Human T-Cell Lymphotropic Viruses (HTLV) type 1 and type 2 account for an estimated 5 to 10 million infections worldwide and are transmitted through breast feeding, sexual contacts and contaminated cellular blood components. HTLV-associated syndromes are considered as neglected diseases for which there are no vaccines or therapies available, making it particularly important to ensure the best possible diagnosis to enable proper counselling of infected persons and avoid secondary transmission. Although high quality antibody screening assays are available, currently available confirmatory tests are costly and have variable performance, with high rates of indeterminate and non-typable results reported in many regions of the world. The objective of this project was to develop and validate a new high-performance multiplex immunoassay for confirmation and discrimination of HTLV-1 and HTLV-2 strains.The multiplex platform was used first as a tool to identify suitable antigens and in a second step for assay development. With data generated on over 400 HTLV-positive blood donors sourced from USA and French blood banks, we developed and validated a high-precision interpretation algorithm. The Multi-HTLV assay demonstrated very high performance for confirmation and strain discrimination with 100% sensitivity, 98.1% specificity and 100% of typing accuracy in validation samples. The assay can be interpreted either visually or automatically with a colorimetric image reader and custom algorithm, providing highly reliable results.The newly developed Multi-HTLV is very competitive with currently used confirmatory assays and reduces considerably the number of indeterminate results. The multiparametric nature of the assay opens new avenues to study specific serological signatures of each patient, follow the evolution of infection, and explore utility for HTLV disease prognosis. Improving HTLV diagnostic testing will be critical to reduce transmission and to improve monitoring of seropositive patients. HTLV viruses are responsible for more than 10 million cases of infection worldwide. The infection is considered as a neglected disease due to lack of vaccines and treatments. Accurate diagnosis is crucial for counselling infected persons and prevention of secondary transmissions. In spite of the development of excellent serological screening assays, many cases of indeterminate and untyped results are still regularly reported and their infection status remain uncertain. To address the need of more precise diagnosis, we have developed a new cutting-edge in-vitro diagnostic confirmation test, named Multi-HTLV, which has been validated on a large panel of HTLV samples. The test is a multiplex immunoassay allowing powerful detection of antibodies against HTLV through combination of a set of selective and validated virus-specific antigens in a blood sample. The Multi-HTLV assay increases the reliability of HTLV diagnostics and strain typing thanks to a high precision mathematical algorithm. Human T-cell lymphotropic viruses (HTLV) were the first human retroviruses to be discovered. They cause blood infections through three transmission routes: mother-to-child transmission (mainly associated with prolonged breast-feeding), sexual transmission and contaminated blood products (blood recipients and drug users) . HTLVs aGag and Env proteins derived from viral lysates or recombinant proteins. Many studies have revealed shortcomings of the WB technique. Indeed, in all regions of the world and specially in high risk populations, a high number of indeterminate and non-typable results have been found [HTLV-1 is divided into four subtypes and circulates mainly in Japan, the Caribbean, West and Central Africa, South America and Australo-Melanesia . HTLV-1-en found ,21. The en found . Some coen found ,23. Hoween found . Therefoen found ,26. The en found ,27,28. Ten found ,26,29. Hen found . Furtheren found ,30. Acceen found .The aim of this study was to develop a new high performing and cost-effective multiplex test to confirm HTLV infection and discriminate the two strains, HTLV-1 and HTLV-2. We used a microplate-based multiplex platform to identify the most relevant antigens (recombinant proteins and synthetic peptides) and developed a readable and easy-to-use assay. The developed assay with updated high-performance antigens offers improved confirmation of HTLV infection and can be performed in routine laboratories with standard lab equipment.The methods section was described in accordance with the Standards for Reporting of Diagnostic Accuracy Studies (STARD) protocol .Written informed consent was obtained from all participants in the United States-based cohort HTLV Outcomes Study (HOST), including use of frozen biospecimens for future research. The study was approved by the University of California San Francisco Committee on Human Research (IRB#s H5901-14117-13D & 11–05747) and other institutional review boards. Written informed consent was obtained by EFS from blood donors after a detailed explanation of how their donation would be used for research purposes.For this study, different sample collections were used .This prospective study was conducted with samples from the United States-based cohort HTLV Outcomes Study (HOST) which includes samples from subjects recruited from five major US blood donation centers ,32. BaseFor the current study, 694 frozen serum specimens were obtained from the cohort repository. We excluded from the study three samples because of labeling errors and two samples because of mismatches, which could lead to misinterpretation. A total of 689 samples from 397 participants were therefore eligible for the analysis. Among them, 60 HTLV-positive patients were followed over time in a longitudinal study and tested to analyze the evolution of their serological profile. However, in order to have independent data for each HOST participant, we included in the study only the first sample tested of each participant, more specifically, 199 HTLV-positive samples from 199 participants (100 HTLV-1 from 100 patients and 99 HTLV-2 from 99 patients) and 198 negative samples from 198 participants .We obtained 140 positive samples (133 HTLV-1 and 7 HTLV-2) and 28 seronegative samples from different French blood donations (transfer agreement 20120224). Samples were collected between 2008 and 2013, and screening was performed by ELISA, using either Prism HTLV-I/HTLV-II or Architect rHTLV-I/II assay , and then HTLV positive samples were confirmed and typed by INNO-LIA HTLV-I/II.We obtained 65 HTLV-2 samples from a commercial source. All patients came from USA and had available data on ethnicity and ages. Samples were collected between 2013 and 2016, screened with an HTLV-1/2 ELISA (brand not specified by Trans-Hit Bio) and subsequently confirmed as HTLV-2 positive by WB (brand not specified by Trans-Hit Bio).The multiplex technology allows multiple parameters to be combined in a single well of a 96-well microplate. The printing process is based on non-contact piezo electric impulsion of a defined volume of an antigenic solution. We used the SciFLEXARRAYER printing system  to print HTLV antigens, at the bottom of each well at precise X-Y coordinates, under controlled humidity and temperature parameters. This technology was used first to make a selection of the most relevant antigens and second for the design and production of the assay, which has already been used in this manner for the development of other confirmatory test for Chagas disease . Each anenv GP21, env GP46 and gag P19; and (ii) three discriminatory antigen spots derived from type-specific immunodominant epitopes of HTLV-1 env GP46, HTLV-1 gag P19 and HTLV-2 env GP46. In contrast to other confirmatory immunoassays, we excluded the detection of anti-P24 antibodies in the Multi-HTLV test because it has been shown in WB assays that indeterminate samples frequently show an isolated P24 band attributable to false reactivity [www.uniprot.org (The Multi-HTLV assay is composed of (i) three confirmatory antigen spots for binding specific HTLV-1 and HTLV-2 antibodies and derived from common-type and type-specific immunodominant epitopes of activity . Relevanprot.org .Each antigen composition was individually optimized to obtain the best performance in terms of sensitivity and specificity, and by calculating the areas under the Receiver Operating Characteristic curve (AUC), using SPSS Statistics software . This optimization step was performed by using a panel of HTLV-infected and seronegative samples from EFS. Further, three positive control (PC) spots were printed in each well to define a precise spatial orientation pattern and to validate the correct sequential addition of all reagents . To facilitate the visual interpretation at the end of the test, two additional control “low”- and “medium”-spots–composed of anti-human-IgG antibodies–were printed and indicate the "minimal" or "intermediate" reactivity for each patient .Following the spotting process, the plates were incubated overnight to allow coating of the antigens at optimal conditions of humidity and temperature. Then, they were dried at 37°C for 2 hrs and washed with PBS-T . A blocking solution containing Bovine Serum Albumin (BSA) was added into the microplate for 2 hrs at room temperature (RT) to avoid nonspecific binding during the testing process. After the saturation step, the microplates were then washed with PBS-T. For long term storage, the blocked microplates were dried at 37°C during 2 hrs, then stored at 4°C in sealed plastic bags with desiccants.The Multi-HTLV test is performed very similarly to a standard ELISA assay enabling simultaneous detection of circulating antibodies to a set of selective and validated antigens. Hundred μL of diluted patient samples in PBS-T  and BSA (1%) (1:50) was added to each well, then microplate was incubated for 1 hr at RT. After three washing steps with PSB-T, 100 μL of anti-human-IgG secondary antibody conjugated to Horseradish Peroxidase, diluted in a defined volume of PBS-T  and BSA (1%) buffer (1:2000), was added and the microplate incubated for 1 hr at RT. To remove unbound secondary antibodies the microplate was washed three times with PSB-T. For the detection of conjugated enzyme activity, a precipitating substrate solution  was added and the microplate incubated in the dark for 20 min at RT. Upon contact of the substrate with the enzyme, it is converted to an insoluble permanent dark-blue precipitate. The reaction was stopped by removing the substrate solution, and drying the microplates at 37°C for 10 min. Each plate was read and analyzed both visually and using a specific microarray reader sciREADER CL2  which acquires high-resolution digital images and an integrated software calculates the pixel intensity for each spot. We calculated the mean value of the duplicated spots to get the net intensity of each antigen. The data were incorporated into a Microsoft Excel 2019 database and analyzed using a specific Multi-HTLV interpretation algorithm to determine the final result .Data from three cohorts  were pooled. Gold standard HTLV type assignments were available for the HOST cohort samples by either PCR or type specific ELISA and INNO-LIA