However, reports on the prognostic impact of various translocations causing KMT2Ar are conflicting. Less is known about associated mutations and their prognostic impact. In a retrospective analysis, we identified 172 adult patients with KMT2Ar AML and compared them to 522 age-matched patients with diploid AML. KMT2Ar AML had fewer mutations, most commonly affecting RAS and FLT3 without significant impact on prognosis, except for patients with ≥2 mutations with lower overall survival (OS). KMT2Ar AML had worse outcomes compared with diploid AML when newly diagnosed and at relapse, especially following second salvage . Therapy-related KMT2Ar AML (t-AML) had worse outcomes compared with de novo KMT2Ar AML . Allogeneic hematopoietic stem cell transplant  in first remission was associated with improved OS . In a multivariate analysis, translocation subtypes causing KMT2Ar did not predict survival, unlike age and allo-HSCT. In conclusion, KMT2Ar was associated with adverse outcomes regardless of translocation subtype. Therefore, AML risk stratification guidelines should include all KMT2Ar as adverse.Acute myeloid leukemia (AML) with rearrangement of the  KMT2Ar leukemias affect the myeloid lineage, lymphoid lineage, or both. They are associated with an adverse prognosis when occurring in infants, children, or adults with leukemia [KMT2A-MLLT3 fusion caused by t is the most common KMT2Ar in adults with AML, but more than 80 different fusion partners have been described [Chromosomal translocations involving 11q23 where the leukemia . The KMTescribed .KMT2Ar leukemias, with a short latency to clinical manifestation [KMT2Ar AML [KMT2Ar AML were classified as adverse [KMT2Ar AML [KMT2Ar AML and their prognostic impact [KMT2Ar in the relapsed or refractory (R/R) setting.Among chemotherapies associated with therapy-related AML (t-AML), topoisomerase II inhibitors are strongly associated with estation . In otheT2Ar AML –9. The E adverse . NonetheT2Ar AML , 12. Genc impact . FinallyKMT2Ar AML treated at our institution to characterize their genomic and phenotypic characteristics further and determine the association of these variables with prognosis and response to various lines of therapy.We conducted a retrospective analysis of patients with KMT2Ar. We excluded patients with 11q23 translocations in whom KMT2Ar was not detected by fluorescence in situ hybridization (FISH)  had mutational analysis done with targeted next-generation sequencing (NGS) panels. These panels included genes frequently involved in hematologic malignancies  had nuscript . Among pP ≤ 0.05 in the univariate analysis were included in the initial multivariate analysis. Backward model selection was used to eliminate variables until all remaining were statistically significant with P < 0.05. Analyses were performed using GraphPad Prism  and SAS version 9.4 .Patient characteristics were summarized using medians and ranges for continuous variables and frequencies or percentages for categorical variables. Continuous variables were compared using the Wilcoxon rank-sum test for pairwise comparisons and the Kruskal–Wallis for multiple comparisons. Categorical variables were compared using Fisher’s exact test. Responses were defined according to the International Working Group recommendations . OverallKMT2Ar AML was 52 years , with a relatively higher proportion of females  (Table KMT2Ar AML commonly manifested as monocytic in 67% of patients compared with 33% in the diploid karyotype group (P < 0.0001), was associated with markers of proliferation such as a higher percentage of bone marrow blasts at diagnosis and a lower platelet count. In this cohort, 69 patients (40%) had t-AML and KMT2Ar.The median age at diagnosis of patients with KMT2A-MLLT3 detected in 97 patients (57%), followed by t/KMT2A-MLLT4 detected in 19 patients (11%), t/KMT2A-ELL in 14 patients (8%), t/KMT2A-MLLT1 in 10 patients (6%), t/KMT2A-EEN in 10 patients (6%), t/KMT2A-MLLT10 in 3 patients (2%), and t/KMT2A-MLLT2 in 2 patients (1%)  and t(q23:v) . No other significant differences in the characteristics were found, including rates of allo-HSCT.The most common translocations were t/1%) Fig. . Seventepatients 7%, folloowed by t;11, which was significantly lower compared to diploid AML  , followed by FLT3 in 14 of 110 patients (13%). These mutations were predominately D835 FLT3 kinase domain mutations , and less commonly FLT3 internal tandem duplications . Mutations in FLT3 and RAS co-occurred in 5 of 110 patients (5%) , TP53 (5%), CEBPA (4%), and IDH1 (3%). Therefore, mutations associated with KMT2Ar most commonly involved the RAS pathway .Among patients with newly diagnosed AML, 117 patients (68%) had targeted NGS assessment of their diagnostic bone marrow specimens  Fig. . The mosKMT2Ar AML significantly differed from that of diploid AML , a TET2 mutation in 1 of 32 patients , and no ASXL1 mutations were detected . Furthermore, there were no NPM1 mutations among 88 patients with KMT2Ar AML, and mutations in FLT3, IDH, and CEBPA were significantly less common when compared to diploid AML , CD13 , and CD34 , and higher expression of CD33   (Table P = 0.9). Notably, patients with KMT2Ar had higher rates of early mortality compared to diploid AML with a 60-day mortality rate of 15 vs 7% (P = 0.004). There was no statistically significant