Understanding the population structure and genetic bases of well-adapted cattle breeds to local environments is one of the most essential tasks to develop appropriate genetic improvement programs.di and iHS approaches, respectively. A series of group-specific and breed-specific candidate genes were identified, which are involved in immune response, sexual maturation, stature related, birth and bone weight, embryonic development, coat colors and adaptation. Furthermore, haplotype diversity and network pattern for candidate genes, including LPGAT1, LCORL, PPP1R8, RXFP2 and FANCA, suggest that these genes have been under differential selection pressures in various environmental conditions.We performed a comprehensive study to investigate the population structure, divergence and selection signatures at genome-wide level in diverse Chinese local cattle using Bovine HD SNPs array, including two breeds from North China, one breed from Northwest China, three breeds from Southwest China and two breeds from South China. Population genetic analyses revealed the genetic structures of these populations were mostly related to the geographic locations. Notably, we detected 294 and 1263 candidate regions under selection using the Our results shed insights into diverse selection during breed formation in Chinese local cattle. These findings may promote the application of genome-assisted breeding for well-adapted local breeds with economic and ecological importance.The online version of this article (10.1186/s12864-019-5822-y) contains supplementary material, which is available to authorized users. Bos t. taurus and the Indian Subcontinent for Bos t. indicus [Cattle, as an important domesticated farm animal, is raised for many purposes, including meat, dairy, leather, and labor. Most of the modern cattle can be categorized into two subspecies: taurine cattle (humpless) and indicine cattle (humped) with dramatic phenotypic differences. The process of domestication was estimated to start approximately 11,000 years ago separately in the Fertile Crescent for  indicus . Selecti indicus , 4.Bos t. taurus ancestry and cattle in southern China are predominantly of Bos t. indicus [Bos t. taurus, Bos t. indicus hybrids [China has rich cattle genetic resources, and these breeds can be divided into different groups based on their locations, morphologies and sex chromosome polymorphisms –7. Previ indicus –11. In c hybrids . Modern  hybrids . Therefo hybrids , 15, pig hybrids , chicken hybrids  and hors hybrids .IGF1, TSHR, PMCH, etc) are associated with growth and metabolic regulation in chicken, and another study demonstrated that NR6A1, PLAG1, and LCORL are associated with morphological changes in pigs during domestication [Selection signature analyses can help to identify numerous candidate genes associated with production traits in domesticated animals. For example, using selection sweep analysis, Rubin et al. revealed that several genes  from eight cattle breeds including Yanhuang cattle (YHC), Menggu cattle (MGC), Caidamu cattle (CDM), Liangshan cattle (LSC), Pingwu cattle (PWC), Zhaotong cattle (ZTC), Wenshan cattle (WSC) and Nandan cattle (NDC). The full name, associated abbreviation for each breed and additional sampling information were presented in Additional file Ne was calculated using the software SNeP v1.1 as described before [Ne based on LD against past t generations, where t = 1/2c and c is the distance between SNPs in Morgans (100 Mb = 1 Morgan was assumed) [Analyses of genetic diversity and effective population size were conducted for eight diverse breeds in China. These include two breeds  from Northern China, one breed from Northwest China (CDM), two breed from Southwest China (LSC and PWC), three breeds from South China   .2 > 0.2). Pairwise genome-wide identity-by-state (IBS) pairwise distances were estimated for samples cluster using PLINK v1.07 (−mds -plot 4). Population admixture was examined using 12,478 SNPs after strict LD-based filter (r2 > 0.02) in STRUCTURE 2.3.4 [N, and IBS1 and IBS2 were the number of loci that share either one or two alleles identical by state, respectively and the N was the number of loci [f3) and four-population (f4) tests implemented in TreeMix were utilized to examine the presence of admixture [We conducted Multidimensional scaling (MDS) analysis using 78,865 SNPs after LD filter  for all retained SNPs were estimated using the PLINK “-ld” option with the default window size of 1 Mb, and the “–ld-window-r2” was set to 0 to generate all pairwise results. The LD decay was further evaluated and fitted against genomic distance using smooth.spline function in R (v3.2.4).We estimated LD across each breed as our previously reported . In briedi statistics based on the unbiased estimates of pairwise FST. We then combined individual pairwise FST between groups  and averaged across all SNPs to obtain the standardized summary di statistic [di value for each group in a nonoverlapping sliding window with 50 neighboring SNPs. Selection regions were defined as those ranking in the top 1% windows with highest average di values [To explore differences among groups from different geographic zones, we estimated the tatistic . Then, wi values , 15, 47.selscan with default settings  [To infer recent selection sweeps, we further utilized the Integrated Haplotype score (iHS) method to measure structure of haplotype, which essentially reflected unusually long haplotypes across genome . iHS was800,000) . To iden800,000) .P-value < 0.05 after Bonferroni multiple test) were considered.Based on the bovine UMD3.1 reference genome assembly, we retrieved genes within the intervals spanning the candidate regions from the UCSC genome browser. The gene enrichment analyses were performed using Database for Annotation, Visualization and Integrated Discovery (DAVID) functional annotation tools . Only clLCORL, LPGAT1, PPP1R8, RXFP2 and FANCA) using the functions haplotype and haploNet from the pegas package [To explore the diversity of haplotypes and evolutionary patterns of candidate genes cross groups/breeds, we first extracted SNP genotypes in each gene as unphased information, then the haplotypes were constructed and their frequencies were estimated using PHASE v2.1 . To obta package .Ho) within breeds  . We observed the distributions of minor allele frequency (MAF) display distinct patterns across breeds, which may reflect the diverse population history, genetic structure and geographic origin of breed formation. We also observed the average MAF ranged from 0.16 to 0.26  and South breeds (primarily originate from indicine), while the second dimension separates the breeds from highland (CDM) and breed from lowland (MGC) Fig. a. The se2 generally decayed rapidly against distances as previously described [We constructed a neighbor-joining tree using pairwise nucleotide genetic distances as described before , 52. We escribed , 24, 53.f3 and f4 statistics. The significant negative f3 statistic values indicated that population A was admixed population and related to B and C. In this study, f3 test results confirmed the admixture of PWC and ZTC. Our results suggested the three most extreme f3 scores  to the geographically proximate populations of YHC|NDC, MGC|NDC and CDM|NDC as sources of admixture for PWC. The similar introgression pattern with extreme f3 scores  was observed for ZTC from three populations . In addition, we did not observe significant result for the f4 test.To further test for evidences of admixture across populations, we assessed the population history with admixture events using maximum likelihood approaches implemented in TreeMix. Our findings also confirmed several features already detected by STRUCTURE Fig. c. As migdi values in the empirical distribution as candidate selection regions for each group. The genome-wide distribution of the standardized di values were generated for these groups , Northwest (CDM), Southwest  and South (WSC and NDC). Totally, we identified the top 1% with highest average ups Fig. . We deteups Fig. , and estIn this study, we identified 107, 103, 100, and 100 regions overlapping with genes in North, Northwest, Southwest and South group, respectively. Based on these regions, we totally detected 901 unique genes overlapped with the top 1% regions. Among them, there were 188, 71, 61 and 125 unique genes for each of North, Northwest, Southwest and South groups. We further performed DAVID analyses on identified genes in group-specific regions for each group. Notably, we found several functional clusters that were significantly enriched into functional importance processes among these breeds. In North group, we observed top three enriched clusters, including aromatic compound catabolic process, arylesterase activity, and response to toxic substance. In Northwest group, we discovered enrichments for ATP binding. In South group, we found most genes were enriched in lipid catabolic process, while no functional enrichment was observed in Southwest group.WIF1, HSPA9 and UBE2D2) were identified under positive selection in four groups   analysis, because this approach could provide important insights into recent selections. We identified a total of 2315 (1263 unique) candidate regions across multiple chromosomes in eight breeds. Among them, we identified 294, 286, 285, 306,298, 289, 305 and 252 candidate regions in YHC, MGC, CDM, PWC, LSC, ZTC, WSC, and NDC cattle, respectively. Totally, we found 1425 genes under selection in 1263 nonredundant regions , was mainly found in North and Northwest cattle  and only minor portions occur in Southwest and South groups   and H2 (19.22%), while three haplotypes  were shared by multiple groups from the North, Northwest, South and Southwest  Fig. a. H2 , followed by the recent introgression of European taurine cattle [We have performed population structure, admixture and phylogenetic analyses on diverse local cattle across a broad latitudinal range in China. Our analyses revealed the population structure was mostly correlated with the geographic distribution. These results were consistent with previous analyses using mitochondrial D-loop sequences and microsatellites in Chinese cattle, which also implied the geographical segregation of local breeds during breed formation , 7, 11. e cattle , 10. In e cattle .di to detect the locus-specific divergence derived from ancient selection events. On the other hand, on the breed level, we utilized iHS based on extend haplotype homozygosity to infer recent selection sweeps. An integrated strategy combining both could facilitate the detection of both ancient and recent selection events [di and iHS approaches, respectively. When compared the identified regions from two methods, we found the 13.5 Mb overlapped regions, corresponding to 221 common genes. However, these two methods are targeting at the different levels (groups or breeds) at different time scales (ancient vs. recent events), therefore the overlaps do not necessarily imply the strongest selection signatures but may reflect the long-lasting selection events. Our combined data suggest that while Chinese cattle breeds share some selected regions, they also retain unique signatures of adaptation in responses to local environments.Based on population structure estimation, we conducted selection signature analyses for four groups in Chinese local cattle, including the North, Northwest, Southwest and South groups. To our knowledge, this is one of the first comprehensive studies on selection signatures using the high density genome-wide SNP array in Chinese local cattle. Our findings may offer valuable insights to understand the genetic mechanism of adaption in different local environmental conditions. In current study, on the group level, we used n events . We totadi method, we observed three genes  under selection in all four groups . WIF1 and HSPA9, related to immune response, were implicated in previous investigations for selection evidences in multiple cattle populations [UBE2D2, which may function as ligase activity and acid-amino acid ligase activity, has also been reported in East African Shorthorn Zebu [RXFP2) [LYN, TGS1, NPR2, SUOX, HBEGF, and NMNAT1) [SAR1B and DGAT1) [RAB5B, PMEL and CDK2) [LCORL, NPR2, and BMP2) and horns (RXFP2) have been identified in cattle, pig, horse and sheep, which could be due to parallel evolution because of similar nature and/or human-imposed selection [Using the