Establishment of allogeneic UTx in cynomolgus macaque requires further exploration of immunosuppression, but the clinicopathological features of uterine rejection are useful for development of human UTx.Uterus transplantation (UTx) is an option for women with uterine factor infertility to have a child, but is still in the experimental stage. Therefore, allogeneic animal models of UTx are required for resolution of clinical issues. In this study, long-term outcomes were evaluated in four recipients (cases 1–4) after allogeneic UTx in cynomolgus macaques. Immunosuppression with antithymocyte globulin induction and a triple maintenance regimen was used. Postoperative ultrasonography and biopsy of the transplanted uterus and immunoserological examinations were performed. All four recipients survived for >3 months after surgery, but continuous menstruation did not resume, although temporary menstruation occurred (cases 1 and 2). All animals were euthanized due to irreversible rejection and no uterine blood flow  and post-transplant lymphoproliferative disorder (case 3). Donor-specific antibodies against MHC class I and II were detected in cases 1, 2 and 4, but not in case 3. Peripheral lymphocyte counts tended to elevate for CD3 Uterus transplantation (UTx) has become a potential option for women with uterine factor infertility to have a child. Data accumulated from animal studies for more than a decade have led to clinical application of UTx. Brännström et al. described the first human delivery after UTx with a living donor in 2014 . TherefoAnimal studies on UTx have been performed in mice, rats, rabbits, dogs, pigs, sheep, and non-human primates . DeliverOur team in Japan has accumulated a large UTx research data archive using cynomolgus macaques , which aWith this background, we decided to move on to establishment of a surgical technique for allogeneic UTx, assuming recovery of a uterus from a deceased donor, to enable procurement of wider and longer vessels; consequently, the surgical procedures and model preparation are more stable, in comparison with a living donor. As described in a previous report , we succMacaca fascicularis, age 5–9 years; average body weight, 3.83 ± 1.12 kg (mean ± standard deviation) after allogeneic UTx [Four female cynomolgus macaques (neic UTx  were usehttp://www.ebi.ac.uk/ipd/index.html) [Total RNA was isolated from peripheral blood cells using TRIzol reagent . Complementary DNA was synthesized using ReverTra Ace  after treatment of isolated RNA with DNase I (Invitrogen/Life Technologies/Thermo Fisher Scientific). Six primer pairs for MHC class I (Mafa-A and Mafa-B), Mafa-DRB, Mafa-DQA1, Mafa-DQB1, Mafa-DPA1 and Mafa-DPB1 loci were used separately for RT-PCR amplification ,16. In aex.html)  using GSex.html) . If shorex.html) . MHC gen®; Custodiol®, Nordmedica, Gentofte, Denmark) via a perfusion catheter  in the femoral or external iliac artery. Then, en bloc retrieval of the uterus with the adnexa, ovarian vein, and proximal vagina was performed, including the iliac vessels (cases 1 and 2) or infrarenal aorta/IVC (cases 3 and 4). The ovarian veins draining to the renal vein or IVC were left in the uterine graft side in cases 3 and 4. In recipient surgery, after entering the peritoneal cavity through a midline incision, a total simple hysterectomy was performed in advance, with preservation of the ovaries. Subsequently, iliac vessels on both sides (cases 1 and 2) or the infrarenal abdominal aorta/IVC (cases 3 and 4) were exposed at a distance of approximately 2 cm. The uterine graft was brought into the operative field and the vagina of the graft was anastomosed with the vaginal vault of the recipient to fix the uterus in the pelvis. Before vascular anastomoses, heparin (100 IU) was administered intravenously. In recipient surgery, end-to-side anastomosis with the recipient’s and donor’s common iliac vessels (cases 1 and 2) or infrarenal aorta/ICV (cases 3 and 4) was performed using continuous sutures . After reperfusion, sutures of the retroperitoneum and round ligaments were performed before closing the abdominal incision. Normothermia was maintained throughout the operation using heating lights and blankets in donors and recipients. Antibiotics and a proton-pump inhibitor were administered after surgery.Operative anesthesia and surgical procedures in donors and recipients were performed as previously described . In brieIntraoperative indocyanine green (ICG)  was injected intravenously to confirm the blood supply in the transplanted uterus and blood flow at the site of vascular anastomosis during surgery and in second-look surgery or autopsy ,21,22. FSevere rejections occurred in the first two transplants (cases 1 and 2) and the immunosuppressive protocol was amended in cases 3 and 4. As induction treatment, animals received antithymocyte globulin (ATG)  intravenously on postoperative day (POD) 0 (the day of surgery) in cases 1 and 2, and 20 mg/kg of ATG on POD 0 and POD 2 in cases 3 and 4. Maintenance treatment consisted of cyclosporine (CyA)  given subcutaneously in cases 1 and 2, and tacrolimus (TAC)  given intramuscularly in cases 3 and 4. These drugs were administered from day 0 in case 1, day –2 in case 2, and day –1 in cases 3 and 4. The target trough levels for CyA and TAC up to 1 month after surgery were planned to be in the ranges of 300–400 ng/mL and 15–20 ng/mL, respectively, and thereafter were adjusted as required. As another maintenance treatment, mycophenolate mofetil (MMF)  was administered orally from recovery of appetite after surgery on POD 11 in case 1, POD 6 in case 3, and POD 6 in case 4. MMF was discontinued on POD 22 in case 1 because the animal did not tolerate the drug. For this reason, MMF was not given in case 2, but MMF was administered forcibly using an orogastric catheter in cases 3 and 4, based on the severe rejections in cases 1 and 2. Methylprednisolone  was injected intravenously on POD 0 and then injected intramuscularly daily starting on POD 1. The dose of methylprednisolone was gradually tapered, and administration was completed on POD 14 in cases 1 and 2. In cases 3 and 4, methylprednisolone was administered at daily doses of 0.5 and 0.2 mg/kg, respectively, until the end of observation. If rejection occurred, steroid pulse therapy of 10 mg/kg methylprednisolone was administered and gradually tapered.After lysing red blood cells with BD Lysing buffer , single cell suspensions of peripheral blood cells were immunostained with fluorescence-conjugated antibodies . Dead ce5 cells per well were cultured in an ELISPOT plate  with 5 × 105 donor splenocytes. The culture was usually performed in triplicate wells, but duplicate culture was used when the number of cells was insufficient. After culture for 3 days, the number of interleukin-2 (IL-2) positive spots was counted by an ImmunoSpot analyzer . Stimulation indices (SI) were calculated by the following formula: the sum of the number of spots in culture of blood cells and donor splenocytes, divided by the number of spots in culture of blood cells only.Heparinized peripheral blood was collected from macaques before and after transplantation. After lysing red blood cells, 5 × 10After heat-inactivation, plasma collected from the transplanted macaques was diluted 10 times with phosphate-buffered saline  before use. The diluted plasma was added to a cell suspension of donor splenocytes as a primary antibody. Fluorescein isothiocyanate (FITC)-conjugated goat polyclonal anti-mouse IgG  was used as a secondary antibody to detect the attached IgG on the surface of the donor cells. Phycoerythrin conjugated anti-CD20 antibody , and allophycocyanin conjugated anti-CD3 antibody  were also used to study the cell type to which the donor specific IgG attached. The cells were analyzed by FCM.After the surgical procedure, animals were separately accommodated in a single cage and the general condition  and laparotomy wound site were evaluated daily. Laboratory assessments including hematology and blood chemistry were performed three times per week for the first two postoperative weeks, two times per week for two months, and weekly thereafter. To monitor for potential rejection after surgery, the size of the transplanted uterus and blood flow in the transplanted uterine artery were determined by transabdominal ultrasonography under anesthesia. Transvaginal biopsy of the transplanted uterine cervix and body and FACS, DSA and MLR analyses were routinely conducted monthly or when considered necessary (whenever graft rejection was suspected). Animals were euthanized if severe weakness, weight loss or abnormal behavior was seen or when determined to be necessary by veterinary staff and investigators. Necropsy was performed immediately after euthanasia to evaluate the graft and extrapelvic organs, including the brain, lung, heart, thymus, thymus, liver, gallbladder, spleen, pancreas, stomach, duodenum, jejunum, ileum, colon, rectum, kidney, bladder, bone marrow and lymph node.Formalin-fixed, paraffin-embedded liver biopsy tissues were sliced to a thickness of 3 μm and stained with hematoxylin and eosin (H&E). Elastica van Gieson (EVG) staining was also performed. An automated staining system  with a Bond Polymer Refine Detection kit  was used for immunohistochemistry and in situ hybridization. Immunohistochemistry was performed using an anti-CD20 monoclonal antibody , an anti-CD3 monoclonal antibody , and an anti-CD8 polyclonal antibody . An EBER Probe  was used for detection of Epstein–Barr virus-encoded mRNA by in situ hybridization. Allograft rejection was evaluated histopathologically using a grading system for monitoring of rejection proposed by a Swedish group ,24, in wChanges of trough levels of CyA (cases 1 and 2) and TAC (cases 3 and 4) in maintenance treatment are shown in All four recipient animals survived for more than 3 months after surgery without any surgical complications. The overall outcomes in these animals are summarized in 2/µL, CRP 11.8 mg/dL) and elevated LDH (1076 IU/L) were found. Swelling of the uterus was found despite blood flow in the uterine artery identified by transabdominal ultrasonography, and rejection was suspected. However, rejection could not be confirmed because samples that were appropriate for evaluation were not obtained from cervical biopsy specimens. The increased inflammatory response and LDH were improved by antibiotics; however, a biopsy specimen from the uterine cervix on POD 70 showed stromal lymphocyte infiltration with capillary endotheliitis, which was consistent with severe rejection  was started on POD 11 because of recovered appetite, but discontinued on POD 22 due to a decrease in appetite. On POD 32, an increased inflammatory response  and elevated LDH (918 IU/L). Rejection was suspected based on the results of case 1. Consequently, second-look surgery was performed on POD 35. The uterus was grossly adhered to the omentum and was markedly swollen A. High aEscherichia coli was identified in a bacterial culture test. A slightly elevated inflammatory response  was found in blood tests, but systemic conditions were good. After that, the uterus gradually shrank and was dissected on POD 126. Similarly to case 1, the intraperitoneal findings were a white shrinking uterus adhered to a wide range of the greater omentum and rectum, with bilateral hydrosalpinx  and elevated LDH (515 IU/L), and