difference in response rates when comparing various 11q23 translocations, although t tended to have a lower CR rate compared to t   . Importantly, none of the patients with long-term remission had KMT2Ar detected on assessments done following induction treatment around Day 30, therefore highlighting the importance of achieving early cytogenetic remission in this setting. Conversely, patients who achieved morphologic remission (CR/CRi) and subsequently relapsed had a higher proportion of KMT2Ar detected at Day 30 following induction (10 of 50 patients or 20%). At the time of relapse, all patients with a cytogenetic analysis done had persistence of the KMT2Ar (55 of 55 patients or 100%). This suggests that KMT2Ar are founding events in this leukemia and that relapse following current treatments is not driven by the acquisition of novel drivers in the absence of KMT2Ar.To assess the validity of KMT2Ar AML had a higher risk of relapse compared to diploid AML with a CIR at 5 years of 66 vs 62% (P = 0.04) . Following the first relapse, patients with KMT2Ar AML had a higher risk of subsequent relapse and worse OS compared to diploid AML with each line of therapy   Fig. . These papy Fig. . The med01) Fig. .Fig. 3CuKMT2Ar AML regardless of age, t was associated with slightly better outcomes compared to other KMT2Ar though these differences were not statistically significant  to other KMT2Ar  and 5-year CIR and OS rates of 80  and 6% , respectively  and 5-year CIR and OS rates of 62%  and 9%  respectively   , TET2 and TP53 mutations in one patient, and JAK2 and CEBPA in the remaining patient.Given that mutational analysis in AML has improved risk stratification by adding to the established value of cytogenetic abnormalities, we analyzed the impact of additional mutations on outcomes of T2Ar AML , 22. MutKMT2Ar AML received an allo-HSCT. Among them, 42 (24%) were transplanted in the first remission as consolidation. We performed a landmark analysis to compare outcomes of patients with KMT2Ar AML who had allo-HSCT in the first remission compared to those who did not. Allo-HSCT was associated with significantly improved OS with a median of 10.4 years  and 5-year OS of 52%   with 01) Fig. . The medKMT2Ar leukemias are frequently associated with central nervous system (CNS) involvement. Among newly diagnosed patients with KMT2Ar AML, 21 patients (12%) had CNS disease, three of whom had it at presentation (2%), whereas the rest developed it later in their treatment (17 patients or 10%). We next assessed the characteristics of these patients and whether there were predictors at baseline of CNS disease. When compared to KMT2Ar AML without CNS involvement (CNS-), we found no difference in variables usually thought to confer an increased risk of CNS involvement such as elevated white blood cell count, a monocytic phenotype  or presence of extramedullary involvement  . Among KMT2Ar patients with CNS involvement, 13 of 21 patients (62%) successfully cleared their spinal fluid from leukemia cells following intrathecal and systemic therapies. There was no statistically significant difference in OS between the KMT2Ar CNS + and CNS− groups, though CNS + patients had a trend for worse long-term survival   and decreased risk of death with an HR of 0.30  , however, this prognostic impact was abrogated by the addition of allo-HSCT to the model. Similarly, having ≥2 mutations in addition to KMT2Ar independently predicted for higher risk of death with an HR of 2.66 , but was not a predictor when allo-HSCT was added to the model. The only other factors identified as independent predictors of the risk of death were age, low platelet count, and an elevated creatinine at diagnosis.In order to assess the impact of confounding variables on prognosis, we conducted univariate and multivariate analyses predicting risks of relapse or death in patients with AML Fig. . Allo-HSKMT2Ar AML is unique and characterized by a relative paucity of mutations, where almost all cells at diagnosis have KMT2Ar by cytogenetic analysis, which persists following resistance or relapse. This confirms previous clinical observations and laboratory studies indicating that KMT2Ar is the founding event and a potent driver in this leukemia with a minimal contribution by additional mutations [RAS or FLT3 genes and did not affect prognosis. These mutations are likely subclonal and occur later in the pathogenesis of the disease but possibly confer some proliferative advantage [KMT2Ar are not preceded by a precursor state, a common feature of non-fusion-driven AML [KMT2Ar which was associated with an adverse prognosis. These patients had co-occurrence of mutations in either RAS, FLT3, or PTPN11.In this study, we show that the mutational landscape of utations –28. Mutadvantage . The rariven AML . HoweverKMT2Ar in this cohort of adults with newly diagnosed AML was 2% (172 of 9465 patients), which is lower than the incidence reported in other studies of 3–7% [KMT2Ar translocations in up to 45% of cases in one estimate [KMT2Ar, while none were detected in the frontline cohort , the target of gemtuzumab ozogamicin (GO). This is in line with a previous report on the immunophenotype of 17 of 19 KMT2Ar AML expressing CD33 (median expression 77%) [KMT2Ar AML in adults. A recent study by the Children’s Oncology Group demonstrated improved