Caenorhabditis elegans (C. elegans); however, the underlying mechanism has not been fully elucidated. In humans, age-dependent loss of muscle function occurs at about 80 years of age and has been linked to dysfunction of ryanodine receptor (RyR)/intracellular calcium (Ca2+) release channels on the sarcoplasmic reticulum (SR). Mammalian skeletal muscle RyR1 channels undergo age-related remodeling due to oxidative overload, leading to loss of the stabilizing subunit calstabin1 (FKBP12) from the channel macromolecular complex. This destabilizes the closed state of the channel resulting in intracellular Ca2+ leak, reduced muscle function, and impaired exercise capacity. We now show that the C. elegans RyR homolog, UNC-68, exhibits a remarkable degree of evolutionary conservation with mammalian RyR channels and similar age-dependent dysfunction. Like RyR1 in mammals, UNC-68 encodes a protein that comprises a macromolecular complex which includes the calstabin1 homolog FKB-2 and is immunoreactive with antibodies raised against the RyR1 complex. Furthermore, as in aged mammals, UNC-68 is oxidized and depleted of FKB-2 in an age-dependent manner, resulting in ‘leaky’ channels, depleted SR Ca2+ stores, reduced body wall muscle Ca2+ transients, and age-dependent muscle weakness. FKB-2 (ok3007)-deficient worms exhibit reduced exercise capacity. Pharmacologically induced oxidization of UNC-68 and depletion of FKB-2 from the channel independently caused reduced body wall muscle Ca2+ transients. Preventing FKB-2 depletion from the UNC-68 macromolecular complex using the Rycal drug S107 improved muscle Ca2+ transients and function. Taken together, these data suggest that UNC-68 oxidation plays a role in age-dependent loss of muscle function. Remarkably, this age-dependent loss of muscle function induced by oxidative overload, which takes ~2 years in mice and ~80 years in humans, occurs in less than 2–3 weeks in C. elegans, suggesting that reduced antioxidant capacity may contribute to the differences in lifespan among species.Age-dependent loss of body wall muscle function and impaired locomotion occur within 2 weeks in  Caenorhabditis elegans (C. elegans has been used as a model to study the genetics of aging and lifespan determination (C. elegans has been attributed to degeneration of the nervous system (2+) release channel homolog, UNC-68, in age-dependent loss of muscle function in C. elegans.Approximately 50% of humans over the age of 80 develop muscle weakness, which contributes to falls and hip fractures, a leading cause of mortality in the elderly . Strikin elegans , both exmination , includimination . Age-deps system  and the s system . Here, w2+ release channel in skeletal muscle required for excitation-contraction (E-C) coupling   , and cal(CaMKII)  are comp(CaMKII) . Calstab(CaMKII) . Calstab(CaMKII)  with pep channel .2+ release channels, the only other member being the inositol 1,4,5-triphosphate receptor (IP3R) . RyRs and IP3Rs are intracellular Ca2+ release channels on the SR/ER and are tetramers that along with associated proteins comprise the largest known ion channel macromolecular complexes  . RyR mayoa clade . Inverteomplexes . Defects failure , cardiac failure , diabete failure , muscle  failure , and neu failure .3R, one of which (SPRY2) allows RyR1 to directly interact with the L-type calcium channel (Cav1.1) in mammalian skeletal muscle  null mutants exhibit severely defective swimming behavior and locomotive characterized by the ‘unc’, or ‘unco-ordinated’ phenotype  null mutants are unaffected by ryanodine  worms, an FKB-2 deletion mutant that results in leaky UNC-68. Competing FKB-2 from UNC-68 with rapamycin or FK506 -generating drug paraquat  RyR1 oxidation depletes calstabin1 from the channels and renders them leaky to Castrength . In the  in mice , we founor FK506  resultedparaquat  caused Fcroscopy . The comC. elegans including progressive oxidation of UNC-68, which depletes the stabilizing binding protein, FKB-2 and, renders the channel leaky within 2 weeks compared to 2 years in mice and 80 years in humans and a potential therapy.Our study provides an underlying mechanism for age-dependent loss of muscle function in UNC-68, the C. elegans intracellular calcium release channel, shares ~40% homology with the human RyR1   as previously described , protein phosphatase 1 (PP1), FKB-2, and phosphodiesterase 4 (PDE-4) using mammalian anti-RyR, anti-PKA, anti-PP1, anti-calstabin, and anti-PDE-4 antibodies, respectively  null mutant  . Based oescribed . The immectively . The preti-PDE-4  was usedl mutant . In the antibody . Taken t2+ transients are reduced in aged mammalian skeletal muscle  (UNC-68 was oxidized (FKB-2 (mdx mice) characterized by impaired muscle function (UNC-68 was significantly more oxidized (day 3–9) in FKB-2 (ok3007) worms compared to WT (RyR1 oxidation has been linked to SR Catrophies . Further muscle) . WT UNC-oxidized  and depld  were mixed with the Ca2+ dye Fluo-4 and baseline fluorescence measurements were taken before adding 1 mM of ATP. By activating the sarco/endoplasmic calcium ATPase (SERCA) with ATP, cytosolic Ca2+ is pumped into the microsomes, resulting in a decrease in Fluo-4 fluorescence. Once the fluorescence level plateaus, thapsigargin (SERCA antagonist) is added to block Ca2+ reuptake into the SR. The rate at which the fluorescence increases directly correlates with the amount of Ca2+ passively leaking into the cytoplasm: a higher increase of fluorescence compared to WT control indicates leaky UNC-68 channels. Our data show that UNC-68 from FKB-2 KO worms had a higher rate of SR Ca2+ leak following thapsigargin administration compared to the WT channels  were treated with rapamycin or FK506. Ca2+ transients were measured in partially immobilized transgenic nematodes expressing the genetically encoded Ca2+ indicator, Pmyo-3::GCaMP2, in the body wall muscle cells (FKB-2 from UNC-68 by rapamycin or FK506 treatment (15 min exposure to each drug) caused reduced body wall muscle Ca2+ transients in WT C. elegans  nematodes, treatment with rapamycin or FK506 had no effect on the Ca2+ transients  worms: the complex I mutant, CLK-1, and the complex II mutant, MEV-1. CLK-1 worms contain a Complex I-associated mutation such that they cannot synthesize their own ubiquinone (UQ), a redox active lipid that accepts and transfers electrons from Complex I or II to Complex III in the ETC. The reduction in Complex I activity of CLK-1 is associated with long-lived worms (UNC-68 oxidation and FKB-2 depletion in the short-lived mutant (MEV-1) compared to WT and long-lived mutant (CLK-1) worms  worms . Indeed,1) worms .2+ leak by reducing the stress-induced depletion of calstabin from the RyR channel complex  to the UNC-68 channel is required to prevent a pathological leak of intracellular Ca2+, similar to the manner observed in mammalian muscle  nematodes and UNC-68 is oxidized at an earlier time point in these mutants relative to WT. Treating aged WT nematodes with the RyR-stabilizing drug, S107, reassociates FKB-2 with UNC-68 and increases the Ca2+ transients, indicating that UNC-68 dysfunction is likely an underlying mechanism of age-dependent decrease in Ca2+ transients in C. elegans body wall muscle. The mechanism causing age-dependent UNC-68 dysfunction involves the loss of UNC-68/FKB-2 from the UNC-68 channel complex due to oxidation of the channel. Of note, this may create a vicious cycle of intracellular Ca2+ leak and oxidative overload in which leaky channels cause mitochondrial Ca2+ accumulation and high levels of ROS production which further oxidize UNC-68 and further exacerbate the Ca2+ leak over the course of the lifespan  the cause for the accelerated kinetics of aging must be determined elsewhere and in an unrelentingly constant manner as exemplified by the rigid control of species lifespan. There is however, only one known case of a significant prolongation of average lifespan in a species: Homo sapiens. Indeed, the average lifespan in the U.S. has doubled in the past century , prevents FKB-2 from co-immunoprecipitating with UNC-68. The aging phenotype that we characterize in WT nematodes  is accelerated in FKB-2 (ok3007). There are eight FKBs that are homologous to mammalian calstabin in the C. elegans genome; FKB-1 and FKB-8 both have ~50% sequence identity to calstabin. Further studies could elucidate the possibility that in the absence of FKB-2, another FKB may stabilize UNC-68, in particular the aforementioned FKB-8 (its gene is in close proximity to that of FKB-2 on chromosome 2) and FKB-1 (most similar to FKB-2 in terms of molecular weight). Such a mechanism could provide transitory compensation for the lack of FKB-2, in which other FKB isoform(s) bind to UNC-68 with lower affinity. Because this binding is weak, and the channel is unstable, this compensation ends up failing at day 7 of age and the Ca2+ leak is exacerbated. This hypothesis is partially supported by the unaltered Ca2+ peaks in FKB2-KO worms at day 5 of age despite a complete depletion of FKB-2 binding protein. Such a compensatory mechanism was not observed with acute rapamycin and FK506 treatment potentially because, first, the Ca2+ leak was acute and there was no time for a compensatory response, and, second, these drugs could act on all FKB isoforms.We also demonstrate that the putative null mutant, UNC-68 becomes oxidized within 2 weeks, whereas the same post-translational modification requires 2 years in mice and 80 years in humans  and serine (7.2%)   comparedUNC-68 is localized to a specific portion of a vesicular network surrounding the myofilament lattice which suggests that the general architecture of the SR is conserved in metazoans. RyRs in vertebrate striated muscle cluster at internal couplings with Ttubules and, peripheral couplings adjacent to the surface membrane, visible as a ‘feet’ in electron micrographs   . However neurons  which maaralysis . myo-3 itraction .C. elegans homolog of RyR, UNC-68, is comprised of a macromolecular complex and regulated by the immunophilin, FKB-2. We have identified age-dependent reduction in body wall muscle Ca2+ transients in nematodes that is coupled to oxidation and remodeling of UNC-68. SR Ca2+ stores are depleted in FKB2-KO worms, suggesting passive UNC-68 leak. This observation is supported by the Ca2+ leak assay results, which show that FKB-2 regulation is critical in preventing UNC-68 channels from aberrantly ‘leaking’ Ca2+ into the cytoplasm. With reduced SR Ca2+, UNC-68 fails to release the burst of Ca2+ required for normal E-C coupling, leading to impaired muscle function. Loss of muscle function is evident in the FKB-2 KO worms during swimming trials, as middle-aged worms performed worse than their age-matched WT controls. Furthermore, our data strongly suggest a role for FKB-2 and UNC-68 in the age-dependent changes in Ca2+ signaling, as treatment with the pharmacological RyR stabilizer S107 increases body wall muscle Ca2+ transients. The advantage of targeting leaky RyR channels rather than using antioxidants would be the avoidance of the adverse effects of blocking beneficial oxidative signals.Taken together, our data indicate that the Escherichia coli at 20°C, as described (fkb-2 (ok3007) were provided by the