic check of the characteristics of the amplifying section, the core of the device. A controlled environment experiment is then reported to detail the behavior of the entire AR, when the internal temperature of the case was changing. GRF), using a vector network analyzer (VNA) (Agilent E8257D) to generate an FMCW signal. The measuring configuration uses a minimum frequency fmin = 5.2 GHz, and a maximum, fMax = 5.6 GHz, with 201 frequency points. This swept bandwidth includes the RF bands used by most of the C-band spaceborne SAR systems. Different values of input power were used to test the linearity of the device (input power range: −80 dBm to −20 dBm), widely including the signal intensity expected for the actual Sentinel-1 input signal. The GRF measured @ 5.4 GHz is 42dB, a value compliant with the design requirements. Once the conditions required to provide an adequate RCS of the AR were verified, the phase stability was first checked, at a stable temperature, using a mixer as a phase detector in a standard homodyne configuration, which provided an IF signal proportional to the phase difference between the input and output of the AR.The first step to evaluate the performances of the AR consists of measurements of its gain  . This apTo evaluate the reproducibility and trend of the thermal effect, we carried out different tests. We first measured the complex transfer function of the AR with different air temperatures, to estimate the stability of the amplifying section response. The AR was positioned inside a thermostatic chamber and air temperature was varied at single steps from 16° to 25°C for 6 h. This procedure guarantees operating in a thermal equilibrium state, moderately reproducing the operating conditions in the field. In 21 parameter measured by a VNA at different temperatures are shown in The results, amplitude, and phase of the SFrom the operational point of view, considering that the main goal of this device is to assist the use of InSAR for deformation monitoring, we tried to estimate the stability of the AR in terms of virtual displacement, i.e., the error introduced by the system in the retrieval of deformation. In SAR interferometry, the differential phase is usually estimated with respect to the wavelength, corresponding to the central frequency of the operated bandwidth, but the phase response, due to the synthetic nature of the pulse, is affected by the amplitude and phase response of the AR over the entire bandwidth. To estimate the effect of possible instabilities, we analyzed the trend of a statistical parameter, D (T (°C)), a virtual displacement, defined through the following Equation (2): D is obtained using the basic formula of radar interferometry to transform the phase to a displacement, stated by the following Equation (3): c/(4π fi), is present in Equation (3). D is calculated at temperature it, averaging the phase, φi, at each frequency, if, over the entire number of frequencies, fN, number of measured frequencies belonging to the swept bandwidth, Nf ∗ (i+1f − if); each phase is normalized to fi; c is the light velocity. This average can represent an estimate of the overall response of the AR to the incoming signal and it allows estimating the stability of the phase retrieved from interferometric processing as a displacement. D versus the temperature measured inside the thermostatic camera is showed in D obtained from the laboratory data is <0.4 mm, for a 15 °C to 25 °C temperature range, the standard deviation is 0.12 mm and the mean value −0.07 mm. Real conditions can usually cover a wider range of temperatures, and these values must be considered preliminary to the following tests, which were carried out with the system in its complete configuration. The variation of A second test of the AR was carried out, arranging a setup to measure the thermal stability of the AR at a short range, and also, in this case, using  an FMCW signal generated by the VNA. The test is based on the air temperature variations, measured through a sensor installed inside the AR. The location is an open-air terrace, which, although does not represent an ideal radar environment, due to the presence of walls and multiple reflections, allowed us to study the AR response, which was, however, clearly detected. This test allowed achieving the goal to characterize the effect of temperature variations on the AR response compatibly with the logistics issues of the environment. The VNA, as introduced in the previous section, is used as step frequency radar, simulating the Sentinel-1 signal. The AR is installed close to the wall, delimiting the bottom of the terrace, about 25 m far from the radar. Here, we refer to the phase measured by the radar in correspondence to the peak associated to the AR position, which represents the phase response of the AR, taking the first acquisition as reference, transformed to displacement, according to Equation (3). The temperature of the AR in this setup is measured every 15 min through a humidity and temperature sensor mounted inside the case. In As far as the phase trend is concerned, the retrieved displacements and the corresponding temperatures are plotted in These results confirm that the amplitude and phase fluctuations are correlated to the temperature trend, as expected, when considering the typical thermal behavior of active components of an RF chain. To discover the type of relationship, we plot the phase data as a function of the temperature, shown in This graph clearly shows that in the “low temperature” range there is a linear trend, well fitted with a rate = 0.51 mm/°C line, and a bias of 11 mm. For temperatures above 20 °C, the variations are of a minor entity and are lower than 2 mm. Separating the data set into two sets, we define a discontinuous function, describing the dependence between the displacement and temperature, represented by Equation (4):It is worth noting that a very similar fit has also been obtained with the data acquired in the two previous data acquisitions.Here we show the results of a field test, where the AR response is analyzed in a real condition, processing Sentinel-1 images; its phase stability is evaluated through a standard interferometric processing, which covers almost a one-year slot. In order to estimate the performances of the AR in a real situation, a prototype of the system and a PCR, rectangular side trihedral with a 0.65 m size, were installed on 22 October, 2019, in an agricultural field close to CTTC. Both reflectors were oriented to receive the Sentinel-1A and -1B signals in the descending orbit geometry. The AR  and the PCR  are still operating in January 2021. Considering that the use of the proposed AR focuses on InSAR applications, the main aim was to compare its phase response with respect to stable points as the PCR and some buildings close to it, representing an optimum phase stable reference. An analysis of the amplitude response is first performed considering that the phase accuracy is also influenced by the SCR of the AR.A quantitative estimate of the RCS along the satellite line of sight LOS was carried out using field measurements, comparing the AR and PCR amplitude response in the acquired SAR images, and assuming that the RCS of the installed PCR corresponds to the expected maximum theoretical value, calculated according to the standard formula for rectangular side trihedral. The assessment of an absolute estimate of the RCS is a task planned for the next steps of the study, during the implementation of the final prototype. The conditions required to perform an accurate radiometric calibration were not guaranteed in the current installation due to the logistics and external factors. We were not able to reduce and evaluate the effect of multiple reflections with unstable characteristics of the agricultural soil, the supporting structure, and the influences of close targets . ® view of the area, where both reflectors are installed. For a quick and easy check, the clear visibility of the AR and the PCR can also be verified by the readers in the SAR images, corresponding to the area where the AR is installed, using the Sentinel Hub browser provided by Synergy for ESA [ for ESA . Figure AR) was estimated considering the responses of the two targets from the amplitude image, shown as PCR = 33.4 dBm2). Calculating ΔAP (dB) = intensity(AR) − intensity(PCR), the estimated value is RCSAR = RCSPCR + ΔAP (dB) = (33.4 + 6.5) dB = 39.9 dBm2. This value is close to the one expected from the design and the laboratory test. The same image was also used in the following. Although in our study we focused on a data-based approach, i.e., evaluating the phase error from the final interferometric phase obtained after processing a temporal series of images, we also estimated the SCR directly form an amplitude image. The SCR was obtained by estimating the signal strength, averaging on 9 pixels distributed in a cross shape, and the clutter signal averaging on the remaining 16 pixels internal to the 5 × 5 square. To perform a quantitative analysis, we process different SLC images and use as reference the PCR amplitude response; the RCS of the AR , Ihan 1 mm , which fThe interferometric processing applied to retrieve the differential phase uses, as reference, the phase response of some pixels corresponding to stable buildings, located tens of meters away from the AR to reduce the atmospheric effect (as lower as possible). In The trend of the phase vs. temperature, as observed in the previous experiments, highlight a linear effect of the temperature on the phase stability. This suggests applying a correction to improve the accuracy of the retrieved phase, using the formula already experienced in the short-range experiments. 2, which fits the design requirements. The stability of the amplitude response shows a variation lower than 2% in the short-range test and can also be considered satisfactory (see y-axis), while the red dots indicate the amplitude values (right y-axis)). It is worth noting that, during the design phase, the choice of the active components was not made through an exhaustive market survey, limiting to the achievement of satisfactory performances of the implemented RF chain. Thus, we cannot presume that a better performance could be obtained with different components, but this task is out of the main goal of this study. The RF gain, and the specifically-designed and implemented antennas, allow achieving a low current consumption, and low-cost, and the transfer function of the device assures a response with a negligible delay and distortion . The results of the several tests analyzed in this study show that the implemented prototype, using a simple correction formula drawn on the bases of experimental data, can improve the performance of the sensor to finally provide a ±2 mm uncertainty, a value that can be considered satisfactory in situation where the installation of conventional PCR or high-cost AR is not advisable. Finally, concerning a rough estimate of the overall cost, in the current version, it is below 1000 euros, but the planned integration of the RF section could reduce to a few hundred euros, the price of commercial development, collocating the device as a low-cost apparatus with fair performance—one that could operate in remote areas with an acceptable risk of losing the device.


