The extracellular matrix (ECM) is critically important for most cellular processes including differentiation, morphogenesis, growth, survival and regeneration. The interplay between cells and the ECM often involves bidirectional signaling between ECM components and small molecules, i.e., growth factors, morphogens, hormones, etc., that regulate critical life processes. The ECM provides biochemical and contextual information by binding, storing, and releasing the bioactive signaling molecules, and/or mechanical information that signals from the cell membrane integrins through the cytoskeleton to the nucleus, thereby influencing cell phenotypes. Using these dynamic, reciprocal processes, cells can also remodel and reshape the ECM by degrading and re-assembling it, thereby sculpting their environments. In this review, we summarize the role of chondroitin sulfate proteoglycans as regulators of cell and tissue development using the skeletal growth plate model, with an emphasis on use of naturally occurring, or created mutants to decipher the role of proteoglycan components in signaling paradigms. Proteoglycans, complex macromolecules that are prominent constituents of the ECM composed of a protein core to which are covalently attached variable length and composition glycosaminoglycan (GAG) chains . BecauseIn contrast to the HSPGs, the interactions of signaling molecules with chondroitin sulfate and dermatan sulfate proteoglycans (CSPG and DSPG), which are the focus of this review and are often the most abundant proteoglycans in tissues, are not as well understood. Chondroitin sulfate (CS) chains are found on multiple proteoglycans; the most common are the hyaluronan- and lectin-binding proteoglycans  which have structural similarity at both the protein and genomic levels. The hyalectan family consists of four members: aggrecan, versican, neurocan, and brevican, which all share a tri-domain structure: an N-terminal globular domain that binds hyaluronan, a central domain bearing the CS chains and a C-terminal region that binds lectins. The CS chains consist of repeating disaccharides of N-acetylgalactosamine  and glucuronic acid (GlcA) decorated with different degrees and patterns of sulfation on the disaccharides: GalNAc may have sulfate on C4 (CS-A), C6 (CS-C) or both C4 and C6 (CS-E), all catalyzed by specific sulfotransferases. The GlcA unit may also be sulfated on the C2 position (CS-B or CS-D) depending on where the sulfate residue is on GalNAc. Dermatan sulfate (DS) derives from chondroitin sulfate by inversion of GlcA to iduronic acid (IdA), catalyzed by an epimerase enzyme.in vitro and in vivo approaches. Although general principles of CSPG interaction with signaling molecules have been shown in several developing tissue systems, we use as example the formation of skeletal structures with a focus on the growth plate of long bones, a transient cartilage template that is, replaced by bone. A complex and highly orchestrated program regulates growth plate cartilage morphogenesis in which chondroblasts proliferate, differentiate to chondrocytes, alter their shape, proliferate in stacks along the longitudinal axis, terminally differentiate to hypertrophic chondrocytes, and elaborate a mineralized vascularized matrix, which is then replaced by osteocytes , a lethal chondrodystrophy of fowl  mouse is due to a 7-bp deletion in exon 5, resulting in a premature stop codon and no aggrecan product  rescue embryos  Sox9 expression in resting and proliferative zones similar to wild type; ii) an increase in Ihh mRNA production in more chondrocytes in the pre-hypertrophic region; iii) relatively normal expression of Ptch1, the receptor of IHH within the bone marrow near the chondro-osteo junction, perichondrium and proliferative zones similar to wild type; iv) strong re-expression of Fgfr3, which encodes the receptor for negative regulators of chondrocyte proliferation such as FGF9 and FGF18 in the proliferative and early hypertrophic zones of the growth plate; all these features closely resemble those found in wild-type embryos. Taken together, the data obtained from RT-PCR, immunochemistry and mRNA in situ analyses confirm that the presence of aggrecan in the growth plate ECM is fundamental to maintaining normal expression and spatial localization of the essential signaling molecules that regulate chondrocyte organization, morphology, and maintenance during growth plate development  type 2, leading to severe chondrodysplasias which suggests a requirement for xylosyltransferase during skeletal development  was identified from an N-ethyl-N-nitrosourea (ENU) mutagenesis screen, and exhibits reduced skeletal element lengths, normal growth plate patterning and no change in chondrocyte proliferation; however, pug mutants display premature maturation and early ossification leading to the disproportionate dwarfism. The mutation in Xylt1 disrupts enzyme activity and leads to reduction in the number of GAG chains in pug mutant proteoglycans. Furthermore, XYTL1 was mislocalized as it was not observed in the cis-Golgi as previously shown in wild type mice . Furthermore, the phenotype described above (delayed chondrocyte maturation) suggests IHH and FGF signaling pathways may be affected. As predicted, an increase in Fgfr3 levels, but no downstream changes in MAPK signaling were observed. Concomitantly, an up-regulation of short-range IHH signaling was observed, while long-range IHH signaling through PTHrP, which inhibits chondrocyte maturation, was not affected. These results suggest that the premature maturation of pug chondrocytes may not be influenced by changes in IHH and FGF signaling. Rather proteoglycans, in addition to regulating diffusion of key signaling molecules, may provide maturation cues to chondrocytes independent of IHH and FGF signaling. Furthermore, since the Xylt1 mutation affects both CSPG and HSPG production, the IHH responses also suggest that these two proteoglycans may function differently in regulating diffusion of IHH through the ECM, with CSPGs expanding the IHH diffusion domain and HSPGs restricting the domain. The premature-maturation pug phenotype, in the absence of signaling changes, suggests a novel proteoglycan cue that influences timing of chondrocyte maturation  spondylodysplastic type 1  of CS, DS, and HS, mutations in the other three enzymes also cause hereditary diseases. Compound heterozygous and homozygous mutations in c type 1  which isc type 1 . Larsen c type 1 . Compounc type 1  which prc type 1 , which pc type 1 . Lastly,c type 1 . Again, Chsy1−/− exhibited brachypodism with a patterning defect in distal phalangeal elements, achondrodysplasia and decreased bone density, caused by a reduction in CS chains and a shift in cell orientation. Transcriptome analyses of candidate genes implicated in joint formation, as well as in vivo analysis of growth factor signaling by FGF, TGFβ, BMP, WNT3A, NOTCH and HH in mouse embryonic fibroblasts (MEPs) and primary chondrocytes, suggested that IHH distribution was altered and that mutant MEPs are more sensitive to HH stimulation. Together these findings suggest that IHH signaling is disrupted, but differences in signaling may be secondary to changes in chondrocyte orientation  mouse model  and Col10a1 mRNA expression were comparable in wild type and bm mutants. In contrast, using a set of antibodies with specificity for all functional sulfate epitopes, immunohistochemistry revealed reduction in CS-4 and CS-6 epitopes and an increase in the CS-0 epitope in the bm growth plate EMC, compared to wild type. In contrast, N-sulfated HS showed comparable staining in wild type and bm growth plate. These data were verified by FACE and 35S-sulfate incorporation experiments; only a reduction of sulfate incorporation into CSPGs of the predominantly CS-4 species and no change in HS-sulfate content in bm cartilage was observed; establishing the bm mouse as an excellent model for investigating the role of under-sulfated CS interactions with signaling molecules during cartilage development , also leads to chondrodystrophies in mice  and humase model  has a muse model . At birtll zones . Aggrecaelopment .Pthr1) was expressed at high levels in the pre-hypertrophic zone in both bm and wild type. In contrast, Fgfr3 and Ihh (expressed in the pre-hypertrophic zone) and its receptor patched (Ptch1) expressed in the proliferative zone exhibited decreases in mRNA levels in bm cartilage by three methods, mRNA in situ, RT-PCR, and immunohistochemistry. In particular, IHH protein was not uniformly distributed between chondrocytes in bm samples, rather a restricted diffusion pattern characterized by protein aggregation was observed. The abnormal IHH distribution was verified by crossing bm mice with LacZ Ptch ± mice and determining the ratio of Gli activator (Gli1/Gli2) to Gli repressor (Gli3) to measure IHH pathway activation  fusion protein and HS and CS GAG chains  showed a gradient of binding affinity (Kd) and binding capacity (Bmax) in order: HS, CS-4, CS-6, CS-0. Since CS-4 is the predominant species in postnatal cartilage and the binding affinity and capacity is higher for the CS-4 to CS-0 motif, a reduction in CS-4 is commensurate with abnormal IHH signaling. Secondly, to demonstrate that IHH interacts with CS specifically and does so through the IHH N-terminal Cardin-Weintraub motif, this motif was mutated which resulted in complete loss of binding to both HS and CS chains, suggesting that the interaction between IHH and CS is primarily mediated through this motif. Lastly, a direct interaction between CSPG and IHH-AP was demonstrated by quantitative immunoprecipitation with a specific aggrecan antibody, demonstrating that the major cartilage CSPG, aggrecan, directly interacts with IHH. Taken together, the biochemical and genetic evidence suggest a biological mechanism whereby undersulfated CSPGs result in restricted IHH diffusion through the ECM leading to a reduction in chondrocyte proliferation, which significantly impacts skeletal growth in the m mutant .nanomelic chick and cmd mouse) leads to lethal phenotypes (Pug) or sulfation (bm) models present with milder chondrodyplasia phenotypes , increased chondrocyte proliferation and expansion of the proliferative zone  leads to accelerated maturation of chondrocytes, while reduction in GAGs in the mature growth plate  leads to changes in morphogen distribution and altered rate of cell division. Lastly, two other mutant mouse models, a Golgi PAP phosphatase  disorders exhibit tissue-specificity for GAG metabolism and function. Those MPSs involving lysosomal storage of CS/DSPG and KSPG families are associated with skeletal disorders . In contrast, HSPGs are usually associated with central