Isolated local failure (ILF) can occur in patients who initially receive definitive radiation therapy for prostate cancer. Salvage therapy for ILF includes high dose rate (HDR) brachytherapy. Prostate Specific Membrane Antigen (PSMA) Positron Emission Tomography (PET) can accurately detect ILF and can exclude extraprostatic disease. Lutetium-177 PSMA Radioligand Therapy (RLT) is a novel treatment for prostate cancer that can target prostate cancer accurately, while sparing radiation dose to normal tissues.ROADSTER is a phase I/II randomized, single-institution study. Patients with an ILF of prostate cancer after definitive initial radiation therapy are eligible. The ILF will be confirmed with biopsy, magnetic resonance imaging (MRI) and PSMA PET. Patients will be randomized between HDR brachytherapy in two fractions  (cohort 1) or one treatment of intravenous Lutetium-177 PSMA RLT, followed by one fraction of HDR brachytherapy (cohort 2). The primary endpoints for the phase I portion of the study (n = 12) will be feasibility, defined as 10 or more patients completing the study protocol within 24 months of study activation; and safety, defined as zero or one patients in cohort 2 experiencing grade 3 or higher toxicity in the first 6 months post-treatment. If feasibility and safety are achieved, the study will expand to a phase II study  where preliminary efficacy data will be evaluated. Secondary endpoints include changes in prostate specific antigen levels, acute toxicity, changes in quality of life, and changes in translational biomarkers. Translational endpoints will include interrogation of blood, urine, and tissue for markers of DNA damage and immune activation with each treatment.ROADSTER explores a novel salvage therapy for ILF after primary radiotherapy with combined Lutetium-177 PSMA RLT and HDR brachytherapy. The randomized phase I/II design will provide a contemporaneous patient population treated with HDR alone to facilitate assessment of feasibility, tolerability, and biologic effects of this novel therapy.NCT05230251 . Prostate cancer is a major male health issue, with over 1.4 million new cases of prostate cancer diagnosed and 375,000 new prostate cancer deaths in 2020 worldwide [There are several potentially curative treatment options for patients with ILF, including salvage radical prostatectomy (RP), as well as prostate directed ablative therapies such as cryotherapy, high intensity focused ultrasound (HIFU), and salvage radiotherapy options such as stereotactic body radiation therapy (SBRT), low dose rate (LDR) brachytherapy or high dose rate (HDR) brachytherapy. In a recent meta-analysis comparing these options, the chances of recurrence-free survival (RFS) were similar, with approximately half of patients with durable disease control with the different salvage techniques . In a muProstate-specific membrane antigen positron emission tomography (PSMA PET) has been used more recently as a tool to detect prostate cancer recurrence after radiation treatments. In this setting, PSMA PET has been shown to change the management plan in a majority of patients when compared to the management plan based on conventional imaging alone . By idenLu-177 PSMA RLT emits beta particles with a maximum energy of 497 keV, which limits the range of these particles in tissue to less than 2 mm . In compWe hypothesized that a theragnostic combination targeting PSMA may improve outcomes and reduce toxicity in men with suspected ILF after definitive radiotherapy. Specifically, use of a diagnostic PSMA-targeting agent will improve our ability to select men with a high probability of ILF. The use of a PSMA-targeting RLT would then allow the selective treatment of the site of ILF as well as potential extra-prostatic disease not detectable even with the diagnostic PSMA PET imaging. In ROADSTER, we aim to assess safety and feasibility of adding Lu-177 PSMA RLT in combination with HDR brachytherapy for ILF after definitive radiotherapy. A randomized phase I/II design will be used to provide a contemporaneous cohort of men treated with our institutional standard salvage therapy of two fractions of HDR. Translational endpoints will include acquisition of liquid and tissue biospecimens for the assessment of response biomarkers and assessment of DNA damage and immune activation among the two cohorts.Patients with suspected ILF after radiation will be recruited. Patients must have a BCF per Phoenix criteria at least two years after initial prostate cancer treatment, an intra-prostatic lesion with PSMA PET avidity  of 3.0 or greater on a diagnostic PSMA PET/CT), and no evidence of extra prostatic disease.9/L, platelet count less than 100 × 109/L, hemoglobin less than 90 g/L, or transfusions required in the previous two weeks prior to enrolment); inadequate renal function (estimated creatinine clearance less than 30 mL/min by Cockroft Gault equation); or inadequate hepatic function  or the alanine aminotransferase greater than 3.5 times the ULN). Patients must not have any ongoing grade 3 or greater GU or GI toxicity (by Common Terminology Criteria for Adverse Events (CTCAE) v4.0) associated with their initial prostate cancer treatment. Patients with previous ablative prostate radiotherapy, i.e., HDR brachytherapy, LDR brachytherapy or SBRT to the prostate, will be excluded. Concurrent or adjuvant ADT at the time of salvage therapy is not permitted; patients given ADT as part of the original primary radiotherapy are eligible.All patients are required to have a prostate biopsy to confirm ILF at the time of enrolment and have no contraindications for HDR brachytherapy, general anaesthesia, PSMA PET/MRI or Lu-177 PSMA RLT. Contraindications for these procedures include having inadequate marrow function  related to prostate cancer by the Expanded Prostate Cancer Index Composite (EPIC), and baseline bloodwork to assess marrow, renal and hepatic function. Baseline bloodwork and urine samples will be collected as liquid biomarkers for future biomarker analysis. A hybrid PSMA PET/MRI will be done prior to randomization. As part of the PET/MRI, delayed PET images of the pelvis will be acquired at least 60 min after the PSMA ligand injection. MR images of the pelvis will be acquired, including T2 weighted, diffusion weighted, and dynamic contrast enhanced sequences. Imaging will be read by a certified Nuclear Medicine specialist (D.L.) experienced in PSMA PET/MRI and PET/CT interpretation using established interpretation schemes , 12. TheThe trial schema is given in Fig. Brachytherapy will be performed using transrectal ultrasound (TRUS) guidance, under general anesthesia in a dedicated brachytherapy suite, with intraoperative planning. Pre-procedure PSMA PET/MR images will be fused with intra-operative TRUS images using an in-house developed surface-based deformable algorithm to accurately delineate the areas of ILF. For each HDR brachytherapy treatment, 10.5 Gy will be prescribed to the entire prostate, with a focal boost to 13.5 Gy to the clinical target volume of the primary tumor (CTVp), as defined by the gross tumor volume (GTV) on all available images with a 5 mm expansion to account for microscopic disease. Details on brachytherapy planning objectives are found in Table Lu-177 PSMA RLT will be delivered in the Department of Nuclear Medicine as per local practice. One dose of 6.8 GBq of Lu-177 PSMA RLT will be administered intravenously, which will deliver an estimated tumor dose of 13.6–40.8 Gy, given the estimated dose of 2–6 Gy per GBq that has been reported in the literature . PatientFigure ROADSTER is a phase I/II, randomized controlled trial with safety and feasibility as primary outcomes. The planned accrual for the phase I portion of the study will be 12 patients: six patients in cohort 1 and six patients in cohort 2. Safety of each cohort will be declared if no more than one patient in each cohort experiences a grade 3 or higher CTCAE V4.0 toxicity at 6 weeks or 6 months post-treatment. Feasibility will be declared if 10 or more of the planned 12 patients are successfully accrued and complete the study as per protocol within 24 months of activation. If the initial accrual of 12 men across both cohorts demonstrates both safety and feasibility, the study will expand to the phase II portion, in which 30 men in total will be enrolled and randomized between these two cohorts. Sample sizes were chosen to support measuring the safety and feasibility of this study in the phase 1 and 2 portions. No formal statistical sample size calculation was performed.Secondary endpoints will include biochemical response, defined as a decrease of PSA by 50% from the baseline level; acute toxicity, defined by changes in CTCAE v4.0 scores; and changes in EPIC QoL scores. These secondary endpoints will be assessed at baseline, 6 weeks after treatment and 6 months after treatment.At the time of the second HDR treatment for cohort 1 and the HDR treatment for cohort 2, patients will receive a research prostate biopsy via a transperineal approach under ultrasound guidance. Targeted biopsies of PSMA PET/MR defined lesions will be obtained along with systematic biopsies of the whole gland. These tissue samples are obtained for further translational studies and will assess the effects of HDR brachytherapy in cohort 1 and Lu-177 PSMA RLT in cohort 2 on the prostate cancer ILF.Blood and urine samples will be collected prior to the first treatment, prior to the second treatment, at 6 weeks after the second treatment and at 6 months after the second treatment, in both cohorts 1 and 2. Patients will have serum PSA, marrow function, renal function and hepatic function bloodwork at 6 months after the second treatment in both cohorts. CTCAE v4.0 toxicity scores and EPIC QoL scores will be done at 6 weeks and 6 months after the second treatment as well.Regarding the translational biomarkers, the research biopsy specimens will undergo panel-based deoxyribonucleic acid (DNA) sequencing analysis of commonly altered DNA repair pathway and prostate cancer genes, tumor-associated T-cell receptor deep-sequencing (TCR-seq), as well as PSMA expression and gamma-H2A histone family member X (gamma-H2AX) expression, a marker of radiation-induced DNA damage, by immunohistochemistry. Blood plasma will also undergo circulating tumor DNA (ctDNA) sequencing as well as peripheral blood mononuclear cell (PBMC) analysis with TCR-seq and flow cytometry assessing markers of immune activation and radiation-induced DNA damage . Finally, urine will be collected for urine tumor DNA (utDNA) sequencing.As a primary outcome, the ROASDTER trial investigates the feasibility and safety of a novel salvage strategy that combines Lu-177 PSMA RLT with HDR brachytherapy in patients with proven radiorecurrent ILF with no signs of metastatic disease. By adding Lu-177 PSMA RLT as a systemic therapy early in the course of cancer relapse, occult metastatic disease may be treated early, potentially reducing rates of systemic progression, which is the most frequent pattern of relapse in this setting . FurtherGiven the physical and biological characteristics of Lu-177 PSMA RLT, we hypothesize this may have an improved toxicity profile compared with other salvage therapy options. Specifically, in a comprehensive meta-analysis of salvage therapy options, there were lower severe genitourinary (GU) toxicity rates amongst the radiation salvage options compared with the non-radiation options. The severe GU toxicity rates for SBRT, LDR brachytherapy, and HDR brachytherapy were 5.6%, 9.1%, and 9.6%, respectively, while the severe GU toxicity rates for cryotherapy, RP and HIFU were 15%, 21%, and 23%, respectively .As a secondary outcome, this trial investigates disease control associated with a standard salvage treatment option, involving two fractions of prostate HDR brachytherapy, in the PSMA-PET era. Most of the salvage data in the literature precedes PSMA PET use and the ROADSTER trial may provide a new benchmark by using PSMA PET as a screening tool to select patients with lower likelihood of having any occult extra-prostatic disease at the time of diagnosing the ILF. This “stage migration” effect may allow for patients who are included in the trial to have a more favourable recurrence-free survival, with adequate treatment of their ILF site. Moreover, PSMA PET and MR data together may allow for more accurate definition of the recurrent disease in the prostate at the time of HDR prostate brachytherapy boost with a focused boost to the lesion and better OAR sparing , 15. TheAdditional outcomes in ROADSTER trial include translational studies, assessing the effects of Lu-177 PSMA RLT and HDR brachytherapy on the radiorecurrent prostate cancer. Tissue, blood, and urine biospecimens will be collected at during the study. These biospecimens will be interrogated to characterize effects on tumor and normal tissue radiation injury and immune activation and to assess for potential biomarkers of response or toxicity after Lu-177 PSMA RLT and HDR brachytherapy.While out of scope for the current trial protocol, which is focused on safety and feasibility endpoints, our practice for men receiving salvage brachytherapy includes long term biochemical and clinical monitoring. Future plans will include examination of long-term disease control and side effects in this population.While there are several Lu-177 PSMA RLT trials ongoing for patients with CRPC, there are only a few studies that are incorporating Lu-177 PSMA RLT in metastatic castrate sensitive prostate cancer (CSPC):  . FurtherIn summary, the ROADSTER trial is a randomized phase I/II trial investigating safety, feasibility and early efficacy of a novel salvage treatment strategy involving Lu-177 PSMA RLT in conjunction with whole prostate HDR brachytherapy with a focused boost. The randomized phase I/II design will provide a contemporaneous patient population treated with HDR alone to facilitate assessment of feasibility, tolerability, and biologic effects of this novel therapy. Access to MR and PSMA PET imaging will allow for localization of the recurrent disease within the prostate, guidance of focal boost delivery at the time of HDR brachytherapy and the precision acquisition of tissue biospecimens from involved and uninvolved prostate for translational science correlative studies.