outcomes with the addition of GO in pediatric KMT2Ar AML [KMT2Ar AML . The NCCN  guidelines recommend a screening lumbar puncture (LP) to assess for CNS involvement in asymptomatic patients with monocytic AML, mixed phenotype acute leukemia, extramedullary disease, WBC >40 × 109/L, or presence of FLT3 mutations [KMT2Ar AML would need a screening LP. We attempted to discriminate further predictors of CNS disease in KMT2Ar AML and found no clear predictors of CNS involvement in this entity.We show that ion 77%) . This fiT2Ar AML . As prevutations , 39. UsiKMT2Ar AML. This is the first study to our knowledge to examine outcomes for these patients with each line of therapy. In this study, patients with KMT2Ar AML had significantly worse outcomes following relapse compared to an age-matched population with a diploid karyotype. We found that the median OS for KMT2Ar AML after second-line therapy was 6 months and 2.4 months only after third-line therapy. This highlights the need to improve outcomes for patients with KMT2Ar AML, and the results of this study could be used when comparing available treatments to promising novel therapies in clinical development. Notably, early results from clinical trials investigating menin inhibitors, which are novel targeted agents for KMT2Ar leukemias or leukemias with other susceptible genotypes are highly encouraging [There is a paucity of data on the outcomes of patients with relapsed ouraging –42.KMT2Ar AML. Therefore, these results should not be applied to the general population with KMT2Ar AML. This study shows that the prognosis of KMT2Ar in adults with AML is adverse, regardless of the translocation, when looking at a diverse  large cohort of patients. Despite a lower risk of relapse in t compared to other KMT2Ar, the difference in OS was not significant albeit a trend and was not an independent predictor of survival in the multivariate analysis. Therefore, risk stratification criteria such as the ones by the NCCN or the ELN, should reflect this data when indicating that among KMT2A rearrangements, t is in the intermediate-risk category [KMT2Ar in the adverse risk category which would better reflect the characteristics of patients seen in the clinic. This is critical as we have shown that allo-HSCT in first CR is associated with significantly improved outcomes in all KMT2Ar AML. Certainly, no prognostic model is perfect, however, these risk models are used in clinical practice to determine which patients should undergo a consolidative allo-HSCT. Including all KMT2Ar in the adverse risk group would emphasize the importance of transplant for these patients. Current risk stratification models were developed largely based on cohorts of younger, fit patients with de novo AML, treated with intensive chemotherapy despite the fact that AML is largely a disease of older age (median age at diagnosis of 68). The advent of highly effective venetoclax-based therapies for older patients with AML invites for more inclusive risk stratification models regardless of age [A recent study by the Cancer and Leukemia Group B (CALGB) found that outcomes of patients with de novo AML, t9;11) and age <60 years, excluding those who underwent allo-HSCT in first CR, were comparatively better than those with other 11q23 translocations  . This wa;11 and acategory , 43. Thes of age .KMT2Ar is associated with adverse outcomes in adults with AML. It has a low mutational burden with a minimal associated prognostic impact. Adverse risk in KMT2Ar AML is worst in older patients and those with t-AML. Consolidation with an allo-HSCT following the first remission is associated with significantly improved outcomes leading to long-term survival. Outcomes of patients with relapsed KMT2Ar AML are dismal, highlighting the need for novel therapeutic strategies.In conclusion, Supplement


They can be externally activated by optogenetic tools, but this requires robust introduction of exogenous optogenetic genes for expression of photosensitive proteins in biological systems. Here we present femtoSOC, a method for direct control of Ca2+ channels solely by ultrafast laser without the need for optogenetic tools or any other exogenous reagents. Specifically, by focusing and scanning wavelength-tuned low-power femtosecond laser pulses on the plasma membrane for multiphoton excitation, we directly induced Ca2+ influx in cultured cells. Mechanistic study reveals that photoexcited flavins covalently bind cysteine residues in Orai1 via thioether bonds, which facilitates Orai1 polymerization to form store-operated calcium channels (SOCs) independently of STIM1, a protein generally participating in SOC formation, enabling all-optical activation of Ca2+ influx and downstream signaling pathways. Moreover, we used femtoSOC to demonstrate direct neural activation both in brain slices in vitro and in intact brains of living mice in vivo in a spatiotemporal-specific manner, indicating potential utility of femtoSOC.Ca For example, Kyung et al. have developed an optogenetic tool, called OptoSTIM1 based on a combination of a plant photoreceptor and the STIM1, which can activate Ca2+ channels solely by ultrafast laser without the need for optogenetic tools or any other exogenous reagents, hence alleviating the requirements which would normally be required for the optogenetic activation of