Caenorhabditis Genetics Center (University of Minnesota). fkb-2 (ok3007) was backcrossed six times. The transgenic strain expressing Pmyo-3: GCaMP2 was kindly provided by Zhao-Wen Wang, University of Connecticut Health Center (myo-3: GCaMP2 was subsequently crossed into fkb-2 (ok3007) for measurement of contraction-associated Ca2+ transients.Worms were grown and maintained on standard nematode growth medium (NGM) plates on a layer of OP50 escribed . N2  for 1 hr at 4°C. The immune complexes were incubated with protein A Sepharose beads  at 4°C for 1 hr, after which time the beads were washed three times with buffer. Proteins were size-fractionated by SDS-PAGE  and transferred onto nitrocellulose membranes for 1 hr at 200 mA . After incubation with blocking solution  to prevent non-specific antibody binding, immunoblots were developed using antibodies against RyR , PKAcat , PDE4 , PP1 , or an anti-calstabin antibody (Santa Cruz 1: 2500). To determine channel oxidation, the carbonyl groups on the protein side chains were derivatized to 2,4-dinitrophenylhydrazone (DNP-hydrazone) by reaction with 2,4-dinitrophenylhydrazine (DNPH) according to manufacturers (Millipore) instructions. The DNP signal on immunoprecipitated UNC-68 was determined by immunoblotting with an anti-DNP antibody . All immunoblots were developed and quantified using the Odyssey Infrared Imaging System  and infrared-labeled secondary antibodies. In addition, immunoblotting and immunoprecipitation of the UNC-68 macromolecular complex were conducted using another anti-calstabin antibody  and the same methods as described.A anti-mammalian RyR antibody (4 μg 5029 Ab ) was use2+ were measured in nematodes expressing GCaMP2 by fluorescence imaging using a Zeiss Axio Observer inverted microscope with an electron-multiplying CCD camera (Photometrics Evolve 512) and an LED light source (Colibri). Nematodes were partially immobilized by placing them individually into a 5–10 μl drop of M9 buffer, suspended between a glass slide and coverslip. 20-s videos of individual nematodes were recorded.Spontaneous changes in body wall muscle Ca2+ transients were analyzed using an interactive data language-based image quantification software that was developed for this purpose in our laboratory. For each 20-s video, signals from the body wall muscles in nematodes expressing GCaMP2 fluorescence were analyzed using an edge-detection algorithm from each frame as ‘line-scan’ images, with the nematode perimeter on the y-axis and time (s) on the x-axis . An additional 170 μl of M9 buffer was applied for a total of 250 μl. The completed preparation was placed on the platform of a Zeiss confocal microscope; after 1 min at baseline, 25 mM of caffeine was added to an equal volume of M9 solution. The resulting body wall transients were recorded for 1 min.Age-synchronized GCaMP2: WT and GCaMP2: FKB-2 KO were grown on NGM plates at 20°C they were separated from their progeny and left undisturbed until day 5. Individual worms were placed in a drop of M9 on a coverslip. The liquid was carefully wicked away using KIMTECH wipes until only a sliver of moisture surrounded the worm. The worm was quickly glued down to the coverslip using a tiny drop of DermaWorm applied to the head and tail of the worm before the worm desiccated. 80 μl of M9 buffer was added immediately afterward to polymerize the glue. Once the worm was secure, a clean lateral cut to the immediate tail region was made using a 20 G 1½ needle  at 45,000× g for 30 min. Pellets were resuspended in lysis buffer containing 300 mM sucrose. Microsomes (5 µg/ml) were diluted into a 20 mM HEPES buffer (pH 7.2) containing 7 mM NaCl, 1.5 mM MgCl2, 120 mM K-gluconate, 5 mM K-phosphate, 8 mM K-phosphocreatine, 1 µM EGTA, and 2 µM CaCl2 mixed with 3 µM Fluo-4 and added to multiple wells of a 96-well plate. Calcium (Ca2+) loading of the microsomes was initiated by adding 1 mM ATP. After Ca2+ uptake and a new Fluo-4 signal baseline was observed, 3 μM Thapsigargin was added to inhibit the calcium uptake by the calcium pump (SERCA). The ‘leak’ of Ca2+ out of the SR is measured by the increase in intensity of the Fluo-4 signal (measured in a Tecan infinite F500 fluorescence plate reader).Microsomes were prepared by centrifuging the Standard M9 buffer was mixed with 2% agar and poured into 96-well plates to create a planar surface for analyzing worm swimming behavior. Once the mixture had polymerized, approximately 180 μl of M9 was pipetted on top of the agar bed and age-synchronized worms from one of two groups (WT or FKB-2 KO) were placed individually into each well. To assess differences in exercise fatigue, worms were allowed to swim freely in M9 buffer for 2 hr; swimming bends and curls  were recAll results are presented as mean ± SEM. Statistical analyses were performed using an unpaired two-tailed Student’s t test (for two groups) and one-way ANOVA with Tukey-Kramer test (for three or more groups), unless otherwise indicated. For survival statistical comparison, we used Gehan-Breslow-Wilcoxon test. p-values <0.05 were considered significant. All statistical analyses were performed with Prism 8.0. C. elegans, with evidence in support of age-dependent oxidation of the C. elegans ryanodine receptor ortholog, UNC-68, causing loss of the calstabin ortholog, FKB-2. This in turn results in calcium leak, reduced body wall calcium transients and muscle weakness, changes that are similar to those that occur in aging human skeletal muscle despite the dramatic differences in the lifespan of the two organisms.This manuscript will appeal to all with an interest in comparative physiology and the molecular biology of age-associated changes in muscle function. The authors draw parallels between aging skeletal muscle in humans and  public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: i) Decision letter after peer review:C. elegans" for consideration by eLife. Your article has been reviewed by 2 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Richard Aldrich as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Role of oxidation of excitation-contraction coupling machinery in age-dependent loss of muscle function in The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:2+, for the benefit of those who may not have a fully nuanced understanding of EC coupling in skeletal (or cardiac) muscle, the authors should modify the statement to include the concept of triggering-influxes of extracellular ca2+.1. While researchers in the field may not be misled by the statement in the Introduction implying that muscle contraction is fully independent of extracellular ca2+ transients and an aging phenotype.2. It is recommended that the authors sort out their thinking on the cause and effect relationships among UNC-68 oxidation, FKB-2 dissociation and UNC-68 leakiness, and present a cogent and compelling argument for them. It is not sufficient to present various lines of evidence showing that these things are interrelated and connected via a fuzzy mechanism to reduced caC. elegans relative to that of mammals and comment on the validity of C. elegans as a model for aging human muscle.3. The authors should discuss differences in EC coupling in 4. Please "dial back" your rhetoric on longevity.5. Presentation is choppy. The presentation of all results shown in Figure 2  should be merged into a single section, eliminating any redundancies in the process.2+ transients, therefore justifying their use in mimicking the aging phenotype. The sentence should be revised to more accurately convey the authors' intended meaning.6. The strangely worded last sentence of the first paragraph of the "Pharmacologically mimicking aging phenotype…" section is internally illogical – the conclusion (second phrase) doesn't logically follow from the premise (first phrase). This muddles the message of this paragraph, which is presumably to convey the idea that it is the ability of rapamycin and FK506 to compete off calstabin (FKB-2 in this case), and not their well-known abilities to inhibit mTOR and calcineurin, respectively, that accounts for their observed effects on ca7. Suggest organizing panels in Figure 3 to mirror organization in Figure 2 since they are similar data, just differing in "stimulus" .8. Need to clarify what time point the "WT+FK506" label corresponds to in all figure panels where it appears.9. Nematode age should be indicated for all experiments/figure panels.10. English quality could be improved .2+." Direct coupling of RyR to sarcolemmal ca2+ channels via SPRY2 domains does not entirely eliminate the need for extracellular ca2+ for EC coupling; it merely reduces it to a very small influx necessary to trigger release from intracellular stores via a ca2+-induced ca2+-release mechanism.11. "This interaction [between RyR1 and CaV1.1] couples excitation of the sarcolemma to muscle contraction and eliminates dependence on extracellular ca12. Figure 2B:2+ transients at this time point?– Is the day-15 bar for the FKB-2-KO missing? Or are there no ca– Are these muscle contraction-induced transients or caffeine-induced transients?2+ transient that drives muscle contraction Not sure why this mini-section is here; recapitulates the first paragraph of the previous paragraph.13. FKB-2 deficiency accelerates age-dependent reduction in ca14. UNC-68 channels are leaky in the absence of FKB-2"Once the fluorescence level plateaus…" Fluorescence level clearly has not plateaued in microsome samples from FKB-2-KO worms at the time thapsigargin is added. If the assay system is capable of truly reaching a plateau from which the resulting leak can be more accurately measured, experiments should be repeated; alternatively, a rationale should be provided for "jumping the gun" with thapsigargin addition.15. UNC-68 channel leak impairs exercise capacity"FKB-2 KO worms exhibit reduced life span compared to WT " – Maximum lifespan was not different; what are the average lifespans for WT and KO worms, and what statistical analysis supports the difference?2+ transient and impairs exercise capacity.16. Pharmacologically mimicking aging phenotype affects ca"…longer treatment of WT worms with FK506 caused oxidation of UNC-68, demonstrating a relationship between depletion of FKB-2 and oxidation of UNC-68" – This implies that depleting FKB-2 causes oxidation of UNC-68. This is causally backwards relative to the proposed model, where oxidation of UNC-68 (associated with aging) causes dissociation of FKB-2, leading to leaky channels and reduced muscle function and exercise capacity. Although in the Discussion, the authors do obliquely address the idea that dissociating FKB-2 could be expected to increase oxidation through increased UNC-68 leakiness, it comes too late in the presentation to do anything but muddle the message.17. Figure 3F:– The only way the labeling for this panel makes sense is if "WT+FK506" and "FKB-2 KO+FK506" labels are switched AND "WT+FK506" corresponds to a measurement at some earlier time point (0 or 1h? Not specified). Labeling in Figure 3D  is consistent with the idea that labels in 3F were switched, but the label "WT+FK506" is still ambiguous.