We also found evidence for positive selection at fifteen sites across cox1, mtMutS, and nad1 in deep-sea octocorals and nad3 of deep-sea antipatharians. These results contribute to our limited understanding of mt adaptations as a function of depth and provide insight into the molecular response of corals to the extreme deep-sea environment.Corals are a dominant benthic fauna that occur across a vast range of depths from just below the ocean’s surface to the abyssopelagic zone. However, little is known about the evolutionary mechanisms that enable them to inhabit such a wide range of environments. The mitochondrial (mt) genome, which is involved in energetic pathways, may be subject to selection pressures at greater depths to meet the metabolic demands of that environment. Here, we use a phylogenomic framework combined with codon-based models to evaluate whether mt protein-coding genes (PCGs) associated with cellular energy functions are under positive selection across depth in three groups of corals: Octocorallia, Scleractinia, and Antipatharia. The results demonstrated that mt PCGs of deep- and shallow-water species of all three groups were primarily under strong purifying selection (0.0474 < ω < 0.3123), with the exception of positive selection in  Corals have a wide geographical and vertical distribution, and species at greater depths have been able to survive in these environments despite added selective pressures that can otherwise restrict the vertical distribution of shallow-water species6. Several coral groups have been documented to have ranges that extend from the intertidal zone to depths > 6000 m9. However, there is still relatively little known about the evolutionary processes in deep-sea corals that enable their success at greater depths10. Investigating underlying differences at the genomic level may elucidate insight into the molecular adaptations of corals to the extreme deep-sea environment.The deep sea (> 200 m) may impose several selective pressures that necessitate biological and physiological adaptation of resident fauna, such as deep-sea corals, to seemingly harsh conditions that include cold temperatures, high hydrostatic pressure, variable oxygen levels, and limited food11. Across Metazoa, mt genomes are typically composed of 13 protein-coding genes (PCGs) that encode many of the subunits responsible for generating energy through oxidative phosphorylation (OXPHOS)12. Genes within the mt genome have a significant impact on the metabolic pathways and functions of an organism and thus environmental pressures associated with depth have the potential to influence nucleotide substitutions and drive adaptation4. Recent studies of deep-sea marine invertebrates have shown evidence for sites of adaptive evolution within the mt genome. These sites of positive selection include the PCGs atp6, cob, cox1, cox3, and nad1-5 in the alvinocaridid shrimp Shinkaicaris leurokolos—endemic to hydrothermal vent and cold seep environments13—and within atp6, cob, cox2, nad2, nad4, and nad5 of mussels in the subfamily Bathymodiolinae, which inhabit chemosynthesis-based ecosystems 14. While research about mt adaptations in the deep sea has been published, only one study was focused on selection in a deep-sea species within the subphylum Anthozoa—the actiniarian sea anemone Bolocera sp.4. These studies demonstrate that the mt genome is under positive selection in deep-sea species when compared to shallow-water relatives of these groups and thus support the hypothesis of adaptive mt evolution to the selective pressures of the deep-sea environment.Mitochondrial (mt) genomes are widely used within phylogenetic and phylogeographical studies to assess the evolutionary history of taxa16. Anthozoans also include a variety of unique characteristics among their mt genomes. The class Octocorallia  includes an additional PCG unidentified in any other metazoan mt genome, the mtMutS gene17, which is involved in DNA mismatch repair18. Selection tests conducted by Muthye et al.19 found mtMutS to be one of the least conserved PCGs in the mt genome and provided evidence that its presence contributes to the low rates of mt sequence evolution in octocorals . Furthermore, Hogan et al.20 described the first report of a circular bipartite mt genome in Cnidaria within an unknown species of Umbellula octocoral . While it remains unclear why or how multipartite genomes arise, their presence in other Metazoa have invoked hypotheses such as greater gene expression efficiency21 and specialized functions that may be selected for and advantageous in extreme environments23. Two orders of the class Hexacorallia included in this study, Scleractinia  and Antipatharia , exhibit group I introns within the genes of cox1  and nad5, further contributing to the unique set of factors to consider when assessing the adaptive evolution of coral mt genomes25.For most metazoans, the mt genome exhibits faster evolutionary rates than nuclear DNA (nDNA) making it useful as a genetic marker for evolutionary studies. However, anthozoans uniquely defy that standard with observed rates of mitochondrial evolution an order of magnitude slower than other invertebrates and slower than that observed within their nDNAIn this study we present a phylogenomic framework to assess whether genes associated with cellular-energy functions are under positive selection across depth. We performed a series of selection tests on the mitochondrial PCGs for deep-sea and shallow-water species of Octocorallia, Scleractinia, and Antipatharia to evaluate mitogenomic differences between shallow (< 200 m) and deep-sea (> 200 m) habitats. In utilizing this approach, results from this study contribute to the limited understanding of the evolutionary response of corals to high environmental stressors in the deep sea and enable assessment of genes that may contribute to their success in that environment.27. Methods of assembly and annotation are described in detail in Quattrini et al. (in review)28 and Muthye et al.19. Additional mt genomes for octocorals (n = 14) and scleractinians (n = 5) were downloaded from GenBank and included in the dataset. The final individual datasets for each group consisted of 84 species of Octocorallia, 44 Scleractinia, and 13 Antipatharia  and is most common at depths > 200 m according to OBIS (https://obis.org). For each taxonomic group, PCGs were aligned using MAFFT v7.48129 with the L-INS-I method to attain the greatest accuracy and then adjusted by eye using AliView30 to ensure sequences were in frame. Loci were then concatenated with phyluce_align_concatenate_alignments (Phyluce v 1.7)31.The majority of mitochondrial genomes (n = 122) included in this study were captured as off-target reads from the target-capture sequence data in Quattrini et al.32 on 14 concatenated mt PCGs for Octocorallia and 13 mt PCGs for Scleractinia and Antipatharia, with ultrafast bootstrap approximation33 using 1000 replicates. The GTR + G substitution model was selected as best fit for the concatenated nucleotide sequences of Scleractinia mt PCGs based on the recent work by Seiblitz et al.34. The TVM + F + G4 and TVM + F + R5 substitution models were determined to be the best fit models for octocoral and antipatharian mt PCG concatenated sequences by ModelFinder35 within IQtree2. Phylogenetic trees were unrooted for use in the selection tests performed within Codeml36. Phylogenetic trees rooted for visualization can be found in the supplementary material  implemented within IQtree v236 to estimate ω—the ratio of nonsynonymous (dN) to synonymous (dS) nucleotide substitutions. Three pairwise model comparisons were selected following Jeffares et al.37 and separately performed for each individual mt PCG region in each group of corals to assess various scenarios of positive selection. Inserted introns within cox1 and nad5 of Scleractinia and Antipatharia were removed and the flanking exonic regions were combined and evaluated as a single block.Adaptive evolution of each mt PCG of deep-sea and shallow-water octocorals, scleractinians, and antipatharians was separately evaluated using Codeml within the PAML packageone-ratio vs. two-ratio) were used to detect whether deep-sea species, set as the foreground, were more likely to have a different ω from the shallow-water species (background), across branches or specific lineages in the phylogeny. This comparison included the one-ratio (M0) model, which estimated one average ω across the entire gene and served as the null against the two-ratio (M2) model which enabled ω to vary across the foreground and background branches37.The “branch” model comparisons  were compared to estimate positive selection and an ω ratio that was variable among codons (sites) but remained the same across lineages. These models differed from the others applied because detection of positive selection was not foreground-specific37. The nearly neutral (M1a) model, which served as the null, only allowed two classes of sites  while the positive selection (M2a) model additionally allowed for positive selection (ω > 1).The “site” models  were implemented to address more specifically whether mt genomes of deep-sea species were more likely to be under positive selection (ω > 1) than shallow-water corals. These models allow for an ω value that is variable across particular sites and branches. Model A (MA) null allowed only two classes of purifying or neutral selection  for both the foreground and background species. This was compared to the MA alternative which allowed for positive selection (ω > 1) as a third class in the foreground species, while the background was restricted to two classes 38.Lastly, the “branch-site” models (39. Adaptive evolution was inferred across each PCG in the “branch” models if the LRT was significant (p < 0.05) and ω was > 1. In the “branch-site” and “site” models adaptive evolution was inferred if the LRT was significant and sites of positive selection with a posterior probability > 95% were identified by the Bayes Empirical Bayes (BEB) output within those models40.Significant differences between the pairwise model comparisons were determined by a likelihood ratio test (LRT) which identified whether the data better fit parameters of the null or alternative model in each comparison41, occur at depths down to 6100 m. Among octocorals, this group includes some of the deepest-occurring species and has the second greatest number of species represented in the deep sea43. Therefore, other deep-sea octocorals (n = 11) were removed and deep-sea sea pens (n = 14) were exclusively selected as the foreground against shallow-water octocorals (n = 59) to assess whether the signal for adaptive evolution may arise at deeper depths. Secondly, the scleractinian mt alignments included three highly divergent species  belonging to the Micrabaciidae family. In phylogenies constructed from mt genes/genomes, these species are recovered as sister to all other scleractinians, and thus have been deemed the “basal clade”45, but this group is recovered as sister to complex corals in nuclear phylogenies28. To determine whether the incongruent placement of this family affected the outcome of these analyses, the three micrabaciids were excluded from the dataset and models rerun. Furthermore, Scleractinia is the only extant order of anthozoans that includes many colonial and solitary species, while Octocorallia and Antipatharia are primarily and strictly colonial46. To determine the influence of adaptive evolution on solitary vs. colonial