nervous system (CNS) pathology (MPS III), and MPS enzymes common to multiple GAG pathways cause both skeletal and CNS pathology . An elegant example of the critical role of CSPG synthetic and catabolic enzymes in growth regulation are the consequences of either deletion of C4ST or MPS VI, both of which lead to skeletal malformations. Although MPS VI and C4ST phenotypes are not identical, there are similarities indicating that both synthesis and degradation of CSPGs cause cellular de-regulation of growth plate development. As discussed earlier, C4ST deficiency hyper-activates TGFβ signaling while down-regulating BMP signaling . MPS VI Understanding functional interactions between components of the ECM and signaling pathways that control synthesis of the ECM components is also critical to degrative diseases like osteoarthritis (OA), a common degenerative disorder with no current disease-modifying therapies. This skeletal disorder is due to degradation of the major CSPG, aggrecan, mostly by specific enzymes, aggrecanases (ADAMTS-5). These aggrecan degradative enzymes are upregulated by mediators associated with joint inflammation or tissue overloading . Interesin vitro studies and clinical trials with plasma have produced mixed results  autologous or xenographic cells from the tissue to be formed; ii) signaling molecules which provide instruction for expressing a desire phenotype; and iii) synthetic scaffolds that hold the cells together and shape the tissue formation  asormation , all witormation . As wellormation . Severalormation , with diormation . Other sormation . Advanceormation , as wellormation . As mentormation  (see preormation . In sum,ormation . AlthougDecades of studies have identified the hierarchical ECM-directed creation of the skeletal growth plate, which involves differentiation and growth of chondrocytes, positioning of cells, matrix and regulators into a highly integrated, bidirectional and temporally orchestrated process. As well, the reciprocity between ECM components, transcription factors, and signaling molecules that coordinate their expression has been revealed by the plethora of chondrodysplasias due to mutations in these regulatory molecules as well as proteoglycans and their biosynthetic enzymes that disrupt growth plate development and maturation summarized in


Human T-cell lymphotropic virus type 1 and 2 (HTLV-1/2) screening is not mandatory in Spanish blood banks. In Catalonia, selective screening was introduced in 2008, followed by universal screening in 2011. We present herein a 10-year experience of HTLV testing in blood donors. HTLV-1/2 selective screening was performed using Ortho-Clinical Diagnostics HTLV-I/HTLV-II Ab-Capture ELISA between February 2008 and May 2009, then Abbott Prism HTLV-I/ HTLV-II assay until December 2010. Abbott Architect rHTLV-I/II assay was then used for HTLV-1/2 universal screening in pooled samples. INNO-LIA HTLV I/II Score (Fujirebio) and in-house HTLV-1/2 proviral DNA real-time PCR were used in reactive samples. Follow-up was offered to confirm HTLV-1/2 donors in Vall d’Hebron Hospital. Between 2008 and 2017, 51 blood donors were confirmed HTLV positive  out of 2,114,891 blood donations . Sixty-nine percent were female, median age was 40 years and most were born in Latin America (69%), followed by Europe (25%), Africa (4%) and Asia (2%). Screening of relatives and partners identified 12 additional HTLV-1 cases. Lookback studies did not show any HTLV-1/2 transmission. HTLV infections found in blood donors mirror epidemiological changes in the population of Spain. Consequently, HTLV should be considered a potential risk for recipients and calls for the design of optimal strategies to ensure transfusion safety. Human T-cell lymphotropic virus (HTLV) was the first oncogenic human retrovirus described . HTLV isHTLV-1 infection remains asymptomatic in most cases, but 5–10% of infected subjects eventually develop disease . InfectiHTLV-1 affects five to ten million individuals worldwide, although this is probably underestimated as many countries do not report prevalence data . The majTransmission of HTLV requires cell-to-cell contact with infected lymphocytes. Three main routes of transmission have been established: (1) vertically from mother to child through prolonged breastfeeding, (2) by sexual contact, and more efficiently from male to female, and (3) by contact with substances of human origin containing infected lymphocytes . Transmission of HTLV-1/2, and especially HTLV-2, is described among injecting drug users ,10,11,12Depending on local epidemiology, various strategies are applied to reduce the risk of transfusion-transmitted HTLV-1/2. In some non-endemic countries, donor suitability is assessed through a pre-donation questionnaire, where individuals from HTLV-1/2 endemic countries are deferred from blood donation. Another strategy is serological HTLV-1/2 screening of blood donations, mandatory in many endemic and some non-endemic countries ,14. FinaIn Spain, leukoreduction by filtration of blood products was introduced in blood banks in 2002, but HTLV-1/2 screening is not mandatory. In Spanish blood banks, donor suitability regarding HTLV-1/2 infection risk is normally assessed through the pre-donation questionnaire; donors with known HTLV-1/2 infection or reporting residence in an HTLV-1/2 endemic country are deferred from donating blood.Catalonia is an autonomous region situated in North-East Spain that, with 7.5 million inhabitants, represents 16% of Spanish population. All blood donors in Spain are volunteer and non-remunerated. The Blood and Tissue Bank of Catalonia (BST) is a government institution and the only blood bank in the region that provides all the hospitals with blood components. BST implemented HTLV-1/2 screening in 2008 as an additional safety measure to leukoreduction, initially in the same blood donors selectively tested for Trypanosoma cruzi (T. cruzi) infection (Chagas disease), mainly from Latin America, then in all blood donors through universal testing using pooled samples .We present herein the results obtained with HTLV-1/2 testing in blood donors from a non-endemic area between 2008 and 2017.Different HTLV-1/2 screening strategies and tests were applied in our blood bank between 2008 and 2017.Between 2008 and 2010, in a first approach, selective individual screening for anti-HTLV-1/2 was performed in the same blood donors at risk for Chagas disease, endemic in continental Latin America. Individuals included belonged to one of the following risk groups: donors born or transfused in an endemic area, donors born of a mother native to a Chagas endemic area or residents in an endemic area for more than 1 month. All Latin America from Mexico to Chile was considered endemic.After identifying the first HTLV-1/2 cases in the selected blood donors and in some of their sexual partners who did not originate from Latin America, the strategy evolved to universal testing. From 2011, HTLV-1/2 testing was performed in all blood donors, independently of their geographical origin, in a 48-sample minipool (MP48).All blood donors answered and signed an ethics committee-approved questionnaire that included questions on risk factors related to HTLV-1/2 acquisition. Information on age, sex and place of birth was registered.Repeatedly reactive donations in routine blood screening were tested with supplementary assays in the Transfusion Safety Laboratory, and the donors were contacted for retesting and counseling.Confirmatory diagnosis, clinical follow-up and testing of family members and partners were offered to anti-HTLV confirmed positive blood donors in the International Health Unit of Vall d’Hebron University Hospital .Three screening methods were successively used between 2008 and 2017. For the individual (ID) testing, serum samples were screened for the presence of HTLV-1/2 antibodies, first with the automated EC-approved assays HTLV-I/HTLV-II Ab-Capture ELISA Test System , in place from February 2008 to May 2009, and then with the Abbott Prism HTLV-I/ HTLV-II chemiluminescent immunoassay , in place from June 2009 to December 2010.The minipool strategy (MP48) in all blood donations was validated between November 2009 and December 2010 using a third screening method, the Abbott Architect rHTLV-I/II chemiluminescent microparticles immunoassay , in parallel with routine testing of individual selective blood donations using the Abbott Prism HTLV-I/ HTLV-II chemiluminescent immunoassay. An analytical sensitivity of 100% was previously observed after 48-fold dilution of positive samples from blood donors obtained in our blood center after HTLV-1/2 ID screening using a panel of 16 well-characterized HTLV-1 or HTLV-2 samples , kindly provided by Vanderleia Barbaro and Simone Kashima . For the validation, 100 µL EDTA plasma from each of 48 different blood donations were mixed to constitute a single 4.8 mL-sample using the automated Tecan liquid handling platform Freedom EVO Clinical , which allowed full traceability of donations included in every MP48. Every reactive result observed in a pooled sample was resolved by individually testing the serum of each of the 48 donations using the Abbott Architect rHTLV-I/II assay.During this validation period (November 2009–December 2010), blood donations considered at risk for Chagas disease were analyzed twice for anti-HTLV-1/2: in ID testing with the Abbott Prism HTLV-I/II assay, and in MP48 with the Abbott Architect HTLV-1/2 assay. The rest of the blood donations, not considered at risk for Chagas disease, were tested only once for anti-HTLV-1/2, in MP48 format.HTLV-1/2 MP48 testing with Abbott Architect rHTLV-I/II was implemented after validation in all blood donations from 2011 to 2017.2O. Five µL were used in each PCR reaction in a 25 µL final volume, using 1x TaqMan Universal PCR master Mix , 0.1x RNase P Primer-Probe , 600 nM each forward (FW) and reverse (RV) primer  and 300 nM TaqMan FAM™ dye MGB probe . Each PCR reaction was performed in a 7500 Real Time PCR System thermocycler (Applied Biosystems) and conditions were the same for both amplified regions: 2 min at 50 °C, 10 min at 95 °C, 40 cycles of 15 s at 95 °C and 1 min at 60 °C. A limit of detection for HTLV-1 proviral DNA of 3.8 HTLV-1 proviral copies/10,000 PBMC was obtained using a quantified HTLV-1 sample kindly provided by Dr. Ana Treviño and Dr. Vicente Soriano, Hospital Carlos III . The results expected in this assay are: negative , positive for HTLV-1 or positive for HTLV-2. A frozen one-use aliquot of whole blood sample from donors previously confirmed as positive for HTLV-1 or HTLV-2  was used as positive control for DNA extraction and HTLV specific detection in each PCR run.Samples