SOCs. Specifically, by focusing and scanning wavelength-tuned low-power femtosecond laser pulses on a small region of the plasma membrane for multiphoton excitation, we directly induced Orai1to covalently bind flavin and polymerize for the formation of SOCs independently of STIM1, which would normally participate in the SOC formation, leading to the activation of Ca2+ influx and downstream signaling pathways. Through a series of experiments and validation tests, we developed the molecular mechanism of femtoSOC including the mechanism of Orai1hexamer formation in the SOC formation and the role of the mitochondria and reactive oxygen species. Finally, to show the potential utility of femtoSOC, we used it to demonstrate direct in vitro and in vivo neural activation in brain slices and an intact living mouse brain, respectively, in a spatiotemporal-specific manner. It is important to note that femtoSOC is highly simple as it can be demonstrated on any two-photon microscope system in which the required laser illumination power is only a few milliwatts . femtoSOC holds promise for various biomedical applications as a much simpler alternative to optogenetic tools.In this study, we present a radically different approach to controlling SOCs. Our method (termed femtoSOC) enables direct control of Ca2+ fluorescent imaging. As shown in Fig. 2+ rise immediately after the femtoSOC laser illumination was evident. As shown in Fig. 2+ concentration was found to start in the femtoSOC laser illumination area and soon diffused to the entire cell in about 10 s, which eventually led to an overall intracellular Ca2+ concentration increase from about 200 nM to about 600 nM , an inhibitor that suppresses the sarco/endoplasmic reticulum Ca2+ ATPase, depletes Ca2+ in ER, and allows an influx of Ca2+ into the cytosol  transfected into cells   laser at 760 nm with 10 times higher power was used to stimulate cells as a control experiment of femtoSOC at the same laser wavelength. No cells showed Ca2+ rise in the control experiment , the probability increased, reached a maximum value, and dropped as the scan area increased. The probability was found to be zero at a scan area of 1 μm2 although the energy density was quite high  2-aminoethoxydiphenyl borate (2-APB) which is an inhibitor of transient receptor potential (TRP) channels and SOCs, (ii) SKF-96365, which is a blocker of voltage-gated Ca2+ channels, transient receptor potential cation (TRPC) channels, and SOCs; and two specific blockers of SOCs, namely (iii) YM-58483 and (iv) GSK-7975A. As shown in Fig. 2+ influx after the femtoSOC laser illumination. Also, YM-58483 and GSK-7975A strongly suppressed Ca2+ influx after the femtoSOC laser illumination  in contrast to a slight level of Ca2+ influx observed in the presence of YM-58483. These results indicate that SOCs were opened by femtoSOC. Third, we performed a similar test to verify if Ca2+ influx is solely through SOCs. Specifically, in the Ca2+ add-back test, YM-58483 was added into Ca2+-free medium 30 s after adding Ca2+ back. As shown in Fig. 2+ influx was found to have already occurred for 30 s, but it was immediately and significantly suppressed by adding YM-58483 to the medium. Moreover, the decay time of the Ca2+ level was found significantly shorter than that in the control. Therefore, these results show that Ca2+ entered cells solely through SOCs.To verify if femtoSOC-induced Caion Fig. . It is iion Fig. . After a2+ influx probability values in the cell groups as shown in Fig. 2+ influx in the Orai1/2/3-KO cells after the femtoSOC  and Mg2+ (1–5 mM) in culture medium separately and observed no fluorescence increase in both cell groups after the femtoSOC laser illumination as shown in Fig. 2+ and Zn2+. Finally, we investigated if femtoSOC also activates transient receptor potential channels (TRPCs), a group of non-specific cation channels. Specifically, we used cells with TRPC1 knockdown and observed very little change in Ca2+ influx probability after the femtoSOC laser illumination as shown in Fig. To verify if Orail1 directly participates in the femtoSOC-induced formation of SOCs, we conducted a series of experiments. First, we used cells with Orai1 knockdown and overexpression and identified decreased and increased CaSOC Fig. . In the lux Fig. , suggeste14 Fig. . Fourth,2+ influx after the femtoSOC laser illumination. Specifically, we used plasmid G-GECO1-Orai1, a fusion of the Ca2+-sensitive green fluorescent protein (GFP) with Orai1 that expressed in HeLa cells to simultaneously visualize both Orai1 activity and Ca2+ influx. As shown in Fig. 2+ influx occurred only in the two-photon scanning area where Orai1 also showed its aggregation at the very beginning after femtoSOC, indicating that Orai1 formed SOCs in the area. Second, to confirm Orai1 aggregation during the femtoSOC laser illumination, we monitored Orai1’s dynamic location with Orai1-YFP and Orai1-mCherry. Figure 2+ influx, we monitored Ca2+ influx in cells cultured in Ca2+-free medium and found no Ca2+ influx under this condition while Orai1-YFP aggregation was present as shown in Fig. 22 we used Tween-20 to disturb the formation of hydrophobic bonds and hence suppress Orai1’s polymerization. As shown in Fig. 2+ influx probability significantly decreased in the presence of Tween-20 (0.025% v/v), further