– Also, the "time course" is really a digital response that goes from ~0 at 2 h to max at 4 h. At minimum, data should be collected from a time point between 2 and 4 hours to demonstrate an actual time course.18. Figure 4D:– What is"WT+FK506" exactly? Is this the 4-h time point, as would make sense given that the comparison is with FKB-2 KO+FK506 , or is this a 0 h (or 1 h) time point, as suggested by the use of "WT+FK506" as the first term in the "WT+FK506", "WT+FK506, 2h" and "WT+FK506, 4h" series?2+ than the FKB-2 KO condition (with or without FK506), since FKB-2 knockout presumably has a greater effect on UNC-68 subunit composition  than competing off FKB-2 with FK506 .– Not clear why the WT+FK506 conditions would show a greater reduction in peak ca19. Figure 4A-D:2+ transients are not terribly impressive  and not all that well correlated with UNC-68 oxidation  or FKB-2 binding . According to the authors' model, it is the loss of FKB-2 that ultimately is responsible for the leakiness of UNC-68 and associated phenotypes. Therefore, complete loss of FKB-2 binding should be more closely correlated with diminished ca2+ transients than UNC-68 oxidation (which presumably causes graded dissociation of FKB-2). By the same logic, FKB-2 KO should be more closely correlated with diminished ca2+ transients than WT+FK506 or paraquat .– Overall effects of FKB-2 KO/KD on ca2+ data.– The idea that the loss of FKB-2 is ultimately responsible for the leakiness of UNC-68 and associated phenotypes appeared to be the proposed model up until the presentation of data hinting that oxidation in and of itself was capable of producing this leakiness. In this context, only the paraquat FKB-2 binding/UNC-68 oxidation data were well correlated with peak ca20. Nematode age is a key parameter that is rarely specified in the text or figures/legends. Note that "young" is uninformative unless specifically defined.21. "Another key question is why UNC-68 becomes oxidized within two weeks, whereas the same post-translational modification requires two years in mice and 80 years in humans (5)." This is not a key question. There are many, many contributors to the longevity of animals. It would be hard to find anyone who would argue that worms would live for years if only they had a greater antioxidant capacity.2+ overload, resulting in ROS production, thus leading to further oxidation of RyR1 and exacerbation of the SR ca2+ leak." Adding to this either/or quality is the apparent bidirectionality of the oxidation-FKB-2 dissociation relationship: not only does UNC-68 oxidation lead to FKB-2 dissociation, FKB-2 dissociation also leads to UNC-68 oxidation . Lost in the presentation is how UNC-68 oxidation causes FKB-2 dissociation and whether (and how) UNC-68 oxidation causes increased UNC-68 leakiness in the absence of FKB-2 dissociation. All in all, the manuscript presents quite the Mobius strip with respect to cause-and-effect relationships.22. The authors go to great lengths to connect UNC-68 oxidation with FKB-2 dissociation and link this relationship to UNC-68 leakiness and the aging phenotype. But it's never really clear whether it is the dissociation of FKB-2 or the oxidation of UNC-68 that underlies the leakiness. The authors clearly believe that FKB-2 dissociation is an important event in this sequence, otherwise, why use FKB-2-KO and WT+FK506 models throughout? But another formulation of their model (Discussion) is all oxidation all the time: "RyR1 leak (due to age-dependent oxidation of the channel) causes mitochondrial ca Essential revisions:2+, for the benefit of those who may not have a fully nuanced understanding of EC coupling in skeletal (or cardiac) muscle, the authors should modify the statement to include the concept of triggering-influxes of extracellular ca2+.1. While researchers in the field may not be misled by the statement in the Introduction implying that muscle contraction is fully independent of extracellular ca2+ induced ca2+ release (CICR), a process in which ca2+ itself activates the channel to release more ca2+. However, in physiological conditions extracellular ca2+ is not necessary for the activation of the mammalian skeletal muscle RyR1 to be activated. Indeed, ca2+ release from mammalian skeletal muscle, RyR1 isoform is essentially triggered by conformational changes to the voltage gated sensor in the DHPR channel, upon depolarization of the T-tubule . This point of view has been supported by studies in which suppression of the extracellular ca2+ failed to blunt muscle contraction. Indeed, myofibers exhibit normal twitching even in a Ringer solution with 80 mM EGTA (Luttgau 1968). Ca2+ release and transients can be also recorded under voltage clamp at large positive potentials under which the slow inward ca2+ current becomes outwards (Brum 1987). Moreover, a series of mutagenesis studies have shown that the amino acid residues 489-503 in the DHPR-β1 subunit directly interact with RyR1 channels and mediate EC coupling . We believe that the Reviewer’s comment refers to the skeletal muscle EC coupling in amphibians , which exhibit a minor dependence of extracellular ca2+ due to the expression of the β-RyR with similar gating properties to mammalian cardiac RyR2 (see Review Takashi 2011). We revised our introduction to clarify that we are describing the mechanism of EC coupling in mammals and in C-elegans (pages 2-3-4-5-6-7-8-10 and 12).We thank the Reviewer for her/his comment. The three mammalian RyRs  exhibit ca2+ transients and an aging phenotype.2. It is recommended that the authors sort out their thinking on the cause and effect relationships among UNC-68 oxidation, FKB-2 dissociation and UNC-68 leakiness, and present a cogent and compelling argument for them. It is not sufficient to present various lines of evidence showing that these things are interrelated and connected via a fuzzy mechanism to reduced ca2+ leak. This ca2+ leak leads to excessive ca2+ accumulation in the mitochondria, generates ROS and further oxidizes UNC-68. In disease conditions (e.g. aging), this mechanism results in a vicious cycle in which oxidation promotes FKB-2 depletion which subsequently promotes further oxidation of UNC-68 or in mammalian muscle, RyR1 . We revised our manuscript to better clarify this mechanism .We thank the Reviewer for her/his comment which helped us to clarify our message. UNC-68 oxidation causes depletion of FKB-2 from the channel, which results in caC. elegans relative to that of mammals and comment on the validity of C. elegans as a model for aging human muscle.3. The authors should discuss differences in EC coupling in C. elegans vs. mammals and commented on the validity of C. elegans as a model for aging human muscle (see revised discussion-page 13).We thank the Reviewer for his comment which helped improve the manuscript. We have discussed the differences in EC coupling in 4. Please "dial back" your rhetoric on longevity.We have largely replaced the word “lifespan” by “age-dependent loss of muscle function” throughout the revised manuscript.5. Presentation is choppy. The presentation of all results shown in Figure 2  should be merged into a single section, eliminating any redundancies in the process.We thank the Reviewer for her/his comment which helped achieve a better organization of the manuscript. In the revised version, we merged the presentation of the results shown in Figure 2 into 1 section and eliminated the redundancies (pages 6 and 7).2+ transients, therefore justifying their use in mimicking the aging phenotype. The sentence should be revised to more accurately convey the authors' intended meaning.6. The strangely worded last sentence of the first paragraph of the "Pharmacologically mimicking aging phenotype…" section is internally illogical – the conclusion (second phrase) doesn't logically follow from the premise (first phrase). This muddles the message of this paragraph, which is presumably to convey the idea that it is the ability of rapamycin and FK506 to compete off calstabin (FKB-2 in this case), and not their well-known abilities to inhibit mTOR and calcineurin, respectively, that accounts for their observed effects on caWe thank the Reviewer for her/his comment which helped clarifying our message on the use of FK506 and rapamycin to compete off FKB-2. We revised these statement in the new version of the manuscript and added references supporting the significance of these experiments .7. Suggest organizing panels in Figure 3 to mirror organization in Figure 2 since they are similar data, just differing in "stimulus" .In the revised manuscript, the panels organization in Figure 3 mirrors the panel organization in Figure 2.8. Need to clarify what time point the "WT+FK506" label corresponds to in all figure panels where it appears.We apologize for not adding this important information. “WT+FK506” label correspond to a short treatment (15min) of WT worms with FK506 drug. We have revised all our figures and figure legends to show the time point for each experiment.9. Nematode age should be indicated for all experiments/figure panels.Again, we thank the Reviewer for her/his comment we apologize for not adding this important information in some experiments. We added the nematode age for all the experiments described in the manuscript.10. English quality could be improved .We revised our manuscript to improve the English quality and remove potentially confusing words.2+." Direct coupling of RyR to sarcolemmal ca2+ channels via SPRY2 domains does not entirely eliminate the need for extracellular ca2+ for EC coupling; it merely reduces it to a very small influx necessary to trigger release from intracellular stores via a ca2+-induced ca2+-release mechanism.11. "This interaction [between RyR1 and CaV1.1] couples excitation of the sarcolemma to muscle contraction and eliminates dependence on extracellular ca1.1 does not eliminate the need for extracellular ca2+ or the activation of RyR1 by ca2+ but it renders it unnecessary to achieve a normal contraction. Please refer to the answer to question 1 for details. In the revised manuscript, we replace “eliminates dependence on extracellular ca2+” by “reduces dependence on extracellular ca2+” (Page 4)We thank the Reviewer for her/his remark. We agree that the physical interaction between RyR1 and Cav12. Figure 2B:2+ transients at this time point?