that tested initially reactive for HTLV-1/2 antibodies in screening were retested twice and, if repeatedly reactive, were analyzed by HTLV-1/2 immunoblot . The INNO-LIA HTLV I/II Score is a line immunoassay that confirms the presence of antibodies against HTLV-1/2 and, additionally, differentiates between HTLV-1 and HTLV-2. The results expected in this assay are: negative, indeterminate, positive for HTLV without typing (HTLV positive), positive for HTLV-1 or positive for HTLV-2. Furthermore, all initially positive samples were assessed for the presence of proviral HTLV-1 (TAX region) or HTLV-2 (LTR region) sequences using an in-house qualitative real-time polymerase chain reaction (PCR) for each HTLV region . Briefly, proviral DNA was obtained from 200 µL whole blood EDTA samples using the QIAamp DNA Mini Kit  and eluted in 100 µL HThe different combinations and interpretation of the results obtained using the supplementary assays are described in the following table .For regular blood donors who were identified in this study as HTLV-1/2 positive, a lookback procedure was initiated in all patients who had been transfused with blood products not tested for HTLV-1/2 or tested as nonreactive. Transfusion services in hospitals that were responsible for the blood component transfusion were contacted and asked to investigate the status of the recipients.For continuous variables, means with standard deviation (SD) were used. Anti-HTLV-1/2 prevalence were calculated with a 95% confidence interval (95% CI) using the Wilson score interval calculation method .In a first approach, blood donors presenting a risk for Chagas disease and tested for anti-Trypanosoma cruzi were screened for HTLV-1/2. The most represented countries of origin of blood donors born in Chagas-endemic areas were Argentina (22%), Colombia (20%) and Ecuador (12%) followed by Brazil (10%), Peru and Uruguay (both 8%), Chile (7%), Venezuela (6%) and Bolivia (4%).Between February 2008 and December 2009, 580,290 blood donations were collected in the blood center; of these, 26,604 were selectively and individually tested for HTLV-1/2  and higher specificity   . Forty-six were positive for HTLV-1, four were positive for HTLV-2 and one blood donor was confirmed as HTLV positive without type differentiation . Mean agDuring this period, 11 blood donors were classified as “Indeterminate”, with an initial reactive screening result and an indeterminate immunoblot result . Follow-up with new blood sample extraction was performed in nine of them. Follow-up was performed by the blood bank in the months or even 1 year following the initial reactive blood donation and, in some cases, follow-up was additionally performed by the International Health Unit of Vall d’Hebron University Hospital. No evolution of HTLV status could be observed in any case that could evidence a seroconversion to HTLV.As for the origin of positive blood donors, Latin American donors accounted for 67% of positive donors, while 25.5% of HTLV-1/2 positive donors were born in Europe. Interestingly, three out of four HTLV-2 cases as well as the non-typed HTLV donor were born in Spain .HTLV proviral DNA was detected in 46 of the 51 HTLV-1/2 blood donations (90% positive PCR). In one of these 46 PCR positive cases, immunoblot was indeterminate but proviral DNA PCR identified the virus as HTLV-2. Donations with undetectable HTLV DNA (5 out of 51) were identified by immunoblot as HTLV-1 in four cases or confirmed as HTLV without type differentiation in one case .Thirty-nine out of the 51 HTLV-1/2 positive blood donors were first-time blood donors. Conversely, 12 blood donors had given blood before the HTLV-1/2 positive screening result. Furthermore, two of them seroconverted to HTLV-1 during the study period. One of these two donors had tested nonreactive for HTLV-1/2 on two occasions using ID-selective screening, and became HTLV-1 positive 3 years after the last nonreactive result when tested using MP48 screening. The second case of seroconversion was detected 6 years after the last blood donation with a nonreactive result.Ten donors out of fifty-one were regular blood donors when HTLV screening was implemented and previous donations had not been tested for HTLV-1/2. As HTLV-1/2 infection could have been acquired previously, all of these blood donations could therefore potentially have transmitted the virus. Additionally, two of the fifty-one positive blood donors seroconverted to HTLV-1 during this study and, although tested HTLV-1/2 nonreactive at the time of previous blood donation, the recipients were investigated. In total, 38 blood donations had been obtained from these 12 HTLV-positive repeat blood donors between 1991 and 2011. Fifty-two components associated with these 38 donations were issued for transfusion  and the lookback procedures were initiated . InformaOf the 51 HTLV-1/2 positive blood donors, 7 could not be traced back and 5 received information about their HTLV-1/2 infection from the blood bank, but no clinical follow-up data was available, as donors declined the appointment arranged in the International Health Unit of Vall d’Hebron University Hospital. Information was available for 39 out of 51 HTLV-1/2 positive blood donors. Blood donors were tested again for HTLV antibodies according to the hospital microbiology laboratory procedure  and confirmed by line immunoassay . HTLV infection was confirmed in all 39 patients (data not shown).None of the donors stated that they were a current or former intravenous drug user and five (12.5%), all from HTLV-1/2 high prevalence countries, declared that they had previously received a blood transfusion in their country of origin, information not disclosed in the blood bank questionnaire. Overall, 54 relatives and sexual partners were tested for HTLV infection, 12 of whom were positive: 7 sexual partners (from 7 different donors), 1 sibling (sister), 1 daughter, 1 father, and 2 mothers. In 6 HTLV-1 positive couples, both partners were born in an endemic country . In one couple, the donor was born in Chile while her partner was born in Spain .The HTLV-1 seroconversion observed in a Colombian blood donor between 2010 (last HTLV-1/2 nonreactive blood donation) and 2013 (first HTLV-1 positive blood donation) was probably associated with the start of a new relationship soon after the last negative blood donation with an HTLV-1 positive Colombian partner. As for the other seroconversion case in an Ecuadorian donor between 2011 and 2017, the only available information was that the sexual partner was also identified HTLV-1 positive during the hospital follow-up. All the blood donors and relatives were completely asymptomatic at the time of the clinical evaluation.This study performed in blood donors and their relatives identified 63 HTLV-1/2 asymptomatic patients in Catalonia in 10 years. HTLV-1/2 infection was probably acquired by vertical or sexual transmission routes in most of the cases, while we cannot rule out transmission by transfusion in the country of origin in five cases. The five blood donors who reported a previous blood transfusion during the clinical follow-up would not have been eligible for blood donation if they had reported this transfusion history in the pre-donation blood bank questionnaire, as a transfusion performed in any of their birth countries  is a reason for deferral in our blood center. In some cases, sexual transmission was suspected but unfortunately the sexual partner could not be tested: this applied to two HTLV-1 positive donors born in a non-endemic area  who reported having had a sexual partner born in an endemic area . Finally, three out of the four HTLV-2 blood donors were Spanish. Most cases of HTLV-2 infection are identified in Spain among intravenous drug users, and most of the cases are actually inmates, coinfected with HIV-1, identified in Spanish prisons ,18. Form6 cells/bag after leukoreduction may be sufficient to transmit the virus from a donor with a very high proviral load, the likelihood of transmitting HTLV-1 by transfusion of leukoreduced components remains very low [The lookback study was performed in 12 out of 51 (23.5%) positive blood donors with previous history of blood donation and did not demonstrate any HTLV-1/2 transmission by blood transfusion. Information was received on 31 out of 52 components (60%). This rather low lookback efficiency may be explained by the difficulty in obtaining information from hospitals many years after transfusion. The recipient’s status was not reported for 21 components, 6 of which had been obtained before 2002 and were transfused between 1991 and 1997 (13 to 19 years before lookback was performed). Only a small number of recipients could be tested for HTLV, as very few of them were still alive at the time the lookback was performed. All the living recipients had received leukoreduced components and tested negative for HTLV. The high efficacy of leukoreduction in reducing the risk of HTLV-1/2 transmission was previously shown in a large study in the United Kingdom [very low .In Spain, new cases of HTLV-1/2 infection are annually reported by the Spanish HIV-2/HTLV Study Group. Between 1985 and 2007, fewer than five cases per year were identified in the entire country (105 cases in 22 years ), most oThis low prevalence appears to contradict the justification for screening for HTLV-1/2 infection in blood banks, especially because universal leukoreduction of all blood components was introduced in Spain in 2002. Leukoreduction reduces the HTLV-1/2 proviral load and HTLV-1/2 transmission risk by retaining infected lymphocytes. Moreover, regional blood banks comply with the Spanish Ministry of Health’s recommendation and defer potential blood donors proceeding from highly endemic countries.Massive immigration from Latin America to Spain began in the 2000s, and prompted Spanish blood banks to take measures to manage the resulting demographic and epidemiological changes. In 2005, a Spanish Royal Decree for blood donation made T. cruzi infection screening mandatory for all individuals born in a Chagas disease-endemic area or whose mother was born in an endemic area . As mostIn non-endemic countries, blood bank HTLV-1/2 screening, as an additional safety measure to leukoreduction, is regularly challenged, as some consider it redundant and expensive . In our Our results highlight an epidemiological change in Spain in terms of HTLV-1/2 prevalence that should be taken into consideration in blood banks, although the transmission risk through transfusion remains relatively low after leukoreduction of blood components. The combination of leukoreduction and pool testing remains perhaps the most optimal option regarding HTLV transfusion safety.