supporting that Orai1 molecules polymerize via hydrophobic bonds.To study the molecular mechanism of femtoSOC, we conducted a series of experiments. First, we investigated the dynamic change of Orai1 associated with Ca2+ level in the ER during the femtoSOC laser illumination. The localized Ca2+ level in the ER was fluorescently indicated by G-CEPIA1ER. After the femtoSOC laser illumination, no fluorescence decrease was observed as shown in Fig. 2+ release from the ER. Next, we observed STIM1’s dynamics, which, according to the classical SOC regulation mechanism, polymerizes if Ca2+ is depleted in the ER, migrates to the cytoplasm–membrane–ER junction, and interacts with Orai1 to form SOCs24 as shown in Fig. 2+ store in the ER. Therefore, STIM1 did not participate in the SOC formation by Orai1. To further confirm these results, we used STIM1-KO HeLa cells obtained by CRISPR and measured their Ca2+ influx probability. As shown in Fig. 2+ influx probability of STIM1-KO HeLa cells incubated in medium with a specific inhibitor of STIM2, G418, and found no change in the Ca2+ influx probability either. Moreover, we performed femtoSOC in STIM1-KO cells in Ca2+-free medium. After 5-s rest, 200 μM Ca2+ was added back into the medium. Immediately Ca2+ influx could be observed in the femtoSOC area. At the first 10 s, the Ca2+ influx stayed in the illuminated region and soon diffused out to the whole cell  and quinacrine dihydrochloride (QCDC), which are quenchers/inhibitors of photoexcited flavin, and diphenyleneiodonium (DPI), an inhibitor of protein-bound flavin, and measured the femtoSOC-induced Ca2+ influx probability in all groups. As shown in Fig. 2+ influx through SOCs 25 to the cell medium for one-day culture. As shown in Fig. 2+ influx probability of these cells increased significantly, while that of cells cultured with flavin adenine dinucleotide (FAD) which suppressed the cellular uptake of RF26 decreased significantly. Taken all data above, flavin is necessary for femtoSOC-induced Ca2+ influx.To further investigate the unique molecular mechanism of femtoSOC which is very different from the classical mechanism of STIM1-triggered Ca28 we treated cells with dithiothreitol (DTT) that could protect the –SH group of cysteine in Orai1 from forming thioether bonds with flavins. The DTT treatment itself did not influence the function of SOC gating  was genetically labeled with YFP and transfected into cells. As shown in Fig. C126S, Orai1C143S, Orai1C195S, Orai1C126S-C195S and Orai1C195S-C143S-C126S, into Orai1-KO cells. Ca2+ influx could occur normally when treated with TG  at complex I. Malonic acid (MA), an inhibitor of ETC at complex II, was also added to the cell medium along with rotenone. As shown in Fig. 2+ influx probability was not influenced in the presence of rotenone or both rotenone and MA. Finally, cells were treated with FCCP, a potent mitochondrial oxidative phosphorylation uncoupler, to terminate the mitochondrial respiration and depolarize the MMP, but the femtoSOC-induced Ca2+ influx probability was also not influenced as shown in Fig. Considering that flavin is involved in intracellular redox reactions and is a vital component of mitochondrial energy production, we investigated a potential influence of femtoSOC on the mitochondria. As shown in Fig. 2DCFDA (DCF) or dihydroethidium (DHE). Also, as shown in Fig. 2+ influx probability was not influenced by any of the ROS scavengers. These results show that the ROS is not related to or influenced by femtoSOC. Taken together, we summarize the total femtoSOC process in Fig. Furthermore, we studied a potential influence of femtoSOC on the reactive oxygen species (ROS). Figure 2+ influx typically activates intracellular signaling pathways, we investigated femtoSOC-induced activation of extracellular signal-regulated kinases (ERKs), one of the most essential signaling pathways that are involved in the regulation of meiosis, mitosis, and postmitotic functions. Specifically, we transfected the plasmid ERK2-GFP fusion protein into HeLa cells to visualize ERK dynamics in the cytoplasm. As shown in Fig. Since Ca2+ indicator Cal-520/AM (6 μM), incubated in a perfusion chamber, and observed by a two-photon confocal microscope . Neurons at about 150-μm depth in the hippocampus were randomly selected for femtoSOC excitation. After the femtoSOC laser illumination, the femtoSOC-excited neuron immediately exhibited high Ca2+ influx (n = 34 neurons from 3 brain slices), followed by Ca2+ rise in the neighboring neurons as shown in Fig. 2+ signal amplitude and response time in the neighboring neurons were related with the distance from the femtoSOC-excited neuron as shown in Fig. 2+ waves. Furthermore, we demonstrated the activation of neurons in an intact living mouse brain. Neurons in the brain were fluorescently labeled by stereotaxic injection of Cal-520/AM after anesthesia for in vivo observation. The brain was imaged by the same two-photon microscope . Target neurons at about 175 μm depth were randomly selected. As shown in Fig. 2+ rise and oscillations after the femtoSOC laser illumination. Two minutes later, a second femtoSOC excitation of the same neuron activated the second Ca2+ response in the neurons. As shown in Fig. To show the utility of femtoSOC, we used it to demonstrate neural activation in a fresh brain