– Is the day-15 bar for the FKB-2-KO missing? Or are there no ca– Are these muscle contraction-induced transients or caffeine-induced transients?2+ in FKB-2-KO worms at day 15 as the mutant already exhibited a significant reduction in ca2+ peak at day 7. The ca2+ transient at day 15 was only measured in WT since their ca2+ peak was not affected at day 7. To be consistent, we removed the WT Day 15 ca2+ transient. We also indicated on the top of panel 2B whether these measurements are contraction-induced ca2+ transients or caffeine-induced ca2+ transients.We thank the Reviewer for her/his comment, and we apologize for not being clear enough in the description of Figure 2B. Indeed, we did not measure ca2+ transient that drives muscle contraction Not sure why this mini-section is here; recapitulates the first paragraph of the previous paragraph.13. FKB-2 deficiency accelerates age-dependent reduction in caWe have removed this redundancy in the revised version of the manuscript.14. UNC-68 channels are leaky in the absence of FKB-2"Once the fluorescence level plateaus…" Fluorescence level clearly has not plateaued in microsome samples from FKB-2-KO worms at the time thapsigargin is added. If the assay system is capable of truly reaching a plateau from which the resulting leak can be more accurately measured, experiments should be repeated; alternatively, a rationale should be provided for "jumping the gun" with thapsigargin addition.We have repeated this leak assay experiment and adjusted the conditions, mainly by using a higher amount of microsomes, which allowed us to reach a fluorescence plateau after adding thapsigargin. The new results are displayed in the new Figure 2F.15. UNC-68 channel leak impairs exercise capacity"FKB-2 KO worms exhibit reduced life span compared to WT " – Maximum lifespan was not different; what are the average lifespans for WT and KO worms, and what statistical analysis supports the difference?The median WT survival was 18 days versus 14 days for FKB-2-KO worms. We performed Gehan-Breslow-Wilcoxon test for survival comparison which gives more weight to death at early time points. Since we do not have censored worms at any time point, we chose this test over the log-rank test to take in consideration the early death of worms. We added these details to the figure 2 legend.2+ transient and impairs exercise capacity.16. Pharmacologically mimicking aging phenotype affects ca"…longer treatment of WT worms with FK506 caused oxidation of UNC-68, demonstrating a relationship between depletion of FKB-2 and oxidation of UNC-68" – This implies that depleting FKB-2 causes oxidation of UNC-68. This is causally backwards relative to the proposed model, where oxidation of UNC-68 (associated with aging) causes dissociation of FKB-2, leading to leaky channels and reduced muscle function and exercise capacity. Although in the Discussion, the authors do obliquely address the idea that dissociating FKB-2 could be expected to increase oxidation through increased UNC-68 leakiness, it comes too late in the presentation to do anything but muddle the message.2+ leak” vicious cycle earlier in the discussion (Page 10).We addressed the concept of “oxidation-ca17. Figure 3F:– The only way the labeling for this panel makes sense is if "WT+FK506" and "FKB-2 KO+FK506" labels are switched AND "WT+FK506" corresponds to a measurement at some earlier time point (0 or 1h? Not specified). Labeling in Figure 3D  is consistent with the idea that labels in 3F were switched, but the label "WT+FK506" is still ambiguous.2+ leak which mitochondrial ca2+ overload and oxidative stress in this strain.We thank the Reviewer for her/his comment, and we apologize for the lack of clarity. Indeed, WT+FK506 are WT worms treated with FK506 for 15 min. The worms exhibit complete depletion of FKB-2 but no oxidation. The oxidation only appears when the WT worms are treated with FK506 for 4 hours. The FKB-2 KO worms have basal oxidation independent of FK506 treatment (no difference between FKB-2-KO treated and untreated), . The basal oxidation in the FKB-2 KO worms could be due to constitutive caThe first line of the gel is not quantified (no bar graphs are shown), it represents an immunoprecipitated RyR1 from mouse skeletal muscle and serves as a positive control for the antibody. The quantification bars reflect only the experiments performed with worms. We revised the figure 3, and 4 legends to better clarify these details.– Also, the "time course" is really a digital response that goes from ~0 at 2 h to max at 4 h. At minimum, data should be collected from a time point between 2 and 4 hours to demonstrate an actual time course.We thank the Reviewer for her/his comment, and we apologize for the lack of clarity regarding the time course experiments. Indeed, our time course includes 0, 15 min, 2 h and 4 h. We have added these missing details to the figure and the legend.18. Figure 4D:– What is"WT+FK506" exactly? Is this the 4-h time point, as would make sense given that the comparison is with FKB-2 KO+FK506 , or is this a 0 h (or 1 h) time point, as suggested by the use of "WT+FK506" as the first term in the "WT+FK506", "WT+FK506, 2h" and "WT+FK506, 4h" series?“WT+FK506” is WT worms treated with FK506 for 15 min. This group exhibits complete depletion of FKB-2 but no oxidation. Of note, the first line of the gel is not quantified (no bar graphs are shown), it represents an immunoprecipitated RyR1 from mouse skeletal muscle and serves as a positive control for the antibody. This detail has been added to the figure 4 legend.2+ than the FKB-2 KO condition (with or without FK506), since FKB-2 knockout presumably has a greater effect on UNC-68 subunit composition  than competing off FKB-2 with FK506 .– Not clear why the WT+FK506 conditions would show a greater reduction in peak ca2+ compared to WT+FK506 at any investigated age. However, this was not the case at day 5. One possibility is that the FKB2-KO worms develop a compensatory mechanism over time due to the chronic ca2+ leak to protect the nematode from severe pathogenic ca2+ overload at an early age. This mechanism ends up failing over time and the ca2+ leak is exacerbated around day 7 of age. However, the adaptation to chronic ca2+ leak is an important question that needs further investigation in future and is beyond the scope in the current study. Please see discussion, first paragraph of page 13 for proposed potential mechanisms.We thank the Reviewer for her/his very interesting comment. We agree with the Reviewer that the FKB-2-KO worms were expected to have a greater reduction in peak ca19. Figure 4A-D:2+ transients are not terribly impressive  and not all that well correlated with UNC-68 oxidation  or FKB-2 binding . According to the authors' model, it is the loss of FKB-2 that ultimately is responsible for the leakiness of UNC-68 and associated phenotypes. Therefore, complete loss of FKB-2 binding should be more closely correlated with diminished ca2+ transients than UNC-68 oxidation (which presumably causes graded dissociation of FKB-2). By the same logic, FKB-2 KO should be more closely correlated with diminished ca2+ transients than WT+FK506 or paraquat .– Overall effects of FKB-2 KO/KD on ca2+ transients until day 7. Again, as stated in response to question 18, this could be due to a compensatory mechanism by which these worms overcome the chronic ca2+ leak early in life but ends up failing at 7 days. In contrast, the WT worms do not develop a compensatory mechanism upon acute oxidation (paraquat treatment) or FKB-2 depletion (FK506 treatment) and therefore rapidly exhibit a leaky ca2+ phenotype (reduced ca2+ transient). We discussed these details and added the age of worms for each experiment in the revised version of the manuscript as requested by the reviewer (pages 13).We thank the Reviewer for her/his comment which help clarifying the results displayed in Figure 4A-D. These experiments were done at 5 days of age. Despite a significant UNC-68 oxidation and absence of FKB-2, the FKB-2-KO worms do not exhibit significant reduction in ca2+ data.– The idea that the loss of FKB-2 is ultimately responsible for the leakiness of UNC-68 and associated phenotypes appeared to be the proposed model up until the presentation of data hinting that oxidation in and of itself was capable of producing this leakiness. In this context, only the paraquat FKB-2 binding/UNC-68 oxidation data were well correlated with peak ca2+ transient at 5 days of age. See answer to question 18 and 19. In WT worms, paraquat treatment causes acute UNC-68 oxidation and subsequently depletes FKB-2 of channel resulting in an acute ca2+. Under these conditions, the WT worms have no time to adapt their ca2+ machinery and as such exhibit a more severe ca2+ leak phenotype at this age (day 5). Again, the pharmacological depletion of FKB-2 is acute, and its consequences are rapidly exacerbated. We discussed these aspects in the revised manuscript (page 13).The FKB-2 KO worms do not exhibit a reduction in the ca20. Nematode age is a key parameter that is rarely specified in the text or figures/legends. Note that "young" is uninformative unless specifically defined.We added the specific age for each experiment in the revised text and in the figures/legends.21. "Another key question is why UNC-68 becomes oxidized within two weeks, whereas the same post-translational modification requires two years in mice and 80 years in humans (5)." This is not a key question. There are many, many contributors to the longevity of animals. It would be hard to find anyone who would argue that worms would live for years if only they had a greater antioxidant capacity.We think this is an important question because the enzymes involved in UNC-68 oxidation are very similar to those causing age-dependent oxidation of RyR1 in mammals which takes orders of magnitude longer. So, if one could answer this question, it would shed some light on the unanswered question of what determines species-specific differences in lifespan. The point is not that the oxidation is the most important issue, but rather the etiology of the tremendous difference in kinetics of the age-dependent signals between species. Despite all of the research on aging this question has yet to be answered.2+ overload, resulting in ROS production, thus leading to further oxidation of RyR1 and exacerbation of the SR ca2+ leak." Adding to this either/or quality is the apparent bidirectionality of the oxidation-FKB-2 dissociation relationship: not only does UNC-68 oxidation lead to FKB-2 dissociation, FKB-2 dissociation also leads to UNC-68 oxidation . Lost in the presentation is how UNC-68 oxidation causes FKB-2 dissociation and whether (and how) UNC-68 oxidation causes increased UNC-68 leakiness in the absence of FKB-2 dissociation. All in all, the manuscript presents quite the Mobius strip with respect to cause-and-effect relationships.22. The authors go to great lengths to connect UNC-68 oxidation with FKB-2 dissociation and link this relationship to UNC-68 leakiness and the aging phenotype. But it's never really clear whether it is the dissociation of FKB-2 or the oxidation of UNC-68 that underlies the leakiness. The authors clearly believe that FKB-2 dissociation is an important event in this sequence, otherwise, why use FKB-2-KO and WT+FK506 models throughout? But another formulation of their model (Discussion) is all oxidation all the time: "RyR1 leak (due to age-dependent oxidation of the channel) causes mitochondrial caOxidation of UNC-68 causes a loss of FKB-2 from the channel. The precise mechanism by which this occurs is unknown and the subject of ongoing structural studies in our laboratory. The result is leaky UNC-68 channels. The same leak can be observed by removing FKB-2 without oxidation (e.g with FK506 or in the FKB-2 KO worms). Conversely, preventing FKB-2 removal with a Rycal drug (S107) can prevent leak even when the channels are oxidized. Thus, we conclude that it is the loss of FKB-2 from the channel that destabilizes the closed state and causes leak, not the oxidation by itself. The oxidation becomes important in the context of lifespan because it is the age-dependent increase in oxidative load in the worms that ultimately leads to UNC-68 oxidation in vivo.


