M61RXrcc4 separation of function mouse line to overcome the embryonic lethality of Xrcc4-deficient mice. XRCC4M61R protein does not interact with Xlf, thus obliterating XRCC4-Xlf filament formation while preserving the ability to stabilize DNA ligase IV. X4M61R mice, which are DNA repair deficient, phenocopy the Nhej1-/- (known as Xlf -/-) setting with a minor impact on the development of the adaptive immune system. The core non-homologous end-joining (NHEJ) DNA repair factor XRCC4 is therefore not mandatory for V(D)J recombination aside from its role in stabilizing DNA ligase IV. In contrast, M61RXrcc4 mice crossed on Paxx-/-, Nhej1-/-, or Atm-/- backgrounds are severely immunocompromised, owing to aborted V(D)J recombination as in Xlf-Paxx and Xlf-Atm double Knock Out (DKO) settings. Furthermore, massive apoptosis of post-mitotic neurons causes embryonic lethality of M61R -Nhej1-/-Xrcc4 double mutants. These in vivo results reveal new functional interplays between XRCC4 and PAXX, ATM and Xlf in mouse development and provide new insights into the understanding of the clinical manifestations of human XRCC4-deficient condition, in particular its absence of immune deficiency.We developed an  Lig4-/- and Xrcc4-/- mice suffer late embryonic lethality caused by apoptosis of post-mitotic neurons (Lig4 gene (Living organisms face DNA double-strand breaks (DSBs), the most toxic DNA lesions, from random or programmed (prDSBs) origins , such as neurons . Likewisig4 gene .Trp53-/-  J recombination defect in combined deficient settings , owing to aborted V(D)J recombination both in humans and animal models . NHEJ deTrp53-/- . Xlf/Cercy (CID) . Howevercy (CID)  or murincy (CID)  settingssettings  as well settings . Consistymphomas .bona fide NHEJ core complexes, the structure of which was recently resolved through cryoelectron microscopy (cryo-EM)  . The V models both in vivo and in vitro J recombination may not always strictly coincide with what we know for the repair of genotoxic DNA breaks, precisely because of the existence of the ‘two-synapses’ mechanism.XRCC4 is a keystone factor, playing two independent roles. First, X4 stabilizes Lig4 through its C-terminal coiled-coil domain . Indeed,in vitro . In addiin vitro . X4-Xlf in vitro . DNA-endin vitro . Severalin vitro . These sXrcc4-/- mice phenocopy Lig4-/- condition, with an embryonic lethality and SCID phenotype  mouse model harboring the M61R missense mutation that abrogates X4-Xlf interaction J recombination, and to expand the picture of NHEJ apparatus in brain and lymphocyte development.Since X4 is compulsory for Lig4 stabilization, henotype . The emb remains . Furtherymphomas . To avoieraction , while kM61R substitution, thus resulting in the loss of X4-Xlf filament (M61RXrcc4 KI mouse model through CRISPR/Cas9 (M61R/M61RXrcc4 mice (M61RXrcc4 mice) were viable arguing against any harmful impact of the M61R substitution during embryonic development, as opposed to the embryonic lethality of Xrcc4 KOs.X4 and Xlf interact through a hydrophobic interface, which is disrupted by the X4filament . We deveSPR/Cas9 . HomozygXrcc4 KO mouse model . At the protein level, X4 expression was barely detectable in spleen, brain, thymus, and mouse embryonic fibroblasts (MEFs) from M61RXrcc4 mice as opposed to their homozygous WT counterparts . The thymocyte maturation program, identified through the various CD4/CD8 stages, was similar in M61RXrcc4 and WT littermates  (Nhej1-/- mice (81.1% ± 1.4 SEM) as described (M61RXrcc4 thymocytes presented an increased apoptosis (7AAD+/AnnV+), similar to that of Nhej1-/- thymocytes   , a traitescribed . Purifie-/-, ns) . This waymocytes . Lastly,ymocytes , with thM61RXrcc4 mice . However, the proportions of immature B cells were decreased in M61RXrcc4 mice .The proportion of mature B lymphocytes in the bone marrow (BM) was not significantly altered in 61R mice , while s61R mice , close tM61RXrcc4 mice, despite the reduced expression of X4M61R. The minor accumulation of DN3A thymocytes and immature B cells suggests a modest decreased V(D)J recombination efficiency, supported by the bias in TCRα usage. This is in sharp contrast with Xrcc4-/- and Lig4-/- mice, which present a complete arrest of lymphocyte maturation and a SCID phenotype. We conclude that when Lig4 is stabilized, the contribution of X4 through Xlf/X4 interaction, disrupted in the context of X4M61R, is not critical for V(D)J recombination in vivo.These results indicate that B- and T-lymphocyte development is not severely affected in M61RXrcc4 onto Paxx-/- and Atm-/- mice (hereafter denoted as M61R-PaxxXrcc4 and M61R-AtmXrcc4). Both double mutant mice were viable but exhibited severe growth retardation and facial dysmorphia as a consequence of either the X4M61R mutation and/or the overall reduction in X4/L4 expression (We previously proposed a model of ‘double DNA repair synapse’ during V(D)J recombination according to which the X4-Xlf ‘filament’ and the RAG1/2 PCC, together with ATM and PAXX, provide two complementary means of DNA end synapsis . To addrpression .M61R-PaxxX4 and M61R-AtmXrcc4 mice , and previously described Atm and Paxx single KO conditions  , which was significantly greater than the one observed for M61RXrcc4 (81.77 ± 1.4 SEM) or Nhej1-/- (81.1 ± 1.4 SEM) animals  and Vβ10-Dβ2Jβ2 (lower panel) rearrangements of the Tcrb locus were present in thymocytes from M61RXrcc4, Atm-/-, and WT mice , as also previously observed in Paxx-/- mice (M61R-PaxxXrcc4 and M61R-AtmXrcc4 thymus.The thymus and spleen cellularity was severely reduced in both Atm mice  as companditions . B cellsaxx mice . B220+ BM in WT) . Likewis animals . The immDKO mice  and sugg-/- mice , they weM61RXrcc4 crossed on Paxx-/- or Atm-/- phenocopy the SCID phenotype of Nhej1-Paxx and Nhej1-Atm DKO conditions owing to impaired V(D)J recombination. Therefore, aside from its essential role in Lig4 stabilization, X4 does participate in DNA coding-end tethering through its interaction with Xlf during V(D)J recombination, and PAXX/ATM are compensatory factors for this later function in vivo, as they are for Xlf. Alternatively, one may consider that the Xlf function being impaired in the context of X4M61R, PAXX/ATM complements this defect as already established.We conclude that M61R in vivo, we introduced M61RXrcc4 on Nhej1-/- background. No M61R-Nhej1-/-Xrcc4 double homozygous mice (hereafter named as M61R-Nhej1Xrcc4) could be recovered out of 166 newborns obtained from various genotype combinations, when 23 were expected , with the rearrangement of the Igh locus and the intracellular expression of Ig-μH chains in CD19+ B220+ CD43+ Pro B cells, which is used as a proxy to evaluate Igh rearrangement completion , which contrasted with the slight reduction observed in M61RXrcc4 and Nhej1-/- mice  . In contrast, M61R-Nhej1Xrcc4 thymocyte development was severely compromised from the DN3A stage on when compared to M61RXrcc4 and Nhej1-/- , and DP thymocytes or mature T cells were virtually absent from these thymii  , arguinge thymii . Dβ2-Jβ2ound E13 , were un-/- mice , furtherM61R-Nhej1Xrcc4 embryonic development phenocopy Nhej1-Paxx DKO, with a profound defect in V(D)J recombination and the apoptosis of post-mitotic neurons leading to embryonic lethality.Altogether, Xrcc4 separation of function allele in mice, which harbor the M61R substitution previously described to abolish X4-Xlf interaction J recombination activity in these mice. Therefore, although X4 is required for V(D)J recombination through Lig4 stabilization, it is not mandatory for coding-ends tethering during V(D)J recombination. These results confirm that X4 exerts a Lig4-independent function through its interaction with Xlf that is decisive for the repair of genotoxic-induced DSBs but compensated for by other DNA repair factors, and possibly RAG2 itself as shown for Xlf J recombination, as noticed in Nhej1-Paxx and Nhej1-Atm DKO. We conclude that PAXX and ATM are compensatory factors for X4M61R during V(D)J recombination. This result further supports our previously proposed model of ‘double DNA repair synapse’ in V(D)J recombination, mediated by the X4-Xlf and RAG2-PAXX-ATM axes, respectively J recombination. This study also unravels novel interplays between X4, PAXX, ATM, and Xlf during development of the brain and the immune system.Altogether, the XRCC4 gene have been reported in humans  complex microinjection in C57BL/6J mouse zygotes pronuclei as described (Xrcc4 exon 3-specific gRNA (5′-GTACCAGCTGCTCCTGACCC-3′) was designed using Crispor (http://crispor.tefor.net/oligonucleotide). The HR template was the ssODN , and 1X protease inhibitor cocktail EDTA free (Roche) on ice for 45 min. Proteins migrated within Tris-Acrylamide gels and Tris-Glycine-SDS buffer and were transferred on PVDF membranes (Immobilon). Membranes were stained with the following antibodies: β-actin , XRCC4 , and DNA ligase IV . Membranes were revealed with appropriate secondary antibodies conjugated with infrared 700 or 800 dyes (LiCor). Immunoblotting was revealed by LiCor CLx analyzer and analyses were performed on Image Studio Lite software.TTGGGCGCATCGGTTTATCT-3′ R1 5′-GCTGCTAAGTTGAAAGCCTG-3′ F2 5′-TGCTTCTGAACCCAACGTACC-3′ R2 5′- AGGTGCTCGTTTTTGGCTTG-3′. F1-R1 PCR amplifies full-length transcript from 5′UTR to 3′UTR (1150 bp) and F2-R2 amplifies exon 2 to exon 4 (400 bp).Total RNAs were obtained from thymocytes with Pure Like RNA mini kit (Invitrogen) and used for RT-PCR . X4 transcripts were amplified with primers F1 5′-Cell phenotyping from 6- to 9-week-old mice was performed on thymus, spleen, and bone marrow using the following antibodies: CD4, CD8, CD25, CD28, CD44, CD69, B220, CD19, CD43, and IgM . Intracellular IgM expression in E18.5 FL cells was performed as previously described  using CDFor phleomycin sensitivity assay, 5000 MEF cells were seeded in triplicates and cultivated with increasing doses (0–300 ng/mL) of phleomycin. Living cells were counted by flow analysis with FACS LSR-Fortessa X-20 after 6 days of culture. Radiation sensitivity of T lymphocytes was performed as described . Ex vivoTaqMan PCR was performed on triplicates of 8 ng of reverse-transcribed RNA from freshly dissected total thymus as described .PCA and HCPC functions of the FactomineR package, respectively  ends (5′RACE) PCR/NGS  from total thymus RNA as described . SequencTcrb rearrangements were analyzed by PCR on genomic DNA from total adult thymocytes or E18.5 fetal thymocytes as described , we determined that a minimum of 47 newborns was necessary to conclude to an embryonic lethality of the M61R-Nhej1-/-Xrcc4 double KO with a power of 80% and a p-value < 0.05.Non-parametric Mann–Whitney test or two-tailed Fisher’s exact test was performed with α-risk = 0.05. p-Values were taken to be significant: *significant 0.05≥p>0.01; **very significant 0.01≥p>0.001; ***highly significant 0.001≥p>0.0001; ****highly significant p≤0.0001. Using G*Power software ; Karla Rodgers (Reviewer #2).Thank you for submitting your article "A XRCC4 mutant mouse, a model for human X4 syndrome, reveals interplays with Xlf, PAXX, and ATM in lymphoid development" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:1) Provide a clearer and more detailed introduction to models of functions of XRCC4, XLF etc in end joining that highlights the fundamental question(s) being addressed via