rejection was suspected based on the results in cases 1 and 2. Consequently, the dose of tacrolimus was increased from POD 62, MMF was readministered at 30 mg/kg twice a day, and steroid pulse therapy was started. The inflammatory response and LDH improved, but the trough concentrations of tacrolimus were poorly controlled and high until POD 116 . This was improved by antibiotics, leading to a good systemic condition.Based on the PTLD in case 3, the dose of MMF on POD 6 was decreased from 40 to 20 mg/kg  and the maintenance dose of methylprednisolone was decreased from 0.4 to 0.2 mg/kg in case 4. When starting administration of ATG, ganciclovir  was also injected intravenously. Transabdominal ultrasonography on POD 13 showed a uterus of normal size and good blood flow in the uterine artery. Histologically, liquefaction degeneration was mild; however, perivascular lymphocyte infiltration and endotheliitis were clearly evident. Based on the endotheliitis, this condition was diagnosed as moderate rejection. No increased inflammatory response or LDH was found in biochemistry tests. Consequently, steroid pulse therapy was used. Transabdominal ultrasonography on POD 25 showed slight uterine swelling. A biopsy specimen showed similar rejection features to those on POD 13, with perivascular fibrosis and edematous changes. Steroid pulse therapy was administered again, ATG was given at 20 mg/kg, and MMF was increased to 30 mg/kg twice a day. In a biopsy on POD 41, obstructive venulitis and endotheliitis of small to middle size arteries were seen, in addition to worsening of capillary endotheliitis, and perivascular fibrosis A. BiocheEscherichia coli was identified in a bacterial culture test. In the resected aorta, the donor-grafted intravascular space in the vascular anastomotic sites was almost stenosed  counts and the CD4+/CD8+ cell ratio are shown in +/CD8+ ratio showed a tendency to decrease. Regulatory T cells (CD4+ FOXP3+) had no relationship with rejection (data not shown).Induction treatment and additional ATG administration decreased lymphocyte counts immediately after surgery . Due to The MLR stimulation index against donor and third-party antigens was determined after surgery . All aniAll animals were seronegative for DSA before transplantation. DSAs against MHC class I and II were detected in conjunction with uterine rejection on PODs 46 (data not shown), 35 and 13 in cases 1, 2 and 4, respectively, after which the DSA titer increased . In caseUTx is now conducted in humans in several countries, but is still considered to be in the experimental stage since there are remaining clinical and technical issues to be resolved . TherefoIn this study, long-term outcomes after allograft were examined in four cynomolgus macaques used in our previous study to establish the allograft procedure. All four macaques developed rejection and three underwent hysterectomy because uterine function could not be recovered due to irreversible chronic rejection. There are case reports of human UTx with rejection ,26,27,28First, an increased WBC and CRP inflammatory response due to rejection and elevated LDH due to cell collapse were found. In rejection or elimination of conventional life-supporting organ transplants, clinical symptoms and biochemical data change immediately. In contrast, the uterus is not a life-supporting organ and rejection is not immediately life-threatening and frequently asymptomatic; therefore, there are no clear findings showing rejection. The biochemical findings above may be one indicator; however, these findings are often caused by irreversible rejection and it may be difficult to recover uterine function in such a case.Second, the uterus is swollen by rejection and then shrinks over time. In the course, inflammatory edema occurs due to rejection, followed by cell injury and necrotic cell death, and granulation tissues and fibrils are formed, resulting in atrophy. In cases 1 and 2, these histopathologic findings were found, but in case 4, the uterus swelled but did not shrink. However, endotheliopathy and vascular occlusion were observed, and the uterus was assumed to have atrophied due to ischemia over time. This assumption is suggested by our previous results showing that the uterus of cynomolgus macaque shrinks after a warm ischemia time of 8 h ,30.E. coli was detected in both. Hygiene control of reared cynomolgus macaques is limited and E. coli from feces probably contaminated the uterus. Also, a UTx patient from Sweden had repeated intrauterine infection and the uterus had to be resected [Third, continuous rejection sometimes promotes intrauterine infection. The uterine cavity is in contact with the outside of the body through the vagina; therefore, intrauterine infection may occur. Since organ transplant recipients are treated with immunosuppressant drugs, they have higher risks for infection than general patients. Furthermore, rejection and ischemia disrupt phylaxis functions in the uterus and enhance the risk for infection. In cases 2 and 4, intrauterine infection occurred, and resected .Fourth, rejection causes thrombus formation. An intravascular thrombus is often caused by surgical procedures of vascular anastomosis and sometimes by blood flow stasis because of occlusion of small and middle-size vessels in the rejected organ due to rejection-induced endotheliopathy. In case 4, thrombi were found from the vascular anastomotic site of the grafted aorta to the iliac artery and severe endotheliopathy occurred. Furthermore, inflammatory cells were detected in the outer membranes of the grafted aorta, which suggests that rejection in vessels is probably the cause of thrombi.Fifth, interestingly, rejection occurred in the oviducts, as well as in the uterus. In cases 1 and 2, lymphocytic infiltration was found in the oviducts of donors. In human UTx, the donor’s oviducts are often removed in transplantation, despite retention of the oviduct in donors having an advantage of maintaining the option of natural pregnancy. However, hydrosalpinx was evoked in cases 1 and 2 due to the remaining oviduct, and this may cause infertility; therefore, surgery with oviduct resection is currently preferable.Finally, the histopathological findings in rejection due to UTx in cynomolgus macaques included frequent onset of endotheliopathy, in addition to those based on clinical criteria for uterine rejection by a Swedish group ,24. SimuPostoperative immunosuppression for prevention of rejection due to organ-specific antigenicity is important in organ transplantation. Antigenicity of the uterus is unclear, but in human UTx many patients have received immunosuppressant protocols similar to those used in renal transplantation. There have been no reports of serious rejection of the uterus; therefore, the uterus is considered to be an organ that is unlikely to develop rejection. However, from our previous results  and the Our results show that studies in non-human primate models have specific problems and limitations, and recognition is required that that results may not always correspond directly to those in humans. However, development of an allogeneic UTx model in a non-human primate is still important for addressing clinical issues in human UTx, and further validation and outcomes of UTx in such models will contribute to successful clinical use of UTx, in which current results indicate exciting developments. The outcomes of these procedures are likely to be improved by accumulation of data from further animal studies, including in non-human primates.In conclusion, allograft of the uterus of cynomolgus macaques resulted in clinicopathological findings of increased inflammatory response, swollen and subsequently shrunken uterus, intrauterine infection, intravascular thrombus formation, oviductal rejection, and a DSA-positive status. Postoperative management and monitoring of macaques are limited, but the uterus of cynomolgus macaques was found to be likely to develop rejection and to have high antigenicity. Therefore, adjustment of protocols for immunosuppressive drugs may be necessary to produce a stable uterine allograft model in cynomolgus macaques. However, the current results are important for resolution of remaining clinical questions in human UTx by producing a scientifically verifiable model of UTx in a non-human primate.


Termitomyces spp.) contain an inverted repeat (IR), a duplicated region half the size of the complete genome. In addition, we found an abundance of sequences capable of forming G-quadruplexes (G4DNA); structures that can disrupt the double helical formation of DNA. G4DNA is implicated in replication fork stalling, double-stranded breaks, altered gene expression, recombination, and other effects. To determine whether this occurrence of IR and G4DNA was correlated within the genus Termitomyces, we reconstructed the mitochondrial genomes of 11 additional species including representatives of several closely related genera. We show that the mtDNA of all sampled species of Termitomyces and its sister group, represented by the species Tephrocybe rancida and Blastosporella zonata, are characterized by a large IR and enrichment of G4DNA. To determine whether high mitochondrial G4DNA content is common in fungi, we conducted the first broad survey of G4DNA content in fungal mtDNA, revealing it to be a highly variable trait. The results of this study provide important direction for future research on the function and evolution of G4DNA and organellar IRs.Mitochondria retain their own genome, a hallmark of their bacterial ancestry. Mitochondrial genomes (mtDNA) are highly diverse in size, shape, and structure, despite their conserved function across most eukaryotes. Exploring extreme cases of mtDNA architecture can yield important information on fundamental aspects of genome biology. We discovered that the mitochondrial genomes of a basidiomycete fungus ( Mitochondria are a vital component of virtually all eukaryotic life, generating energy in the form of ATP and regulating the respiratory metabolism. Mitochondria maintain their own genome, a legacy of their endosymbiotic origin. However, despite their common ancestry and conserved function, mitochondrial genomes (mtDNA) of major lineages such as plants, fungi, and metazoans differ considerably not only in size, content, and structure but also in mutability, inheritance, and replication . Fungi bTermitomyces , a clade of obligate mutualistic symbionts of termites, are an exception. During mating in Termitomyces, nuclei of both parents enter a shared cytoplasm from which offspring cells grow, rather than one parent adopting a nucleus from the other as is typical for most basidiomycetes  that generally occupies half of the genome, duplicating several key genes and increasing the mtDNA size significantly. In addition, we found an abundance of G-quadruplex (G4DNA) motifs, prompting the question whether these two structural features are functionally correlated. We also sequenced the mtDNA of five other Lyophyllaceae: Blastosporella zonata, Tephrocybe rancida, Myochromella boudieri, Asterophora parasitica, and Tricholomella constricta, to estimate the origin of the IR and the G4DNA increase.Like the vast majority of eukaryotes, in most fungi mtDNA transmission during sexual reproduction is uniparental. Species of the genus omycetes . This imbisporus , anotherG-quadruplex DNA, or G4DNA, is