Citation per paper correlated with publication count among the authors and institutions. Mandibular cortical width was the most frequently used and most cited measurement index. References published during the 1970s and 1980s have built the foundation for the development of research that investigates the potential associations between osteoporosis and radiographic measurements on panoramic radiographs. Osteoporosis detection by dental panoramic radiographs is a perennially investigated research topic with global contributions. Panoramic radiographs are considered early detection and screening tools for osteoporosis by worldwide research.This bibliometric study evaluated the scientific impact of papers dealing with osteoporosis detected by dental panoramic radiographs by performing citation analysis and cited reference analysis. Retrospective data was extracted from the Web of Science Core Collection database and imported into VOSviewer, CRExplorer, and CitNetExplorer for analyzing semantic contents, cited references, and temporal citation network. The 280 relevant papers identified were cited 4874 times, having an h-index of 38 and 17.4 citations per paper. The top five major contributing countries were Japan ( Analyzing bibliometric data helps us understand research trends and hot topics, which are also important and getting attention in the medical fields ,2. It waTherefore, the primary aims of this study are to reveal the overall citation performance of papers dealing with osteoporosis detection by panoramic radiographs and identify the major contributors, the most cited papers, and the important references and hot topics. The secondary aim is to reveal the main indices used for mandibular bone measurements as early detection and screening tools for osteoporosis.Retrospective data was extracted from the Web of Science (WoS) database hosted by Clarivate Analytics. IRB or ethical approval was not required for this study.The search strategy was defined as Topic=(osteoporosis) AND Topic=(panoramic). The search included papers with both the words osteoporosis and panoramic in the title, abstract or keywords. Only articles and reviews were considered. The background data of the papers were analyzed descriptively, which included author, country, institution, language, journal, and publication year. The top 10 most cited papers were identified.We downloaded the full records of the papers yielded from the search and imported into a bibliometric software called VOSviewer  for furtCited references of all included papers were analyzed by CRExplorer , which ip < 0.05.We first performed descriptive data analysis. Analytical statistics, namely Pearson’s correlation tests, were subsequently performed to analyze whether there existed a significant correlation between citation per paper and publication count with regard to authors, institutions, countries, and journals. Data were analyzed with the Statistical Package for Social Sciences . Test results were statistically significant if n = 274, 97.9%). The 280 papers were cited 4874 times in total, having an h-index of 38 and 17.4 citations per paper.The search in WoS yielded 280 papers: 263 of which were articles and 17 were reviews. They were published in 113 journals from 1991 to 2018 by 969 authors with 312 affiliations from 42 countries. Most of the papers were published in English  among the five. When examining all contributors, publication count and citation per paper had a significant correlation for authors  and institutions  but not for countries  and journals .The top five most prolific authors were from Hiroshima University (Professor Taguchi has since moved to Matsumoto University) and the University of Manchester, and, thus, these two institutions were among the most prolific research bases . Brazil The top 10 most cited papers are listed in From As said, the first of the 280 papers were published in 1991. When their references were analyzed, the largest positive peaks shown in the RPYS before 1991 were 1974, 1977, and 1982–1983 . The resTo better visualize how the abovementioned six representative references have influenced the subsequent references, a temporal citation network was synthesized. They were the earliest ones among the 100 most cited references by the 280 papers . ApparenOsteoporosis International or specialized oral and maxillofacial radiology journals within dentistry. It demonstrates that most of the relevant literature were published in specialized journals. The earliest work in This bibliometric study has affirmed that papers on osteoporosis detection by panoramic radiographs have received a large number of citations per paper (17.4). The analyses mainly concerned top-cited papers. The correlational results were consistent with the stated hypothesis for the authors and institutions but not for the countries and journals. The topic has received global contributions from Asia, the United States, Europe, and South America. Notably, Japan was the most prolific country on this topic, despite the fact that the United States has been the traditional outstanding dominating power in terms of publishing scientific papers such as in the fields of osteoporosis  and radiFrom A previous meta-analysis published by Calciolari et al.  identifiIn the current study, six important references were published before 1991, when the first of the 280 osteoporosis papers analyzed were published. One of these six references was a statistics paper that presented a methodology of using kappa statistics to test inter-observer agreement . This suThe current study has several limitations. Firstly, relevant papers not indexed in WoS, preprints uploaded to servers such as arXiv and bioRxiv, and unpublished papers were not identified and included in the current analysis. However, WoS is often considered as the “gold standard” for extracting bibliometric data for analysis because it is more accurate and better at tracking data from older publications . MeanwhiOn average, papers dealing with osteoporosis detection by panoramic radiographs have received 17.4 citations per paper.These papers have received global contributions from Asia, the United States, Europe, and South America, with Japan and Brazil among the largest contributors.Most of the papers and references were published in journals specialized in osteoporosis or oral and maxillofacial radiology.Periodontitis was one of the topics with many citations per paper.Mandibular cortical width was the most frequently used and most cited measurement index relative to mandibular cortical index and panoramic mandibular index.Important references were published during the 1970s and 1980s that have built the foundation for the development of research that investigates the potential associations between osteoporosis and radiographic measurements on panoramic radiographs.Though, currently, panoramic radiograph is not a recognized tool for diagnosing osteoporosis, its reliability in screening / detecting osteoporotic patients has been frequently investigated and cited.Within the constraints of the limitations of the study, the following can be concluded:


DENND1A locus is associated with polycystic ovary syndrome (PCOS), a disorder characterized by androgen excess. Theca cells from ovaries of PCOS women have elevated levels of a DENND1A splice variant (DENND1A.V2). Forced expression of this variant in normal theca cells increases androgen biosynthesis and CYP17A1 expression, whereas knockdown of the transcript in PCOS theca cells reduced androgen production and CYP17A1 mRNA. We attempted to create a murine model of PCOS by expressing hDENND1A.V2 using standard transgenic approaches. There is no DENND1A.V2 protein equivalent in mice, and the murine Dennd1a gene is essential for viability since Dennd1a knockout mice are embryonically lethal, suggesting that Dennd1a is developmentally critical. Three different hDENND1A.V2 transgenic mice lines were created using CMV, Lhcgr, and TetOn promoters. The hDENND1A.V2 mice expressed hDENND1A.V2 transcripts. While hDENND1A.V2 protein was not detectable by Western blot analyses, appropriate hDENND1A.V2 immunohistochemical staining was observed. Corresponding Cyp17a1 mRNA levels were elevated in ovaries and adrenals of CMV transgenic mice, as were plasma steroid production by theca interstitial cells isolated from transgenic ovaries. Even though the impact of robust hDENND1A.V2 expression could not be characterized, our findings are consistent with the notion that elevated hDENND1A.V2 has a role in the hyperandrogenemia of PCOS.The  DENND1A locus has been associated with PCOS in diverse populations in genome-wide association (GWAS) and replication studies -TRE-hDENND1A.V2, or an empty vector control (pCMV-BAM) using Continuum . Cells were harvested 48 h after transfection.COS-I, CHO, and MA-10 cells were cultured in tissue culture dishes  for Western blot studies. COS-I and CHO were grown in DMEM, high glucose pyruvate medium  supplemented with 10% fetal bovine serum , 1% glutamine, and 1% penicillin/streptomycin . MA-10 cells were cultured on Waymouth’s media supplemented with 1.1g/l NaHCOCHO cells transfected with pCMV-BAM-hDENND1A.V2 vector were fixed with 4% formalin for 1 h, washed with PBS three times and incubated for 1 h at room temperature with blocking solution containing 10% goat serum , 3% BSA , and 0.2% Triton X-100 . After blocking, cells were incubated overnight at 4 °C with anti-hDENND1A.V2 rabbit polyclonal antibody (dilution 1:100) ,9. AfterImmunohistochemical (IHC) analysis for hDENND1A.V2 was performed on 5 μm-thick sections of formalin-fixed, paraffin-embedded ovarian tissue collected from PMSG/hCG-stimulated wild-type and transgenic mice.2O2 in methanol for 30 min to remove endogenous peroxidase. Antigen retrieval was performed in citrate buffer at 95 °C for 20 min. After washing several times in PBS, slides were incubated with blocking solution containing 10% goat serum and 0.2% triton X-100 in 1X PBS for 1 h at room temperature. A primary anti-hDENND1A.V2 rabbit polyclonal antibody was used for overnight incubation at 4 °C, 1:200 dilution. Then, slides were incubated with biotinylated secondary anti-rabbit antibody  for 1 h at room temperature. Following the incubation, the slides were washed with PBS and incubated with Vectastain Avidin-Biotin-Complex (ABC) reagent  for 30 min at room temperature. Following several washes with PBS, the staining was developed with ImmPACT Diaminobenzidine (DAB) for 2 min and rinsed with tap water to stop the reaction. Wild-type and transgenic samples were processed in parallel to ensure the same conditions for these samples. Lastly, the stained slides were dehydrated using an increasing concentration of ethanol and xylene and were mounted with Vectastain  mounting media. The primary antibody was neutralized by preincubation with the hDENND1A V2 specific peptide  for 30 min at room temperature. The peptide concentration used was 10 μg/mL.Slides containing the sectioned ovarian tissue were deparaffinized in 100% xylene and then rehydrated through decreasing the concentration of the ethanol. The slides were then treated with 3% H2, and 95% humidity in McCoy’s 5a medium supplemented with 5% FBS and 1% penicillin/streptomycin . The remaining ovary tissue was washed with media and digested for 60 min at 37 °C in collagenase-DNAse solution containing 4 mg/mL collagenase IV , 10 μg/mL DNAse , and 10 mg/mL BSA  in M199 medium  to isolate TIC. After digestion, cells were centrifuged at 1000 rpm for 5 min and washed several times in McCoy’s 5a medium . Finally, TICs were resuspended and cultured for 48 h at 37 °C, 5% CO2, and 95% humidity in McCoy’s 5a medium supplemented with 5% FBS and 1% penicillin/streptomycin .TIC and GC were isolated from F3 wild-type and CMV-hDENND1A.V2 Tg mouse ovaries and cultured as described by Tian et al. . BrieflyELISAs for P4, 17OHP4, and adione were performed without organic solvent extraction using kits from DRG International, Inc.  as described by the manufacturer’s protocol. ELISA for P4 was performed on sera collected from WT and CMV-hDENND1A.V2 Tg mice. In addition, P4, 17OHP4, and adione ELISAs were performed with cell culture media collected from ovarian theca interstitial and granulosa cells from F3 WT and CMV-hDENND1A.V2 Tg mice, treated with and without 20 μM forskolin for 48 h, and normalized by cell protein. The P4 antibody displayed 100% cross reactivity with P4; 17OHP4, 0.3%; corticosterone, 0.2%; pregnenolone 0.1%; <0.01% for DHEA, DHEAS, estriol, estradiol-17β, DHT, cortisol, testosterone (T), and adione. The 17OHP4 ELISA antibody displayed 100% cross-reactivity with 17OHP4; 11-desoxycortisol, 1.4%; P4, 1.2%; deoxycortisol, 0.05%; corticosterone, <0.05%; and <0.01% for DHEA, DHEAS, estriol, estradiol-17β, DHT, cortisol, aldosterone, and adione. The adione antibody displayed 100% cross-reactivity to adione; progesterone, 0.01%; and < 0.01% to 17OHP4; 17OHP5, DHEA, androsterone, cortisol, T, dihydrotestosterone, estriol.p-value was < 0.05. InfoStat software [Statistical analysis was performed with Prism 8.3 by GraphPad Software . A t-test was used for comparison of two groups of data, and a one-way ANOVA with Bonferroni corrections when multiple groups of data were compared. Data were presented as means ± standard errors. Normality of data was assessed by the D’Agostino and Pearson omnibus normality test. Samples were considered significantly different when the software  was also