generation of the mouse model described in this manuscript.2) Both reviewers list several inconsistencies in the figures, such as labeling of axes, percentages of cell populations, splenic cellularity in certain mutant mouse lines, scales in Figure 4, mean/standard deviations etc. these should be carefully checked and made uniform throughout the manuscript.3) The protein levels of X4 and Lig4 in the double knock-out mice/embryos should be shown.4) The Xlf-/- result is not shown in Figure 3E. Either the relevant citation needs to be included on this line, or the Xlf-/- result needs to be shown in Figure 3E.The detailed comments from the reviewers are also appended below for providing context.Reviewer #1 (Recommendations for the authors):XRCC4 deficient mouse model exhibits late embryonic lethality and defective lymphogenesis. However, no clinically significant immunodeficiency was observed in human patients with XRCC4 (X4) hypomorphic mutations, even in those with no detectable X4 in the fibroblasts. The contradictory phenomena observed in mouse and human make the roles of X4 in lymphocyte development unclear. Roch et al., developed a unique X4 mutant mouse model and investigated the roles of X4 in V(D)J recombination. The authors first generated a viable X4M61R knock-in mouse model using CRISPR/Cas9 technique. The M61R mutation disrupts the interaction between X4 and XLF, but not the interaction of X4 and LIG4. The expression of X4 protein is severely inhibited in the homozygous X4 M61R/M61R (X4M61R) mice, while LIG4 can be partially stabilized by this mutant. The separation-of-function of this mutant could help identify the functions of Lig4 stabilizing activity of X4 and the roles of X4-XLF interaction. The authors further show that MEFs from X4M61R mice show mild sensitivity to IR and phleomycin, and X4M61R does not severely affect B- and T-lymphocyte development in mice. The authors therefore suggest that the X4-XLF interaction is not critical for V(D)J recombination in vivo. The authors further investigated the functional redundancies between PAXX, ATM, and X4 in lymphoid development by generating X4M61R-PAXX-/- and X4M61R-ATM-/- double mutant mice. The X4M61R-PAXX-/- and X4M61R-ATM-/- double mutant mice exhibit impaired V(D)J recombination, causing SCID phenotype. The authors further suggest that PAXX and ATM are compensatory factors of X4M61R in immune development. The authors also introduced X4M61R on XLF-/- background mice. They found that X4M61R-XLF-/- mice experience late embryonic lethality caused by massive neuronal apoptosis, and exhibit defect in V(D)J recombination.1. In the Introduction section, the authors propose their idea of a "double DNA repair synapsis" model for repair of broken ends specifically caused by RAG1/2 complex during V(D)J recombination. However, the synapsis model is not clear here. The authors might want to provide more details about the model in this section, so readers could easily understand the model. The authors suggest that RAG2, PAXX, and ATM signaling mediate the first synapse to stabilize the RAG-cut broken ends; XRCC4-XLF filaments mediate the second synapse to tether the broken ends. The "two synapsis" model was proposed based on the observations of functional redundancy in V(D)J recombination between XLF and RAG2, PAXX, and ATM kinase activity. In the current manuscript, the authors seek to understand the contributions of XRCC4 to this model. However, the "two synapse" model is not very consistent with other studies. PAXX and XLF were reported to stabilize the synaptic complex of broken ends by interacting with Ku70/Ku80 complex . This suggests that PAXX and XLF have overlapping functions in mediating end synapsis, and this does not mean PAXX and XLF are within different synaptic states (first synapse and second synapse). Moreover, if XRCC4 and XLF are in the same "second synapse" pathway, X4M61R-XLF-/- double mutant mice then should perform V(D)J recombination to some extent; this would lead to at least modest lymphocyte development, because the "first synapse" by either RAG2 or ATM, or PAXX could compensate for the "second synapse" as suggested by the authors. But X4M61R-XLF-/- mice reported by the authors are actually immune deficiency. The interaction of XLF and XRCC4 is indeed important for end synapsis, but it does not rely on the XRCC4-XLF filament. While the results here are interesting, the authors might need to rephrase the Introduction and frame the scientific question of this manuscript in a way that reconciles the above contradictions.2. The authors conclude that PAXX/ATM are compensatory factors for X4 in immune development based on the SCID phenotype observed in X4M61R-PAXX-/- and X4M61R-ATM-/- double mutant mice. However, the results may also suggest that PAXX/ATM are compensatory factors for XLF but not directly for X4, because M61R mutation disrupts the interaction of X4 and XLF, which might result in XLF being unable to function in the X4M61R mice.3. In lines 69-71, the authors cite Wang's paper to support the roles of XRCC4-XLF filaments in broken DNA end synapsis. However, in the Wang paper, XRCC4 and XLF are suggested to play roles mainly at the ends of the DNA  rather than through a XRCC4-XLF filament structure. Moreover, the statement of "X4 and PAXX binds DNA broken ends and stimulates X4-XLF DNA ends tethering in vitro" is not true. PAXX alone cannot bind dsDNA . It is true PAXX can stimulate end-to-end synapsis and ligation . The stimulation by PAXX is through the interaction with Ku70/80, but not through the XRCC4-XLF filament.4. The authors need to carefully examine their figures and related labels and legends to make sure they are correct. For example, one label is missing on Figure 1D; the x-axis labels of Figure 1G are incorrect; Figure 1 legend, line 134, "X4M61R vs X4-/- (bottom)" is inconsistent with those shown on Figure 1G.5. The percentages of specific cell populations are shown on some flow cytometry panels but not on others. The ratios of the cell populations would make the flow cytometry data easier for readers to understand.6. In line 164, the complete loss of viability was caused by 2 but not 1 Gy as shown on Figure 1G.7. In line 228, the cellularity of X4M61R single mutant littermates in the spleen is missing.8. The protein levels of X4 and Lig4 in the double knock-out mice/embryos could provide more information about the roles of X4 in lymphocyte development.9. In the Introduction, the authors should also mention and consider the substantial literature supporting end-to-end synapsis models, which involve XLF interaction with XRCC4 and Ku. Both end-to-end synapsis and X4-XLF filament formation may be occurring.10. The authors should consider that IR  produces ~30 DSB per cell, whereas during V(D)J in T or B cells, there are likely to be only a few DSB per cell. One possible explanation for the difference between DNA repair roles and V(D)J is the number of events per cell and titration out of the XLF and X4 repair factors. Also, the chemistry and structure of the DNA ends for IR (and for phleomycin) are different than for V(D)J coding ends. The authors may want to consider that this could be an important reason for the difference in repair versus V(D)J for the role of X4.Reviewer #2 (Recommendations for the authors):XRCC4 (X4), a core component of the NHEJ DNA double strand break (DSB) repair pathway, functions to stabilize DNA Ligase IV (Lig4). X4 also associates with the XRCC4-like factor (Xlf) to form filaments that bridge and tether broken DNA ends. Both X4-/- and Lig4-/- mice show late embryonic lethality with massive neuronal apoptosis, as well as complete defects in V(D)J recombination. It was not known the extent that X4, aside from Lig4 stabilization, contributed to NHEJ in general DNA repair versus repair of programmed breaks during V(D)J recombination. To delineate differing functions of X4, a knock-in X4(M61R) mouse line was generated, where the M61R mutation was previously shown to disrupt interaction with Xlf while maintaining and stabilizing Lig4. A potential weakness of this model system is the reduced levels of X4(M61R) and Lig4 expression levels. Nevertheless, the overall results from this well-designed study are largely consistent with the authors' model and conclusions. Through combined genetic, immunological, and biochemical characterizations, the authors' illustrated three main points regarding X4 contributions to NHEJ. First, unlike X4-/-, X4(M61R) mice were viable with Lig4 stably expressed. Notably, while isolated X4(M61R) mouse cells showed deficiencies in repair of exogenously produced DNA DSBs, there were only relatively modest defects in B and T lymphocyte development, a marked contrast to that found with X4-/- mice. Second, The X4(M61R) mice phenocopy Xlf-/- mice by showing only minor defects in lymphocyte development, but with severe defects in V(D)J recombination when crossed with ATM or PAXX deficient mice. These results are consistent with the authors' model in which a "double DNA repair synapse" tethers the DNA ends in V(D)J recombination in a compensatory manner with one synapse mediated by the X4-Xlf filament and a separate synapse mediated by ATM signaling, PAXX, and the C-terminal region of RAG2. In a third point, X4(M61R) crossed with Xlf-/- mice showed late embryonic lethality due to post-mitotic neuronal apoptosis, with severe defects in V(D)J recombination in fetal hepatocytes and thymocytes. This latter point suggests that X4-Xlf filament cannot be compensated by a second DNA repair synapse in the fetal environment, perhaps due to the low expression of X4M61R. Alternatively, Xlf may function in an, as yet, unknown manner to rescue DNA repair defects of X4(M61R). In summary, using a novel separation of function X4 mutant, this study demonstrates that aside from its role in stabilizing Lig4, X4 is not requisite for B and T lymphocyte development likely due to functional redundancy of DNA end tethering in V(D)J recombination. Significantly, these results help to explain how patients with hypomorphic X4 mutations present with clear signs of DNA repair deficiency, but with no evident defects in development of the adaptive immune system.This paper describes the development and characterization of a XRCC4 mutant mouse model, which helps to elucidate separate functions of X4 in NHEJ. Overall, this well-presented work demonstrates novel insights into the interplay between different NHEJ factors. Essential revisions:1) Provide a clearer and more detailed introduction to models of functions of XRCC4, XLF etc in end joining that highlights the fundamental question(s) being addressed via generation of the mouse model described in this manuscript.The Introduction was rephrased and reorganized as requested. We added in particular 3 references of recently published articles describing the cryo-EM structure of the NHEJ core complex, thus improving our knowledge on DNA synapsis during NHEJ mediated DNA repair. We hope it increased the understanding of the scope of our study and readability of the manuscript.2) Both reviewers list several inconsistencies in the figures, such as labeling of axes, percentages of cell populations, splenic cellularity in certain mutant mouse lines, scales in Figure 4, mean/standard deviations etc. these should be carefully checked