results. For the two other studies, sample type assignment was only available based on INNO-LIA test results. The pooled dataset was split into a development and validation set. All data from EFS and Trans-Hit Bio were allocated to the development set and the HOST samples were allocated randomly (p = 0.5) to either the development or the validation dataset. Only HOST samples were included in the validation set because of PCR-based HTLV classifications. PCR has high confirmation power and strain discrimination, which in turn leads to more accurate validation analysis. The validation set consisted of 96 positive samples (53 HTLV-1 and 43 HTLV-2) and 103 negative samples.The addition of EFS and Trans-Hit Bio data to HOST data for the development set allowed a larger sample size, which is beneficial for statistical methods and machine learning algorithms. It comprised 308 positive samples (180 HTLV-1 and 128 HTLV-2 samples) and 123 negative samples. For both HTLV detection and typing, a logistic regression (LR) model was trained. Outliers were expected in the development set due to possible imperfect INNO-LIA labeling of EFS and Trans-Hit Bio samples. Sample deviances were used to detect outliers, which were omitted if the human interpretation of the Multi-HTLV assay did not match the INNO-LIA label. Due to the gold standard labeling of HOST samples, only samples with deviances higher than the maximum deviance of HOST samples were checked. Deviances below this threshold were expected to arise from regular variability but not necessarily from mislabeling.arm package (version 1.11–2) [Logistic regression was used as a baseline positive/negative classification method using GP21-I/II (Confirmation), P19-I/II (Confirmation), GP46-I/II (Confirmation), P19-I, GP46-I and GP46-II as independent variables (features). To prevent (quasi-) complete separation problems, Bayesian analysis with non-informative prior assumptions was applied, by use of the R  1.11–2) . A backw 1.11–2) . A RandoThen, we developed an algorithm based first on counting the number of positive antigens per sample and then applying logistic regression formulas. The algorithm is as follows: if the number of confirmatory antigens is zero, then the sample is directly considered to be negative; if the number of antigens is equal or higher than one, then a first logistic regression formula for confirmation is applied. It permits classification of samples with one reactive antigen among negative (LR probability (p)<0,5) or indeterminate categories , and samples with number of antigens higher than one in indeterminate  or positive  categories. Thereafter, only positive samples are typed as HTLV-1 and/or HTLV-2 by applying a second logistic regression formula . The IntAfter application of the interpretation algorithm, sensitivity  and specificity  with corresponding confidential interval (CI) of 95%, were used for diagnostic accuracy and performance evaluations of the Multi-HTLV assay. Indeterminate results obtained were included for the calculation of sensitivity and specificity. In this case, a PCR-negative sample that was indeterminate with Multi-HTLV, was treated as a false positive. Conversely, a PCR-positive sample that was indeterminate, it was counted as a false negative.First, we evaluated the Multi-HTLV confirmatory assay by analyzing the immunoreactivity of each antigenic marker for all tested patients. By visual analysis of images recorded with a specific image reader, we could clearly separate the majority of HTLV negative and positive results. Next, a mathematical algorithm was developed to facilitate a faster and accurate analysis of the confirmation and typing results. In addition, the algorithm enables precise determination of atypical profiles, for which visual interpretation is more complicated . The traBy analyzing the classification of the samples compared to the PCR gold standard, the specificity did not attain optimal performance as two negative samples in each set were classified in the indeterminate category .To further investigate the 4 indeterminate samples obtained, we looked at their serological profiles . In bothTyping performance showed a strain specificity of 99,03%  for the development set and 100%  for the validation set. In comparison, the INNO-LIA assay has a strain specificity of 94.17%  for the samples in the development set and 95.83%  for the samples in the validation set. Among these untyped or falsely typed samples with INNO-LIA, there was a higher proportion of HTLV-2 PCR samples (n = 7/10) .In our study, 3 HTLV-1/HCV and 12 HTLV-2/HCV co-infected samples from the HOST cohort were also included. Importantly, all these samples were correctly confirmed and typed with Multi-HTLV . We noteThen, we studied the creation of a predictive model for proviral load depending on the serological profile of HTLV positive patients. In 246 HTLV-1 and 245 HTLV-2 samples from the entire HOST cohort, linear and rank correlations between intensities of each antigen of the Multi-HTLV and proviral load were highly significant. Based on these observations, two multivariable models (one for HTLV-1 and one for HTLV-2) were created using the logarithm of the proviral load (log (PVL)) as dependent variable and the antigen intensities as independent variables. Models showed linear Pearson’s correlation coefficients of 0.56 and 0.54 respectively, between observed and predicted proviral load . AlthougFinally, we examined longitudinal patient samples from the HOST cohort using the Multi-HTLV assay. We observed in 35 of 60 patients a certain dynamic in antibody levels between the initial donation and 10–11 years of follow-up. These changes were sometimes visible on the INNO-LIA assay, but measuring the intensities of individual spots with the Multiplex reader provides more accurate values and facilitates visualization of the variations in serological signatures ; A. HoweHere we have developed an innovative and high-performance Multi-HTLV assay solving the major limitations associated with the currently used WB confirmatory test. The multiplex assay allows monitoring of different serological profiles for each patient first by visual analysis, and second through a combination of highly specific synthetic antigens as well as a custom-designed high-precision algorithm that ensures the most reliable confirmatory and typing results. The multiplex technology can be performed in any routine laboratory with simple equipment and a small sample volume.This study targets an important medical need which is to develop a cutting-edge diagnostic tool to improve counselling and reduce viral transmission of HTLV. Over the past 30 years and since the development of the INNO-LIA assay, no significant progress has been made to further improve HTLV diagnostics, thus the current strategy is to simply select the best screening method ,39. Our Despite the imperfect classification of PCR-negative samples with Multi-HTLV, it is preferable to categorize them as indeterminate rather than giving an incorrect result, as they may be subject to further investigation to better understand their status. Furthermore, it would be important to test more co-infected samples and in particular HIV/HTLV samples, which currently cause many difficulties .The Multi-HTLV assay is thus not only a useful tool to accurately diagnose HTLV infection but also a good tool to understand indeterminate samples, which represent a major challenge in HTLV diagnostics, particularly in endemic areas . Indeed,Plasmodium falciparum [gag as well as an Env antigen. The multiparametric aspect of the Multi-HTLV assay and its associated confirmation algorithm takes into account the acquisition of at least two reactivities to highly sensitive and specific Env and Gag antigens and therefore enhances the analysis and provides a more reliable diagnostic of HTLV infection.In our study, we also obtained indeterminate results for PCR-negative blood donors, due to an isolated GP21-I/II. Unfortunately, these individuals were not tested for proviral load and follow-up sampling was not performed, making it difficult to understand whether they represent seroconverting cases. For the other indeterminate results, we obtained intensities on antigens other than GP21-I/II that are very close to the minimal intensity value. It is possible that these intensities would vary when the test is repeated and that the final result may change. In a future study, it would be important to track individuals with similar profiles to better understand the role of seroconversion or cross-reactivity. It is also interesting to note, that several studies have identified two serological profiles of WB indeterminate subjects from African and Caribbean populations, called "N" and "HGIP". However, INNO-LIA and PCR tests revealed that these reactivities were probably not caused by HTLV-1 infection but were likely due to cross-reactivity with antigens of other microbial agents, in particular the parasite lciparum ,43. ThesOur study also shows that typing of HTLV strains using the Multi-HTLV assay shows promise. Only one sample in the development set was not correctly classified. The proposed method shows higher performance with regard to HTLV type assignment as compared to the INNO-LIA. This would be necessary due to the appearance of new strains in certain endemic areas and in high-risk populations. It is also interesting to note that in our study, INNO-LIA non- or falsely-typed results were related to the HTLV-2 strain. These observations are reminiscent of reported problems with WB, which lacks typing sensitivity particularly for HTLV-2 strains . For INNAccuracy in distinguishing HTLV strains remains important for prognosis of associated diseases and in cases of some co-infections . For exaenv and proviral DNA levels in HTLV-1 has been observed in patients who develop HAM/TSP compared to asymptomatic patients [Another medical need related to HTLV infection concerns the understanding of associated diseases because currently no vaccine is available and treatment options for HAM/TSP and ATLL are inadequate ,46. The patients . Howeverpatients . With thIn summary, our data demonstrate that the Multi-HTLV assay is a novel and reliable assay to detect and type HTLV-infected patients, as well as a research and discovery tool to perform serological profile studies, as recently performed for Chagas disease ,50. SincS1 Appendix(DOCX)Click here for additional data file.S2 Appendix(DOCX)Click here for additional data file.S3 AppendixIn a very few cases, sample can be declared weakly positive by visual analysis, but correctly classified as indetermined with the algorithm. In other cases, samples can be declared both HTLV-1 and HTLV-2 or untyped, but the specific serological profile of the respective type was recognised by the algorithm which assigns the correct result.(PDF)Click here for additional data file.S4 Appendix(A) Antigen reactivities obtained with Multi-HTLV and INNO-LIA assays for one HTLV-1 & two HTLV-2 patients. (B) Plots showing comparative evolution of proviral load and the sum of 6 antigens intensities measured by Multi-HTLV for one HTLV-1 and two HTLV-2 patients over 10–11 years.(PDF)Click here for additional data file.