But AML-DRG model was not associated with NRM (p = 0.072). Univariate analysis showed that the AML-DRG model could better stratify AML patients into different risk groups compared to the AML-HCT-CR model. Multivariate analysis confirmed that prognostic impact of AML-DRG and AML-HCT-CR models on post-transplant OS was independent to age, sex, conditioning type, transplant modality, and stem cell source . AML-DRG and AML-HCT-CR models can be used to effectively predict post-transplant survival in patients with AML receiving AHCT. Compared to AML-HCT-CR score, the AML-DRG score allows better stratification and improved survival prediction of AML patients post-transplant.We aimed to validate and prove the novel risk score models of acute myeloid leukemia (AML)-specific disease risk group (AML-DRG) and AML-Hematopoietic Cell Transplant-composite risk (AML-HCT-CR) in patients with acute myeloid leukemia (AML) after allogeneic hematopoietic stem cell transplantation (AHCT). Among the 172 AML patients analysed, 48.3% (n = 83) were females. Median age was 31.5 years (range 14 to 62 years), two patients was more than 60 years old (1.2%). Median follow-up was 44 months (range 1 to 94 months). According to the AML-DRG model, 109, 49 and 14 patients were in low-, intermediate- and high-risk group, respectively. According to the AML-HCT-CR model, 108, 30, 20 and 14 patients were in low-, intermediate-, high- and very high-risk group, respectively. Our results showed that the AML-DRG and AML-HCT-CR models significantly predicted cumulative incidence of relapse ( Allogeneic hematopoietic stem cell transplantation (AHCT) is a curative treatment for AML patients3. Disease relapse and transplant-related mortality (TRM) are important for long-term survival of AML patients, especially the disease relapse5. How to prevent post-AHCT relapse on the basis of controlling the toxicities of conditioning regimen was important to improve the outcomes of AML patients7. Survival of patients after AHCT is largely dependent on disease- and patient-related factors12. Pre-transplant risk assessment is the key to optimizing transplant outcomes.Acute myeloid leukemia (AML) is a clonal malignancy characterized by genetic heterogeneity due to recurrent gene mutations. Long term overall survival has stagnated in the past few decades. Even with an array of new gene mutation-targeted agents available for AML treatment14, representing disease-related factors, can predict overall survival (OS) but can’t integrate patient’s comorbidities and overall conditions. The hematopoietic cell transplantation-specific comorbidity index (HCT-CI) is also predictive of outcome but only includes comorbidities16. The disease risk comorbidity index (DRCI) and haplo-DRCI, integrating the DRI and HCT-CI, can effectively predict outcomes after AHCT17. Although previous studies have proved that minimal residual disease (MRD) is an independent predictor of survival in patients with AML20, MRD is excluded from most models. Therefore, two comprehensive new prognostic scores, AML-specific disease risk group (AML-DRG) and AML-Hematopoietic Cell Transplant-Composite Risk (AML-HCT-CR), have been shown to be predictive of OS and PFS in patients with AML received AHCT21. However, there are no published reports of the AML-DRG and AML-HCT-CR models in China. In this retrospective study, we aimed to verify the clinical effectiveness and generalizability of the AML-DRG and AML-HCT-CR models in a cohort of patients with AML receiving AHCT.Several prognostic models have been developed in recent years. The disease risk index (DRI)A total of 172 adult patients diagnosed with non-M3 acute myeloid leukemia who underwent the first AHCT from January 1, 2013 to December 30, 2018 in the First Affiliated Hospital of Zhengzhou University and Shandong Provincial Hospital Affiliated to Shandong First Medical University were enrolled in this study. Secondary AML was defined as AML developed after treatment with systemic chemotherapy and/or radiation therapy. All enrolled subjects in this study provided written informed consent. The study was conducted in accordance with the Declaration of Helsinki. All patients were followed up through our outpatient clinic, medical records in hospital, or by telephone cells. The follow-up endpoint was December 30, 2020.21. The AML-HCT-CR score assignment: AML-DRG score with the addition of 1 score for age ≥ 60 and 1 score for HCT-CI ≥ 321. DRI was applied as described by Armand et al.13. HCT-CI and HCT-CI/Age were applied as published by Sorror et al.16. ELN2017 genetic risk group was applied as described by Döhner, et al.22. MRD was assessed pre-HSCT by flow cytometry. MRD < 0.1% was judged to be negative23.AML-DRG score assignment: 1 for secondary AML, 1 for adverse European Leukemia 2017 genetic risk (ELN2017 genetic risk), 2 for CR with MRD positive or unknown MRD status, 4 for active disease2/day, day − 9), busulfan , cyclophosphamide . For HLA-haploidentical and HLA-matched unrelated donor transplants, the major conditioning regimen as follows: cytarabine , busulfan , cyclophosphamide , anti-thymocyte globulin (ATG) . Four patients received total body irradiation (TBI)-based regimen: cyclophosphamide  , total body irradiation . The GVHD prevention scheme used cyclosporine combined with mycophenolate mofetil and methotrexate.All patients received myeloablative conditioning. The major conditioning regimen for the identical sibling donor (ISD) group as follows: hydroxyurea , cytarabine  at 3 years after transplantation. The secondary endpoints were progression-free survival (PFS) at 3 years, non-relapse mortality (NRM) at 3 years, and cumulative incidence of relapse at 3 years. OS was defined as time from transplantation until death from any cause. PFS was defined as time from randomization to disease progression. Non-relapse mortality was defined as death from any cause not subsequent to relapse. Relapse was defined as either reappearance of leukemic blasts in the peripheral blood or at least 5% blasts in the bone marrow aspirate or biopsy specimen not attributable to any other cause, or reappearance or new appearance of extramedullary leukemia. Acute GVHD and chronic GVHD were diagnosed and graded according to the standard international criteriaOS, PFS were estimated using the Kaplan–Meier method and compared using the Log-rank test. Cumulative incidences of relapse, non-relapse mortality, and GVHD were calculated by accounting for competing risks. Competing risks for GVHD included death without GVHD and relapse. Relapse was a competing risk for non-relapse mortality, and non-relapse mortality was a competing risk for relapse. The comparison of the cumulative incidence in the presence of a competing risk was done using the Fine and Gray model. The impact of the AML-DRG and AML-HCT-CR models on survival outcomes were determined using univariable and multivariable Cox proportional hazards regression models. The discriminative ability of the models was assessed by Harrell’s C-statistics. p < 0.05 was considered significant. SPSS version 23.0 and R version 3.6.2 were used for data analysis.Informed consent was obtained for study participation from all patients, parent and/or legal guardian for minors and the protocol was approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University.Among the 172 AML patients analysed, 48.3% (n = 83) were females. Median age was 31.5 years (range 14 to 62 years), two patients was more than 60 years old (1.2%). Median follow-up was 44 months (range 1 to 94 months). According to the AML-DRG model, 109, 49 and 14 patients were in low-, intermediate- and high-risk group, respectively. According to the AML-HCT-CR model, 108, 30, 20 and 14 patients were in low-, intermediate-, high- and very high-risk group, respectively. The basic clinical data of the patients were shown in Table p = 0.605). The incidence for all-grade chronic GVHD at 2 years was 23.2% (95% CI 19.4–27.0), 22.4% (95% CI 15.5–29.3) and 7.1% (95% CI 0–22.1), respectively (p = 0.430)  and chronic GVHD developed in 38 patients (22.1%). For the entire cohort, the cumulative incidence of grade II to IV acute GVHD at day 100 was 22.7% (95% CI 20.0–25.4), the cumulative incidence of all-grade chronic GVHD at 2 years was 21.7% (95% CI 19.0–24.4). The cumulative 100-day incidence of grade II to IV acute GVHD for the low-, intermediate- and high-risk AML-DRG groups was 22.0% (95% CI 18.3–25.7), 18.4% (95% CI 11.8–25.0) and 7.1% (95% CI 0–21.1), differences are not statistically significant ( p = 0.582). The incidence for all-grade chronic GVHD at 2 years was 23.4% (95% CI 19.6–27.2), 30.3% (95% CI 19.8–40.8), 10.0% (95% CI 0–21.7) and 7.1% (95% CI 0–22.7), respectively ( p = 0.287) , 13.3% (95% CI 4.3–22.3), 30.0% (95% CI 15.8–44.2) and 7.1% (95% CI 0–21.1), respectively  and 24.8% (95% CI 22.1–27.5), respectively. We found that relapse and NRM occurred in 37 (21.5%) and 44 (25.6%) of 172 patients. Reasons for relapse were hematological in 30 patients (17.4%), extramedullary in 4 patients (2.3%), and hematological plus extramedullary in 5 patients (2.9%).p < 0.001)    , 32.7% (95%CI 25.4–40.0) and 50.0% (95% CI 30.2–69.8), respectively (The 3-year OS and PFS of the entire cohort were 56.9% (95% CI 49.9–64.9) and 53.1% (95% CI 45.9–61.3). Patients in low-, intermediate- and high-risk AML-DRG groups had median OS of 33.0 (1 ~ 94), 16.0 (1 ~ 73) and 4.5 (1 ~ 69) months, respectively (p < 0.001), with the corresponding 3-year OS of 69.6% (95% CI 61.0–79.6), 38.6% 95% CI 27.1–55.0) and 7.1% (95% CI 1.1–47.2), respectively (p < 0.001). The 3-year PFS were 67.3% (95% CI 58.8–77.2), 34.5% (95% CI 23.4–50.8) and 7.1% (95% CI 1.1–47.2), respectively (p < 0.001)  and 7.08 , respectively when compared with the low-risk group. Also, the risk of death was higher in high-risk group compared with the intermediate-risk group , confirming the ability of the AML-DRG model in post-transplant survival prediction. For AML-HCT-CR groups, patients with high  and very high-risk groups  had significantly increased risk of death than low-risk group, while there was no difference between low and intermediate-risk  and between high and very high group . Similar results were found in a univariable analysis for PFS as summarized in Table In univariable analysis for OS, patients with intermediate and high-risk AML-DRG groups had a significantly increased risk of death with hazard ratio (HR) of 2.62 , 0.71 (95% CI 0.63–0.79), 0.61 (95% CI 0.52–0.69), 0.52 (95% CI 0.43–0.60) and 0.59 (95% CI 0.50–0.67), respectively. Compared with the DRI model and the HCT-CI/Age, the AML-DRG and AML-HCT-CR models had significantly better discrimination ability on OS prediction with C-index. The risk assessment ability of AML-DRG and AML-HCT-CR may be better than that of ELN2017 genetic risk, DRI and HCT-CI/Age models.21. In this study, we examined the effect of the AML-DRG and AML-HCT-CR models on clinical outcomes of AHCT. The results demonstrated that both AML-DRG and AML-HCT-CR models could significantly predict the OS, PFS and relapse. The AML-HCT-CR model could significantly predict the NRM. While the GVHD did not reach statistical significance.Risk stratification is essential to predict the prognosis of patients with AML receiving AHCT. Recently, the AML-DRG and AML-HCT-CR models, which combines DRI, HCT-CI, ELN2017 risk classification, MRD and other important prognostic factors, was published and has demonstrated a significant impact in terms of OS and PFS21 reported that the OS at 5 years for low-, intermediate- and high-risk AML-DRG risk groups were 62.8%, 33.1% and 12.6%, respectively, and 5-year PFS were 60.4%, 31.1%, and 7.9%, respectively. The OS at 5 years for low-, intermediate-, high- and very high-risk AML-HCT-CR risk groups were 71.1%, 53.6%, 37.4%, and 12.7%, respectively, the corresponding PFS at 5 years were 67.4%, 51.7%, 36.2%, and 9.6%, respectively. We also performed pairwise comparisons in survival analysis among different groups. The results showed that for AML-DRG, all pairwise comparisons were statistically significant. However, unlike the reference publication, we did not find the difference of OS between AML-HCT-CR low-risk and intermediate-risk group , high-risk and very high-risk group . Fist, the sample of high- and very high-risk AML-HCT-CR patients was relatively small. Second, in our study, the conditioning regimens included busulfan (Bu)- and total body irradiation (TBI)-based regimens in patients, while the reference publication included busulfan (Bu)- and Melphalan-based regimens. Third, unlike the reference publication, only two patients in our sample were older than 60 years. Perhaps these reasons caused our results