MYC, and supraphysiological androgen is effective in some patients. Here, we show that this repression is independent of AR chromatin binding and driven by coactivator redistribution, and through chromatin conformation capture methods show disruption of the interaction between the MYC super-enhancer within the PCAT1 gene and the MYC promoter. Conversely, androgen deprivation in vitro and in vivo increases MYC expression. In parallel, global AR activity is suppressed by MYC overexpression, consistent with coactivator redistribution. These suppressive effects of AR and MYC are mitigated at shared AR/MYC binding sites, which also have markedly higher levels of H3K27 acetylation, indicating enrichment for functional enhancers. These findings demonstrate an intricate balance between AR and MYC, and indicate that increased MYC in response to androgen deprivation contributes to castration-resistant PCa, while decreased MYC may contribute to responses to supraphysiological androgen therapy.Androgen receptor (AR) in prostate cancer (PCa) can drive transcriptional repression of multiple genes including  Androgen receptor in prostate cancer (PCa) transcriptionally represses multiple genes including MYC. Here, the authors suggest that increased MYC in response to androgen deprivation contributes to castration-resistant PCa, while decreased MYC may contribute to responses to supraphysiological androgen therapy. AR gene and an upstream AR enhancer, which occurs in the majority of cases3. Significantly, this upstream AR enhancer is activated primarily in CRPC2. A second frequent genomic alteration in CRPC is amplification of the MYC gene. Interestingly, the enhancer driving MYC expression in CRPC is also developmentally regulated and prostate-specific, and is strongly activated in CRPC5. The precise basis for the activation of these enhancers is not clear, but may be related to alterations that occur in the AR cistrome and transcriptome with PCa development and progression8, and to a broad reactivation of developmental epigenomic programs found with progression to CRPC9.The androgen receptor (AR) plays a central role in prostate cancer (PCa) development, and androgen deprivation therapy  to suppress AR activity is the standard treatment for metastatic PCa, but tumors invariably recur . Many respond to agents that further suppress androgen synthesis such as abiraterone, or to direct AR antagonists , but most men still relapse within 1–2 years. A subset of these relapsed tumors appear to be AR-independent, but the majority have persistently high levels of AR expression and activity. One mechanism driving this increased AR expression is amplification of the 10. In contrast, AR has been found to increase MYC expression in several PCa cell lines, and in AR-positive apocrine breast cancer cells12. However, most studies have found that AR stimulation in PCa cells decreases expression of MYC, and this appears to be a direct effect on MYC transcription15. The molecular basis for this AR repression of MYC, and more broadly for AR-mediated repression of multiple additional genes, is less clear and may be through diverse mechanisms17.Interactions between AR and MYC proteins may also contribute to PCa development and progression. AR stimulation has been reported to suppress the expression of MYC in normal prostate epithelium, which may be a physiological mechanism through which AR drives terminal differentiation18. The extent to which this is related to restoration of AR function is unclear as MYC overexpression has been reported to broadly suppress AR activity19. However, further data suggest that increased MYC in CRPC may alter the AR cistrome, as MYC overexpression causes some gain in AR binding sites, and the MYC motif is enriched in a subset of AR binding sites that are acquired in CRPC19. Finally, MYC has been reported to modulate AR splicing20.MYC overexpression can override androgen-mediated cell differentiation in normal prostate cells and drive androgen-independent proliferation in PCa cellsMYC super-enhancer within the PCAT1 gene and the MYC promoter. Together these findings demonstrate an intricate link between AR and MYC function in PCa, wherein they both compete for coactivators and function cooperatively to maintain stable expression of genes regulating multiple cellular functions. Clinically, these findings indicate that increased MYC in response to androgen deprivation contributes to the development of CRPC, while decreased MYC may contribute to tumor regression in response to supraphysiological androgen therapy in men with CRPC.In this report, we show that increased AR expression markedly enhances androgen-mediated transcriptional repression, and that this repression is largely independent of AR binding to chromatin and is associated with coactivator redistribution. MYC is rapidly and dramatically downregulated by androgen in cells expressing high AR levels, and a subset of androgen-repressed genes are MYC regulated and suppressed due to reduced MYC. Conversely, suppression of androgen activity in vitro and in vivo increases MYC expression. We further find that MYC binding is also associated with a subset of androgen-stimulated genes, and that a substantial fraction of MYC binding sites overlap AR binding sites. Notably, these AR/MYC binding sites have markedly higher levels of H3K27 acetylation (H3K27ac) relative to AR alone binding sites, indicating that the AR/MYC sites are enriched for functional enhancers. Global AR activity is also enhanced or repressed by MYC downregulation or overexpression, respectively, also consistent with coactivator redistribution. Finally, we establish that androgen treatment represses MYC expression by disrupting the interaction between the prostate-specific AR gene and higher levels of AR expression), and LNCaP cells stably overexpressing exogenous AR (LA) , the VCaP cell line  Fig. . RNA-seqLA) Fig. . DHT-stiLA) Fig. .Fig. 1GlExamination of overlapping DHT-stimulated versus DHT-repressed genes in the microarray and RNA-seq data also showed that the number of DHT-repressed genes was increased in the VCaP and AR-overexpressing LNCaP cells Fig. . Similar21. To further assess for direct effects we used binding and expression target analysis (BETA) to integrate the DHT-driven gene expression changes with genome-wide AR binding data, which was assessed by ChIP-seq in VCaP cells after 12 h of DHT stimulation22. This showed that AR binding (as assessed by ChIP-seq at 12 h after DHT-stimulation) was highly correlated with DHT-stimulated genes, but not with DHT-repressed genes , suggesting indirect mechanisms, although this likely also reflects transcript stability for some genes Fig. . Transcrnes Fig. . AR bind23. Consistent with the BETA showing an indirect effect of AR, expression of DHT-repressed genes that are associated with H3K27ac sites that do not overlap AR sites (H3K27ac only) decreased most significantly in response to DHT One mechanism that could contribute to indirect transcriptional repression is redistribution of transcriptional co-factors such as BRD4. Indeed, global analyses demonstrated that DHT increased BRD4 binding at AR sites that were not associated with H3K27ac under basal conditions (AR unique peaks), but decreased its binding globally at H3K27ac sites that were not AR associated (H3K27ac unique peaks) Figs. , S1f. BR24.We also performed motif analyses around basal H3K27ac peaks in loci of genes that are stimulated versus repressed by DHT at the 2 h time point to determine whether AR may be altering recruitment of other transcription factors (TFs) at DHT-repressed genes. ETV6, ETS1, and RUNX2 motifs were enriched at H3K27ac sites in DHT-stimulated gene loci, while an enrichment of CBX3, JUN, HSFY2, and MYC/MAX motifs was identified in DHT-repressed loci Figs. , S1j. Of25. RNA-seq showed that MYC was amongst the most significantly decreased TF at all time points  Fig. . This trlls Fig. . As expells Fig. , c.q value < 0.05, 14,697 MYC-centralized peaks were called under vehicle condition, and this decreased to 8076 upon 4 h of DHT induction, with 2060 of these being new peaks , we then performed RNA-seq analyses in VCaP cells after MYC knockdown by siRNA Fig. , h. AnnoWe then determined the overlap between genes that were decreased by MYC siRNA (MYC_upregulated) and downregulated by DHT (DHT_downregulated), which indicated that reduced MYC could account for ~8% of DHT-repressed genes  Fig. . ConversThe data above are consistent with the hypothesis that reduced MYC binding is a contributor to DHT-mediated transcriptional repression. However, basal MYC binding was also associated with DHT-stimulated genes see Fig. , and theThis MYC association with DHT-stimulated genes suggested that a subset of these genes may be co-regulated by MYC and AR. Consistent with this hypothesis, comparison of MYC and AR ChIP-seq data showed that ~25% of MYC binding sites in vehicle-treated VCaP cells overlapped AR binding sites  Fig. . Convers27.MYC binding at these 3716 common AR/MYC sites, as well as at MYC specific sites, was markedly decreased by DHT Fig. . We thenNotably, for AR and MYC common sites, baseline H3K27ac was high and showed a transient decrease at 2 h of DHT