Bone morphogenetic protein (BMP) is a phylogenetically conserved signaling pathway required for development that is aberrantly expressed in several age-related diseases including cancer, Alzheimer’s disease, obesity, and cardiovascular disease. Aberrant BMP signaling in mice leads to obesity, suggesting it may alter normal metabolism. The role of BMP signaling regulating cancer metabolism is not known.C. elegans harboring BMP gain-of-function (gof) and loss-of-function (lof) mutations were examined for changes in activity of catabolic and anabolic metabolism utilizing Western blot analysis and fluorescent reporters. AMP activated kinase (AMPK) gof and lof mutants were used to examine AMPK regulation of BMP signaling. H1299 (LKB1 wild-type), A549 (LKB1 lof), and A549-LKB1 (LKB1 restored) lung cancer cell lines were used to study BMP regulation of catabolic and anabolic metabolism. Studies were done using recombinant BMP ligands to activate BMP signaling, and BMP receptor specific inhibitors and siRNA to inhibit signaling.To examine BMP regulation of metabolism, C. elegans and cancer cells is responsive to nutrient conditions. In both C. elegans and lung cancer cell lines BMP suppressed AMPK, the master regulator of catabolism, while activating PI3K, a regulator of anabolism. In lung cancer cells, inhibition of BMP signaling by siRNA or small molecules increased AMPK activity, and this increase was mediated by activation of LKB1. BMP2 ligand suppressed AMPK activation during starvation. BMP2 ligand decreased expression of TCA cycle intermediates and non-essential amino acids in H1299 cells. Furthermore, we show that BMP activation of PI3K is mediated through BMP type II receptor. We also observed feedback signaling, as AMPK suppressed BMP signaling, whereas PI3K increased BMP signaling.BMP signaling in both C. elegans. These studies suggest that aberrant BMP signaling causes dysregulation of metabolism that is a potential mechanism by which BMP promotes survival of cancer cells.These studies show that BMP signaling suppresses catabolic metabolism and stimulates anabolic metabolism. We identified feedback mechanisms where catabolic induced signaling mediated by AMPK negatively regulates BMP signaling, whereas anabolic signaling produces a positive feedback regulation of BMP signing through Akt. These mechanisms were conserved in both lung cancer cells and  Bone morphogenetic proteins (BMP) are phylogenetically conserved, orchestrating essential developmental processes from metazoans to mammals . BMP sigThere are over 20 BMP ligands that are divided into four distinct subtypes based on structure similarity and function . BMP ligGenetic and evolutionary analysis suggests that the increased expression of BMPR2 mRNA in adipose tissue of obese patients has been selected because it is a metabolic “thrifty” gene that is contributing to present day obesity . Recent C. elegans, we examined BMP signaling for a role in catabolic and anabolic metabolism. We find that in addition to promoting anabolic signaling through BMPR2 activation of PI3K, BMP signaling also suppresses AMP activated kinase (AMPK), the master regulator of catabolism. Furthermore, feed-back regulation by AMPK suppresses BMP signaling, whereas feed-forward regulation by Akt enhances it. BMP2 ligand suppressed expression of tricarboxylic acid cycle (TCA) intermediates and non-essential amino acids. These studies provide evidence that aberrant BMP signaling alters normal metabolic signaling events, which has implications as a potential contributing cause of the metabolic dysfunction in cancer and other age-related diseases.Utilizing lung cancer cells that have aberrant BMP signaling and C. elegans strains are derived from the Bristol strain N2 [E. coli. Nematode cultures, genetic crosses, and other C. elegans husbandry were performed according to standard protocols [All train N2  and wererotocols .C. elegans at L4 stage were mounted on 2.5% agarose pads with 10 µM Levamisole. Fluorescence images were captured on a standard epifluorescent microscope. Quantification was carried out using the Fiji suite [ji suite . At leasC. elegans at 48 h after the L4 stage. C. elegans were synchronized by bleaching [nhr-49 were forward primer TCCGAGTTCATTCTCGACG and reverse primer GGATGAATTGCCAATGGAGC [spp-9 were forward primer GTTCTCTTTCTGGTTGCGGT and reverse primer GCTCTACAAACATCTTCTGGTGCA. Primer sequences for act-1 were forward primer CCATCATGAAGTGCGACATTG and reverse primer CATGGTTGATGGGGCAAGAG [dbl-1 and spp-9 to the housekeeping gene act-1 (Actin) (Applied Biosystems by Thermo Fisher Scientific). Fold-change in gene expression between experimental sample and the wild-type control was determined by this software using the formula: 2(−ΔΔCt). Experimental ΔCt values were compared with wild-type ΔCt values using the unpaired t test.Total RNA was extracted from leaching . Total Rleaching . After RAATGGAGC . Primer GGCAAGAG . QuantStC. elegans were synchronized by alkaline bleaching and arrested at L1 stage on unseeded NGM plates overnight. Arrested L1s were transferred to seeded plates and grown on standard NGM plates until L4 stage at 20 °C. Fifty synchronized L4 stage nematodes for each strain were placed in 15 μl M9 buffer and 15 μl NextGel protein sample loading buffer (4x)  was added, flash frozen in liquid nitrogen and stored at − 80 °C until used for western blotting. Protein samples were boiled for 5 min with sample buffer (Bio-Rad), then centrifuged at 13,000 rpm for 1 min. Western blot analysis were performed as previously reported [C. elegans studies . Quantification of band intensity was measured using ImageJ Software (NIH) and statistical comparisons were made using one-way ANOVA.A549 and H1299 cell lines obtained from patients with non-small cell lung cancer (ATCC) were cultured in Dulbecco’s modified Eagle’s medium  with 5% fetal bovine serum. Normal Human Primary Bronchial/Tracheal Epithelial Cells were from ATCC (PCS-300-010) and cultured in Airway Epithelial Cell Basal Medium (PCS-300-030) supplemented with Bronchial Epithelial Cell Growth Kit (PCS-300-040). JL5 was synthesized by John Gilleran and Jacques Roberge from Rutgers Molecular Design and Synthesis, and DMH1 (S7146) was purchased from Selleckchem. AICAR (A9978) was purchased from Sigma-Aldrich.Immunofluorescence imaging was performed as previously described . BrieflyFor studies examining response to BMP2 ligand (R&D Systems # 355-BM), lung cancer cells were incubated overnight with media without FCS. In the morning media was replaced with fresh media without FCS then treated with recombinant BMP2 (20 ng/ml) for designated times. In starved experiments, DPBS was added in the morning for 20 min then treated with BMP2 (20 ng/ml) or media with FCS for 20 min Fig. C. In staLung cancer cells treated with insulin were incubated with media overnight without FCS, starved with DPBS for 20 min then treated with insulin (100 ng/ml) for 60 min  were used for BMPR2 knockdown. Negative control was Silencer Select negative control siRNA . H1299 lung cancer cells were transfected with siRNA using Lipofectamine® RNAiMAX Reagent . 750,000 cells were plated in a 6 well plate and were grown overnight. Next morning the cells were transfected with 6 nM BMPR2 or 6 nM of siRNA control for 48 h. according to manufacturer’s protocol. Knockdown experiments were performed 4 times.H1299 and A549 cells were incubated overnight in media without FCS. Cells were then treated with DMSO or BMP2 20 ng/ml for 40 min in triplicate. Cells were harvested with a 40:40:20 buffer of methano: acetonitrile: water and 0.5% formic acid. LC − MS analysis of the cellular metabolites was performed on the Q Exactive PLUS hybrid quadrupole-orbitrap mass spectrometer (Thermo Scientific) coupled to hydrophilic interaction chromatography (HILIC) as previously reported , 33. TheIn lung cancer studies, paired student t-test assuming unequal variances was used to compare means. The mean of control was compared with the mean of each treated group. Differences with p values < 0.05 were considered statistically significant. The following signified *p < 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.C. elegans to two different nutrient stress paradigms and monitored the expression of a BMP-regulated gene, spp-9 [spp-9p::GFP transcriptional reporter would indicate suppression of BMP signaling [C. elegans for 8 h leads to a decrease in BMP signaling  [AMP Associated Kinase (AMPK) is a conserved, central regulator of nutrient sensing and energy homeostasis in many organisms. In aradigms , 40. Upoaradigms  and Thr2elegans) . Phosphoaak-2 was activated in these animals. Western blot analysis identified that AMPK phosphorylation is enhanced in various BMP loss-of-function (lof) mutants of BMPR2 daf4(e1364), BMPR1 (SMA-6), and Smad transcription factor (SMA-2) compared to wild-type animals, even in well-fed conditions  were utilized as a control. If AMPK were activated, we would expect that downstream targets of AMPK would also be upregulated in BMP mutants. One such target is the nuclear hormone receptor-49 (nhr-49). We show that a lof BMP mutant SMA-6 exhibits increased expression of an nhr-49p::nhr-49::GFP transgene, whereas gain-of-function (gof) BMP mutants lon-1(e185) and lon2 (e678) exhibit decreased expression of this AMPK target  was crossed to BMP transcriptional reporter spp-9p::GFP to determine how BMP signaling is regulated when AMPK is absent. In fed conditions, we find that BMP signaling is suppressed in AMPK lof mutants, suggesting that AMPK promotes BMP signaling during nutrient-stable conditions  animals fail to increase spp-9::GFP expression under nutrient-deprived conditions when animals are starved for 8 h  (AMPK) transgenic strain in which a truncated version of the AAK-2 protein has a gain-of-function phenotype [aak-2(gof) mutants are smaller in body-size compared to wild-type animals  gof mutants. These results suggest that the regulation of body-size and energy homeostasis by BMP-AMPK cross-talk is uncoupled; other BMP-regulated genes are likely responsible for the body size regulation.ody-size ; BMP lofhenotype . We findaak-2(gof) animals, nhr-49 mRNA levels are upregulated as would be expected for a downstream target of AMPK activation. However, Type 1 BMP receptor (sma-6) and Type 2 BMP receptor (daf-4) mRNA levels are unchanged compared to wild-type, but spp-9 mRNA levels are increased, showing that AMPK regulation of BMP signaling is post-transcriptional.Finally, using a qRT-PCR approach, we show that AMPK inhibition of BMP signaling is through post-transcriptional mechanisms Fig. E. In aakTaken together, these data show that nutrient-dependent energy homeostasis is regulated by BMP-AMPK cross-talk.We examined whether BMP regulates AMPK/LKB1 signaling in lung cancer cells, which have aberrant expression of BMP signaling , 46. FulBMPR2 siRNA was used to further evaluate the role of BMP signaling suppressing LKB1. BMPR2 siRNA knockdown in H1299 cells did not change the levels of pAMPK (Thr172) but did significantly increase pLKB1(Ser428) levels Fig. F. BMPR2 Next, we examined whether BMP signaling regulated metabolism in lung cancer cells by examining changes in tricarboxylic acid cycle (TCA) intermediates and amino acids following treatment with BMP2 ligand. We examined TCA cycle intermediates and amino acids because suppression of AMPK would decrease TCA cycle activity , which wOur prior studies showed that JL5 induces the mislocalization