Urolithin A (UA) is produced by gut microflora from foods rich in ellagitannins. UA has been shown to improve mitochondrial health preclinically and in humans. Not everyone has a microbiome capable of producing UA, making supplementation with UA an appealing strategy.n = 100 participants) and to characterize differences in gut microbiome between UA producers from non-producers.This is the first detailed investigation of the prevalence of UA producers in a healthy population and the ability of direct UA supplementation to overcome both microbiome and dietary variability. Dietary intake of a glass of pomegranate juice (PJ) was used to assess UA producer status (Subjects were randomized (1:1) to either PJ or a food product containing UA (500 mg). Prevalence of UA producers and non-producers were determined in the PJ group. Diet questionnaires and fecal samples were collected to compare differences between UA producers and non-producers along with plasma samples at different time points to assess levels of UA and its conjugates between the interventions.p < 0.0001).Only 12% of subjects had detectable levels of UA at baseline. Following PJ intake ~40% of the subjects converted significantly the precursor compounds into UA. UA producers were distinguished by a significantly higher gut microbiome diversity and ratio of Firmicutes to Bacteroides. Direct supplementation with UA significantly increased plasma levels and provided a >6-fold exposure to UA vs. PJ (Differences in gut microbiome and diet that dictate natural exposure to UA can be overcome via direct dietary UA supplementation. Urolithin A (UA) is produced endogenously by human gut bacteria exposed to dietary polyphenolic compounds that include ellagic acid (EA) and ellagitannins (ET), such as punicalagin . These pUA has previously been shown to activate mitophagy, the recycling of defective cellular mitochondria, and to improve mitochondrial health in pre-clinical models of aging  and in aA few interventional studies have investigated the presence of urolithin metabolites after intake of dietary fruits (strawberry and raspberry) and nuts  in different study populations , 12. InvThis report details the results from a clinical study in American adults (aged 20–80 years) that estimates the relative prevalence of UA producers and non-producers following a standardized dietary challenge with PJ. Metagenomic profiles of the fecal samples of volunteers distinguish the gut microbiome of UA producers from non-producers. The primary objective of the study was to compare the levels of UA and its conjugates (UA glucuronide being the main metabolite) in the circulation over time during a randomized, crossover study in which participants were orally administered either 100% PJ containing a natural profile of UA precursor compounds to a nutritional product containing a calibrated dose of 500 mg of UA. The rationale of doing such a comparison was to document the variability and low levels of UA observed via natural exposure to diet rich in precursors vs. the uniform and consistent exposure obtained via direct supplementation with UA.The trial was a single-center, two-period, crossover, randomized, open label study. It was approved by an independent institutional review board  to be conducted at a clinical research site  and is registered as NCT04160312. Volunteers were recruited (from October 2019 to January 2020) through the clinical site database, social media platforms, general practitioners’ offices, and advertisements in local newspapers. From 136 screened participants, 100 successfully screened subjects were randomized on site to either the PJ or Mitopure intervention group Fig. . All subTo be eligible for inclusion, subjects needed to be in general good health defined as the absence of any chronic ailments and as determined by the study medical doctor, and to have given voluntary, written, informed consent to participate. Subjects also needed to be willing to consume the investigational products, complete screening questionnaires, and complete all clinic visits during the study. Subjects were excluded if they had presence of any chronic medical conditions and clinically significant abnormal laboratory results at screening. Individuals deemed to be cognitively impaired and unable to give informed consent were excluded as well. Women who were pregnant, breastfeeding, or planning to become pregnant during the course of the trial, and subjects who had taken antibiotics within the previous 30 days of the study screening, were also excluded. Subjects were instructed to follow their usual diet and exercise routine and to not consume any other dietary supplements that could interfere with the assessment of the study products for the duration of the study. Subjects completed a food-frequency questionnaire (FFQ) during the screening visit to assess their dietary intake. The FFQ used for this purpose was the DHQ-III .Group 1: Mitopure – Pomegranate JuiceGroup 2: Pomegranate Juice – MitopureSubjects who successfully passed the screening procedures were randomized on a 1:1 basis to receive either Mitopure (500 mg UA) oral supplementation or 8 fl. oz (~240 mL) of PJ. Natural precursors to UA  and UA levels were assessed in each batch of the PJ and Mitopure administered using calibrated and validated HPLC methods. Randomization was carried out by a study-independent statistician using a computer-generated program to create a block randomization list. Each subject was assigned a unique randomization number. Participants were randomized to one of the following sequence groups as follows:The investigation products were the Mitopure powder, a fruit-flavored powder containing 500 mg of pure UA and ingredients that include blueberry, raspberry fruit powder, pomegranate natural flavoring, and fiber and 8 fl. oz (~240 ml) of a commercially available 100% PJ . Intake happened onsite and was supervised by the research staff to ensure 100% compliance. Mitopure powder was admixed into a vanilla-flavored commercial yogurt (4 fl. oz) and the PJ was served chilled. Participants were asked to consume the investigational product within 10 min. The time of the first sip of PJ and spoonful of yogurt intake containing admixed Mitopure was recorded as the time of product ingestion in the study database.Plasma and dried blood spot (DBS) samples were collected from all subjects at baseline (T0), at 6 h (T6), and 24 h (T24) after the intake. During the screening visit, approximately 16 mL of blood was collected to assess clinical, laboratory, and hematology parameters. Following that, at each visit and time point, an 8 mL blood sample was drawn into K2-EDTA coated tubes  over different batches. Each batch included eight calibration samples, at least two blank samples, one double blank plasma sample, as well as three different quality control samples in duplicate. The inter-batch precision was between 3.1 and 12.3%, whereas the inter-batch accuracy was in the range from 96.6 to 99.3% of nominal concentration. Measurements were performed according to the FDA Guidance for Industry and EMA guidelines on bioanalytical method validation as previously described . The quaValidated HPLC analytical methods were used to detect EA and punicalagin. For determination of the parent UA levels in commercial grade food products and dietary supplements, a validated HPLC analytical method was developed with a limit of quantification of 1.17 ng/ml. The quantification was performed by column separation with reversed phase liquid chromatography followed by detection with a diode array detector.® 96 Soil kit (Macherey Nagel). A minimum of one negative control was included per batch of samples from the DNA extraction and throughout the laboratory process (including sequencing). A ZymoBIOMICS™ Microbial Community Standard (Zymo Research) was also included in the analysis as a positive (mock) control. Before sequencing, the quality of the DNA samples was evaluated using agarose gel electrophoresis, and the quantity of the DNA was evaluated by Qubit 2.0 fluorometer quantitation. The prepared DNA libraries were evaluated using Qubit 2.0 fluorometer quantitation and Agilent 2100 Bioanalyzer for the fragment size distribution. Quantitative real-time PCR was used to determine the concentration of the final library before sequencing. The library was sequenced using 2 × 150 bp paired-end sequencing on an Illumina platform. A total of 99 fecal samples were sequenced to an average depth of 19.9 M read pairs (Illumina 2 × 150 PE) per sample. On average, 96.5% of the high-quality microbiome reads from a sample were mapped to a reference human gut gene catalog, and on average 200 metagenomic species (MGS) were detected per sample. Shotgun sequencing was successful for all samples, and metagenomic analysis was performed to provide taxonomical and functional abundance profiles. For MGS abundance profiling, a set of 1273 MGS, which have highly coherent abundance and base composition in a set of 1776 independent reference human gut samples, was detected. The analysis is based on the MGS concept [U test. To assess differences in the overall species community composition between the study groups (beta diversity), we used permutational multivariate analysis of variance (PERMANOVA) with 1000 permutations. When performing statistical testing on multiple hypotheses, the Benjamini–Hochberg (BH) method was used to control the false discovery rate (FDR) at a level of 10%.DNA was extracted from ~0.1 g aliquots of the fecal samples using the NucleoSpin concept . To taxoDuring the study, all adverse events (AEs) reported by the subject were documented with respect to onset, severity, resolution, and relatedness to the investigational products by a qualified medical investigator. All AEs occurring during the conduct of the clinical study were recorded in the electronic Case Report Form. All AEs coded via MedDRA and recorded during the study are reported in Supplementary Table t-test was used to determine if there was a difference in AUC between Mitopure and PJ supplementation for UA glucuronide, UA sulfate, and parent UA. For microbiome sequencing results, statistical comparisons between groups were performed using the two-sided Mann–Whitney U test. To assess differences in the overall species community composition between the study groups (beta diversity), PERMANOVA was used. When performing statistical testing on multiple hypotheses, the study used the BH method to control the FDR at a level of 10%.An independent statistician performed sample size estimates. The study was powered on the primary objective of showing superiority of UA supplementation to PJ consumption to increase UA levels from baseline. With a sample size of 100, the estimated power to detect a 3.5 times higher UA concentration after UA supplementation as compared with PJ consumption was set at 80%. Following the ICH guideline E9, a blind data review meeting was held prior to locking the study database to define the Intent-to-treat (ITT) and Per-Protocol (PP) statistical populations. ITT population was defined as all randomized participants with at least one consumption of either placebo or treatment product. PP population was defined as all randomized participants with complete data for the primary outcome and at least 95% compliance in terms of amount of product consumed with no major protocol deviations as agreed during the blind data review. Participants who dropped out prior to receiving any treatment were excluded. The primary endpoint was the absolute change in UA glucuronide plasma levels from T0 to T24 in the UA group as compared with the PJ group. Primary outcome data were assessed to identify any confounders and carry-over effects from period 1 to period 2 of the trial. If there were no confounders, repeated measures ANOVA was used to detect any difference in UA glucuronide plasma levels between active (Mitopure) and comparator (PJ) over time. Absolute change was measured by comparing UA glucuronide plasma levels at the two time points. For the area under the curve (AUC) analysis, unpaired To identify the most suitable interventional product that contained dietary precursors and would lead to the generation of UA, the study measured the levels of UA precursors  in the matrix of several commercial grade pomegranate extract dietary supplements and juice-based products  and high producers (p = 0.001), as shown by the clear segregation of the groups. No clear separation