Fibroblast-like synoviocytes (FLS) play a critical role in the pathogenesis of rheumatoid arthritis (RA). Chronic inflammation induces transcriptomic and epigenetic modifications that imparts a persistent catabolic phenotype to the FLS, despite their dissociation from the inflammatory environment. We analyzed high throughput gene expression and chromatin accessibility data from human and mouse FLS from our and other studies available on public repositories, with the goal of identifying the persistently reprogrammed signaling pathways driven by chronic inflammation. We found that the gene expression changes induced by short-term tumor necrosis factor-alpha (TNF) treatment were largely sustained in the FLS exposed to chronic inflammation. These changes that included both activation and repression of gene expression, were accompanied by the remodeling of chromatin accessibility. The sustained activated genes (SAGs) included established pro-inflammatory signaling components known to act at multiple levels of NF-kappaB, STAT and AP-1 signaling cascades. Interestingly, the sustained repressed genes (SRGs) included critical mediators and targets of the BMP signaling pathway. We thus identified sustained repression of BMP signaling as a unique constituent of the long-term inflammatory memory induced by chronic inflammation. We postulate that simultaneous targeting of these activated and repressed signaling pathways may be necessary to combat RA persistence. Key features of these cells during homeostasis include the production of extracellular matrix (ECM) components and providing nutrients to the synovial fluid (SF)2. Healthy synovium is composed of multiple layers of FLS, which forms the synovial lining and sublining through cell–cell contacts4. Inflammatory and pro-resolving mediators are tightly regulated to maintain normal synovium functioning1. However, in inflammatory and autoimmune diseases such as rheumatoid arthritis (RA), an imbalance between these signals causes homeostasis disruption leading to synovial tissue damage, cartilage destruction and bone degeneration.Fibroblast-like synoviocytes (FLS) are synovial tissue-resident and specialized mesenchymal cells critical for homeostasis4. The transformed FLS are major source of proinflammatory cytokines, cartilage degrading enzymes as well as the osteoblast activating factor and thereby possess an ability to induce synovitis, arthritis and osteoporosis4. Recent genome wide studies have established that multiple signaling and transcriptional factors with potential pathogenic activities exhibit epigenomic reprogramming in the RA FLS7. The inflammatory milieu in RA synovium promotes an abnormal epigenetic landscape, modulates chromatin accessibility and favors the pathogenic processes observed in RA FLS such as invasion, migration and production of proinflammatory mediators9. The vital role of tumor necrosis factor-alpha (TNF) in triggering and maintaining an aggressive FLS phenotype has been well documented both in the in vivo and in vitro studies13. Hence, manipulating TNF-induced sustained chromatin activation in RA FLS represent a treatment opportunity that might aid in combating RA persistence and therapeutic resistance.In the course of RA presentation, persistent inflammatory signals drive the development of an active inflammatory state, which leads to a persistent aggressive phenotype in FLS. These aggressive FLS are often referred to as transformed FLS15. Recent studies suggest that treatment with TNF prolonged chromatin accessibility and histone modifications for up to 72 h in the RA-FLS10. Other studies have also shown that FLS gain a long-term inflammatory memory due to the exposure to TNF15. However, the identity of the genes and the pathways that attain a long-term inflammatory memory remain undefined. Furthermore, the duration for which the inflammatory memory persists is also not well understood. In the present study, using an integrative analysis of our own and publicly available epigenomic and transcriptomic data, we determined the identity of the key signaling pathways that are part of the inflammatory memory and likely contribute to the persistent inflammatory phenotype of RA-FLS. We compared the short-term and chronic TNF-induced transcriptomic and epigenetic reprogramming in mouse and human FLS derived from healthy and chronically inflamed arthritic joints. Our results suggest that chronic exposure to TNF contribute to the development of long-term inflammatory memory and thereby switching FLS from passive responders to imprinted aggressors through the activation and repression of critical signaling networks.Previous studies comparing human RA-FLS to macrophages showed that TNF treatment could induce sustained gene expression in RA-FLS but not in macrophages16. These primary FLS from the Tg197 mice expressed high levels of the human TNF transgene  data from multiple sources; where normal primary mouse FLS (mFLS) between passages 3–5 were treated with or without recombinant human TNF for 16, 24 and 48 h from independent data sets generated in our laboratory and public data sets available on NCBI-Sequence Read Archive  and prostaglandin I2 receptor (PTGIR), pro-angiogenic vascular endothelial growth factor C (VEGFC), a member of the poly(ADP-ribose) polymerase protein family (PARP14) as well as the Fas cell surface death receptor (FAS). We next performed a prediction analysis to determine the transcription factors that are likely to regulate the 52 SAGs, using the transcription factor enrichment analysis ChEA318. This analysis prioritizes transcription factors based on the overlap between given lists of differentially expressed genes based on the putative targets as determined by ChIP-seq experiments from ENCODE projects. The results revealed RELA, STAT1, STAT2, JUN and JUND among the top 10 transcription factors predicted to regulate the expression of SAGs , Birc3 , RELB (encodes for p50 NF-kB transcription factor), FAS (encodes for TNF superfamily cell surface receptor involved in programmed cell death) and IRF1  are examples of components of various proinflammatory signaling cascades. The RNA-seq peak intensities at the exons of each of these genes was higher in both short-term and chronic TNF conditions, compared to the untreated controls. Concomitantly, the intensity of ATAC-seq and DNase-seq peaks at the promoters of these genes was also increased by both short-term and chronic TNF-exposure.In order to determine whether the genes exhibiting sustained differential expression between short-term and chronic TNF-exposure also exhibited sustained changes in chromatin accessibility, we compared the genes assigned to the chromatin accessibility peaks with that of the mSAGs Fig. A. We obsily PARP1 as well ily PARP1 as well BMP4), its downstream transcriptional effector SMAD9  and the BMP signaling target and transcriptional regulator from the family of the inhibitor of DNA binding and cell differentiation proteins (ID4) were a part of this predicted network. CheA3 transcription factor enrichment analysis tool predicted that the transcriptional repressor CCCTC-Binding Factor (CTCF)19 is one of the top candidates likely to downregulate the expression of hSRGs in response to chronic inflammation  mSRGs contained accessible chromatin within 5 kb of their TSS in both short-term and chronic-TNF exposure conditions Fig.  and foun5 mSRGs c20. Epigenetics reprogramming of FLS in particular is suggested to be a major mediator of FLS transformation, RA persistence and therapeutic resistance8. Recent large-scale genome-wide studies have incontrovertibly established that FLS transformation is associated with epigenetic-remodeling. However, which of these changes are reversible and which of them are permanent is not fully understood16. A recent study identified that multiple pro-inflammatory genes in human RA FLS escaped transcriptional repression for at least up to period of 72 h of TNF treatment. These escaped genes were suggested to contribute to the persistence of synovitis observed in RA patients. This study also reported that the reprogramming seems to be restricted to RA FLS and was not observed in macrophages due to their tolerogenic mechanisms, which suppress the expression of TNF-inducible genes14. We aimed to determine whether the absence of tolerogenic mechanisms persists for a long-term and if escape from transcriptional repression can also be observed in FLS from TNF-transgenic mouse model of inflammatory arthritis. The irreversible transformation of FLS behavior directly depends on the signaling cascades and transcriptional networks that are reprogrammed due to the epigenetic mechanisms5. We set out to identify the signaling and transcriptional networks that are specifically susceptible to long-term modulation. It was previously reported that the genomic responses of FLS from TNF-transgenic mice greatly resemble those of RA FLS from humans, suggesting that molecular mechanisms underlying long-term inflammatory memory overlap between mice and humans16. However, FLS from the Tg197 mice differ from human RA FLS, as they continue to express human TNF from the transgene, even under culture condition, where as the human primary RA FLS are not under the direct influence of TNF. We therefore performed a comparative analysis of next-generation sequencing data from 28 different mFLS samples that were subjected to TNF either for short periods or for chronic exposure in the inflamed joints and compared these results with the genome-wide data from 9 human RA FLS samples treated with TNF for varying time periods. Some of these data were generated in our laboratory and others were accessed from the public repositories16. This exercise provided a critical insight into the molecular understanding of the sustained epigenetic remodeling. Importantly, it also led to the identification of the signaling cascades both activated and repressed by these epigenomic changes  and intermediate components such as NF-κB subunit 2 , NF-κB inhibitor alpha  and NF-κB inhibitor epsilon . Other SAGs that directly and indirectly contribute to the sustained and exacerbated NF-κB signaling included the TNFAIP3, which encodes the deubiquitinating enzyme A20, that plays a role in the NF-κB non-canonical activation23. The C–C motif and multiple C-X-C motif chemokine ligands24 and prostaglandin synthesis pathway components were other additional pro-inflammatory pathways that show sustained activation in the RA FLS25. Together these data have uncovered that FLS attain a robust long-term inflammatory memory in response to chronic inflammation. The SAGs identified to possess “long-term inflammatory memory” are likely to govern the aggravated and sustained responses to inflammatory stimuli in the transformed RA-FLS.Our pathway and functional prediction analysis for mSAGs also revealed their strong association with inflammatory diseases and pathways, which aligns with the established roles for pro-inflammatory signaling and transcriptional networks in autoimmune joint diseases. Hence, our data showing that NF-κB, STAT and AP-1 signaling pathways exhibit sustained activation was expected27. A previous study reported that BMP4 is downregulated in the synovial tissue of RA patients28. Others reported that BMP4 plays a critical role in upregulating the expression of SMAD9 due to multiple BMP-responsive elements located at the promoter region of SMAD9 gene29. Further, treatment with exogenous BMP ligands was beneficial as it inhibited the pro-inflammatory phenotype of RA FLS30. Here, we identified that BMP4 and SMAD9 are a part of the long-term inflammatory memory that downregulates their expression. Taken together, we speculate that the sustained repression of BMP signaling may be critically required to ensure the persistently transformed phenotype of the RA FLS.Notably, we observed that chronic TNF