slice and an intact living mouse brain in vivo. Specifically, we demonstrated the activation of any target neuron in a fresh brain slice of normal mice (C57BL/6) within the microscope’s field of view without the need for optogenetics. The brain slice  was labeled with Ca2+ influx spectrum shown in Fig. 2+ influx probability shown in Fig. 2+ influx. The low laser power, high dependence of femtoSOC on the laser wavelength, and high cell viability (membrane integrity) indicate that femtoSOC-induced photoporation by breakdown or ionization is not present and hence does not contribute to Ca2+ influx. Nevertheless, there might still be other possible mechanisms underlying femtoSOC-induced Ca2+ influx. Other potential methods of flavin excitation are also worth investigation. For example, blue light might activate flavin and further SOC channels under certain conditions. The photoexcited flavins covalently bind Cys126 in TM2 and Cys195 in TM3 of Orai1 via thioether bonds, respectively, to create a hydrophobic core. The Orai1 molecules might polymerize by hydrophobic bonds to form the hexameric SOC channels.It is important to note that the illumination power of femtoSOC is as low as 1–4 mW, lower than the typical laser power used in two-photon microscopy. Also, this is much lower than the femtosecond laser power used for two-photon excitation of optogenetic proteins (20–150 mW). The Ca32 that does not work for the formation of SOCs under normal conditions, but when SOCs are inhibited, Orai3 may form SOCs to induce very small Ca2+ influx. In the presence of Orai1, the contribution of Orai2 to SOCs is mostly suppressed.31 Previous reports show that even if Orai2 is knocked down by siRNA, Ca2+ influx through SOCs is totally unaffected.34 Moreover, Orai2 in cells without Orai1 provides very small Ca2+ influx.35 Recently, it has been found that Orai2 expression in some types of Orai1-KO cells is significantly upregulated,36 which accounts for very small Ca2+ influx in Orai1-KO cells. Figure 2+ influx. The expression pattern of Orai1 in different cell lines varies a lot. As shown in Supplementary information, Fig. Orai1 plays a dominant role in femtoSOC since SOCs are mainly composed of Orai1 and STIM1 although there are other Orai family units such as Orai2 and Orai3. Orai3 usually works as an exceptional OraiThe general drawback of femtoSOC comes from the two-photon excitation that requires tight focusing of femtosecond laser. Usually that needs microscope systems instead of direct illumination by laser or even LED. Therefore, it is not easy to simultaneously activate a large amount of cells or neurons of free-running mice. The drawback can be overwhelmed by combining other optical technologies like spatial light modulation for automatic multi-focusing and implantable miniaturized objectives.2+ bursts in neurons in two-photon microscopy of mouse brains with an unknown mechanism.38 More importantly, as shown in our experimental demonstration, femtoSOC has potential to enable optogenetics-free single-neuron activation for neuroscience research and is fully compatible with two-photon microscopy systems used in previous optogenetics research. For example, Tank Group and Hausser Group have reported a two-photon microscope with two femtosecond lasers at different wavelengths for simultaneous two-photon microscopy analysis of neurons and two-photon excitation of neural Ca2+ signals by optogenetics.40 Recently, Harvey Group has found the feature-specific competition of neurons in the V1 (vision) area in mouse brains by using single-neuron optogenetic perturbation.41 Deisseroth group and Yuste Group have demonstrated that the activation of several target neurons is sufficient for triggering perception42 and controlling the specific behavior of mice,43 respectively, by two-photon optogenetic excitation. In these reports, single neurons were stimulated one by one by using two-photon excitation of optogenetic proteins. femtoSOC is expected to be employed in conjunction with the existing two-photon optogenetic excitation systems or significantly simplify them by virtue of no need for introducing exogenous optogenetic genes into biological systems. Such single-cell activation guarantees the cell specificity in this way.femtoSOC can account for the previously reported phenomenon of occasional Ca2+ imaging, cells were incubated with 1.5 μM Fluo-4/AM  for 30 min at 37 °C or with 10 μM Rhod-3/AM in the presence of PowerLoad™ and probenecid  for 60 min at room temperature. For Ca2+ imaging of the mitochondria, cells were incubated with 2 μM Rhod-FF/AM  for 55 min at 37 °C. For Zn2+ indication, cells were incubated with 5 μM FluoZin™-3 AM  (1–7 μM Zn2+ cell buffer) for 30 min at 37 °C. For Mg2+ indication, cells were incubated with 4 μM Magnesium Green (M3735) (5 mM Mg2+ cell buffer) for 30 min at 37 °C. For ROS imaging, cells were incubated with 75 μM carboxy-H2DCFDA  for 60 min at 37 °C or with 5 μM dihydroethidium  for 30 min at 37 °C. For MMP indication, cells were incubated with 0.2 μM TMRM  for 30 min at 37 °C. To measure cell viability, cells were incubated with 75 μM propidium iodide  for 10 min at 37 °C. Cells were protected from ambient light during incubation. After staining with dyes, cells were washed and the buffer was replaced with fresh culture medium. For fluorophores