and an overlap were observed between the groups of low producer and high producer. In summary, the alpha and beta diversity results indicate that the ability to convert UA from its precursors is significantly associated with a higher microbiome richness and overall composition.Shotgun sequencing determined microbiome composition from the fecal samples of individuals producing UA at different levels following PJ intake. This metagenomic analysis compared the abundance of MGS and genes in subjects from the no, low, and high producer groups. Metagenomic profiles determined the microbiome alpha diversity, which indicates variation of microbes in a single sample. Alpha diversity was assessed both as microbiome richness (number of species or genes observed in a sample) and microbiome variability, and was quantified using the Shannon index . The ShaClostridiales and Ruminococcaceae family was found in the high UA producer group. Of note was also the increased abundance of Akkermansia muciniphilia in the microbial high UA producer group compared to the low and non-producers  compared with non-producers. At the phylum level, abundancy of Firmicutes (F) and Bacteroidetes (B) was assessed, as increased F/B ratio is associated with several markers of gut and organismal health –20. Bothp < 0.0001; 2.4-fold higher mean level) with Mitopure supplementation compared with drinking PJ  as assessed by the 6 and 24 h time points  mean UA glucuronide levels were 5.48 ± 19.97 ng/mL, slightly increasing to 12.84 ± 36.34 ng/mL at the 6 h time point and rising to 110.47 ± 131.6 ng/mL 24 h following PJ intake. In comparison, during Mitopure supplementation, the mean baseline (T0) levels of UA glucuronide were 9.57 ± 47.78 ng/mL; at 6 h UA glucuronide concentration peaked in circulation to 480.75 ± 238.03 ng/mL and then declined to 255.52 ± 129.38 ng/mL 24 h after Mitopure intake  than high producers . Equol is a metabolite generated when gut microflora metabolizes dietary isoflavones such as daidzein , 29. Stup ≤ 0.0001) higher iAUC to UA glucuronide when compared with PJ  of PJ that contained the necessary dietary precursors. The results observed pose an important question in terms of nutritional practices: is an optimal diet sufficient by itself? And how can someone know whether they are likely to harness the appropriate nutrients from the diet via their gut microbiome? Equally important would be to understand better how to harness the various gut microbiome species that would confer the UA producer capacity to an individual, and measure precisely if one was a natural UA producer or not with minimally invasive methods. Advanced nutrition approaches that allow the delivery of a nutritional bioactive such as UA in a calibrated manner will likely play a key role in filling the gap created by the natural heterogeneity of the gut microbiome to deliver health benefits.Supplementary Figures and Tables


Objective: This study aimed to evaluate the potential association between uric acid (UA) lowering and cardiovascular risk reduction among UA-lowering therapies in adults.Methods: A systematic search for randomized controlled trials (RCTs) was conducted according to the protocol pre-registered in PROSPERO (No. CRD42020199259). We search for RCTs in PubMed, Embase, Web of Science, the Cochrane Library, and ClinicalTrials.gov up to July 1, 2020. A meta-analysis was performed using a fixed- or random-effects model.Results: In total, 30 studies involving 18,585 hyperuricaemic patients were included. Xanthine oxidase inhibitor (XOI) therapy produced a 6.0% reduction in relative risk (RR) for major adverse cardiovascular events (MACEs). The use of febuxostat was associated with a higher risk of cardiovascular events (CVEs) , but the difference was not statistically significant. Allopurinol treatment was associated with a lower CVE risk . Among the UA-lowering therapies, the drug treatments were associated with all-cause mortality . The subgroup with a UA endpoint <7 mg/dl was not associated with a higher CVE risk , and in the subgroup with a UA endpoint <5 mg/dl group, a lower risk of CVEs was not observed .Conclusions: UA reduction caused by XOIs reduced the incidence of MACEs. UA-lowering medicines were associated with changes in all-cause mortality but not cardiovascular outcomes. The lower UA endpoint was not associated with reduced cardiovascular risk. UA is a widely accepted risk factor for cardiovascular disease (CVD) as demonstrated by epidemiological and Mendelian randomization studies , 2. The Hyperuricemia is defined as serum UA levels >7.0 mg/dl in men and >5.7 mg/dl in women; the therapeutic target for gout is <6.0 mg/dl . HoweverTherefore, we performed this study to further explore the relationship between UA-lowering therapies and cardiovascular risk. The main purpose of this meta-analysis was to evaluate the association between UA lowering and cardiovascular risk reduction using different UA-lowering agents and to determine whether a cut-off value for the greatest cardiovascular benefit exists. We also discussed the effects of different UA-lowering agents on cardiovascular outcomes and mortality in patients with hyperuricaemia.ClinicalTrials.gov, published from inception to July 1, 2020, were considered. We read the references of the included studies to identify additional relevant articles. The primary search terms we used included the following: (hyperuricemia) or (uric acid-lowering) and (xanthine oxidase inhibitors or uricosurics or uricases or purine nucleotide phosphorylase inhibitors) and  and . The specific search strategies are shown in This study was conducted according to a pre-specified protocol registered at the International Prospective Register of Systemic Reviews (CRD42020199259). Relevant randomized controlled trials (RCTs) in databases, including PubMed, Embase, Web of Science, the Cochrane Library, and We used the following prespecified inclusion and exclusion criteria for the trials. We included trials if they were (1) RCTs that included more than 50 patients who received UA-lowering medicines, with at least a 4-weeks follow-up period; (2) phase II-IV clinical trials in which the participants were adults (age>18 years); and (3) clinical trials that reported the serum UA levels and clinical outcomes. The clinical outcomes included CVD morbidity and mortality, hospitalization, and all-cause mortality. We excluded trials in which (1) the main pharmacological effects of the drugs were not UA lowering and the drugs had certain effects on cardiovascular outcomes ; (2) the enrolled participants did not suffer life-threatening diseases ; and (3) the studies involved benzbromarone, due to serious adverse effects, including liver dysfunction.As shown in In our study, the cardiovascular outcomes were divided into two categories, including major adverse cardiovascular events (MACEs) and cardiovascular events (CVEs). We defined MACEs as cardiovascular death, non-fatal myocardial infarction, and non-fatal stroke . The numWe defined intervention group as a more potent pharmacological UA-lowering strategy, whereas control group which regard as a less intensive UA- lowering therapy corresponded to placebo/usual care or the active control group of the trial. Among febuxostat studies, we defined allopurinol treatments or placebo as control groups. Among allopurinol studies, treatments with placebo were considered as control groups. Among more potent pharmacological agents like lesinurad, verinurad, oxypurinol, and pegloticase, treatments with febuxostat or allopurinol were used as control groups.Two independent reviewers used the Cochrane Risk-of-Bias Assessment Tool to assess the potential risk of bias in all RCTs. If disagreement occurred, arbitration by another person was sought.The data were analyzed according to the intention-to-treat principle, and the results are presented as a fitted line in a meta-regression and as an RR with a 95% confidence interval (CI). Regarding the cardiovascular and clinical outcomes, we performed a meta-analysis using a fixed-effects or random-effects Mantel-Haenszel model according to the level of heterogeneity. Meta-regression analyses were used to evaluate the association between UA reduction and cardiovascular risk. These analyses were conducted using random-effects models with the restricted maximum likelihood estimation. The Knapp and Hartung adjustments were applied to estimate the standard errors of the estimated coefficients to calculate the summary effect estimates. Regrading to cut-off values for the cardiovascular benefits, we conducted subgroup analyses by categorizing the endpoint level of serum UA. Sensitivity analyses were also performed, including a meta-analysis performed by the “leave-one-out” method, analyses of studies with subjects with cardiovascular comorbidities, analyses of high-quality studies with sample sizes ≥200 and follow-up periods ≥24 weeks, and analyses of hyperuricaemic subjects with and without symptoms.I2 more than 50%); otherwise, a fixed-effect model was used. Publication bias was assessed using funnel plots, and Egger's weighted regression was used to test for asymmetry. The statistical analysis was performed by Metafor package version 2.40  and RevMan version 5.3 (Cochrane Collaboration).In our study, the between-group heterogeneity was quantified using the Cochrane Q statistics, with the values of 25, 50, and 75% representing low, moderate, and high degrees of heterogeneity, respectively. We used a random-effects model if there was considerable level of heterogeneity , 7 trials involving allopurinol , and 2 studies involving verinurad (375 patients) simultaneously reported the serum UA level and cardiovascular outcomes. Regarding MACEs as cardiovascular outcomes, of the 19 XOIs included in the trials, each 1 mg/dl (59.52 μmol/l) reduction in the UA level was associated with an average 6.0% CV risk reduction , 7 trials involving allopurinol , 4 studies involving lesinurad , 2 studies involving verinurad (375 patients), 1 study involving oxypurinol (405 patients), and 1 study involving pegloticase (212 patients) that reported the cardiovascular outcomes. The results were divided into the following two categories: MACEs and CVEs. Compared with the control groups, the groups treated with different UA-lowering medicines were not associated with greater incidences of MACEs . No difference was found between the groups treated with UA-lowering medicines and the control groups in terms of heart failure hospitalization  or all-cause hospitalization . The results of clinic outcomes are shown in In total, 7 trials involving febuxostat , 4 trials involving allopurinol (544 patients), 1 study involving lesinurad (324 patients), 1 study involving verinurad (204 patients), and 1 study involving oxypurinol (405 patients) that reported the clinical outcomes. The clinical outcomes included the following three components: all-cause mortality , heart fI2 = 0.0%, P = 0.06), whereas in the subgroups with a serum UA endpoint <5 mg/dl, the intervention group did not show a lower risk of MACEs . Similarly, as presented in I2 = 0.0%, P = 0.02), and in the subgroups with a serum UA endpoint <5 mg/dl, the intervention group did not show a lower risk of CVEs .The analysis of the UA endpoint included 12 trials involving febuxostat , 7 trials involving allopurinol , and 2 studies involving verinurad (375 patients). In our study, the lower UA endpoint was not associated with a lower risk of either MACEs or CVEs. As shown in I2 = 65.0%, P = 0.028, I2 = 57.0%, P = 0.024, We performed sensitivity analyses according to the “leave-one-out” method, analyses of studies with subjects with cardiovascular or cardiorenal comorbidities, and analyses of high-quality studies. As shown in Since the first discovery of the association between UA and cardiovascular risk in 1967 in the Framingham cohort, the importance of UA in CVD has become increasingly evident. The increased serum UA level has a similar prognostic impact in CVE and all-cause mortality, whether caused by increased generation or reduced excretion . SeveralThe present comprehensive study evaluated the association between UA and cardiovascular outcomes. The study was divided into three parts, and each part addressed one clinical conundrum. In the first part, we performed meta-regression analyses to explore the relationship between lowering UA and cardiovascular risk reduction. As shown in I2 = 0.0%, P = 0.56), and the use of