to be different from the reference publication. Therefore, a multi-centre clinical trial is required to confirm the findings from the reference publication. The multivariable analyses have shown that prognostic prediction of the AML-DRG and AML-HCT-CR models on post-transplant survival was independent to age, sex, conditioning type, transplant modality, and stem cell source . This means that the model can be applied in patients transplanted using both HLA-matched and unmatched donors.In our retrospective study including 172 patients with AML, we confirmed that the AML-DRG and AML-HCT-CR models have a prognostic prediction on OS and PFS . This is consistent with the reference publication. Kongtim et al.7. Therefore, how to prevent relapse after AHCT on the basis of controlling the toxicity of the conditioning regimen is important to improve the clinical outcome of patients with very high-risk AML-HCT-CR group.Since the AML-DRG and AML-HCT-CR models have been developed and validated their utility in mortality prediction, we further identified the specific cause of mortality. Our study found that the AML-DRG and AML-HCT-CR models were associated with the 3-year OS, mainly due to relapse. The AML-DRG model categorized patients into 3 distinct relapse risk groups, with 3-year cumulative incidence of relapse ranging between 12.2% for the low-risk group to 50.0% for the high-risk group. Similar results were found for the AML-HCT-CR model (12.3%for the low-risk group and 42.9% for the very high-risk group). The results suggest that for the patients in the intermediate and high-risk groups of AML-DRG and AML-HCT-CR models, MRD and other related indexes should be closely monitored after transplantation, and maintenance treatment or pre-emptive treatment should be given, so as to reduce the risk of recurrence and improve the efficacy of the transplantation. Patients in the very high-risk AML-HCT-CR group had higher recurrence and NRM rates, especially high NRM rates (42.9%for relapse rate and 50.0% for NRM rate). These patients usually had advanced-stage disease and/or high comorbidities burden before AHCT, which suggested that they had a higher risk of disease relapse and may be vulnerable to drug toxicities and transplant complications. A lower intensity conditioning regimen may help to prevent the transplant-related toxicity and mortality. However, this may lead to high relapse rates after AHCT, particularly for those with relapse/refractory leukemia21. It demonstrated that AML-DRG and AML-HCT-CR models provide better tools for risk stratification of patients.Our study found that the AML-DRG and AML-HCT-CR models had a significantly better discrimination ability on OS prediction with Harrell C-index of0.69 and 0.71, respectively, when compared with the DRI model (C-index0.61), HCT-CI/Age (C-index0.59) and ELN2017 risk classification (C-index0.52). This was partially in accordance with the reference study in which the AML-DRG and AML-HCT-CR models significantly better predicted risk of death after transplant with C-indices of 0.672 and 0.715, respectively27, we did not find AML-HCT-CR be prognostic for GVHD. A possible explanation for our results is that combining AML-DRG and HCT-CI probably weakens the weight of HCT-CI in GVHD prognostication.The AML-DRG model had no prognostic for NRM. In addition, both AML-DRG and AML-HCT-CR models were not prognostic for GVHD. Those data are also very important for HSCT choice. Further incorporation of NRM- and GVHD- related specificity indicators may help to achieve a more comprehensive scoring system. Although HCT-CI had been considered be prognostic of NRM and GVHDIn conclusion, our data confirm results similar to the reference publication and provide useful information on OS, PFS, relapse, and NRM prediction. Compared to AML-HCT-CR model, the AML-DRG allows better stratification and improved survival prediction of AML patients post-transplant. Considering the absence of prognosis for NRM and GVHD, we recommend using the AML-DRG and HCT-CI separately to obtain more accurate and relevant information to guide transplant choice.


In plants, Ca2+ signaling is central to both pattern‐ and effector‐triggered immunity, with the generation of characteristic cytoplasmic Ca2+ elevations in response to potential pathogens being common to both. However, despite their importance, and a long history of scientific interest, the transport proteins that shape Ca2+ signals and their integration remain poorly characterized. Here, we discuss recent work that has both shed light on and deepened the mysteries of Ca2+ signaling in plant immunity.Calcium ions function as a key second messenger ion in eukaryotes. Spatially and temporally defined cytoplasmic Ca This review summarizes how calcium ions, and the proteins which transport them, are integrated into plant immune responses. All eukaryotes use immune systems to protect themselves against potential pathogens. The plant immune system consists of two characterized perception layers: one that utilizes cell‐surface pattern recognition receptors (PRRs) to perceive extracellular immunogenic patterns, and another that relies on intracellular nucleotide‐binding leucine‐rich repeat (NLR) receptors that recognize pathogenic effectors inside the cell  or the host plant, as in the case of macromolecules released upon cell damage (DAMPs) or secreted peptide phytocytokines  or immune‐modulating peptide phytocytokines are recognized by PRRs, which leads to defense responses termed pattern‐triggered immunity (PTI) , mitogen‐activated protein kinase (MAPK) cascades, transcriptional reprogramming, and phytohormone biosynthesis  levels in living cells, and thus Ca2+ is sequestered in intracellular stores  or the apoplast via active transport, generating enormous electrochemical potential gradients across membranes , decoding (via Ca2+ sensor proteins), and responses (via regulation of downstream cellular processes).CaClapham, . Owing t2+ signaling is involved in all aspects of life, including growth regulation, development, abiotic stress responses, and reproduction , a single member, RBOHD, is responsible for ROS production in response to elicitors  involved in plant immunity remained comparably elusive for many years  greatly expanded the possibilities for real‐time, kinetic analysis of Ca2+ fluxes in intact tissues upon infection or elicitor treatment. The first GECI deployed in plants was aequorin (AEQ) from Aequoria victoria ‐LIKE (PBL) family members BOTRYTIS‐INDUCED KINASE 1 (BIK1) and PBL1  and intensiometric  sensors , as a putative ROS sensor  remain unclear. Interestingly, AEQ‐measured calcium signals in response to H2O2 were reduced in cngc2 and cngc4 mutants , autoinhibited Ca2+‐ATPases (ACAs) and ER Ca2+‐ATPases    in plant proteomes is still under debate and will not be discussed in detail here. Indeed, while studies suggest multiple plant proteins, including RKs, to display GC activity , with one or more CaM‐binding domains (CaMBDs) found within the cytosolic C termini of all CNGCs examined to date  regulation  or HR‐like lesion mimic (hlm) mutants dnd1 and dnd2/hlm1  , spontaneous cell death, and dis‐regulated auxin signaling  was reported to be reduced in cngc2 mutants compared to WT plants after treatment with the PAMP lipopolysaccharide (LPS) ‐type protein to be required for this process. Given the lack of mammalian‐type NOS enzymes in land plants  cause cngc2 (and cngc4) phenotypes, though this will require further study to resolve fully. Given that PTI is not affected in cngc2 and cngc4 mutants grown at low Ca2+ concentrations  recently identified loss‐of‐function mutants of OsCNGC9 (a group III CNGC and homolog of Arabidopsis CNGC18) that displayed compromised resistance to rice blast disease and lesion‐mimic phenotypes after flowering   , which is caused by an N‐terminal glycine to glutamic acid mutation in the Lotus homolog of Arabidopsis CNGC19  dominant gain‐of‐function mutations that trigger autoimmunity such as et al, et al, et al, et al, et al, 2+ influx and cell death  .CNGC20 has also recently been identified as a positive regulator of a specific form of autoimmunity  and alipathic glucosinolate production  , which are ligand‐regulated, homo‐ or heterotetrameric cation channels functioning in animal nervous systems. In Arabidopsis, GLRs form a 20‐member family that is sub‐divided into three clades: GLR1s, GLR2s and GLR3s; , products of hydrolyzed host cell walls that act as DAMPs during H. arabidopsidis colonization  are required for effective long‐distance signal propagation in plants  GLRs are also direct or indirect targets of PRR‐activated signaling pathways. It has been proposed that OG perception involves RKs of the WALL‐ASSOCIATED KINASE (WAK) family of epidermal growth factor (EGF)‐motif containing RKs  genes, which were transcriptionally upregulated in response to a panel of elicitors but not abiotic stresses  family was only recently identified. OSCAs have nine transmembrane helices, with a short extracellular N terminus and a larger C terminus, and constitute a 15‐member family in Arabidopsis / LYSM‐CONTAINING RECEPTOR‐LIKE KINASE 5 (LYK5) complexes, PBL27 directly phosphorylates the anion channel SLAH3  caused enhanced steady state Ca2+ signals and additionally failed to generate stimulus dependent stomatal Ca2+ oscillations due to retarded Ca2+ efflux after initial influx  are small proteins occurring in both prokaryotes and eukaryotes and form a family of eight members in Arabidopsis ANN1 was identified as a positive regulator of local and systemic Ca2+ responses that are induced upon mechanical wounding and perception of S. littoralis oral secretions, with ANN1 loss‐of‐function or overexpression lines displaying enhanced or decreased susceptibility toward S. littoralis, respectively  , is a positive regulator of cell death, as multiple acd6 alleles were found to induce varying degrees of autoimmunity and have been subject of research for over 20 years . As outlined previously, PTI and ETI induce qualitatively similar signaling outputs, some of which  have been shown to involve the same molecular components in both pathways  activated helper NLRs, massive ion fluxes and rapid cell death have been documented   was recently found to produce 2′,3′‐cAMP/cGMP through hydrolysis of RNA and DNA molecules  proteins  responsible for the early Ca2+ transient during PTI is/are still largely unknown.The molecular basis of Ca2+ signaling continues to benefit from tool development, and the modern, ever‐growing GECI repertoire has allowed for ever‐more detailed analyses of Ca2+ signals in vivo. However, we must remember that our conceptualization of Ca2+ signaling is at least partially defined by the GECIs we use, and may be too broad. It is possible that loss of individual Ca2+ channels evokes loss of individual Ca2+ signals within micro‐ and nanodomains, which are simply not resolved even by state‐of‐the‐art Ca2+ measurements.The study of immunity and Ca2+ signal upon elicitor perception. These results beg the question of whether PRR‐mediated signaling cascades indeed target and regulate such a high number of individual channels. One possibility is that such a dividing and reunifying signaling architecture may allow for genetic robustness, although this remains to be explored. It is also possible that individual channels function in cell type‐specific manners, as has been at least suggested in the case of OSCAs in guard cells. It is also possible that each of the Ca2+ channels currently identified are indeed either quantitative contributors and/or regulators, while the channel(s) mediating the major influx still await identification. Indeed, the recent identification of channel families  or novel characterization of known proteins as potential channels  indicates that there remains much to be discovered regarding Ca2+‐permeable channels in plants.It is remarkable that numerous channels from different families appear to contribute quantitatively to the rapid Ca2+ signals that evoke specific downstream responses, or if their channel activity rather represents the loss of membrane impermeability, with Ca2+ influx just being a fellow traveler of cell death’s onset. In either case, it will be as well critical to resolve the role of “classical” Ca2+ channels in ETI signaling.With regards to NLRs functioning as cation channels in ETI, future studies will have to find if they indeed generate CaPhilipp Köster: Visualization; Writing—original draft; Writing—review & editing. Thomas A DeFalco: Visualization; Writing – original draft; Writing—review & editing. Cyril Zipfel: Funding acquisition; Writing—review & editing.The authors declare that they have no conflict of interest.