corals in the deep sea, the foreground was modified to exclusively test solitary-deep (n = 8) scleractinians against shallow-water Scleractinia (n = 33) by removing colonial-deep representatives (n = 3) from the analysis. The reverse was accomplished by removing the solitary-deep representatives, while the colonial-deep scleractinians exclusively made up the foreground against shallow-water species. The list of sequences involved in these datasets can be found in Supplementary Table Additional analyses were performed to test potential factors influencing the detection of positive selection in mt genomes by modifying the foreground taxa or excluding specific taxa from the alignments before running the same models. Firstly, sea-pens within the superfamily PennatuloideadN/dS) than shallow-water species, revealed all PCGs in the mt genome of octocorals were under purifying selection (ω < 1). Average omega values of each gene ranged from 0.0697 to 0.2964 in the null, M0 model , applied to determine whether deep-sea species were more likely to have a different ω ratio  were performed to identify positive selection along lineages and codons particularly of deep-sea taxa. In deep-sea octocorals, 14 sites of positive selection with a posterior probability > 95% were identified across three genes cox1, mtMutS, and nad1  analysis  were applied to identify sites of positive selection (ω > 1) by assessing whether the data had a better fit to the M2a model, which allowed ω > 1. Sites of positive selection were inferred by the BEB analysis output of PCGs that were significantly different in the site models. Eight positively selected sites across a vs. M2anad4 and nad6, which were no longer significant based on the LRT  when excluding other deep-sea octocorals, with a ω ratio > 1 in deep-sea sea pens  and the background to shallow-water octocorals. The branch models found that all PCGs that were significant in the full Octocorallia analysis, remained significantly different  between deep-sea sea pens and shallow octocorals with the same trends except for LRT Fig. . In addiatp6 was the only gene to be significantly different  between deep-sea and shallow-water antipatharians  in deep-sea species against shallow-water species (ω = 0.0560) undergoing purifying selection. Omega values ranged from 0.0001 to 999 (otherwise infinity) in deep-sea species, with the extreme high value (999) resulting from no synonymous substitutions in atp8 and the extreme low value (0.0001) resulting from no nonsynonymous substitutions in cob, cox3, and nad4l  and cox3  but displayed the opposite relationship, with shallow-water species having significantly higher ω values than deep-sea species  and site-model (M1a vs M2a) analyses signifying that positive selection was undetected even at the codon level. Excluding the highly divergent, basal species  did not affect the outcome of these analyses  in the branch-model analysis. Omega values ranged from 0.0620 to 0.3123 in the M0 model  and cox1  remained significantly likely to display different ω ratios (the same trend) among deep-sea and shallow-water species  higher ω ratio (0.3870) in shallow-water species when deep, solitary Scleractinia were set as the foreground species  mtDNA16 might create a discontinuity between what we know of selection pressures in other metazoan mt genomes versus its influence in corals. By using phylogenies constructed from mt genomes generally congruent with previously published mt-based phylogenies34, we address this knowledge gap and broaden our understanding of adaptation in the deep sea by providing insight into mitochondrial evolution as a function of depth in three diverse coral groups.The geographical expanse of corals from coastal reefs to the abyssal depths of the deep sea allude to their molecular diversity. This is especially true of their mitochondrial genomes which encode for metabolic functions supportive of their success under extreme conditionsatp6) underwent strong purifying selection. The low ω (dN/dS) values along branches in the phylogeny indicated a greater average number of synonymous substitutions revealing that overall evolution favored mutations that did not alter the encoded amino acids. Given the functional metabolic importance of the mt genome in OXPHOS, this strong purifying selection likely acts to prevent the fixation of deleterious mutations55. Additionally, among octocorals deep-sea species displayed higher ω values in all mt PCGs and therefore underwent more relaxed purifying selection in comparison to their shallow-water counterparts.Branch-model analyses indicated that all mt PCGs of deep-sea and shallow-water species within Octocorallia, Scleractinia, and Antipatharia  than in non-Tibetan species50. The observation of purifying selection, yet higher numbers of nonsynonymous mutations, in deep-sea octocorals provides additional evidence for mitochondrial adaptation towards a relaxed purifying selection, even amongst the slow evolution of anthozoan mt genomes that sets them apart from other Metazoa15. Perhaps these nonsynonymous substitutions give selective advantages in deep-sea lineages under extreme environmental conditions.Of the 14 mt PCGs in octocorals, 10 were better fit to the M2 model that allowed for positive selection, indicating that this difference in ω values in deep-sea corals is not random. A similar trend of overall purifying selection, but higher nonsynonymous mutations in deep-sea lineages, has been observed in other deep-sea invertebrates such as bathymodioline mussels, vesicomyid clams, and alvinocaridid shrimp, all of which had representatives from hydrothermal vent environments included in their studiescob, cox1, and cox3) and one (atp6) of the 13 mt PCGs were significantly different between deep and shallow-water species. cob and cox3 had significantly higher ω values in deep-sea lineages of Scleractinia which is consistent with the trend in octocoral PCGs. However, in Scleractinia, cox1 displayed a greater ω value in the shallow-water species, opposing the previous observations in octocorals. Similarly, cox1 also had a higher ω ratio in shallow-water mussels—the only exception to the trend previously described in the bathymodioline mussel mt genome14. cox1, a subunit of cytochrome c oxidase complex IV, functions in facilitating an electron transfer to oxygen as the ultimate acceptor molecule in the generation of ATP through oxidative phosphorylation47. Repetition of this finding in these two studies highlights that cox1 may potentially be under relaxed purifying selection in shallow water lineages because of other stressors not as common in the deep sea, such as oxidative stress, which has been noted to impact shallow scleractinians57. Nevertheless, future studies could explore selection pressures on cox1 in shallow-water scleractinians.In contrast to octocorals, branch-model analysis for Scleractinia and Antipatharia indicated that only three , which transfers the energy created by the proton electrochemical gradient to the F1 region to phosphorylate ADP to ATP58. atp6 was found to have sites of positive selection in studies previously mentioned for the deep-sea anemone Bolocera sp., bathymodioline mussels, and alvinocaridid shrimp14. However, our finding is unique in that it shows deep-sea black coral species are more likely to be under positive selection across atp6 in contrast to only at a few select codon sites. This result encourages further exploration of the evolutionary differences in the atp6 gene in black corals as they were the only group to show positive selection within the branch-models. Notably, black corals are some of the longest living and deepest occurring corals in the deep sea59.The atp8 and nad6. We also found evidence of positive selection more specifically in deep-sea taxa of Octocorallia at 14 sites across cox1, mtMutS, and nad1, and at one site in nad3 of deep-sea Antipatharia. Thus, we present the first evidence for adaptive evolution of octocoral and black coral mt genes in the deep sea through selection analyses. These positively selected sites may influence physiological changes in the bioenergetic functions they encode. nad1, nad3, and nad6 are three of the seven mt-encoded subunits that comprise four membrane-bound proton pumps within the NADH dehydrogenase enzyme (complex I), the first and largest complex in the respiratory chain60. Positively selected sites along the NADH dehydrogenase genes have been detected in all of the mt selection studies of deep-sea invertebrates previously mentioned and in a deep-sea crab, holothurian, and asteroid mt genome54. Combined with our results, there is strong evidence that these genes have important metabolic functions for deep-sea fauna because of continued detection of positively selected sites across diverse taxa. Moreover, positively selected sites in cox1 may be advantageous in deep-sea lineages to support ATP production by OXPHOS to meet high metabolic demands of the deep sea61. The atp8 gene, the second PCG for ATP synthase, further supports the assembly of the F0 region of the enzyme and therefore facilitates ATP production61 Because of its function, substitutions in genes encoding for complex V may have significant impacts on the metabolic capacities of an organism to adapt to the bioenergetic demands of extreme environments.Although purifying selection was depicted as the predominant driver of mitochondrial evolution in the branch-models of octocorals, the site-models indicated that positive selection occurred at eight sites within mtMutS in mt genomes of octocorals are of growing interest among researchers63. Most recently, using the same data sequences applied here  to capture mt genomes, Muthye et al.19 demonstrated a putative loss of mtMutS in Pseudoanthomastus sp. 1 and a higher rate of mt-sequence evolution compared to closely related species with mtMutS. This supports the ongoing case for the function of mtMutS in DNA repair63. The results of our study further demonstrate that the mtMutS gene is significantly more likely to contain sites of positive selection in deep-sea lineages of octocorals based on a significant LRT of the branch-site model. Perhaps the metabolic requirements of the deep sea demand greater DNA repair capabilities and/or functional requirements of mtMutS.Lastly, the unique presence and functionality of 65. This would support why genes with positively selected sites also displayed strong purifying selection in the branch-models. However, the atp6 gene of Antipatharia was significantly more likely to have an average ω > 1 across deep-sea lineages based on the branch-models. atp6 was therefore expected to have sites of positive selection in the branch-site models since the gene overall had an average ω under positive selection. The BEB analysis identified one site of positive selection  in deep-sea Antipatharia with a posterior probability > 95%, yet the LRT was not significant. A statistical study on accurately detecting sites of positive selection revealed that when 10% of sites are under strong positive selection, a 30-taxon tree input within Codeml correctly identifies over 75% of positively selected sites while a 5-taxon tree only detects about 20% of positively selected sites66. This suggests that our smaller sample size for black corals (n = 13) could be limiting the detection of positively selected sites in the branch-site model. However, there are only an estimated 273 species within the order Antipatharia25 in comparison to the over 3500 and 1500 species within