ulations , 30, 56.orn Zebu . Previouorn Zebu . Several [RXFP2) , 34, 58, NMNAT1) , 59–63, d DGAT1) , and immnd CDK2) , 64–67. election .LCORL were identified as a candidate, and this gene has been previously reported in human, cattle, horse and pig and other domestic animals [LCORL has been associated with height or stature in human genome-wide association study [PPP1R8, CPNE7, FANCA and SPG7 displaying groups-specific selection pattern which were related to reproduction, lipid metabolic, process cardiac system and nervous system development [BMP2 gene showed selection signatures in cattle genome [LPGAT1 gene was identified as group-specific, which is supported by the previous evidence [LPGAT1 play an essential role in lipid synthesis in mice [LPGAT1 may play a significant role in influencing BMI, percent body fat and hepatic triacylglycerol secretion in humans [Our study also revealed a list of genes within group-specific selection regions. These unique genes under selection can be responsible in shaping particular characteristics of the population, thus resulting in the origin and maintenance of that breed. For instance, in the North group,  animals , 27, 68.on study , horses on study  and dogson study . As Norton study , 73. In elopment , 34, 74.e genome , 55, ande genome . In the evidence . A recen in mice . Also, Ln humans , 78. ThuZFHX4 and LPCAT1  [TNNI2, EPHX1 and ROBO1  [UBE2E1 and GNA14  in PWC, LSC and ZTC within Southwest groups, respectively [In addition, using the iHS approach, we totally found 1425 genes under selection in 1263 nonredundant regions  –81 in YHtiation) , 82–85 aectively , 87. TheSLC16A7, PTPN4, NFE2L3, ENPP2, KITLG, CAPN2, KLHL29, ABHD15, FRMD4A, ALDH5A1, and AREG), which are involved in growth, morphology, metabolism, meat quality and immune response. These identified genes have previously been suggested under selection in cattle or other farm animals [Notably, we observed numerous candidate genes displaying breed-specific selection in Chinese local cattle. For instance, we detected 28 candidate regions with the high iHS values on multiple chromosomes in the CDM breed from Northwest  cattle, while separate distinct haplotypes were detected in the Southwest and South (indicine-derived) cattle, suggesting different evolutionary history for these populations. Also, haplotype diversity and their network pattern for positive selection genes, including nditions , 73. As nditions . Also, nnditions , 96. In LPGAT1, LCORL, PPP1R8, RXFP2 and FANCA, suggest that these genes have been under differential selection pressures in various environmental conditions.Our results provided an important glimpse into diverse genomic selection during breed formation in Chinese local cattle. We identified a series of group-specific and breed-specific candidate genes, which are involved in immune response, sexual maturation, stature related, birth and bone weight, embryonic development, coat colors and adaptation. Furthermore, haplotype diversity and their network pattern for positive selection genes, including Additional file 1:Table S1. The full names, associated abbreviation for each breeds and additional information on the locations of the sampling areas for eight populations. (XLSX 12 kb)Additional file 2:Table S2. Candidate selective signature and genes under positive Selection using di approach for four groups including North, Northwest, South and Southwest. (XLSX 31 kb)Additional file 3:Table S3. Candidate selective signature and genes under positive Selection using iHS approach for eight populations including YHC, MGC, CDM, PWC, LSC, ZTC, WSC and NDC. (XLSX 83 kb)Additional file 4:Figure S1. The effective population sizes (Ne) of Chinese native cattle analyzed in this study. Ne of YHC, MGC, CDM, LSC, PWC, ZTC, WSC and NDC is plotted separately. X and Y axis represents generations and Ne respectively. (TIF 1662 kb)Additional file 5:Figure S2. Neighbor-joining tree of eight populations. The tree was constructed using genetic sharing distances. (TIF 2514 kb)Additional file 6:Figure S3. LD decay patterns in the eight breeds. Pairwise LD measures for all retained SNPs were performed using the PLINK “-ld” option. We used the default window size of 1 Mb, and set “–ld-window-r2” to 0 in order to get all pairs reported. The LD decay along genomic distance was fitted by smooth.spline function in R. (TIF 1673 kb)Additional file 7:Figure S4. Haplotype networks of two loci. (A) LPGAT1 and (B) PPP1R8, (C) RXFP2 for four cattle groups. Each node represents a different haplotype, with the size of the circle proportional to frequency. Branch lengths are proportional to the number of nucleotide differences. Circles are color coded according to group . (TIF 1362 kb)


Actinomyces bacteria. Actinomyces israelii is diagnosed in the overpowering majority of reported cases. Abdominopelvic manifestation forms 10% to 20% of all actinomycosis, and may be misdiagnosed as either a malignancy or chronic inflammation due to the lower correct preoperative diagnostic rate (<10%).Renal actinomycosis is a rare clinical infection, subacute to chronic presentation caused by the A 38-year-old man with alcoholic liver cirrhosis experienced right flank pain, abdominal pain, and fever for 3 days. Leukocytosis, acute kidney injury, and impaired liver function were found. A computed tomographic scan demonstrated multiple renal cystic lesions, along with fluid accumulation at the right subphrenic and retroperitoneal spaces.A israelii and the pathological findings of multiple renal abscesses of actinomycosis with the characteristics of sulfur granules.Renal actinomycosis was confirmed via cultures of both the abscess and nephrectomy specimen which grew A nephrectomy was performed for an inadequate percutaneous drainage of renal abscess.A full course of antibiotics with intravenous penicillin G (3 million units every 4 hours) was prescribed for 2 weeks, followed by oral penicillin V given at a dose of 2 grams per day for 6 months at our out-patient facility.A precise diagnosis of primary renal actinomycosis depends on any histopathological findings and/or cultures of specimens. A high dose of intravenous penicillin G is the first choice, followed by oral penicillin V, with the duration of each being dependent upon the individual condition. Actinomyces israelii is diagnosed in the overpowering majority of reported cases.–4 Abdominopelvic manifestation forms 10% to 20% of all actinomycosis cases. Primary RA is a rare clinical entity and may be misdiagnosed as either a malignancy or chronic inflammation due to the lower correct preoperative diagnosis rate (<10%).,4,5 It has been reported in fewer than 20 cases in English medical publications since 1990.,5–12The clinical presentations include pyelonephritis, renal or perirenal abscess, and masses. A computed tomography (CT) scan is a reliable diagnostic tool in offering adequate clues on the anatomical extent of RA.–3 The diagnosis of primary RA is based on a histopathological evidence of biopsy, aspiration, or nephrectomy, or a microbiological culture of Actinomyces, or both.,3,5 Clinical practice has shown that primary RA can be cured through antibiotics, with high doses of intravenous penicillin G for 1 to 2 months, followed by oral penicillin V for 6 to 12 months.–4,6Renal actinomycosis (RA) is a rare subacute to chronic infection caused by the 23, with segmented neutrophils at 96.6%, hemoglobin at 10.8 g/dL, platelet counts of 407 × 103/mm3, blood urea nitrogen at 50 mg/dL, creatinine at 2.7 mg/dL, lactate at 16.3 mg/dL, albumin at 2.3 g/dL, glutamic-oxaloacetic transaminase at 122 U/L, glutamic-pyruvic transaminase at 49 U/L, lactate dehydrogenase at 486 U/L, alkaline phosphatase at 432 U/L, C-reactive protein at 40.4 mg/dL, blood glucose at 106 mg/dL, along with other test result levels which were unremarkable. A CT scan demonstrated fluid accumulation at the right subphrenic and retroperitoneal spaces, multiple cystic lesions within the right kidney, along with an irregular surface of the kidney adjacent to retroperitoneal fluid  of 31,700/mm2.1Written informed consent was obtained from the patient for publication of this case report and all accompanying images. Ethical approval was not required, but the patient's informed consent and agreement of images for publication were completed.3Actinomyces are Gram-positive anaerobic bacteria that exist as normal flora in the oral cavity, tonsillar crypts, gastrointestinal, respiratory, and genitourinary tracts in humans. Actinomycosis is a rarely subacute to chronic clinical infection caused by the Actinomyces bacteria. A israelii is diagnosed in the overpowering majority (80%) of reported cases.–4Actinomyces. Abdominopelvic manifestation forms 10% to 20% of all actinomycosis cases. RA may be classified as primary and secondary, and hematogenously spread from other infectious lesions.,5,6 Primary RA is a rare clinical entity, with fewer than 20 cases being reported in English medical publications since 1990.,5–12 The manifestations of primary RA include pyelonephritis, renal or perirenal abscess, and masses. Risk factors associated with the acquisition of actinomyces may involve diabetes mellitus, malignancy, immunosuppression , alcoholism, liver cirrhosis, and local tissue damage . Primary RA is known to be easily misdiagnosed as a malignancy or another chronic inflammatory disease due to the lower correct preoperative diagnostic rate (<10%).,4,6 Clinical presentation of primary RA includes nonspecific symptoms such as fever, weight loss, and pain. Laboratory data are nonspecific, including results regarding anemia, leukocytosis, increased erythrocyte sedimentation rate, raised high-sensitivity C-reactive protein, and elevated alkaline phosphatase in hepatic actinomycosis.Any tissue or organ in the human body can be infected with –3A CT scan is a reliable imaging tool used for both locating lesions and providing sufficient information on the anatomical involvement of RA. This includes an enlarged kidney infiltrated with multiple cystic lesions or with the presence of an inflammatory process.Actinomyces, or a combination of such. Microscopically, sulphur granules are colonies of bacteria that appear as intertwined branching filament masses on a hematoxylin-eosin stain.,3,5A definitive diagnosis of RA is based upon the histopathological findings of a percutaneous biopsy, needle aspiration or nephrectomy, or a culture of Staphylococcus aureus or Gram-negative anaerobic enterobacteria in colesional actinomycosis.–4,6Clinical practice has shown that actinomycosis can be treated through high doses of antibiotics, involving 18 to 24 million units a day of intravenous penicillin G for 1 to 2 months, followed by oral penicillin V at a dose of 2 to 4 g per day for 6 to 12 months, depending upon the location of infection site, severity of disease, and the patient's response to management. Additionally, β lactams, doxycycline, clindamycin, erythromycin, and clarithromycin are alternative drugs which are considered. β lactamase inhibitors provide additional coverage against potential β lactamase producers such as 4In conclusion, primary RA is diagnostically challenging due to it being easily misdiagnosed as a malignancy or chronic inflammation. A clear-cut diagnosis depends upon histopathological findings and/or cultures of specimens. Depending upon each patient's susceptibility to antibiotics, the first choice is a high-dose of intravenous penicillin G, followed by oral penicillin V, with the duration of each treatment dependent upon the individual condition.Many thanks for performing the percutaneous drainage by the radiologist and the postoperative critical care by the team of intensive care unit. This study was approved by the Institutional Review Board of Taichung Veterans General Hospital (No. CE18102A).Conceptualization: Sung-Yuan Hu, Che-An Tsai.Data curation: Wei-Kai Liao, Shih-Che Huang, Ren-Ching Wang, Che-An TsaiFormal analysis: Wei-Kai Liao, Shih-Che Huang, Ren-Ching Wang.Investigation: Wei-Kai Liao, Shih-Che Huang, Sung-Yuan Hu, Che-An Tsai, Ren-Ching Wang.Supervision: Sung-Yuan Hu.Writing – original draft: Wei-Kai Liao, Shih-Che Huang.Writing - review & editing: Sung-Yuan Hu.All authors contributed to the interdisciplinary interpretation of the clinical, radiological and histopathological findings, edited the manuscript for important intellectual content and approved the final version.