a naturally occurring conformation of DNA that folds in a four-stranded conformation rather than the common double helix. Although originally considered as an in vitro curiosity, G4DNA is now known to exist in living cells of ciliates, humans, yeasts, nematodes, and others . The forG4DNA can have many effects depending on the location at which they occur and the shape they form. Effects associated with G4DNA include: replication fork stalling, altered gene expression, recombination, double-stranded break (DSB) formation, and genome instability . In addiSaccharomyces cerevisiae, and found an ∼10-fold increase of G4DNA motif content in mtDNA compared with nuclear DNA. However, a broad, comparative survey of G4DNA content of fungal nuclear and mtDNA is still lacking.Recent studies have employed predictive algorithms to map potential G4DNA in sequenced genomes based on sequence patterns . This reCandida albicans, the mitochondrial IR is centered on a replication origin, and appears to regulate genome replication through recombination-driven replication with the IR as an initiation site stability, we hypothesize that the IR is correlated with enriched G4DNA in Termitomyces strains were selected to include at least two strains for each of the three most species-rich associated termite host genera  . The Termitomyces isolates were grown at 25 °C in the dark. Tricholomella constricta, Ast.parasitica, T.rancida, and M. boudieri isolates were grown at 15 °C in the dark. For Termitomyces sp. DKA19, no culture was present and hyphal nodules collected from a termite mound were used instead. Nodules were stored in pure ethanol at −20 °C. Prior to extraction, five nodules were rinsed with clean pure ethanol and dried on filter paper.We selected species for this study based on their phylogenetic position  and ecol2, T159) . TermitoDNA was extracted using a cetyltrimethylammonium bromide (CTAB) protocol  online. To reconstruct mitochondrial genome sequences from whole-genome sequencing data, we took a reference-based iterative read baiting approach using the IOGA-pipeline  was done manually by performing a BLAST search of the genome against closely related genomes such as ious v11 , and theious v11  to identsp. T132 , which wsp. T132  with Bowsp. T132  using thsp. T132 .A, Termitomyces species: T132 and T13. The master mix and PCR program used can be found in the B), To confirm the presence of the mitochondrial IR in vitro, a touchdown PCR was conducted. The primers that were used were designed manually based on the mitochondrial genome assembly A,  online. cox1, cox2, cox3, cob, and atp6). We used RevTrans  to account for the inherent uncertainty of in silico detection of G4DNA. Because G4Hunter occasionally reports sequences with significant overlap, we eliminated sequences that overlapped a previously reported sequence by >50% of their length. This was done to avoid counting the same potential G-quadruplex twice or more.We used G4Hunter  to scan To identify potential locations of replication origins, we created cumulative GC skew graphs  of our mTermitomyces covering the known diversity of the genus with strains from the three known genera of fungus-growing termites Macrotermes, Microtermes, and Odontotermes . The mtDNA of B. zonata, T. rancida, and all Termitomyces specimens featured a large IR region and therefore duplication of all genes contained within, though the gene content of the IR varied from species to species. All species except Ast. parasitica had introns in one or more mitochondrial genes. In particular, cox1 often featured several introns, most of which harbor LAGLIDADG or GIY-YIG endonucleases, a phenomenon commonly found in basidiomycete fungi . Such plasmids are commonly found in fungal mitochondria, for example, maranhar/kalilo in Neurospora  matches with the host mtDNA, as well as occasional matches with mtDNA of other species, suggesting these plasmids were present in the common ancestor of other strains as well, and inserted parts of their DNA in the host mtDNA.When BLASTing plasmid sequences against our mtDNA assemblies, we found significant Termitomyces  and single copy region 2 .We detected the mitochondrial IR in our assemblies by a 2-fold increase in read coverage in the duplicated region, due to the assembler merging the identical copies. The inverted nature of the duplication was suggested by inverted read pairings between the ends of the assembled sequence and the borders of the duplicated region. To confirm the presence of the IR, we designed primers around the predicted border regions of the IR and the single copy regions. We succeeded in amplifying products for all four border regions in two strains of atp6, nad2, nad3, nad4, cox2, and several tRNAs , and several species showing distinctively high G4DNA content. These include the model species N. crassa, to see if G4DNA was located differentially in these regions between species , as well as the ribosomal subunits, than expected by chance (P < 0.05), whereas in human mtDNA, there was no bias.The high G4DNA motif content found in our mtDNA assemblies is similar to the high content found in human mtDNA . However species . We compt-test  showed the difference in G4DNA motif content between IR and SC was statistically significant across species (P = 0.02). To exclude the possibility that this increase in G4 is simply due to a higher concentration of noncoding DNA in the IR, we calculated what percentage of DNA in the IR and SC regions was part of exons of conserved protein-coding genes or the ribosomal subunits. We found that in all but two cases, the percentage of exonic DNA was in fact higher in the IR than in the SC regions  or even zero , but therevisiae , and frorevisiae . As suchAllomyces macrogynes and others  mitochondrial IRs, while rare in fungi, occur among others in rough HR . Consistrough HR , which crough HR .Termitomyces could also be due to the colocalization of replication origins in the repeats. The association of G4DNA with replication origins is supported in other organisms , often encompass the ribosomal subunits. This mimics the multitudinous tendency of nuclear ribosomal DNA, and it may be selectively beneficial to harbor the highly conserved rDNA in a region of increased genomic stability. As ribosomal DNA may be associated with G4DNA  the IR helps repair DSBs caused by G4DNA; 2) both structures are involved in the replication of mtDNA. We also provide the first comparative analysis of G4DNA content in mtDNA in fungi. The large discrepancy in mitochondrial G4DNA content between fungal species raises an opportunity for examining the effects of G4DNA on genome stability, replication, recombination, and transcription. Such studies would benefit from comparing similar species with different G4DNA content. In addition, some of the fungi with high mitochondrial G4DNA content are already established model species, N. crassa in particular. These species would be ideal candidates for experimental studies on G4DNA function and evolution, and could improve our insight in how G4DNA affects our own  DNA.We report a large IR and an apparent increase in G4DNA content in the mitochondrial genomes of Finally, we have shown that in fungal mtDNA coding regions are significantly depleted in G4DNA motifs, in contrast to humans. This suggests that fungi have stronger selection against G4DNA in exons, as they might lack the means to regulate G4DNA formation posttranscriptionally.Genome Biology and Evolution online.Supplementary_Material_evz122Click here for additional data file.


TRIB3 was reported to associate with pig growth, GRP, TTR, CNR1, GLP1R, BRD2, HCRTR2, SEC11C, and ssc-mir-122 were reported to associate with growth traits in human and mouse. The newly detected candidate genes will advance the understanding of growth related traits and the identification of the novel variants will suggest a potential use in pig genomic breeding programs.Improvement of the growth rate is a challenge in the pig industry, the Average Daily Gain (ADG) and Days (AGE) to 100 kg are directly related to growth performance. We performed genome-wide association study (GWAS) and genetic parameters estimation for ADG and AGE using the genomic and phonemic from four breed  populations. All analyses were performed by a multi-loci GWAS model, FarmCPU. The GWAS results of all four breeds indicate that five genome-wide significant SNPs were associated with ADG, and the nearby genomic regions explained 4.08% of the genetic variance and 1.90% of the phenotypic variance, respectively. For AGE, six genome-wide significant SNPs were detected, and the nearby genomic regions explained 8.09% of the genetic variance and 3.52% of phenotypic variance, respectively. In total, nine candidate genes were identified to be associated with growth and metabolism. Among them,  Growth rate is a vital economic trait and significantly affected pig production  in pure Duroc population  were reported that associated with ADG and AGE  has been widely used for mapping candidate genes of complex traits  for mapping candidate genes underlying ADG and AGE traits. Estimation of genetic parameters, such as heritability was also carried out. The detected candidate genes, potential breeding markers, and the better understanding on genetic architectures of ADG and AGE will benefit breeding programs.Animals used in this study were from two isolated farms, and composed of four pure breeds, including Duroc, Yorkshire, Landrace, and Pietrain . ADG andwhere correction factors are different for sire and dam, and the formulas are shown below:ADG was calculated by following equation:In total, 31,173 phenotypic observations from farm 1 and 24,374 phenotypic observations from farm 2 were recorded. Tail and ear tissue samples were collected and preserved with 75% alcohol and were stored in −20°C freezers.ng/μl, quantity ≥ 1 μg, and passed gel electrophoresis test were used for genotyping. A total of 4,865 DNA samples, including 1,595 samples from farm 1 and 3,270 samples from farm 2 were genotyped by Illumina PorcineSNP50 Bead Chip, which includes 50,697 SNPs, and all SNP markers were mapped to Sus scrofa genome build 11.1  in an R procedure  . The genomic locations were downloaded from https://www.animalgenome.org/pig/ and genes within the candidate region are considered as candidate genes.The candidate genes nearby the genome-wide significant SNPs were identified by Ensembl database using the gene annotation information of Sus scrofa genome version v11.1 , and ssc-mir-122 were identified in the nearby candidate regions on chromosome 1. GRP is a regulatory neuropeptide that stimulates gastric G cells to release gastrin and regulate gastric acid secretion and intestinal movement, which affects the food intake and may lead to anorexia, bulimia, and obesity if lacked of the genes  and plays an important role on growth and energy metabolism  and FTO (Fat mass and obesity-associated protein) that located on chromosome 6 significantly affected muscle development and obesity, respectively . The science ethics committee of the HZAU approved all the animal experiments that conducted in this study.XiaL, SZ, and XinL conceived the study. ZT, LY, and JX wrote the codes and did the data analysis. XiaL and ZT drafted the manuscript and all authors contributed to finalizing the writing.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.