Octocorallia and Scleractinia67. Although our black coral tree included the fewest taxa, it had the highest sampled proportion of species (4.76%) among these groups and nonetheless conveyed the bathymetric expanse of the order by including the shallowest and deepest species for which the mt genomes were available. The impressive depth distribution of Antipatharia and limited knowledge of this group encourages further analysis of their adaptive evolution as more evidence may be uncovered with the inclusion of more black coral mt genomes.Incongruence was evident between the results of the branch-site and site models applied in this study. However, this can be attributed to episodic selection, meaning positive selection may only be acting on specific sites and lineages which could be overshadowed in the branch-models by the overall purifying selection occurring across the genome68. It is possible that signals for positive selection may arise at deeper depths and the inclusion of the upper bathyal fauna may obscure their detection. We selected a subset of octocorals that included 14 deep-sea sea-pens, generally regarded as deeper dwelling species within the group43, to test for variability in the results. Results revealed that nad4l had an average ω > 1 in deep-sea sea-pens, which was not detected when other deep-sea octocorals were included in the foreground. Additionally, there were eight more sites of positive selection detected within Pennatuloidea in branch-site analysis, but three fewer in the site-model analysis. This presents a future avenue for positive selection analyses to be conducted across various depth ranges to map changes in adaptive evolution associated with environmental extremes, such as in the abyss.In this study, deep-sea corals were generalized as species inhabiting > 200 m depth, yet we know that corals can be found down to abyssal depthsatp6 in black corals and nad4l in sea pens were exceptions to this trend with average ω values indicative of positive selection. Sites of positive selection were located at 14 codon sites in cox1, mtMutS, and nad1 of deep-sea octocorals and at one site in nad3 of deep-sea antipatharians supporting the influence of selective pressures in the deep-sea environment acting on these genes. Lastly, analyses presented within this study emphasize the variability among adaptive evolution in the mt genome in different taxonomic groups of corals. The ability of corals to inhabit such an extensive range of depths is in large part due to the metabolic functions encoded by the mitochondrial genome. This study provides further insight into specific mitochondrial PCGs under adaptive evolution in Octocorallia, Scleractinia, and Antipatharia inhabiting the deep sea.The results of this study strongly support that the evolution of mt PCGs of deep and shallow- water species of Octocorallia, Scleractinia, and Antipatharia is mainly driven by purifying selection, suggesting that mitochondrial function within corals is highly conserved. However, Supplementary Information.Supplementary Table 1.


When brain cancer relapses, treatment options are scarce. The use of molecularly matched targeted therapies may provide a feasible and efficacious way to treat individual patients based on the molecular tumor profile. Since little information is available on this strategy in neuro-oncology, we retrospectively analyzed the clinical course of 41 patients who underwent advanced molecular testing at disease relapse.BRAF-V600E mutation. In selected patients, whole exome sequencing was conducted.We performed Sanger sequencing, targeted next generation sequencing, and immunohistochemistry for analysis of potential targets, including programmed death ligand 1, cyclin D1, phosphorylated mechanistic target of rapamycin, telomerase reverse transcriptase promoter mutation, cyclin-dependent kinase inhibitor 2A/B deletion, or IDH) wildtype glioblastoma. Molecular analysis revealed actionable targets in 31 of 41 tested patients and 18 patients were treated accordingly (matched therapy group). Twenty-three patients received molecularly unmatched empiric treatment (unmatched therapy group). In both groups, 16 patients were diagnosed with recurrent IDH wildtype glioblastoma. The number of severe adverse events was comparable between the therapy groups. Regarding the IDH wildtype glioblastoma patients, median progression-free survival (mPFS) and median overall survival (mOS) were longer in the matched therapy group .The investigation included 41 patients, of whom 32 had isocitrate dehydrogenase (These encouraging data provide a rationale for molecularly matched targeted therapy in glioma patients. For further validation, future study designs need to additionally consider the prevalence and persistence of actionable molecular alterations in patient tissue.The online version contains supplementary material available at 10.1007/s00432-022-04050-w. Novel systemic treatment options are urgently needed in the field of neuro-oncology. For glioblastoma patients, clinical outcome remains poor and effective systemic treatment options beyond temozolomide are scarce. Especially at disease recurrence, the standards of care are not well defined  Classification of Tumors of the Central Nervous System, a set of molecular markers has been implemented in clinical routine  at the Institute of Neuropathology of the University Hospital Essen or by an external provider of genetic diagnostics and sequencing services.All patients with recurrent disease under oncologic treatment at the Division of Clinical Neurooncology, Department of Neurology at the University Hospital Essen, who underwent advanced molecular testing from January 2017 until December 2020 were considered eligible for this retrospective analysis. The following inclusion criteria had to be met:Decisions to perform advanced molecular analysis for patients with recurrent glioma were made by treating physicians. Data were discussed in the molecular tumor board of the University Hospital Essen and treatment recommendations were based on the criteria for molecularly matched targeted therapy (see below). As most targeted treatments do not have a label for brain cancer, approval for reimbursement by health insurances was requested for each individual patient, which requires an additional medical review by insurances. Before starting therapy, we extensively informed patients about their individual baseline situation, the side effect profile of the corresponding drug, and the available treatment alternatives in detailed individual visits. In a few patients, Karnofsky Performance Score (KPS) was 50% and almost exclusively driven by motor deficits (such as hemiparesis or nonfluent aphasia), whereas the cognitive function was well preserved allowing for the patients to provide reliable informed consent. These patients were considered to receive a further line of treatment, if an estimated life expectancy of at least six months was to be assumed. Treatment response was determined according to the updated response assessment criteria for high-grade gliomas .TERT) promoter, FISH for cyclin-dependent kinase inhibitor 2A/B (CDKN2A/B) deletion, and panel-based NGS including key genetic alterations associated with tumor proliferation such as BRAF-V600E mutation. Furthermore, whole exome sequencing (WES) using Illumina’s NovaSeq 6000 system with a read length of 2 × 100 base pairs and an output of 12 GB per sample with matched WES from blood (germ-line control) were conducted in 18 patients.We performed immunohistochemistry on programmed death ligand 1 (PD-L1), phosphorylation of mechanistic target of rapamycin (p-mTOR), and cyclin D1, Sanger sequencing for telomerase reverse transcriptase  had been reported in the past  and overall survival (OS), the time period of interest extended from the time point of latest MRI-defined recurrence before onset of investigated therapy until the next MRI indicating repeat recurrence. Before the initiation of treatment, we ruled out putative pseudoprogression by subsequent MRI and/or positron emission tomography (PET) imaging according to the updated response assessment criteria for high-grade gliomas  wildtype glioblastoma. All patient characteristics are listed in Tables IDH wildtype glioblastoma patients, 56% in the matched therapy group versus 63% in the unmatched therapy group received no additional treatment during the therapy line of interest.In total, 41 patients received advanced molecular analysis, of whom 18 (44%) received a molecularly matched targeted treatment recommendation (henceforth defined as the matched therapy group) and 23 (56%) were treated according to a molecularly unmatched empiric treatment decision (henceforth defined as the unmatched therapy group). In both groups, 16 patients were diagnosed with recurrent isocitrate dehydrogenase  and MET was used in patients with MET amplification, as it was shown to be effective in the treatment of lung cancer  and ROS1, was administered in patients with ALK rearrangement pursuant to the findings observed in lung cancer  inhibitor, was administered in patients with homozygous CDKNA2/B deletion due to published evidence from its use in metastatic breast cancer  , was used in the presence of an EGFR mutation as had been used previously in the successful treatment of lung cancer  patients had an actionable molecular target and 18 (44%) were treated with a molecularly matched targeted therapy, whereas 23 (56%) were treated according to a molecularly unmatched empiric treatment decision instead  were diagnosed with IDH wildtype glioblastomas and received treatment with the combination of dabrafenib plus trametinib at fourth disease recurrence (patient with the second longest treatment duration) and at first disease recurrence (patient with the third longest treatment duration).There were three patients in the matched therapy group with treatment durations of at least 24 weeks (six months). Two of these patients received treatment with the combination of dabrafenib plus trametinib and one patient received treatment with cabozantinib. The patient treated with cabozantinib had the longest duration of treatment in the matched therapy group (48 weeks), he was diagnosed with an IDH wildtype glioblastoma patients was 3.8 months in the matched therapy group and 2.0 months in the unmatched therapy group  for the IDH wildtype glioblastoma patient at first recurrence. The poorest outcome was noted for patients treated with palbociclib. Two of three patients who received a combination of dabrafenib plus trametinib and all patients who received a single-drug treatment with cabozantinib or osimertinib had longer PFS durations than mPFS (3.8 months) in the matched therapy group.As shown in Fig. IDH wildtype glioblastoma patient after treatment with cabozantinib at second disease recurrence (+ 10.1 months). All cabozantinib-treated patients and two of three patients treated with the combination of dabrafenib plus trametinib had a longer PFS under molecularly matched targeted therapy. The most prominent PFS shortening could be observed for a patient after treatment with eribulin (-10.1 months). The individual PFS differences for the matched therapy group are shown in Fig. Individual PFS differences between the treatment line in which the molecularly matched targeted therapy was administered and the treatment line immediately before revealed a longer PFS under molecularly matched targeted therapy in seven patients with the largest extension in an Treatment-related death occurred