induces certain genes to undergo sustained repression, which we refer to as SRGs. At the functional level, the SRGs were predicted to function in pathways regulating stem cell proliferation, growth and differentiation. Interestingly, our study also suggests that SRGs may regulate BMP signaling pathway. In fact, BMP signaling was found to be active in several rheumatic diseases such as oligoarticular juvenile idiopathic arthritis (JIA) and RA. However, the expression of key molecules involved in this signaling pathway seems to differ with disease context and its implications in RA are not completely understoodID4 gene as an SRG. ID4 belongs to the family of the inhibitor of DNA binding domain (ID) containing transcriptional regulators with pivotal roles during development, adult tissue homeostasis and tumorigenesis31. However, their roles in FLS and RA progression are not known. ID proteins are targets of the BMP signaling32, and BMP-SMAD-ID signaling network was previously reported to play a role in tumor growth and angiogenesis35. Interestingly, the BMP-SMAD-ID axis was reported to suppress p16/INK4A-mediated cell senescence during the reprogramming of fibroblasts into pluripotent stem cells36. Since cellular senescence is known to contribute to the increased release of catabolic and inflammation promoting molecules by several cell-types, including the FLS37, our data suggest that the sustained repression of the BMP4-SMAD9-ID4 axis may function as another unique means to bolster the catabolic phenotype of the RA FLS.Another interesting result in our study is the identification of In summary, this study uses bulk RNA-seq data of primary FLS from RA patients and mouse models to show that multiple signaling networks are irreversibly modified due to the TNF-mediated long-term epigenetic and transcriptomic reprogramming. These genetic factors may together contribute to the persistence in FLS transformation and inflammatory joint diseases. Large-scale single cell transcriptomic and epigenetic studies on normal and patient synovial biopsies will further aid in confirming the pathological significance of these findings. The identification of similar mechanisms in mouse and human FLS suggest that mouse models serve as valuable tools to study at least some of the aspects of therapeutic resistance in inflammatory joint diseases. Future studies such as genetic manipulation of the identified SAGs and SRGs in mice will help establish their pathophysiological significance in RA. Therapeutic targeting of the signaling pathways identified in this study could provide a safer alternative to the use of epigenetics modifying drugs, which are often associated with off-target side effects.11. Hind paws from wild-type mice with a C57/B6 background were deskinned, followed by digestion with 1 mg/ml collagenase IV (Sigma-Aldrich) for 2 h at 37 °C under sterile conditions36. Cells were cultured in DMEM with 10% fetal bovine serum (Invitrogen) and utilized at passage-4.All methods were carried out in accordance with the institutional guidelines of Emory University. Animal experiments were approved by the institutional care and use committee (IACUC) at Emory University. FLS were prepared from wild type C57BL/6 mice. FLS were isolated by digesting interphalangeal joint synovium according to previously described protocolp-value < 0.05 (Audic Claverie test) followed by Benjamin-Hochberg multiple testing corrections for determining false discovery rate.Mice fibroblast cells (FLS) treated with 5 ng/mL recombinant human TNF (R&D Systems) for 16 h were harvested at 95% confluence. Total RNA was extracted and purified using the RNeasy Mini Kit (Qiagen). Only samples with an RNA integrity number (RIN) > 7.5 were used. Libraries were generated from 250 ng RNA using TruSeq Stranded Total RNA Sample Prep Kit (Illumina). Sequencing was carried out using Illumina Hi-Seq 2500 System . Except for mouse FLS treatment, all other RNA-seq data were downloaded as FastQ files  by PCR amplification using Illumina sequencing adaptor primers (Illumina). Paired-end sequencing was performed on a Hi-Seq 2500 System (University of Georgia Genomics Core) to generate data in FastQ file format. In order to study chromatin accessibility changes due to chronic inflammation, we downloaded FastQ files from a DNase I hypersensitivity sequencing (DNase-seq) experiment of wild-type FLS and inflamed FLS from inflammatory arthritis mouse model  for 16 h and were processed according to standard ATAC-seq protocol described previously39. Functional protein–protein analysis was performed using STRING protein–protein interaction plugin on Cytoscape, an open source software platform for visualizing complex networks pathway analysis tool41. Open-source tool, ChEA3 that uses ChIP-seq experiments from the ENCODE database was used to predict transcription factors associations with SAG and SRGs18.Ingenuity Pathway Analysis tool was used to predict the canonical regulatory pathways and diseases and functions associated with the SAGs and SRGsSupplementary Information.


The external beam radiotherapy (EBRT) applied for prostate cancer (PCa) has been one of the most important and hottest research fields over recent decades. This study aimed to explore the research hotspots of EBRT in PCa and help the researchers have a clear and intuitive reference basis for later researches.The literature scientometric analysis related to “EBRT applied for PCa” was conducted via the Web of Science Core Collection from 2010 to 2019. The Microsoft Office Excel 2019 and CiteSpace V. 5.7.R1 software were introduced for visualizing and analyzing the data.A total of 7860 relevant papers were extracted and downloaded. A total of 7828 papers were extracted and analyzed after data cleansing by CiteSpace. The tendency of published papers was comprehensively increasing from 2010 to 2019. Among all 73 countries/regions, USA published the most papers, accounting for 39%, which was the most active contributor with most publications. Australia , England  were cooperating most cohesively with other countries. Univ Toronto was the most productive institute (229), while Harvard Univ  had extensive collaborations with other institutes. The International journal of Radiation Oncology Biology Physics had the largest number of publications and the highest number of co‐citations. Briganti A had the largest volume of publications. D'Amico AV had the highest number of co‐citations. Four latest and largest clusters were identified as oligometastases, salvage therapy (SRT), prostate‐specific membrane antigen (PSMA), and hypofractionation. Thirteen references became strongest burst citations lasting until 2019. The studies of “oligometastases,” “SRT,” “PSMA,” “hypofractionation,” “postoperative radiotherapy,” and “dose and fraction regimen changes” were prevailing in the recent years.The “oligometastases,” “SRT,” “PSMA,” “hypofractionation,” “postoperative radiotherapy,” and “dose and fraction regimen changes” may be the state‐of‐art research frontiers, and related studies will advance in this field over time. We forecast the research tendency in the field of EBRT applied for prostate cancer: The “oligometastases”, “SRT”, “PSMA”, “hypofractionation”, “postoperative radiotherapy” and “dose and fraction regimen changes” may be the state‐of‐art research frontiers, and related studies would advance in this field over time. Then, the Boolean operation Appendix  was usedThe WoSCC covered one of the largest worldwide databases of peer‐reviewed publications,2.2All 7860 papers were extracted with full records and cited references in the WoSCC, the retrieval results were exported to both Microsoft Office Excel 2019 and plain texts for analyzing. The Microsoft Office Excel 2019 was used for analyzing the distribution of publication types and the trend of the numbers of annual publications. The CiteSpace V. 5.7.R1 software33.1In total, 7860 articles from 2010 to 2019 were retrieved and analyzed. As shown in Figure After importing data to CiteSpace V. 5.7.R1 software for removing duplications and data cleansing, zero duplication was found, and the total 7828 papers were extracted and analyzed through data cleansing.3.2All 7860 papers were published in 73 countries/regions (WoSCC). Figure 3.3All 7860 papers were published in 7034 institutes. Figure 3.4A total of 7860 papers were published across 1139 journals, 27% of the total papers published by the top 10 journals with high outputs. Among the top 10 journals with high outputs Table , the Int3.5A total of 7860 papers were totally published by 30 377 authors, with 9% of the total papers published by the top 10 productive authors. Briganti A ranked the first  among the top 10 productive authors, followed by Nguyen PL , and Karnes RJ  Table . Each of3.63.6.1All papers from 2010 to 2019 were loaded into CiteSpace V. 5.7.R1 software for analyzing the paper co‐citation network, and the time slice was selected for 1 year with pathfinder. Figure 3.7The top four latest clusters , prostate‐specific membrane antigen (PSMA), and hypofractionation) are presented in Table 3.7.1The citation bursts were considered as papers that were received drastic augments in the references, could partially response the research dynamics and hotspots of a field. The whole papers were exported by CiteSpace V. 5.7.R1 software each year from 2010 to 2019, with a total 58 references tagged as citation bursts Table , which h44.1The field of EBRT for PCa has been obtained great attention for decades, and the total papers are increasing over time. Nine countries of the top 10 productive countries are from developed countries except China. Cooperation among countries is extensively close, especially in the western countries. In particular, the value of centrality of Australia is the highest, which may indicate that it has an obvious advantage over other countries in a certain research direction, resulting in a high degree of cooperation with other countries. China and Japan are the only two Asian countries that have entered the top 10 productive countries, but their cooperation intensities are weaker than those of European, North American, and Oceanian countries. The research institutes of this field are also keeping close cooperation with each other. The top 10 research institutes with most publications are all derived from developed countries, with nine institutes from USA, one institute from Canada.4.2Among all the 1139 academic journals, the International Journal of Radiation Oncology Biology Physics ranks first concerning the number of published papers. And the number of citations of this journal also ranks first, showing the profound influence in this field. Each of the top 10 authors contributes at least 51 papers. As a result, they are classified as “prolific authors.” D'Amico AV, Zelefsky MJ, and Bolla M are the most prominent authors with high co‐citations, indicating that the three authors have high‐quality papers.4.34.3.168Ga‐PSMA‐11 PET/CT (positron emission tomography/computed tomography) has been exhibited superior results for the early detection of oligometastases, especially at low PSA levels.The early detection for PCa oligometastases4.3.2p = .67) and superior outcome than late SRT (LSRT) (PSA > 0.5)  for high‐risk PCa patinets using high‐dose radiotherapy.Ghadjar et al4.3.368Ga‐PSMA‐11 PET/CT was significantly more sensitive than standard imaging 68Ga‐PSMA‐PET can detect the recurrent PCa lesions at the serum PSA levels low enough to cause changes in the stage of primary TNM, which may alter the treatment regimen,68Ga‐PSMA could be used for detecting early biochemical recurrence setting, which allowed the early identification of potential metastatic lesions and provided a trustworthy method for pre‐SRT staging.68Ga‐PSMA PET.68Ga‐PSMA PET/CT might make modifications of the first proposed treatment strategy in about 99/131 (76%) men, what predominantly included continuing surveillance, SRT, SBRT, hormonal manipulations, etc.4.3.4Moderately HFRT for PCa is well tolerated and feasible in the localized4.44.4.1p < .0001). However, there may be overtreating for patients never experience relapse by implementing with ARTEven though the tumors are removed completely by radical prostatectomy, it still remains controversial whether radiotherapy of postoperative patients with PCa is required. Although not all PSA relapsing will change to clinical progression,4.4.2The dose range of the non‐DE‐CFRT is limited to 64–70 Gy, due to the long‐term toxicity risks of threatening the rectum and bladder.4.4.3Proton therapy and heavy ion therapy have the characteristics of Bragg peak, which contribute a higher dose ratio between