Brassica napus (rapeseed) is one of the most important oil crop in the world, therefore, increasing rapeseed yield through breeding is inevitable in order to cater for the high demand of vegetable oil and high-quality protein for live stocks. Quantitative trait loci (QTL) analysis is a powerful tool to identify important loci and which is also valuable for molecular marker assisted breeding. Seed-yield (SY) is a complex trait that is controlled by multiple loci and is affected directly by seed weight, seeds per silique and silique number. Some yield-related traits, such as plant height, biomass yield, flowering time, and so on, also affect the SY indirectly. This study reports the assembly of QTLs identified for seed-yield and yield-related traits in rapeseed, in one unique map. A total of 972 QTLs for seed-yield and yield-related were aligned into the physical map of B. napus Darmor-bzh and 92 regions where 198 QTLs overlapped, could be discovered on 16 chromosomes. Also, 147 potential candidate genes were discovered in 65 regions where 131 QTLs overlapped, and might affect nine different traits. At the end, interaction network of candidate genes was studied, and showed nine genes that could highly interact with the other genes, and might have more influence on them. The present results would be helpful to develop molecular markers for yield associated traits and could be used for breeding improvement in B. napus.Worldwide consumption of oil is increasing with the growing population in need for edible oil and the expansion of industry using biofuels. Then, demand for high yielding varieties of oil crops is always increasing.  Brassica napus) is an important crop utilized globally for vegetable oil production, animal feed and biofuel. B. napus  is a polyploid species, which was derived from the hybridization between turnip rape  and cabbage   . In termtication . Spring ing type . Currenting type .B. napus. Application of molecular marker techniques for quantitative trait loci (QTL) is a trending strategy , candidate genes for PH (31 genes), branch initiation height (15 genes), and BN (17 genes) were found  via arrangement into the physical map of B. napus “Darmor-bzh”, the candidate genes for SY and yield-related traits in B. napus were also searched inside the overlapping QTLs regions and a genetic network was constructed to understand the interaction of candidate genes.Therefore, the present study aimed to compare yield and yield-related QTLs in 22 populations which were developed in four different countries , some QTLs could not be aligned on the map. Also, it happened that only one flanking marker could be placed, and some literatures gave only one flanking marker; therefore, we took a uniform area of 1 cM to delimit all the QTLs, so that QTLs region could be located on the map even with one marker. Note that apart from related literatures, marker sequences could also be found on some database, such as Brassica.info1, NCBI2, Brassica IGF Project3, Gramene Markers Database4. The map was build using Circos software , on C07 (2.53 Mb) and on C08 (36.35 Mb). They could be located in separate regions or they could overlap with other populations’ QTLs. Thus, 92 regions that contained 198 overlapping QTLs were observed, they were located on 16 chromosomes and involved 11 traits and 17 populations which were developed in China, America and Germany , it is obvious that Express617 × R53 and Express617 × V8 shared a common parental line in Express617, as well as MF216 × P1804, RV128 × P1804, RV289 × P1804, and TO1141 × P1804 which had P1804 in common. Thus, overlapping QTLs from these populations were not surprising . Further analysis revealed that regions of overlapping QTLs were mainly located on chromosome A03 (24/92), then on C02 (11/92), A10 and C01 , A7 and C06 , A01 (6/92), A02 (5/92), A05 (3/92), A06, A09, C03, C05, C07 and C09 , and C08 (1/92). The most frequently detected overlapping QTLs for each traits are presented on Figure 2A, among 92 regions of overlapping QTLs, 30 regions were for FT, 20 regions were for SW, 13 regions were for PH, 11 regions were for SY, 4 regions were for SPUA, 3 regions each were for NPB, PN, SN and TW, and 1 region each for BY and SL.The present results revealed that QTLs from the Canadian population RV289 × P1804 and Tapidor × Ningyou7 for FT trait on A02), eight regions of overlapping QTLs involved five populations , six regions of overlapping QTLs involved three populations , and four regions of overlapping QTLs involved four regions  . Moreover, 53 regions of overlapping QTLs were of environmental fixed QTLs: 28 among them were from the mapping population of Chinese environments, 13 and 12 among them were from German and American environments, respectively. The remaining 39 regions of overlapping QTLs were of mixed populations: QTLs from China and America overlapped the most (27/39 overlapping QTLs), and eight and two implied QTLs from China and Germany, and America and Germany, respectively, also since “Sollux × Gaoyou” was developed both in China and Germany, two overlapping QTLs implied the populations developed from America, China and Germany . Finally, 13 regions of overlapping QTLs might affect multiple traits simultaneously: three regions were found on each of A03  and C02 , two regions were found on each of A10 (FT/SW and SY/TW) and C01 (both SN/SW), and one region was found on each of A05 (NPB/PH/SN), A07 (PH/SW/SY) and C06 (BY/PH/SW)  that might affect NPB, PH and SN traits, six other genes on C2  that possibly affect FT, PH and PN traits. Besides, candidate genes detected in B. napus often fell into QTL intervals of traits which were different from related function in A. thaliana orthologous genes. For example, B. napus BnaC09g36060D is homologous of A. thaliana RAX2 gene AT2G36890 which was related to BN, however, B. napus BnaC09g36060D fell into overlapping QTLs of FT. These findings showed genes that might be responsible for one or multiple traits. Besides, homologous genes might affect dissimilar traits.A total of 1562 genes in B. napus that might offer maximum benefit might correspond to a genome enriched with regions where QTLs of multiple traits overlapped, as seen on chromosomes A03, A05, A07, A10, C01, C02, and C06 where QTLs for two or three traits could overlap in the same regions, as mentioned previously  × Ceres. The remaining populations mentioned in this study had no common ancestry, in our knowledge. Otherwise, 53 regions of environment fixed QTLs could be deduced from the alignment map , despite the genotype difference of populations, similar QTL regions could be found due to the environment impact. Nevertheless, overlapping QTLs could be observed from populations of different environments , these regions might be enriched with related gene variations, but it is also possible that they share common ancestry. Besides, we could find QTLs which could not overlap with other QTLs , they were specified QTLs which could not be on the same region as the others, probably due to the above-mentioned factors , but also the density of genetic map of which they were deduced. A region on A03 (from 4.25 to 6.05 Mb) had overlapping QTLs for FT, implying five populations, which were stable QTLs from populations of two different environments. Also, several regions of overlapping QTLs with multiple traits were found, these regions might be advantageous to develop for rapeseed breeding in targeting multiple traits simultaneously.In the present study, position of QTL in different populations were different, probably due to the genetic difference between parental lines, the type and size of the populations used, and the environments where they were developed . OverlapBrassica genomes, candidate gene identification for controlling SY, and knowledge of the location of these specific genes in the genome and their roles will extend their importance in molecular-marker technology in plant breeding. This is because development of allele molecular markers from the gene itself can be synthesized. Findings of the current analysis are valuable to enhance breeding programs. Genes found within these QTLs intervals could be potential genes that might affect these traits and consequently could be manipulated to get the desired traits, notably producing high SY.Besides, our analysis supported the importance of quantitative traits analysis in identification of SY and yield components. However, identification of the right gene combination in various loci is a challenging task, particularly when several loci with multiple alleles are associated. In this case, molecular plant breeding and genomics rather than conventional breeding will play a very significant role in increasing breeding efficiency. For example, sequencing of the B. napus: first, n = 4), seed weight (n = 2), and PH (n = 1), by comparative mapping between A. thaliana, B. rapa, B. oleracea and B. napus, in 348 DH lines from cross between KenC-8 and N53-2, they were grown in Dali, Shannxi for 5 years (Winter type) and Sunan, Gansu Province for 3 years (Spring type). Second, A. thaliana and located on flanking regions of up to 450 kb on either side of significant SNPs markers, and then used them to GO annotations, with auxin, GA, IAA, SL, CK, and FT taken as the most likely candidate. Finally, n = 6), PN per plant (n = 7), branch PN (n = 7), branch yield (n = 3), and thousand seed weight (n = 1), from a study led on 520 rapeseed accessions grown in Chongqing and Yunnan, by using GWAS and RNA-seq tools. Comparing our results with these cited papers, BnaA02g02560D was a candidate for SY in both our analysis and BnaA02g02560D was a candidate for SY in our analysis, however, it was a candidate for PH in BnaA02g12130D and BnaA02g12260D were candidates for FT in our analysis, and they were candidates for PH and branch initiation height in In the current study, a total of 147 candidate genes were found in 65 regions of overlapping QTLs, and several candidate genes might affect multiple traits at the same time. As mentioned above, three independent works were published on candidate genes for yield traits in A. thaliana as a model plant to map and discover genes in related species. For instance, BnFLC010, candidate gene underlying qFT10-4 QTL, which was the key gene that controls differentiation of spring or winter rapeseed types based on these comparative mapping analyses. Furthermore, A. thaliana. Advancement of these studies in order to ascertain the effects of the QTLs on different agronomic traits by use of comparative mapping technique have been undertaken by many research groups  is available on STRING database but is not enough to provide more information for this study compared to A. thaliana. First, homologous genes in B. rapa would represent only a part of detected candidate genes in B. napus and this would distort the results. Second, annotated and cloned genes were rare in B. rapa than in A. thaliana, thus taking A. thaliana as reference would help greatly in comprehension of the interaction of candidate genes based on their functions. This strategy was used previously to investigate on proteins interaction in other studies  which are involved in important functions  might have higher influence on the other genes which possibly dependent on these nine genes. CO, FT, FLC and AGL20 were four key genes in the network interaction of our analysis and they are both implicated in plant flowering development. CONSTANS (CO) encodes a transcription factor that induces flowering in long exposure to daylight and stimulates the expression of FLOWERING LOCUS (FT) in phloem of leaves . It has A. thaliana, five main pathways were found to control flowering: the gibberellin pathway in which flowering needs the activity of gibberellic acid; the endogenous pathway which modulates flowering according to the age of plant; the photoperiod and the vernalization pathways which control flowering according to day length and temperature fluctuation, respectively; and the autonomous pathway which works independently of gibberellin and photoperiod pathways (Actually, appropriate FT greatly influences the potential of rapeseed yield , it coulpathways . The keypathways .A. thaliana but not B. napus, which indicated that findings might be imprecise. Nevertheless, construction of this network gave new insight on interaction of candidate genes.Otherwise, these nine above-mentioned genes might affect variation of traits, and quantitative traits are affected by structure and dynamism of genetic regulatory networks . In the B. napus, which could be aligned and combined in one physical map. Unluckily, some QTLs could not be aligned in the map. However, stable QTLs which corresponded to overlapping QTLs could be predicted, but need confirmation. Nevertheless, region enriched with QTL were found which could help to understand the process of close linkage in traits and to identify varied or conserved area. Then, this map could serve as reference to understand rapeseed genome characteristic, especially while studying yield traits. Additionally, new candidate genes could be detected in these hypothetical stable QTLs, but still need to be confirmed as well, even if they were carefully chosen due to their presupposed yield related function and their position within QTL regions. Based on our current study, markers could be developed and potential candidate genes could be tested for breeding improvement purpose.This study reported QTLs for yield and yield-related traits in NR and HC wrote the manuscript. NR, HC, HD, SP, and WY performed the analysis of QTL alignment map. NR analyzed the candidate genes and interaction network. ML, BW, and LY supervised the work, and revised 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.