neither in the matched therapy group nor in the unmatched therapy group. The number of adverse events of CTCAE grade III or higher was comparable in both groups. Table Our pilot data indicate that molecularly matched targeted brain tumor treatment is well tolerated and associated with prolonged disease control and longer survival compared to patients who received molecularly unmatched empiric therapy or patients with tumors lacking those molecular markers and genomic alterations that were selected for this study. Our retrospective analysis considered in an unbiased way every local glioma patient at disease recurrence whose tumor tissue received advanced molecular diagnostics from January 2017 until December 2020. We observed actionable molecular alterations in 76% of the cases, of whom 44% received molecularly matched targeted treatment. For other solid cancer entities, actionable targets were detected in 26% (pancreatic cancer), 47% (extrahepatic cholangiosarcoma), and 40%  of investigated patients  recurrent tumors  inhibitor approved for the adjuvant treatment of melanoma with BRAF-V600E or -V600K mutations. There are early indications from other reports that these two drugs act favorably in glioblastoma patients  and recurrent  tumor tissue samples from 50 patients with Lastly, limitations inherent to the performed analysis should be mentioned. The retrospective study design, small sample size and heterogeneous cohort, inconsistent disease stages, lack of randomization, and diverse types of administered drugs cannot allow simplified conclusions on efficacy. Furthermore, it has to be questioned if the presence of a distinct actionable molecular target is linked per se with a favorable prognosis in brain cancer. For future studies, a putative selection bias has to be ruled out by a matched-pair control cohort. Furthermore, it remains unknown whether a specific target represents the real tumor driver in a specific tumor entity.However, we have observed a thorough effect of molecularly matched targeted therapy. The data from our pilot study provide a very reasonable rationale for follow-up of a larger cohort of molecularly stratified glioblastoma patients in a prospective controlled trial.Supplementary file1 (DOCX 27 kb)Supplementary file2 (DOCX 29 kb)Below is the link to the electronic supplementary material.


Genome-wide identification of cystathionine beta synthase genes in wheat and its relationship with anther male sterility under heat stress”, it should be “Genome-wide identification of genes encoding cystathionine beta synthase domain-containing proteins in wheat and its relationship with anther male sterility under heat stress”.In the published article, there was an error in the article title. Instead of “In the published article, there was an error in the legend forTaCBS members in wheat.“aLength of the amino acid sequence. bMolecular weight of the amino acid sequence. cIsoelectric point of the TaCBS proteins. dNumber of transmembrane domains, as predicted by the TMHMM server. eProtein subcellular localization prediction by the BUSCA web server. The bold values indicate the selected transcript representing this gene. All TaCBS genes in the table indicate genes encoding cystathionine beta synthase domain-containing proteins.”In the published article, there was an error in the legend for TaCBS members are mapped to 21 chromosomes . Green colored lines represent the connections between duplication events. Megabase pairs (Mb) are used to measure the scale.”“In the published article, there was an error in the legend for TaCBS. On the chromosome’s outermost side are the TaCBS members names.”“In the published article, there was an error in the legend for “In the published article, there was an error in the legend for “In the published article, there was an error in the legend for TaCBS members and brownish yellow round shapes corresponding to predicted miRNAs.”“In the published article, there was an error in the legend for “Supplementary Material. As requested by the reviewer, we have modified Figure S1 and the legend of Figure S1 within Presentation 1. The correct figure and legend appears below.In the published article, there was an error in the “Figure S1 Results of classification and conserved domains predicted using the SMART web site.The CBS domain, the transmembrane region, the Phox and Bem1 (PB1) domain and the complexity region are represented by the pentagonal element, the blue column, the triangular element and the pink rectangular element, respectively. Other domains include DUF21, voltage chloride channel (Voltage CLC), and Carbohydrate binding domain (CBD). The classification according to one pair or two pairs of CBS domains (CBSX and CBSCBS). Other classifications are named by the other structural domains they contain for labeling, such as CBSCLC, CBSCBSCBD, CBSDUF1 and CBSCBSPB1. The 66 identified members corresponding to this protein are shown in parentheses.”Abstract. As requested by the reviewer, we have modified the text. This sentence previously stated:A correction has been made to the TaCBS gene has a very important connection with the heat-stressed sterility process in wheat, and these data provide a basis for further insight into their relationship.”“Together with anther phenotypes, paraffin sections, starch potassium iodide staining, and qRT-PCR data, we hypothesized that the The corrected sentence appears below:“Together with anther phenotypes, paraffin sections, starch potassium iodide staining, and qRT-PCR data, we hypothesized that the genes encoding CDCPs has a very important connection with the heat-stressed sterility process in wheat, and these data provide a basis for further insight into their relationship.”Introduction, paragraph 4. As requested by the reviewer, we have modified the text.A correction has been made to These sentences previously stated:“In addition, research on the CBS gene family has mainly concentrated on soybean, rice, cotton and Arabidopsis , but there is no report on wheat. Therefore, considering the above studies, we speculate that CBS gene may play an important role in regulating fertility, especially in indehiscence of sterile anther in wheat, and these issues are worthy of further exploration. In this study, the genome-wide members of the wheat CBS gene family were identified by bioinformatics methods, and the physiological and biochemical properties, conserved motifs, cis-elements, gene collinearity and gene expression patterns of all family members were comprehensively analyzed. qRT-PCR was performed for the expression patterns of 6 TaCBS genes in wheat anther indehiscence under high-temperature stress conditions. These results provide a theoretical basis and technical reference for further analysis of the functional roles of the CBS gene family in wheat male sterility.”The corrected sentence appears below:Arabidopsis , but there is no report on wheat. Therefore, considering the above studies, we speculate that genes encoding CDCPs may play an important role in regulating fertility, especially in indehiscence of sterile anther in wheat, and these issues are worthy of further exploration. In this study, the genome-wide members of the wheat gene family encoding CDCPs were identified by bioinformatics methods, and the physiological and biochemical properties, conserved motifs, cis-elements, gene collinearity and gene expression patterns of all family members were comprehensively analyzed. qRT-PCR was performed for the expression patterns of 6 TaCBS genes in wheat anther indehiscence under high-temperature stress conditions. These results provide a theoretical basis and technical reference for further analysis of the functional roles of the gene family encoding CDCPs in wheat male sterility.”“In addition, research on the gene family encoding CDCPs has mainly concentrated on soybean, rice, cotton and Materials and Methods, Phylogenetic tree analysis of wheat CBS gene family heading. The heading has been corrected to Phylogenetic tree analysis of wheat gene family encoding CDCPs.A correction has been made to Materials and methods, Phylogenetic tree analysis of wheat gene family encoding CDCPs. As requested by the reviewer, we have modified the text.A correction has been made in This sentence previously stated:“MEGA-X software was used to construct a phylogenetic tree of the CBS gene family from the above plant species and wheat.”“The corrected sentence appears below:“MEGA-X software was used to construct a phylogenetic tree of the gene family encoding CDCPs from the above plant species and wheat.”Materials and methods, Analysis of protein domain, exon and intron structure, and conserved motif of CBS gene family heading. This heading has been corrected to Analysis of protein domain, exon and intron structure, and conserved motif of CDCPs encoded by gene family.A correction has been made to the Materials and methods, Cis-element prediction of CBS gene family heading. The heading has been corrected to Cis−element prediction of gene family encoding CDCPs.A correction has been made to the Materials and methods, Transcriptomic data analysis of wheat CBS gene family heading. The heading has been corrected to Transcriptomic data analysis of wheat gene family encoding CDCPs.A correction has been made to the Materials and methods, Transcriptomic data analysis of wheat gene family encoding CDCPs.In the published article, there was in error in The sentences previously stated:“TBtools software was used to perform log normalization on the downloaded TPM (Transcripts Per Kilobase Million) values, and to draw the expression heatmap of wheat CBS genes.”The corrected sentence appears below:“TBtools software was used to perform log normalization on the downloaded TPM (Transcripts Per Kilobase Million) values, and to draw the expression heatmap of wheat genes encoding CDCPs.”Materials and methods, Prediction of putative miRNAs targeting TaCBS genes heading. The heading has been corrected to Prediction of putative miRNAs targeting genes encoding CDCPs.A correction has been made to the Materials and methods, Prediction of putative miRNAs targeting genes encoding CDCPs.A correction has been made in This sentence previously stated:TaCBS genes was generated by Cytoscape_v3.9.1 .”“The miRNA target network map for The corrected sentence appears below:“The miRNA target network map for genes encoding CDCPs was generated by Cytoscape_v3.9.1 .”Results, Identification characterization and phylogenetic analysis of CBS genes in wheat heading. The heading has been corrected to Identification, characterization and phylogenetic analysis of genes encoding CDCPs in wheat.A correction has been made to Results, Sequence features of gene structure, motifs and conserved domains of the TaCBS gene family, paragraph 1.A correction has been made to These sentences previously stated:TaCBS gene family proteins showed that these proteins can bedivided into two major branches;”“The phylogenetic tree of 66 TaCBS genes, with 8 being the most , CBSCBS (11proteins), CBSCBSCBD (6 proteins), CBSCLC (5 proteins), CBSDUF1 (1protein) and CBSCBSPB1 (18 proteins). More details are available in The corrected sentences appear below:“The phylogenetic tree of 66 CDCPs showed that these proteins can be divided into two major branches;”TaCBS members, with 8 being the most , CBSCBS1-11 (11proteins), CBSCBSCBD1-6 (6 proteins), CBSCLC1-5 (5 proteins), CBSDUF1 (1protein) and CBSCBSPB1-1-18 (18 proteins). More details are available in Results, Chromosome distribution and gene duplication of wheat TaCBS gene family, paragraph 1.A correction has been made to These sentences previously stated:TaCBS genes are uneven on 21 wheat chromosomes. Chromosomal localization of TaCBS genes is displayed in “The distribution and density of “These homologous genes have homologous sites on three or two partial homologous chromosomes, indicating that the wheat TaCBS gene has a large number of homologous sites, showing a high homology retention rate. The conserved positions of these fragment replication regions located on different chromosomes suggests that fragment replication events play an important role in the expansion of the number of TaCBS genes in wheat.”The corrected sentences appear below:TaCBS members were tandemly distributed. There are 1 , 2 (in 4D), 3 , 4 , 5  and 6  TaCBS members in different chromosomes, respectively.”