In patients with chronic kidney disease (CKD), adverse outcomes such as systemic inflammation and anemia are contributing pathologies which increase the risks for cardiovascular mortality. Amongst these complications, abnormalities in mineral metabolism and the metabolic milieu are associated with chronic inflammation and iron dysregulation, and fibroblast growth factor 23 (FGF23) is a risk factor in this context. FGF23 is a bone-derived hormone that is essential for regulating vitamin D and phosphate homeostasis. In the early stages of CKD, serum FGF23 levels rise 1000-fold above normal values in an attempt to maintain normal phosphate levels. Despite this compensatory action, clinical CKD studies have demonstrated powerful and dose-dependent associations between FGF23 levels and higher risks for mortality. A prospective pathomechanism coupling elevated serum FGF23 levels with CKD-associated anemia and cardiovascular injury is its strong association with chronic inflammation. In this review, we will examine the current experimental and clinical evidence regarding the role of FGF23 in renal physiology as well as in the pathophysiology of CKD with an emphasis on chronic inflammation and anemia. Cardiovascular disease is the leading cause of mortality across all stages of chronic kidney disease (CKD), and common occurrences such as fibroblast growth factor 23 (FGF23) excess, chronic inflammation and iron deficiency have been shown as prominent risk factors ,2,3,4. OFGF gene family expanded from three to six genes via gene duplication, during their first phase of the metazoan linage [FGF gene family was subjected to two large-scale gene duplications during their second phase [Nearly 50 million years ago, life on earth was flourishing due to the re-population from terrestrial creatures, which progressed life beyond their aquatic habitats ,7,8,9. Tn linage . During nd phase . This rend phase ,14,15. + channels and the electrical excitability of neuronal cells [The highly conserved FGF family consists of 22 members in mammals and is divided into three classifications: intracellular, paracrine and endocrine FGFs, based on their mechanisms of action ,16. Theial cells ,23,24,25al cells ,13. Unlial cells ,27,28. Tal cells ,30,31. AFgf23 gene in patients with autosomal dominant hypophosphatemic rickets (ADHR) was identified thereafter [FGF23 is a member of the FGF19 subfamily, which utilizes αKlotho to carry out its physiological functions, targeting the kidney to promote phosphate excretion and enabling the suppression of active vitamin D synthesis ,33,34,35ereafter ,38. Undeereafter ,41,42,43176-X-X-Arg179 and is not present in other FGF family members [Prior to FGF23 secretion from bone, post-translational modifications allow the peptide to circulate in the bloodstream as a full-length mature form , which possess biological activity, or circulate as proteolytic cleaved fragments  38,40,44,45. Thi members .178 via polypeptide N-acetylgalactosaminyltransferase 3 , which protects FGF23 from proteolytic cleavage and helps support its intact structure [As a circulating biologically active peptide, FGF23 is O-glycosylated at several residues, such as Thrtructure ,47,48,49tructure ,51,52. 180 via secretory protein kinase family with sequence similarity-20 member C (FAM20C), which impedes O-glycosylation at Thr178 by GalNT3 and results in its biologically inactive form by separating the respective FGFR and αKlotho binding regions [Furthermore, as proteolytic cleaved fragments in circulation, FGF23 is phosphorylated at multiple amino acid residues, such as Ser regions ,54. A fi regions ,57,58,59Regarding the metabolic rate of FGF23, renal extraction appears to play a critical role in its metabolism, yet only a minor contribution of FGF23 excretion via the kidney may contribute since FGF23 is not detectable in urine under physiological conditions . HoweverFgfr genes (Fgfr1–4) have been identified [Fgfr gene family endured only one expansion in chordate evolution via two large scale gene duplications, which transpired during early vertebrate evolution in the second phase [Fgfr genes contain eighteen coding exons, which have undifferentiated exon-intron organizational structure and are the principal receptors which mediate the biological effects of FGFs to their respective target tissues [The mammalian proteome encodes 58 distinct receptor tyrosine kinases and from this superfamily, four functional entified ,61,62. Tnd phase . Human F tissues ,19,31. In addition to being ubiquitously expressed throughout the genome, their monomeric structure constitutes three extracellular immunoglobulin (Ig)-like domains, containing the ligand-binding pocket formed by Ig-like domains II and III, a single transmembrane helix and a split intracellular tyrosine kinase domain ,20,31. W2+ levels and leads to the activation of the calcineurin/nuclear factor of activated T-cells (NFAT) pathway [FGFR signaling is predominantly transduced by the cytoplasmic effectors, phospholipase Cγ (PLCγ) and FGF receptor substrate 2α (FRS2α) ,31,61,67 pathway ,65,71,72 pathway ,31,67,73 pathway ,74. FRS2 pathway ,76. Canonical FGF23-mediated signaling employs αKlotho as its co-receptor to conduct its physiological activity  73,77,7,79,80. BIn the kidney, FGF23 binds αKlotho and FGFR1c to promote phosphaturia via the downregulation of the sodium-dependent phosphate co-transporters, NaPi-2a and NaPi-2c, at the apical surface of the proximal tubule ,84,85. I2+ levels in cultured cells such as cardiac myocytes, hepatocytes and injury-primed renal fibroblasts [Prevailing research has elucidated the existence of non-canonical FGF23-mediated signaling, which occurs in an FGFR-dependent, αKlotho-independent manner  65,90,9,94,95,96roblasts ,98,99. Troblasts ,102,103.2-integrin on the cell surface [Despite these notable pathological conditions, it is feasible for cells that are lacking αKlotho, but are FGF23 responsive, to express alternative co-receptor(s) for FGF23 binding such as cell adhesion proteins of the cadherin and immunoglobulin superfamilies, which might interact with FGFR4 ,108,109. surface . As a re surface .In addition to downstream signaling events resulting from intact FGF23, furin-mediated cleavage results in the separation of the respective FGFR and αKlotho binding domains, generating N-terminal and C-terminal fragments ,44. ThesIt is plausible to speculate that C-terminal FGF23 competitively blocks the action of intact FGF23, due to binding αKlotho and not activating FGFRs, thus contributing to the inhibition of canonical FGF23-mediated signaling by acting as an antagonist for the FGFR:αKlotho complex. The existence of such a mechanism is supported by an in vitro study which shows that in the presence of C-terminal FGF23, the FGF23-mediated reduction of phosphate uptake by proximal tubule cells can be diminished . NeverthThe kidneys are part of an inextricable network known as the bone-kidney-intestinal axis . The cruOrganic phosphate is predominantly found in foods that are rich in protein (non-phytates) such as meat, fish and dairy products, which have a bioavailability of 40–80% ,121,122.To prevent phosphotoxicity, FGF23 levels rise and fall in correlation with the amount of dietary phosphate absorbed ,140. WheDuring the early stages of CKD, the expression of the renal FGF23 co-receptor, αKlotho, decreases in response to kidney damage and progressively declines along with the loss of functional nephrons, promoting partial resistance to FGF23′s physiological actions ,143,144.These metabolic disturbances primarily contribute to associated pathologies that are observed in CKD, such as immune dysfunction, systemic inflammation, anemia, vascular calcification, skeletal muscle atrophy and cardiac hypertrophy, resulting in premature death ,153,154.Systemic immune activation and alterations in iron trafficking are interlinked in CKD, as the inflammatory milieu contributes to the pathology of functional iron deficiency ,160,161.Fgf23 mRNA levels in bone and serum levels of C-terminal FGF23 protein, yet serum intact FGF23 levels were unaltered [Many clinical studies have reported associations between FGF23 and inflammatory markers in disease states ,177,178.naltered . Moreovenaltered . This dinaltered ,179.Equally, IL-6 has been shown to be a novel regulator of FGF23. In the state of acute and chronic inflammation, IL-6 elevates the synthesis and secretion of FGF23 from bone by inducing the phosphorylation of signal transducer and activator of transcription 3 (STAT3) through soluble IL-6 Receptor (sIL-6R)-mediated trans-signaling, prompting STAT3 to bind its response element on the Fgf23 promoter . UtiliziAdditional mediators of the inflammatory milieu, such as tumor necrosis factor-α (TNF-α) and OSM, have also been shown to stimulate FGF23 synthesis and secretion. In cultured bone cells and following a recent study utilizing animal models of renal and extrarenal inflammation, Glosse et al. showed that increasing concentrations of TNF-α elevates the production of FGF23 in a dose dependent-manner in UMR106 cells, while Egli-Spichtig et al. revealed the contribution of TNF-α stimulation to the renal production of FGF23, respectively ,182. In In contrast to functional iron deficiency and its mediators regulating FGF23 production, absolute iron deficiency has been shown to regulate FGF23, along with clinical studies demonstrating an association between FGF23 and erythropoiesis ,188,189.Collectively, these data suggest inflammation and iron deficiency are both potent stimuli of FGF23 production. Additional studies are required to investigate the disarranged processing events of FGF23 following conditions of acute and chronic inflammation, absolute iron deficiency and elevated EPO levels. C-terminal and intact FGF23 levels are elevated at dissimilar magnitudes, where during acute settings more biologically inactive FGF23 enters the circulation, yet during chronic settings more biologically active FGF23 exists . AltogetWith alterations in phosphate homeostasis, it has been recognized that widespread tissue injury and accelerated CKD progression can be attributed to the presence of chronic inflammation ,195,196.Indeed, Singh et al. unveiled by the use of in vitro experiments and various animal models of FGF23 excess, that FGF23 can directly target the liver to enhance the inflammatory milieu . HepatocMoreover, FGF23 can also stimulate the production of inflammatory cytokines from other known reservoirs. In patients with chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF), serum FGF23 levels are significantly elevated ,203,204.Apart from these studies, the use of genome wide-analysis has indicated that many pro-inflammatory genes are regulated by FGF23 . Since FCollectively, these data suggest FGFR4 blockade might be effective in reducing systemic inflammation in patients. It is possible that FGF23-induced cytokine production is not initially pathological, but more so representative of a beneficial effect which transitions into a detrimental consequence due to the prolonged duration of exposure to increased circulating FGF23, rather than the degree of FGF23 elevations per se. Apart from this postulation, it is important to indicate other factors besides circulating FGF23, such as elevated serum phosphate levels, might be culpable for elevations in inflammatory cytokines and perhaps bestows inflammatory tissue damage ,210. TheCurrently, 40 million adults in the United States suffer from CKD, which corresponds to ~10–15% of the adult population and accounts for 24% of the annual Medicare budget ,212. WitCKD is a state of systemic inflammation, characterized by low to