of BMPR2 from the plasma membrane to the cytosol , 52. SinTaken together, these results show the conserved crosstalk between BMP and AMPK across phyla.Our prior studies showed that BMP2 ligand activated mTORC1 p-p70S6) signaling in lung cancer cells through phosphatidylinositol-3-kinase (PI3K) . Whether signalin/daf-16 nuclear localization in C. elegans in BMPR2/daf-4 lof mutants. Insulin-like proteins signal through DAF-2 receptor activating PI3K/Akt signaling. The DAF-2/PI3K/Akt signaling pathway phosphorylates FoxO/DAF-16, inhibiting its entry into the nucleus thereby decreasing its activity [daf-2(e1370) lof mutants exhibit strong DAF-16::GFP nuclear localization (Fig. daf-4 significantly increases DAF-16::GFP nuclear localization compared to wild type, although this activation is not as strong as loss of insulin signaling (Fig. C. elegans.To assess if BMPR2 signaling through PI3K is conserved, we tested FoxOactivity . As expePrior studies have shown that BMP7 enhances insulin signaling through PI3K, suggesting an interaction between BMPR2 and insulin signaling. We examined if insulin regulated BMPR2 in lung cancer cells. Unlike BMP2, insulin activated Akt in lung cancer cells during starvation (Fig. Fig. BMP ligand and receptor expression is increased in cancer and several other age-related diseases. Normally there is little signaling BMP in the mature lung ; howeverAberrant BMP signaling is pathological. Studies in cancer show that BMP signaling enhances cell survival, migration, cancer stem cell self-renewal, and metastasis, and increased BMP expression is associated with a worse survival , 56–60. AMPK is a master regulator of catabolism, and like BMP, is phylogenetically conserved in metazoans . AMPK caC. elegans and lung cancer cells that BMP suppresses AMPK. Suppressing BMP signaling in lung cancer cells and C. elegans led to the activation of AMPK. These data demonstrate that pharmaceutical targeting of BMP receptors can induce AMPK activation. Inhibition of either BMP type 1 or BMP type 2 receptors led to the activation of AMPK. This suggests that inhibition of BMP type 1 receptors is required, but AMPK activation can be achieved by inhibiting BMPR2. Further studies are needed to determine whether it is best to target BMP type 1 or type 2 to promote activation of AMPK. Our studies suggest that BMP signaling inhibits AMPK by inhibiting LKB1. The regulation might be reciprocal, as prior studies have shown that LKB1 binds to BMP type 1 receptors, inhibiting their signaling by inducing degradation [77 inhibiting its binding to transcriptional targets of BMP and TGFβ [We show in both radation . LKB1 haand TGFβ . FurtherC. elegans. Activated AMPK suppressed BMPR2 in both H1299 and A549 cells. Since A549 cells that do not express LKB1, it suggests that AMPK and not LKB1 mediates the downregulation of BMPR2. Although LKB1 expression may not be required to suppress BMPR2, it is required for full activation of AMPK. Therefore lung tumors with LKB1 lof would have less AMPK activity and less suppression of BMP signaling. LKB1 is mutated in 20–30% of NSCLC and is associated with a more aggressive phenotype. LKB1 mutated tumors having a less suppressive effect on BMP signaling in cancer is suggested by a prior report demonstrating that NSCLC with LKB1 lof had higher expression of BMP2 [AMPK induced negative feedback regulation of BMP signaling in both lung cancer cells and  of BMP2 .C. elegans are consistent with BMP signaling activating PI3K and downstream anabolic signaling pathways Akt and mTOR by BMPR2 Smad-independent mechanisms. BMP induction of anabolic signaling pathways was responsive to nutrient conditions. During starvation BMPR2 expression is decreased and lung cancer cells were no longer responsive to BMP2 ligand. Furthermore, Akt-mediated insulin signaling prevented the decrease in BMPR2 expression during starvation. Since a number of tyrosine kinases and oncogenic mutations, including K-ras, mediate cell growth through PI3K/Akt signaling, multiple oncogenes may utilize Akt to enhance BMPR2 signaling.Prior studies have suggested that BMP signaling activates PI3K through BMPR2 independent of Smad activation. In mesenchymal progenitors, BMP2 mediates chemotaxis by inducing binding of BMPR2 to PI3K regulatory p55γ subunit . Our stuC. elegans. Aberrant BMP signaling in lung cancer is growth promoting and has been shown to be pathological in other age-related diseases. These studies suggest the aberrant BMP signaling causes metabolic dysfunction, which has implications in causing pathological responses in cancer and potentially other age-related diseases.We demonstrate that BMP signaling is integrated with the master regulators of anabolic and catabolic metabolism in cancer, which is conserved in


Among the HTLV-1/2 positive sera, there were co-infections with viral , bacterial  and parasitic  pathogens related to HTLV-1/2 co-morbidities that can contribute to inconclusive diagnostic results. Sera positive for HIV were included among the HTLV-1/2 negative samples. Considering both HTLV-1 and HTLV-2-infected samples from all states and different groups , mono or co-infected and HTLV-/HIV+, the test specificity ranged from 90.09 to 95.19% and the sensitivity from 82.41 to 92.36% with high accuracy (ROC AUC = 0.9552). This multi-epitope protein showed great potential to be used in serological screening of HTLV-1 and HTLV-2 in different platforms, even taking into account the great regional variation and different profile of HTLV-1 and HTLV-2 mono or co-infected individuals.A multi-epitope protein expressed in a prokaryotic system, including epitopes of Env, Gag, and Tax proteins of both HTLV-1 and HTLV-2 was characterized for HTLV-1/2 serological screening. This tool can contribute to support the implementation of public policies to reduce HTLV-1/2 transmission in Brazil, the country with the highest absolute numbers of HTLV-1/2 infected individuals. The chimeric protein was tested in EIA using serum/plasma of HTLV-infected individuals and non-infected ones from four Brazilian states, including the North and Northeast regions  and Southeast region  depicting different epidemiological context of HTLV-1/2 infection in our country. We enrolled samples from Pará ( The human T-lymphotropic viruses 1 and 2 (HTLV-1 and HTLV-2) are endemic in Brazil that presents the highest number of infected people around the world. According to the recent report of the Brazilian Ministry of Health . Rates fRecently Rosadas and Taylor  showed aOne study conducted in Salvador, Bahia, evaluated the performance of four commercially available serological screening tests for HTLV-1 infection in Brazil; three enzyme immunoassays (EIA) and one chemiluminescence assay (CMIA), and concluded that all commercial assays could be safely used . However, the high sensitivity of some kits may lead to false-positive results, which could increase the testing cost due to the need for confirmation , 10, andThe International Health Policy Forum for the elimination of HTLV in 2021  pointed out the necessity to improve the diagnostic tests, including the production of rapid tests (point of care) to fill the HTLV-1/2 diagnostic gaps and to promote the biotechnology independence in countries with high rates of HTLV-1/2 infection, as in Brazil. In addition, in this period of the COVID-19 pandemic, we are experiencing a shortage of HTLV EIA kits available on the market, joint to the fact that those ready to use were not been tested with samples from different geographic regions and populations in Brazil. Therefore, considering the reasons above and the necessity to provide screening diagnostic tests for HTLV-1/2 in public health services in Brazil, this work aimed at the development of a diagnostic tool based on a multi-epitope recombinant protein containing antigens for HTLV-1 and HTLV-2 that can be used in different diagnostic platforms.Treponema pallidum), and parasitic  pathogens related to HTLV-1/2 comorbidities and that might induce inconclusive diagnostic results. The study population consisted of 551 samples of peripheral blood from mono-infected or co-infected individuals with HTLV-1, HTLV-2, or HTLV-untyped, also with other viral and parasitic pathogens, in addition to HTLV seronegative and HTLV seronegative/HIV positive individuals (HTLV-/HIV+) from four different states from Brazil (n = 225) were screened at Fundação HEMOMINAS, a blood center in Belo Horizonte, Minas Gerais, by EIA  or microparticle chemiluminescence immunoassay  and confirmed by Western blot . Serum samples from São Paulo (SP) were provided by Instituto Adolfo Lutz (IAL), a central public health laboratory in São Paulo, and screened by EIA  and confirmed by WB 2.4. In addition, 114 serum samples from Pará (PA) provided from Universidade Federal do Pará (UFP), and 153 sera from Maranhão (MA) provided from Universidade CEUMA de São Luiz were also tested. Serum samples from Pará were screened by EIA Murex HTLV-I+II, DiaSorin, Dartford, UK, and the positive ones were confirmed by qPCR or nested PCR (tax and env gene region). Samples from Maranhão were screened by CMIA  and confirmed by WB 2.4  and nested PCR (tax region). The project was approved by the Ethics Committee for Research of Universidade Federal de Minas Gerais (UFMG) and Fundação HEMOMINAS, as well as by IAL, UFP, and CEUMA, receiving the Ministry of Heath protocol numbers CAAE # 55618516.1.0000.5149 and # 55618516.1.3001.5118.We enrolled samples from the states of Pará, Maranhão, Minas Gerais and São Paulo, Brazil, from blood donors' candidates (Pará and Minas Gerais), pregnant women (Maranhão) and HIV+/high risk for sexually transmitted infection . Among the HTLV-1/2 positive sera, there were coinfections with viral , bacterial  with Brazilian sequences deposited at GenBank (https://www.ncbi.nlm.nih.gov/genbank/). The chosen epitopes of Env, Gag, and Tax protein were disposed of in tandem, separated by flexible proline and glycine rings (Escherichia coli Rosetta-gami 2(DE3) cells  were transformed and induced for 5 h with 1 mM IPTG . The protein was purified with affinity chromatography using sepharose His-Trap HP  column and Äkta Start system  and following tested in-house. The multi-epitope protein (1 μg) was transferred to a PVDF membrane . The membrane was incubated for 16 h at 4°C with a pool of HTLV-1, seronegative, or HTLV-2 infected samples (1:100). A commercial mouse anti-histidine antibody  was used as a positive control. As a secondary antibody was used anti-mouse IgG  or anti-human IgG . The detection was performed using a TMB substrate .A multi-epitope protein was designed for expression in a prokaryotic vector and was based on proteins validated all around the world in EIA and Western blot (WB) tests for HTLV-1 and HTLV-2: Env (gp46) and Gag (p19). In addition, based on our previous studies, Tax protein epitopes were also included for both viruses . The chipubmed/; –17]. Latne rings  and inseThe sensitivity and specificity of the recombinant multi-epitope protein were tested using an indirect ELISA. The 96-well high-binding flat-bottom microplates  were coated with the purified recombinant protein  followed by blocking -−4 h at room temperature (RT). The microplates were incubated with the sera samples shown in J statistic, Cohen's Kappa coefficient, and likelihood-ratios were also calculated to evaluate the performance of the multi-epitope recombinant protein to recognize HTLV-1 and HTLV-2 antibodies.Statistical