allopurinol decreased the CVE risk . The patients receiving UA-lowering agents showed a higher risk of all-cause mortality . However, these differences were not statistically significant. In the sensitivity analyses using by the “leave-one-out” method . Second, the differences in the patients' backgrounds may lead to different cardiovascular outcomes. Some serious non-fatal diseases, including heart failure and chronic kidney diseases, may cause interference effects. Third, several definitions of CVEs were used in the included studies. In our studies, we defined MACEs as cardiovascular death, non-fatal myocardial infarction, and non-fatal stroke; the symptoms of the events are typical and rarely missed. However, according to our definition, CVEs include a larger range of events, and we extracted information according to study outcomes and treatment of emergent adverse events. Non-specific symptoms, including transient ischaemic attack, thrombotic events, and arrhythmia, can easily be neglected. Therefore, the incidence of CVEs may have been underestimated.In our study, the UA reduction caused by XOIs reduces the incidence of MACE but not the incidence of CVEs. The use of UA-lowering medicines was associated with a significant risk of all-cause mortality but not total RR for cardiovascular outcomes. The subgroup analyses provided evidence that febuxostat may be associated with a higher cardiovascular risk and that allopurinol treatments may decrease cardiovascular risk. Regarding the serum UA endpoints, a lower UA endpoint was not associated with reduced cardiovascular risk.The original contributions presented in the study are included in the article/CJ and HYi were involved in the conception and design of the study. HYi and HYu collected the data. HYi, HYu, XT, WG, and RJ analyzed and interpreted the data and performed the meta-analysis. HYi, RJ, and CJ prepared the manuscript. All authors have approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Physiologia Plantarum 157, 264-279). Pyruvate activation kinetics were similar between the two GmAOX2 isoforms but differed substantially from those of GmAOX1, suggesting that selective expression of AOX1 and 2 could determine the level of AOX activity. However, this alone cannot completely explain the differences seen in sub-mitochondrial particles isolated from different legume tissues and possible reasons for this are discussed.Alternative oxidase (AOX) is an important component of the plant respiratory pathway, enabling a route for electrons that bypasses the energy-conserving, ROS-producing complexes of the mitochondrial electron transport chain. Plants contain numerous isoforms of AOX, classified as either AOX1 or AOX2. AOX1 isoforms have received the most attention due to their importance in stress responses across a wide range of species. However, the propensity for at least one isoform of AOX2 to accumulate to very high levels in photosynthetic tissues of all legumes studied to date, suggests that this isoform has specialized roles, but we know little of its properties. Previous studies with sub-mitochondrial particles of soybean cotyledons and roots indicated that differential expression of GmAOX1, GmAOX2A, and GmAOX2D across tissues might confer different activation kinetics with pyruvate. We have investigated this using recombinantly expressed isoforms of soybean AOX in a previously described bacterial system ( It is well documented that the plant mitochondrial electron transport chain (mETC) is branched, with phosphorylating and non-phosphorylating pathways coexisting see . A functin vivo activity, or engagement, of alternative oxidase (AOX) in plants reflects many levels of regulation, including transcriptional and post-transcriptional control. AOX capacity may be limited by protein abundance , at least partly determines AOX activity  contains three AOX genes GmAox1, GmAox2a, and GmAox2d and these are preferentially expressed in different tissues. The most abundant AOX protein of soybean photosynthetic tissues, GmAOX2a, is absent in roots and this pattern of expression is conserved for orthologs in other legumes , GmAox2a , GmAox2d . PCR products were amplified from G. max leaf cDNA, inserted into pGEMtEasy via A-T ligation  then digested and cloned into pET22b(+) using standard restriction enzymes  and T4 ligase . Resultant constructs: pET22b(+)GmAox1, pET22b(+)GmAox2a and pET22b(+)GmAox2d were confirmed via Sanger sequencing , then transformed into E.coli strain BHH8 , a new start codon was introduced via the NdeI restriction site within the F primers. Consequently, the translated peptide sequences contained a methionine that would not typically occur in the mature GmAOX proteins.Mitochondrial target peptide sequences of the  GmAOX2D  were pre GmAOX2D . Primersain BHH8  via heatMethods for bacterial culture, protein synthesis, cell harvesting, and membrane vesicle isolations were carried out as previously described . Protein2). Before beginning the assay, 5 mM DTT, 1 mM KCN and a 10 μl aliquot of inverted membrane vesicles  were added, followed by 10 μl of the “effector.” Final pyruvate concentrations ranged between 0.5 and 5,000 μM. After inserting the lids and equilibrating for 3 min, 2.5 mM NADH was used to initiate the reaction, which was inhibited by 1 mM SHAM after 4 min and the residual rate was allowed to proceed for a further 4 min. Each pyruvate concentration was measured in a separate assay; incremental additions were not possible because the activity of membrane vesicles subsided over time. A control reaction containing no pyruvate was run alongside each assay for the same reason, and results were presented as a percentage activation relative to the control. Prior to each set of assays, both chambers were tested with 5 mM pyruvate to ensure the electrodes yielded equivalent rates.Assays were carried out with a Clark type oxygen electrode , essentially as described by Sauromatum guttatum AOX in mouse , 30% (v/v) glycerol, 5% (v/v) β-mercaptoethanol, 2% (w/v) SDS, and 0.002% (w/v) bromophenol blue] containing 25 mM DTT. After boiling at 95°C for 2 min and centrifuging briefly, a further 25 mM DTT was added to the supernatant. Samples of 2.5 μg equivalent protein were loaded directly onto 10% SDS-PAGE gels and resolved at 60 V for approximately 60 min, before being transferred to a 0.45 μm nitrocellulose membrane . The membrane was incubated with “AOA” antibody raised against in mouse  then a ppost hoc Tukey test.Data were analyzed using SPSS version 25 (IBM). Michaelis Menten constants were determined by non-linear regression. Specific activities of different AOX isoforms were compared using one-way ANOVA with E. coli BHH8 contains a stable, highly reduced ubiquinone pool due to the lack of cytochrome bo quinol oxidase and cytochrome bd-1 quinol oxidase, making it ideal for the investigation of heterologously expressed AOX protein kinetics  were much higher than that for GmAOX1 (4 μM)  . With sa1 (4 μM) , and wit1 (4 μM) , suggest1 (4 μM) .To ensure that enzyme activities were not influenced by reduction state, DTT was included in all isolation solutions and assay mixtures. Monomeric AOX subunits were confirmed using immunoblots from non-reducing SDS-PAGE . GmAOX1,The immunoblots showed that GmAOX1 protein was less abundant in membrane vesicle preparations than were the GmAOX2 isoforms . Such diPYR1/2 for GmAOX2D measured here in bacterial membrane vesicles (40 μM) was strikingly similar to that measured previously in sub-mitochondrial particles from soybean root  was only moderately higher than that of AOX2D (40 μM) in our vesicle system, the KPYR1/2 for AOX activation in cotyledon sub-mitochondrial particles was much less  and have a higher inherent capacity than constitutively expressed isoforms such as GmAOX2A that may carry out more ‘house-keeping’ functions in the tissues in which they are expressed.The fact that GmAOX1 has higher specific activity and requires less pyruvate for its activation, compared to its more highly expressed AOX2 counterparts in soybean, may be significant  legumes . GmAOX2Din planta and how they vary with tissue type and growth conditions. Organic acid concentrations have been estimated in whole tissue extracts from numerous plant species, but for AOX activation it is the intramitochondrial concentrations that are important, and these are very difficult to measure, especially pyruvate given that its import and generation within mitochondria can occur via multiple pathways (in vivo estimation of pyruvate levels (in situ biosynthesis (While it is clear that different AOX isoforms have tissue-specific expression eg , differepathways . The adve levels  may be oynthesis .This article is dedicated to the memory of Jim Siedow, a pioneer of the field of plant respiration, a highly valued colleague, and a very good friend: DD.The original contributions presented in the study are included in the article/DD conceived the project. CS, JS, and TM designed and carried out all experimental procedures. JW contributed to experimental design and provided facilities and guidance. CS drafted the manuscript. JS, JW, and DD edited the manuscript. All authors approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The Pellino family is a novel and well-conserved E3 ubiquitin ligase family and consists of Pellino1, Pellino2, and Pellino3. Each family member exhibits a highly conserved structure providing ubiquitin ligase activity without abrogating cell and structure-specific function. In this review, we mainly summarized the crucial roles of the Pellino family in pattern recognition receptor-related signaling pathways: IL-1R signaling, Toll-like signaling, NOD-like signaling, T-cell and B-cell signaling, and cell death-related TNFR signaling. We also summarized the current information of the Pellino family in tumorigenesis, microRNAs, and other phenotypes. Finally, we discussed the outstanding questions of the Pellino family in immunity. Immune responses are mainly divided into innate immunity and acquired immunity. Innate immunity can respond rapidly to pathogens as the first line of defense mediated by macrophages, dendritic cells, neutrophils, epithelial, and endothelial cells. It utilizes germ-line encoded pattern recognition receptors (PRRs) to detect conserved microbial components known as pathogen-associated molecular patterns (PAMPs) or endogenous ‘alarmins’ released during infection and inflammation. Toll-like receptors (TLRs), nucleotide-binding oligomerization domain-like receptors (NLRs), retinoic acid-inducible gene I-like receptors (RLRs), C-type lectin receptors (CLRs), DNA sensors, and melanoma 2-like receptors are not part of the PRRs (AIM-2-like receptors). Mammalian TLRs recognize bacteria and nucleic acids and sense inflammation caused by bacteria through binding to ligands on the cell surface and in the nuclear body. NLRs and RLRs, on the other hand, detect nucleic acids in the cytosol –3. PRRs Ubiquitination, a posttranslational modification involving the conjugation of the 76 amino acid proteins to the lysine residue of other proteins, is catalyzed by the sequential action of ubiquitin-activating (E1), ubiquitin-conjugating (E2), and ubiquitin-ligating (E3) enzymes. Ubiquitin contains seven lysine residues and one N-terminal methionine (M1) residue, each of which can be attached to another ubiquitin moiety. The presence of these lysine residues and the M1 forms a variety of ubiquitin chains , which are recognized by substrate proteins with linkage-specific ubiquitin-binding domains to trigger multiple biological functions such as K48- and K11-linked chains for protein degradation, M1 or K63-linked chains for signal transduction . The subDrosophila Peli, Human PELI, Mouse Peli), first discovered in Drosophila, is a novel and evolutionarily conserved protein with 424 amino acid residues and an estimated molecular weight of 47 kDa , and other phenotypic changes of cells and tissues   by inter tissues –47.IL-1R, TLRs, and NLRs were involved in innate immunity to mediate the production of inflammatory cytokines , 49 and Drosophila genome, Pellino interacts with and regulates plasma membrane MyD88-K48-Ub turnover to balance Toll-mediated immune signaling positively or negatively  domain which can allow the recruitment of the adapter MyD88 for the transduction of signals . In thisvia IL-1R and IL-1R-accessory proteins to recruit MyD88-dependent signaling cascades, namely, IRAK4, IRAK1, IRAK2, TRAF6, and