moderate levels of circulating inflammatory markers which have been acknowledged since the late 1990’s ,224,225.2-), hydrogen peroxide and hydroxide radicals [2- at the site of inflammation and injury, which promotes the activation of pro-inflammatory gene programs and further ROS production [Multiple factors contribute to the etiology of the inflammatory state in CKD. One such factor is oxidative stress, which occurs when there is a disproportion of the production to degradation ratio in reactive oxygen species (ROS) generating superoxide anions  results from a reduction in the body’s total iron content, where the rate of dietary iron absorption cannot compensate for the increased demand for iron or blood loss . What chEPO is a kidney-derived hormone which stimulates the production of erythrocytes by regulating the differentiation of hematopoietic progenitor cells, such as pro-erythroblasts, in bone marrow . ExperimAs AID poses a major risk factor for CKD-associated pathologies, traditional treatments such as erythropoietin stimulating agents, intravenous iron, oral iron supplementation and blood transfusions have been widely employed to alleviate the condition. Clinically, the etiology of AID should be addressed early since the improper diagnosis between AID and functional iron deficiency could be detrimental. An example of this issue is whether a CKD patient with TSAT levels below 20% and serum ferritin levels above 500 ng/mL would benefit from iron supplementation . PatientLikewise, CKD patients have high likelihoods of developing functional iron deficiency (FID) . This coThis hepcidin-FPN interaction triggers the internalization of FPN from the surface of cells such as hepatocytes, macrophages and enterocytes, which leads to FPN degradation by the lysosome ,273. As Furthermore, approaches tackling hepcidin-induced internalization of FPN have been subjected to examination. Agents such as FPN stabilizing antibodies have been utilized to block FPN degradation in phase II clinical trials ,304. In Anemia in CKD is a multifactorial process and with traditional origins, such as impaired erythropoiesis and iron deficiency, studies have devoted considerable attention towards unraveling the contribution of FGF23 to renal anemia ,305,306.The formation of bone, also known as osteogenesis, is essential for erythrocyte and platelet production in bone marrow . ExperimAs for the indirect actions of FGF23, the interconnection between FGF23 and pro-inflammatory cytokines, such as in CKD, provides further insight on how FGF23 indirectly contributes to anemia. As previous alluded, excess serum FGF23 levels directly stimulate the synthesis and secretion of various pro-inflammatory cytokines such as IL-6 and IL-1β, which triggers the formation of a vicious cycle that promotes the uncontrolled production of both factors. As a consequence of this enhanced inflammatory milieu, cytokines upregulate the production of hepcidin in the liver. These pathologic events foster excessive serum hepcidin levels, which in turn, promotes functional iron deficiency. In summary, abnormalities in mineral metabolism and the metabolic milieu are associated with chronic inflammation and anemia, and FGF23 is a risk factor in this context. Recent investigations have shown that inflammation and alterations in both EPO and iron status, are potent inducers of FGF23 production. As for the multi-directional relationship between factors, FGF23 exerts pleiotropic actions as a positive mediator of the inflammatory response and a negative regulator of erythropoiesis. Clinical and laboratory findings have revealed novel insights into the pathomechanisms of CKD and the establishment of a feed-forward cycle promoting the dysregulation between factors has led to the identification of novel therapeutic opportunities, such as targeting non-canonical FGF23-mediated signaling.However, further studies are necessary to investigate the disarranged processing events of FGF23 following conditions of inflammation and anemia, where C-terminal and intact FGF23 levels are elevated in the circulation at dissimilar magnitudes. Moreover, these studies should aim to answer a key question, whether N-terminal and C-terminal cleaved fragments possess biological activity and if so, whether these activities are alike or disparate to the functions of intact FGF23.


Since the viruses partially hijack their hosts’ cellular machinery for proliferation, we examined the PQS motif size, loop length, and nucleotide compositions of 7370 viral genome assemblies and compared viral and host PQS motifs. We studied seven viral taxa infecting five distant eukaryotic hosts and created a resource providing a comprehensive view of the viral quadruplex motifs. Overall, short-looped PQSs are predominant and with a similar composition across viral taxonomic groups, albeit subtle trends emerge upon classification by hosts. Specifically, there is a higher frequency of pyrimidine loops in viruses infecting animals irrespective of the viruses’ genome type. This observation is confirmed by an in-depth analysis of the Herpesviridae family of viruses, which showed a distinctive accumulation of thermally stable C-looped quadruplexes in viruses infecting high-order vertebrates. The occurrence of viral C-looped G4s, which carry binding sites for host transcription factors, as well as the high prevalence of viral TTA-looped G4s, which are identical to vertebrate telomeric motifs, provide concrete examples of how PQSs may help viruses impinge upon, and benefit from, host functions. More generally, these observations suggest a co-evolution of virus and host PQSs, thus underscoring the potential functional significance of G4s.A subset of guanine-rich nucleic acid sequences has the potential to fold into G-quadruplex (G4) secondary structures, which are functionally important for several biological processes, including genome stability and regulation of gene expression. Putative quadruplex sequences (PQSs) G The imps are altial PQSs ,10. Thisial PQSs .PQSs are also present in viral genomes ,12, and 15+ sequences in plants and invertebrates [Within a quadruplex, the length of the G-tracts as well as the length and the base composition of the loops are critical to determine the conformation of the G4s and their stability ,34,35,36Homo sapiens), invertebrates, protozoa, fungi, and plants. Here, we provide a large comparative view of the quadruplex motif loop content at nucleotide-level resolution, with particular focus on the Herpesviridae family, ubiquitous large dsDNA (linear double-stranded DNA) viruses that are amongst the best characterized host-adapted viruses.Given that viruses utilize the hosts’ cellular machineries for replication and transcription, especially in large DNA viruses , we wishTo analyze the G4 motifs in a large panel of viruses, we retrieved the 7370 viral genome assemblies from the viruSITE  database3+N1–7G3+N1–7G3+N1–7G3+ sequences by regular expression matching , as previously described for eukaryotic genomes [Homo sapiens.First, we analyzed several viral genome metrics: genome size , which varies from 0.2 kbp to over 2400 kbp; GC content (%), which varies from 17.8% to 76.1%; and PQS densities (PQS/kbp), that allow to compare the quadruplex content of each assembly independently of the genome lengths, as well as their presence on the positive (G-rich) or negative (C-rich) strand . To identify the G4 motifs, we searched the canonical G genomes ,10,36. Arho = 0.38, non-parametric test for association between paired samples p < 2.2 × 10−16; Caenorhabditis elegans, 0.002 G4/kbp in Plasmodium), or bacteria (0.001 to 0.02 G4/kbp)  .Pandoravirus salinus genome. Intriguingly, the Mimiviridae viruses, which exhibit a low GC content (≈28%), are exceptionally G4-poor with only three G4 motifs found in the Mimivirus terra2 assembly . Globally, there is a significant positive correlation between the PQS and GC content, although the relationship is weak  Deinococcales and Thermales bacterial orders), and correlated it with oxidation-dependent regulation of transcription [Finally, in our broad viral set, we detected a significant enrichment for PQSs in the negative (C-rich) strand of dsDNA, ssRNA, RT, and unclassified viruses but not for the ssDNA virus . The ovecription .The distribution of genome size, PQS density, GC content, and strand biases of the viruses with respect to their various hosts are reported in P < 0.01; upper panel P < 0.01 except Fungi-Plants adjP = 0.542; lower panel To examine in more detail the nature of the quadruplex motifs, we inspected loop lengths and nucleotide compositions (irrespective of their position) for the dsDNA , ssDNA (102 PQSs), ssRNA (553 PQSs), RT (85 PQSs), and bulk unclassified (206 PQSs) viruses. Unfortunately, the few PQSs identified in the dsRNA and satellite viruses were insufficient to pursue such in-depth analyses. As shown in P = 0.681; upper panel P = 0.789; lower panel Thus, based on the rather short length of the loops, there is an overall bias for the most thermodynamically stable G4 motifs in viral genomes, similar to other genomes ,34,35,36rho = 0.73, non-parametric test for association between paired samples P = 1.2 × 10−15; rho = 0.10, P = 0.38; P = 0.00619; Since herpesviruses infect different animal hosts, including mammals, birds, reptiles, fish, amphibians, and invertebrate animals , we examC content  are related to their potential to form a G4 structure, to the fact that they constitute a one-dimension target sequence for the binding of host’s transcription factors, or to both. Consistent with this view, we observed that 6 of the 10 most common viral PQS motifs matched several vertebrate transcription factor sites, notably SP1 and SP3 . AlthougAnother remarkable feature of the most frequent PQS loop composition is the excess of TTA loops that are frequent in viruses infecting mammals, birds, reptiles, and fish A, a loopThe full-length sequences were retrieved from the viruSITE resource . A totalFor each of the 7370 assemblies, genome size  and total GC content (GC content was defined as the sum of G and C nucleotides in the respective assembly) were evaluated using bash and Perl scripts. Spearman’s rank correlation tests were used to assess correlations between the different variables.fasta file of each of the retrieved assemblies, in both DNA/RNA strands, as originally described by Huppert and Balasubramanian [We define a G-quadruplex motif as a sequence with at least four runs of 3+ guanines, separated by loop sequences containing one to seven nucleotides, that may themselves be guanines. Terminal guanines were excepted as loop sequences . Nevertheless, single G loops were allowed in the search. We searched, by regular expression matching, for the motifs previously defined -{3,}G{3,}- in the ramanian . Then, tramanian  for furtbalAcu1, Marmoset calJac3, Dog canFam3, Green monkey chlSab2, Kangaroo rat dipOrd1, Wallaby macEug2, Crab-eating macaque macFas5, Mouse lemur micMur2, Mouse mm10, Gibbon nomLeu3, Bushbaby otoGar3, Baboon papAnu2, Orangutan ponAbe2, Rhesus macaque rheMac8, Golden snub-nosed monkey rhiRox1, Squirrel monkey saiBol1, Tarsier tarSyr2, Tree shrew tupBel1);18 mammals ;9 birds/reptiles ;8 fish ;3 amphibians .We also retrieved 52 eukaryote genome assemblies from the UCSC Genome Browser portal. These included:fasta file of each of the retrieved assemblies. We performed the same subsequent analyses as described for viral sequences. Canonical PQS content {3,}G{3,}-) for the 12 species reported in We searched for short-looped quadruplex sequences, by regular expression matching, -{3,}G{3,}-, in the A, PQST, PQSC, PQSG; where PQSX is a quadruplex motif containing at least one X1–7 loop, X = {A,T,C, or G}), after normalizing the data matrix (variables were centered and reduced). Correlation between PQSA (PQST or PQSC) loop content and PQSG loop content was estimated by calculating Pearson correlation coefficients. Finally, pattern discovery within the quadruplex sequences found in mammalian herpesviruses or in promoter regions of the human reference genome hg38 was performed using the RSAT software suite [Principal component analysis (PCA) was implemented using the FactoMineR and factoextra packages in the R environment. The analysis was performed on loop composition information , taking into account the number of motifs per genome, the length of the nucleotide loops separating the G-tracks and their base composition. Remarkably, there is a predominance of single-nucleotide loop motifs in the paired viruses and animal host genomes. These G4s are the most thermodynamically stable quadruplexes, suggesting a high folding potential and stabilization in cells. We had previously observed a strong compositional bias in these sequences in eukaryotic genomes, disfavoring pyrimidine loops while resulting in the accumulation of less stable structures (carrying single A or G loops) . Here, u