and made uniform throughout the manuscript.We carefully checked the Figures and Figure legends for mistakes and inconsistency.3) The protein levels of X4 and Lig4 in the double knock-out mice/embryos should be shown.Unfortunately, we will not be able to address this issue in a reasonable time frame. Because of the successive lockdown due to covid19 pandemic, we have been asked to sacrifice most of our living animals. Since the study was essentially finished and the manuscript was being prepared we had no choice but kill the mice related to this project.4) The Xlf-/- result is not shown in Figure 3E. Either the relevant citation needs to be included on this line, or the Xlf-/- result needs to be shown in Figure 3E.Citation has been added.The detailed comments from the reviewers are also appended below for providing context.Reviewer #1:XRCC4 deficient mouse model exhibits late embryonic lethality and defective lymphogenesis. However, no clinically significant immunodeficiency was observed in human patients with XRCC4 (X4) hypomorphic mutations, even in those with no detectable X4 in the fibroblasts. The contradictory phenomena observed in mouse and human make the roles of X4 in lymphocyte development unclear. Roch et al., developed a unique X4 mutant mouse model and investigated the roles of X4 in V(D)J recombination. The authors first generated a viable X4M61R knock-in mouse model using CRISPR/Cas9 technique. The M61R mutation disrupts the interaction between X4 and XLF, but not the interaction of X4 and LIG4. The expression of X4 protein is severely inhibited in the homozygous X4 M61R/M61R (X4M61R) mice, while LIG4 can be partially stabilized by this mutant. The separation-of-function of this mutant could help identify the functions of Lig4 stabilizing activity of X4 and the roles of X4-XLF interaction. The authors further show that MEFs from X4M61R mice show mild sensitivity to IR and phleomycin, and X4M61R does not severely affect B- and T-lymphocyte development in mice. The authors therefore suggest that the X4-XLF interaction is not critical for V(D)J recombination in vivo. The authors further investigated the functional redundancies between PAXX, ATM, and X4 in lymphoid development by generating X4M61R-PAXX-/- and X4M61R-ATM-/- double mutant mice. The X4M61R-PAXX-/- and X4M61R-ATM-/- double mutant mice exhibit impaired V(D)J recombination, causing SCID phenotype. The authors further suggest that PAXX and ATM are compensatory factors of X4M61R in immune development. The authors also introduced X4M61R on XLF-/- background mice. They found that X4M61R-XLF-/- mice experience late embryonic lethality caused by massive neuronal apoptosis, and exhibit defect in V(D)J recombination.1. In the Introduction section, the authors propose their idea of a "double DNA repair synapsis" model for repair of broken ends specifically caused by RAG1/2 complex during V(D)J recombination. However, the synapsis model is not clear here. The authors might want to provide more details about the model in this section, so readers could easily understand the model.The Introduction was rephrased and reorganized to take into account these comment. We hope it increased the understanding of the scope of our study and readability of the manuscript.The authors suggest that RAG2, PAXX, and ATM signaling mediate the first synapse to stabilize the RAG-cut broken ends; XRCC4-XLF filaments mediate the second synapse to tether the broken ends. The "two synapsis" model was proposed based on the observations of functional redundancy in V(D)J recombination between XLF and RAG2, PAXX, and ATM kinase activity. In the current manuscript, the authors seek to understand the contributions of XRCC4 to this model. However, the "two synapse" model is not very consistent with other studies.Nat. Com. 2016 and Betermier et al., TCB 2020). We believe it's the reason why it may appear sometimes inconsistent with purely DNA repair studies performed in vitro or in cellulo following genotoxics. The most striking inconsistency being the fact that although Xlf is a critical NHEJ factor as shown by the impressive radiosensitivity of Xlf deficient cells, the consequence on V(D)J recombination is almost null in contrast to what was well established for most NHEJ deficiencies.As we mentioned in the introduction, the "two synapse" model is a strict specificity of the V(D)J recombination process (as opposed to random genotoxic-induced DSBs) because of the presence of the R1/2 post cleavage complex remaining on broken DNA ends as we proposed in our previous papers . This suggests that PAXX and XLF have overlapping functions in mediating end synapsis, and this does not mean PAXX and XLF are within different synaptic states (first synapse and second synapse). Moreover, if XRCC4 and XLF are in the same "second synapse" pathway, X4M61R-XLF-/- double mutant mice then should perform V(D)J recombination to some extent; this would lead to at least modest lymphocyte development, because the "first synapse" by either RAG2 or ATM, or PAXX could compensate for the "second synapse" as suggested by the authors. But X4M61R-XLF-/- mice reported by the authors are actually immune deficiency. The interaction of XLF and XRCC4 is indeed important for end synapsis, but it does not rely on the XRCC4-XLF filament. While the results here are interesting, the authors might need to rephrase the Introduction and frame the scientific question of this manuscript in a way that reconciles the above contradictions.The Introduction was rephrased and reorganized. We hope it increased the understanding of the scope of our study and readability of the manuscript.2. The authors conclude that PAXX/ATM are compensatory factors for X4 in immune development based on the SCID phenotype observed in X4M61R-PAXX-/- and X4M61R-ATM-/- double mutant mice. However, the results may also suggest that PAXX/ATM are compensatory factors for XLF but not directly for X4, because M61R mutation disrupts the interaction of X4 and XLF, which might result in XLF being unable to function in the X4M61R mice.We thank the Reviewer for this thought and added a sentence accordingly.3. In lines 69-71, the authors cite Wang's paper to support the roles of XRCC4-XLF filaments in broken DNA end synapsis. However, in the Wang paper, XRCC4 and XLF are suggested to play roles mainly at the ends of the DNA  rather than through a XRCC4-XLF filament structure. Moreover, the statement of "X4 and PAXX binds DNA broken ends and stimulates X4-XLF DNA ends tethering in vitro" is not true. PAXX alone cannot bind dsDNA . It is true PAXX can stimulate end-to-end synapsis and ligation . The stimulation by PAXX is through the interaction with Ku70/80, but not through the XRCC4-XLF filament.The Introduction was rephrased and reorganized to take into account these comment. We hope it increased the understanding of the scope of our study and readability of the manuscript.4. The authors need to carefully examine their figures and related labels and legends to make sure they are correct. For example, one label is missing on Figure 1D; the x-axis labels of Figure 1G are incorrect; Figure 1 legend, line 134, "X4M61R vs X4-/- (bottom)" is inconsistent with those shown on Figure 1G.We carefully checked the Figures and Figure legends for mistakes and inconsistency.5. The percentages of specific cell populations are shown on some flow cytometry panels but not on others. The ratios of the cell populations would make the flow cytometry data easier for readers to understand.The % of cell population was shown only on Fig2D. For consistency with all other FACS panel we removed the % values on Fig2D. The actual values of gated cell population are computed on side histograms..As suggested, we tried representation of the data by ratio rather than true values. As shown in 6. In line 164, the complete loss of viability was caused by 2 but not 1 Gy as shown on Figure 1G.Corrected.7. In line 228, the cellularity of X4M61R single mutant littermates in the spleen is missing.Mean+/-SEM were checked for consistency.8. The protein levels of X4 and Lig4 in the double knock-out mice/embryos could provide more information about the roles of X4 in lymphocyte development.Unfortunately, we will not be able to address this issue in a reasonable time frame. Because of the successive lockdown due to covid19 pandemic, we have been asked to sacrifice most of our living animals. Since the study was essentially finished and the manuscript was being prepared we had no choice but kill the mice related to this project.9. In the Introduction, the authors should also mention and consider the substantial literature supporting end-to-end synapsis models, which involve XLF interaction with XRCC4 and Ku. Both end-to-end synapsis and X4-XLF filament formation may be occurring.We added a paragraph on DNA end synapsis with reference to Zhao et al., for further detailed reading and the 3 references on resolution of NHEJ core complexes through cryo-EM.10. The authors should consider that IR  produces ~30 DSB per cell, whereas during V(D)J in T or B cells, there are likely to be only a few DSB per cell. One possible explanation for the difference between DNA repair roles and V(D)J is the number of events per cell and titration out of the XLF and X4 repair factors. Also, the chemistry and structure of the DNA ends for IR (and for phleomycin) are different than for V(D)J coding ends. The authors may want to consider that this could be an important reason for the difference in repair versus V(D)J for the role of X4.cc) double KO conditions. So we do not think that the number of breaks or their nature is the central issue, but rather the existence of a V(D)J specific backup system for the repair of prDSB.We agree that there are major qualitative and quantitative differences beetween VDJ and IR generated DSBs. Nevertheless, again taking Xlf deficiency as an example the one V(D)J break is repaired in Xlf KO mice but not anymore in many Xlf/NHEJ (or RAG2