stimulation, but was restored to baseline at the 24 h time point Figs. . ConsistAs noted above, relative to AR unique sites, AR/MYC common binding sites, as well as MYC unique sites, are associated with high H3K27ac Fig. . This inAs expected, GSEA showed that the set of genes that were decreased after MYC depletion was enriched for DHT-repressed genes, consistent with MYC downregulation contributing to the DHT-repressed genes Fig. . SurprisAnalysis of a previously reported data set (GSE82223) confirmed that the MYC-activated gene set was repressed by DHT in VCaP cells, and this repression was prevented by treatment with AR siRNA Fig. , h. In cKLK3, TMPRSS2) versus regulated by MYC alone  or with shared AR and MYC binding sites (NKX3-1)  and LAM cells (LNCaP cells overexpressing AR and MYC), which were maintained in medium containing basal androgen (FBS medium) Fig. . We firs-1) Fig. –c. As ex-1) Fig. . MYC oveion Fig. c–e. Nextlls Fig. .Fig. 4MYKLK3 and TMPRSS2, that were repressed in the LAM versus LA cells , and then shifted them to FBS medium without adding DHT  or FBS medium with the AR antagonist enzalutamide . Both removal of the DHT and addition of ENZ induced MYC expression Fig. , b. We aels Fig. b, c.Fig.Next, we globally assessed the effects of AR blockade with ENZ on expression of all TFs, and identified MYC and AR as among the most significantly induced TFs Fig. . Further26. This markedly activated AR-dependent transcription (as assessed by PSA mRNA), but substantially repressed MYC, and both effects were effectively reversed by ENZ co-treatment , but is significantly higher in CRPC, and MYC signature genes are also dramatically overexpressed in CRPC as compared with primary PCa . As PCa Fig. . ConsistPCa Fig. . FinallyPCa Fig. . TogetheMYC gene is embedded in a topologically associated domain (TAD) containing multiple distal enhancers that engage with the MYC promoter in a tissue-specific manner29. The MYC promoter in PCa cells has been found to interact with a centromeric prostate-specific super-enhancer (SE) overlapping PCAT15. Global analysis of SE distribution in VCaP cells, based on H3K27ac, confirmed that the region spanning PCAT1-PRNCR1 was the top-ranked amongst all 1244 SEs, and showed that the telomeric PVT1 region TAD contains a weaker SE . Its PCa-specific interaction with the MYC gene promoter has been demonstrated in LNCaP cells4, and CRISPR/Cas9-directed knock-out of the PCAT1 SE decreases MYC gene expression in VCaP cells30. The 8q24 TAD also harbors lncRNAs that have been implicated in MYC regulation and its oncogenicity31. Therefore, we explored whether these were modulated by AR similarly to MYC. Indeed, RNA-seq analysis showed that DHT treatment in VCaP cells led to reduction of transcripts in the entire 8q24 locus  on the left and right boundaries of 8q24 locus were not repressed, or were only modestly transiently repressed, by DHT   Fig. . These rPCAT1 SE interaction with MYC, this may be direct or indirect, and previous studies indicate that AR may have a transcriptional activation function at this site in some contexts36. Indeed, there is a positive correlation between AR and MYC mRNA in primary PCa, but not in benign prostate  to primary (T) and then to metastatic CRPC (M)  Fig. . These agen Fig. .Fig. 7CoPCAT1 gene is only accessible in PCa  with perturbed dimerization function, and C562, 595A (M3) that would entirely lose DNA binding and transactivation capacities . However, we found that ARs with mutations that abrogate DNA binding could still repress MYC expression, supporting the conclusion that redistribution of coactivators including BRD4 is driving the MYC repression 19. One basis for this effect, similarly to the effect of androgen on MYC expression, may be coactivator redistribution. However, given the large number of genes modulated by MYC, an alternative mechanism is certainly MYC induction of one or a series of genes that more directly suppress AR activity.As expected, we found that MYC depletion by siRNA decreased the expression of androgen-repressed genes, consistent with a subset of these genes being MYC regulated. However, we found that MYC depletion also increased the expression of androgen-stimulated genes, and particularly androgen-stimulated genes lacking a MYC binding site. Conversely, MYC overexpression markedly suppressed the expression of androgen-stimulated genes. A previous study similarly found that MYC overexpression suppressed AR transcriptional activityPCAT1 overlapping MYC SE for PCa cells, a further understanding of how it is regulated may yield strategies to selectively suppress MYC expression in PCa.As noted above, one clinical implication of these findings is that androgen deprivation therapies for PCa result in a compensatory increase in MYC, which may be important for initial tumor cell survival. Therefore, agents that can suppress MYC expression or activity may be most effective when used early in combination with androgen deprivation. Such agents may include a new generation of BET protein inhibitors and MYC-targeting compounds. Moreover, given the specificity of the 2. LNCaP AR and MYC overexpression stable line (LAM) was generated by lentiviral infection of LA cell line with the pCDH-MYC vector  using the Lenti-Pac HIV Expression Packaging system , followed by hygromycin B and puromycin selection. LNCaP stable lines overexpression of AR WT and mutants were generated using AR mutants that were constructed based on DNA mutagenesis , followed by lentiviral infection and hygromycin B selection.LNCaP AR overexpression stable line (LA) was from Dr. Matthew L Freedman , which was generated based on lentiviral infection of LV_AR_orf vector that expresses Flag-tagged AR and hygromycin B selectionAntibodies used in these studies are as follows: MYC , AR , β-tubulin (mouse mAb clone KMX-1 EMDMillipore MAB3408), PSA , Vinculin (mouse mAb clone hVIN-1 Sigma V9264), H3K27Ac , BRD4 , MED1 , Flag , β-actin (mouse mAb clone AC-15 Abcam Ab6276). For immunoblotting, antibodies were used at 1:000 dilution. For IHC antibodies were used at 1:2500. For ChIP, antibodies were used at 4 μg per sample, or 5 μg per sample for HiChIP. MYC siRNA-1 was from Dharmacon (LU-003282-02-0002ON-TARGETplus Human MYC (4609) siRNA (#26). MYC siRNA-2 targeting sequence GCTTGTACCTGCAGGATCT was from Dharmacon (#1094). DHT was used at final concentration of 10 nM unless specified otherwise.2. Briefly, 4 × 15 cm dishes (two dishes for each assay) of VCaP cells in 5% CDS medium were treated with vehicle or DHT  and then fixed with 1% formaldehyde for 10 min, followed by quenching the reaction by glycine. Cells were lysed and pelleted nuclei were resuspended in 100 μl of 0.5% sodium dodecyl sulfate (SDS) and incubated at 62 °C for 10 min, followed by dilution in Triton X-100 to quench the SDS. Chromatin was then digested overnight at 37 °C with 500 U of PstI , followed by 80 °C for 20 min. Each sample then underwent ligation in 1 ml with 8000 U of T4 DNA Ligase  at room temperature. Proteinase K (30 μl of 20 mg/ml)  was then added followed by incubation overnight at 65 °C. RNase A (15 μl)  was then added followed by incubation for 45 min at 37 °C. Samples were then purified by phenol:chloroform:isoamyl alcohol  extraction and then ethanol precipitated.3C was performed as previously describeddsDNA was quantified using Qubit . For quantitative 3C droplet digital PCR (ddPCR) analyses, 400 ng of VCaP-PstI 3C library DNA was used as template. The MYC-promoter was used as bait (constant) that was paired with primer specifically located in target regions. Primer sequences are in Supplementary Table Each 3C library DNA sample was digested with Dpn II . The 4C ligation was in 10 ml with 50 μl T4 DNA ligase  at room temperature overnight. DNA was then ethanol precipitated and purified with the QIAquick PCR purification kit . The 4C-DpnII library was then amplified on the Veriti 96-Well Thermo Cycler (Applied Biosystems). Limited PCR (round-1) was conducted with the MYC-promoter region as the bait. The PCR products were purified using PCR purification kit and re-amplified by limited PCR (round-2) with primers containing Illumina sequencing adapters . Primer sequences are in Supplementary Table 3, 1% SDS and Proteinase K) for 8–16 h at 65 °C. ChIPed DNA was purified by phenol–chloroform extraction and then used for ChIP-qPCR or ChIP-seq library preparation.Cells were crosslinked in 1% formaldehyde for 10 min and quenched by glycine. Cell nuclear fraction was extracted by LB1 buffer  followed by washing in LB2  and LB3 buffer . Chromatin was sheared to 300–500 bp by Diagenode bioruptor sonicator and then incubated with antibody-conjugated protein A and G beads overnight at 4 °C with rotation. The antibodies for ChIP-seq were anti-MYC  or anti-H3K27ac . Antibodies for ChIP-qPCR were anti-MYC (Abcam Ab32072), anti-AR (Abcam Ab74272), or anti-H3K27ac (DIAGENODE C15410196). All antibodies were used at 4 μg per sample. After washing in RIPA buffer , the beads were eluted in elution buffer  and digested by MboI restriction enzyme (NEB-R0147). The biotin-dATP (Thermo 19524016) was incorporated to DNA in a fill-in master mix containing DNA polymerase I (NEB-M0210). After ligation and sonication, the