CDH1, CDH2, SNAI1, SNAI2, VIM, TWIST1) in clear cell renal cell carcinoma (ccRCC). A total of 533 ccRCC patients with RNASeq data from The Cancer Genome Atlas (TCGA) cohort were included for analysis. Gene expression of these EMT markers was compared between tumor and normal tissues based on Oncomine database and TCGA cohort. Their correlations with progression-free survival (PFS) and overall survival (OS) were also examined in both TCGA cohort and FUSCC (Fudan University Shanghai Cancer Center) cohort. Cox proportional hazards regression model and Kaplan-Meier plot were used to assess the relative factors. Functional enrichment analyses were utilized to describe biologic function annotations and significantly involved hallmarks pathways of each gene. We found that Epithelial marker, CDH1 expression was lower, while mesenchymal markers  expression was higher in ccRCC primary tumors. In the TCGA cohort, we found that patients with higher expression of VIM, TWIST1 or lower expression of CDH1 had worse prognosis. Further, in the FUSCC cohort, we confirmed the predictive ability of mesenchymal markers and epithelial marker expression in PFS and OS of ccRCC patients. After generating Cox regression models, EMT markers  were independent prognostic factors of both PFS and OS in ccRCC patients. Our preliminary EMT prediction model can facilitate further screening of EMT biomarkers and cast a better understanding of EMT gene function in ccRCC.Epithelial-to-mesenchymal transition (EMT) is important in tumor invasiveness and metastasis. We aimed to determine prognostic value of six key EMT markers ( Kidney cancer is one of the most common urological tumors worldwide, and nearly 73,820 new cases and 14,770 deaths were estimated in the United States in 2019 , and RCCTumor stage is the most powerful, but still a relatively crude predictor of patients survival with ccRCC . Since 2Epithelial-to-mesenchymal transition (EMT), first described in the 1980s, is involved in physiological embryogenesis as well as in some pathological processes, such as solid organ fibrosis . EMT is CDH1, CDH2, SNAI1, SNAI2, VIM, TWIST1 in transcriptional and protein level according to datasets hosted on the Oncomine and TCGA platform; in the second stage, survival analysis based on distinct comparison expression of six hub genes have been evaluated in TCGA cohort and validated in FUSCC cohort; in the third stage, significantly involved hub genes were selected, and functional annotation of hub genes were elaborated.This study consisted of three stages. In the first stage, we assessed differential expressed CDH1, CDH2, SNAI1, SNAI2, VIM, TWIST1 in 20 types of cancers between tumor and normal tissues. Transcriptional expressions of VIM were significantly elevated in cancer tissue compared with normal tissues, while expression of CDH1, a key epithelial marker, was significantly decreased in cancer tissue in multiple datasets.As shown in CDH1, CDH2, SNAI1, SNAI2, VIM, TWIST1 between ccRCC tumor samples and adjacent normal tissues respectively based on RNA-sequence data from TCGA database. Consistent with Oncomine data, CDH1 expression was lower in ccRCC primary tumors in comparison with adjacent normal tissues  male patients and 179 (34.69%) female patients. The median age of the 516 ccRCC patients was 60.5 years, with a range from 26 to 90 years. Information of TNM stage, AJCC stage, ISUP grade, laterality was shown in The FUSCC cohort consisted of 248 (67.57%) male patients and 119 (32.43%) female patients. The median age of the 367 ccRCC patients was 56 years, with a range from 21 to 86 years. The detailed clinical data are shown in CDH1, had worse PFS  + 1.360×SNAI1 expression (ref. Low) + 1.905×VIM expression (ref. Low) + 2.179×TWIST1 expression (ref. Low) + 1.274×T stage (ref. T1-T2) + 1.919×M stage (ref. M0) + 2.021×AJCC stage (ref. I-II) + 2.013×ISUP grade (ref. 1-2) for PFS, and another formula: = -0.564×CDH1 expression (ref. Low) + 1.532×SNAI1 expression (ref. Low) + 1.804×VIM expression (ref. Low) + 1.714×TWIST1 expression (ref. Low) + 1.226×T stage (ref. T1-T2) + 1.778×M stage (ref. M0) + 2.515×AJCC stage (ref. I-II) + 1.954×ISUP grade (ref. 1-2) for OS. The Kaplan–Meier method was used to determine the significant survival outcomes , shown in p<0.001) for PFS (p<0.001) (p<0.001) for PFS (p<0.001)  . To furt for PFS  and 0.68p<0.001) .p<0.001). In addition, CDH1 showed negative association with stromal score (r2=-0.191), while stromal score positively correlated CDH2 (r2=0.337), SNAI1 (r2=0.199), SNAI2 (r2=0.201) and TWIST1 (r2=0.305) mRNA expression in ccRCC patients from TCGA cohort.As seen in CDH1, CDH2, SNAI1, SNAI2, VIM, TWIST1 was enriched in hemophilic cell adhesion and cell-cell adhesion of GO: BP, adherens junction, anchoring junction and cell junction of GO: CC, RPTP-like protein binding, phosphatase binding, protein phosphatase binding and enzyme binding of GO: MF. Participating upstream or downstream signaling pathways enrichment include adherens junction, cell adhesion molecules (CAMs) of KEGG pathways. The details were illustrated in CDH1, CDH2, SNAI1, SNAI2, VIM, TWIST1 from FUSCC cohort was performed in In this study, PPI network, activation and indirect relation was predicted in CDH1, CDH2, SNAI1, SNAI2, VIM, TWIST1. Results suggested that the most involved significant pathways included apical junction, epithelial mesenchymal transition, estrogen response, hypoxia, kras signaling pathway up, inflammatory response, myogenesis, TNF-alpha signaling via NF-κB, etc. The details are shown in A total of 100 significant genes were obtained by GSEA with positive and negative correlation. Importantly, GSEA was used to perform hallmark analysis for The primary value of the expression of EMT related markers would be prediction of post-surgery prognosis when ccRCC patients seek guidance from clinicians. Although better understanding of the mechanism of ccRCC progression has been established with emerging evidence and studies, tumor stage and grade remain the most significant predictors of recurrence and survival. In other hand, clinicopathological characteristics also showed limitations after taking into consideration of the molecular diversity in ccRCC. In the study, we examined the differential expression of six key EMT markers in ccRCC tumors from Oncomine and TCGA databases. After further confirming their prognostic value of progression-free survival and overall survival of ccRCC patients from TCGA database, we validated it in ccRCC patients from FUSCC cohort. ROC curves were also generated to validate the ability of the logistic model to predict prognosis. Our results suggested that EMT related markers might be a good predictor of prognosis of ccRCC patients.Since 2001, many prognosis models and nomograms for ccRCC have been developed and reported. Frank et al. proposed the SSIGN score in 2002, which predicted the outcome of unilateral ccRCC patients treated with radical nephrectomy and was composed of 1997 TNM stage, tumor size, nuclear grade, and histological tumor necrosis . One yeaCDH1 (encoding E-cadherin) is one the main epithelial marker, whose downregulation will reinforce the destabilization of adherens junctions and is a hallmark of EMT [SNAI1 (encoding Snail) and SNAI2 (encoding Slug) are two master regulatory transcription factors which can directly repress E-cadherin expression by binding to the specific E-boxes of E-cadherin's proximal promoter [TWIST1 (encoding TWIST1) is another important transcriptional activator that upregulates N-cadherin and downregulates E-cadherin [VIM (encoding Vimentin) functions as a positive regulator of EMT, whose upregulation is thought to be a prerequisite for EMT induction [CDH1, SNAI1, TWIST1, and VIM were significant predictors for either progression-free survival or overall survival in both TCGA and FUSCC cohorts. After multivariate Cox regression analysis, these four EMT markers were confirmed to be independent prognostic factors for ccRCC patients in FUSCC cohort.In the study, we focused on EMT related markers. k of EMT . SNAI1  mouse model to test whether inhibition of EMT pathway would influence ccRCC progression. Third, there are surely other EMT markers which play crucial roles in ccRCC progression, such as β-catenin [There are several limitations of the study should be considered. First, this is a retrospective study of FUSCC cohort. Based on the current promising result, we aim to generate a more precise survival prediction panel of ccRCC in prospective cohorts using both tissue arrays and RNA-Seq. Second, the current study lacks of functional -catenin , MMP2  [t-test. Cut-off of p value and fold change were as following: p value=0.01, Fold Change=1.5, gene rank=10%, Data type: mRNA.In this study, transcriptional expression profiles of ine.com) . Differehttps://genome-cancer.ucsc.edu/proj/site/hg Heatmap/). Differential transcriptional expression profiles of CDH1, CDH2, SNAI1, SNAI2, VIM, TWIST1 was measured between ccRCC tumor samples and adjacent normal tissues, respectively.A total of 533 ccRCC patients with available RNA-sequence data from TCGA database were consecutively recruited in analyses . The genCDH1, CDH2, SNAI1, SNAI2, VIM, TWIST1 and their frequently altered neighbor genes was constructed using cBioPortal, an open-access online resource accessing the TCGA genomics data [In this study, significant co-regulated network of genomic profiles of ics data . HierarcCDH1, CDH2, SNAI1, SNAI2, VIM, TWIST1 were analyzed in ccRCC patients. The primary end point for patients was progression-free survival (PFS), and overall survival (OS) was the secondary end point, which was evaluated from the date of first therapy to the date of death or last follow-up. X-tile software was utilized to take the cut-off value of mRNA expression of six hub genes, in concordance of which overall participants were divided to two groups, respectively [CDH1 expression (ref. Low), CDH2 expression (ref. Low), SNAI1 expression (ref. Low), SNAI2 expression (ref. Low), VIM expression (ref. Low) and TWIST1 expression (ref. Low). All hypothetical tests were two-sided and p-values less than 0.05 were considered significant in all tests.Among the 533 ccRCC patients, 516 patients had complete clinicopathologic information. Phenotype and expression profiles of hub genes in the 516 ccRCC patients from TCGA were analyzed and displayed. Survival comparison between distinct mRNA