TAK1, that leads to the activation of the MAPKs and NF-κB  and Smad7 can bind to Pellino1 via mad homology (MH2) domains to mediate growth factor-β (TGF-β). It inhibited IL-1R signaling by preventing Pellino1 from forming a complex with MyD88, IRAK1, IRAK4, and TRAF6, which further suppressed IL-1β induced NF-κB activation and production of proinflammatory cytokines  in mouse embryonic fibroblasts between wild type (WT) and Pellino1 knockout (KO) mice, Pellino1 is overlooked or unnecessary for the IL-1R pathway  from the nucleus to the cytoplasm and activates the transcription factor CREB in a p38 MAPK-dependent manner  like IL-1R. Upon stimulation of ligands, several TLRs such as TLR2 and TLR4 recruit MyD88, IRAKs, TNF receptor-associated factor 3 (TRAF3), and TRAF6 to activate TAK1, leading to the activation of MAPK and NF-κB , LPS, and the RNA virus in the CNS (in vitro and in vivo (via IRAK1/IRAK4/Pellino3/scavenger receptor-A1(SR-A1) dependent mono-ubiquitination of TRAF family member associated NF-κB activator (TANK)  . In detar (TANK) .In the TRIF-dependent interferon induction signaling Figure 3via a RIP homology interaction motif (RHIM) in TRIF-dependent signaling  activation and apoptosis ignaling , 91, 92 ignaling , 94 and ignaling , 96 leadignaling . Both TNvia K48-linked polyubiquitylation  are involved in the anti-infection process by activating the NF-κB signaling pathway, type I IFN signaling pathway, autophagy-related pathway, and pyroptosis pathway , 101.via NOD2 in chemical drugs induced models of colitis  in response to LPS , Pellino1 induces the AKT and MAPK activated protein kinase 2 (MK2) phosphorylation to restore cell migration potential, proangiogenic responses and the wound healing ability with VEGFR2 deficiency  in vivo . Further in vivo . In mous in vivo . Transge in vivo . Pellino in vivo –120. In  in vivo . Pellinotination  and p53 h as p21 . Furtherh as p21 . IAP mayh as p21 , 122. Hih as p21 , 123. Fuh as p21 . This maAs a positive regulator of inflammatory factors, Pellino1 induces the production of inflammatory factors followed by the change of inflammatory microenvironment leading to the transformation of normal cells to tumor cells. So it is necessary to study the inflammatory microenvironment induced by the Pellino family in normal cells, tumor cells, and even cell co-culture systems.MicroRNAs (microRNAs) are small non-coding RNAs with the capability of modulating gene expression at the post-transcriptional level either by inhibiting messenger RNA (mRNA) translation or by promoting mRNA degradation . Severalin vitro. It induces immune dysregulation by lowering umbilical cord mesenchymal stem cells (UC-MSCs) proliferation, migration, and mitigates the decrease in T follicular helper (Tfh) cells and increases T regulatory (Treg) cells (SLE) (A positive correlation between microRNA-21 and Pellino1 suggests that microRNA-21 and Pellino1 might be associated with autoimmune primary ovarian insufficiency (POI) patients . MicroRNls (SLE) . MicroRNls (SLE) . MicroRNls (SLE) , microRNls (SLE) , microRNls (SLE) , and micls (SLE)  can all ls (SLE) . In non-ls (SLE) . It is cDrosophila  and the Shandong Provincial Natural Science Foundation (No. ZR2019MH001).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Recently, fertility services have started resuming since COVID-19 was declared a pandemic, but there remains significant uncertainty in the way this care will be delivered in the United Kingdom. The objective of our study was to explore the impact of COVID-19 on individuals using fertility services in the United Kingdom. The study was conducted in two phases between May 2020 and July 2020: an online questionnaire involving 1212 participants and subsequent individual semi-structured telephone interviews with 15 participants. Through thematic analysis, we learned from the questionnaire findings that 74% of individuals identified as White British, 21% as Black and Minority Ethnic (BAME) and 2.6% as male. Ninety-six per cent of individuals from the questionnaire explained that COVID-19 had a ‘negative impact’ on their fertility treatment, namely ‘delay in care’. Eighty-two per cent of participants discussed concerns about the ‘uncertainty’ they felt about fertility services; these included the ‘unknown impact of COVID-19 on pregnancy outcomes’, the ‘unknown impact on general gynaecology services’ and the ‘unknown impact of COVID-19 on fertility success’. Through semi-structured telephone interviews with 15 participants, we learned about the ‘cultural pressures’ individuals from BAME backgrounds faced in relation to care. Participants were mindful about the ‘pressures on the service’ when reopening, and therefore ‘advancing maternal age’, ‘socio-economic background’ and ‘previous unsuccessful fertility treatment’ were the main factors individuals considered important when ‘prioritising’ fertility care. Our findings can be used by fertility service providers to appreciate the patient perspective when considering the reopening of fertility services nationally and internationally.The impact of COVID-19 on patients seeking or undergoing fertility treatment is not entirely known. Many patients have had their treatment postponed during the pandemic. As fertility services begin to recommence, it is important to understand how the pandemic has affected this group of patients. In addition, it is vital to appreciate and understand the patient’s voice in order to ensure services take into account the patients’ concerns as they begin to offer certain fertility treatments. Our study was conducted in two phases and involved an online questionnaire and individual interviews with people. We found that people were worried about services restarting and how care would be prioritised. People also discussed some of the perceived barriers to seeking fertility healthcare. Our findings highlight the importance of understanding the patient’s voice when recommencing fertility services. The COVID-19 pandemic has had an unprecedented impact on the world. Fertility services in the United Kingdom were temporarily suspended when the pandemic first started. In addition, routine gynaecological workload was also stopped throughout the country. Consequently, patients who were about to undergo fertility treatment, or indeed, midway through it, had to have it stopped.Previous public health crises including H1N1 swine flu, HIV/AIDS and the West African Ebola  epidemicThe regulatory authority of IVF clinics in the United Kingdom  published General Direction 0014, instructing clinics to complete active treatments by 15 April 2020. Concurrently communication from the Chief Executive and Chief Operating Officer of the NHS directed all NHS providers to, amongst other measures, prepare to ‘postpone all non-urgent elective operations from 15 April at the latest, for a period of at least 3 months’.On Friday, 1 May, the Secretary of State for Health and Social Care announced that fertility clinics would be allowed to open from May 11. The HFEA allowed clinics to apply from May 11 to resume licenced activities. A small number of clinics were able to recommence treatments shortly thereafter, with appropriate measures to reduce risk with distancing and reduced footfall through clinics. From the 15 April 2020, all non-urgent elective operations were postponed for at least 3 months (British Fertility Society).The resumption of services has been variable and slow, as some clinical spaces were used for other purposes and many staff were redeployed to work in other parts of the health service . Even by August 2020 some clinics were yet to reopen.The impact of the shutdown on service users is not exactly known and therefore this study was designed to explore this impact. The objective of the study was to explore participants’ experiences of fertility care during COVID-19 and any perceived barriers to care and to gain insight into the way participants felt about fertility care upon resumption of services. The study was conducted in two phases: phase one being an online questionnaire and phase two being individual semi-structured telephone interviews.This study involved an exploratory qualitative approach through two phases. Phase one involved the use of a context-setting online questionnaire . In addition to demographic data, the questionnaire content included participants’ experiences of fertility care in the United Kingdom, their perceptions of COVID-19 and its impact on their fertility care and perceived barriers to fertility care. Consent was obtained online by participants clicking on the questionnaire link and ticking the respective disclosure.The findings from the questionnaire study were used to inform the second phase of the study that involved individual semi-structured interviews with participants selected through purposive sampling. Interviews are a common method of obtaining qualitative data . In addiwww.qsrinternational.com/nvivo-product). NVivo is a software that is used commonly in qualitative research to organise large amounts of qualitative data. Thematic analysis involved a number of stages including (1) familiarising with the data set , (2) generating initial codes (ascribing labels to the open-ended responses), (3) generating and refining themes (grouping the different codes together under common themes) and (4) relating the themes to the context of the study  There is so much information about Covid and how it affects people. But, there just doesn’t seem to be much about the way Covid can affect pregnancy and if there are any known future complications from it. My partner and I just really worry. Eighty-eight per cent of participants from the questionnaire perceived fertility care changing in the way services will be delivered once they resume again. Participants had concerns about how their care would resume once services recommenced, if clinics will be taking any extra precautions in the form of personal protective equipment and what measures services will have in place to ensure participants do not contract COVID-19. Participants also discussed concerns as to whether certain fertility services will ever resume in the way they were previously.I know that things will have to change due to Covid. I get that. But that doesn’t stop me from worrying whether I will be able to have my IVF. I just don’t know how things will ever get back to normal. It just makes everything feel so hopeless. I have read so many news articles saying that people just don’t have the right sort of PPE. This worries me a lot. I am so keen to get on with my treatment, but I am also worried that I will be putting myself at risk of catching Covid from hospitals. Participants were asked to comment on their thoughts about the 10-year storage limit on frozen embryos, eggs and sperm. In the questionnaire findings, over 80% of participants felt the additional 2-year storage limit was a positive decision, but some participants discussed their anxieties.Eight participants from the semi-structured interviews explained that an extension implied to them that future fertility care will be delayed upon resumption. One participant felt that the extension was implying that fertility services cannot cope with the patient demand and needed ‘more time to help people’. Participants discussed the following:I mean, it’s great that people are allowed to have their eggs for example stored for two years longer, but the fact that the government have done this makes me anxious that there will be a delay in restarting my treatment as the extra two years may be needed. The two-year extension is all very well, but does that mean that I will have to wait longer before I am seen again to start my treatment? I just think that the government have introduced this new policy as they can’t cope with the patient demand. More and more people want fertility care, but how can they fit everyone in? This is their way of pretending that they are helping patients, but I don’t think they are really. Ninety-one per cent of participants from the questionnaire were concerned about what fertility treatments will be offered and to whom and when services will recommence. Participants in the interviews expanded on this:I am worried what sort of fertility treatments my centre will be able to restart again. Even when they do restart, I wonder if they be prioritising it to certain people. There was already stigma in my community from being childless already and this has made it