Low pathogenic avian influenza (LPAI) H7 subtype viruses are infrequently, but persistently, associated with outbreaks in poultry in North America. These LPAI outbreaks provide opportunities for the virus to develop enhanced virulence and transmissibility in mammals and have previously resulted in both occasional acquisition of a highly pathogenic avian influenza (HPAI) phenotype in birds and sporadic cases of human infection. Two notable LPAI H7 subtype viruses caused outbreaks in 2018 in North America: LPAI H7N1 virus in chickens and turkeys, representing the first confirmed H7N1 infection in poultry farms in the United States, and LPAI H7N3 virus in turkeys, a virus subtype often associated with LPAI-to-HPAI phenotypes. Here, we investigated the replication capacity of representative viruses from these outbreaks in human respiratory tract cells and mammalian pathogenicity and transmissibility in the mouse and ferret models. We found that the LPAI H7 viruses replicated to high titre in human cells, reaching mean peak titres generally comparable to HPAI H7 viruses. Replication was efficient in both mammalian species, causing mild infection, with virus primarily limited to respiratory tract tissues. The H7 viruses demonstrated a capacity to transmit to naïve ferrets in a direct contact setting. These data support the need to perform routine risk assessments of LPAI H7 subtype viruses, even in the absence of confirmed human infection. Low pathogenic avian influenza (LPAI) viruses of the H7 subtype have been associated with periodic documented outbreaks in gallinaceous birds in North America for decades ,2. DurinIn 2018, two LPAI H7 subtypes were associated with poultry outbreaks in the United States: H7N1 virus in turkeys and chickens in Missouri and Texas, respectively, and H7N3 in turkeys in California . While LHere, we evaluated the mammalian pathogenicity and transmissibility of representative viruses from LPAI H7N1 and H7N3 outbreaks that occurred in North America in 2018. Viruses from both outbreaks replicated efficiently in human respiratory tract cells, productively infected mice and ferrets without the need for host adaptation and exhibited a capacity for virus transmission between ferrets in the presence of direct contact. These findings illustrate the continued pandemic potential of North American H7 subtype influenza viruses.Viruses. The North American lineage LPAI H7N1 viruses, A/chicken/Texas/18-007912-2/2018 (ck/TX) and A/turkey/Missouri/18-008108-11/2018 (tky/MO), the LPAI H7N3 virus, A/turkey/California/18-031151-4/2018 (tky/CA), and the LPAI H7N9 virus, A/goose/Nebraska/17096-1/2011 (gs/NE), were propagated in the allantoic cavity of 10-day-old embryonated hens’ eggs at 35°C for 48 h. The North American lineage HPAI H7N3 virus, A/Mexico/InDRE7218/2012 (Mex/7218), and Eurasian lineage HPAI H7N7 viruses, A/Netherlands/219/2003 (NL/219), A/Netherlands/230/2003 (NL/230), and A/Italy/3/2013 , were propagated similarly at 37°C for 26–30 h. For each virus, allantoic fluid was pooled from multiple eggs, clarified by centrifugation, and frozen in single-use aliquots at −70°C. Stocks were serially titrated in eggs to determine a 50% egg infectious dose (EID50) by the method of Reed and Muench [Sequence analysis. Sequence data for tky/MO (EPI_ISL_429249), ck/TX (EPI_ISL_429251), and tky/CA (EPI_ISL_429250) were deposited into the Global Initiative on Sharing All Influenza Data (GISAID) database and alignments of full-length coding sequences were performed using BioEdit software.Ethics statement. All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the Centers for Disease Control and Prevention and were conducted in an Association for Assessment and Accreditation of Laboratory Animal Care International-accredited facility.Cell culture. The human bronchial epithelial cell line, Calu-3 (ATCC), was cultured and grown to confluence on 24 mm membrane inserts  under submerged conditions as previously described [50 titre for one hour before washing and were then cultured at 37°C or 33°C as indicated. The supernatant was collected at the times specified post-inoculation and frozen at −70°C until serial titration in eggs for detection of infectious virus . Statistical significance of viral replication kinetics was assessed using a two-way analysis of variance with a Tukey post-test or unpaired t test correcting for multiple comparisons using the Holm-Sidak method.escribed . Cells wMouse infection. Female BALB/c mice (Jackson Laboratories), 6 weeks of age, were anesthetized by vaporized isoflurane and inoculated with 106 (n = 11) or 103 EID50 (n = 6) of virus by the intranasal (i.n.) route in a 50 µl volume. All mice were observed daily for clinical signs of disease. Five mice per group inoculated with 106 EID50 of virus were weighed daily for morbidity and mortality assessments; three mice per group inoculated at each virus dose were euthanized at day 3 or 6 p.i. for collection of nose and lung tissues. Eight additional mice per virus were inoculated with 106 EID50 of virus by the ocular route in a 5 µl volume [1.5 EID50/ml).l volume , and fouFerret infection. Male Fitch ferrets (Triple F Farms), 5–10 months of age and serologically negative to currently circulating H1, H3, and B influenza viruses by standard hemagglutination inhibition assay, were used in this study. Ferrets were housed in Duo-Flo Bioclean mobile units (Lab Products) for the duration of each experiment. Ferrets (six per virus) were inoculated by the intranasal route with 106 EID50 of each virus in a 1 ml volume and observed daily for clinical signs and symptoms of infection. Nasal washes, conjunctival washes, and rectal swabs were collected from three ferrets on alternate days, and three additional ferrets were euthanized on day 3 p.i. for collection of tissues to evaluate systemic spread of virus, as previously described [escribed ,23. For escribed .Virus-induced syncytium formation assay. Vero cell cultures (ATCC) were inoculated with influenza viruses to determine the pH at which syncytia formation occurs, as described previously [eviously  Briefly,Poultry outbreaks of LPAI H7 viruses in North America in 2018. LPAI H7N1 viruses have been sporadically detected in North America, primarily from migratory birds and specimens collected during routine surveillance of domestic poultry [ poultry ,27. Howe poultry . SequencIn contrast to H7N1 subtype viruses, outbreaks of LPAI H7N3 viruses have been reported with increased frequency in North America, with outbreaks in Texas, Missouri, New Jersey, and California over the past 15 years . In SeptLPAI H7 virus replication in Calu-3 cells. The human bronchial epithelial cell line, Calu-3, supports replication of a wide range of both human and zoonotic influenza A viruses [p < 0.01). This difference was most striking at the lower MOI tested (0.01); mean peak titres were 2.5–3.3 logs higher for 2018 H7 viruses than for the 2011 gs/NE virus, while at MOI 1 mean peak titres were 1.5–2.1 logs higher. viruses , and has viruses ,29,30. T9.5–10.3 EID50/ml in this model, generally higher, albeit only slightly so, than the mean peak titres among all 2018 LPAI H7 viruses examined (>108 EID50/ml); differences between LPAI and HPAI viruses were not uniformly statistically significant. These data show that the 2018 LPAI H7 viruses possess a robust capacity for efficient replication in human respiratory tract cells, reaching titres generally comparable to those of HPAI H7 viruses.In a previous study, an LPAI H7N9 virus associated with a poultry outbreak in 2017 replicated to significantly higher titrs in Calu-3 cells than an HPAI virus from the same outbreak , underscBeyond overall viral load, efficient replication at the cooler temperature of the human upper airways represents an additional measure of zoonotic influenza virus adaptation to human hosts . InfectiMammalian pathogenicity of LPAI H7 viruses. North American LPAI H7 viruses typically replicate efficiently in mice without the need for prior host adaptation, and are generally associated with low to moderate morbidity in this species, depending on the inoculating strain [6 EID50 challenge dose, reaching mean peak titres ≥105.5 EID50/ml day 3 p.i. , a capacity shared by other LPAI H7N3 North American viruses [3 EID50 inoculating dose with both viruses is in agreement with other contemporary LPAI H7 North American strains [g strain ,33. To ay 3 p.i. . Unlike  viruses ,33. The  strains ,31,34. MNext, we employed the ferret model, which more closely emulates humans, to examine the capacity of LPAI H7 viruses isolated in 2018 to cause disease. Overall, ferrets inoculated by the intranasal route with either ck/TX or tky/CA viruses exhibited a mild infection. Inoculated ferrets did not exhibit clinical signs such as lethargy, sneezing, or rhinorrhea; transient rises in body temperature did not exceed 1°C above baseline for any ferret. Weight loss was observed in only 1/3 ferrets inoculated with either virus (maximum weight loss of 5.2–5.3%).5 EID50/ml or g . Infectious virus was also present in pooled intestinal tissue and rectal swabs from 1/3 and 2/3 ferrets, respectively. However, days 1 and 5 p.i. rectal swabs were negative in both H7N1 and H7N3 virus-infected ferrets.Both LPAI H7 influenza viruses replicated efficiently throughout the ferret respiratory tract, including the lung, in agreement with other North American lineage viruses from this subtype ,32. Amon/ml or g . Infecti4.3 EID50/ml or g following H7N3 virus infection (3 EID50/ml) among virus-positive conjunctival samples for both H7N1 and H7N3 viruses, in the absence of virus detection in eye tissue or conjunctival washes collected days 1–5 p.i.  in nasal wash specimens on day 1 p.c., which was likely reflective of virus on the surface of the nose in the absence of productive virus infection. Collectively, these results support that selected LPAI H7 viruses are capable of limited transmission between mammals, despite the absence of confirmed human infection.The H7N1 ck/TX virus transmitted to 3/3 contact ferrets, with transmission kinetics associated with the timing of peak viral titres detected in nasal wash specimens among inoculated ferrets . All conRecent studies have indicated a link between influenza virus HA stability and increased adaptation to human hosts ; a lowerH7 subtype viruses originating from wild bird reservoirs have caused numerous outbreaks in land-based poultry on multiple continents, occasionally resulting in human infection . As riskSelected H7 subtype LPAI viruses from North America have been previously shown to replicate to high titre in human respiratory tract cells and induce innate immune responses early after infection ,29,30. HWhile both LPAI and HPAI H7N1 viruses have caused documented outbreaks in Europe and Africa in recent years , H7N1 vi6 EID50 inoculation dose. However, compared with the H7N1 ck/TX virus, the H7N3 tky/CA virus replicated to higher mean peak titres in the lung on day 3 p.i., maintained high viral titres in the lung through day 6 p.i., and was detected in the nose tissue of all tested mice , and other epidemiologic considerations likely contribute to this phenomenon . However