analyses were performed using GraphPad Prism 8.0.2, with Kruskal–Wallis and Mann–Whitney test when appropriate. A receiver operating characteristic (ROC) curve, a statistical tool to evaluate the global accuracy of the test was applied to evaluate the test performance using the same software. Positive and negative predictive values, calculated through the reported prevalence in each state, Youden's The multiepitope protein was designed using HTLV-1 and HTLV-2 p19, gp46, and Tax sequences, and a histidine tail for protein purification by nickel affinity chromatography was added. After the protein purification, a Western blot was conducted. This assay showed that the multi-epitope protein is recognized by sera from individuals infected by HTLV-1 or HTLV-2, which was not observed for the seronegative individuals .n = 92). First, we evaluated the results considering all samples from the four states clustered by viruses: HTLV-1, HTLV-2, and HTLV with or without co-infections . The untyped and indeterminate samples were slightly recognized by the in house-ELISA, with one untyped sample from Minas Gerais State (14.3%) showing a positive results. According to the multi-epitope ELISA, the seronegative controls (n = 297) showed 5.1 and 4% of positive and indeterminate results, respectively, whereas these rates were both 6.5% for HTLV-SN/HIV (n = 92). Analysis of the negatives samples in a joined group (HTLV-SN and HTLV-SN/HIV+) showed statistically significant differences from HTLV-1 and HTLV-2 , 30 HTLV-2 positive samples , two HTLV untyped and five samples with the indeterminate result by serology and 389 HTLV-negative samples, including HIV positive ones  and Pará (0.9772), followed by São Paulo (0.8309) and Maranhão (0.6926). Youden's J statistic index showed the lowest result in São Paulo (0.51) and the highest in Minas Gerais (0.93). Considering the global result (from the four Brazilian states), the AUC  and Youden's  the AUC  and the Likelihood ratios are used for assessing the value of performing a diagnostic test . Values The Index sample's distribution in the ELISA of each state is shown in The sensitivity and specificity's results varied among the states, as already shown in In regard to HTLV-2, the sensitivity detection was overall 57.14%, with lowest number presented by Minas Gerais (37.50%), followed by São Paulo (53.85%) and Pará (85.71%). The specificity was overall 94.61%; 86.06% in Pará samples, 97.05% in São Paulo, and 98.36% in Minas Gerais.Serological diagnostic for HTLV-1/2 infections include viral antigens encoded by structural and regulatory genes and methods available fall in two categories: the screening tests and those with the capacity to confirm and discriminate the HTLV-1 and HTLV-2 infections , 28. Altin-silico analysis to be incorporated in the multi-epitope protein developed.The diagnostic systems widely used are based on Gag and Env proteins of HTLV-1 and HTLV-2. Gag protein is cleaved into the proteins p15, p19, and p24 that are well characterized as antigens and are present in many commercial assays. Although central and C-terminal portion of Env gp46 showed to be highly immunogenic –34, recoVaccinia virus WR/pLW44 followed by flow cytometric assay showed that the C-terminal portion of Tax protein is highly antigenic and recognize antibodies anti-HTLV-1 in individuals presenting HAM/TSP or rheumatologic diseases and also in asymptomatic carriers  and parasitic  pathogens related to HTLV-1/2 co-morbidities and that can contribute to inconclusive diagnostic results  and Southeast region . Nonetheless, to beyond of this regional scope, we enrolled 551 samples from individuals with different profiles, such as blood donors' candidates (Pará and Minas Gerais), pregnant women (Maranhão), and a vulnerable HIV positive individual with a high-risk for sexually transmitted infection (São Paulo). In addition, many of these individuals  are co-infected with viral , bacterial , mono or co-infected and those HTLV seronegative (including HTLV-/HIV+), the EIA test using the chimeric protein showed a general specificity ranged from 90.09 to 95.19%, and a sensitivity ranged from 82.41 to 92.36% with high accuracy (ROC AUC = 0.9552). The specificity of the assay for HTLV-1 detection was 92.55%, ranging from 89.31 to 94.78%, and the sensitivity was 99.19%, ranging from 99.54 to 99.96 (ROC AUC 0.992). The specificity of the assay for HTLV-2 detection was 94.61%, ranging from 90.99 to 96.82% (ROC AUC 0.8953).Schistosoma mansoni. However, this sample was the only one among the 28 co-infected samples that was not positive in the ELISA multi-epitope. On the other hand, there were more discordant results for HTLV-2 positive samples: 6 (20%) and 9 (30%) showed indeterminate and negative results in the ELISA multi-epitope, respectively. Of note, the HTLV seronegative controls (n = 297) and HTLV-SN/HIV (n = 92) showed 27 and 12 discordant samples, respectively. The Southeast region (Minas Gerais and São Paulo) considered the area of intermediate prevalence in Brazil had 3 and 1 discordant samples, respectively; however, the states Maranhão and Pará that represent the Brazilian North region, the area of highest prevalence in Brazil, presented 20 and 15, respectively. Regarding the discordant results presented, the false-negative samples may be related to their quality . About the false-positive samples, the developed multi-epitope in-house test was compared with commercial tests used for a long time around the world  and the necessary test adjustment is in course.Related to HTLV-1 discordant samples, 1.6% (2/125) positive samples showed indeterminate results in the developed assay. Both are asymptomatic individuals from Minas Gerais . The female is co-infected with About the false-positive samples, the cross-reactions due to co-infections are already reported by others, and also the developed multi-epitope in-house test was compared with commercial tests used for a long time around the world  and the necessary test adjustment is course.TM HTLV-1/2 score . All of them informing sensitivity and specificity with scores higher than 99% and many reports confirmed around 95% of efficiency in samples from Peru, Sweden, the United States, French, Switzerland, Spain, and Japan , Avioq HTLV1/2 (Avioq Inc.), the CMIA Architect rHTLV-1/2 (Abbott Laboratories), and Elecsys® HTLV-1/2 Immunoassay , and as confirmatory assays the WB HTLV Blot 2.4  and the INNO-LIAnd Japan –44.In Brazil, da Silva Brito et al.  comparedRecently, a multiplex immunoassay for serological confirmation and differentiation of HTLV-1 and HTLV-2 using samples from French and USA (United State-based cohort HTLV Outcomes Study—HOST) blood banks and also from a Canadian commercial company was reported. This experimental assay is based on GP21, GP46, and p19 having three antigens to detect both viruses and three others to discriminate them. It was developed an algorithm followed by logistic regression formula to characterize each sample and sensitivity and specificity were calculated based on PCR tests and results showed high performance either for diagnostic confirmation or discrimination .Although the current knowledge, the different new approaches developed and in development around the world, and flowcharts for diagnosis based on serological and molecular complementary tests, the HTLV-1/2 diagnostic is still challenging. Our next step, already in course, is test other peptides for the development of a discriminatory rapid tests for HTLV-1 and HTLV-2 using these same gene regions of the multiepitope. The challenge is to obtain a sensitive, specific, low-cost, and easy-to-perform test that can be applied in a variety of health services.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by the Ethics Committees for Research of Universidade Federal de Minas Gerais (UFMG) and Fundação HEMOMINAS, as well by IAL, UFP, and CEUMA, receiving the Ministry of Heath Protocol Numbers CAAE # 55618516.1.0000.5149 and # 55618516.1.3001.5118. The patients/participants provided their written informed consent to participate in this study.EB-S, MM, and GF conceived and designed the experiments. GF, LC, AR, DD, and DS performed the experiments. EB-S, GF, LC, and LR analyzed the data. EB-S, MM, AC-A, MB, RI, AV, JC-R, and FF contributed reagents, materials, and analysis tools. EB-S, GF, MM, and AC-A wrote the paper. All authors contributed to the article, reviewed the manuscript, and approved the submitted version.This work was supported by grant from PPSUS , Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG-APQ-00907-20), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazilian Ministry of Health and Pro-Reitoria de Pesquisa da UFMG (PRPq-UFMG). EB-S was supported by PQ#302157/2019-0, RI PQ#312979/2018- 5, and AV PQ#301869/2017-0). FF and GF received fellowship from CNPq. LC and DD (PNPD/CAPES) received fellowship from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Fertility plans are a prominent area for agency research, and are a clear example of a misalignment between resources and agency capacity. We relied both on the idea of conversion factors of the Capability Approach and the pragmatist tradition of temporal-oriented agency to propose a framework for the study of fertility agency as the conversion process of resources into plans and behavior. We outlined said framework by using a unique dataset on fertility plans composed of open and closed questions from an Italian sample. Economic factors and imaginaries related to children and family represented the vast majority of (hindering and enabling) conversion factors. The notion of conversion factors is crucial for disentangling the network of heterogeneous elements involved in fertility agency: it allows focus to be shifted from structural factors related to social position and psychological characteristics to more situated elements that enable agency capacity. As a prominent example of agency, fertility plans ought to receive greater attention than they have thus far been given in agency research. Indeed, the widespread use of the concept of agency focuses on the human capacity to be the “perpetrator” of the course of action Giddens, , p. 9.1IFertility plans are a turning point in life trajectories. While many decisions have long-lasting consequences to one's life course , childbearing decisions embody a definitive and irrevocable commitment to a long series of costs, burdens, and sleepless nights – but also expected happiness. Accordingly, childbearing decisions cannot fairly be compared with other choices.In contemporary societies, the freedom of choice in several life-course domains has become normative Giddens, , and manEmpirical research on life-course agency has typically foregrounded the analysis of the socio-psychological characteristics able to account for the heterogeneity of individual success across the life-course Elder, . Howeverconverted into actual fertility plans and behavior. Sen has emphasized that capabilities and achievements vary depending on the institutional, societal, and cultural context, as well as on the research question. In the CA, the factors that convert resources into agency are defined as conversion factors . Our data includes both open and closed questions which we examined through a qualitative analysis augmented with frequency distributions across different groups of respondents. We have focused on fertility plans as the first step of fertility agency. The analysis considered the hindering