sheared chromatin was incubated together with Protein A beads and anti-H3K27ac antibody  or AR antibody  for overnight at 4 °C with rotation. ChIPed DNA was purified from the eluted chromatin using Zymo DNA Clean & Concentrator kit. Biotin-labeled DNA was captured by Streptavidin C-1 bead  and fragmented by Tn5 enzyme to generate libraries for high throughput sequencing. Next-generation sequencing was conducted on a HiSeq4000 machine with 150 bp paired-end reads.HiChIP was performed as previously describedRNA isolation was carried out using the TriZOL reagent (Ambion) and the qRT-PCR analysis on gene expression was performed on the TaqMan StepOnePlus Real-Time PCR Master system (Applied Biosystems). The following TaqMan primer-probe sets were purchased as inventoried mixes from Applied Biosystems: MYC (Hs00153408_m1), AR (Hs00171172_m1), PSA/KLK3 (Hs02576345_m1), TMPRSS2 (Hs01120965_m1), NKX3-1 (Hs00171834_m1), LMNB1 (Hs01059210_m1), RAD51AP1 (Hs01548891_m1), PVT1 (Hs01069041_m1), PCAT1 (Hs04275836_s1), CASC8 (Hs03666772_g1), FAM84B (Hs00326521_m1), LINC00977 (Hs01596349_m1), GSDMC (Hs00937071_m1). The CCAT1 gene TaqMan primer set is forward primer: GGCCAGCCCTGCCACT; reverse primer: CAGTTTTCAAGGGATTTTAGGAGAA; and probe: ACCAGGTTGGCTCTGTATGGCTAAGCGT. The inventoried internal control was GAPDH .LNCaP was grown in RPMI-1640 containing 10% FBS . LNCaP cell lines overexpressing AR  were maintained in medium containing hygromycin B (200 μg/ml). LNCaP cell lines overexpressing AR and MYC  were maintained in medium containing hygromycin B and puromycin. VCaP cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) medium containing 10% FBS. For androgen-starving conditions, cells were grown in medium containing 5% CDS . Plasmids and siRNA transfections were carried out using the Lipofectamine 2000 (Invitrogen), following the manufacturer’s directions. Cell proliferation was performed using the CellTIter-Glo Luminescent cell Viability Assay Kit .2 asphyxiation using an AVMA approved method and apparatus.ICR/scid male mice  were used to generate xenografts. VCaP xenografts were established in the flanks of male SCID mice by injecting ~2 million cells in 50% Matrigel. When the tumors reached ~1 cm, biopsies were obtained and then the mice were castrated. Groups for testosterone test are: control castrated VCaP xenografts (C); castrated VCaP xenografts with testosterone (5 mg/mouse/day for 3 days) (T); and castrated VCaP xenografts with testosterone and enzalutamide  (T + E). For IHC, additional biopsies were obtained 4 days after castration, and the tumors were harvested at relapse. Frozen sections were examined to confirm that the samples used for RNA extraction contained predominantly nonnecrotic tumor. Fixed sections were subjected to IHC staining for MYC (Santa Cruz sc-764) or PSA (BioDesign K92110R), both at 1:2,500 dilution. All animal experiments were approved by the Beth Israel Deaconess Institutional Animal Care and Use Committee and were performed in accordance with institutional and national guidelines. Tumor-bearing mice were sacrificed before the tumors reach the size considered to be distressing (2 cm). Animal stress and discomfort were minimized by the use of methods recommended by the AVMA Panel on Euthanasia and approved by the Institutional Animal Care and Use Committee. Mice were monitored daily by animal facility staff for tumor size and overall health. When necessary, euthanasia was be carried out by personnel in the animal facility by COhttps://github.com/FelixKrueger/TrimGalore) with parameters “-q 20–phred33 –stringency 4 –length 20 -e 0.1”. The clean reads were then aligned to hg19 human genome using STAR (version 2.4.2a)45 with parameters “–outSAMattributes NH HI NM MD –outSAMstrandField intronMotif –quantMode GeneCounts”. Reads per kilobase per million mapped reads (RPKM) were then calculated based on gene read count and GENCODE v24 GRCh37 annotation. R package clusterProfiler46 was used to perform KEGG/GO enrichment analysis and Gene Set Enrichment Analysis (GSEA). To identify the differentially expressed genes in Fig. Total RNA was extracted from VCaP cells using TriZol reagent and whole transcriptome sequencing was conducted on Novaseq6000 S4 flowcell for PE150 sequencing . Raw sequencing reads were first trimmed by Trim Galore  and heatmap analysis by deeptools (version 3.3.1)49. Cistrome SeqPos50 and R package PWMEnrich were used to identify enriched transcription factor binding motifs in a set of peaks. Binding and Expression Target Analysis (BETA) was used to assess the regulatory capacity of the given ChIP-seq peaks on gene expression51. Super-enhancers were recognized from H3K27ac ChIP-seq peaks by Ranking Of Super Enhancer52.To generate ChIP-seq library, 5 ng ChIPed DNA was processed with the Rubicon ThruPLEX-FD kit and sequenced as 75 bp single-end reads. ChIP-seq reads were first trimmed by Trim Galore in the same option setting with RNA-seq and then aligned to hg19 human genome by Bowtie2 (version 2.2.1)37. Samples of tissue adjacent to the tumor from four of the prostate cancer patients were also included. The data processing was conducted as previously described. Briefly, after trimming by Trim Galore (version 0.4.4_dev), trimmed reads were mapped to the hg19 genome reference using Bowtie2 and Bismark v0.19.053. Another 208 pairs of primary PCa and adjacent tissue WGBS data were from the Genome Sequence Archive for Human under the accession number PRJCA00112438. Output files of Bismark were downloaded for downstream analysis. R package “dmrseq” was used to process the count data and plot average methylation signal of super-enhancer regions54.Two PCa tissues whole-genome bisulfite sequencing (WGBS) data sets were used in this study. The paired-end WGBS data set from the prostate tissue of four healthy donors and five prostate cancer patients were obtained from the authors55. A fragment library was created by the function “createVirtualFragmentLibrary” using the parameter firstCutter = “ctgcag”, secondCutter = “gtac”. 4C bam files were then loaded to initiate a Data4Cseq object and the viewpoint was set at MYC promoter region. The raw read count was RPM-normalized for the comparability between different samples. A near-cis plot was finally generated to cover the MYC promoter and PCAT1 super-enhancer regions.The PE reads were first aligned to hg19 human reference genome using Bowtie2 (version 2.2.1). The resultant bam files were then used for downstream analyses by R package “Basic4Cseq”56. The default settings  were used to filter experimental Hi-C artefacts and other uninformative di-tags. The resultant bam file was used to generate valid pairs by command “samtools sort -n hicup.bam | bamToBed -i–bedpe | awk ‘OFS = “\t” {print $7,$1,int(($2 + $3)/2), $9, $4, int(($5 + $6)/2), $10}’ > HiChIP.allValidPairs” for downstream loop calling. HiChIP loops were identified by hichipper pipeline57 using valid HiChIP read pairs and pre-determined ChIP-seq peaks. The corresponding ChIP-seq peaks were used to locate HiChIP loop anchors during loop calling.Pair-end HiChIP reads were first trimmed by Trim Galore and then aligned to hg19 human reference genome by HiCUP pipelinehttps://portal.gdc.cancer.gov/) were processed by STAR2/RSEM pipeline to generate TPM values for each sample. TPM values of CRPC RNA-seq data were downloaded from http://davidquigley.com/prostate.html1. Before gene expression level comparison, the AR or MYC amplified samples were removed. MYC-upregulated gene z-score of each participant was defined as the sum of z-scores of 136 MYC-upregulated genes. For each gene, z-score = (x − μ)/σ; x indicates pre-normalized gene expression level, μ indicates study mean of gene expression and σ indicates study standard deviation of gene expression.TCGA PCa RNA-seq data . The bam files were then subjected to MACS2 for peak calling with the parameter “–SPMR” on and–keep-dup = 1. UCSC bedGraphToBigWig tool was used to convert Resultant bedgraph files to bigWig files. The bigWig files of each group were averaged and merged to a single bigWig file by a custom shell script for further visualization.Data in bar graphs represent mean ± SD of three technical replicates and are representative of at least three independent experiments. Results for immunoblotting are representative of at least three independent experiments.Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