E) and molecular networking  was used to elucidate the metabolic profiles and chemical structures of the secondary metabolites produced by pigmented (P1) and non-pigmented (NP1) Serratia marcescens (S. marcescens) strains. Tandem mass spectrometry-based molecular networking guided the structural elucidation of 18 compounds for the P1 strain  and 15 compounds for the NP1 strain  using the MSE fragmentation profiles. The serratamolide homologues were comprised of a peptide moiety of two L-serine residues (cyclic or open-ring) linked to two fatty acid chains . Moreover, the putative structure of a novel open-ring serratamolide homologue was described. The glucosamine derivative homologues  consisted of four residues, including glucose/hexose, valine, a fatty acid chain (lengths of C13 – C17 and varying from saturated to unsaturated) and butyric acid. The putative structures of seven novel glucosamine derivative homologues and one glucosamine derivative congener (containing an oxo-hexanoic acid residue instead of a butyric acid residue) were described. Moreover, seven fractions collected during RP-HPLC, with major molecular ions corresponding to prodigiosin, serratamolides , and glucosamine derivatives , displayed antimicrobial activity against a clinical Enterococcus faecalis S1 strain using the disc diffusion assay. The minimum inhibitory and bactericidal concentration assays however, revealed that prodigiosin exhibited the greatest antimicrobial potency, followed by glucosamine derivative A and then the serratamolides . These results provide crucial insight into the secondary metabolic profiles of pigmented and non-pigmented S. marcescens strains and confirms that S. marcescens strains are a promising natural source of novel antimicrobial metabolites.An integrated approach that combines reverse-phase high-performance liquid chromatography (RP-HPLC), electrospray ionization mass spectrometry, untargeted ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS Serratia species are facultatively anaerobic, Gram-negative rods that are classified into the Enterobacteriaceae family (Serratia marcescens (S. marcescens), is widely known to produce a distinctive red pigment referred to as prodigiosin (Serratia plymuthica (S. plymuthica), Serratia rubidaea (S. rubidaea), and Serratia nematodiphila (S. nematodiphila) have also been reported to synthesize this pigment, while non-pigmented strains of these species have been identified  gene cluster comprised of 14 genes (pigABCDEFGHIJKLMN) , serrawettin W2 and serrawettin W3 (8 to C14) of the fatty acyl chain and may be saturated or unsaturated  that is produced by several bacterial genera, such as g others . Prodigig others  and is cg others . ResearcHIJKLMN) . In contettin W3 , while vettin W3 . Serrataaturated . In compaturated .N-butylglucosamine ester derivatives) are non-ribosomally synthesized diacylated peptoglucosamine derivatives (Serratia species. They have not been extensively studied and to date only S. marcescens SHHRE645 strain. The identified glucosamine derivative homologues (A to C) were comprised of four residues, including glucose, valine, butyric acid and a saturated or unsaturated β-fatty acid residue of varying length , liquid chromatography (LC), gas chromatography (GC), and nuclear magnetic resonance (NMR) (S. marcescens strain MSRBB2 using high performance LC (HPLC) linked to high resolution MS (HRMS) and HRMSn analysis. The characteristic fragmentation patterns produced by HRMSn analysis allowed for the identification and elucidation of 33 compounds, including 7 prodiginines and 26 serratamolides  . Chromatce (NMR) . Moreovece (NMR) . For exaamolides .http://gnps.ucsd.edu) as an open-access tool that employs a computational algorithm to compare the degree of similarity between tandem MS spectra and generates a molecular network comprised of clusters of structurally related metabolites. Thus, a visual representation of the structural relationships between natural compounds within crude extracts is generated, where a node within the network represents a compound and the relatedness between two compounds (nodes) is referred to as an edge. Moreover, GNPS allows for the comparison of the tandem MS spectra to the publicly available spectral library for rapid identification of known compounds  molecular network platform (ompounds .S. marcescens strain using reverse-phase high performance liquid chromatography (RP-HPLC), electrospray ionization mass spectrometry (ESI-MS), untargeted ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MSE) and molecular networking (using the GNPS molecular network platform) analysis. In addition, the susceptibility of a clinical Enterococcus faecalis  strain to fractions collected during RP-HPLC analysis was evaluated using disc diffusion assays  and broth microdilution assays to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of selected fractions.Here, we present an integrated approach to identify and structurally elucidate the naturally produced complex of secondary metabolites of a pigmented (P1) and non-pigmented (NP1) S. marcescens strains from an oil refinery effluent sample and a river water sample, respectively as well as the molecular identification of the strains. The two S. marcescens strains were streaked from the glycerol stocks onto Nutrient agar  and were incubated at 30°C for 18–24 h. The S. marcescens P1 and NP1 strains were deposited in the South African Rhizobium Culture Collection . The clinical E. faecalis S1 strain, used for the disc diffusion and MIC assays, was streaked from the glycerol stock onto tryptone soy agar  supplemented with 6 g/L yeast extract  (TSAYE0.6%) and was incubated at 37°C for 18–24 h. The clinical E. faecalis S1 strain is curated and accessible in the Water Resource Laboratory culture collection in the Department of Microbiology at Stellenbosch University.A previous study by S. marcescens P1 and NP1 strains into 10 ml Luria Bertani broth  in triplicate and incubating on a test tube rotator  at 30°C. Each seed culture was subsequently inoculated into a 2 L baffled flask containing 500 ml Peptone Glycerol  broth [composed of 5 g peptone powder and 10 ml glycerol  in 1 L distilled water] in triplicate, which was incubated on an orbital shaker  at 30°C for 120 h at 120 rpm. Following a 5 days incubation period, the P1 and NP1 broth cultures were centrifuged at 10,000 rpm for 20 min at 4°C to obtain the cell free supernatant. The cell free supernatants were lyophilized and dissolved in 70% HPLC-grade acetonitrile  in analytical quality water (milliQ water) (v/v). The acetonitrile soluble fraction was transferred into a sterile McCartney bottle and lyophilized. This step was repeated thrice to further purify the crude extracts. The triplicate crude extracts were then pooled, lyophilized, analytically weighed and stored at −20°C until further use.The production and partial purification of secondary metabolites was performed as described by v/v) to a concentration of 10.00 mg/ml and were injected into a Discovery BIO Wide Pore C18 HPLC column . Liquid chromatography was conducted with milliQ water containing 0.1% trifluoroacetic acid  as solution A and acetonitrile containing 0.1% TFA (v/v) as solution B. An isocratic flow at 40% B from 0 to 2 min for sample loading was followed by a linear gradient from 40 to 95% B from 2 to 36 min. The column was washed for 2 min with 95% B and then regenerated with a reversed gradient from 95 to 40% B from 38 to 45 min. Chromatography of compounds was followed by continuous monitoring of the absorbance at 230 and 254 nm.The P1 and NP1 crude extracts were subjected to RP-HPLC  analysis, in the Department of Microbiology, in order to obtain purified fractions. The lyophilized P1 and NP1 crude extracts were dissolved in 40% acetonitrile in milliQ water (v/v)] was injected into the ionization source at a flow rate of 0.3 ml/min. The analytes were subjected to a capillary voltage of 2.5 kV, cone voltage of 15 V, a source temperature of 120°C, desolvation gas (N2) flow of 650 L/h and desolvation temperature at 275°C. Data acquisition in the positive mode was performed by MS scanning a second analyser through the mass to charge (m/z) range of 300–1,500 in centroid mode. The high resolution mass calibration was done with sodium formate and in-analysis single point lock spray calibration using leucine enkephalin (m/z = 556.2771). The ESI-MS data was processed using MassLynx software version 4.1 . The accurate masses and molecular formula of the detected compounds were used to search online databases, such as Norine (https://bioinfo.lifl.fr/norine/) and PubChem (https://pubchem.ncbi.nlm.nih.gov/), of known natural products and an extensive literature search was conducted for the putative identification of the metabolites.The absorbing peak fractions collected from the RP-HPLC were analyzed using a Waters Synapt G2 high resolution mass spectrometer fitted with a Z spray electrospray ionization source  at the LCMS unit at the Central Analytical Facility . For direct mass analysis, 3 µl of the sample + (compound 2) corresponding to the peak observed at 8.48 min in the UPLC-MSE data for the P1 strain , 541.3485 [C28H48N2O8 + H]+ (compound 9), 543.3644 [C28H50N2O8 + H]+ (compound 13), 561.3749 [C28H52N2O9 + H]+ (compound 6), 559.3600 [C28H50N2O9 + H]+ (compound 3), and 571.3931 [C30H54N2O8 + H]+ (compound 18), which corresponded to peaks observed in the UPLC-MSE data for the P1 and NP1 strains (m/z 587.3908 [C30H54N2O9 + H]+ (compound 11) corresponding to the peak observed at 13.76 min in the UPLC-MSE data for the NP1 strain  correspond to cyclic or open-ring serratamolide homologues that have previously been reported  coupled to two saturated hydroxydecanoic acyl groups (C10) (18 was comprised of two serine residues (cyclic peptide moiety) linked to two saturated hydroxydodecanoic acyl groups (C12), which corresponded to a serratamolide homologue reported by 9 and 13 were comprised of two serine residues (cyclic peptide moiety) coupled to a 3-hydroxydecanoic acyl (C10) and a hydroxydodecanoic acyl (C12) (13 thus corresponded to the previously described serratamolide C (9 had a double bond in the hydroxydodecanoic acyl (C12:1) residue (5 position (from the C=O group) of the fatty acyl chain, as this corresponded to the position previously reported in literature  predicted using the MassLynx 4.1 software package, in the compound suggested that both lipopeptides were open-ring structures and corresponded to the fragmentation pattern of the previously described open-ring serratamolides B and C (11 was comprised of one serine residue coupled to one saturated hydroxydodecanoic acyl (C12) and one serine residue coupled to one unsaturated hydroxydodecanoic acyl (C12:1). However, the addition of a hydroxyl group [based on the experimental molecular formula ] in the compound suggested that the lipopeptide was an open-ring structure and corresponded to the fragmentation pattern previously described by Compound ps (C10)  confirmips (C10) . In addiyl (C12) . Compounmolide C . However residue  confirmi residue . The douterature . Compoun B and C   did not cluster in the molecular network; however, it was detected at 12.46 min in the UPLC-MSE data for the NP1 strain . A major fragment at m/z 284.185 was then observed, representing a dehydrated serine residue linked to an unsaturated hydroxydodecanoic acid (C12:1). In addition, the dehydration product of the m/z 284.185 fragment was detected at m/z 266.176. Similar to the previously described open-ring structures, the addition of a hydroxyl group [based on the experimental molecular formula ] in the compound suggested that the lipopeptide was an open-ring structure; however, this compound has not previously been reported in literature or databases (Norine and PubChem). The novel 8 homologue was thus proposed to comprised of two L-serine residues coupled to two unsaturated D-3-hydroxydodecanoic acyl groups (C12:1), correlating to described serratamolides  and 559.3953 [C29H54N2O8 + H]+ (compound 14) corresponding to the peaks observed in the UPLC-MSE data for the P1 and NP1 strains, while m/z 573.4136 [C30H56N2O8 + H]+ (compound 19) corresponded to a peak observed in the UPLC-MSE data for the P1 strain  correspond to glucosamine derivative homologues that have previously been reported .The molecular network of the F2 cluster and corresponding structures for the known and novel compounds in the F2 cluster are presented in 1 strain . The frareported . For allvative A  , 583.3947 [C31H54N2O8 + H]+ (compound 15) and 589.4056 [C30H56N2O9 + H]+ (compound 12) had similar fragmentation profiles to 14, 16, and 19 and corresponded to peaks observed in the UPLC-MSE data for P1 and NP1 strains (m/z 575.3902 [C29H54N2O9 + H]+ (compound 5), 587.4268 [C31H58N2O8 + H]+ (compound 