expressions groups of ectively . The folTo further validate prognostic implications of six hub genes in real world, a total of 367 ccRCC patients, who have undergone radical nephrectomy in the Department of Urology of Fudan University Shanghai Cancer Center (FUSCC)  from Aug. 2008 to Sept. 2016, were consecutively recruited in analyses, with electronic medical records or pathology reports available. Clinical and pathological parameters, specifically age at surgery, clinical manifestation, tumor laterality, TNM stage, ISUP grade classification were summarized. Tissue samples, including ccRCC and normal tissues, were collected during surgery and available from FUSCC tissue bank. A central review of pathology was performed by an experienced pathologist. Clinicopathological characteristics were obtained from electronic records. Patients were regularly followed up by telephone, mail, or in the clinic once every 3 months. All the study designs and test procedures were performed in accordance with the Helsinki Declaration II. The Ethics approval and consent to participate of the current study was approved and consented by the ethics committee of FUSCC.CDH1, CDH2, SNAI1, SNAI2, VIM, TWIST1 were listed in Transcription level of six hub genes was measured using RT-PCR analysis in 367 ccRCC patients from FUSCC cohort. Total RNA sequence was extracted using TRIzol reagent  from 367 paired tumor samples. Total RNA reverse-transcribed reaction was performed using the SuperScript First-Strand cDNA Synthesis System . ABI Prism 7900 Sequence Detector (Applied Biosystems) was utilized to realize Real-time PCR reactions. Forward and reverse PCR primers of Kaplan-Meier method with 95% confidence intervals (95%CI) and log-rank test was applied to analyze different survival outcomes in separate curves in 367 ccRCC patients from FUSCC cohort. Univariate analyses were performed with Cox logistic regression models to find independent variables, including pT stage, pN stage, M stage, AJCC stage, and ISUP grade.Integrated score was identified as sum of the weight of each significant hub gene. X-tile software was utilized to take the cut-off value. All hypothetical tests were two-sided and P-values less than 0.05 were considered significant in all tests. The receiver operating characteristic curve (ROC) was constructed by predicting the probability of a diagnosis being of high or low integrated score of significant hub gene expression. Area under curve (AUC) analysis was performed to determine the diagnostic ability. In addition, validation of diagnosis and prognostic value of integrated score was constructed using mRNA data from TCGA.ESTIMATE algorithm, available from "estimate" R package, was obtained and utilized to measure to measure stromal components in ccRCC tumor microenvironment. Transcriptional expression levels of EMT markers and stromal scores were matched to sample identification names in the TCGA database. Pearson correlation coefficient analysis was used to reflect the degree of linear correlation between two random variables. The value of r is between -1 and 1. When the value is 1 or -1, the two random variables are completely negatively or positively correlated.http://string-db.org) (version 10.0) online database was used to predict PPI network of DEGs and analyzing the functional interactions between proteins [CDH1, CDH2, SNAI1, SNAI2, VIM, TWIST1 from FUSCC cohort was performed in a heat map. Color gradients suggest high (red) or low (blue) expression level.In the present study, protein-protein interaction (PPI), activation, indirect relation and activation expression modular was predicted using Search Tool for the Retrieval of Interacting Genes  using Benjamini and Hochberg (BH) false discovery rate (FDR) method by default were applied to correct the occurrence of false positive results . The sigCDH1, SNAI1, VIM, and TWIST1 were found to be independent PFS and OS of ccRCC patients.We preliminarily tested six key EMT markers and validated their prognostic value in both TCGA cohort and USCC cohort. Among these six EMT markers, Supplementary FiguresSupplementary Table 1


Ebolavirus (EBOV) life cycle involves interactions with numerous host factors, but it remains poorly understood, as does pathogenesis. Herein, we synthesized 65 siRNAs targeting host genes mostly connected with aspects of the negative-sense RNA virus life cycle . We produced EBOV transcription- and replication-competent virus-like particles (trVLPs) to mimic the EBOV life cycle. After screening host factors associated with the trVLP life cycle, we assessed interactions of host proteins with trVLP glycoprotein (GP), VP40, and RNA by co-immunoprecipitation (Co-IP) and chromatin immunoprecipitation (ChIP). The results demonstrate that RNAi silencing with 11 siRNAs  decreased the replication efficiency of trVLPs. Co-IP revealed nine candidate host proteins  potentially interacting with trVLP GP, and four  potentially interacting with trVLP VP40. Ch-IP identified nine candidate host proteins  interacting with trVLP RNA. This study was based on trVLP and could not replace live ebolavirus entirely; in particular, the interaction between trVLP RNA and host proteins cannot be assumed to be identical in live virus. However, the results provide valuable information for further studies and deepen our understanding of essential host factors involved in the EBOV life cycle. Ebolavirus (EBOV) is a single-stranded, negative-sense RNA virus with a heterogeneous filamentous structure  that wasAlthough numerous studies have been devoted to EVD therapeutics, such as immune-based treatments and small molecule inhibitors, and considerable progress has been made , the molTo model the EBOV life cycle using a safe method, a series of trVLPs have been developed . These tIn the EBOV life cycle, GP is critical for EBOV entry, uncoating and fusion processes , while VL-glutamine , and 1% penicillin- streptomycin  at 37°C with 5% CO2. Plasmids pCAGGS-VP30, pCAGGS-VP35, pCAGGS-NP, pCAGGS-L, p4cis-vRNA-RLuc, pCAGGS-Tim1, and pCAGGS-T7 were kindly provided by Drs. Heinz Feldmann and Thomas Hoenen, Rocky Mountain Laboratories, National Institute of Health (NIH). Plasmid p4cis-vRNA-RLuc containing EBOV non-coding regions, a reporter gene, and three genes  involved in morphogenesis, budding and cell entry, was used to produce trVLPs. In the following experiments, all tests were carried out with EBOV trVLPs.Human embryonic kidney (HEK) 293T cells were cultured in DMEM  containing 10% FBS , 2 mM g. The supernatant was then used to infect target 293T cells (p1) previously transfected with RNP components   by tapping gently about 100 times, and trVLPs were stored on ice or in a refrigerator at 4°C until use.Producer 293T cells (p0) in 6-well plates were transfected with plasmids encoding each EBOV structural protein , and pCAGGS-T7 (250 ng) encoding a Renilla luciferase reporter . Cell suGS-Tim1) . Target -1 to 10-10. Attenuated trVLPs (100 μl) were added to eight wells in each row of a 96-well plate, and 293T cell suspension (100 μl) was added to each well to a final cell density of 2 × 105 cells/ml. Additionally, 293T cells not infected with trVLPs were included as controls. CPEs were observed and recorded each day for 7 days, and TCID50 values were calculated according the Reed–Muench method, giving a value of 103.76/0.1 ml  were resuspended in NTE buffer a final volume of 1 ml and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate  was added and mixed thoroughly. The mixture was shaken gently for 1 h at room temperature then passed through a 0.22 μm filter . HEK 293T cells cultured in 8-well chamber slides with removable wells were mixed with 4′,6-diamidino-2-phenylindole  at 10 μg/ml for 10 min to stain nuclei, and 293T cells were then incubated with DiI-labeled trVLPs at 4°C for 10 min and washed with PBS. The internalization of DiI-labeled trVLPs in 293T cells was imaged at different times from 10 to 60 min at 37°C on the stage of a fluorescence microscope.To select host factors that are potentially related to various stages of the EBOV life cycle , we chose host genes that are associated with membrane traffic machinery, endoplasmic reticulum-Golgi recycling, inhibition of secretion, and lipid droplet formation. For example, the knockdown of FLT4, RAB11B, and PLCH1 resulted in high-level secretion inhibition; GOSR1, LTK, and CNKSR1 are linked to endoplasmic reticulum-Golgi recycling; VAMP2, RAB11B, and COPG are membrane traffic machinery-related molecules ; HSPA8, -ΔΔCt method, and the smaller the value, the higher the knockdown efficiency. The most efficient siRNAs were chosen for RNAi screening tests  for 48 h. Negative control siRNA  served as a control.SiRNAs targeting the 65 selected genes were designed and three sequences were synthesized for each gene. To choose the most efficient sequence for RNA interfere (RNAi), the gene knockdown efficiency of each sequence was evaluated by quantitative PCR, with the 18sRNA gene as a control. Values were calculated using the 2After trVLP infection, 293T cells in 24-well plates were washed with PBS, and total RNA was extracted from cells using TRIzol  according to the manufacturer’s instructions. qRT-PCR was performed with a EBOV nucleic acid test kit  on an ABI 7500 qPCR system . The positive control, provided in the kit, was serially diluted over three orders of magnitude, and the absolute quantity of EBOV RNA (copy/ml) was calculated using the comparative Ct method and a standard curve. All qRT-PCR experiments were performed in triplicate and repeated independently three times.Antibodies recognizing HSP70 , HSP90β , MEK1/2 (Cat# 9122S), ARFGAP1 (Cat# 14522S) and Trk  were purchased from Cell Signaling Technology . Antibodies recognizing MAPK11 (Cat# ab183208), VEGF Receptor 3 , Annexin V , GRP78 , GAPDH (Cat# ab8245), and Ebola virus GP (Cat# ab1918) were purchased from Abcam . Antibodies recognizing 14-3-3 zeta/beta  and Hsc70  were purchased from Novus Biologicals . Antibody recognizing Ebola virus VP40 (Cat# sc-51872) was purchased from Santa Cruz Biotechnology . Antibodies against HSPA1A, HSP90AB1, MEK1/2, ARFGAP1, NTRK1, MAPK11, FLT4, and GRP78 were rabbit-derived IgGs. Antibodies against ANXA5, YWHAZ, and HSPA8 were mouse-derived IgGs. Normal mouse IgG (Cat# SC-2025) and normal rabbit IgG (Cat# SC-2027) were obtained from Santa Cruz Biotechnology and used as isotype controls in immunoprecipitation tests.3.76/0.1 ml) for 48 h and whole-cell extracts were prepared followed by incubation overnight with appropriate antibodies recognizing host proteins plus Protein G beads . Antibodies mixed with Protein G beads and normal cell/extracts without trVLPs, and Protein G beads mixed with trVLP-treated cell extracts without antibodies, were included as control groups. Beads were then washed five times with low-salt lysis buffer, and immunoprecipitates were eluted with 2× SDS Loading Buffer and resolved by SDS-PAGE. Proteins were transferred to PVDF membranes and further incubated with anti-GP or VP40 antibodies. Proteins of interest were detected with the Super Signal West Pico chemiluminescent substrate . To confirm that siRNAs interfered with the expression of target proteins in a specific manner, immunoblot analysis of target host proteins was performed after siRNA transfection. Meanwhile, to confirm the specificity of the antibodies, target host proteins, GP, and VP40 proteins from the above extracts were tested before and after Co-IP separately . The housekeeping protein GAPDH was used as an internal control for immunoblot analyses.HEK 293T cells in 12-well plates were infected with trVLPs (TCID50 = 102.76/0.01 ml) for 48 h, HEK 293T cells