The use of robotic systems in physical rehabilitation protocols has become increasingly attractive and has been given more focus in the last decade as a result of the high prevalence of motor deficits in the population, which is linked to an overburdened healthcare system. In accordance with current trends, three robotic devices have been designed, called ParReEx Elbow, ParReEx Wrist, and ASPIRE, which were designed to improve upper-limb medical recovery . The three automated systems were tested in a hospital setting with 23 patients (12 men and 11 women) suffering from motor deficits caused by various neurological diseases such as stroke, Parkinson’s disease, and amyotrophic lateral sclerosis (ALS). The patients were divided into three groups based on their pathology . Objective clinical measures, such as the Medical Research Council (MRC) scale, goniometry, and dynamometry, were used to compare pre- and post-rehabilitation assessments for both robotic-aided and manual physical rehabilitation therapy. The results of these tests showed that, with the exception of a few minor differences in muscular strength recovery, the robotic-assisted rehabilitation methods performed equally as well as the manual techniques, though only minor improvements were validated during short-term rehabilitation. The greatest achievements were obtained in the goniometric analysis where some rehabilitation amplitudes increased by over 40% in the vascular group, but the same analysis returned regressions in the neuromuscular group. The MRC scale analysis returned no significant differences, with most regressions occurring in the neuromuscular group. The dynamometric analysis mostly returned improvements, but the highest value evolution was 19.07%, which also in the vascular group. While the results were encouraging, more research is needed with a larger sample size and a longer study period in order to provide more information regarding the efficacy of both rehabilitation methods in neurological illnesses. In neurological diseases, motor recovery is often deficient, with many patients experiencing long term impairments that have a negative effect on their quality of life and their capability of performing daily living activities. The increased survival of the population and chronic morbidities create the premises for a higher incidence of motor dysfunction, regardless of the lesion topography, involving either the pyramidal, extrapyramidal, or the peripheral motor system.Physical therapy consists of specific exercises that have been developed to reestablish the functionality of the affected segment of the motor system and acts as an adjuvant to medical treatment. These movements can be easily taken over by automated systems  and roboPrevious studies have compared the results of robotic physical therapy to those of classical physical therapy.Masiero et al.  analyzedSingh et al.  conducteTaravati et al.  conducteThe efficacity of robotic physical therapy was also tested in ,7,8,9, wASPIRE, ParReEx Elbow, and ParReEx Wrist are a family of modular devices targeting the rehabilitation of the shoulder, elbow and wrist and were developed in a modular configuration with a unified, centralized, control system and a user interface. As it is a robotic system that uses a unified control system, it makes it possible to treat 1, 2, or 3 patients at the same time under the supervision of a single physician. Each robotic module is fast and easy to setup and configure. The targeted cost of the rehabilitation robotic system  is under EUR 50,000, making its implementation in various hospitals in Eastern Europe a feasible low-cost solution, enabling a larger number of people to access rehabilitation as well as requiring a lower number of therapists needed to perform the rehabilitation, given the fact that a therapist can work with up to three patients at the same time using the robotic system.Within this study, the validation of the robotic system through a first set of clinical trials is targeted.The prototype of the robotic system validated through the clinical trial is only configured for the rehabilitation of the right arm: before preparing the current clinical study, an analysis regarding the hand dominance and side of stroke was completed.In 2005 Waller et al.  examinedA 2006 study by Harris et al.  determinIn 2014, Nam et al.  evaluateTo test and validate the performance of the previously mentioned robotic-aided physical therapy devices, an exploratory, multimodal evaluation was conducted in the Neurology Department of the Municipal Clinical Hospital Cluj-Napoca (SCMCJ). On the one hand, Neurophysiological tests were conducted, and given the extent of the obtained data, these results were separated and have already reported by the research group ; on the In the field of neurology, pathologies are often characterized by serious motor deficits. Motor deficits might occur either suddenly, such as in cases of cerebrovascular diseases, or gradually, such as in cases of neurodegenerative pathologies, for example, motor neuron diseases. Sometimes, motor deficit is absent, but the lack of proper regulation/modulation has a huge impact on movement quality. Starting from the above premises and by judging the most frequently occurring disease for each category, the target pathologies for the current study were ischemic stroke, Parkinson’s disease, and amyotrophic lateral sclerosis (ALS). Stroke patients have an extensive cerebral lesion and present deficiencies corresponding to that territory. Extrapyramidal diseases are characterized by degenerative lesions of the basal ganglia, and the deficiencies mainly involve inappropriate movement modulation. In motor neuron diseases, in different parts of the brain, both the central–motor cortex and the peripheral–corticospinal tracts, the peripheral motor neurons might suffer degeneration. The motor deficits are composed of an upper and lower motor neuron syndrome.Physical rehabilitation, besides the preservation of joint integrity and direct influences on muscle trophicity, also represents an important trigger for plasticity . IschemiTo clinically evaluate the efficiency of the rehabilitation protocols, both the movement forces and amplitudes should be evaluated. The Medical Research Council scale (MRC) is a widely accepted and validated tool for the evaluation of motor deficit. It appreciates the strength of a muscle group in a semi-quantitative manner, while goniometry and dynamometry are more objective methods that permit an exact, quantitative evaluation of neurological diseases ,23. The Our initial exploratory study has the following objectives: (1) to assess the benefits of physical therapy for different neuromotor disorders(this objective was described in ); (2) toThe robotic systems ASPIRE ,26,27, PThe devices were developed to be adaptable to different limb characteristics, thus being able to cover a wider range of patients. Of these, there are also mechanically adaptable mechanisms to compensate for different limb segment lengths as well as the usage of textile straps, allowing limb segment girth adaptation.The study protocol was approved by the Ethics Committee of the SCMCJ. It was developed in accordance with the principles of the Declaration of Helsinki regarding biomedical research. All of the participants signed an informed consent detailing the scope and details of the medical procedure, the functionality of the robot, and the environment in which said process would take place. The duration of the study was 3 months, from October to December 2019. A total of 23 patients were admitted (12 men and 11 women). The participants showed various degrees of upper limb motor deficit.Inclusion criteria sought patients within the age range of 18–80 years; suffering from ischemic/hemorrhagic stroke, Parkinson’s disease or, ALS; having suffered a neurological disorder within the last 2–24 months; have a mini-mental scale(MMS) score of 18–23 (mild cognitive impairment) or 24–30 (no cognitive impairment), as long as the mild cognitive impairment does not influence the patient’s capability to perform the physical therapy; be Brunnstrom stage (BS) 3 , 4 , 5 , or 6 ; have a modified Ashworth scale (MAS) score of 1  when the affected limb is moved in flexion or extension), 1+ , or 2 ; and have left arm impairment, right arm impairment, or bilateral impairment.Inclusion criteria regarding rehabilitation with the robotic system  required the patient to present right arm impairment , be able to maintain a sitting position without any aiding devices other a than wheelchair, be of a stable state , and have no wounds on the surface of the right arm.The exclusion criteria were minor patients or patients older than 80 years, health conditions that were incompatible with the physical therapy , social constraints , and economic constraints . Exclusion criteria regarding rehabilitation with the robotic system implied left arm impairment, the patient being unable to maintain sitting position, the unstable state of the patient (patient was unable to understand that he was going to perform rehabilitation with a robot instead of a human), or the patient presenting wounds or cannula on the right arm.The participants were grouped according to their pathology, resulting in a Vascular, an Extrapyramidal, and a Neuromuscular group.The selection and distribution of the patients between the manual and robotic therapy is illustrated in The time spent by each of the patients enrolled in the trial was 15 ± 2 min with the ASPIRE system for shoulder rehabilitation, 15 ± 3 min with the PArReEx elbow system for elbow rehabilitation, and 10 ± 2 min with the PArReEx Wrist system for wrist rehabilitation. Each patient preformed two rehabilitation sessions daily. Conventional rehabilitation was performed by the physiotherapist with the patient seated on the side of a hospital bed, and the entire rehabilitation motion was performed manually by the physiotherapist. Times recorded during the conventional rehabilitation were lower .The amount of the