21), and 599.4265 [C32H58N2O8 + H]+ (compound 17) also had similar profiles to glucosamine derivative A to C and corresponded to peaks observed in the UPLC-MSE data for the P1 strain, while another node at m/z 545.3787 [C28H52N2O8 + H]+ (compound 10) corresponded to peaks observed in the UPLC-MSE data for the NP1 strain  predicted by the MassLynx 4.1 software package provided further indication of the addition of the hydroxyl group for both 5 and 12  and fragmentation patterns did not correspond to previously identified glucosamine derivatives (N-butylglucosamine ester derivatives) and are hypothesized to be novel glucosamine derivatives homologues. The F2 nodes with m/z 607.3924, 581.3781, 579.3623, and 567.3618 were found in the UPLC-MSE data and corresponded to the sodiated adducts of compounds 16, 14, 7, and 10, respectively,  exhibited a unique fragmentation profile and corresponded to a peak at 16.28 min observed in the UPLC-MSE data for the P1 strain (20 did not have a major fragment of m/z 232.116 (corresponding to a hexose residue linked to butyric acyl group) that was common in all of the previously described glucosamine derivatives. Instead, a major fragment at m/z 274.129 was observed, which suggested a modification in the fragment corresponding to the hexose and butyric acyl moiety , and a dehydration product of the m/z 274.129 fragment was observed at m/z 256.117. Furthermore, a second major fragment of m/z 354.302 was observed, which corresponded to an unsaturated C16:1 fatty acid chain. In comparison to the glucosamine derivative A (16) which also had an unsaturated C16:1 fatty acid chain, this glucosamine derivative had an additional C2H3O based on the experimental molecular formula  predicted using the MassLynx 4.1 software package. It is thus feasible that oxo-hexanoic acid was incorporated into the glucosamine derivative instead of butyric acid. In addition, 20 did not cluster with the glucosamine derivatives in the molecular network; rather, the compound formed an individual cluster and this further suggests the reduced relatedness of this compound to the glucosamine derivatives. Although NMR is required to confirm the structure, the putative novel congener of 20 was proposed to consist of glucose, oxo-hexanoic acid, valine and an unsaturated C16:1 fatty acid chain, which is distinctive from that of the previously described glucosamine derivatives  to the fractions corresponding to the major compounds  4, 9, 13, 7, 14, and 16 (for both P1 and NP1), and the fraction of compound 2 collected from P1 crude extract (results not shown). Thereafter, the MIC and MBC assays were performed using five fractions (selected based on activity and quantity available), including fractions of the major compounds  2, 4, 9, 13, and 16. A summary of the MIC and MBC assay results are outlined in 4, 9, and 13)  exhibited identical activity against E. faecalis S1 with a MIC of 3 mg/ml and MBC of >3 mg/ml. In comparison, glucosamine derivative A  was found to have a lower MIC and MBC of 0.75 mg/ml and 3 mg/ml, respectively. Overall, however, the lowest MIC and MBC recorded against E. faecalis S1 was observed for prodigiosin , at <0.75 and 1.5 mg/ml, respectively, , prodigiosin (2), serratamolide homologues  and glucosamine derivative congeners   and 11 glucosamine derivatives (including the putative structures of eight novel glucosamine derivative congeners) were identified in this study and their structures were elucidated.This work reports on the identification of four molecular families of secondary metabolites associated with refer to –5. The srefer to  and serrrefer to . The strrefer to  and glucrefer to . Moreove10 to C12) and the presence or absence of double bonds (saturated or mono-unsaturated) in the fatty acyl chains and cyclization (open-ring or cyclic) of the peptide moiety. The glucosamine derivative congeners, comprised of glucose, valine, butyric acyl (or oxo-hexanoic acyl for 20) and a fatty acyl residue, similarly varied in the length (C13 to C17), as well as the presence or absence of double bonds (saturated or unsaturated) or a hydroxyl group in the β-fatty acyl residue. During UPLC-MSE analysis, the elution time (Rt) of the serratamolide and glucosamine derivative homologues was, as expected, dependent on the length of the β-hydroxy fatty acid chains of the compound. For instance, the serratamolides with shorter saturated fatty acid moieties, such as serratamolide A (C10 + C10) (4), eluted from the column before the serratamolides with longer saturated fatty acid moieties, such as serratamolide 571 (C12 + C12) (18) (refer to 16) (C16:1) eluted before the corresponding saturated novel glucosamine derivative K (21) homologue (C16) with the same residues and fatty acid chain length  (13) eluted from the column after the open-chain serratamolide 561 (C10 + C12) (6) with the same fatty acid chain length. Furthermore, it was observed that the glucosamine derivatives with an additional hydroxyl group within the fatty acyl moieties were retained in the column for a shorter time period than the corresponding glucosamine derivative without a hydroxyl group, due to the increased polarity of the compound. For instance, the glucosamine derivative C  with a C14 fatty acyl chain eluted from the column after the corresponding novel glucosamine derivative D homologue  with a C14 fatty acyl chain that has an additional hydroxyl group. Overall, it was observed that the serratamolide and glucosamine derivative homologues separated as expected according to hydrophobicity (fatty acyl chain length) and chain saturation.It is well-known that certain bacteria naturally produce various lipopeptide congeners which vary in fatty acid chain length, fatty acyl saturation or in the amino acyl residues of the peptide moiety . This warefer to . It was refer to . Previourefer to  and the tty acid . It has tty acid , which cE analysis in the current study revealed that the P1 strain produced four metabolic classes, including prodigiosin, serratiochelin A, serratamolide homologues (n = 6) and glucosamine derivative congeners (n = 10), while NP1 produced three metabolic classes, including serratiochelin A, serratamolide homologues (n = 8) and glucosamine derivative congeners (n = 6). It is hypothesized that the combination of RP-HPLC and UPLC-MSE analysis was an effective approach to identify the minor metabolic constituents and elucidate the microbial metabolic profile of the environmental Serratia strains.E analysis may have contributed to the detection of the minor metabolites; it is hypothesized that the altered culture conditions and media composition employed, may have activated “silent” NRPS clusters (such as the glucosamine derivatives in the P1 strain and the serratiochelin in both strains), thereby increasing the metabolic constituents produced by both the pigmented and non-pigmented S. marcescens strains.Genetically, secondary metabolites are synthesized by a number of gene clusters that can be identified using a bioinformatics approach . CertainS. marcescens SM6 strain and used the antiSMASH 5.0 prediction tool to identify biosynthetic gene clusters involved in secondary metabolism within the strain. The authors identified five biosynthetic gene clusters involved in secondary metabolite production, including gene clusters responsible for the production of two siderophores (serratiochelin and chrysobactin), a thiopeptide, a serratamolide and a glucosamine derivative , serratamolide A (4), B (9), and C (13) homologues and glucosamine derivative A (16) and C (14) homologues, as well as the novel glucosamine derivative E (7) homologue exhibited activity against a clinical Gram-positive bacterial strain. The MIC and MBC assays further revealed that serratamolide A (4), B (9), and C (13) exhibited identical activity against E. faecalis S1, suggesting that the presence of a double bond or the increased length of one of the two fatty acid chains from C10 to C12 exhibited no additional antimicrobial potency against the Gram-positive strain. In comparison, glucosamine derivative A (16) was found to exhibit a lower MIC and MBC in comparison to the serratamolides. This indicates that E. faecalis S1 was more susceptible to the glucosamine derivative A (16) than the serratamolides. Overall, however, E. faecalis S1 was found to be the most susceptible to prodigiosin (2), as a lower MIC and MBC was obtained against the clinical strain in comparison to glucosamine derivative A (16) and the serratamolides .Results from this study indicated that prodigiosin (Staphylococcus aureus (S. aureus) and Bacillus subtilis (B. subtilis) , membrane permeability or cell growth inhibition was observed at this concentration  . It has proteins . Furtherproteins . Serrata strains . It has ntration . Currentabolites . Moreoveg strain . MoreoveE and molecular networking analysis allowed for the identification and elucidation of 21 secondary metabolites , including serratamolide homologues, glucosamine derivative homologues, prodigiosin (P1) and serratiochelin A. Moreover, the putative structure of an open-ring serratamolide homologue and putative structures of eight novel glucosamine derivative congeners were elucidated. The structural relatedness of the novel open-ring serratamolide and glucosamine derivative congeners to the known serratamolides and glucosamine derivatives, respectively, was revealed using molecular networking and fragmentation patterns of the UPLC-MSE data. This approach thus proved vital for metabolite identification and characterization. The identified metabolic families exhibited structural diversity and well-ordered chromatographic behaviors due to structural modifications of fatty acid chain lengths, open-ring or cyclic structures and the degree of saturation (saturated or mono-unsaturated) of the fatty acid chain. Moreover, seven fractions collected during RP-HPLC, with major molecular ions corresponding to prodigiosin (2), serratamolide A (4), B (9), and C (13) homologues and glucosamine derivative A (16), C (14), and E (7) homologues, displayed antimicrobial activity against the clinical E. faecalis S1 strain using the disc diffusion assay. However, the MIC and MBC assays revealed that the three serratamolides  exhibited the lowest antimicrobial potency  of the metabolites tested, and highlighted that presence of a double bond or the change in length of a fatty acid chain (C10 to C12) of the serratamolides provided no additional antimicrobial potency against the Gram-positive strain. Prodigiosin (2) however, exhibited the greatest antimicrobial potency, followed by glucosamine derivative A (16). This study thus highlights the importance of Serratia species as a source of novel antimicrobial compounds for potential therapeutic application. It is recommended that future research investigate the mode of action and synergism of the bioactive secondary metabolites produced by these two S. marcescens strains. In addition, the use of whole genome sequencing of the P1 and NP1 strains and subsequent genome mining of NRPS gene clusters is a promising future endeavour to identify additional secondary metabolites not actively being produced under typical cultivation conditions.The use of RP-HPLC for chromatographic fractionation, high resolution ESI-MS, UPLC-MS