“The distribution and density of genes encoding CDCPs are uneven on 21 wheat chromosomes. Chromosomal localization of genes encoding CDCPs is displayed in TaCBS members in wheat.”“These homologous genes have homologous sites on three or two partial homologous chromosomes, indicating that the wheat genes encoding CDCPs has a large number of homologous sites, showing a high homology retention rate. The conserved positions of these fragment replication regions located on different chromosomes suggests that fragment replication events play an important role in the expansion of the number of Results, Localization and synteny of the TaCBS genes in the wheat genome heading. The heading has been corrected to Localization and synteny of the genes encoding CDCPs in the wheat genome.A correction has been made to the Results, Localization and synteny of the genes encoding CDCPs in the wheat genome, paragraph 1.A correction has been made to These sentences previously stated:“We analyzed the collinearity of these TaCBS genes in the wheat genome by using the Bio-linux system with the two-way blast comparison analysis and the MCScanX tool, and a total of 52 pairs of collinearity genes were identified.”The corrected sentence appears below:“We analyzed the collinearity of these genes encoding CDCPs in the wheat genome by using the Bio-linux system with the two-way blast comparison analysis and the MCScanX tool, and a total of 52 pairs of collinearity genes were identified.”Results, Strong purifying selection for the TaCBS gene pairs in wheat.A correction has been made to This sentence previously stated:“A further analysis of these replicated gene pairs revealed the Ka/Ks values of all wheat TaCBS genes were less than 1, with a maximum value of 0.451.”The corrected sentence appears below:TaCBS members were less than 1, with a maximum value of 0.451.”“A further analysis of these replicated gene pairs revealed the Ka/Ks values of all wheat Results, Synteny analysis of TaCBS genes between wheat and four representative pplant species heading. The heading has been corrected to Synteny analysis of genes encoding CDCPs between wheat and four representative plant species.A correction has been made to Results, Expression pattern analysis of wheat CBS gene in different tissues heading. The heading has been corrected to Expression pattern analysis of wheat genes encoding CDCPs in different tissues.A correction has been made to the Results, Expression pattern analysis of wheat genes encoding CDCPs in different tissues, paragraph 1.A correction has been made to This sentence previously stated:TaCBS genes showed different expression patterns in roots, stems, leaves, spikelets, and grains.”“According to the expression data of the heatmap, we found The corrected sentence appears below:“According to the expression data of the heatmap, we found genes encoding CDCPs showed different expression patterns in roots, stems, leaves, spikelets, and grains.”Results, Expression pattern analysis of wheat genes encoding CDCPs in different tissues, paragraph 2.A correction has been made to These sentences previously stated:TaCBS genes of group 1 showed high expression in all tissues, especially in roots, stems, spikelets and grains.”“The TaCBS genes play an important role in wheat growth and development, which also implies that there may be a certain degree of biofunctional differentiation among different TaCBS members.”“This suggests that The corrected sentences appear below:“The genes encoding CDCPs of group 1 showed high expression in all tissues, especially in roots, stems, spikelets and grains.”“This suggests that genes encoding CDCPs play an important role in wheat growth and development, which also implies that there may be a certain degree of biofunctional differentiation among different TaCBS members.”Results, Identifying miRNA targets for TaCBS genes throughout the genome, paragraph 1, These sentences previously stated:A correction has been made to Supplementary Table 10).”“In order to better understand the posttranscriptional alteration of TaCBS genes by miRNAs, we identified 29 miRNAs that target 41 genes .”“In order to better understand the posttranscriptional alteration of Results, The connection between wheat CBS family and another sterility initiated by high temperature and qRT-PCR investigation, paragraph 2.A correction has been made to These sentences previously stated:TaCBS gene in wheat, we investigated the expression levels of TaCBS gene in Normal and HT-ms anthers at the mononuclear and trinuclear stages.”“To further explore the function of TaCBS8 gene showed elevated expression at both the mononuclear and trinuclear stages of HT-ms anthers compared with Normal anthers (“In contrast, the The corrected sentence appears below:“To further explore the function of genes encoding CDCPs in wheat, we investigated the expression levels of these genes in Normal and HT-ms anthers at the mononuclear and trinuclear stages.”TaCBS8 member showed elevated expression at both the mononuclear and trinuclear stages of HT-ms anthers compared with Normal anthers (“In contrast, the Discussion, paragraph 4.A correction has been made to These sentences previously stated:“The 66 CBS genes in this study were unequally distributed on 21 chromosomes, with chromosomes 3A, 3B, and 3D being the most abundant Figure 3“These results suggest that the TaCBS genes shared by these several species may have been highly conserved during evolution.”TaCBS38 and TaCBS35, which are homologous pairs in all three species except barley, showed a highly expressed state in roots, stems, leaves, spikelets, and grains (“In addition, the genes of d grains Figure 7The corrected sentences appear below:TaCBS members in this study were unequally distributed on 21 chromosomes, with chromosomes 3A, 3B, and 3D being the most abundant (“The 66 abundant Figure 3TaCBS members shared by these several species may have been highly conserved during evolution.”“These results suggest that the TaCBS38 and TaCBS35, which are homologous pairs in all three species except barley, showed a highly expressed state in roots, stems, leaves, spikelets, and grains (“In addition, the members of d grains Figure 7Discussion, paragraph 5.A correction has been made to This sentence previously stated:“In the present study, most of the CBS genes identified had these JA and IAA-related cis-acting elements in the upstream region Figure 6The corrected sentence appears below:TaCBS members identified had these JA and IAA-related cis-acting elements in the upstream region (“In the present study, most of the m region Figure 6Discussion, paragraph 6.A correction has been made to This sentence previously stated:TaCBS genes being targeted by these miRNAs.”“In the present study, a total of 29 putative tae-miRNAs have been identified, with 41 The corrected sentence appears below:TaCBS members being targeted by these miRNAs.”“In the present study, a total of 29 putative tae-miRNAs have been identified, with 41 Discussion, paragraph 7.A correction has been made to These sentences previously stated:“According to qRT-PCR results, five CBS genes were underexpressed in the mononuclear and trinuclear anthers of HT-ms compared with Normal anthers. The TaCBS8 gene showed an elevated expression trend in both the mononuclear and trinuclear stages of sterile anthers compared with the same period in Normal anthers, especially showing a highly significant difference in the mononuclear stage (TaCBS35 and TaCBS38 had almost identical expression trends (“Another example is that the genes n trends Figure 2TaCBS genes in the expression of HT-ms anthers in the present study suggests an association of CBS genes with sterility caused by high-temperature induction in wheat.”“Similarly, the down-regulation of the majority of The corrected sentences appear below:TaCBS members were under-expressed in the mononuclear and trinuclear anthers of HT-ms compared with Normal anthers. The TaCBS8 member showed an elevated expression trend in both the mononuclear and trinuclear stages of sterile anthers compared with the same period in Normal anthers, especially showing a highly significant difference in the mononuclear stage (“According to qRT-PCR results, five TaCBS35 and TaCBS38 had almost identical expression trends (TaCBS8 member is not involved in synteny, whereas the TaCBS35 and TaCBS38 members are a pair of paralogous homologs and are orthologous homologous to other species (“Another example is that the members n trends Figure 2“Similarly, the down-regulation of the majority of genes encoding CDCPs in the expression of HT-ms anthers in the present study suggests an association of genes encoding CDCPs with sterility caused by high-temperature induction in wheat.”Conclusions.A correction has been made to These sentences previously stated:“In this study, we identified 66 CBS genes in wheat.”TaCBS genes are conservative and diversified.”“Based on protein motifs, gene structure, chromosomal location, Ka/Ks analysis, cis-acting elements, putative miRNAs analysis, synteny analysis and expression pattern analysis, TaCBS genes were identified, and the analysis of the regulatory relationships between these miRNAs and TaCBS gene interactions further increased our understanding of TaCBS genes.”“Twenty-nine miRNAs targeting 41 “These outcomes indicate that the wheat CBS gene family may have some relationship with high temperature-induced male sterility.”“In addition, the abnormal expression of these CBS genes may be one of the reasons why HT-ms anthers develop small and with no dehiscence, which may be one of the factors that eventually lead to anther abortion.”The corrected sentences appear below:“In this study, we identified 66 genes encoding CDCPs in wheat.”TaCBS members are conservative and diversified.”“Based on protein motifs, gene structure, chromosomal location, Ka/Ks analysis, cis-acting elements, putative miRNAs analysis, synteny analysis and expression pattern analysis, TaCBS members were identified, and the analysis of the regulatory relationships between these miRNAs and TaCBS gene interactions further increased our understanding of TaCBS genes.”“Twenty-nine miRNAs targeting 41 “These outcomes indicate that the wheat gene family encoding CDCPs may have some relationship with high temperature-induced male sterility.”TaCBS members may be one of the reasons why HT-ms anthers develop small and with no dehiscence, which may be one of the factors that eventually lead to another abortion.”“In addition, the abnormal expression of these The authors apologize for these errors and state that these do not change the scientific conclusions of the article in any way. The original article has been updated.