physical therapy delivered for the left hand was identical to the therapy delivered for the right hand.When joining the study, each patient was informed of the robotic system and saw the robotic system working, had time to individually evaluate the potential benefits, and signed an informed consent. A baseline clinical, gonio-, and dynamometric evaluation was performed. Goniometry evaluates the motion ranges of each assessed joint, and the gathered data are expressed in degrees. The serial investigation provides information regarding the possible gain in joint flexibility after seven consecutive days of physical therapy, which is important for both the spastic and plastic hypertonic states, such as in our patient groups. Below are the exact measurement protocols for each investigated joint.For the measurements, the patients should ideally be at rest in a standing position, with the upper limb in supine position next to the body. Under the conditions of the investigated pathologies, this is not possible, so each measurement tends to be as close to the resting position as possible but is adapted to the individual medical condition.For shoulder flexion, the center of the goniometer  is fixedElbow flexion is measured with the upper limb in 90-degree abduction while keeping the hand, elbow, and shoulder in the same plane during the movement. The center of the goniometer is positioned on the olecranon, the fixed arm is kept parallel to the humerus, and the mobile arm is kept parallel to the ulnaris bone. The patient executes forearm flexion on the arm, with the measured angle being in the range of motion for elbow flexion. In cases where elbow flexion is being measured, the degrees of freedom also permit pronation and supination. With the elbow next to the torso and in 90-degree flexion and with the interior face of the forearm oriented medially, the patient holds a wooden stick in his/her hand. The arms of the goniometer are positioned parallel to the stick. Then, the patient executes the movement, the fixed arm is kept in position, the mobile arm follows the movement, and the resulting angles are registered as ranges of motion for pronation/supination.The flexion and extension of the radio-carpal joint was evaluated in the present study. For both radio-carpal flexion and radio-carpal extension, the center of the goniometer is positioned on the lateral epicondyle of the ulna, the fixed arm of the goniometer is parallel to the ulna, and the mobile arm moves together with the hand. More detailed information and imagery is described in the literature .Muscle strength is another parameter being investigated in the current study, with motor deficit being the most important factor for vascular and neuromuscular diseases. It is frequently measured for each movement. For this purpose, to reduce the subjectivism of the evaluation, measurement scales were developed. This study uses such a scale, with the Medical Research Council scale (MRC) being used in the current study, which is reproduced in Using this technique, one of the investigators performed all of the examinations as follows: abduction, adduction, flexion, and extension of the shoulder; elbow flexion and extension; flexion and extension of the hand; supination and pronation of the forearm; finger extension of the metacarpal-phalangeal and inter-phalangeal joints; and finger adduction and abduction. The measurements were performed on a wider number of joints than those included in the rehabilitation protocol in order to study the influence of the exercises on other joints of the upper limb. The other, more objective method for the evaluation of muscle strength is dynamometry. This is a process that uses a specialized device—in the present study, a Baseline Push-Pull Dynamometer—to measure quantifiable data for muscular force (expressed in kgf), power, and resistance. As with goniometry, in this case, the positioning of the patient, or the limb and the limb segment is incredibly important in order to obtain reproducible data.Strength during shoulder flexion is preferably measured on sitting patients by positioning the dynamometer on the distal third of the antero-medial surface of the arm, with the shoulder being stabilized by the therapist. The recording starts with the flexion of the elbow against the resistance of the therapist and the in-between device. To measure the strength during shoulder extension, the patient position is similar; there is a need to stabilize the shoulder, and the dynamometer should be put on the distal third of the arm but this time on the postero-lateral surface. The measurement starts with the extension of the shoulder. Strength measurements during shoulder abduction and adduction are performed similarly, except the dynamometer should be positioned on the lateral or medial surface of the distal third of the arm, and the performed movement should be executed accordingly.To measure strength during elbow flexion, the body is still in a sitting position, the shoulder is flexed 45 degrees, and the elbow is flexed at 90 degrees with the palm facing upwards. The therapist stabilizes the arm and places the dynamometer on the distal third of the forearm medially and maintains resistance against the flexion performed by the patient. The same is steps are performed to measure the strength during extension, but the device should be positioned on the lateral surface of the forearm, and the data recording is made during extension. Measuring strength during elbow pronation and supination requires the same patient position, but the subject is asked to hold a wooden stick in his/her hand. The dynamometer is placed on the lateral part of the stick, and the patient is asked to perform the movement.Measuring the radio-carpal joint flexion/extension strength requires the same position from the patient, but the forearm must be stabilized by the therapist. Then, for the measurement, the dynamometer is placed either on the palmar or on the outer side of the hand and the appropriate movement is performed against the therapists’ resistance.Regardless of the deficit, the muscles to be tested need to be isolated by stabilizing the patients’ position in a personalized manner; the angle of different joints should be chosen in a way that is suitable to start a particular movement. Even if optimal, the measurements should be repeated three times; if they are not quantifiable, then after a few minutes, the whole procedure should be repeated; it is useful to start on the unaffected side first. The evaluation is correctly executed, reproducible, and comparable if it is always performed by the same examiner. All of the baseline measurements were conducted by the same investigators/method in order to further reduce the bias induced by subjectivism. After the baseline, a 7 day long, 2 times/day rehabilitation program was implemented , and thep < 0.05.The obtained data were gathered in a database, and afterwards, it was statistically analyzed using IBM SPSS Statistics 20 . After the descriptive statistics were determined, the chosen analysis was non-parametric given the low number of participants. Since the data rows constituted their own control for the second and third groups, the Wilcoxon Matched Pairs test was used. An exception was the Vascular group, where the control treated by the physical therapist was an individual group with left hemiparesis. In this case, the test used for the analysis was the Mann–Whitney U test for independent variables. Each data row for every individual parameter was tested for the type of distribution using the Kolmogorov–Smirnoff test, and since normal distribution was found, the best evaluation method was applied. The significance threshold was considered Each test was performed at the beginning and at the end of the rehabilitation process, with the first test representing the baseline clinical evaluations.Demographic data and some characteristics of the patients enrolled in the clinical trials are shown in The assessment of the patients started with goniometry, which was expressed in degrees. The results were compared with the baseline assessment of the patients. The results are presented in The evolution of the MRC data with respect to the baseline are presented in The results of the dynamometric analysis are presented in p-values of each pathology group and each rehabilitation motion. In order for the null hypothesis to be accepted, no significant difference the p-values must score above the previously defined threshold (p > 0.05). p-values for each group and rehabilitation motion, even if there were differences recorded between baseline assessment and final evaluation in terms of percentage, there was no p-value that scored under 0.05.The results presented above offer a preliminary assessment regarding functional changes following the rehabilitation process in the studied pathologies. The results are discussed from the premise of not being statistically significant, with this being a consequence of either the low number of investigated cases or the reduced duration of the rehabilitation process. The discussion considers tendencies in order to sketch possible evolutions at an exploratory level. In the below discussion, the