and enabling conversion factors involved in the fertility decision-making process. Unlike most related studies, we used an inductive approach , but they are also oriented by contingent factors that shape the course of action.Recent developments have translated into a more sociological formulation of Sen's conversion factors that convert resources into agency achievement Sen , 2008, cThe iterative dimension of fertility agency is embedded in the elements belonging to the “shadow of the past” and the “present condition,” despite often deploying their effects on future expectations. Projectivity agency is influenced by the “shadow of the future” and synthesized in the “personal narrative of the future,” which also includes fertility decisions  theory – perhaps the most influential theory on fertility dynamics developed in post-war Europe – individuals (and secularized women in particular) reprioritise their own careers and self-actualisation over family and childbearing  uncertainty as a consequence of globalization and neoliberal policies  have dramatically declined in several European countries, dropping to far below the replacement level. In Southern Europe, Italy, after having witnessed a fertility rebound at the beginning of the new millennium, is now experiencing a constant fertility decline, officially re-entering the lowest-low category in 2019, with a TFR of 1.29  who were not exposed to any lab treatment and only answered the survey questions. Our participants consisted of 276 respondents aged between 20–40 in stable relationships . We ensured a balanced participation by sex, as well as between the jobless, permanently employed, and temporarily employed. The participants were recruited through the services of a specialized survey agency, which gave no information as to the content of the research . The agency was also asked to try to ensure a distribution by education and parity that would mimic that of the national population distribution, but without imposing strict quotas. Participants were selected from panel participants available to the agency, boosted with additional participants recruited through advertisements distributed in public places, again without providing any information about the experiment's content.We asked the participants to answer a large array of demographic, socio-economic, and psychological questions. The first part of the questionnaire was dedicated to fertility intentions, followed by open questions about the role of uncertainty in their lives and the personal narratives associated with fertility intentions  was able to encourage the respondents to consider both micro and meso aspects. This strategy allowed the respondents to separately consider the micro and meso aspects while also giving them the freedom to report on one or both aspects, thereby enabling us to understand the similarities and differences of their personal narratives. While in-person interviews on family planning may be oriented toward social desirability, social norms, and interviewer expectations, we attempted to overcome these biases by collecting the answers to the open-ended questions through an anonymous form completed on a computer , and the hindering factors among the respondents with fertility intentions lower than the value attributed to the expected happiness of a(nother) child . We assumed that this group of respondents had faced some obstacles to their fertility intentions that we hoped to explore in their answers to the open questions. The two groups partly overlapped by construction: The achieved capability group was used to code the factors enabling fertility intentions, and the unachieved capability group to code the factors hindering it. The descriptive statistics of the sample and the two subsamples can be seen in Drawing distinct measures between fertility intentions and the expected happiness that having a(nother) child could bring allowed us to more closely focus on analyzing the enabling and hindering conversion factors of fertility intentions within the respondents' personal narratives. Accordingly, the analysis explored two distinct subsamples of respondents. We analyzed the enabling factors in the answers to the open questions for respondents with fertility intentions from 5 to 10  has the advantage that the analyst has access to the respondents' ideas in their own words. This opens up possibilities of using content analysis to search for subtler patterns within the responses compared with closed questions . Regarding education's impact on fertility, we distinguished the respondents' education levels between low , medium (2 = high school), and high (3 = tertiary or higher). The respondents' religious affiliation was classified by distinguishing between those who felt themselves as belonging to a specific religion and those who did not. The overwhelming number of religious respondents (88%) declared themselves to be Catholics. We conducted the analysis for first-birth, second-birth, and higher-order parity separately, and divided the respondents between the childless and parents with one, two, and three children for the unachieved capability subsample. Due to their limited number, we did not consider respondents with two or three children  as being part of the achieved capability subsample.We interpreted the results of the analysis of the distribution of codes among different groups of respondents as the presence of different/similar conversion factors in personal narratives associated with fertility intentions  to self-employment with a relatively developed career. Uncertainty and generic economic troubles often refer to – without specifying an exact meaning – an “uncertain personal economic situation” or “employment situation.”At the micro-level, the most frequently reported hindering factors were perceptions and expectations concerning the economic situation and imaginaries related to children and family  by those with permanent work contracts  child and hardships in work-family conciliation were often reported. Regarding the latter, people reported that they expected to be unable to successfully meet their children's needs or have sufficient time to work. Conciliation problems were associated both with the need for career development and the absence of flexibility in the working schedule. “I wish I had achieved a more stable and better paid position, lack of flexibility regarding working hours – I am not allowed to work remotely” . In some cases, school timetables did not coincide with parents' working hours. “I already have two children, no help  close other than my husband. The management is all in my hands. At the slightest hitch, there is a problem. School hours do not coincide with work hours. My contract was not renewed when it expired after returning from my second maternity leave” .Imaginaries related to children and family were frequently cited as hindering factors to fertility intentions. Reported hindering imaginaries refer to expected parental duties and the fulfillment of personal aspirations outside of the family. The desire for self-realization and freedom was not an exclusive feature of highly-educated, career-oriented respondents, but was also observed in those with lower levels of education . “The current state of my relationship and the work situation of my partner, who is often away on business. Before having a child, I want to make sure I can create the best possible family environment around him or her” .Despite the fact that the open question analyzed thus far concerned personal situational factors, some respondents reported macro-level factors. However, we were able to more accurately analyse macro-economic hindering factors with the answers to the open question on macro-economic aspects.The lack of family policies and childcare services was cited by over one-third of the respondents. They referred both to the need for economic support for couples with children and the high cost, or complete lack of, childcare services. “The lack of services to families. The lack or high cost of day-care centers. Poor economic support for families and high taxes that have a very negative impact on family income” . The references to lack of family policies were equally distributed among different parities, with a slight increase among respondents with two children  showed no particularly different distribution of factors among these different groups.We analyzed the enabling factors to fertility intentions within the sub-sample of respondents with a high level of fertility intentions (5–10). As with hindering factors, the economic factors were also prominent, but as enabling factors. While a precarious job was regarded as a significant obstacle, the presence of a stable job has the same, though beneficial, salience for positive fertility intentions . This factor was reported mostly by irreligious respondents, women, and the childless  child from the household income. Parenthood is a life-changing experience that shapes the meaning and salience of different aspects of life and personal identities and, in this case, reduces the importance of economic factors to further fertility plans. These findings seem to contradict the “relative income hypothesis”  deserves greater attention from sociological research on agency. Fertility plans are a huge space of hyperprojectivity capable of shaping entire life-courses and transforming daily life into a long-term binding process. Despite the fact that the outcomes of fertility plans and parenthood can never be consistently predicted with accuracy, their narratives may help stabilize uncertainties, orient decisions in several life domains, and provide reasons for the enduring efforts of fertility agency.giacomo.bazzani@unifi.it.The datasets presented in this article are not readily available because Identifiable data. Requests to access the datasets should be directed to GB, The studies involving human participants were reviewed and approved by the University of Florence. The patients/participants provided their written informed consent to participate in this study.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.This work was supported by the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme, grant agreement n° DLV-72596 under the EU-FER project (PI: DV).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Cell division cycle associated 3 (CDCA3) mediates the ubiquitination WEE1 kinase at G2/M phase. However, its contribution to cancer immunity remains uncertain.  We first evaluated the effect of CDCA3 on the prognosis of patients with renal cell carcinoma (RCC). The results of bioinformatics analysis were verified by the tissue microarray, immunofluorescence (IF) staining, CCK-8 assay, colony formation, cell cycle, and Western blot. + T cells and Tregs. Tissue microarray demonstrated that CDCA3 was strongly associated with poor prognosis and positively relevant to CD8+ T infiltration. In vitro experiments showed that exgenomic interference of CDCA3 could attenuate cellular proliferation, arrest cell cycle, and blockade accumulation of CDK4, Bub3, and Cdc20 in mitosis process.  