the target and adjacent normal tissue compared with photon therapy. Studies have shown that PCa patients are acceptable by treating with post‐prostatectomy proton therapy, which has the favorable acute gastrointestinal and genitourinary toxicity rates through the minimum 3 months follow‐up.The strategy to administer radiotherapy in PCa patients, therefore, needs to be carried out by the patient and multidisciplinary treatment group with full consideration of a series of patient/clinical relate factors  to achieve better prognosis.5This scientometric investigation on the application of EBRT for PCa is helpful for researchers to grasp the hotspots and trends of this field. By retrieving and collecting documents from the WoSCC, the data analysis is more objective, comprehensive, and reproducible, which can offer large information in this field, even helping researchers obtain massive data in a short time.However, there are also some limitations in this study. First of all, the WoSCC is the only data source of this study, some databases, like PubMed, Scopus, and Google Scholar, are not involved and analyzed. Second, the majority of papers are published in English and the few non‐English papers are not included. Third, a deviation may exist in the results of study measurement analysis and the actual status of the studies, because the cited times of some papers published recently is not high. Forth, there may be some missing items in the plain texts downloaded from WoSCC, which makes the total results of CiteSpace 7828, while WoSCC is 7860. But the missing papers were only 32, which might not affect the total results of this study. Finally, all papers were retrieved on 20 August 2020, but the literatures published in 2020 were not included in this analysis. Nevertheless, this study covered an overwhelming majority in the documents published since the year of 2010. The small number of papers may not change the entire tendency in this study.In conclusion, the “oligometastases,” “SRT,” “PSMA,” “hypofractionation,” “postoperative radiotherapy,” and “dose and fraction regimen changes” may be the hottest research frontiers in radiotherapy field. The four clusters and 13 references with strongest burst citations should be considered carefully for the fresh researchers in this field. This study will be of great significance in the field of EBRT for PCa, particularly for clinical decision‐making and management of PCa patients.The authors made no competing interests.RL conducted the search strategy, analyzed the data, wrote and revised the manuscript. XL and BY conceived and designed the study, critically revised the manuscript. JQ critically revised the manuscript and provided the final approval of the manuscript. All authors read and approved the final manuscript.The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.This study is a bibliometrics analysis, which does not contain any studies with human or animals performed by any of the authors.Data and material can be obtained upon request.Table S1‐S12Click here for additional data file.Appendix S1Click here for additional data file.


It is postulated that CS is mainly responsible for critical COVID-19 cases, including acute respiratory distress syndrome (ARDS). Recognizing the therapeutic potential of Spirulina blue-green algae (Arthrospira platensis), in this in vitro stimulation study, LPS-activated macrophages and monocytes were treated with aqueous extracts of Spirulina, cultivated in either natural or controlled light conditions. We report that an extract of photosynthetically controlled Spirulina (LED Spirulina), at a concentration of 0.1 µg/mL, decreases macrophage and monocyte-induced TNF-α secretion levels by over 70% and 40%, respectively. We propose prompt in vivo studies in animal models and human subjects to determine the putative effectiveness of a natural, algae-based treatment for viral CS and ARDS, and explore the potential of a novel anti-TNF-α therapy.An array of infections, including the novel coronavirus (SARS-CoV-2), trigger macrophage activation syndrome (MAS) and subsequently  The novel coronavirus disease (COVID-19) is an emerging contagious respiratory tract illness caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)  -α and interleukin (IL)-6, to cause a Ishikawa .An influx of TNF-α, as part of the CS, destabilizes endothelial cell networks and induces damage of vascular barrier, capillary damage, diffuse alveolar damage (DAD), apoptotic cell death, and multi-organ failure. Furthermore, a recent analysis indicated higher systemic levels of IL-2, IL-7, IL-10, monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1A (MIP-1A), and TNF-α, among critically ill COVID-19 patients  Shimizu . ARDS reConsidering the role of TNF-α in triggering COVID-19-related cytokine storm syndrome (COVID-CS) and ARDS, it is necessary to develop new approaches for anti-TNF therapy. Indeed, since the outbreak of the pandemic, TNF-α blockers have shown promising outcomes in treating, and mitigating, severe illness  extract.Herein, a novel approach is proposed for TNF-α inhibition, based on a treatment with the blue-green algae Spirulina  to different doses of Spirulina extracts, cultivated in either full-range solar spectrum or controlled light conditions. We report that an aqueous extract of a photosynthetically controlled Spirulina (LED Spirulina) inhibits TNF-α secretion by over 70% from LPS-activated macrophages and over 40% from LPS-activated monocyte cells.Cell lines for measuring anti-inflammatory activity human monocyte (THP-1) and murine macrophages (RAW 264.7) were used. All were provided by the American Type Culture Collection . Additionally, Rosewell Park Memorial Institute (RPMI) 1640, Dulbecco’s Modified Eagle Medium (DMEM) were purchased from ATCC. Fetal bovine serum (FBS), penicillin-streptomycin, sodium pyruvate, and glutamine were purchased from Biological Industries . 2-Mercaptoethanol was purchased from BIO-RAD and ELISA kits were purchased from PeproTech. LPS was purchased from Sigma-Aldrich .Spirulina (UTEX 3086) was cultivated in Zarrouk medium , here referred to as “Solar Spirulina”, and (B) red/blue/UV at irradiance of 750 μmol/(m2s) , here referred to as “LED Spirulina”.Temperature was kept at 31 ± 2 °C and pH was maintained at 10.8 ± 0.2 °C. Cultures were grown under two illumination conditions: (A) full-range solar spectrum at irradiance of 750 μmol/ and a Li-Cor quantum sensor. Both cultures were water-extracted using physical freeze-thawing for cellular disruption  DDW with 0.1% formic acid and (B) acetonitrile containing 0.1% formic acid. The gradient started with 2% B then increased to 30% B in 4 min, then increased to 40% B in 1 min and kept isocratic at 40% B for 3 min. Then increased to 98% B in 6 min and kept isocratic at 98% B for 9 min.Phase B returned to 5% in 3 min and the column allowed to equilibrate at 5% B for 5 min before the next injection. The flow rate was 0.4 mL/min. MS/MS analysis performed with heated electrospray ionization (HESI-II) source connected to a Q Exactive™ Plus Hybrid Quadrupole-Orbitrap™ Mass Spectrometer Thermo Scientific™. ESI capillary voltage was set to 3500 V, capillary temperature to 300 °C, gas temperature to 350 °C, and gas flow to 35 mL/min..The mass spectra (m/z 67–1000) acquired in negative and positive-ion mode with high resolution . Data-dependent MS/MS was performed with collision energy of 15, 50, and 100 eV5 cells/well) were inoculated into a 96-well plate and cultured for 48 h in DMEM 5% FBS (v/v) and penicillin-streptomycin (100 IU/mL and 1 µg/mL) at 37 °C with 5% CO2. Various concentrations of the Spirulina extracts were added 20 min prior to LPS (100 ng/mL) stimulation for additional 4 h of culturing. The concentrations of the cytokines were measured in the ELISA reader infinite M200 PRO  at 450 nm with correction at 620 nm.The amounts of TNF-α and IL-6 secreted from mouse macrophage cells were measured with TNF-α and IL-6 ELISA kits (PeproTech) according to manufacturer instructions. First, RAW264.7 cells (1 × 105 cells/well) were inoculated into a 96-well plate and cultured for 48 h in RPMI 1640 culture medium supplemented with 10% (v/v) heat-inactivated FBS, 1% sodium pyruvate (11.0 mg/mL (100 mM)), 0.05 µM 2-mercaptoethanol, and 1% penicillin-streptomycin (100 IU/mL and 1 µg/mL) at 37 °C with 5% CO2. Various concentrations of Spirulina extracts were added 20 min prior to LPS (100 ng/mL) stimulation for additional 2 h of culturing. The concentrations of the cytokines were measured in the ELISA reader infinite M200 PRO  at 450 nm with correction at 620 nm.The amount of TNF-α secreted from human monocyte cells was determined with TNF-α ELISA kit (PeproTech) according to manufacturer instructions. THP-1 cells . Compound putative identification was performed based on MS spectral data using MzCloud database and HRMS and isotope profile data using ChemSpider database.p < 0.05.All in vitro experimental data was obtained from three separate experiments performed in three replicates, and statistical significance was determined by one-way analysis of variance (ANOVA) and post hoc Tukey, using the Statistical Analysis System program, PRISMA 8. The difference between the significance levels was set to P value of 0.01 and 7.8×10–10, respectively.The metabolomics profile from Spirulina extracts was determined using un-targeted LC/MS/MS. PC analysis of the 109 detected features (metabolites) is displayed in Fig. In addition, C-phycocyanin (CPC) bioactive compound was also significantly increased in the LED extract, by 4.7-fold.Monocytes and macrophages play a pivotal role in COVID-related CS; therefore, the inhibitory effect of the extracts related to CS was tested in both macrophages and monocyte cells.Our results illustrate the effects of the two extracts (Solar Spirulina and LED Spirulina) on LPS-induced secretion of TNF-α from mouse macrophages. LED Spirulina shows a 70% reduction in TNF-α levels at the concentration of 0.1 μg/mL Fig. b, while Since monocytes contribute to COVID-CS . Such cultivation conditions may be realized in a fully controlled, indoor system, uninterrupted by diurnal or seasonal variations.We suggest at least three advantages of this novel approach of a natural, algae-based CS shield, and note that it requires further research.First, as TNF-α is a key player in many acute inflammatory reactions, acting as an amplifier of inflammation, algae-based anti-TNF blockade has the potential to assist in treating other virus-induced CS, such as COVID-19, influenza, and autoimmune-related inflammatory diseases.Second, and on condition that in vivo experiments establish the effectiveness of LED Spirulina in reducing CS, our proposed novel treatment could be dispensed extensively without putting the health of patients at risk, since Spirulina is FDA approved as a dietary supplement, and since its administration is non-intrusive, and can be orally absorbed.Third, if animal and clinical trials confirm the efficacy of this anti-TNF therapy at the rates reported above, and the substance is available to the general population, a robust therapeutic intervention can be expected that is independent of vaccines, their requisites and potential ramifications, including skilled healthcare workers in clinics and possible side effects, especially in vulnerable populations with chronic diseases, which could only be detected in phase IV clinical trial years after administration.Concomitantly, an algae-based anti-TNF therapy should be unaffected by variations in SARS-CoV-2 genome across major mutation clusters (Toyoshima et al. An ancillary, yet substantial benefit, such as therapeutic intervention—should it be proven efficient in human subjects—will free up capacity of ICUs, enable patients to receive over-the-counter affordable treatment option.Drawing on our in vitro results, we stress the need for prompt in vivo studies to assess the effectiveness of various Spirulina extracts among animal models, as well as in clinical trials.In addition, future research should note that the effect on TNF-α expression was greater than on IL-6. This finding suggests that the extract targets TNF-α secretion selectively and further validation with a more detailed mechanism of the anti-inflammation cascade within macrophages and monocyte cells should be explored.It should likewise be noted that the extract from the LED-controlled cultivation process was significantly more effective at suppressing release of TNF-α. To date, no natural compound has been proven to suppress specifically TNF-α. Therefore, LED Spirulina could pave the way for novel algae-based bioactive compounds as anti-TNF treatment.