obtained data are grouped according to the evaluated pathologies.Ischemic stroke produces a typical lesion pattern characterized by the death of directly affected cell groups located in the middle of the affected area and by the suffering but potential survival of cells at the outer limit of the lesion, the penumbra area. Under favorable circumstances, this region might regain function, sometimes leading to spectacular recovery. According to one trend, commencing physical rehabilitation as quickly as possible after the patient is table frequently produces quite visible effects. The physiopathology is probably proprioceptive signaling and primary somatic-sensory to motor cortex triggering, and this enhances neuroplasticity .As our study showed, there was no difference in terms of whether the rehabilitation was conducted using robotized or therapist-implemented protocols.The baseline assessment revealed lower ROM in the Vascular group, where the biggest improvement was also recorded, but the ROM of the Extrapyramidal group and the Neuromuscular group were still higher than the ROM of the Vascular group, even after the rehabilitation. There were no significant differences between the groups regarding the MRC scale and dynamometry, either in baseline or at the final assessment.Vascular group: the effect of rehabilitation on this group is quantifiable using the proposed methods, as demonstrated for the selected pathology by several studies [ studies ,33,34. TGoniometric evaluation of an ischemic stroke patient makes sense when spasticity is already installed or is in course of being installed in order to alleviate its effect on the patient’s movements and ranges of motion. Our recruited patients were subacute to chronic patients, making them suitable for the evaluation. The approach holds quite a well-defined methodology. The evaluation being conducted by the same evaluator is a necessary condition to preserve objectivity. Rehabilitation led to a slight increase in the ranges of motion at the investigated joints, underlining an otherwise proven effect of physical therapy on spasticity.Extrapyramidal group: in this case, one cannot speak about motor deficit; the symptom is rather definable and is motor dysfunction resulting from the lack of or disturbed modulation of motor activity. Both the direct and indirect pathways involved in Parkinson’s disease led to reduction of the excitatory feed-back loop on the motor cortex. This result is produced via the direct pathway by the lack of D1 receptor signaling on the globus pallidus internus (GPi) and the consequent reduction of ventro-lateral and ventro-anterior thalamic nuclei excitatory influences on the motor cortex [r cortex . A similr cortex .however, the long-term effects have not been evaluated [When compared to the other investigated pathologies, the Extrapyramidal group showed clear, consequent movement towards an increase in all of the investigated parameters. This increase is not significant and is probably caused by the mentioned limitations; still, the visual tendency is obvious. The most pronounced effect is seen for the MRC scale evaluation, and the least modified parameter is the dynamometric assessment. Physical strength theoretically remains the same, but the correct allocation of motor resources leads to more increased peripheral activation. The ranges of motion were expected to show a tendency towards an increase by means of a reduction in the rigidity and plastic hypertonia. The proprioceptive input increased the excitatory cortico-striatal influences, which, in turn, in the relative absence of dopamine, improve the activity of the indirect pathway, inhibiting the thalamic excitatory loop. The overall effect seems to be beneficial in the short term; valuated .Neuromuscular group: The whole motor system is affected by the degeneration process, including the upper and lower motor neurons and the in between pathways, leading to muscle atrophy and mixed motor deficit. Through clinical testing, both muscle strength and goniometry show disappointing results when compared to the results seen in the other pathologies. The increase in the muscle strength is only seen for a few distal muscle groups in the dynamometric measurements, which were not proved by the MRC testing, where there is not even a visual tendency towards improvement. Moreover, when investigating joint mobility, the post-rehabilitation data are even less favorable than the baseline data. The stimulating effect of rehabilitation is not beneficial centrally, at least theoretically, considering the already over-activated, excitotoxic effect of glutamate, which has been previously described in motor neuron diseases. It seems that the peripheral atrophic muscles and the individual extensively affected unstable muscle fibers show exhaustion and cell death rather than regaining strength after rehabilitation. The effect of physical therapy might be limited to the preservation of joint flexibility and to the maintenance of actual muscle performance without any performance improvement tendencies, which might overwhelm the affected motor systems. Even though the effects are limited, other clinical studies have also illustrated slight improvements in the quality of life of patients with neuromuscular disorders [isorders , motivatAs mentioned at the beginning, the present study has several limitations at this stage. First, there were no long-term monitoring evaluations; thus, it was not possible to determine the long-term beneficial effects, even for short-term physical therapy. Secondly, this study had a modest sample size. Third, the duration was short, so it is not possible to make any unequivocal conclusions about the efficacy of the method, but surely completing long-term robotic/physical therapeutic rehabilitation programs can lead to clear favorable results. On the other hand, as the current study presented short, exploratory research, it provides the possibility to fine-tune the robotic systems and to set new goals for more thorough clinical research, which will include a larger number of patients.The results of our study have also been confirmed also in a systematic review and meta-analysis  that ass(1)General benefits of physical therapy in neuromotor diseases: besides the Vascular group, which is known to benefit from physical therapy, there are also promising results in the other study groups that could improve the quality of life of chronic patients and delay the effect of degenerative diseases;(2)Comparative assessment of robotic versus manual physical therapy: our results illustrate that robotic therapy can be used for the treatment of neuromotor diseases with similar results as manual therapy, which, in the face of the upcoming crisis caused by the ageing of the population, represents a valid option to enable the treatment of more patients requiring physical therapy;(3)Possible improvements of the robotic devices: each module of the upper limb robotic system has been carefully analyzed based on clinical data, and our team is working on improving the overall system. For the shoulder rehabilitation device, ASPIRE, our team has recently completed processing the clinical data [cal data , while PWith respect to the defined objectives of this exploratory study, our results point out the following:The addition of supplementary stimuli , such asThe improvement of the graphical user interface to investigate the possibility of working with more patients simultaneously under the supervision of a single therapist;Data recording using different sensors ,40, bothAdditionally, in terms of structural improvements, the results of this clinical study provided several areas of improvement for the modular robotic system, which will aim to increase the efficiency with respect to the medical act of providing rehabilitation:p-values obtained in this study revealed the validation of the null hypothesis on a medium-sized sample of patients. However, this result does not mean that a clinical difference is not achievable. As the percentual comparison of Statistically there was no difference between the robotic rehabilitation and conventional rehabilitation, as the This initial exploratory study conducted on a group or patients with different neuromotor diseases comparatively assessed the use of robotic and manual physical therapy for rehabilitation. Our study demonstrated that there are no relevant differences between physical therapy performed by robotic devices and the conventional physical therapy that is manually performed by a therapist, encouraging future work and a more extensive clinical study. The clinical approach—MRC, gonio-, and dynamometry—remains a useful and valid technique to evaluate the results of rehabilitation and follow-up. Short term physical therapy only shows a tendency towards a quantifiable effect on the investigated pathologies, and even this is not always positive; it raises the necessity of only recommending exercises that have been carefully evaluated on physio pathological grounds.For future work, there is a need to extend the number of participants and the duration of the study in order to be able to formulate conclusions based on significant differences and not only tendencies.