Bioinformatics analysis predicated CDCA3 was an independent predictor of poor prognosis in RCC and was associated with poor TNM stage and grade. CDCA3 was related to the infiltration of CD8 CDCA3 presents as a good biomarker candidate to predict the prognosis of RCC patients and potentiates the immune tumor microenvironment (TME) of RCC. The firt of ICI . Traditit of ICI , 6. In cκB/cyclin D1 signaling axis [Cell division malfunctions trigger tumor development and antitumor immune response . CDCA3 hing axis . There aing axis . Moreoveing axis . HoweverIn this paper, we try to evaluate the predicable performance of CDCA3 in RCC and figure out the attribution of CDCA3 to TME of RCC. Finally, we endorse that targeting CDCA3 would be a potential therapeutic way to flight RCC.https://portal.gdc.cancer.gov/) portal. Patients were divided into high expression group and low expression group based on the median of gene expression. We first drew Kaplan-Meier (KM) survival curve, receiver operating characteristic (ROC) curve, and risk curve to study the prognosis of patients in terms of overall survival (OS). Next, we analyzed the differences in clinical data including gender, clinical stage, TNM stage, and grade among different expression groups of CDCA3. In addition, we used univariate and multivariate COX regression to analyze the prognostic significance of CDCA3 expression and clinical data. At the same time, we drew a nomogram diagram and calibration curve to better interpret the prognostic significance of CDCA3. Moreover, fold change = 2 was used to compare the differences of gene expression among different expression groups of CDCA3, and a heat map of differentially expressed genes was drawn to show the expression trend in different groups. Finally, considering that CDCA3 can be used as an oncogene to affect the progression of tumor, we performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis on the upregulated genes of CDCA3 in different expression groups to identify CDCA3 functional pathway localization in tumors.The RNA-seq data, clinical information, somatic mutation data, and microsatellite instability (MSI) status of 881 RCC were all from The Cancer Genome Atlas  is a scRNA-seq database focusing on TME. We obtained the relationship between CDCA3 and RCC TME at single-cell level in TISCH.Cell type Identification By Estimating Relative Subsets Of RNA Transcripts (CIBERSORT) algorithm was used to estimate the infiltration proportion of 22 kinds of immune cells in normal kidney and RCC samples to describe the profile of immune cell infiltration in RCC. The abundance of immune cells infiltration and the expression of 8 important immune checkpoints  among different CDCA3 expression groups were compared. Finally, we also analyzed the correlation between CDCA3 expression with tumor mutation burden (TMB) and MSI. Tumor Immune Single-cell Hub . Then, Caki-1 and 786-O were transfected with the lentivirus, according to the manufacturer's instructions. Two days later, puromycin was added for screening. Knockdown efficiencies of CDCA8 were assessed by Western blot.Caki-1 and 786-O were purchased from the Type Culture Collection . Cells were cultured in RPMI-1640 medium  with 10% fetal bovine serum . The culture was maintained in a humidified incubator with 37°C, 5% COCultured cell lysates were prepared using a Column Tissue & Cell Protein Extraction Kit . Then total proteins were then separated on 10% SDS polyacrylamide gels. After overnight incubation with various primary antibodies, including anti-CDCA3 , CDK4 , Cdc20 , Bub3 , and anti-GADPH  at 4°C, membranes were washed thrice for 5 min each time, using TBST (in 0.1% Tween20). Then, they were incubated in the presence of a secondary rabbit antibody  for 1 h and washed thrice using TBST for 5 min each time. Signals were detected using the chemiluminescence system.μl CCK-8  to each well and incubating at 37°C for 2 h, the absorbance at 450 nm was measured by the Rayto-6000 system .The cells were seeded in 96-well plates  and cultured for 1, 2, and 3 days. After adding 10 For cell proliferation, we seeded 200 cells to each well of 6-well plates for 14 days, then fixed with 4% paraformaldehyde (PFA) and stained with crystal violet. The cells were photographed, and the numbers of colonies were counted.μl of permeate were added to the cell suspension and vortexed to mix. Finally, cells were stained in the dark at 4°C for 30 min and analyzed by flow cytometry. The stained cells were assessed by flow cytometry , and analysed by FlowJo vX.0.7 software.Cell cycle analysis was performed using a Cell Cycle Staining Kit , as instructed by the manufacturer. Cells were washed using PBS, after which 1 ml of DNA staining solution and 10 The RCC tissue microarray was purchased from Outdo  and contains 150 RCC tissues and 30 paired paracancer tissues along with their survival, clinical information, etc. Samples were collected from the National Human Genetic Resources Sharing Service platform (2005DKA21300). All points on the chip were detected by Immunofluorescence (IF). The expression of CDCA3, CD8, CD4, CD68, FOXP3, and PD-1 was detected by intensity and positive number of IF. We divided 150 RCC patients into two groups based on the optimal CDCA3 cut-off value and plotted survival curves to identify their prognostic significance. Finally, we analyzed the correlation between CDCA3 and CD8, CD4, FOXP3, CD68, and PD-1.Tissue microarray were deparaffinized by graded alcohol and then washed three times with phosphate-buffered saline (PBS), permeabilized with 0.4% Triton X-100 for 30 min, and blocked with goat serum working liquid  for 2 hours after antigen retrieval. The sections were then incubated overnight with mixed primary antibodies at 4°C, washed in PBS to remove unbound primary antibodies, and incubated with secondary antibodies in the dark at room temperature (RT) for 1 hour. The sections were counterstained with 4′, 6 diamidino-2-phenylindole (Sigma-Aldrich) for 5 minutes and washed with PBS. The primary antibodies included CDCA3 . The fluorophore-conjugated secondary antibodies used were goat anti-rabbit Alexa Fluor 488  and goat anti-mouse Alexa Fluor 549 . Images were captured by confocal laser scanning microscopy . The fluorescence intensity was analyzed by using the ImageJ software.T test. Chi-square test was used for categorical variables, and Wilcoxon test was used for continuous data. Survival differences were calculated using Kaplan-Meier and logarithmic rank tests.In this study, R (version 4.0.2), GraphPad Prism 8, and SPSS 20.0 software were used to analyze the data. Survival, survminer, timeROC, rms, Limma, ggplot2, pheatmap, and ClusterProfiler R package were used in this study. The significance of differences between groups was assessed by the student p < 0.001, n = 881). ROC curves suggested a good accuracy of CDCA3 expression in predicting RCC prognosis , Tregs (p < 0.001), memory B cell (p < 0.001), follicular helper T cell (p < 0.001), activated NK cell (p < 0.05), and M0 macrophage (p < 0.01) was upregulated in the patients with high expression of CDCA3, while naive B cell (p < 0.001), resting NK cell (p < 0.05), Monocyte (p < 0.001), and M2 macrophage (p < 0.001) was downregulated. TMB and MSI levels reflect tumor surface neoantigen abundance and can stimulate antitumor immune response. CDCA3 was also positively correlated with TMB , PDCD1LG2 , and SIGLEC15 (p < 0.05) were downregulated in patients with high expression of CDCA3, while CTLA4 (p < 0.001), LAG3 (p < 0.001), PDCD1 , and TIGIT (p < 0.01) were upregulated (egulated . The disegulated .Further, we conducted tissue microarray to try to prove the above results. β signal pathway, NF-κB signal pathway, and JAK-STAT signal pathway. (κB axis and other important immune-related pathways.The distribution of different genes among patients with different CDCA3 expression groups was shown in the volcano map . The heapathway. . GO enripathway. . These rTo further understand the effect of CDCA3 on the biological behavior of RCC, we constructed CDCA3-knockdown cell lines for functional experiments. Lentiviruses carrying CDCA3 shRNA were used to obtain CDCA3-knockdown Caki-1 and 786-O. The Western blot results showed that the expression of CDCA3 was significantly decreased in the RNAi group, indicating that the CDCA3-knockdown cell lines were successfully constructed . Meanwhi+ T cells and Tregs. Importantly, we verified our results in vitro.Our study suggested that CDCA3 can independently predict prognosis and affect tumor progression in RCC. CDCA3 may also be involved in the regulation of immune-related pathways, and stimulated the infiltration of immune cells, such as CD8Scholars revealed that CDCA3 influence many tumor progression and treatment through a variety of pathways and is associated with poorer prognosis , 15. Our+ T cells in endogenous and exogenous data. Previous studies have shown that CD8+ T cells can recognize tumor-specific antigens and played a role in tumor control [+ T cells has been proved to be associated with a good prognosis of most cancers [+ T cells in RCC was associated with a poor prognosis [Infiltrating immune cells directly affect the occurrence, development, and treatment of tumors. It has been reported that CDCA3 is closely related to immune infiltration in hepatocellular carcinoma . Our res control . The hig cancers , but therognosis , this isAs we know, immune checkpoint is a key molecule in tumor immune escape pathway. There were a lot of evidences showed that immune checkpoints were related to the benefit degree of ICIs treatment, which can be used as biomarkers for ICIs treatment –22. Our κB pathway to mediate tumor progression [κB is involved in the regulation of inflammation and innate immunity in tumor development. P53 also plays an important role in immune system. P53 mutation in cancer triggers B cell antibody response and CD8+ killing T cell response [β can inhibit the proliferation, activation, and effector function of T cells. In addition, TGF-β further enhances immunosuppression in TME by promoting Tregs differentiation and destroying T cell immunity [In summary, reactive TME is the key to immunotherapy, and CDCA3 helps to evaluate this phenomenon. Furthermore, enrichment analysis was performed to evaluate the actual molecular mechanism of CDCA3 in RCC. CDCA3 has been suggested to influence the NF-gression . Our resresponse . TGF-β cimmunity . These e+T cells and its influence on immunotherapy of renal cell carcinoma need to be further explored; second, we have proved that CDCA3 can block the cell cycle, but there is no further study on the biological mechanism.TME is recognized as a complex dynamic ecosystem, which is composed of malignant tumor cells, various infiltration immune cells, fibroblasts, and a variety of cytokines. In this ecosystem, immune response plays an important role in tumorigenesis and development. RCC has always been regarded as an immunogenic malignant tumor –28, and + T cell infiltration in RCC. Targeted therapy against CDCA3 is a promising new therapeutic modality, and focusing on it may help to improve the management of therapeutic resistance in the combination of ICI and TKI, but this needs further research to confirm.CDCA3 can be used as an oncogene to affect the prognosis of RCC patients. Downregulation of CDCA3 causes cell stagnation in G2/M phase, promotes cell apoptosis, and reduces proliferation ability. More importantly, the immunological implications of CDCA3 have also been preliminarily evaluated. CDCA3 may participate in the regulation of immune infiltration in tumor microenvironment by affecting the expression of many immune regulatory factors and TMB, which is expected to provide valuable reference for clinical ICIs treatment. Overall, CDCA3 can be used as a biomarker to evaluate prognosis and CD8