SF3B1)-mutated Uveal Melanoma (UM) in the literature (n = 146). Missense mutations in the spliceosome gene SF3B1 result in an altered splice site recognition and aberrant mRNA transcripts. The SF3B1-mutated UM show early- and late-onset of metastatic disease for which, currently, no distinguishing biomarkers exist. Using a cutoff of 60 months for stratification, we found that a largest basal tumor diameter was more prevalent in the early-onset metastatic disease group. Furthermore, using differential gene expression and the detection of aberrant transcripts, we found that the expression of alpha/beta-Hydrolase domain containing 6 (ABHD6) is associated with early-onset metastatic SF3B1 and aberrant transcripts that are associated with early-onset SF3B1-mutated UM. Our results provide more accurate prognostication and targets for future functional studies in an effort to elucidate pathogenesis of SF3B1-mutated UM.This study describes clinical and genetic characteristics of the largest aggregated cohort of Splicing Factor 3 Subunit B1  or long PFS (PFS ≥ 60 months) for SF3B1-mutated (SF3B1mut) UM patients. We collected 146 SF3B1mut UM from our Rotterdam Ocular Melanoma Studygroup (ROMS) database and external published datasets. After stratification of all SF3B1mut UM using short PFS vs. long PFS, only largest tumor diameter (LTD) was significantly larger (mean: 17.7 mm (±2.8 SD) in the short PFS SF3B1mut group vs. the long PFS group . Combined ROMS and The Cancer Genome Atlas (TCGA) transcriptomic data were evaluated, and we identified SF3B1mut-specific canonical transcripts  or distinct expression of SF3B1mut UM aberrant transcripts, indicative of early- or late-onset or no metastatic SF3B1mut UM.Approximately 25% of all uveal melanoma (UM) contain driver mutations in the gene encoding the spliceosome factor  Mutations in secondary UM driver genes are strongly associated with prognosis of UM patients and affect BRCA1-associated protein 1 , splicing factor 3b subunit 1 , and eukaryotic translation initiation factor 1A X-Linked . BAP1 is an enzyme involved in deubiquitination and interacts with different proteins such as DNA damage repair protein breast cancer type 1 (BRCA1). Splicing Factor 3b Subunit 1 (SF3B1) mutations occur in 15–29% of UM [SF3B1-mutated UM were allocated in cluster two comprising disomy chromosome 3 and chromosome 8q gain [Uveal melanoma (UM) is a highly malignant tumor with metastatic capacity. Metastatic disease is detected either early <60 months) or late during follow-up. Staging of primary UM has been performed using American Joint Committee on Cancer (AJCC)  criteria0 months 9% of UM ,8,9,10 a9% of UM . In The  8q gain . Moreove 8q gain . Finally 8q gain . These USF3B1 in UM occur mostly at the gene position that encode the amino acid (AA) residues 625, and more rarely affected AA-residues are 666, 700, 783, 781, 742, and 1123 [SF3B1 mutations occur in a heterozygous state and are change-of-function mutations that result in a broad range of aberrantly spliced transcripts due to the mutant SF3B1 protein, as encoded by the mutant allele. The wild-type allele remains active to produce the canonical spliced transcripts. The aberrant transcripts are the result of the use of alternative recognition sites by the mutated spliceosome complex and thereby utilizing (or preferring) a non-canonical splice site due to genomic mutations encoded within the Heat Domains of SF3B1 [SF3B1 amino acid substitutions more frequently affect residue K700 and K666, respectively. In the TCGA-Uveal Melanoma cohort (TCGA-UVM) [SF3B1 mutations detected reside within the residue R625. In the remaining four cases, SF3B1 mutations reside twice within the residue K666 and once within T663 and H662 and therefore account for 22% of non-R625 SF3B1 mutations.Mutations in and 1123 . SF3B1 iof SF3B1 . Somaticof SF3B1 . This peSF3B1 mutated (SF3B1mut) UM, we first set out to describe clinical characteristics of SF3B1mut UM from the updated ROMS cohort (n = 48) [SF3B1mut UM. Finally, we hypothesize that canonical gene expression or aberrantly spliced transcript expression can be used to discriminate between early-onset metastatic disease  < 60 months) and late-onset metastatic disease defined as PFS ≥ 60 months SF3B1mut UM patients.To predict early- (PFS < 60 months) or late- (PFS ≥ 60 months) onset metastatic disease in the (n = 48) ,17 and cGNAQ/GNA11 gene mutation status, SF3B1 mutation status, and corresponding amino acid changes and/or nucleotide changes were included. The rationale behind PFS cutoff of 60 months was based on a previously observed bimodal metastatic potential [t-test was applied for continuous variables, whereas Fisher’s Exact test was applied for categorical variables. p-value < 0.05 was considered statistically significant.We combined clinical and genetic variables from 10 publicly available datasets: Alsafadi et al. , Royer-Botential  and due BAP1, SF3B1, GNAQ, GNA11, and EIF1AX was determined for all UM with either BAP1 immunohistochemistry (IHC), Sanger sequencing, and/or next-generation sequencing, as previously described [BAP1, SF3B1, or EIF1AX mutations, which were acquired as described previously by Smit et al. [SF3B1 mutation and for which we exclude UM with concomitant mutations in either BAP1 or EIF1AX (n = 10).Mutation status of escribed ,25,26. At et al. . FurtherProgression-free survival (PFS) was determined as the interval from treatment until metastasis or metastasis and subsequent death due to UM or until last follow-up. If the interval from treatment to metastatic disease and interval from treatment to death due to UM was reported, we used the interval from treatment to death due to UM or development of metastasis as study endpoints. Patients were censored when they were lost to follow-up or when death from other cause than UM occurred, or occurrence of death was reported without any cause.We used whole-transcriptome sequencing data from 26 UM; information on sample acquisition, preparation, and sequencing has been described previously by Smit et al. . In brien = 80) were downloaded from NCBI dbGaP website in 2019, under phs000178.v10.p8. Sample acquisition, library preparations and sequencing protocols of the TCGA-UVM cohort were described previously by The Cancer Genome Consortium [The whole-transcriptome BAM files for the TCGA-UVM cohort  and converted back to unmapped paired-end reads with Samtools   and Sambn = 41140).Raw read counts for GENCODE (v30) transcripts were used as input for DESeq2 (v1.24.0) , with thSF3B1mut-only samples and SF3B1wildtype (wt) samples (excluding any double-mutants and/or SRSF2mut samples) was performed. An additional Wald test between SF3B1mut-only samples based on PFS < 60 months with metastatic disease and PFS ≥ 60 months was performed on the combined (ROMS and TCGA-UVM) cohort. Hereafter, PFS < 60 months refers to UM patients with early metastatic disease and late PFS (PFS ≥ 60 months) refers to patients with late or without metastatic disease. During differential gene expression analysis, we corrected for gender batch-effect in all analyses and additionally for cohort batch effect (ROMS/TCGA-UVM) in the combined cohort. To correct for multiple hypothesis testing after DESeq2 analysis, we employed independent hypothesis weighting  [2) were shrunk using their respective coefficient using apeglm (v1.6.0) [2 fold change ≥ |0.5|, adjusted p (q) ≤ 0.05, and average read counts of ≥10 over all samples.For both the ROMS and TCGA-UVM cohort separately, a Wald test between v1.12.0) . Fold-ch(v1.6.0) . A princn = 186) and HALLMARK (n = 50) gene sets (version 7.0), which contain 7732 distinct genes in all of the chosen gene sets obtained from the Molecular Signatures Database (MSigDB) [Using the R package fgsea (v1.10.0)  with 100(MSigDB) , for staSF3B1-related alternative splicing, we performed a strategy using a custom in-house workflow designed around STAR and DEXSeq [https://github.com/vivekbhr/Subread_to_DEXSeq accessed on 25 November 2019) script to further process our custom annotation (GFF3) and create a flattened version, in which each exon is split into its constituent non-overlapping exonic portions. Subsequently, this custom-flattened GFF3 file was used to count the reads per overlapping exonic portion using featureCounts (v1.6.3) [SF3B1mut and SF3B1wt samples and SF3B1early and SF3B1late samples for both ROMS and the TCGA-UVM cohort.To detect d DEXSeq . This wo(v1.6.3) . These e(v1.6.3)  to detecp-value ≤ 0.05 and a log2 fold-change of the splicing event ≥|0.5|. In downstream analysis, we denoted the exonic regions (acceptor/donor) not-yet-present within the GENCODE v30 annotation as novel (acceptor/donor) splicing aberrations.To determine statistically significant differentially expressed exonic regions, we used the following criteria: adjusted ABHD6, CSRNP1) derived from the aforementioned DGE analyses were validated with quantitative PCR (qPCR). Then, a 500 ng (nanogram) RNA input was used in cDNA conversions, except for one (PFS < 60 months) SF3B1mut sample, for which a maximum input of 450 ng RNA was possible due to concentration restraints. A real-time PCR (RT-PCR) reaction mix consisted of 5- or 7-times diluted cDNA, 10 μL iTaq Universal SYBR Green Supermix , and 10 μM forward and reverse primers, which were placed in a CFX96 real-time system . Delta cT values of genes of interest were calculated relative to the control transcript CHMP2A. All qPCR reactions were successfully performed in technical triplicates, except for sample 7, for which only two cT values for CHMP2A could be used. Differential gene expression was calculated using the threshold cycle (Ct) method [SF3B1mut UM sample with 45.5 months PFS with development of metastatic disease was used as a control during qPCR experiments.Within our cohort consisting of 48 ROMS samples, there was tumor material available of 31 Formalin Fixed Paraffin Embedded (FFPE) UM to validate in silico results on with IHC. Of these samples, 20 fresh frozen UM patient samples were available for RNA isolation, as described previously by Smit et al. , of whic) method . A SF3B1ABHD6 expression. An ophthalmic pathologist independently evaluated the histopathological characterization of the tissue sections and the immunohistochemistry staining. For every section, an immunoreactive score (IRS) was determined. We first determined the intensity of the cytoplasmatic staining . Next, the percentage of stained cells that showed the predominant intensity, was determined; no positive cells were scored as 0% and less than 10%, 10% to 50%, 51% to 80%, and more than 80% were scored as 1, 2, 3, or 4, respectively. Then, IRS was calculated by multiplying the score for percentage of stained cells with the score for the intensity of the staining [Immunohistochemistry was performed with an automated immunohistochemistry staining system  using the alkaline phosphatase method and a red chromogen. In brief, following deparaffinization and heat-induced antigen retrieval for 64 min, the tissue sections were incubated with a rabbit polyclonal antibody raised against synthetic peptide of human ABHD6  for 1 h at 37 °C, followed by red detection and counterstain with hematoxylin II and bluing reagent according to the manufactures instructions (Ventana). Kidney, tonsil, and the retinal pigment epithelium were used as positive controls for