PD-AVB sequence was induced by: Premature atrial contraction (8/14), premature ventricular contraction (5/14), His extrasystole (1/14), conduction block in a branch (1/14), and atrial tachycardia termination (1/14). All patients with PD-AVB received a dual-chamber pacemaker during hospitalization. Conclusion: The prevalence of PD-AVB was 14%, and may be underestimated. PD-AVB episodes were more likely associated with syncope in comparison with patients without PD-AVB.Background: In patients with complete atrioventricular block (AVB), the prevalence and clinical characteristics of patients with pause-dependent AVB (PD-AVB) is not known. Our objective was to assess the prevalence of PD-AVB in a population of patients with complete (or high-grade) AVB. Methods: Twelve-lead electrocardiogram (ECG) and/or telemonitoring from patients admitted (from September 2020 to November 2021) for complete (or high-degree) AVB were prospectively collected at the University Hospital of Nice. The ECG tracings were analyzed by an electrophysiologist to determine the underlying mechanism of PD-AVB. Results: 100 patients were admitted for complete (or high-grade) AVB . Arterial hypertension was present in 68% of the patients. Baseline QRS width was 117 ± 32 ms, and mean left ventricular ejection fraction was 56 ± 7%. Fourteen patients (14%) with PD-AVB were identified, and presented similar clinical characteristics in comparison with patients without PD-AVB, except for syncope (which was present in 86% versus 51% in the non-PD-AVB patients,  Pause-dependent atrioventricular block (PD-AVB) can be defined as a paroxysmal and complete AVB whose mechanism is “bradycardia-dependent”. An early description of bradycardia-dependent AVB was made in 1971 by Coumel et al. . AlthougPD-AVB is considered a rare entity, but the prevalence of 4AVB is not known. The purpose of this study was to assess the incidence and the clinical characteristics of patients with PD-AVB in a population of patients admitted into a cardiac care unit for complete (or high-grade) AVB.All patients admitted at the cardiac care unit of the University Hospital of Nice from September 2020 to November 2021 for complete (or high-grade) AVB were included in this single-center prospective observational study, and their electrocardiograms (ECGs) were systematically collected. Complete AVB but also high-grade (including 2 to 1) AVB were confirmed on 12-lead surface and/or telemetry monitoring by one experienced electrophysiologist (S.-S.B.). Patients with vagally-mediated AVB  defined AVB were classified as permanent if complete (or high-grade) AVB was present all along the period of the hospitalization stay during cardiac monitoring, or intermittent if complete (or high-grade) AVB was observed in alternation with 1:1 conduction . To avoid any confusion with previous studies, the term “paroxysmal” will not be used in our study and will be discussed later.The clinical and electrocardiographic characteristics of the patients were analyzed; episodes of PD-AVB were classified according to their initiation mechanism. All patients gave their written informed consent for the pacemaker implantation (if needed). The study was approved by the institutional review board.The statistical analysis was made with Excel . Categorical variables are described as numbers and percentages. Continuous variables are described as mean ± SD for variables with normal distributions or as median with range for variables not normally distributed.n = 43) of patients with intermittent complete AVB. Baseline conduction disturbances in patients with intermittent complete AVB are presented in p = 0.31).During the inclusion period, 100 patients were admitted for complete (or high-grade) AVB . Arterial hypertension was present in 68% of the patients. Baseline QRS width was 117 ± 32 ms, and mean left ventricular ejection fraction was 56 ± 7%. Fourteen patients (14%) with PD-AVB were identified, and presented similar clinical characteristics in comparison with patients without PD-AVB except for syncope (present in 86% versus 51% in the non-4AVB patients) . PD-AVB p = 0.01). To the best of our knowledge, our series is the second largest described with PD-AVB to date. Of note, a recent case reported the possibility for conduction block to occur within a bundle branch, and not within the AV node itself [Our study is the first to report the prevalence of PD-AVB in a population of patients admitted into a cardiac care unit for complete (or high-grade) AVB. While 4AVB was considered a rare phenomenon, our study demonstrates a prevalence of 14%, reaching one third if considering only patients with intermittent complete (or high-grade) AVB. Our cohort of patients is representative of the prevalence of complete (or high-grade) AVB in a University center with medium-to high-volume activity. Isolated descriptions or case reports/case series have been published concerning PD-AVB, without further information about its prevalence . In our e itself . All pree itself .Interestingly, our study is the first to report double distinct mechanisms recorded within the same patient. Four patients presented with two distinct mechanisms of PD-AVB: One patient had PVC-induced PD-AVB and a second episode without significant increase in the PP intervals before AVB (from 690 to 710 ms), and without PR prolongation ; anotherLee et al. reported the largest bicentric series to date of paroxysmal AVB. Nevertheless, both tachycardia-dependent AVB  and idioTo summarize, our suggestion for paroxysmal AVB classification may be as follows:(1)“Extrinsic” vagally-mediated AVB characterized by significant PR prolongation or Wenckebach before initiation of AVB, gradual slowing of the sinus rate , resumption of AV conduction with sinus acceleration, PP interval prolongation during ventricular asystole. Often, a shortening of the PR interval compared to the last PR interval with AV conduction before an AV block can be observed . A clinical history suggestive of heightened vagal tone is present.(2)“Idiopathic” paroxysmal AVB involving a younger population (mean age 55 ± 19 years) without cardiac and ECG abnormalities, without progression to persistent forms of AVB, and with efficacy of cardiac pacing. AVB occurs with abrupt onset and delayed emergence of an adequate escape rhythm without PP cycle lenghthening or PR interval prolongation. A low baseline adenosine plasma level was found in this specific population. This entity should remain a diagnosis of exclusion.(3)“Intrinsic” AVB (suggesting AV conduction disease), which may be divided into four categories: Congenital heart block; tachycardia-dependent AVB; PD-AVB; and finally other acquired AVB when the preceding features/conditions are lacking (non-PD-AVB group in our study) as shown in The main differences that may be seen between our population and Lee’s report are that their population was significantly younger than ours (69 versus 84 ± 6 years-old) with thinner QRS duration (123 ± 32 versus 132 ± 27 ms in our series). The authors also reported that PAC was the main trigger of PD-AVB in 30% of the cases, PVC in 23%, His extrasystole in 10%, and other variable mechanisms in 37%. Our results are in line with the literature, because PAC were also found to be the most prominent trigger (two thirds).Of note, in our series, two patients out of 14 with PD-AVB underwent a transcatheter aortic valve intervention (TAVI) in their past medical history. According to the latest European guidelines, early permanent cardiac pacing may be recommended in the case of transient high-grade AVB during TAVI in patients with pre-existing RBBB ,16. ActuThis is a monocentric study with a limited number of patients. True prevalence of PD-AVB could have been underestimated. No electrophysiological study was performed in this elderly population with an indication of permanent cardiac pacing (syncope present in 80% of the cases). Electrophysiological study is usually not recommended in the setting of symptomatic complete AVB .Of note, all our patients with PD-AVB received a dual-chamber pacemaker . PD-AVB (once identified) usually occurs in diseased His–Purkinje systems. Further studies are needed to confirm the usefulness/superiority of conduction system pacing in these patients, as recently reported by Du et al. .The prevalence of 4AVB was 14%, and may be underestimated. PD-AVB episodes were more likely associated with syncope in comparison with patients without PD-AVB. These patients may elicit multiple triggers of PD-AVB.