Introduction: The complexity of understanding cancer risk in MS is increased by inconsistencies in study design, and the lack of age-, sex-, and ethnicity-specific risk estimates. Aims of our study were to estimate the incidence of cancers in the MS population of Catania  and to evaluate the impact of disease-modifying treatments (DMTs) in cancer risk.Materials and Methods: We screened 2,730 PwMS according to the MS criteria of Mc Donald 2010 referring to MS center of Catania in the period between 2003 and 2013. We matched database of MS patients with the Integrated Cancer of Catania-Messina-Siracusae-Enna. We calculated age and sex specific standardized incidence ratios (SIR) and the relative risk (RR) of developing cancer in MS patients treated with at least two different DMTs compared to who received one or no treatment.Results: Out of 2,730, 1,180 MS patients  were enrolled. We found 36 cancers. Global SIR was 1.18 (CI95% 0.78–1.58), with a significantly higher risk in men with a range age of 20 to 50 years  and in women over 50 years [1.82 (CI95% 1.08–2.55)]. RR of developing cancer was 1.99 (CI95% 1.14–3.45) in MS patients switching one DMT and 3.38 (CI95% 1.83–6.22) in who switched at least twice.Discussion: Our results demonstrated that cancer risk was not increased in our MS population; but age and sex different distribution may partly drive cancer risk. Higher cancer risk in MS patients switching more than two DMTs should take into account in treatment decision making. Multiple Sclerosis (MS) is a severe acquired autoimmune neurodegenerative disease of the central nervous system (CNS) with extremely variable disease course, that usually affects persons in their third/four decades of life, even if late onset is described . Women hIt was suggested that MS and cancer may share some aberrant functions of immune system .Indeed, in MS there is an alteration of the function of regulatory T cells (Tregs), or suppressor T cells, modulating the immunoreaction by suppressing the activation of the immune system . RegardiIn the last 10 years, we have witnessed to great changes in the therapeutic scenario of the MS . CurrentWe aimed to compare cancer incidence among persons affected by MS to that in the general population settled in the geographic area of Catania . Moreover, we evaluated any correlation among DMTs use and cancer development.2) (www.interno.gov.it)An observational retrospective analysis of prospectively collected data of patients with MS was performed at our tertiary MS center of Catania, Italy. Catania, Italy, is the second populated  and largest province of Sicily, Italy . We screened all patients who received their diagnosis between January 1st, 2003 and June 31th, 2013. Once entered the clinics, patients were followed up prospectively with at least one scheduled visit per year.Inclusion criteria were: (a) age ≥18 years; (b) lived in the city of Catania; (c) diagnosis of MS according to the Mc Donald criteria ; (d) at “no DMTs” group including patients not treated with any DMT in their MS history;“no switch” group including patients treated with only one DMT in their MS history;“1 switch” group including patients who experienced one therapeutic switch;“≥2 switches” group including patients who experienced at least two switches.About the DMTs use, we stratified our MS cohort according to the number of switch MS treatment :The study protocol was approved by the local ethics committee (Comitato Etico Catania 1) (19/2017/PO on 6th July 2017). All patients provided written informed consent. The study was conducted in accordance with the ethical principles of the Declaration of Helsinki and with the appropriate national regulations.At the time of first visit in our center, in our clinical practice for each patient, we collected the following clinical data: data of disease onset, MS clinical course, type and duration of DMTs, different switch strategy, total number of relapses, disability level assessed by Expanded Disability Status Scale (EDSS).Oncological data regarding cancers occurred after diagnosis. We included in our analysis oncological diagnosis occurred at least 2 years after the MS diagnosis. Main groups of cancers were considered: brain and nervous system, breast, bones and joints, digestive system, endocrine system, eye and orbit, female genital system, male genital system, oral cavity and larynx, respiratory system, skin, urinary system and lymphoma-myeloma-hematopoietic or lymphatic cancer of MS were collected. We matched such data with the data of Integrated Cancer Registry of Catania-Messina-Siracusae-Enna, considering all during the observation period. We calculated age and sex specific standardized incidence ratios (SIR), as measure of the relative cancer risk.We aimed to investigate the cancer incidence among persons affected by MS to that in the general population settled in the geographic area of Catania and the relative risk (RR) of developing cancer in MS patients treated with at least two different DMTs compared to who received one or no treatment .We also examined whether the cancer risk was different in patients with different treatment strategy: induction strategy where immunosuppressive (IS) is followed by immunomodulatory (IM) treatment, and escalation strategy where IM drugs where followed by IS treatment. Those who used interferon beta and glatiramer acetate were classified into the IM group, and those who used methotrexate, mitoxantrone, fingolimod, and azathioprine were classified into IS group. Natalizumab were included in the IS group even if its mechanism of action is not typically immunosuppressive, but because it is considered as second-line therapy.Skewness and kurtosis tests were applied to the continuous variables to confirm a normal distribution and results were expressed as means and standard deviations (SD). The Mann-Whitney test was used to compare continuous and categorical variables, and Pearson's chi-square test or Fisher's exact test was used to evaluate the categorical variables.Overall, sex-specific and age-specific estimates were reported expressed in rates per 100.000 residents associated to their relative confidence intervals, or all cancers and for main groups of cancers. These estimates were compared to those present in the Integrated Cancer Registry, available for the same period through the calculation of the rate ratios.p-value < 0.10). The selection of the multiple regression model will be done with the “all possible models” approach, then a regression analysis will be performed for all possible combinations. The Likelihood ratio test will be used to compare the log-likelihood of the regression model in which the aforementioned variable has been excluded. In the same way, the possible interaction (Joint effect) between two variables  will be evaluated. The “test for linear trend” and the “test for departure from linear trend” will be used for quantitative variables in order to evaluate a possible dose dependent effect (“linear or trend effect”).SIR was calculated by dividing the number of observed cases of cancer by the numbers of expected cancer cases, represented a measure of the relative risk of cancer. Wald's test assuming a Poisson distribution of the observed cases was used for determining 95% confidence interval (CI) for SIR. All statistical analysis was performed by SPSS software . Unconditional logistic regression analysis were performed using binomial “cancer yes/no” as outcome; we considered age, sex, disease duration, MS type, baseline EDSS, DMT duration (in months) and number of switches as independent factors. In order to investigate the independent effect of a risk or protective factor after adjustment for one or several other factors or to adjust for confounding variables, we carried out a multivariate model. Multivariate analysis was performed with conditional logistic regression, adjusting in the model for clinical and genetic covariables that will be significantly different to univariate analysis between cases and controls  satisfied the inclusion criteria and were enrolled for the study . The meaWe found 36 cancers in 36 patients (3.1% of enrolled population). Global SIR of 1.18 (CI 95% 0.78–1.58) was obtained. Stratifying for age and sex, we found a significantly higher risk in men with the range age 20–50 years  and in women over 50 years  .About the 36 cancers, 8 (22.2%) were primary located in the genitourinary system , 6 (16.7%) thyroid cancers, 6 (16.7%) were colon cancers, 5 (13.9%) were of the CNS, 5 (13.9%) were lymphomas, 4 (11.1%) were breast cancers and 2 (5.6%) were melanomas. Further analyses with respect to the cancer sites in MS patients resulted in an increased risk of genitourinary cancer [SIR = 1.65 ], and for thyroid cancer [SIR = 1.32 ], but not for breast cancers [SIR = 1.17 ] and cancers which are located in nervous system [SIR = 1.03 ].p < 0.001), with higher age at MS onset . Moreover, MS patients with cancer had longer MS disease duration, worse disability (higher mean EDSS), that is an increased percentage of patients who got EDSS 4.0  which means a limitation in ambulation , in “no switch” group was 1.17 CI 95% (1.02–2.34), 1.99 (CI 95% 1.14–3.45) in MS patients who switched one DMT and 3.38 (CI 95% 1.83–6.22) in MS patients who switched at least twice.The multivariate analysis showed that age, age at onset, disease duration, and more that 2 switches were associated with a higher risk of developing cancer .Our results showed that incidence of cancer in our MS cohort was not higher than general population. However, we found a significantly higher incidence of cancer in men in the range age 20–50 years and in women over 50 years. Our MS patients who experienced at least two therapeutic switches DMTs had a higher risk for the development of cancers. Moreover, in our multivariate analysis, age, disease duration and more than 2 switches were associated with a higher cancer risk. As described above, data about MS and cancer risk are conflicting with a majority of these evidences suggesting that patients with MS are at reduced or at least not overall increased risk for the development of cancer , 26, 28.Our finding of a higher risk in younger men and older women in our MS population deserves attention. For most MS cohorts reported in the literature, there is no relevant difference in cancer risk between men and women , 29, 30.About cancer site, our data are in agree with the current literature, showing that, although the overall risk for malignancies in the MS patients does not seem to be increased, a possible raising of certain cancers such as breast cancer , 15 cancAn interesting data is the higher incidence of thyroid cancer in our cohort, not confirmed by other reports , 37. It A small but significantly increased risk of breast cancer in patients with MS was reported in a few studies; in detail a 1.6-fold (RR = 1.56) increased risk of breast cancer was found among a cohort of 11,817 MS patients , 41. TheIn our cohort, we demonstrated that the risk of cancer in patients who had received only one DMT was not increased, in line with other studies , 42, 44.Finally, we observed that MS patients with cancer diagnosis had a worse EDSS and got EDSS 4.0 faster compared to other MS patients. Indeed, it has been well-demonstrated that MS course depend not only on disease characteristics but also on several factors including comorbidities, as cancer , 46, 47.Our multivariate analysis showed that age, age at onset, disease duration and >2 switches, are associated with a higher cancer risk . As for Our study had several limitations. Firstly, the restricted area in which it was conducted may lead to a number of cancer cases that is too small to obtain conclusions for each cancer subtype. Moreover, we cannot exclude also the underestimation of the number of cancers due to the possible absence of follow-up or non-registered cancers. Secondly, we did not take into account data about the duration of DMTs exposure, as the cancer risk could be time-dependent. In addition, the intensity and duration of exposure of other factor as alcohol consumption, tobacco use, sun exposure, hormone therapy etc., need to be evaluated in the next studies, as