HT vector and infiltrated into Nicotiana benthamiana leaves. The plant-expressed chimeric HBcHEV ORF2 551–607 protein was recognized by an anti-HBcAg mAb and anti-HEV IgG positive swine serum. Electron microscopy showed that plant-produced chimeric protein spontaneously assembled into “knobbly” ~34 nm diameter VLPs. This study shows that HBcAg is a promising carrier platform for the neutralizing epitopes of hepatitis E virus (HEV) and the chimeric HBcAg/HEV VLPs could be a candidate for a bivalent vaccine.The core antigen of hepatitis B virus (HBcAg) is capable of self-assembly into virus-like particles (VLPs) when expressed in a number of heterologous systems. Such VLPs are potential carriers of foreign antigenic sequences for vaccine design. In this study, we evaluated the production of chimeric HBcAg VLPs presenting a foreign epitope on their surface, the 551–607 amino acids (aa) immunological epitope of the ORF2 capsid protein of hepatitis E virus. A chimeric construct was made by the insertion of 56 aa into the immunodominant loop of the HBcAg. The sequences encoding the chimera were inserted into the pEAQ- Every year according to the World Health Organization, hepatitis E virus (HEV) causes approximately 20 million infections worldwide and 44,000 deaths related to hepatitis E. HEV infection is a self-limiting disease but can progress to chronic hepatitis. Pregnant women, as well as immunocompromised and immunosuppressed patients, are the high-risk groups with higher mortality rates and susceptibility to chronic HEV infection . The morHEV is a single-stranded, positive-sense RNA virus that belongs to the Hepeviridae family . Seven mVLPs are highly immunogenic structures and can induce protective immunity. The hepatitis B capsid protein (HBcAg) has been shown to be capable of self-assembly into VLPs when expressed in a number of eukaryotic heterologous systems including mammalian cells , plants Nicotiana benthamiana plants of chimeric HBcAg VLPs carrying this epitope and evaluate the particles as candidate HEV vaccines. Transient expression with the pEAQ-HT vector of a HBcHEV ORF2 551–607 construct led to the production of chimeric VLPs. We examined the ultrastructure of the particles by electron microscopy and showed that they consisted of particles 28–38 nm in diameter, typical of HBcAg VLPs, with a “ragged” surface, which was most likely due to the location of the HEV ORF2 56 aa on the top of the HBc spikes. Furthermore, the chimeric protein was recognized by an anti-HEV IgG positive swine serum and retained HBcAg reactivity. From this study, we believe that HBcHEV ORF2 551–607 protein could be used for future immunological studies. Amino acids 551–607 from HEV ORF2 are immunogenic epitopes from the main immunogenic region of the ORF2. To improve the immunogenicity of this sequence, HBcAg VLPs have been used as particulate carriers. Here, we describe the efficient expression in SalI and AseI restriction sites , which were used for sub-cloning of the PCR fragment. The HEV ORF2 551–607 peptide was cloned into the MIR of the monomeric HBcAg construct in the previously described pEAQ-mEL [Escherichia coli XL1-Blue, putative clones harboring the expression vector (pEAQ-HT:HBcHEV ORF2 551–607) were verified by sequencing . The 56 amino acid residues of HEV ORF 2 551–607 consensus sequences were based on HEV genotype 3 nucleoprotein HEV ORF2 1–660 (GenBank accession number DQ079627.1). Amplification of DNA fragment for the production of the HEV ORF2 501–607, was carried out by PCR. The target HEV ORF2 551–607 sequences were amplified using the synthetic master gene HEV ORF2 1–660 . The PCRpEAQ-mEL . The resHT:HBcHEV ORF2 551–607 was transformed into the electrocompetent A. tumefaciens strain LBA4404. Then, transformed colonies were selected from agar plates containing 50 μg/mL kanamycin and 50 μg/mL rifampicin. Agrobacterial suspensions were cultured at 28 °C and shook (200 rpm) for 48 h. The agrobacterial cells were pelleted by centrifugation (4000× g centrifugation for 10 min.). Following resuspension in 10 mM MES 2-(N-morpholino) ethansulfonic acid, pH 5.6, 10 mM MgCl2 and 100 µM acetosyringone to an OD600 of 0.4 and incubation of 3 h at room temperature, bacterial suspensions were syringe-infiltrated into the leaves of 5–6-week-old N. benthamiana plants. Leaf tissue was harvested 7 days post infiltration (dpi). Control agrobacterial suspensions containing the empty pEAQ-HT vector [HT:HBcAg were included to compare with pEAQ-HT:HBcHEV ORF2 551–607 for observation of protein expression and VLPs formation.The recombinant vector pEAQ-T vector  without g for 10 min and the supernatant was kept as soluble protein fraction. Nevertheless, the insoluble protein (IP) fraction could be extracted from the pellet through boiling with protein denaturing buffer  mixed 3:1 with 2-mercaptoethanol) and centrifuged at 13,000× g for 10 min. Proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 12.5% (w/v) polyacrylamide-SDS or NuPAGE Bis-Tris gels  and the gels stained with InstantBlue . For Western blot analysis, the electrophoresed proteins were electroblotted onto a nitrocellulose membrane  via wet transfer at 100 V for 1 h. Blotted membrane was blocked with 5% (w/v) milk powder in phosphate-buffered saline (PBS) containing 0.05% (v/v) Tween 20 (PBST) for at least 1 h. The membrane was subsequently incubated with either mouse anti-HBcAg monoclonal antibody  (1:4000 dilution) or HEV immunoglobulin G (IgG) positive swine serum (dilution 1:200) for 1 h. The membrane was washed three times with PBST at 5 min intervals. Then, the membrane was incubated with a goat anti-mouse IgG horseradish peroxidase (HRP)-conjugated secondary antibody  diluted 1:30,000 for detection of 10E11, and secondary anti-pig IgG antibody-HRP  diluted 1:8000 for detection of swine serum, respectively. Washing steps were repeated three times before the blot was soaked in SuperSignal™ West Dura Extended Duration Substrate . The emitted luminescence was detected with the ImageQuant LAS 500 system  and on X-ray film. Small-scale protein extraction was conducted to test for HBcHEV ORF2 551–610, and mHBAg expression and accumulation. Approximately 90 mg of the infiltrated leaf was harvested at 7 dpi and homogenized with a ceramic bead for 30 s at speed 4  in 3× volume of extraction buffer ). Then, samples were centrifuged at 13,000× w/v) dry milk) and incubated for 1 h at room temperature. Then, 100 µL anti-HEV IgG positive and negative swine serum diluted 1:100 in blocking buffer was added and incubate for 1 h at 37 °C. Plates were washed again with PBST before the addition of 100 µL/well of an HRP-conjugated anti-swine secondary antibody 1:10,000 . After incubation with the secondary antibody, plate wells were washed three times before adding 50 µL/well of the substrate solution of OPD Substrate Tablets . Plates were incubated, in the dark, at room temperature from 10 to 20 min, and then the reaction was stopped by the addition of 50 µL/well of 1M H2SO4, and the plates were read at 492 nm in a plate reader Epoch Microplate Spectrophotometer . Previously characterized negative swine sera by using commercial kits from PrioCheck HEV Ab  were used as controls.Microtiter plates (Greiner 96-well flat bottom) were coated with 100 µL/well of serial dilutions of purified protein in duplicate wells (100 ng/well to 12.5 ng/well) in PBS (pH 7.4) and incubated overnight at 4 °C. After three washes with PBST, plates were incubated with 200 µL/well of blocking solution  at 4 °C for 20 min using an SS34 rotor . The clarified supernatant was filtered over 0.45 µm syringe filters. The VLP samples were subjected to a two-stage purification process, as described by Peyret 2015 [w/v) and 2 mL of 70% (w/v). The sucrose cushions were prepared in UltraClear ultracentrifuge tubes  and centrifuged in a Surespin 630/36 rotor  at 167,000× g, 4 °C, for 2.5 h. The gradient was fractionated by piercing the bottom of the tube with a needle and recovering the bottom and interface fractions. Then, these fractions were dialyzed thoroughly against 200 mM ammonium bicarbonate buffer (pH 8.0) overnight. The samples were concentrated using a SpeedVac vacuum concentrator , and loaded onto a Nycodenz step gradient  extending from 60 to 20% (w/v), with 2 mL of each concentration in UltraClear ultracentrifuge tubes . High-speed centrifugation was operated at 274,000× g using the TH-641 rotor  for 20 h, at 4 °C. The mHBcAg and mHBcAg-HEV ORF2 551–607 proteins were dialyzed against PBS using a Float-A-Lyzer .To purify assembled particles derived from constructs pEAQ-ret 2015 . FirstlyTo determine if particles incorporated RNA, equal protein amounts of purified mHBcAg VLPs and chimeric HBcHEV ORF2 551–607 VLPs were loaded on 1% of agarose gel. The RNA content of the different protein fractions was analyzed on ethidium bromide-stained 1% agarose gels followed by Coomassie blue staining.w/v) uranyl acetate. As an experimental control, the monomer HB core particles without a gene insert were also examined. The grids were examined using an FEI Tecnai 20 transmission electron microscope .Approximately 10 µL of the VLPs sample was adsorbed on copper-palladium grids, washed with sterile distilled water, and negatively stained with 2% (According to Peyret (2015) , we consN. benthamiana were used as templates for PCR amplification of HEV ORF2 551–607 to construct the pEAQ-HT:HBcHEV ORF2 551–607 (The nucleotide sequences of the whole HEV genotype 3 nucleoprotein (HEV ORF2 1–660)  codon-op 551–607 . PrimersA. tumefaciens and used for expression of the recombinant proteins in N. benthamiana.The constructs pEAQ-HT:HBcAg and pEAQ-HT:HBcHEV ORF2 551–607 were transformed into N. benthamiana leaves agroinfiltrated with the recombinant vector pEAQ-HT:HBcAg and pEAQ-HT:HBcHEV ORF2 551–607 were harvested on the 7th day post infiltration (dpi) and analyzed by SDS-PAGE and Western blot to check protein expression. Western blot analysis with an anti-HBc mAb (10E11)  or HBcAg at the same concentrations as controls for specific interactions. An anti-HEV IgG negative swine serum was used as a control for background signal. The anti-HEV Ig G positive swine serum specifically binds the HBcHEV ORF2 551–607 VLPs, when the concentration of the VLPs is 100 ng/well . The absA purified chimeric HBcHEV ORF2 551–607 VLPs and HBcAg VLPs fractions were loaded on an agarose gel and after electrophoresis, the gel was stained with ethidium bromide (EtBr) followed by staining with Coomassie blue for identification of nucleic acids incorporation into VLPs. The VLP fractions migrated as a band that stained positive in either EtBr , panel bN. benthamiana preference to boost translation by using codons preferred by the host expression system. In addition, (GGS)4 linkers were inserted between HBcAg and HEV ORF2 551–607, to stabilize the conformation of the chimeric VLPs and compose the multidomain protein. The development of a recombinant vaccine against HEV, in which the ORF2 capsid protein or neutralizing epitopes of ORF2 are presented as multimeric structures, is highly