worse. Surely things will take a while to return to normal and even with the different treatments, they can’t possibly get everyone in on time. There will be some criteria I bet on who gets what first. From both the questionnaire and interview findings, participants expressed concern about the potential variation in treatments amongst fertility centres. They were also worried about how fertility services would be prioritised amongst patients. Participants discussed factors that they felt were important when deciding how services should be prioritised. These included ‘advancing maternal age’, ‘socio-economic background’ and ‘previous unsuccessful fertility treatment’.Participants were worried about how centres would begin the process of restarting their treatment. During semi-structured interviews, participants discussed their anxieties about routine gynaecology care:I am due to have a keyhole test on my fallopian tubes to check if they are working properly, but at the same time, they said I can have some endometriosis treated. I wonder how long this keyhole surgery will now take because of COVID. I have been waiting for a laparoscopy for such a long time. I guess this will never happen now. Participants through both phases of the study expressed concern about being diagnosed with COVID-19. They were worried about the impact the virus would have on any developing pregnancy as well as long-term implications on a live baby. Concerns were also expressed as to whether a miscarriage was more likely or not in a COVID-positive patient.From both aspects of the study, participants discussed concerns about the way male fertility services may be delivered. Male participants in the interviews explained that they perceived official communication from services provides as ‘female orientated’ and that there was less focus on the needs of male patients.It is great that all the official organisations are trying to keep everyone updated about fertility treatment etc, but I have found that there doesn’t seem to be much focus on men who are undergoing fertility treatment specifically. I have had cancer before and so there just doesn’t seem to be much information on how my fertility care might be affect or how I might be feeling. Participants from a Black and Minority Ethnic (BAME) background discussed the ‘cultural pressures’ they faced during COVID-19 and the implications on their wider social circumstances. Through semi-structured interviews, they expressed concerns about being ‘disadvantaged’ from a health equalityt perspective and felt ‘more at risk’ from pregnancy-related complications. Many had read about health outcomes in those individuals from a BAME background who were diagnosed with COVID-19. Participants in the semi-structured interviews expressed concerns as to whether ethnicity in general influenced fertility outcomes.As routine gynaecology work was also suspended, participants discussed this as a barrier to fertility treatment. Some participants were waiting for benign gynaecological surgery prior to fertility care. Other barriers included ‘not being kept updated about service resumption’ by some fertility centres, perceived ‘lack of interest in fertility care by the government’ and the influence of the media. To expand on this, participants felt that the media focussed primarily on how COVID-19-related care was delivered as opposed to fertility care. As a result of the uncertainty in the way services may be delivered, some participants considered ‘postponing’ their care until the pandemic was ‘more stable’.Through 1212 questionnaires and 15 semi-structured interviews, our findings not only provide insight into the negative impact COVID-19 has had on participants’ fertility care but also highlight the professional support they have or, in the majority of cases, have not received during the closure of services. During the semi-structured interviews, we gained in-depth understanding and awareness of the barriers and concerns participants perceived to their care as services began to slowly resume particularly for those patients who were identified as male. In addition, some participants discussed how they were impacted by the cessation of routine benign gynaecological surgery. In addition, we demonstrate the absolute need for clinicians and other fertility care providers to be mindful of the changing needs of this group of patients as they may require further emotional, medical and psychological support during their treatment(s). Our findings highlight the importance of providing information that is tailored to specific concerns related to fertility and COVID-19 to patients undergoing fertility care.in situ for the way fertility services will be delivered. The latest WHO report on COVID-19 also does not make reference to the resumption of fertility services. As routine gynaecological surgery was also postponed during the pandemic, the government and secondary care services will need to explore how they will be able to manage this workload once services resume.During COVID-19, fertility clinics responded in different ways by alerting their patients through websites, social media, and in some cases, through personal telephone calls to keep them updated on their respective services. Furthermore, some clinical spaces were utilised for other medical functions and staff were redeployed to other clinical and non-clinical duties. Patients found this information useful as they also felt informed. Whilst a position statement released by the British Fertility Society provided insight into the resumption of fertility services in the United Kingdom, uncertainty in its exact delivery still remains. In addition, should a second wave of COVID-19 occur, there do not appear to be contingency plans Numerous reports were released through the British Fertility Society on how fertility services can be reintroduced. However, to our knowledge, there are no other published studies in the United Kingdom involving semi-structured interviews that explored the impact of COVID-19 on individuals who are undergoing or about to undergo fertility treatment. Our study provides significant insight into the way participants perceived COVID-19 to have influenced their care and the support they received during the pandemic from their respective fertility centre. Participants also discussed the perceived barriers to care and their concerns about resuming fertility services. The findings from our study highlight the importance of the patient’s voice when considering the resumption of fertility services.Due to COVID-19 and the social restrictions imposed by the government, recruitment to the study was via online media. As a result, our findings may have captured those individuals who are social media active and therefore may under-represent the findings from those who are not. Whilst attempts were made to ensure a fair representation from individuals from a variety of demographics, the overall majority of participants were from a White British background and therefore this may have influenced the findings.The number of replies from individuals who identified as male were also low, and as such the views of this group were under-represented. The findings from this UK study may not necessarily represent the views from those individuals throughout the world. Finally, the study was conducted during May 2020 to July 2020 and therefore the findings are representative of a time where our knowledge on COVID-19 was limited. Since this time, knowledge in this area in relation to pregnancy in particular has advanced.Our findings echo similar messages from other studies. Our study provides the basis for further qualitative work. Undoubtedly, it may take some time before services resume back to the ‘pre-COVID-19’ era and therefore, services may need to be prioritised amongst patients. How this prioritisation is done is a question for stakeholders, clinicians and academics to decide with the engagement of patient and public involvement.The impact of COVID-19 has been far reaching for many individuals. For those undergoing fertility care, it has been a particularly challenging and unpredictable time. This study has truly enabled participants’ voices to be heard. The findings from this study can be used by fertility service providers to appreciate the patient perspective when considering the reopening of fertility services nationally and internationally and be mindful of patient concerns.E P M has received a grant from Gedeon Richter as the UK Chief Principle Investigator for the PREMIUM study. E P M has also received consultancy fees from Chugai Pharma. E P M has been an adviser for Pfizer in 2018 and Gedeon Richter in 2019. No other conflict of interest for any of the other authors.The data underlying this article will be shared on reasonable request to the corresponding author.B K conceived the study, obtained ethical approval and was involved in the design, execution, analysis, manuscript drafting and critical discussion. A S was involved in the analysis, manuscript drafting and critical discussion. A B and E P M were involved in the design, manuscript drafting and critical discussion. All authors approved the final version of the manuscript.


In addition to the β3-adrenergic signaling, numerous other hormones and secreted factors have been reported to affect thermogenesis. In this review, we discuss several major pathways, β3-adrenergic, insulin/IGF1, thyroid hormone and TGFβ family, which regulate thermogenesis and browning of WAT.Obesity, an excess accumulation of white adipose tissue (WAT), has become a global epidemic and is associated with complex diseases, such as type 2 diabetes and cardiovascular diseases. Presently, there are no safe and effective therapeutic agents to treat obesity. In contrast to white adipocytes that store energy as triglycerides in unilocular lipid droplet, brown and brown-like or beige adipocytes utilize fatty acids (FAs) and glucose at a high rate mainly by uncoupling protein 1 (UCP1) action to uncouple mitochondrial proton gradient from ATP synthesis, dissipating energy as heat. Recent studies on the presence of brown or brown-like adipocytes in adult humans have revealed their potential as therapeutic targets in combating obesity. Classically, the main signaling pathway known to activate thermogenesis in adipocytes is β Brown adipose tissue (BAT) is specialized in heat production through non-shivering thermogenesis by burning fuels such as fatty acids (FA) and glucose . BAT is Of all tissues, BAT expresses the highest levels of uncoupling protein 1 (UCP1) and UCP1 plays a central role in non-shivering thermogenesis . UCP1 ac+/PAX7+/EN1+ progenitor cells can give rise to other cell types, such as skeletal myocytes, they commit to brown adipocyte lineage upon early B cell factor 2 (EBF2) expression , paired box 7 (PAX7) and myogenic factor 5 (MYF5) . While tpression . Additiopression . Ewing Spression . Committpression , 15. ZFPpression , 17. EBFpression . In addipression . PPARγ cpression . Some of3-adrenergic receptor  to induce adrenergic signaling  reaction, in supporting robust glycolysis. In addition, electrons generated from AIFM2 action are transferred to CoQ for ETC activity of mitochondria. AIFM2 KO mice exhibited higher body weight, lower oxygen consumption, and lower BAT temperature whereas AIFM2 overexpression resulted in the opposite phenotypes. In addition, the expression of AIFM2 was induced upon refeeding with high carb diet when examined at thermoneutrality, suggesting that glucose oxidation appears to be especially important in the fed state to promote thermogenesis when the blood glucose level is relatively high.We recently discovered a BAT-specific NADH oxidase, AIFM2, which is required for cold- and diet-induced thermogenesis . Upon co3-AR signaling plays a dominant role in regulating thermogenesis. Besides those mentioned above, there are many more proteins that are involved in and downstream of β3-AR signaling. Further studies will elucidate more components related to β3-AR signaling that regulate thermogenesis at various steps such as substrate metabolism, mitochondrial homeostasis, gene expression as well as signal transduction.Collectively, there is no doubt that βin vitro with no increase in the expression of PPARγ and CEBPα, indicating that insulin/IGF-1 signaling is critical for BAT development. Since the addition of PPARγ agonist, rosiglitazone, did not rescue the impaired differentiation, insulin/IGF-1 