the impact of dose is well-known for carcinogenesis. Moreover, we carried out our study in the period between 2003–2013, when dimethyl fumarate, teriflunomide, cladribine, alemtuzumab, and ocrelizumab were not available yet; hence, this could prevent us to understand the possible impact of these new drugs on carcinogenesis. A recent review showed that for dimethyl fumarate and teriflunomide an increased cancer risk was not demonstrated, whilst the use of alemtuzumab and cladribine require caution because of their potential risk of developing malignancies . HoweverIn conclusion, our results suggest that some MS subpopulations may be more susceptible to the risk of develop cancer. The relationship between MS and risk of cancer is complex- Although the immune-mediated pathogenesis of MS enhancing the immune surveillance due to activation of inflammatory cells , may “prFurthermore, the finding of higher cancers risk in patients who switched at least twice may have significant implications on MS management, influencing treatment decision making process. Indeed, our results raised the question of long-term toxicity of MS drugs, highlighting the importance of taking into account the consequences of a succession exposure to many different molecules, in particular with the development of new MS therapies more and more powerful.More prospective researches are needed via epidemiology and experimental study designs to confirm these data.This study was carried out in accordance with the recommendations of the Ethics Committee of Catania (Catania 1) with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Ethics Committee of Catania (Catania 1) (No. 19/2017/PO).The results described in this paper were partially presented as poster presentation at ECTRIMS meeting, 25th−28th October 2017 Paris, France.ED and CC contributed to data curation, and drafting, reviewing and editing the manuscript. SA contributed to data curation and investigation. AZ, ST, SLF, and DM contributed to data curation. MC contributed to data curation, methodology and formal analysis. SS contributed to data curation and formal analysis. MZ performed conceptualization, supervision and validation. FP contributed to the methodology, supervision, and reviewing, editing and validation of the manuscript.ED received personal fees by Biogen and Sanofi Genzyme. He also received travel funding from Bayer, Biogen, TEVA, Sanofi Genzyme and Merck. CC received personal fees by Sanofi Genzyme. She also received travel funding from Bayer, TEVA, Sanofi Genzyme, Almirall, Novartis, Roche, Biogen, and Merck. AZ received travel funding from Bayer and Sanofi Genzyme. ST received travel funding from Biogen. SL received travel funding from from Bayer, TEVA, Sanofi Genzyme, Novartis, Biogen, and Merck. DM served on the advisory board for Bayer, Biogen, Merck, Novartis, Roche, Sanofi, TEVA, and also received personal fees for speaking activities at congresses or sponsored symposia. MZ has received honoraria for speaking activities by Bayer Schering, Biogen, Merck, Novartis, TEVA and Sanofi Aventis; he also served as advisory board member the following companies: Bayer Schering, Biogen, Merck, Novartis; he received grant for congress participation from Bayer Schering, Biogen Idec, Merck, Novartis, Sanofi Aventis and TEVA. FP has received honoraria for speaking activities by Bayer Schering, Biogen, Merck, Novartis, Roche, TEVA and Sanofi Aventis; he also served as advisory board member the following companies: Bayer Schering, Roche, Biogen, Merck, Novartis; he was also funded by Pfeizer and FISM for epidemiological studies; finally he received grant for congress participation from Bayer Schering, Roche, Biogen, Merck, Novartis, Sanofi Aventis, and TEVA. The remaining 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.


Brassica napus at different stages during induction of embryogenic microspores and development of microspore-derived embryos (MDEs) by using a series of monoclonal antibodies specific for cell wall components, including arabinogalactan-proteins (AGPs), pectins, xyloglucan and xylan. We used JIM13, JIM8, JIM14 and JIM16 for AGPs, CCRC-M13, LM5, LM6, JIM7, JIM5 and LM7 for pectins, CCRC-M1 and LM15 for xyloglucan, and LM11 for xylan. By transmission electron microscopy and quantification of immunogold labeling on high-pressure frozen, freeze-substituted samples, we profiled the changes in cell wall ultrastructure and composition at the different stages of microspore embryogenesis. As a reference to compare with, we also studied in vivo microspores and maturing pollen grains. We showed that the cell wall of embryogenic microspores is a highly dynamic structure whose architecture, arrangement and composition changes dramatically as microspores undergo embryogenesis and then transform into MDEs. Upon induction, the composition of the preexisting microspore intine walls is remodeled, and unusual walls with a unique structure and composition are formed. Changes in AGP composition were related to developmental fate. In particular, AGPs containing the JIM13 epitope were massively excreted into the cell apoplast, and appeared associated to cell totipotency. According to the ultrastructure and the pectin and xyloglucan composition of these walls, we deduced that commitment to embryogenesis induces the formation of fragile, plastic and deformable cell walls, which allow for cell expansion and microspore growth. We also showed that these special walls are transient, since cell wall composition in microspore-derived embryos was completely different. Thus, once adopted the embryogenic developmental pathway and far from the effects of heat shock exposure, cell wall biosynthesis would approach the structure, composition and properties of conventional cell walls.Microspore embryogenesis is a manifestation of plant cell totipotency whereby new cell walls are formed as a consequence of the embryogenic switch. In particular, the callose-rich subintinal layer created immediately upon induction of embryogenesis was recently related to protection against stress. However, little is currently known about the functional significance of other compositional changes undergone by the walls of embryogenic microspores. We characterized these changes in  Upon chromosome doubling, haploid embryos then transformed into doubled haploid (DH) and therefore fully homozygous plants in a single generation, placing this developmental switch in the center of numerous genetic and trait discovery breeding applications. Described more than 50 years ago, a deep mechanistic understanding of the changes undergone by induced microspores is still lacking.The plant kingdom is characterized by a high level of developmental plasticity and totipotency. Microspore embryogenesis is an androgenic form of totipotency in which haploid vacuolated microspores or young pollen grains are Brassica napus, the androgenic switch is induced by a heat stress treatment of at least one day at 32°C  are a type of cell surface glycoproteins enriched in arabinose and galactose residues . They apeggplant . These feggplant . HoweverB. napus in vivo anther development  at the suitable stage for embryogenesis induction, and in vivo maturing pollen grains. To study them, we produced and analyzed a collection of samples of in vivo and in vitro cultured cells processed for transmission electron microscopy (TEM) by high pressure freezing and freeze substitution (HPF-FS), acknowledged as the best procedure to maintain plant cell ultrastructure as close to the native state as possible during in vivo and in vitro processes  were grown in the greenhouses of the COMAV Institute  under controlled temperature conditions and natural light during spring months.4 microspores per ml. Adjusted microspore suspension was distributed in sterile culture dishes. Dishes were incubated in darkness for 24 h at 32°C to induce embryogenesis, and then continuously at 25°C for MDE progression up to the time of sample collection and processing.Flower buds containing mostly vacuolated microspores were selected as previously described . Brieflyin vivo materials, we processed anthers excised from 3.0 mm-long buds, containing microspores at the suitable stage for induction of embryogenesis (vacuolated microspores), and anthers excised from 3.7 mm-long buds, containing mid and mature pollen grains. They, together with B. napus microspore cultures, were processed as described in in vivo anthers and in vitro materials were high-pressure frozen in a Leica EM HPM-100 high-pressure freezer  and transferred to LN2. All samples were freeze substituted using a Leica AFS2 system (Leica Microsystems) with 2% OsO4 in anhydrous acetone at -80°C for 7 days, followed by slow warming to room temperature over a period of 2 days. After rinsing in several acetone washes, they were removed from the holders, incubated in propylene oxide for 30 min, rinsed again in acetone, and infiltrated with increasing concentrations of Epon resin  in acetone according to the following schedule: 4 h in 5% resin, 4 h in 10% resin, 12 h in 25% resin, and 24 h in 50, 75, and 100% resin, respectively. Polymerization was performed at 60° for 2 days. Six to eight resin blocks containing microspore cultures and MDEs at different developmental stages were obtained. Using a Leica UC6 ultramicrotome, thin sections (1 μm) were obtained for light microscopy observation, and ∼80 nm sections were obtained for electron microscopy. Sections were mounted on formvar-coated, 200 mesh copper grids, stained with uranyl acetate and lead citrate, and observed in a Jeol JEM 1010 electron microscope.For the B. napus -β-oligosaccharide segments in (1→3)-β-glucans. For the detection of AGPs we used the following primary antibodies: JIM13, a rat IgM monoclonal antibody (mAb) recognizing the AGP2 epitope present in different plant exudates such as gum Arabic and gum ghatti ; JIM8, aB. napus ; JIM14, B. napus ; JIM16, B. napus . For theB. napus ; LM5, a B. napus ; LM6, a B. napus ; JIM7, aB. napus ; JIM5, aB. napus ; LM7, a B. napus . For theB. napus ; LM15, aB. napus ; For xylB. napus . For mor4-treated, epoxy-embedded samples. This processing is not compatible with immunolocalization of protein epitopes, because OsO4 is likely to alter protein epitopes. However, we demonstrated that this procedure can be successfully used to specifically detect carbohydrate epitopes  onto Formvar and carbon-coated, 200-mesh nickel grids (Electron Microscopy Sciences). Sections were hydrated with PBS during 1 min. Non-specific binding was avoided by applying PBS + 0.2% BSA + 3% skimmed milk for 30 min. Afterwards sections were incubated with the corresponding primary antibody diluted according to Immunogold labeling was performed in HPF-fixed, OsOepitopes , not so 2). This number of particles was determined by hand counting particles over the compartments under study. These compartments, determined a priori, were (1) the cytoplasmic Golgi stacks and vesicles, (2) the cell wall, differentiating between middle lamella and the rest of the cell wall when differences between both regions were detected, (3) the intine for microspores and pollen, (4) the inner walls in embryogenic microspores and MDEs; and (5) the epidermal walls in MDES. As an estimation of background noise, we determined the particle labeling density over regions where the presence of the studied carbohydrates is not expected. These regions included the cytoplasm (excluding the Golgi stacks and Golgi-derived vesicles), cytoplasmic organelles and nucleus. Background noise never exceeded 5%, compared to the quantified signal. The area in μm2 was measured using a square lattice composed of 11 × 16 squares of 15 × 15 mm each.From each developmental stage, three different blocks were randomly selected and micrographs were taken systematically always at the same magnification. Sampling was carried out over a number of micrographs taken randomly from all the cells of interest on each grid. The minimum number of micrographs was determined using the progressive mean test , with a in vivo microspores, in vivo pollen, first dividing embryogenic microspores, 2-4 celled embryogenic structures, multicellular structures, defined as