desirable. Here, we present a strategy for vaccine design and characterization of the HEV ORF2 551–607 epitopes that may be useful for further HEV vaccine design. The HEV ORF2 551–607 peptide DNA sequences were synthesized by PCR and cloned into HBcAg coding sequenced between amino acid 78 to 83. The HBcAg gene and HEV ORF2 551–607 sequences expressed in this study were codon-optimized based on The inserted HEV ORF2 551–607 sequences into HBc immunodominant loop (between 73 and 83 aa) is expected to be displayed on the HBcAg VLP surface. Protein modeling analysis of the chimeric VLPs , panel dIn many cases, the immunogenicity of small synthetic peptides can be increased after being attached to a carrier molecule such as VLPs. In a previous study, HBcAg carrying linear epitope located at 423–437 aa could self-assemble into VLPs and these particles could induce anti-ORF2 Abs, but the synthetic 423–437 aa peptide could not . This stN. benthamiana does not lead to necrosis or apoptosis (data not shown) which allowed us to harvest the plants at the peak of recombinant protein accumulation. In this case, the optimal harvest time for the chimeric HBcHEV ORF2 551–607 VLPs was determined to be around 7 dpi. During this study, a constant OD 600 of 0.4 for the agrobacteria infiltration suspension was used, as reported previously [N. benthamiana (Because HBcAg is an immunodominant immunogen , and inseviously . HBcAg cthamiana , panel cthamiana  and chimthamiana . Their lthamiana , panel bthamiana . The facN. benthamiana plants have been reported to encapsidate random cellular nucleic acid [The VLPs of HBcAg produced in eic acid . The preeic acid ,50.N. benthamiana plants has emerged as an alternative to other expression systems such as yeast, bacterial, and mammalian cells. Plant expression systems have many advantages in terms of safety as plant pathogens do not infect humans and also owing to their scalability and their ability to perform a eukaryotic post-translational modification [N. benthamiana plants [Transient expression of recombinant proteins in fication . Some lification . In our a plants . Plants a plants .E. coli as VLPs, including p179 and p239 [E. coli and the baculovirus-insect cell system. In our previous studies, we showed that the HEV ORF2 110–610 recombinant protein formed unstable nanosized virus-like particles with non-uniform sizes ranging from 10 to 100 nm [Currently, three HEV vaccine candidates have been under evaluation in clinical trials. Two of them have been produced in and p239 , the thiand p239 . Developo 100 nm ,25. The o 100 nm  demonstrWe report the expression and the self-assembly of chimeric HBcHEV ORF2 551–607 VLPs in the plants, representing the first report of the successful transient expression of chimeric HBV/HEV VLPs in this expression system. Hence, plants may be a novel source for the cost-effective production of multivalent vaccines. Currently, we do not know if the HEV ORF2 551–607 epitope is a neutralizing epitope. Future studies of the immunogenicity of chimeric HBV/HEV ORF2 VLPs in animal models and their ability to protect against HEV infection are ongoing.


Cirrhotic patients who were consecutively hospitalized between January 2016 and March 2019 were screened. Serum cardiac biomarkers at admission, including N-Terminal pro-B-type natriuretic peptide (NT-pro BNP), high-sensitivity cardiac troponin T (hs-cTnT), creatine kinase (CK), creatine kinase MB (CK-MB), and lactate dehydrogenase (LDH), were collected. Acute decompensating events at admission, primarily including ascites, acute gastrointestinal hemorrhage, and acute-on-chronic liver failure (ACLF), were recorded. P < 0.001). The NT-pro BNP level significantly correlated with ascites, acute gastrointestinal hemorrhage, and ACLF. The hs-cTnT level was significantly higher in cirrhotic patients with acute decompensating events than in those without decompensating events . The hs-cTnT level significantly correlated with acute gastrointestinal hemorrhage, but not ascites or ACLF. LDH , CK , and CK-MB  levels were not significantly different between cirrhotic patients with and without acute decompensating events.  The NT-pro BNP level was significantly higher in cirrhotic patients with acute decompensating events than in those without any decompensating events (median: 140.75 pg/mL versus 41.86 pg/mL,  The elevated NT-pro BNP level seems to be closely related to the development of acute decompensating events in liver cirrhosis. Liver cirrhosis is a state of systemic hyperdynamic circulation characterized by increased cardiac output and decreased peripheral resistance, especially in the presence of decompensating events . This diBNP and N-Terminal pro-B-type natriuretic peptide (NT-pro BNP) are secreted in response to increased myocardial stress –8. The NTherefore, this study is aimed at exploring the relationship between these cardiac markers and decompensating events in cirrhosis.2), (3) cardiac diseases , (4) ischemic stroke, and (5) absence of data regarding serum cardiac markers detected during the hospitalizations. Repeated admissions of the same patient were not deliberately excluded, because this study focused on the in-hospital outcome and decompensating events during the hospitalizations, but not on long-term follow-up outcomes. If a patient had multiple measurements of serum cardiac markers during the same hospitalization, we selected the data obtained at the first time of blood collection. The study protocol was approved by the Medical Ethics Committee of our hospital. We primarily collected demographic data, etiology of liver cirrhosis, decompensating events at admissions, and laboratory tests including serum cardiac markers.We have prospectively collected the demographic, clinical, and laboratory data of cirrhotic patients who were consecutively admitted to the Department of Gastroenterology of the General Hospital of Northern Theater Command  and treated by an attending physician (XQ) since January 1, 2016. Until March 31, 2019, there were a total of 761 admissions. We retrospectively screened the patients who had undergone the evaluation of laboratory data regarding serum cardiac markers during the hospitalizations. Exclusion criteria were as follows: (1) confirmed or suspected diagnosis of malignancy, (2) severe renal insufficiency (estimated glomerular filtration rate (eGFR) < 30 mL/min/1.73 mAcute gastrointestinal hemorrhage was defined as previously described , 14. The+ < 125 mmol/L, it is set to 125; if Na+ > 137, it is set to 137.If Na2 [The grade of ascites was defined according to the consensus of the International Ascites Club . Patient2 . The eGF2 , as follWe divided cirrhotic patients into 5 groups: (1) cirrhotic patients without acute decompensating events, (2) cirrhotic patients with acute decompensating events, (3) cirrhotic patients with ascites, (4) cirrhotic patients with acute gastrointestinal hemorrhage, and (5) cirrhotic patients with ACLF.All serum cardiac markers were measured at the Department of Laboratory of our hospital. They included NT-pro BNP detected by enzyme-linked immunosorbent assay (ELISA) (double antibody sandwich method) with a normal range of 0-125 pg/mL, hs-cTnT by ELISA (double antibody sandwich method) with a normal range of 0-0.05 ng/mL, CK by coupled-enzyme assay with a normal range of 38-174 U/L, CK-MB by immune inhibition assay with a normal range of 0-24 U/L, LDH by the spectrophotometric method with a normal range of 109-245 U/L, and hs-CRP by latex immune turbidimetry with a normal range of 0-3 mg/L. Only the data obtained at the first time of measurement were selected, thus avoiding the influence of drugs used during hospitalization.P < 0.05 was considered to be statistically significant. SPSS statistics software version R23.0.0.0 was employed to perform all statistical analyses.Continuous data were expressed as mean ± standard deviation and median (quartiles) and were compared by using the Wilcoxon rank-sum test. Categorical data were expressed as frequency (percentage) and were compared by using the chi-square test. Considering that age and gender are important factors influencing serum NT-pro BNP and hs-cTnT levels –26, partn = 96, 71.64%), acute gastrointestinal hemorrhage , and ACLF .Overall, 176 patients with liver cirrhosis were included , of whomP < 0.001) and hs-cTnT (P = 0.007) than those without decompensating events  and hs-cTnT (P = 0.002) than those without decompensating events  and hs-cTnT (P = 0.003) than those without decompensating events  than those without decompensating events  the NT-pro BNP level was significantly higher in decompensated cirrhosis. (ii) The NT-pro BNP level also significantly correlated with Child-Pugh and MELD scores in cirrhosis with acute decompensation, but not in those without decompensation. (iii) The hs-cTnT level was elevated in cirrhosis with acute decompensation but was unrelated to the liver disease severity. (iv) The LDH level significantly correlated with Child-Pugh and MELD scores in cirrhosis, but was unrelated to decompensating events. (v) CK and CK-MB levels were neither significantly increased in cirrhosis with decompensation nor correlated with Child-Pugh and MELD scores.NT-pro BNP, a prohormone of BNP, is secreted into the systemic circulation by cardiac ventricles in response to myocardial hypertrophy and is involved in the regulation of cardiac volume homeostasis –29. ThusThe serum NT-pro BNP level is significantly higher in patients with liver cirrhosis , 12, 33,In addition, BNP is a natriuretic hormone released from myocardial cells in response to volume expansion, end-diastolic wall stress, and possibly increased intracardiac filling pressures , 37. Hyphs-cTnT, a protein complex regulating the contraction of striated muscle, is released when myocardial ischemia induces nonreversible injury of myocardial tissue . hs-cTnTWe did not find any significant difference in CK and CK-MB levels, comparing compensated and decompensated cirrhotic patients. Moreover, there was no correlation of CK and CK-MB levels with Child-Pugh and MELD scores in cirrhosis. These analyses were performed in the groups with and without decompensating events. Traditionally, CK-MB is helpful for estimating the infarct size in acute myocardial infarction and is highly specific to heart tissue , 56 whilLDH, a cytoplasmic enzyme, exists in a wide range of tissues and is elevated when cells are damaged. LDH is not specific for the diagnosis of a disease. There are five types of serum LDH isoenzymes. Among them, LDH1 is mainly derived from the heart and LDH5 from the liver . HoweverFirst, the number of patients with ACLF was small in our cohort and the relationship between serum cardiac markers and ACLF needs further clarification. Second, a reasonable and convenient approach for quantifying blood loss volume during acute gastrointestinal hemorrhage and volume of ascites was unavailable. Third, healthy controls may make the results more comprehensive. Further studies should further consider the effects of the severity of such decompensating events on the long-term prognosis.An elevated NT-pro BNP level might be useful to identify the cardiac volume overload caused by acute decompensating events in advanced cirrhosis. Additionally, the hs-cTnT level was elevated in cirrhosis with acute decompensating events.