The use of rabbit hemorrhagic disease virus (RHDV) as a biocontrol agent to control feral rabbit populations in Australia, in combination with circulating endemic strains, provides a unique environment to observe the interactions between different lagoviruses competing for the same host. Following the arrival of RHDV2 (GI.2) in Australia, it became necessary to investigate the potential for immunological cross-protection between different variants, and the implications of this for biocontrol programs and vaccine development. Laboratory rabbits of various immune status—(1) rabbits with no detectable immunity against RHDV; (2) rabbits with experimentally acquired immunity after laboratory challenge; (3) rabbits immunised with a GI.2-specific or a multivalent RHDV inactivated virus prototype vaccine; or (4) rabbits with naturally acquired immunity—were challenged with one of three different RHDV variants . The degree of cross-protection observed in immune rabbits was associated with the variant used for challenge, infectious dose of the virus and age, or time since acquisition of the immunity, at challenge. The immune status of feral rabbit populations should be determined prior to intentional RHDV release because of the high survival proportions in rabbits with pre-existing immunity. In addition, to protect domestic rabbits in Australia, a multivalent RHDV vaccine should be considered because of the limited cross-protection observed in rabbits given monovalent vaccines. Oryctolagus cuniculus) populations [Rabbit hemorrhagic disease virus (RHDV), a member of the genus Lagovirus in the family Caliciviridae, is used effectively as a biocontrol agent in Australia to reduce the significant environmental and economic impact of feral European rabbit . This vaccine offers complete protection against both the GI.1c and GI.1a variants used in biocontrol programs [Rabbit biocontrol programs are very successful in Australia, with the resulting feral population decline delivering an estimated $1 billion AUD in economic and environmental benefits annually ,9. Neverprograms , but GI.programs . In addiprograms . To dateprograms . ConsequCross-protection between different RHDV variants was investigated previously in the context of both natural immunity acquired after challenge and vaccinal immunity ,15. AustThe objectives of this research were to establish whether prior immunity to different RHDV genotypes could offer cross-protection against GI.2. Three types of immunity were investigated: firstly, rabbits with experimentally acquired immunity after primary infection with the current GI.1a biocontrol variant or the non-pathogenic GI.4 virus A; secondYoung domestic rabbits with various immunity and infection statuses were obtained from three geographically distinct regions in Australia. Both the cross-protection experiment on experimentally acquired immunity  and vaccNew Zealand White rabbits of both sexes were obtained from a colony known to be free of benign and pathogenic lagoviruses. Prior to both experimental challenges, all rabbits were confirmed to be seronegative to Australian lagoviruses by ELISA .50) of a commercially available GI.1a infectious virus preparation (GenBank acc. Number MF598301.1) [50 of the GI.1a challenge was titrated as part of the quality control tests for commercial preparations of RHDV. To obtain rabbits with GI.4c immunity, five-week-old rabbits were given a 1 mL oral dose (with an estimated 2300 genome copies) of a clarified homogenate of duodenal tissue from a GI.4c-infected rabbit (GenBank acc. Number KX357705.1). Titration to determine RID50 of GI.4c was not performed. Therefore, ‘genome copies’ is used as an in vitro approximate to quantify the GI.4c challenge.To obtain rabbits with GI.1a immunity, five-week-old rabbits were orally inoculated with 100 ‘50% Rabbit Infectious Doses’ (RID98301.1) . The RIDper os (PO) with 50 RID50 of GI.1bP-GI.2 (GenBank acc. Number MW467791)  A.Rabbits from a commercial breeding facility that were seronegative to GI.1 and GI.2 were vaccinated with a single dose of either a prototype, inactivated GI.2-specific vaccine or a prototype inactivated multivalent RHDV vaccine . Rabbits were vaccinated by subcutaneous injection at 10–12 weeks of age and then challenged PO with a heterologous lagovirus 28 days after vaccination B.Rabbits with naturally acquired immunity to lagoviruses were obtained from three commercial rabbit farms in Australia. These rabbits were initially obtained for other purposes. Following the detection of anti-GI.1 or anti-GI.2 antibodies based on serology, or GI.2 disease, these rabbits were then serendipitously selected for inclusion in this study. Although rabbits were not tested for anti-GI.4 antibodies, given the widespread distribution of this virus in Australian rabbit farms, these rabbits could possibly also have had anti-GI.4 antibodies . New ZeaTo mimic a natural route of infection, all rabbits were challenged by the oral administration of a 1 mL volume of virus solution. To assess cross-protection between circulating Australian lagoviruses, rabbits were challenged with various doses  of (1) t50 was selected as the challenge dose for rabbits with experimentally or naturally acquired immunity. This was based on an estimate of the amount of RHDV a rabbit may be exposed to during a control program [50 . All vaccinated rabbits were given a high 1500 RID50 PO dose  on arrival; (2) prior to vaccination or intentional exposure to RHDV to obtain natural immunity; (3) prior to challenge; and (4) after euthanasia.Rabbits were monitored twice daily during the seven-day or fourteen-day challenge period for the development of clinical signs of RHDV . While it was anticipated that moribund rabbits would be euthanised with an overdose of intravenous barbiturate, euthanasia was not required during the challenge period . All surviving rabbits were humanely killed after this challenge period with an overdose of intravenous barbiturate; however, the rabbits with immunity to GI.2 that survived heterologous challenge were repurposed into other research projects.p-value less than 0.05.Survival analyses was conducted with R Version 4.0.4 . PackageThree specific ELISAs were used to detect antibodies against GI.1, GI.4 and GI.2 to determine the immune status of rabbits prior to challenge. Details of GI.1 and GI.4 antibody ELISAs are as published ,28. A blTo help clarify the immune status of individual rabbits, where cross-reactivity between the different antigens was suspected, samples were also tested in three separate hemagglutination inhibition (HI) tests against GI.1a, GI.1c and GI.2 antigens .The presence of viral RNA in swabs of the cut surface of livers was determined using reverse-transcription quantitative PCR (RT-qPCR) assays specific to GI.1, GI.2 and GI.4c ,29,30. D50) dose of GI.2) remained seronegative after challenge and GI.2 was not detected by RT-qPCR testing on collected liver swab, indicating that it did not become infected. No seronegative rabbits challenged with a 1500 RID50 dose of GI.2 (0/12), GI.1a (0/12) or GI.1c (0/12) survived, with all succumbing to infection within 4 days post challenge (dpc), 6 dpc and 8 dpc, respectively. Median survival for a 50 RID50 dose of GI.2 was 4 dpc, compared to 3 dpc for a 1500 RID50 dose of GI.2. Median survival times for a 1500 RID50 dose of GI.1a and GI.1c were 3.5 and 3 dpc, respectively. These differences were not statistically significant (p = 0.3).Following RHDV challenge, all seronegative rabbits challenged with GI.1a or GI.1c died, and 7/8 challenged with GI.2 died within 14 days of infection . The sur50, all (7/7) rabbits with GI.1a acquired immunity survived, as did 7/9 rabbits with GI.4c acquired immunity (p = 0.2). Notably, there was strong evidence for cross-protection against GI.2 challenge for both variants, with a statistically significant survival probability compared to seronegative controls (p < 0.0001) given the same 50 RID50 GI.2 challenge.Following a GI.2 challenge of 50 RIDimmunity . The sur50. No GI.2-vaccinated rabbits survived infection with either 1500 RID50 of GI.1a (0/6) (50 of GI.1c (0/6)  A or 15001c (0/6) B. All ra0/6) (50 D–F.p = 0.002). That is, at 33 weeks of age and when given 1500 RID50, all GI.1-immune rabbits survived challenge with GI.1a (7/7) but only one survived challenge with GI.2 (1/7)  A.50 survived challenge but only five rabbits (5/9) given 1500 RID50 survived challenge (p = 0.08).There was a trend towards a dose-dependent difference in survival following GI.2 challenge in rabbits with GI.1 immunity. At 12 weeks of age, when given GI.2, nine rabbits (9/10) given 150 RIDhallenge A. Howeve50 dose of GI.2, at 12 weeks of age, five rabbits (5/9) survived challenge; but at 33 weeks of age, only one rabbit (1/7) survived (p = 0.03).The cross-protection against GI.2 challenge in rabbits with GI.1 immunity was found to reduce with age and/or time since acquisition of GI.1 immunity. When given the same 1500 RIDsurvived A. This d50 of either GI.1a, GI.1c or GI.2. One rabbit survived GI.1a challenge (1/4), two rabbits survived GI.1c challenge (2/3) and three rabbits survived GI.2 challenge (3/4) (p = 0.3); however, sample sizes were small in this experiment due to the serendipitous nature of acquiring these rabbits. Future robust studies to assess the relationship between GI.2 immunity and heterologous RHDV challenges should be undertaken.Rabbits from farm 2, with naturally acquired GI.2 immunity, showed varying degrees of cross-protection against infection with 1500 RIDge (3/4) B. There 50 dose  surviving a 1500 RID50 challenge. There was no statistical difference in the observed survival between these different challenge doses (p = 0.7).Rabbits from farm 3, that had survived an RHDV (GI.4cP-GI.2) outbreak, had complete cross-protection against a heterologous GI.2 (GI.1bP-GI.2) challenge, with all (13/13) rabbits surviving a 150,000 RID50 dose C. ModeraImmunological cross-protection between GI.1a, GI.1c and GI.2 lagoviruses has implications for both the use of these viruses as biocontrol agents in feral rabbit populations and the composition of vaccines to provide optimal protection for domestic rabbits in Australia. Here, we present a series of experimental studies in rabbits with various immune statuses and their subsequent response to challenge to Australian lagoviruses.As expected, all seronegative rabbits succumbed to disease following infection with GI.1c, GI.1a, or GI.2 viruses, demonstrating a very high case fatality rate for all viruses investigated. This is consistent with previous virulence studies with these variants ,20. In cThe origin of the naturally acquired immunity for the rabbits from farm 1, with detectable GI.1 antibodies, or for the rabbits from farm 2, with detectable GI.2 antibodies, remains unknown. The GI.1 ELISA is unable to distinguish between GI.1a or GI.1c antibodies, and between antibodies derived from natural infection or GI.1 vaccination. The known presence of GI.4 virus circulating at farm 2 also complicates the serological picture in these rabbits. Rabbits from farm 3 arrived at EMAI unvaccinated and infected with GI.4cP-GI.2. Therefore, the origins of their immunity may be assumed to be a consequence of surviving GI.4cP-GI.2 infection.The source of the naturally acquired immunity may be due to maternal antibodies. A breeding doe may confer maternal antibodies to her kittens for up to one year after RHDV vaccination . The rab50) with GI.1a at 33 weeks of age, suggesting that this immunity was not waning. Therefore, it is more likely that these rabbits acquired immunity due to natural exposure to GI.1 at farm 1.Alternatively, the naturally acquired immunity could be from previous sub-lethal exposure to these viruses. Both suppliers had no recent reports of disease; but given that GI.1 does not generally cause disease in rabbits less than nine weeks of age, natural infection with these viruses cannot be excluded. GI.1a is known to circulate naturally in the geographic region from which these rabbits were obtained . A cohorLikewise, this same scenario could exist for the rabbits with GI.2 immunity from farm 2. GI.2 can cause disease in younger rabbits from four weeks of age, and the pathogenicity of Australian GI.2 variants is close to 100% in susceptible animals . However50 dose, all rabbits that had survived a GI.4cP-GI.2 infection did not succumb to a heterologous GI.1bP-GI.2 challenge. As both viruses share the same GI.2 capsid protein, it is expected that previous infection with GI.4cP-GI.2 should protect against a lethal challenge of GI.1bP-GI.2 [Despite an extremely high 150,000 RID1bP-GI.2 . This im1bP-GI.2 . It is eThe prototype inactivated GI.2-specific vaccine did not offer any cross-protection against GI.1a or GI.1c  in contrThe results presented here demonstrate that, in laboratory rabbits, there is cross-protective immunity, even between heterologous strains, that protect against lethal disease. The extent of cross-protection conferred depends on the source of immunity , the virus genotype that has induced the immunity , the timing between acquisition of immunity and challenge, and in some cases the infectious dose of the challenge virus. Consequently, for biocontrol programs, the immunity of feral rabbit population should be established prior to intentional RHDV release. For optimal protection of domestic rabbits from unintentional infection with lagoviruses, a multivalent vaccine providing broad immunity to the recognized viruses should be considered.