structures with 6–10 cells, pollen-like structures, globular MDEs differentiating between the embryo proper and the suspensor domains, heart-shaped, torpedo and cotyledonary MDEs. Specific differences among these stages were found for JIM13 immunogold labeling. However, the rest of antibodies showed comparable results for embryogenic microspores from the 2 to the 10 cell stages, and also for the embryo proper and suspensor domains of the globular MDE. In these cases, we grouped together the comparable stages for simplicity. For each stage and tissue, labeling density (number of gold particles/μm2) was expressed as the mean labeling density of all micrographs ± SD. Comparisons between mean labeling densities among stages and among tissue and subcellular regions were performed by one-way ANOVA tests using StatGraphics software. Means were separated using a Tukey’s test with p ≤ 0.05. Labeling densities and statistical comparisons were shown as independent histograms for each antibody and tissue. In order to facilitate comparisons between tissues, all the histograms were built using the same Y-axis scale, although in some cases, this led to the occurrence of empty or almost empty charts.Initially, we considered the following stages/cell types for our study: B. napus DH4079 microspore culture system, fresh (in vivo) vacuolated microspores , the vesicles produced by fragmentation of the TGN and, in embryogenic microspores, the growing cell plate  in many cases, including the two  studied . Since ncles/μm2 . During cles/μm2 . JIM8 epcles/μm2 . Althouge grains , the gene grains .in vivo microspore/pollen development, and highly dynamic, changing dramatically as the microspore becomes embryogenic and then develops as an MDE. However, among the AGP epitopes studied, the most abundant by far were those recognized by JIM13, which accumulated principally in walls of in vivo microspores and in vitro embryogenic microspores and MDEs during the first stages of their development.Together, these results demonstrated that the AGP composition of the cell walls produced during microspore embryogenesis is unique, remarkably different from in vivo microspores and pollen, and of embryogenic microspores, but clearly found in the intine of in vitro pollen-like grains  were almost undetectable in any compartment of e grains . During e grains .LM5 epitopes ((1-4)-β-D-galactan residues from rhamnogalacturonan-I) were in general very scarcely found . Very loin vivo micropores and pollen grains (data not shown). Indeed, the labeling density of the intine of pollen-like structures was nearly twice that of embryogenic microspores -α-L-arabinans) were abundantly found in the thickened intine of pollen-like structures  and, to rospores . Howeverrospores  and innerospores  of embryrospores . In innerospores . Accordiin vitro than in in vivo structures, and with levels in pollen-like structures  presented very low levels in ructures . Howeverructures .in vivo and in vitro (embryogenic) microspores was similar, but it differed between in vivo and in vitro pollen grains. We also deduced that the pectin levels of the intine differed in pollen-like and embryogenic microspores. In addition, the first cell walls formed in embryogenic microspores were dramatically different from MDE walls. In embryogenic microspores, the most abundantly detected pectins were those containing arabinan. During MDE development, pectin deposition seemed to be highly dynamic, each of the pectin epitopes studied having a specific temporal and spatial pattern of deposition.Based on these results we concluded that, in terms of pectin composition, the intine of in vivo pollen grains and in vitro pollen-like structures , and pectin epitopes, with the exception of JIM7 epitopes (highly methyl-esterified HG). However, both intines are remarkably different in terms of AGP epitopes. Thus, it seems that the change in developmental fate implies the formation of new walls and the delivery of particular AGPs to both the existing intine and the newly formed inner walls and subintinal layer. This is in line with other observations of different AGP profiles in somatic cells of the same species but with different in vivo and in vitro developmental fates  the formation of new walls with a unique structure and composition, and (2) remodeling of the existing microspore intine. One of the first signs of embryogenesis induction is the er cells . We hereal fates , and wilin vivo maturing pollen grains and in vitro pollen-like grains  are remarkably similar not only in terms of ultrastructure, but also of AGP, pectin and xyloglucan composition, and different from existing walls (the intine) and walls of MDEs. This adds to our previous observations on callose and cellulose composition  the compositional remodeling of the preexisting intine walls of microspores, and (2) the formation of unusual walls with a unique structure and composition. The cell walls formed position . These sposition . Second,position .All datasets generated for this study are included in the manuscript and/or the supplementary files.JS-S performed the sample production and processing and manuscript writing. PC-M performed the immunolabeling and TEM observations. AD did the TEM observations and manuscript revision.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.


Of the tested models, an Exploratory Structural Equation Model (ESEM) fit the data best. Multigroup ESEM and ESEM-within-CFA generally supported the measurement invariance of the questionnaire across groups. Internalizing symptomatology, rumination and low happiness were related mainly to low emotional stability across countries, while low agreeableness and low conscientiousness were related chiefly to externalizing symptomology . Some correlational differences arose across countries and are discussed. Our findings generally support the BFPTSQ as an adequate measure to assess the Big Five personality domains in Spanish- and English-speaking young adults.The present study examined the measurement invariance of the Big Five Personality Trait Short Questionnaire (BFPTSQ) across language (Spanish and English), Spanish-speaking country of origin (Argentina and Spain) and gender groups . Evidence of criterion-related validity was examined via associations  between the BFPTSQ domains and a wide variety of mental health outcomes. College students ( According to a biopsychosocial model of psychopathology, several biological, psychological and social variables have been indicated to impact health outcomes . One of The Five-Factor Model  is one of the most widely accepted structural personality models . The FFMTraditionally, measures to assess personality traits encompass many items and are, therefore, time-consuming. Several shorter alternatives, including the Big Five Questionnaire-Children version BFQ-C; , the BigAmong these short personality measures, the BFPTSQ  has a nuOriginally, the psychometric properties of the BFPTSQ were examined in French-speaking adolescents from Quebec . The FreThe Spanish BFPTSQ was adapted and validated in a sample of Spanish adults . FindingIn addition, although there is currently an increasing demand for short scales, their construction is not exempt of difficulties . FollowiSpecifically, in the present study we: (a) test the BFPTSQ structure in two different Spanish-speaking countries (Argentina and Spain); b) test the structure of the English BFPTSQ version; (c) test the measurement invariance across countries (Argentina vs. Spain), languages (English vs. Spanish) and gender groups; (d) explore the internal consistency of the scales among groups; (e) examine the associations among the five BFPTSQ domains and a large number of psychological constructs  in college students from the USA, Argentina and Spain . We focused on this set of variables because substance use ,23 and mn = 29), and the five cases who failed to report their gender, the final sample included 2158 undergraduate students. College students from one university in Spain, one university in Argentina, and two universities in the USA completed an online survey on personality traits, personal mental health and marijuana use behaviors [for more information see Before the assessment of the participants, the ethic committee of the Universidad de Córdoba and Universitat Jaume I approved the study, as well as the Collaborative Institutional Training Initiative (CITI program) in the USA universities (ID: 21636999 and 21637000).At all the university sites, the participants were administered the questionnaires below.Big Five Personality Trait Short Questionnaire [BFPTSQ; Strongly Disagree, 4 = Strongly Agree): openness, extraversion, agreeableness, conscientiousness and emotional stability. In the present study, all the reversed items were indicated with an r after the item number . Responses were summed on all five scales and divided by the number of their items [Personality traits were assessed with the 50-item  BFPTSQ;  at the U BFPTSQ;  at the sir items . Thus, tnone, not at all, 1 = slightly or rarely, less than a day or two; 2 = mild, several days; 3 = moderately, more than half the days, 4 = severely, nearly every day). A score of 2 or higher in most domains, except substance use (score of 1 or higher), is suggestive of clinically-relevant mental health problems [Past 2-week psychopathology was assessed using the 23-item DSM-5 Self-Rated Level 1 Cross-Cutting Symptoms Measure-Adult . For Spaproblems . The meaproblems  and collproblems  samples.The Cronbach’s alpha of the total scale in the current total sample was .92 . In the case of the subscales with more than one item the Cronbach’s alphas were: depression .78 , mania .55 , anxiety .80 , somatic distress .65 , psychosis .83 , repetitive thoughts and behaviors .76 , and personality functioning .79 .The prevalence rates of potential symptom presentation for the 13 domains  of the DSM-5 Cross-Cutting Symptoms Measure in the whole sample were as follows: depression (34.60%), anger (37.17%), mania (25.40%), anxiety (24.06%), somatic distress (17.51%), suicidal ideation (10.50%), psychosis (4.60%), sleep disturbance (29.55%), memory problems (15.79%), repetitive thoughts and behaviors (12.22%), dissociation (16.60%), and personality functioning (22.48%). For substance use, the rates for specific substance are presented: alcohol use (31.72%), tobacco (24.14%), and illicit drug use (19.20%).2(2) = 26.06, p < .001), anger  = 7.18, p < .05), mania  = 19.70, p < .001), anxiety  = 19.70, p < .001), suicidal ideation  = 7.85, p < .05), psychosis  = 20.60, p < .001), memory problems  = 7.98, p < .05), alcohol use  = 31.62, p < .001), and illicit drug use  = 7.36, p < .05).Significant differences in the prevalence rates of the following symptoms between countries were found: depression . Summing the responses to all the items provides a total score. A previous project undertaken by the research team translated the ABS into and adapted it to English. The preliminary results revealed that the scores for the Spanish and English ABS versions displayed good internal consistency. The various differential item functioning analysis indicated that items generally operate similarly across the three participating countries [Antisocial behavior was assessed with the Antisocial Behavior Scale ABS; . The ABSountries . The Crono, 1 = yes). The total score reflects all the consequences that individuals experienced in the last 30 days. Previous research supports the test-retest reliability, as well as the discriminant and convergent validity, of the B-MACQ [The 21-item B-MACQ was employed to assess negative marijuana-related consequences . All thee B-MACQ , and hase B-MACQ . The CroFrequency of marijuana use was assessed by this question: “How many days in the last 30 days have you used marijuana?” If the participants responded 1 or higher, they completed the marijuana quantity measure. To report the consumed amount of marijuana, the participants were administered a visual guide indicating several amounts of marijuana in grams. Their typical weekly marijuana use in the last 30 days was assessed by the Marijuana Use Grid MUG; . The parNot at all, 7 = Very Well). In Argentina and Spain, the