Although often asymptomatic and detected incidentally, varicocele is a relatively common problem in patients who seek medical attention for infertility problems. Ultrasound (US) is the imaging modality of choice for evaluation, but there is no consensus on the diagnostic criteria, classification, and examination technique. In view of this uncertainty, the Scrotal and Penile Imaging Working Group of the European Society of Urogenital Radiology (ESUR-SPIWG) undertook a systematic review of the available literature on this topic, to use as the basis for evidence-based guidelines and recommendations. This paper provides the results of the systematic review on which guidelines were constructed. Varicocele is defined as dilation of the pampiniform venous plexus draining the testicle, with reflux of venous blood , 2. AlthVaricocele is detected and graded clinically using the criteria introduced by Dubin and Amelar in 1970, a subjective evaluation which is highly dependent on the expertise of the physician . Colour There is a large, but extremely heterogeneous body of published investigations on US imaging of varicoceles. Even though variability makes it impossible to perform a metanalysis, a systematic literature review is possible.The Scrotal and Penile Imaging Working Group of the European Society of Urogenital Radiology (ESUR-SPIWG) attempted this task. The group performed a systematic review of the available literature and released a guideline and recommendation paper with the aim to standardize US imaging for varicoceles in Europe. After having formulated 15 relevant clinical questions Table  6].Tabl.Tabl6].TThe systematic literature review at the root of these recommendations is presented in its complete form in this work.The ESUR-SPIWG believes that standardization was necessary not only to improve clinical practice, but also to define the methodological basis for future studies and metanalyses, with the hope of a better understanding of the relationship between varicoceles and infertility.Data for this systematic review were collected according to the PRISMA statement 2009 guidelines . EligibiESUR-SPIWG proceeded with full awareness of the limitations of the varicocele literature. These limitations include heterogeneous patient groups, small sample sizes, lack of studies with diagnostic accuracy data, lack of randomized controlled trials or controlled studies with patient outcome data, and use of a variety of outcome measures. Overall, these difficulties precluded use of meta-analytical procedures. Instead, narrative syntheses were used to summarize the evidence for the questions of interest.When review of the literature revealed insufficient publications to address a recommendation from an evidence basis, the recommendation was obtained from expert opinion based on clinical practice, experience, knowledge and judgment. If differences of opinion among the expert emerged, consensus was achieved using the modified Delphi technique .Understanding whether the alleged correlation between impaired spermatogenesis, infertility and varicocele is evidence based is fundamental in making a recommendation for US as a valuable investigation in infertile patients. Infertility is defined as “failure to achieve a clinical pregnancy after 12 months or more of regular unprotected sexual intercourse” . ApproxiBoth clinical and laboratory studies show a detrimental effect of varicoceles on spermatogenesis , 16. TesSeveral mechanisms have been proposed to explain the link between varicoceles and impaired spermatogenesis. A multi-factorial aetiology is likely, involving both heat, oxidative stresses and androgen deprivation . The temAlthough sperm quality will frequently improve following treatment, there is conflicting evidence regarding the value of varicocele repair in male infertility –34. A syGuidelines for varicocele repair in subfertility are inconsistent. In the UK, the National Institute for Health and Clinical Excellence guidelines advises that surgery for varicocele should not be offered for fertility treatment as there is no definite evidence that it improves pregnancy rates. Opinion from the American Society for Reproductive Medicine and the Society for Male Reproduction and Urology concludes that varicocele repair may be offered to the male partner of an infertile couple where there is evidence of abnormal semen parameters and no/minimal identified female factor .Evaluation of the diagnostic potential of US imaging in patients with varicoceles is markedly hampered by difficulties in comparing studies obtained with different US classifications and different imaging parameters. A consensus should play a key role for obtaining more reliable results in future studies.There is general agreement in Europe that valuable information about varicocele can be obtained using colour flow Doppler, and that evaluation of the presence and characteristics of venous reflux is useful when physical examination alone is insufficient to determine whether treatment is required. Additionally, colour Doppler evaluation plays a key role in the assessment of persistence or recurrence of varicoceles after surgical treatment; veins often remain dilated and clinical examination alone cannot fully assess the presence or absence of persistent venous reflux.Patients with reflux have significantly impaired semen analysis parameters compared to patients without reflux . A prospConsequently, a number of sonographic classifications have been proposed to establish firm criteria for the diagnosis, treatment and prognosis of varicocele , 42–51. Some classifications grade varicoceles according to the characteristics of venous reflux only. The patient is evaluated either supine, upright, or in both positions. Hirsh et al. classify varicocele in three grades according to reflux duration while standing during the Valsalva manoeuvre. Cornud et al. classify varicoceles in three categories according to the duration of reflux during the Valsalva manoeuvre measured with spectral Doppler interrogation; there is no standardisation as to whether the examination should be performed with the patient supine or standing . Oyen anOther classifications use both morphological and Doppler parameters. Hoekstra and Witt score varicoceles by measuring the size of the dilated veins and the presence of reflux during the Valsalva manoeuvre in the upright position. Chiou et al. developed a system which assigns a maximum score of 9 points incorporating the maximum venous diameter (scores 0 to 3), the presence of venous plexuses (scores 0–3) and changes in flow direction during the Valsalva manoeuvre (scores 0–3). Diagnosis of varicocele requires a total score of at least 4 points . All parThe Sarteschi’s classification, first published in Italian in 1993  and publThe purpose of every classification system is to provide useful morphological and hemodynamic information for treating varicoceles and predicting treatment outcomes. Unfortunately, a clear consensus has not been reached, and there is no universally recognized system to classify varicocele severity. The thresholds used to differentiate between normal and pathological findings are often different and even conflicting, resulting in US examinations that can be interpreted as positive or negative for varicocele diagnosis and severity depending on the classification system used. The result is that all grading systems have a low predictive value in terms of the effect of varicoceles on impairment of spermatogenesis as well as on sperm quality improvement after varicocele correction.Since no classification system for varicoceles is universally recognized, the ESUR-SPIWG recommends recording all the parameters of the different classifications, to allow comparison among the results of different studies, regardless of which classification system is used , and how . A standardised method to measure the veins is necessary to compare results obtained in different studies.The size threshold of the veins that meets the definition for varicoceles varies in different studies, as does the examination technique employed. In particular, venous diameter is evaluated in the supine position or while standing, either at rest or during the Valsalva manoeuvre in different papers , 53–62. There is no consensus on the threshold values used to define varicocele by the maximum venous diameter. A diameter of 3 mm or more is commonly considered diagnostic for varicocele Fig. , but lowGiven the existing wide methodological variability, it is evident that standardization is needed to obtain reproducible results in future studies. For measurement of all quantitative parameters, including venous diameter, it is critically important to document the patient’s position and define the sampling site unequivocally. Non-evidence-based recommendations can be offered advising how the study should be performed. Reviewing the literature, the majority of investigators examine the patient in both the supine and upright positions during the Valsalva manoeuvre, and accept a size threshold of 3 mm during the Valsalva manoeuvre as diagnostic for varicocele , 56–59. When measuring venous diameter, it is critically important to indicate the patient’s position, measurement site (relative to the testis or to the cord), and whether the measure was obtained during the Valsalva manoeuvre or at rest. The ESUR-SPIWG recommends measuring the largest vein, irrespective of location, while standing and during the Valsalva manoeuvre. When the venous diameter is measured in this way, 3 mm or more is considered diagnostic for a varicocele  [V = L × 2W × 0.52). A third formula, introduced by Lambert et al., multiplies the product of the three diameters by 0.71 (V = L × W × H × 0.71) [US assessed testicular volume is obtained from the three linear diameters of the testes—length (L), width (W) and height (H), and varies according to the mathematical formula applied for calculation. Three different formulas are used in the literature. The most common is the ellipsoid formula, which is also the one most widely used for automated volume calculation by US systems. Using this formula, the volume is obtained by multiplying the product of the three testicular diameters by 0.52 ( × 0.52) . In anot × 0.71) . Mbaeri  × 0.71) . The gol × 0.71) .There are no uniform reference values for the normal testicular volume in different populations, probably because measurements obtained in different studies are biased by a variety of circumstances such as the geographic areas and other environmental factors, nutritional status, and ethnicity –73. At UStudies in infertile men have shown a direct correlation between semen parameters, sex hormone levels and testicular volume measured at US . An averIn patients with idiopathic hypogonadotropic hypogonadism and micropenis, Sun-Ouck et al. and Rajendra et al. found a