Costimulatory molecules have been found to play significant roles in anti-tumor immune responses, and are deemed to serve as promising targets for adjunctive cancer immunotherapies. However, the roles of costimulatory molecule-related genes (CMRGs) in the tumor microenvironment (TME) of acute myeloid leukemia (AML) remain unclear. In this study, we described the CMRG alterations in the genetic and transcriptional fields in AML samples chosen from two datasets. We next evaluated their expression and identified two distinct costimulatory molecule subtypes, which showed that the alterations of CMRGs related to clinical features, immune cell infiltration, and prognosis of patients with AML. Then, a costimulatory molecule-based signature for predicting the overall survival of AML patients was constructed, and the predictive capability of the proposed signature was validated in AML patients. Moreover, the constructed costimulatory molecule risk model was significantly associated with chemotherapeutic drug sensitivity of AML patients. In addition, the identified genes in the proposed prognostic signature might play roles in pediatric AML. CMRGs were found to be potentially important in the AML through our comprehensive analysis. These findings may contribute to improving our understanding of CMRGs in patients with AML, as well as provide new opportunities to assess prognosis and develop more effective immunotherapies. Acute myeloid leukemia (AML) is one of the most prevalent hematological tumors. It is defined by the increase of undifferentiated myeloid progenitor cells in the hematopoietic system . For decThe tumor microenvironment (TME) plays a crucial role in AML growth, development, and therapy . In TME,via using CIBERSORT and ESTIMATE algorithms. First of all, expression levels of CMRGs were used to divide a total of 242 AML patients into two clustered costimulatory molecule subgroups. AML patients were then classified into CMRG-related gene subtypes according to those chosen differentially expressed genes between two costimulatory molecule subtypes. In addition, we constructed a signature that accurately predicted the clinical outcomes of AML patients and characterized the AML immune landscape. Our work, in a nutshell, systematically describes the landscape of costimulatory molecules and highlights their potential applications clinically, so aiding the creation of a rationale to guide AML patient care and treatment.This work systematically assessed the expression patterns of CMRGs and obtained a complete picture of the intra-tumoral immunological landscape https://portal.gdc.cancer.gov/) and the Gene Expression Omnibus (GEO) (https://www.ncbi.nlm.nih.gov/geo/). One GEO AML cohort named GSE10358 and the TCGA-AML cohort were obtained for further relative analyses. The fragments per kilobase million (FPKM) values from the TCGA-AML set were converted to transcripts per kilobase million (TPM) and were then assumed to be the same as those from microarrays (https://ocg.cancer.gov/programs/target) as an external validation cohort. Whole blood cohorts from GTEx downloaded from the UCSC Xena database (https://xenabrowser.net/datapages/) served as control samples for the analyses on the TCGA-AML and TARGET-AML samples.Gene expression and relevant clinicopathological information on AML were from databases named The Cancer Genome Atlas (TCGA)  and a testing AML set (n = 121). Based on CMRG-related genes with prognostic value, we carried out the Lasso Cox algorithm to minimize the over-fitting risk in the training AML set. We next analyzed each independent variable’s change and established a risk model by using 10-fold cross-validation. Candidate genes were further analyzed and chosen in the training AML set based on the using multivariate Cox regression results. The costimulatory molecule signature was calculated using the data of each gene’s coefficient and expression. Then, based on the median score of whole samples in the AML training set, samples of two sets were respectively divided into low- or high-risk groups. Survival analysis and the creation of receiver operating characteristic (ROC) curves were performed on AML patients belonging to two risk categories.The CMRG-related risk model was generated, and the score was estimated for each AML sample to quantify the costimulatory molecule patterns. Differentially expressed genes were subjected to univariate Cox analysis. Using an unsupervised clustering technique, AML patients were divided into three distinct CMRG-related gene subtypes  based on the findings of the univariate Cox analysis. The chosen 242 patients with AML were then randomly categorized into two sets at a ratio of 1:1, a training AML set  of drugs using the “pRRophetic” package in R software.p < 0.05.All statistics were analyzed in R software (version 4.1.0). Statistical significance was all set at This research contained 60 CMRGs in total. We firstly investigated the somatic copy number variations in the 60 CMRGs. A total of 455 patients from two AML cohorts (TCGA-AML and GSE10358) were integrated into our study at the beginning. Since only 242 AML samples had information on clinical results, we extracted their data of them for further analysis. The univariate Cox regression and Kaplan-Meier analyses revealed that 44 CMRGs were found might be related to the prognosis of AML . A costiWe found that subtype B was enriched in activated immune pathways, such as cytokine receptor interaction, cell adhesion molecules, graft versus host disease, antigen processing and presentation, B cell receptor signaling pathway, allograft rejection, Toll-like receptor signaling pathway, regulation of actin cytoskeleton, and chemokine signaling pathway . To inveThe biological role of costimulatory molecule patterns in AML remains unknown. We used the “limma” package in R software to identify costimulatory molecule subtype-related differential expressed genes and then carried out the functional enrichment analysis based on them . KEGG ann = 121) and a testing group (n = 121) at a ratio of 1:1. Following, LASSO and multivariate Cox analyses for costimulatory molecule subtype-related differentially expressed genes were carried out to identify CMRG-related genes with prognostic value in AML patients. According to the minimum partial probability of deviance (The CMRG-related prognostic signature was then established. First, we randomly divided the AML patients into two groups, including a training group (deviance , hub ovedeviance . We thendeviance . Hence, To validate the predictive value of the CMRG-related risk model in patients with AML, we separately divided AML patients in each set into two different risk groups according to the cutoff, which was the median value of those in the AML training set. In detail, AML patients whose scores were larger than the chosen cutoff were grouped as low risk, while others whose scores were smaller than the chosen cutoff were grouped as high risk. The survival curves revealed that AML patients in the training set with lower scores significantly had a favorable overall survival time when they were compared to those with higher scores . In the To investigate the difference in TME and expression of checkpoints between two risk groups divided by constructed signature, we divided all the 242 AML patients into two groups according to cutoff and perform the CIBERSORT algorithm, by which we could assess the correlations between risk scores and immune cell abundance. As the scatter diagrams in We then selected chemotherapy drugs to evaluate the treatment sensitivities of patients in different risk AML groups. Interestingly, we found that the patients with higher costimulatory molecule scores had lower IC50 values for WZ-1-84, WO2009093972, SL 0101–1, S-trityl-L-cysteine, Roscovitine, Rapamycin, Parthenolide, NVP-TAE684, Kin001-135, GNF-2, Dasatinib, CGP-082996, CGP-60474, Bortezomib, and AZ628, while IC50 values of chemotherapeutics such as VX-702, Vorinostat, Thapsigargin, SB 216763, Plx4720, PF-562271, OSI-906, MK-2206, Mitomycin-C, Midostaurin, Gemcitabine, Embelin, Cytarabine, BX-795, Bosutinib, BI-D1870, AZD7762, Axitinib, AP-24534, AKT inhibitor III, AG014699 and ABT263 were significantly lower in the patients with lower costimulatory molecule scores . MoreoveThe involvement of costimulatory molecule signal in innate immunity and anticancer effects has been shown by research . NonetheThe clinical outcome of AML after conventional chemotherapy is still poor . DespiteCheckpoints in the immune system play crucial roles in the immunosuppression of most cancers . In hemain vivo and in vitro need to be performed to confirm the findings in this study. Also, the number of clinical characteristics that both datasets in this study contained is too small. Some common and crucial clinical variables for AML patients were unavailable for further analysis, which may affect the confirmation of the prognostic value of constructed signature in the immune response in AML.Absolutely, this study had several limitations. First of all, the analyses were all carried out on the samples obtained from the public databases with retrospective data. Large-scale prospective studies Our analysis based on the CMRGs revealed a potential regulatory mechanism in AML, by which they might affect the TME, clinical features, drug susceptibility, and prognosis of patients with AML. These findings highlight the applications of CMRGs in AML in clinics and provide ideas for applying personalized immunotherapy to AML patients.