in 12-well plates were fixed with 1% formaldehyde for 10 min, and glycine  was added to terminate the cross-linking reaction. Whole-cell extracts were then prepared using a Simple ChIP Enzymatic Chromatin IP Kit  following the manufacturer’s instructions with appropriate antibodies recognizing host proteins, and normal mouse or rabbit IgGs served as isotype controls. Beads were then washed and cross-linking substances were unlocked according the ChIP kit instructions. Purified nucleic acids were then amplified by qPCR with a qPCR Probe Kit . Primers and probe were designed according to the sequence of Zaire ebolavirus isolate Homo sapiens-wt/GIN/2014/Gueckedou-C05 (GenBank No. KJ660348.2) as follows: EN-Z-F, ATGATGGARGCTACGGCG; EN-Z-R, TGCCCTCTGTATGCTGGCCCT; EN-Z-P, CCARAGTTACTCGGAAAACGGCATG.After infection of trVLPs  for allelic discrimination.t-tests , and multiple comparisons were performed where appropriate. All analysis was performed with Graphpad Prism 5  and differences between group means with p < 0.05 were considered statistically significant.Data in normal distributions are presented as the mean ± standard deviation . StudentEBOV-trVLPs synthesized as described above displayed a filamentous-like morphology with a diameter of 100–500 μm as visualized by EM Figure . Meanwhip < 0.05; Figure To identify factors associated with the life cycle of trVLPs, siRNAs targeting 65 selected genes were tested in 293T cells infected with trVLPs in triplicate by qRT-PCR. The results showed that siRNA knockdown of ANXA5, ARFGAP1, FLT4, GRP78, HSPA1A, HSP90AB1, HSPA8, MAPK11, MEK2, NTRK1, and YWHAZ efficiently inhibited trVLP replication , four are involved in plasma and/or endosome membrane trafficking , and three play important roles in signal transduction pathways . Further bioinformatic analysis by GeneMANIA revealed that these candidate host factors were markedly enriched in gene categories associated with 14-3-3 protein families , annexin families , heat shock protein (HSP) families , and MAP kinase families  interacted with GP, whereas ARFGAP1 and ANXA5 did not appear to interact with GP  were consistent with A better understanding of the mechanisms of the EBOV life cycle and host–virus interactions (including protein–protein and protein–RNA interactions) would greatly assist the elucidation of EBOV pathogenesis and therapeutic development. Herein, we identified 11 candidate host proteins by RNAi silencing that appear to function in the EBOV-trVLP life cycle among 65 host factors presumed to be associated with the virus life cycle and secretary pathways. Meanwhile, we measured candidate host protein levels after RNAi by western blot analysis, and the results revealed significant interference effects and confirmed the specificity of RNAi silencing. Bioinformatic analysis by GeneMANIA indicated that these candidate host proteins belong to the following families: (1) HSPs that participate in a wide variety of cellular processes, including activation of proteolysis, protecting the proteome against stress, infection, and transport of newly synthesized polypeptides ; (2) 14-Heat shock protein family proteins are known to be associated with many viruses. For example, the Hsp70 chaperone network mediates the dengue virus life cycle, GRP78 is a potential defensive molecule against hepatitis A virus replication, and Hsp90 controls reactivation of human immunodeficiency virus type 1 from latency . MeanwhiTo confirm the interactions of candidate host proteins with EBOV, Co-IP, and Ch-IP were performed. The results revealed that HSPA1A, HSP90AB1, HSPA8, MAPK11, NTRK1, MEK2, FLT4, GRP78, and YWHAZ interacted with EBOV GP. Meanwhile, immunoblot analyses of the 11 target host proteins before and after IP confirmed the high degree of specificity for those antibodies, and specific interactions between GP and candidate host proteins. Four of these proteins belong to HSP families, which are involved in a variety of cellular functions, and some of the others function in plasma and/or endosome membrane trafficking and signal transduction pathways. These results are consistent with the functions of EBOV GP, which is critical for EBOV cell entry, and uncoating and fusion processes occurring at the cell membrane through the endosomal pathway. Protein interactions can involve various mechanisms including subunit polymerization, cross polymerization, and molecular recognition. However, the detailed mechanisms of the interactions between GP and host proteins remain unclear. Meanwhile, it is worth noting that additional factors likely act as a bridge between the candidate host proteins and GP.Co-immunoprecipitation and immunoblot analyses also revealed that ANXA5, GRP78, HSPA1A, and HSP90AB1 interact with VP40, and bioinformatic analysis indicated that ANXA5 engages in phospholipid binding, while GRP78 and HSP90AB1 are associated with the COP9 signalosome, which is believed to act as a scaffold to facilitate spatial sequestration for multiple interacting molecules that regulate protein stability, transcription, protein phosphorylation and intracellular distribution . The funInteractions of host proteins with trVLP RNA were assessed, and the results indicated that HSP90AB1, HSPA1A, GRP78, ANXA5, ARFGAP1, FLT4, MAPK11, MEK2, and NTRK1 interact with trVLP RNA. Protein-RNA interactions are important for various cellular process including transcriptional and post-transcriptional regulation, and activating immune responses against virus infection . In the There were some limitations to our study. Firstly, all the experiments were based on EBOV trVLPs and HEK 293T cells, the interactions may not reflect the physiology of live EBOV. Further experiments using EBOV are therefore required to confirm these current findings. Secondly, the interaction patterns are unclear, which may work either through direct binding or through the indirect action of one or more intermediate molecules, or through the formation of protein complexes by bridge proteins. Furthermore, it is unclear which region or segment of the GP or VP40 protein interacts with the host proteins. Finally, as ebolavirus probably infects different cell types via different mechanisms, the interactions we found in 293T cells may not be relevant in other host cells, such as monocytes and hepatocytes . SubsequAnalysis of EBOV life cycle-associated host factors by RNAi silencing identified four chaperones , four plasma and endosome membrane trafficking molecules  and three signal transduction elements  that may be essential for EBOV replication. Among these host proteins, nine  potentially interact with trVLP GP, four  potentially interact with trVLP VP40, and nine  participate in host protein-trVLP RNA interactions. Although EBOV trVLP cannot replace live ebolavirus entirely, and these interactions cannot be assumed to be identical in live virus, the study provides valuable foundations for further research.D-SY, T-HW, C-YH, Z-GW, Y-HL, and L-FC performed the experiments. H-PY and D-SY performed the statistical analysis. H-PY, N-PW, and L-JL designed the study and drafted the manuscript. All authors participated in writing the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Dioscin is a typical saponin with multiple pharmacological activities. The past few years have seen an emerging interest in and growing research on this pleiotropic saponin. Here, we review the emerging pharmacological activities reported recently, with foci on its antitumor, antimicrobial, anti-inflammatory, antioxidative, and tissue-protective properties. The potential use of dioscin in therapies of diverse clinical disorders is also discussed. Natural products have been an important source of drugs for antifungal, antibacterial, antitumor, and other pharmacological interventions , 2. ReceO-α-L-rhamnopyranosyl-β-D-glucopyranoside) could be isolated from various kinds of vegetables and herbs, most of which belong to the family of Dioscoreaceae  serve as starchy staple food in undeveloped countries around the world [As a steroidal saponin, dioscin  and six of eight Cytochrome P450 (CYP450) enzymes tested . Even for the two enzymes (CYP2C19 and CYP2D6) inhibited by dioscin, the IC50 values were higher than 150 μM [Using pooled human liver microsomes (H161) as the reaction system, dioscin, at a high concentration of 200 n 150 μM . Since CAs the end product with poor solubility of purine catabolism catalyzed by xanthine oxidase (XO) in human, uric acid in blood is mainly excreted through urine and feces –31. ResuAnd this recently published study identified that it is the metabolite of dioscin, tigogenin, that contributes to the uric acid-lowering effects of dioscin through suppressing URAT1 and promoting ABCG2  . Conside Candida albicans could cause a series of infections that are associated with oral cavity, urogenital tracts, as well as the increasingly used indwelling devices. In some cases, C. albicans could cause life-threatening bloodstream infections [ C. albicans was first reported by Sautour et al. in 2004 [ C. glabrata, C. tropicalis were also reported simultaneously [ C. albicans was attributed to its ability to induce damage to plasma membrane [ C. albicans biofilms, a microbial lifestyle with complex structures and high resistance to antifungal agents [As an important human fungal pathogen,fections . The ant in 2004 , 39 and  in 2004 , and inhaneously , 39, 40.membrane . The celmembrane . Recentll agents , 42. Saprolegnia parasitica, a pathogenic fungus in freshwater ecosystem [ Pyricularia oryzae, one famous fungal pathogen of rice [Dioscin could also cause increased membrane permeability and excessive ROS generation incosystem . In addi of rice . In a wo in vitro antiviral activities against adenovirus, hepatitis B virus and vesicular stomatitis virus, although the efficacy varies in different phases of infections [Viruses are important human pathogens that could cause a broad spectrum of diseases. Recently, dioscin was reported to havefections .2+ release and increased endogenous reactive oxygen species (ROS) production are common to be found [Dioscin has demonstrated antitumor activities against many kinds of tumors, such as lung cancer, esophageal cancer, gastric cancer, colon cancer, glioblastoma, cervix carcinoma, ovarian cancer, breast cancer, prostate cancer, and leukemia. The target tissues listed can be referred to the review on saponins in 2016 . Recentlbe found , 53–56. be found , 57, 58.be found . In somebe found , 59, altα gene [2/M cell cycle arrest was found in multiple kinds of tumor cells through downregulation of cyclin B1, checkpoint kinase CHK2 and cyclin-dependent kinase CDK1 [Besides, the apoptosis-inducing effects of dioscin also involves the activation of ERK1/2 and AIF pathway , 61, theα gene , potentiα gene . Meanwhiα gene , 53, 63.ase CDK1 , 63, 64,ase CDK1 . in vivo studies also confirmed that dioscin could attenuate invasion and migration, decrease tumor size, and extend the lifespan of rats [Antitumor effects of dioscin also involves inhibition of migration, invasion and angiogenesis. Dioscin could decrease the CAP-1 related cell migration and invasion, and markedly down-regulate MMP2 and MMP9 expression , 58. DNM of rats , 54.