The present study conducts a comprehensive study on heat-treatment-induced cracking of Inconel 738 (IN738) alloy fabricated by laser powder bed fusion (LPBF) using scanning electron microscopy (SEM), energy dispersion spectrum (EDS), and electron backscatter diffraction (EBSD). The results indicate that the macroscopic crack is dominantly triggered by the strain-age cracking mechanism and propagates along grain boundaries. The initiation of cracking is facilitated by the superimposition of residual stress induced by the LPBF process and contraction stress induced by precipitation, while the reopening of compress pores at grain boundaries weakens the grain boundaries and provides fast channels for cracking. These results revealed the coupling effects in triggering heat-treatment-induced cracking, offering a fundamental guideline for crack control during heat treatment of additively manufactured IN738 alloy. The IN738 alloy is widely used in aerospace gas turbine components in place of heat-resistant steels and IN718 ,3. The rThe primary additive manufacturing technologies adopted for IN738 include laser metal deposition (LMD), electron powder bed fusion (EPBF), and LPBF. The LMD is a process that involves producing melt tracks via relative movement between the deposition source and the workpiece . Althoug3 to 139 J/mm3 by evading the keyhole effect and lack of fusion [Although high-alloyed IN738 exhibits advanced mechanical properties, it also endures high crack sensitivity, which is further exacerbated by the high residual stresses and temperature gradients introduced by the LPBF process ,17. Mostf fusion . During f fusion . Thus, hf fusion . Other rf fusion . Furtherf fusion , while hf fusion ,24. HoweThe primary cracking modes in LPBF IN738 include solidification cracking, liquation cracking, ductility dip cracking, and strain-age cracking ,26,27. SThis study investigated the cracking behavior of the IN738 alloy that underwent heat treatment at 1130 °C for 3 h. A detailed microstructure characterization before and after heat treatment was systematically conducted, with extra care taken on the heat treatment induced cracking. Based on the results, mechanisms of heat treatment induced cracking in LPBF IN738 alloy and the effects of precipitate, pores, and residual stress on the cracking were discussed.In this study, spherical IN738 powders with diameters of 15–45 μm were used as raw materials. The chemical composition of powders is shown in 3, were subjected to mechanical grinding and polishing. The vertical planes of the AB and HT samples were ground with sandpaper ranging from 600 to 4000 grit and then polished to a smoothness of 0.05 μm on polishing clothes. The grain and precipitate characteristics were observed using a TESCAN scanning electron microscope (SEM), coupling with a backscattered electron (BSE) detector. The energy dispersive X-ray spectroscopy (EDS) analysis was performed with SEM equipment to analyze the chemical composition and distribution of elements. The electron backscatter diffraction (EBSD) measurements were performed with EDAX equipment mounted on the SEM equipment with a step size of 1 μm. The EBSD test results were processed and analyzed by Aztec software (Version 2.1). The specimens for transmission electron microscopy (TEM) analysis were prepared by the twin-jet electropolisher (TJ100-SE) with a solution of 10 vol.% perchloric acid and 90 vol.% ethanol at the temperature of −30 °C. The TEM characterization was carried out on JEOL JEM-F200 with a voltage of 200 kV. For the evaluation of mechanical properties, the Vickers hardness of the samples was measured using QNESS Q10 CHD Master+ with a load of 5 kg. The residual stresses in the samples were determined by X-ray diffraction using Proto LXRD, with the utilization of Mn Kα radiation operated at 30 kV and 25 mA.To observe the microstructure, the samples, with dimensions of 10 × 10 × 10 mmThe heat treatment for IN738 is generally designed for dissolving the Laves phase and generating precipitates. The chemical compositions of AB and HT samples were further characterized by EDS and listed in It is worth mentioning that compared to the AB sample, a number of spherical pores can be observed in the HT sample with random distribution with respect to BD, as shown in the inset of 14 mm−2, while the HT sample exhibited an average GND of 2.3 × 1014 mm−2, suggesting that the heat-treatment process does not significantly alter the GND density. Furthermore, the high dislocation density regions are mainly localized at grain boundaries in grains with relatively high length-to-width ratios and present a tendency to be parallel to the BD.To clarify the mechanism of the heat-treatment-induced crack in Considering the features of crack propagation along the grain boundaries, the uncracked grain boundaries in the HT sample were further characterized, as shown in The AB sample was crack-free and had low porosity in the present study. After heat treatment at 1130 °C for 3 h, which is generally utilized as a solid solution process to dissolve Lave phases, macroscopic cracking occurred in the HT sample with the porosity increased simultaneously. The crack microstructure did not exhibit features of liquation or resolidification. Thus, solidification cracking and liquation cracking mechanisms can be ruled out. The results showed that cracking propagated along tortuous grain boundaries, accompanied by large-sized γ’ phase and elemental enrichment of Ti and Al on the crack surface. It is suggested that the current cracking could be attributed to strain-age cracking. The reason and the dominant factor that affected the cracking will be discussed in the following paragraphs.3 and exhibits a low lattice parameter mismatch (coherent) with the nickel matrix, allowing fast precipitation kinetics. The effect of γ’ phase on crack susceptibility involves the following aspects: (i) Degradation of deformation capability. Despite the limited strengthening capacity reported for the large-sized γ’ phase [The γ’ phase plays a vital role in IN738 alloy as a primary strengthening source. The phase has a nominal chemical composition of Niγ’ phase , the preγ’ phase . Moreoveγ’ phase . It is wγ’ phase , leadingγ’ phase . It coulOn the one hand, pores at grain boundaries in the HT sample reduced the contact area between grains, lowered grain boundary strength, and provided fast channels for crack propagation, leading to the degradation of crack resistance. On the other hand, these pores facilitated the formation of tortuous grain boundaries. The tortuous grain boundaries could obstruct grain boundary sliding by interlocking, avoiding drastic strain localization at the triple junction of grain boundaries, as supported in The mechanism for the increasing porosity of the HT sample is interpreted as follows: The laser additive manufacturing is processed in a flowing argon atmosphere. The melt pool undergoes directional motion driven by Marangoni convection, folding the liquid metal over the surface while simultaneously entraining argon bubbles . During Both γ’ phase precipitation and pore formation are spatially randomly distributed, independent of BD , yet mac2(Psi) relationship shown in Residual stress is acknowledged as a crucial factor promoting strain-age cracking, which is widely reported in welding ,44,45,46(i)The heat-treatment-induced cracking in the present study is attributed to strain-age cracking.(ii)The crack propagated was driven by the superimposition of contraction stress induced by precipitation and residual stress induced by the LPBF process. The former is facilitated by local enrichment of Ti and Al in the early stage of heat treatment when the residual stress is not fully relieved by heat treatment.(iii)The formation of large-sized pores after heat treatment is attributed to the reopening of compressed argon bubbles. The pores weaken the grain boundaries and provide fast channels for cracking.In this study, microstructure variations before and after heat treatment at 1130 °C for 3 h were systematically characterized to clarify the mechanism for heat-treatment-induced cracking in LPBF IN738. The following conclusions have been reached:To avoid heat-treatment-induced cracking, careful modifications are required for fabrication and heat-treatment processes to reduce residual stresses, and the sluggish kinetics of γ’ phase precipitation or eliminate pores.