Spanish RTSQ version was utilized [see the translating and adaptation procedures in Rumination was measured by the Ruminative Thought Style Questionnaire RTSQ; . The parCompletely Unhappy to 10 = Completely Happy).One question was about general happiness. The participants had to respond about how happy they felt in general that day (by attempting to ignore any feelings they had yesterday) on a 10-point scale  of the relevant estimates were calculated and reported. Two model types were employed to assess factor validity: the ICM-CFA  and the ESEM  with target loading rotation.All the analyses were done with version 25 of SPSS and version 7.4 of Mplus . The roba priori correlated uniquenesses, employed to reflect that some items relate to the same primary trait or subdomain, and they share either a similar content, but reversed scores, or contain the same word. Twenty-seven a priori correlated uniquenesses were posited. Specifically, the correlated uniquenesses introduced for openness were: 1 with 21, 11 with 36, 16 with 21, 26 with 41r, 26 with 46, 1 with 16, 41r with 46; for extraversion: 7r with 32r, 2 with 22r, 12 with 42, 2 with 27, 17 with 27; for agreeableness: 18 with 23, 8 with 33, 23 with 33, 23 with 43, 18 with 43; for conscientiousness: 29 with 39, 19r with 24r, 19r with 39, 9r with 19r, 4 with 14; and for emotional stability: 10 with 35, 10 with 15r, 5r with 25, 5r with 45r, 30r with 50r. A detailed description of the conducted ESEM and ICM-CFA models can be found in Morizot [In line with Marsh et al.  and MoriThe model fit assessment was made according to various indices . The chiAfter identifying the best factor model, factor structure was tested in each country, and measurement invariance was tested between the Spanish-speaking groups (Argentina and Spain), the Spanish and English versions, and across gender groups using multi-group ESEM. These models were assessed with a series of increasingly stringent multiple-group models (see ): configIf there is evidence for noninvariance of the factor loadings across groups, as partial factor loading invariance cannot be tested in ESEM ,45, an ETo assess changes in the model fit tests, the Satorra-Bentler scaled chi-square test  was compIn both the Spanish and English questionnaire versions, sources of reliability were explored by resorting to Cronbach’s alphas and ordinal omegas . The souds were below .50).When studying the BFPTSQ structure in the total sample, the best fitting model was the ESEM model, in which correlated uniquenesses were allowed (M2b). See the fit indices of all the models performed in Upon finding the best factor solution, an ESEM was performed in each country. Fit indices were acceptable for the Spanish sample. In the Argentinian sample, the CFI and TLI were close, but lower than .90. However, the RMSEA, RMSEA 90% CI values and SRMR were adequate (≤.05). In both samples, factor loadings were salient and significant in its hypothesized factor, except for items 31 and 41 in the openness to the experience factor see . All theThe fit indices of the English version were adequate. The factor loadings of the English version are presented in A few minor differences emerged across groups when studying the invariance of the Spanish questionnaire version between the Argentinian and Spanish participants. Constraining the intercepts across Spanish speakers resulted in a ΔRMSEA below .015, and ΔCFI was -.017 (MG3). Hence a model with a partial invariance of intercepts (MG3b) was estimated. Based on the modification indices, four items across groups were freed: 13r (“provokes quarrels or arguments with others”), 38r (“can sometimes be rude or mean to others”); 43 (“likes to cooperate with others”) (Arg > Sp) from the agreeableness factor; 49r (“can do things impulsively without thinking about the consequences”) (Arg > Sp) from the conscientiousness factor. This model gave a better fit than the model with the fully invariant intercepts and ΔCFI ≤ .01. When further constraints were included (MG4 to MG7), ΔCFI was ≤ .01 and ΔRMSEA was ≤ .015, which suggested reasonable invariance across groups. Considering that the structure of the Spanish BFPTSQ had been previously studied [To test the measurement invariance of the English and Spanish (Spanish and Argentinian combined samples) versions, a configural invariance model was performed (MG1). This model showed acceptable fit indices as it can be seen in The results of the invariance analyses done across gender are also presented in The correlations between personality domains and criterion variables in all three countries are presented in SD = .055) across 330 possible comparisons. The results were interpreted using the following: differences <1 SD were small, differences between 1 SD and 2 SD were medium, those between 2 SD and 3 SD were large, and any over 3 SD were substantial. Results presented in In order to determine if personality dimensions were related differentially to distinct criterion variables across countries, the absolute value of the differences in the magnitude of the correlations for pairs of countries was computed and is presented in The present study examined different sources of validity of the English and Spanish versions of the BFPTSQ ,17 in coNot surprisingly, when considering previous work that have examined complex structures such as the Big Five ,17,39, tOur findings also indicated an improved model fit when correlated uniquenesses were allowed. Despite including the correlated uniquenesses between the items that were reversed-coded within the same factor or shared the same words being conceptually defensible and increasing the model’s fit, they also inevitably reduced the size of the factor loadings as factors had less variance left to explain. This was salient for the openness factor, which allowed seven correlated uniquenesses and provided lower factor loadings. However, it was noteworthy that the ESEM model’s fit was acceptable even when correlated uniquenesses were allowed, but was still far from excellent according to the typical criteria suggested for practical fit indices . Morin eAll the items presented significant factor loadings on their intended target factor, except items 31  and 41 (“has few artistic interests”) of the openness factor (in both the whole sample and the Spanish- and English-speaking samples). A previous study conducted with a general Spanish population sample indicated the primary factor loadings of items 31 and 41 respectively on the openness factors of .38 and .58. The factor loadings for French-speaking  were alsWith the extraversion dimension, all the items showed salient factor loadings on their intended factor , except for item 42 (“likes exciting activities that provide thrills”). This sensation seeking-related item had a factor loading of .27 on extraversion, and a factor loading of .38 on openness. Previous studies have indicated that sensation seeking tends to be openness-related ,54.All the emotional stability and conscientiousness items showed salient factor loadings on their intended factor, but some items in the agreeableness dimension also cross-loaded on the openness factor. As expected, the reverse-coded items that indicated antagonism or low agreeableness loaded on the agreeableness factor, while the positively worded items of this domain cross-loaded on the openness factor. Future revisions of the scale should consider these findings, and the fact that the use of positively worded items and reversed forms in the same scale  to reduce response bias has been questioned. Suárez-Alvarez et al.  illustraIn line with previous research, our findings supported the measurement invariance of the BFPTSQ across gender groups . The preOur measurement invariance results across languages  revealed that all the agreeableness items loaded primarily on their intended factor in the Spanish-speaking sample. However, in the English-speaking sample, the positively-worded agreeableness items had similar factor loadings on the agreeableness and openness factors as it was shown in The addition of constraints between intercepts only led to a few noninvariant intercepts across languages. Spanish speakers tended to obtain higher scores than English-speakers for one conscientiousness item tapping the self-discipline facet (4) and for two openness items tapping the intellectual inquisitiveness facet (11 and 36). Compared to the Spanish-speaking participants, English speakers scored higher for: a) two extraversion items tapping the expressiveness (22r) and sociability (32r) facets; b) one for the agreeableness item tapping the compassion facet (28r); c) one conscientiousness item tapping the order facet (9r); and d) one emotional stability item tapping the worry facet (20r). Although a few intercepts and noninvariant loadings were observed across groups based on CFI differences, the RMSEA differences still suggested that the model was completely invariant across languages. The noninvariance of some intercepts and loadings was based mainly on the proposed typical criteria of changes in the fit indices. Nevertheless, these criteria are rough guidelines  and someThe present study aimed to examine the association between the BFPTSQ scores with a wide diversity of outcome variables by particularly focusing on substance use (or substance-related variables) and poor mental health outcomes. As already noted, these behaviors are highly prevalent in college students around the world and a valid, yet brief, version will most likely facilitate both cross-cultural studies and routine interventions to detect students at high risk of developing substance use and/or mental health problems. As in previous studies, low emotional stability, low agreeableness, low conscientiousness and low extraversion were related to mental health outcomes ,60. InteSD and 3 SD above the average difference in the magnitude of the correlations) was between conscientiousness and marijuana-related problems in the USA and Argentina. Lack of a significant negative association between marijuana-related problems and conscientiousness in the Argentinean sample was somewhat unexpected, as in the case of low conscientiousness and low agreeableness with alcohol, tobacco and illicit drug use. Future research should replicate this finding to know if the disinhibition-related domains assessed within the FFM framework could influence drug outcomes in Argentinian college students.Other externalizing behaviors, such as drug use, also showed significant correlations with low agreeableness and low conscientiousness, as in previous meta-analysis ,61, at lIn line with previous studies conducted using the BFPTSQ and other measures to assess the FFM, happiness was mainly related to both emotional stability (or low neuroticism) and extraversion ,66,67, wOur research is not without its limitations. First, even though the BFPTSQ’s psychometric properties have been previously explored in a Spanish population , this isDespite its limitations, the present research supports the BFPTSQ’s factor validity, the reasonable invariance of the measure across genders, across two Spanish-speaking populations, and between Spanish and English speakers. It also evidences the scales’ reliability and criterion validity . Taken together, our results suggest the BFPTSQ is a useful short measure for assessing the FFM broad domains between English and Spanish speakers, at least for young adults from the USA, Argentina and Spain.S1 TableJournal of Personality and Social Psychology, 75, 729–750; and Morizot, J. (2014). Construct validity of adolescents’ self-reported big five personality traits: importance of conceptual breadth and initial validation of a short measure. Assessment, 21, 580–606.A) English, B) Spanish. In bold are marked the items that are identical in both questionnaires. The items can also be found in: Benet-Martinez, V., & John, O. P. (1998). Los Cinco Grandes across cultures and ethnic groups: Multitrait multimethod analyses of the Big Five in Spanish and English. (DOCX)Click here for additional data file.S2 TableThe columns “Argentina” and “Spain” correspond to the factor loadings of the M2b model see . Bold de(DOCX)Click here for additional data file.