signaling seemed to act on the upstream of PPARγ/CEBPα. Consistently, phosphorylation of CEBPβ at Thr188, which is required for DNA binding and therefore transcriptional activation of PPARγ/CEBPα, was reduced. FIGIRKO mice were also protected from diet-induced obesity, possibly due to impaired adipose development. FIGIRKO mice failed to maintain their body temperature during cold exposure, although they had higher energy expenditure/basal metabolic rate with no change in activity, this paradox remaining to be explained. In this regard, the study utilized aP2 promoter to express Cre recombinase for gene KO, which could have affected not only adipose tissues but also other cell types, such as macrophages.Insulin/IGF1 signaling plays important roles in adipose tissue development and thermogenesis. Mice with fat-specific double knockout of insulin receptor (IR) and IGF1 receptor (IGF1R) (FIGIRKO) exhibited decreased adiposity with more than 85% reduction in BAT, having lower body weight . Indeed,The effects of insulin signaling and IGF-1 signaling were further investigated by comparing fat-specific IR single KO (F-IRKO) and IGF1R single KO (F-IGFRKO) to IR/IGF1R double KO (F-IR/IGFRKO), which were all generated by Cre driven by adiponectin promoter . F-IGFRKBAT-specific IR KO mice created by using UCP1 promoter-Cre, however, showed impaired BAT development . Since tInsulin/IGF1 signaling plays a role in browning of WAT as well. PTEN antagonizes the action of PI3K by converting PIP3 back to PIP2 and, therefore, insulin/IGF1 signaling. PTEN knockdown (KD) by direct injection of Rec2-Cre in iWAT of PTEN floxed mice increased AKT signaling and induced browning . These e3-adrenergic signaling. β3-adrenergic signaling pretreatment reduced the activation of IR, IRS1 and IRS2. In addition, β3-adrenergic signaling mitigated AKT activation and IRS1-associated activation of PI3K upon insulin treatment, while it did not affect MAPK activation and IRS2-associated activation of PI3K. Moreover, β3-adrenergic signaling abolished insulin-mediated glucose uptake in brown adipocytes  is classically known to regulate thermogenesis , was reported to affect thermogenesis as well . T2 admivia adenovirus injection increased the expression of PRDM16 and UCP1 in BAT, energy expenditure and body temperature, while decreasing body weight. In mature brown adipocytes, BMP7 increased mitochondrial activity through p38-ATF2 and SMAD1/5/8 pathways  superfamily have been shown to regulate thermogenesis Figure 2pathways . BMP7-trxidation . Furtherxidation . BMP7 trBMP8b was found to be highly expressed in BAT and its expression increased during differentiation and during cold exposure . BMP8b K2, a product of COX2, upregulated thermogenic gene expression, such as CIDEA1 and PGC1α, in the presumptive beige precursor cells, which was further augmented by the addition of SB431542, implying the possible interaction between TGFβ/SMAD3 and COX2/PGE2 pathways. These presumptive beige precursor cells were isolated and selected from WAT by FACS using a series of cell surface markers and were termed inducible beige stem/progenitor cells (iBSCs). iBSCs were then transplanted subcutaneously into immunodeficient nude mice on HFD with IPTT-300 transponder. During the cold exposure, iBSCs from TβR1 KO mice maintained higher temperature and showed higher expression of thermogenic genes with multilocular lipid droplet morphology, suggesting that the KO iBSCs underwent beige adipogenesis. Altogether, TGFβ/SMAD3 signaling negatively regulates WAT browning although further research will help to clarify the contributions of different downstream pathways.TGFβ/SMAD3 signaling pathway has been reported to suppress browning of WAT . TGFβ trThe discovery of brown or brown-like adipocytes in human adults has made targeting adipocytes an attractive strategy to fight against obesity and its associated diseases. Activation of existing thermogenic adipocytes and/or induction of new thermogenic adipocytes can be mediated by various hormones and their signaling cascades, including those important pathways we describe in this review. Understanding the precise molecular mechanisms underlying regulation of thermogenesis and browning of WAT is critical for advancement of the future anti-obesity therapeutics.CT drafted and HS revised the manuscript. All authors contributed to the article and approved the submitted version.The work was supported in part by DK120075 to HSS.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Androgen receptor (AR) signaling mainly controls prostate cancer (PCa) growth. Hence, the conventional regimen for PCa includes androgen deprivation therapy (ADT) or antiandrogen in order to reduce PCa recurrence. However, castration-resistant prostate cancer (CRPC) is insensitive to environmental androgen via self-supplementation of androgen or inactivation of AR signaling. Accordingly, CRPC has limited treating options and their outcome is poor. Previous studies reveal that forming CRPC needs to maintain cell growth under low AR signaling conditions and to mitigate AR dependency by differentiation. Previous studies show that the epidermal growth factor (EGFR) and signal transducer and activator of transcription 3 (STAT3) participate in the maintenance of PCa growth under androgen ablation. We have found a novel mechanism in which EGFR cooperates with STAT3 and initiates neuroendocrine differentiation. This review aims to summarize the recent findings on EGFR and STAT3 in CRPC induction and discuss the unsolved issues therein.Prostate cancer (PCa) is one of the most common cancers in the world and causes thousands of deaths every year. Conventional therapy for PCa includes surgery and androgen deprivation therapy (ADT). However, about 10–20% of all PCa cases relapse; there is also the further development of castration resistant adenocarcinoma (CRPC-Adeno) or neuroendocrine (NE) PCa (CRPC-NE). Due to their androgen-insensitive properties, both CRPC-Adeno and CRPC-NE have limited therapeutic options. Accordingly, this study reveals the inductive mechanisms of CRPC (for both CRPC-Adeno and CRPC-NE) and fulfils an urgent need for the treatment of PCa patients. Although previous studies have illustrated the emerging roles of epidermal growth factor receptors (EGFR), signal transducer, and activator of transcription 3 (STAT3) signaling in the development of CRPC, the regulatory mechanisms of this interaction between EGFR and STAT3 is still unclear. Our recent studies have shown that crosstalk between EGFR and STAT3 is critical for NE differentiation of PCa. In this review, we have collected recent findings with regard to the involvement of EGFR and STAT3 in malignancy progression and discussed their interactions during the development of therapeutic resistance for PCa. MYCN) such as chromogranin A (CHGA), synaptophysin (SYP), and γ-enolase (ENO2)) instead of AR response genes (prostate-specific antigen (PSA)/KLK3, prostate-specific membrane antigen (PSMA)/FOLH1, SAM-pointed domain containing ETS transcription factor (SPDEF), and NK3 Homeobox 1 (NKX3-1)) [Prostate cancer (PCa) is a leading cause of new cancer cases in men, which accounts for 6.8% of all cancer deaths and 14.1% of new deaths . IntermeNKX3-1)) ,8,9. CRPNKX3-1)) ,11,12,13de novo CRPC-NE (CRPC-NE derived from NE cells) only occurred in approximately 1% of PCa cases and about 20% of CRPC cases [In order to overcome androgen dependency, PCa cells undergo several signaling changes that are grouped into the following strategies: 1. enhancing cell survival and growth; 2. mitigating androgen dependency; and 3. undergoing the epithelial-to-mesenchymal transition (EMT) and further NE differentiation (NED) . To overPC cases ,19, in wPC cases ,21,22. NEpidermal growth factor (EGF) receptor  signaling controls various physiological roles from the cellular level to the organ level, such as organ development and wound healing . In pathAs described in previous studies, PCa cells activate complementary growth signaling (GR and EGFR) and AR alternative splicing to overcome the absence of AR signaling in an ADT condition ,35. The In the upstream of EGFR signaling, several transducers were identified, including the scinderin (SCIN)/ubiquitin-specific peptidase 39 (USP39)/protein-tyrosine-phosphatase of regenerating liver 1 (PRL1) as activators and microRNA (miR)-146a/miR-27a-5p/CKLF-like MARVEL transmembrane domain containing 5 (CMTM5) as inhibitors ,49,50,51The second aim of the EGFR in CRPC-Adeno and CRPC-NE induction involves androgen independency. Androgen independency is caused by the constitutive activation of intracellular androgen signaling  or the repression of AR expressions . As andrUpstream from EGFR, several activators, including fucosyltransferase 8 (FUT8), YB-1/ErbB2, and MMP9, stimulated EGFR signaling and pushed PCa cells into becoming androgen-independent ,73. DuriThe EGFR is distributed in plasma membranes in order to bind with environmental EGF signals . In non-NED is a complex progression in which cellular reprogramming and epigenetic modulation are involved ,91,92. EIn addition to the above-mentioned mechanism, EGFR mutations are also thought to participate in NED; they are not mentioned in CRPC-NE development. Miyoshi et al. and Baglivo et al. reported that activated mutations for EGFR were found in large-cell NE lung cancer tissues, which helped lung adenocarcinoma cells acquire resistance to EGFR tyrosine kinase inhibitors (TKIs) and NED ,101. TheThe JAK/STAT3 signaling cascade is an intracellular signaling mediator of cytokines and interferons that is a part of pathogen-associated molecular patterns (PAMPs) . Immune twist [pim-1) [PTTG1) [VCP) [PD-L1) [clusterin [Due to EMT-related transcription factors (SNAI1/2 and TWIST1) being the targets of STAT3, the activation of JAK/STAT3 signaling in cancer cells is commonly believed to initiate the EMT ,108. STAtwist , the Pim [pim-1) , the PTT [PTTG1) , valosin1) [VCP) , program [PD-L1) , and clulusterin . Intereslusterin . Activatlusterin ,119,120.lusterin . The preWhen involved in NED, STAT3 is no longer activated by JAK. Zinc finger and BTB domain containing 46 (ZBTB46)/leukemia inhibitory factor (LIF) signaling, pigment epithelium-derived factor (PEDF)/Ras homolog family member A (RHOA)/inhibitor of κB kinase (IKK)/NF-κB/IL-6, and LncRNA-p21/polycomb repressive complex 2 (PRC2)/AKT are known signaling mediators ,124,125.DAB2IP) is a tumor-suppressor gene that blocks various survival-essential signaling pathways like PI3K/AKT, AR, JAK/STAT, and Wnt [In addition to cell metamorphosis, STAT3 maintains PCa cell survival under ADT conditions. Previous studies have revealed that IL-6 and phospholipase Cε (PLCε) activated STAT3 via direct phosphorylation or non-canonical Hedgehog signaling that activates the AR, survivin, and NF-κB/promatrilysin expressions to maintain cell survival under ADT conditions ,129,130. and Wnt . Zhou et and Wnt . By comb and Wnt . That re709-phosphorylated STAT3 is translocated into nuclei and initiates antiapoptotic and proliferation-related gene expressions such as c-Myc, Bcl-xL, and cyclin-D1 [From the previous discussion, we have concluded that EGFR signaling helps to maintain PCa cell growth under ADT conditions, and that JAK/STAT3 signaling drives CRPC-Adeno cells into the EMT, which further fostered transdifferentiation into CRPC-NE. These two signaling pathways with distinct biological activities sometimes cooperate and trigger different cell outcomes in cancer cells. In fact, STAT3 is one of the downstream signaling transducers of EGFR signaling via the PI3K/AKT/mechanistic targets for the mammalian target of rapamycin (mTOR) cascade . When acyclin-D1 ,134,135.yclin-D1   overexpression . Lapatin709 and facilitated PCa cell proliferation, not differentiation [A newly discovered EGFR translocation mechanism elicited new advancements in CRPC development. However, many unanswered questions remain unresolved with regard to the regulation of EGFR/STAT3 interplay. One attractive aspect is switching the EGFR between proliferation and differentiation. From previous discussions, we found that either EGFR or STAT3 proliferation and differentiation signaling cannot coexist. Differentiation seems to be stress-dependent; as a result, EGFR- and STAT3-mediated survival signals inhibited the repression of differentiation ,119,120.ntiation . Does nuntiation . This abntiation . Anotherntiation , becausentiation ,158. Suc