To date, 13 patients from nine families with biallelic NHLRC2 variants have been published. In all of them, the recurrent missense variant p.(Asp148Tyr) was detected on at least one allele. Common manifestations included lung or muscle fibrosis, respiratory distress, developmental delay, neuromuscular symptoms and seizures often followed by early death due to rapid disease progression.FINCA syndrome [MIM: 618278] is an autosomal recessive multisystem disorder characterized by NHLRC2 variants identified by exome analysis. All patients described here presented with moderate to severe global developmental delay and variable disease progression. Seizures, truncal hypotonia and movement disorders were frequently observed. Notably, we also present the first eight cases in which the recurrent p.(Asp148Tyr) variant was not detected in either homozygous or compound heterozygous state.Here, we present 15 individuals from 12 families with an overlapping phenotype associated with nine novel We cloned and expressed all novel and most previously published non-truncating variants in HEK293-cells. From the results of these functional studies, we propose a potential genotype-phenotype correlation, with a greater reduction in protein expression being associated with a more severe phenotype.NHLRC2-related disease should be considered in patients presenting with intellectual disability, movement disorders, neuroregression and epilepsy with or without pulmonary involvement.Taken together, our findings broaden the known phenotypic and molecular spectrum and emphasize that  NHL repeat containing 2 (NHLRC2) gene (HGNC: 24731) encodes a ubiquitously expressed and well conserved protein consisting of an N-terminal thioredoxin domain (TRX) and a large NHL repeat domain. The exact function remains elusive and only few functional studies have been conducted [NHLRC2 plays a role in reactive oxygen species (ROS)-induced apoptosis and that the loss of NHLRC2 results in increased sensitivity to ROS-induced cell death [NHLRC2 as a regulator of phagocytosis involved in actin polymerization and filopodia formation [NHLRC2 knockout mouse model revealed embryonic lethality due to gastrulation failure [The HGNC: 2471 encodesNHLRC2 in three individuals from two non-consanguineous families of Finnish descent as the cause of a cerebral-pulmonary disorder [MIM: 618278]  . They naNHLRC2 variants detected in 15 additional individuals with various neurological symptoms, thereby expanding the allelic series and broadening the phenotypic spectrum of NHLRC2-related disease.Here, we present nine novel NHLRC2 variants were detected by exome sequencing (ES) and classified according to the ACMG guidelines ;[Phe462Ser], tended to have a milder clinical course. In four individuals from two unrelated families with progressive neurological manifestations and intractable seizures, the same homozygous disease haplotype containing a start-loss variant was detected  were associated with an early onset multisystem phenotype including pulmonary disease. When the sum of NHLRC2 protein levels from both alleles appears to exceed a certain critical level, a phenotype without progressive respiratory symptoms was observed . The small number of pathogenic NHLRC2 variants described so far and the lack of knowledge about the function of NHLRC2 are likely to hinder a molecular diagnosis in unsolved cases. In this regard, we consider it useful to apply the protein modeling approach presented here and to perform a comprehensive clinical characterization of the affected individual. We recommend that AlphaFold mutant protein modeling and MaSIF prediction be calculated to check whether the variant is localized to a potentially functionally relevant region or substantially alters the tertiary structure of the protein. The criterion PM1 could then be applied at the level of supporting evidence (PM1_sup). However, we urgently need basic knowledge of the physiological function and interaction partners or substrates of NHLRC2 to validate the predicted effects and to establish a possible functional readout.Currently, it is difficult to classify non-truncating variants as (likely) pathogenic according to the ACMG scoring framework, especially in autosomal recessive disorders when the second variant is not known to be pathogenic. In this situation, the criteria PM2 and PP3 are likely to be assigned to most In addition, the specificity of the phenotype criterion (PP4) could be upgraded to moderate level of evidence, if additional symptoms besides NDD/ID are present and comprehensive genetic testing has been performed. In our opinion, this should include symptoms from different systems, such as neurological, pulmonary, gastrointestinal or hematological.NHLRC2 variants should be considered relevant in patients with NDD/ID, movement disorders, neuroregression and epilepsy even in the absence of pulmonary findings. To explain this variable phenotypic spectrum, we propose a genotype-phenotype correlation of residual NHLRC2 protein level and function with phenotype severity. Follow-up studies reporting the clinical course of affected individuals would be important to understand whether all affected individuals have a progressive multi-organ disease with variability in age of onset and severity of clinical manifestations, or whether there is indeed a distinct genotype-phenotype correlation. In this respect, our proposed model is limited and needs to be critically evaluated in further publications.Taken together, our data broaden the hitherto known phenotypic spectrum, extend the allelic series and emphasize that rare biallelic Supplementary File 1Supplementary File 2Supplementary Figure 1Supplementary Figure 2Supplementary Figure 3Supplementary Figure 4Supplementary Figure 5Supplementary Table 1Supplementary Table 2


DNA ligases are essential enzymes involved in DNA replication and repair processes in all organisms. These enzymes seal DNA breaks by catalyzing the formation of phosphodiester bonds between juxtaposed 5′ phosphate and 3′ hydroxyl termini in double-stranded DNA. In addition to their critical roles in maintaining genomic integrity, DNA ligases have been recently identified as diagnostic biomarkers for several types of cancers and recognized as potential drug targets for the treatment of various diseases. Although DNA ligases are significant in basic research and medical applications, developing strategies for efficiently detecting and precisely quantifying these crucial enzymes is still challenging. Here, we report our design and fabrication of a highly sensitive and specific biosensor in which a stable DNA hairpin is utilized to stimulate the generation of fluorescence signals. This probe is verified to be stable under a wide range of experimental conditions and exhibits promising performance in detecting DNA ligases. We anticipate that this hairpin-based biosensor will significantly benefit the development of new targeting strategies and diagnostic tools for certain diseases. Deoxyribonucleic acid (DNA) ligase is an essential and ubiquitous enzyme involved in DNA replication, repair, and recombination in prokaryotic and eukaryotic cells. This enzyme catalyzes the formation of phosphodiester bonds between the juxtaposed 5′ phosphate end and 3′ hydroxyl end in double-stranded DNA in the presence of ATP ,2,3,4. ROn the other hand, the concept of an “extraordinarily stable hairpin” was introduced to the research fields of nucleic acid chemistry in 1989 ,16, whicDeionized water and Tris-HCl buffer solution  were purchased from Sangon Biotech . Lithium chloride was purchased from Shanghai Aladdin Bio-Chem Technology . T4 DNA ligase and 10 × T4 DNA ligase reaction buffer  were purchased from Sangon Biotech .All oligonucleotides used in this study were synthesized by Sangon Biotech . Nucleotide sequences and the modification details of these oligonucleotides are given in A 60 μL solution containing DNA probes with or without DNA ligase was placed in a micro quartz cuvette  at room temperature for fluorescence spectroscopic analysis using F-7100 Fluorescence Spectrophotometer (Hitachi). Fluorescence emission spectra were measured using 515 nm excitation light at an angle 90° and recorded every nanometer from 530 nm to 650 nm. Both excitation and emission slits were set to 5 nm.A solution containing 900 nM (unless otherwise stated) of Oligo 1, Oligo 2, Oligo 3, and Oligo 4 , 40 mM TAs shown in The unique thermodynamic properties of extraordinarily stable mini-hairpins (ESMHs) ,16 were m (5′ CGCGAAGCG 3′) 88.5 °C > Tm (complete duplex) 74 °C > Tm (the double-stranded structures formed by Oligo 1 and Oligo 2) 44 °C > Tm (the double-stranded structures formed by Oligo 3 and Oligo 4) 38 °C > 37 °C, Oligo 1 and Oligo 3 can be ligated in the presence of DNA ligase to form a long single-stranded Oligo 5, in which an extraordinarily stable hairpin was induced to stimulate the dehybridization of Oligo 5 from the double-stranded structures (m (melting temperature) values were simulated using the Tm Calculator from Thermo Scientific Web Tools, provided by Thermo Scientific. This thermodynamic-driven spontaneous process causes the efficient dissociation of fluorophores and quenchers and produces remarkable fluorescence signals. Without DNA ligases, the double-stranded structures formed by Oligo 3, Oligo 4, and Oligo 5 remain stable upon heating and thus produce no fluorescence signals. The details of our designed biosensors are depicted in In consideration of the facts that Tructures . The aboThe design of our biosensor leverages the DNA ligase’s most universal property, which is its ability to form phosphodiester bonds between the juxtaposed 5′ phosphate end and 3′ hydroxyl end of dsDNA containing cohesive ends in the presence of ATP. Among these ligases, the T4 DNA ligase stands out as the most extensively utilized enzyme in molecular biology and biochemical experiments. Its ubiquity and ease of acquisition, as well as the comprehensive understanding of its properties and activities, facilitate the prediction of its performance under specific conditions. Moreover, the T4 DNA ligase exhibits wide adaptability and high efficiency under varying reaction conditions. Consequently, these factors make the T4 DNA ligase a suitable representative of DNA ligases for our purposes. Additionally, since the detection strategy for DNA ligases primarily relies on the inherent catalytic activity of these enzymes, our current experimental findings can be extrapolated to other DNA ligases as well.A pilot study was carried out first to demonstrate the feasibility of our newly designed DNA probes for detecting the presence of T4 DNA ligase. As depicted in the fluorescence spectroscopic results  process,To achieve better sensing performance, several investigations were subsequently carried out to optimize two main factors: incubation time and probe concentration. As shown in In addition to the incubation time, the DNA probe concentration was also optimized in our study. In theory, it is evident that fluorescence intensity increases with an increase in probe concentration, particularly in the presence of a relatively high enzyme concentration. As depicted in At optimal conditions, the fluorescence emission spectra of our DNA probes were determined with T4 DNA ligases at different concentrations , the results of which are given in A regression analysis was performed with the fluorescence intensities collected at 561 nm, as shown in Fit model: Logarithm (Log3P1)Fitting equation: y = A − B × ln(x + C)A = 34.70, B = −83.88, C = 0.89blank = 0.00017 U/mLMeanblank = 0.028 U/mLSDlow concentration sample = 0.0053 U/mLSDLimit of Blank: LoB was defined as the highest apparent analyte concentration expected to be found when replicates of a sample containing no analyte are tested. LoB is estimated by measuring replicates of a blank sample and calculating the mean result and the standard deviation (SD):Limit of Detection: LoD is determined by utilizing both the measured LoB and test replicates of a sample known to contain a low concentration of analyte. The mean and SD of the low-concentration sample are then calculated according toIn this study, we designed a highly sensitive and specific method for detecting DNA ligases, taking advantage of the unique properties of extraordinarily stable DNA mini-hairpins. The catalytic activity of DNA ligases was utilized to rejoin the nick sites that occurred within the nucleotide sequences of these hairpins, which stimulated the separation of the fluorophores and quenchers and further induced fluorescence signals from the DNA probes. It is the first time that the detection of DNA ligases with particular hairpin sequences has been achieved in a simple and fast manner, requiring only one reaction and mild conditions. This versatile detection method can be further applied to the real-time monitoring of intracellular DNA ligase activity, offering valuable insights for biomedical research. Currently, the insufficient cellular affinity of this biosensor prevents it from effectively entering cells. Our next steps involve transforming the dual-molecular probes into single-molecule ones and further integrating our fundamental principles with a variety of nanocarriers , which could help the probes extravasate into cells and tissues via enhanced permeability and retention effect. In addition, the native deoxyribonucleic acid backbones of the non-extraordinarily stable hairpin structure can be replaced with methyl- or phosphorothioate-modified backbones to improve its metabolic stability from nuclease-mediated degradation in serum. As research on cellular detection techniques advances, this approach could potentially evolve into diagnostic tools for various diseases and target identification in drug discovery in the future.