significant increase of the testicular volume after medical treatment , 80.A relationship between the testicular volume, measured at US, and varicocele has been documented. Zampieri et al. reported that venous reflux is associated with testicular atrophy and varicocele repair is associated with testicular volume increase –83.The ESUR-SPIWG recommends measuring testicular volume in all patients with varicoceles as a surrogate evaluation of testicular function. The formula used to calculate the volume from the three diameters should be reported. Lambert’s formula is preferred  venous structures demonstrated. The patient is then placed in the upright position and the largest vein (irrespective of the location) is identified and measured during the Valsalva manoeuvre. The second part of the study is evaluation of reflux. The equipment is set to detect slow flow. The pulse repetition frequency is minimized, wall filter set to minimum and gain to the maximum below noise threshold; this can be obtained by increasing the gain up to the level at which artefacts are visible and then decreasing it to a level at which they just disappear.Colour Doppler and spectral analysis should be performed and interrogation should be obtained at the level of the inguinal canal, in the supratesticular region and in vessels at the level of the testis.The ESUR-SPIWG recommendations emphasise that reflux evaluation is the most important component in the Doppler US examination of varicoceles. Therefore, spectral analysis should supplement colour Doppler interrogation. The essential parameter to measure is the duration of reflux; measuring the reflux peak velocity is not necessary Diameter of the largest vein (irrespective of the location) measured while standing and during the Valsalva manoeuvreChanges of flow at colour Doppler interrogation and spectral analysis in the spermatic veins according to the patient’s position and before and during Valsalva .The following information should be included in the US report:Several studies have investigated intratesticular Doppler waveforms to predict parenchymal damage in infertile patients with varicoceles –105. As Several studies have investigated whether changes in testicular vascularization were appreciable, since they could represent (indirect) confirmation of this theory. The purpose of these investigations was to provide insight into the pathophysiology of varicoceles, not to identify new parameters for clinical use in patient management. Earlier studies failed to demonstrate changes in testicular blood flow in varicocele patients , 102, a .Calculation of testicular volume blood flow, however, requires estimating the cross-sectional area of the testicular artery, a difficult evaluation in a millimetric vessel. Other authors used peak systolic velocity (PSV) and resistive index (RI) or pulsatility index (PI) as more reproducible and easily obtained parameters to estimate indirectly perfusion abnormalities. Biagiotti et al. found higher PSV and RI in the testicular arteries of patients with varicoceles . Ünsal eThere is a widely held belief that a unilateral right-sided varicocele should prompt investigation for a retroperitoneal process causing obstruction of the right internal spermatic vein. While this may be justified if a varicocele appears suddenly, be it right left, or bilateral, in the majority of cases the literature does not support this approach.With an incidence of about 10% of varicoceles, bilateral clinical varicocele is relatively uncommon . HoweverAs clinical diagnosis is neither sensitive nor accurate, a right-sided varicocele is, in the majority of cases, identified at colour Doppler US when a study is requested to evaluate a clinically evident left-sided one –113.Several authors consider varicocele a bilateral disease, although on the right side, the condition is often subclinical. This explains, in their opinion, bilateral testicular dysfunction in patients with unilateral left clinical varicocele , 114. PaIn adults, there is some evidence that simultaneous repair of left clinical and right subclinical varicocele is beneficial, as even a small subclinical unrepaired varicocele continues to have a detrimental effect on testis function –120. ConIsolated right-sided varicocele is found in less than 1% of patients . In the A variety of causes can result in right-sided clinical varicocele, including impaired venous drainage from the testis by venous thrombosis, tumour invasion or compression. Isolated and/or palpable right-sided varicocele may occur in otherwise healthy patients with venous anatomical variations , such asBased on these considerations, the ESUR-SPIWG recommends undertaking colour Doppler US on both sides in patients with left-sided varicoceles, as a subclinical right-sided varicocele will be often revealed. In patients with an isolated clinical right-sided varicocele, it also recommends extending the US examination to the abdomen, to look for congenital anomalies and abdominal/retroperitoneal pathology  onwards and that this adverse effect increases with the grade of the varicocele [The need for follow-up in patients with subclinical varicoceles is debated. Current literature supports the thesis that a varicocele has a detrimental effect on testicular size and function from childhood  and adolescence  presenting with subclinical varicoceles and both normal testicle volume and semen analysis. In these patients, annual follow-up is advised by means of physical examination, scrotal US including measurements of testis size and, if patients are willing to comply, semen analysis , 131.Based on the currently available literature, the ESUR-SPIWG recommends follow-up of subclinical varicoceles for untreated adolescents and for young adults with normal testicular volume and normal semen analysis , if there is evidence of recurrence on clinical examination or if postoperative sperm analysis is unsatisfactory. In other centres both sperm analysis and US are performed 3 months after varicocele repair. In other centres sperm analysis and US are performed 3 months and 1 year after varicocele repair, with the rationale that recurrent varicocele may only appear one year or later after the procedure , 138.If varicocele repair is performed for treatment of subfertility, then sperm analysis must form the basis of follow-up. If semen analysis improves, scanning is usually unhelpful for the patient, while if semen analysis remains unsatisfactory, US can be considered to look for persistent reflux or recurrent disease.It is commonly thought that US should be extended to the abdomen in newly discovered varicoceles to identify tumours. This practice, however, is not fully supported by the available literature. The classic triad of symptoms of renal cell carcinoma (RCC) is haematuria, flank pain, and a palpable abdominal mass. Other paraneoplastic signs such as fever or general malaise may also be present with retroperitoneal tumours. A number of case reports and small case series suggest that varicoceles are a possible mode of presentation of retroperitoneal tumours –152. ThiThe mechanism for the development of a symptomatic varicocele is not always extension of tumour/thrombus along the renal vein. Pathological extrinsic compression of the venous drainage of the pampiniform plexus by the primary tumour or enlarged lymph nodes, or an increased blood supply to the tumour may also lead to a local increase in venous pressure, or an increase of reflux of blood into collaterals .Besides advanced retroperitoneal tumours, a variety of non-tumour lesions have been associated with secondary varicoceles including non-neoplastic enlarged lymph nodes, non-neoplastic renal vein thrombosis, large aneurysmal dilatations, pancreatic pseudocysts, renal arteriovenous malformations, and the nutcracker  syndrome –158.Anecdotally, a secondary varicocele is often considered when the disease is found solely on the right side. Isolated right-side varicocele occurs in a minority of patients with clinical varicoceles , 115. TuThere are no data that currently support extended US examination to the abdomen in all patients with a newly diagnosed varicocele. An exception to this is in children less than 9 years of age presenting with a varicocele. Epidemiological studies have demonstrated that varicoceles develop at puberty. Oster et al. observed that no varicoceles were detected in 188 boys 6–9 years of age . Akbay eIn conclusion, suspicion of a secondary varicocele can arise when a clinically evident varicocele presents acutely, and remains tense in a supine position. Secondary subclinical varicocele has not been reported. The ESUR-SPIWG believes that US examination should always be extended to the abdomen in children less than 9 years presenting with an acute varicocele, to rule-out a renal or retroperitoneal tumour. In adults, the alleged association between varicoceles and retroperitoneal masses is based on a limited number of series and case reports in which the varicocele was a late sign of a symptomatic, palpable lesion , 166–169.Although the possibility of missing an undetected retroperitoneal lesion during a Doppler investigation for varicocele when the abdomen is not evaluated in an adult is very unlikely, it cannot be completely excluded Fig. . Patient. Not all tubular extratesticular structures, either palpated or identified at US, are varicoceles. Colour Doppler interrogation is important to differentiate between varicocele and other tubular structures on grey-scale US, such as spermatoceles and cyst clusters, tubular ectasia and postvasectomy changes.The ESUR-SPIWG recommend considering other pathologies when performing US in patients with clinical diagnosis of varicoceles (Table Patients presenting with uncommon conditions , 171, asZinner syndrome has also been described as a mimic for varicocele. It is a rare developmental anomaly of the mesonephric duct consisting of unilateral renal agenesis, ipsilateral seminal vesicle cyst, and ipsilateral ejaculatory duct obstruction resulting in dilatation of the epididymis and vas deferens. In Zinner syndrome, the dilated vas deferens and epididymis can simulate venous dilatation. Reflux can be artefactually recorded at colour Doppler interrogation, due to sperm movement during the Valsalva manoeuvre .A few uncommon pathological conditions can mimic intratesticular varicocele . The ValGuidelines have increasingly become a familiar part of clinical practice. They can improve the quality and consistency of care, promoting use of procedures of proved benefit and discouraging use of ineffective ones . ImagingThis systematic literature review of US imaging for varicocele shows significant variability. There is, however, good evidence to support the contention that an accurate imaging diagnosis of patients with varicoceles is important to guide clinicians in making effective treatment decisions. The recommendations and guidelines released by the ESUR-SPIWG are aimed to produce a framework that allows standardisation across future studies with the intention of clarifying the role of US in varicocele assessment.