staining .Analysis was performed using the R statistical platform language (v3.6.3). All used custom R code can be freely requested by contacting the authors.SF3B1mut samples. One UM sample classified as SF3B1mut UM in the Yavuzyigitoglu et al. set was excluded due to melanoma originating from Nevus of Ota. We hypothesized clinical or genetic variables could be indicative for either early-onset metastatic disease SF3B1mut UM (PFS < 60 months) or SF3B1mut UM patients with late-onset metastatic disease (PFS ≥ 60 months). Therefore, we acquired 761 UM from a total of 11 large-scale UM studies (SF3B1mut UM (Updating the ROMS database  resulted studies  and geneB1mut UM .n = 48) consisted of 21 males and 27 females with mean age at diagnosis of 56.7 years (±16.8 standard deviation (SD)). The mean largest tumor diameter (LTD) was 13.6 mm (±3 SD), and mean tumor thickness was 6.8 mm (±2.6 SD). Age at diagnosis did not differ between the ROMS and non-ROMS cohorts (p = 0.836). However, LTD, tumor thickness, inflammation, and necrosis were significantly different  between the ROMS and non-ROMS UM patients (p < 0.001) compared to the mean PFS in the non-ROMS cohort of 45.4 months (±33.6 SD). Demographic and genetic variables per dataset are shown in The ROMS cohort  larger (17.7 mm (±2.8 SD) in the PFS < 60 group compared to the PFS ≥ 60 months group (mean: 14.7 mm (±3.7 SD) (SF3B1mut UM at risk (n = 113) was 131.5 months (95% CI: 101.0–195.4) using Kaplan–Meier survival analysis , and 52 UM patients had PFS ≥ 60 months. Only the mean largest tumor diameter was significantly (±3.7 SD) . The medanalysis .n = 8). However, two patients developed both liver and lung metastasis. Two patients showed liver and ossal metastases, and one other patient was diagnosed with only ossal metastasis. One patient had liver and pancreatic metastases. Finally, there was one patient that developed lung, liver, kidney, and subcutaneous metastases  showed decreased expression in the PFS <60 UM group and increased expression in the PFS ≥ 60 months SF3B1mut UM group  (p = 0.11)  A, and th = 0.11) B. The RTSF3B1mut and SF3B1wt UM within the ROMS cohort, whilst again using the UVM-TCGA with the same design as validation cohort. Comparing SF3B1mut vs. SF3B1wt samples within the ROMS revealed 2107 differentially expressed exonic regions from 1353 distinct genes, of which 397 exonic regions from 257 distinct genes were also found differentially expressed within the TCGA-UVM  exon usage to discriminate between TCGA-UVM . Of theson usage . Using twildtype .SF3B1mut and SF3B1wt UM, we hypothesized whether similar patterns could be indicative of early- or late-onset of metastatic disease in SF3B1mut UM (PFS < 60 months vs. PFS ≥ 60 months) within the ROMS cohort.In line with the discriminatory value of using differential exon usage to distinguish SF3B1mut samples , and in total, four genes were known onco- and tumor suppressor genes . These splicing events supplement the full-length transcript markers shown previously to differentiate between early- and late-onset metastatic disease of SF3B1mut UM  in the literature and report on the prognostic value of clinical and genetic variables, whilst distinguishing between early (PFS < 60 months) and late (PFS ≥ 60 months) metastatic onset SF3B1mut UM. This could provide us with tools to suggest a more stringent treatment protocol or inclusion in specific clinical trials. The question remains if these potentially more stringent screening programs will result in the prolonged overall survival of SF3B1mut UM patients. However, after validation of our results in external cohorts, low expression of ABHD6 could potentially be used as a biomarker to detect and select high-risk SF3B1mut UM.In the current study, we describe the largest number of ABHD6 is indicative of a long progression-free survival (PFS ≥ 60 months), whereas a low expression is indicative of early-onset metastatic disease (PFS < 60 months). As described in previous reports, UM with somatic alterations within the spliceosome have a distinct CNV profile and are characterized by aberrant transcripts due to a mutated allele of SF3B1. Considering survival, we have shown that, in particular, SF3B1mut UM gives rise to late-onset metastases [SF3B1mut UM patients may also show a less favorable prognosis with early-onset metastatic disease. Although, the majority of the samples with a PFS > 60 months originated from our cohort (n = 9), these UM patients were also present within the Alsafadi et al. (n = 1), Rodrigues et al. (n = 3), Shain et al. (n = 1), and Harbour et al. (n = 2) UM cohorts. Even though this constitutes only a limited number of UM patients within each external cohort, it does illustrate that late-onset metastatic disease occurs in SF3B1mut UM, but only when study follow-up is sufficient. From the clinical parameters, only LTD was significantly larger in the early-onset metastatic UM group compared to the late-onset metastatic UM, which corroborates with earlier study findings, where tumor size was shown to be an independent predictor of UM aggressiveness [SF3B1mut to the other studies for metastases, we did not observe significant differences between clinical and pathological parameters. Interestingly, the ROMS patients have a longer follow-up in contrast to the other studies, possibly explaining difference in median PFS. Regarding metastatic location, there were no associations between extrahepatic metastatic locations and high risk or low risk SF3B1mut UM, most likely due to a restricted number of metastatic samples.A high expression of tastases . Neverthsiveness ,42. All BAP1 mutations, SF3B1 mutations are change-of-function mutations rather than loss-of-function mutations, with the R625 alteration being the predominant hotspot in UM. However, mutations at other locations encoding K666 or K700 also occur within smaller proportions of UM. At present, it is not clear whether mutations outside the common SF3B1 hotspots are merely passenger mutations, or if these contribute to UM pathogenesis warranting the need for an unbiased diagnostic approach capable of detecting compromised SF3B1 function. One such method could be based on a straightforward PCR primer assay, designed to detect SF3B1mut-specific aberrant transcripts indicative of the transcriptomic hallmark of SF3B1mut UM. This could broaden our understanding and inventory of SF3B1mut UM.In contrast to SF3B1mut allele are probably vast due to the amounts of aberrant transcripts that are formed. For example, the tumor suppressor BRD9 that is linked to melanomagenesis was recently shown to be downregulated in SF3B1mut UM due to the incorporation of a poison exon, which leads to the non-sense mediated decay of BRD9 [ABHD6 expression can be used to discriminate between early-onset and late-onset metastatic SF3B1mut UM. ABHD6 is a lipase involved in endocannabinoid signaling and possibly has other effects in inflammation, metabolic syndromes, and insulin secretion. In contrast to earlier publications [ABHD6 is associated with high-risk SF3B1mut, which we validated using qPCR and IHC in nearly all UM cases tested. However, in order to implement this biomarker in clinical settings, more samples should be tested in another study with sufficient follow-up. For now, the results are promising, and it is possible to identify early onset metastatic SF3B1mut UM.Downstream effects of the  of BRD9 . Most liications ,45, our SF3B1mut and SF3B1wt UM (concordant in both the ROMS and TCGA-UVM cohort), of which 37 are known cancer genes. In addition, GSEA revealed perturbations within spliceosome machinery and inflammatory responses. Moreover, downregulation of chemokine signaling, T-cell receptor signaling, and natural killer cell-mediated cytotoxicity pathways within SF3B1mut UM could indicate a decreased inflammatory function, reflecting potential immunological differences [SF3B1 mutations, we could identify 2107 SF3B1mut-specific or significantly perturbed splicing aberrations with donor and/or acceptor extensions or shortenings.A limitation of our differential expression analyses to draw convincing conclusions are the low number of samples. However, 617 differentially expressed genes were observed between ferences . FurtherSF3B1mut-specific alternatively spliced transcripts between early- and late-onset SF3B1mut, we identified putative biomarkers between these groups of patients. We detected four canonical transcripts  with significant up-regulated expression within the late-onset UM compared to early-onset UM. We set out to validate the expression of ABHD6 using IHC and rt-qPCR on secondary UM samples. The expression of ABHD6 varies between cancer types and is increased in Ewing sarcoma, prostate cancer, Burkitt lymphoma, and leukemia [ABHD6 expression in early metastatic UM was significantly downregulated compared to primary non-metastatic uveal melanoma, which corroborates our study findings within SF3B1mut UM [SF3B1mut UM cases should be included to draw a convincing conclusion that ABHD6 expression is a prognostic biomarker of PFS in SF3B1mut UM. Due to a lack of IHC staining in samples 3 and 7, we cannot fully conclude ABHD6 IHC is a reliable test to differentiate between early- and late-onset metastasis in SF3B1mut UM, and unfortunately, these results do not corroborate the more reliable qPCR results. An explanation for discrepancy between qPCR and IHC staining in sample 3 and 7 is the fact that the RNA was isolated from a different (N2 stored) part of the tumor, and we cannot exclude some heterogeneity in the tumor specimen. However, pooled data of qPCR results and IHC signaling do support our conclusion that high ABHD6 expression is characteristic for late-onset metastatic disease in SF3B1mut UM. The ABHD6 gene maps to chromosome 3 and the possible involvement of  chromosome 3 deletions could play a role in the ABHD6 deficiency and shorter progression-free survival. However, the log2fold changes are much more than expected from simply losing a copy of the chromosome, nor did we observe a significant change of expression in other chromosome 3 genes such as BAP1. We also detected 34 significantly perturbed splicing aberrations with donor and/or acceptor extensions or shortenings between early- and late-onset metastatic SF3B1mut UM. These findings warrant further research in utilizing such biomarkers to develop classification schemes to distinguish SF3B1mut UM patients with potential early- and late-metastatic onset.Using differential expression analysis on canonical transcripts and detection of leukemia ,47. MoreB1mut UM . PreferaSF3B1 mutated UM in literature (n = 146). Patients with SF3B1-mutated UM show early- and late-onset metastatic disease (defined as before or after a follow-up time of 60 months), for which, currently, no biomarkers exist, and for which we identified several promising candidates with potential distinguishing characteristics when implemented within an expression-based classifier. In addition, the largest tumor diameter (LTD) was found to be increased in the early-onset UM. Our results provide more accurate prognostication and targets for future functional studies in an effort to elucidate pathogenesis and clinical stratification of SF3B1-mutated UM.This study describes clinical and genetic characteristics of the largest aggregated cohort of