κB signaling pathway (via blocking inhibitor κB-α (IκB-α) degradation), thus strengthening drug absorption in adriamycin- (ADR-) resistant erythroleukemic K562/ADR cells, HepG2/ADR cells [ in vivo samples, dioscin could upregulate the expression of the major components of gap junctions connexins Cx26 and Cx43, resulting in more efficient herpes simplex virus thymidine kinase/ganciclovir- (HSV-tk/GCV-) induced bystander killing [Dioscin could overcome multidrug resistance and enhance antitumor activity of other drugs. Multi-Drug Resistance (MDR) gene encodes p-glycoprotein (p-170), which is located on the cell membrane and pumps drugs out of cancer cells, resulting in drug resistance. Dioscin was found to significantly inhibit MDR1 mRNA and protein expression and inhibit the NF-DR cells  and humaDR cells , 36, 37.DR cells . Another killing .Autophagy also participates in dioscin-induced apoptosis, which could be detected 12 hours after low-dose dioscin exposure and earlier than apoptosis in human lung cancer A549 and H1299 cells and hepatoma Huh7 cells. Dioscin-induced autophagy via ERK1/2 and JNK1/2 pathways. When autophagy was inhibited, dioscin-induced cell apoptosis was significantly enhanced, hence dioscin may provide some benefits for cell survival and act as a cytoprotector , 68. It α (C/EBPα), which is a critical factor for myeloid differentiation and induce the differentiation of promyelocytes to granulocytes and monocytes [α-MSH-induced melanogenesis [Dioscin also induces differentiation of promyelocytes and melanogenesis in melanoma cells , 59. In ogenesis .Given its significant antitumor activities, dioscin may be a promising candidate for developing anticancer therapies. Moreover, its resistance-sensitizing activity may make dioscin an adjunctive to current anticancer pharmacological therapies.As the major organ in human for metabolism (detoxification and assimilation), liver often faces multiple stresses and thus can undergo various pathogenesis in response to those stresses. These pathological changes can manifest as fibrosis, injuries, cholestasis, and fatty liver diseases.γ, Nrf2, HO-1, and SOD was increased, while TGF-β1/Smad, Wnt/β catenin, and MAPK signaling pathways could be suppressed by dioscin to mitigate oxidative stress and alleviate hepatic fibrosis [Hepatic fibrosis, featured by excessive accumulation of extracellular matrix , is a long-term process that gradually causes damaged architecture and impaired function of liver, resulting in cirrhosis associated with high mortality and morbidity . Via PI3fibrosis , 111, suDioscin could attenuate the acute liver injury induced by DMN and CCl4 through inhibiting apoptosis, necrosis, oxidative stress and inflammation , 113. ThFeatured by abundant fat storage in liver, NAFLD can progress into cirrhosis and hepatocarcinoma . In NAFLα-naphthylisothiocyanate (ANIT), dioscin could clean toxic bile constituents from the liver through upregulation of multidrug resistance-associated protein 2 (Mrp2) and bile salt export pump (Bsep) [+-taurocholate cotransporting polypeptide (Ntcp)) caused by ANIT could also be prevented by dioscin treatment. Moreover, dioscin could increase the levels of antioxidative powers  and antiapoptotic proteins in animals, as well as in primary cultured cells, to mitigate oxidative stress and to reduce apoptosis in cholestasis [Cholestasis, caused by excessive accumulation of bile salts in liver and featured by primary sclerosing cholangitis, biliary cirrhosis and atresia, could result in increased oxidative stress and apoptosis , 118. Inp (Bsep) , 118. Ab in vitro [ in vivo antiapoptotic activity was exerted through upregulating Bcl-2 and Bcl-x and downregulating Bak, CYP2E1, caspase 3, caspase 9, p53, and PARP [Ischemia/reperfusion (I/R) can engender damage to tissues in surgery, transplantation and stroke, while the underlying mechanism involves oxidative stress, inflammation and apoptosis , 120. Alin vitro , 54, 61,in vitro . The in and PARP . The proand PARP . In a woand PARP –123.β/Smad3 signaling to delay fibrosis [In mouse model of pulmonary fibrosis induced by crystalline silica, dioscin could inhibit TGF-fibrosis . Throughfibrosis . In addifibrosis .β1, which plays an important role in renal fibrosis, could also be inhibited by dioscin via Sirt3 to mitigate renal fibrosis [Chronic kidney disease (CKD) severely influences the people's health, with 10-15% of the adults in the world affected, imposing a heavy global burden . CKD alsfibrosis .In inflammatory kidney injury induced by LPS, TLR4/MyD88 pathways was suppressed by dioscin via upregulating miR-let-7i . MoreoveSince renal damage could be caused directly by hyperuricemia, given the anti-hyperuricemic effect of dioscin, dioscin may offer another kind of nephroprotection , 30. The In vivo protective effects of dioscin against myocardial I/R injury in rats were exerted through upregulation of connexin 43 [β/mTOR pathways, improving the impaired cardiac function [Clinically, the therapeutic use of doxorubicin for cancer is impeded by its cardiotoxicity, while treatment with dioscin (at the level of ng/mL) could increase the viability of cardiocytes that were exposed to doxorubicin. This kind of protective effects was associated with decreased oxidative insults mediated by downregulation of miR-140-5p in H9c2 cells and rodents . Moreovenexin 43 . In addiDuring treatment of ischemic stroke, which causes a large proportion of morbidity and mortality worldwide, reperfusion can cause damage to brain . Dioscinα, through inhibiting NF-kB nuclear translocation and activation [Adhesion molecules-mediated interaction between monocytes and endothelial cells plays an important role in the initial phase of atherosclerosis, which is an inflammation-associated vascular disease . Dioscintivation .β, IL-6, and TNF-α via modulation of NK-kB can attenuate the inflammatory response in atherosclerotic plaque [Meanwhile, the secretory inhibition of IL-1c plaque . Anotherc plaque . The forc plaque . In rat c plaque . Dioscinc plaque . This maα, IL-1β and IL-6, which are secreted by immune cells recruited to injury sites. Sustained inflammatory cytokines release and reaction result in chronic inflammation, which is featured by excessive immune cell infiltration and is the major cause of many diseases [Inflammation, as the host response to detrimental stimuli, are mediated by pro-inflammatory cytokines such as TNF-diseases .α, IL-1β, and IL-6 secretion in HUVECs, macrophages, NRK-52E and HK-2 cells, can be inhibited by dioscin [α, IL-1β and IL-6 can also be suppressed in mice and rat model of multiple diseases, such as acute liver injury [TNF- dioscin , 147. Thr injury , liver fr injury , obesityr injury , cerebrar injury , 141, anr injury , 132, thAcute neuroinflammation caused by endotoxemia could be mitigated by dioscin treatment, which is associated with improved 5-HT metabolism . ProtectIn adipocytes, dioscin could also increase the expression of adiponectin, a hormone known to own anti-inflammatory effects, thus possibly exerting another kind of indirect anti-inflammatory effect . in vitro inhibitory effects of dioscin on synovial cell hyperplasia induced by IL-1β has been reported [As a chronic inflammatory disease caused by disorder of individual immune system and featured by overgrowth of synoviocytes and fibroblast-like synoviocytes, rheumatoid arthritis may engender articular degeneration and damage, affecting heavily public health given its prevalence , 153. Threported . In collreported . in vivo therapeutic effects of dioscin may be accomplished by inhibiting the increase in stromal cell-derived factor-1 (SDF-1), CXCR4 (receptor of SDF-1) and p38 MAPK signaling in synovial tissue of rats [In gouty arthritis induced by monosodium urate crystal, the of rats . Conside of rats , 155.β-catenin signaling, which activates catabolism in chondrocytes, could also be downregulated by dioscin, while PPARγ, whose activation can lower the production of catabolic factors and ameliorate the oxidative stress, could be upregulated, thus contributing to the protecting effects of dioscin against osteoarthritis [Osteoarthritis, emanating from damage to cartilage and underlying bones, can cause pain, stiffness, and deformity and affect millions of people all around the world , 157. Inrthritis .Obesity is often associated with increased risk for a series of diseases including type 2 diabetes mellitus (T2DM), certain cancers, osteoarthritis, cardiovascular, and respiratory diseases. Worldwide, the incidence of obesity is growing while the drug is limited, highlighting the necessity for developing new drugs . in vitro with an IC50 of 20 μg/ml (thus inhibiting the absorption of fat) while Dioscorea nipponica Makino powders containing dioscin inhibits the rise of blood triacylglycerol concentration induced by oral gavage of corn oil emulsion in mice [Dioscin inhibits porcine pancreatic lipase in mice . In rats in mice . This ef in mice , 151. In in mice .At concentrations without obvious cytotoxicity, dioscin can inhibit insulin exposure-initiated differentiation by delaying cell cycle progression and suppressing the phosphorylation of ERK1/2 and p38 rather than c-JNK in differentiating preadipocytes. Dioscin can also inhibit adipogenesis through inducing phosphorylation of AMPK, which stimulates the catabolic process of fatty acid to facilitate the energy production .β cell proliferation through Wnt/β-catenin pathway and attenuate the impairments (decreased viability and increased apoptosis) of β cells induced by high-glucose treatment [Besides its anti-obesity effects, dioscin might exert beneficial effects on diabetes, which represents a heavy burden on public health system. In a study published in 2018, dioscin has been shown to promote reatment . Moreovereatment . Taken tOxidative stress plays a considerable role in the pathogenesis of a variety of diseases and injuries such as pulmonary fibrosis , CKD 12, inflammβ1 on ROS production and myofibroblast differentiation could also be suppressed by overexpression of Sirt3 [ in vivo expression of other antioxidant enzymes such as HO-1, Nrf2, and GSH-Px in rats and lower the levels of MDA, NO and iNOS [SOD2, which contributes to removing superoxide during oxidative stress , could bof Sirt3 . In cardof Sirt3 . Dioscinand iNOS , 149. Inand iNOS , 141.Osteoporosis, featured with loss of bone tissue that associated with raised risk of fracture, is caused by imbalanced bone homeostasis towards bone resorption by osteoclasts . This imDioscin could facilitate osteoblastic proliferation and differentiation through low-density lipoprotein receptor related protein 5 (Lrp5) and estrogen receptor (ER) pathways in mouse pre-osteoblast-like MC3T3-E1 cells and in human osteoblast-like MG-63 cells . Apoptos in vitro, suggesting its potential use as cosmetic whitening agent and for preventing melanin hyperpigmentation [α-melanocyte-stimulating hormone (MSH), and this inhibition was due to the decreased expression of tyrosinase, tyrosinase-related protein-1 (TRP-1) and TRP-2 via downregulation of phosphor-CREB and microphthalmia-related transcription factor (MITF) [ In vitro inducing effect of dioscin on growth hormone release in rat pituitary cells has been reported to be strong, with an approximately 17-fold stimulation of release [Dioscin could synergize with oxyresveratrol in suppressing tyrosinase , using L-tyrosine and L-DOPA as substratesentation . Later, r (MITF) . In vitr release . Dioscin release . Dactylogyrus intermedius, with an EC50 of 0.44 mg/L, which is more effective than the positive used [Dioscin also showed strong anthelmintic activity against the aquacultural ectoparasiteive used .Recent years have seen a lot of reports on the beneficial effects of dioscin, including antitumor, hepatoprotective, antiviral, nephroprotective, cardioprotective, antifungal, anti-hyperuricemia activities. Although dioscin falls out of the rule of five due to its large molecular weight, it does fall into the sweet scope where the balance between protein-protein interaction (PPI) inhibition and oral bioavailability may be found . Moreove