with acetoxymethyl (AM) ester, cells were incubated for 10 min to allow complete de-esterification. To test if there is any photoporation of the plasma membrane, 300 μM PI was added to the cell buffer and kept during and after femtoSOC. All green and red fluorescence signals were detected in spectral bands of 500–530 nm and 553–618 nm, respectively.For Caz-projection imaging was used to observe Orai1’s dynamics, especially Orai1 puncta or clusters. It is important to note that Orai1’s dynamics was not identified if the confocal microscopy plane was in the middle of cells because Orai1 was mostly expressed in the plasma membrane. Therefore, z-projection imaging of the cell bottom (membrane surface) was used to provide Orai1 images.For confocal microscopy, 35-mm dishes containing cells were transferred to a custom-built fixator  on the stage of the Nikon upright two-photon confocal microscope . Cells were observed using a 60×, 1.0 NA, water immersion objective lens. Experiments were performed at room temperature. The experimental duration for a single dish of cells was limited within 2 h to keep cells in good conditions. A laser at 488 mm was operated at about 0.1 mW for excitation of Fluo-4, STIM1-YFP, DCF, DHE, and TMRM, and about 3 mW for excitation of flavin’s autofluorescence. For flavin autofluorescence measurements, cells were localized by dishes with a grid at the bottom . For Rhod-3 and PI imaging, a laser at 543 nm was operated at about 0.1 mW. Images were acquired for 2.2 μs/pixel with 512 × 512 pixels or 2.4 μs/pixel with 1024 × 1024 pixels and processed by ImageJ for cropping, rotation, and contrast adjustment. Confocal microscopy for 2+-free medium or buffer was composed of 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 10 mM glucose , 10 mM HEPES, and 20 μM EGTA (both from Sigma). CaCl2 was purchased from the Sinopharm Chemical Reagent Co., Ltd. To block SOCs, high-concentration 2-APB was incubated with cells for 30–60 min to provide a moderate block effect since it is a broad-spectrum blocker. SKF-96365, a blocker of voltage gated Ca2+ channels, TRPCs, and SOCs and YM-58483, a specific blocker of SOCs, were also used for the inhibition of SOCs. Cells were incubated with SKF-96365 for 5–10 min and YM-58483 for 15 min, respectively, before experiments.2-aminoethyl diphenylborinate , YM-58483 , thapsigargin , potassium iodide , diphenyliodonium chloride , riboflavin , flavin adenine dinucleotide disodium salt hydrate , rotenone , malonic acid , carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone , MitoTEMPO , TEMPO , α-tocopherol , and muscimol hydrobromide (G019) were purchased from Sigma-Aldrich. SKF-96365  was purchased from Abcam. Tween-20  was purchased from Sangon Biotech. DTT  was purchased from Tanon. The Ca2) for only one-time use in a short duration (63–500 ms). At 700 nm, the femtosecond laser power was operated at about 1.5 mW at the sample.The two-photon scan with the femtosecond laser  in a two-photon microscope was defined by the typical two-photon microscope frame and shared the same galvanometric mirrors of the confocal microscope. It can be inserted into any pre-designed confocal microscopy sequences as a single two-photon microscopy frame. Therefore, it allowed automatic femtoSOC at any pre-defined time and could be achieved by any commercial two-photon microscopy drivers. Once target cells were determined, confocal microscopy was at first stopped. A new confocal microscopy sequence was then defined in the following steps: (1) define a confocal microscopy sequence  in the same field of view; (2) define the femtoSOC laser illumination duration and area in the target cell; (3) define the start time slot of each femtoSOC laser illumination, determined by how long femtoSOC should be before and after the laser illumination; (4) define the femtosecond laser parameters of the femtoSOC laser illumination such as the laser wavelength and power; (5) start the confocal microscopy sequence for automatic recording of femtoSOC-induced cellular activity. During confocal microscopy, the femtosecond laser was blocked by an optical shutter. During femtoSOC, the CW lasers for fluorescence excitation were blocked by another optical shutter. The two-photon scan area was defined to be a small region of the target cell  were finally plated in a 35-mm dish (Corning) pre-coated with poly-D-Lysine (Sigma). The medium was replaced with Neurobasal medium (Gibco) supplemented with 2% B27 (Gibco) 3 h after cell plating. The identities of neurons and astrocytes were distinguished based on their morphological features. All cells were cultured on 35-mm dishes at 37 °C in a humidified 5% CO2 atmosphere and reached 60%–80% confluent at the time of femtoSOC.HeLa, HEK293T, and MCF-7 cells were cultured in Dulbecco’s Modified Eagle’s Medium . Jurkat T cells were cultured in RPMI 1640 medium (Hyclone). All cells were supplemented with 10% fetal bovine serum  and 1% penicillin/streptomycin (Gibco). To culture primary neurons and astrocytes, the cortex was isolated from C57BL/6 mice postnatal 0 days, digested in 0.25% trypsin (Gibco) for 10 min at 37 °C, and terminated with 10% FBS. After rinsed with phosphate-buffered saline , the tissue was dissociated and suspended in DMEM supplemented with 10% FBS, followed by filtering with 40-μm strainers (Corning). The cells (~5 × 10NheI and BamHI. RNA interference of Orai1 or TRPC1 was performed using the plasmid encoding Orai1 or TRPC1 shRNA (Genechem), respectively, which was constructed by subcloning the target sequence into GV298 vector (U6-MCS-Ubiquitin-Cherry-IRES-puromycin) between AgeI and BamHI. The target sequence for Orai1 shRNA was TGTCCTCTAAGAGAATAAG, the sequence for TRPC1 shRNA was ATCGTTACTTGACTTCCAT, and the control sequence was TTCTCCGAACGTGTCACGT.44 HeLa cells were transiently transfected with the plasmids through jetPRIME (Polyplus-transfection) according to the manufacturer’s instructions. In brief, ~3 × 105 cells were seeded on a 35-mm dish in 2 mL of DMEM supplemented with 10% FBS and 1% penicillin/streptomycin 24 h prior to the transfection, which enabled cells to reach 60%–80% confluent at the time of the transfection. Then, 1 μg plasmids were dilute into 200 μL buffer, followed by adding with 2 μL jetPRIME and incubation for 10 min at room temperature. The mixed buffer was added into the dish drop wise and replaced with refresh culture medium after 4–5 h. For femtoSOC experiments, shRNA-transfected cells were prepared 24–48 h after the transfection, while Orai1-transfected cells were prepared 72 h after the transfection.GCaMP6s, CEPIA3mt, and G-CEPIA1ER used in this study were generous gifts from Dr. Franck Polleux. NES-jRGECO1a (Addgene plasmid #61563), G-GECO1-Orai1 (Addgene plasmid #73561), ERK2-GFP (Addgene plasmid #37145), Orai1-YFP (Addgene plasmid #19756), and STIM1-YFP (Addgene plasmid #18857) were purchased from Addgene. Lck-GCaMP5G was cloned and purchased from GeneChem, Shanghai. Orai1-mCherry was cloned and purchased from BioFeng, Shanghai. The plasmids encoding Orai1 (Genechem) were constructed by subcloning human Orai1 into GV417 vector (CMV-MCS-IRES-Cherry-SV40-Neomycin) between Orai1 gene ). The sgRNA sequences were used in conjunction with a Cas9-coding plasmid, while Lenti-U6-sgRNA-EFS-CRISPR-PGK-cherry was transformed from lentiCRISPR v2  to generate Orai1-KO single-cell clones. The virus was enriched, filtered, and transfected to HeLa cells. The mCherry-positive cells were sorted by flow cytometry and cloned. The effectiveness of the knockout was tested by PCR-Sanger sequencing. The results show that the sequences 5’-GGCGG-3’ in the Orai1-KO cells were absent compared to WT cells, indicating that they are a homozygote of the Orai1 gene knockout. Furthermore, the absent expression of Orai1 was verified by western blotting and the result was shown in Fig. 45A set of three CRISPR sgRNA sequences was designed to target the STIM1 gene ). This knockout process was identical to that for Orai1-KO cells. The results of PCR-sanger sequencing show that the sequences 5’-GTGTGGGCGGCTGCTGGTTTGCCTATATCCAGAACCGTTAC-3’ in STIM1-KO cells were absent compared to WT cells, indicating that they are a homozygote of the STIM1 gene knockout. Furthermore, the absent expression of STIM1 was verified by western blotting and the result was shown in Fig. A set of three CRISPR sgRNA sequences was designed to target the Orai1/2/3-KO cells were a gift from the lab of Prof. Mohamed Trebak from Penn. State. Uni. The absent expression of STIM1 was verified by western blotting and the result was shown in Supplementary information, Fig. Cells were dissected and homogenized in lysis buffer (RIPA: protease inhibitor cocktail = 100:1). Whole cell lysates were analyzed in a western blot experiment according to standard protocols. Antibodies for immunoblotting were as follows: Orai1 rabbit polyclonal antibody , rabbit anti-ORAI1 , TRPC1 rabbit polyclonal antibody , GAPDH mouse monoclonal antibody , Peroxidase AffiniPure goat anti-mouse IgG (H + L) , Peroxidase AffiniPure goat anti-rabbit IgG (H + L) . Proteins were visualized using enhanced chemiluminescence. The images were acquired by Tanon 4200SF (Tanon). Immunofluorescence microscopy of eIF4E-p was performed 24 h after femtoSOC. Cells were fixed with 5% paraformaldehyde and permeabilized with 0.5% Triton X-100 . The cells were then incubated with an anti-eIF4E antibody  overnight at 4 °C. The sample was washed with PBS for 5 min and incubated with diluted secondary antibody anti-Rabbit IgG (H + L)  for 1.5 h at room temperature.2 atmosphere. After 6, 15, or 25 h, cells were stained with PI and observed by the confocal microscope to record the number of viable and dead cells.To measure cell viability and proliferation rate after femtoSOC, dishes with a grid at the bottom  were adopted to locate cells for femtoSOC. In brief, all cells within the grid of Grid-500 dishes were scanned by the femtosecond laser and then cultured at 37 °C in a humidified 5% CO2 and 5% CO2 with artificial cerebrospinal fluid (ACSF) which contains 125 mM NaCl, 1.25 mM KCl, 25 mM NaHCO3, 1.25 mM KH2PO4, 25 mM D-Glucose, 2 mM CaCl2, 1 mM MgCl2, supplemented with 2 mM Na-pyruvate and 0.5 mM L-ascorbic acid during loading of Cal-520/AM  and observed with a multiphoton confocal microscope .Brain slices of C57BL/6 mice were prepared by cutting brain tissue from an area around the hippocampus after sacrificing the mice immediately . The brain slices were 300-μm thick and incubated in the perfusion chamber at room temperature in an atmosphere of 95% O2) of the neuron as a single frame of 0.5 s at a wavelength of 700 nm and an average laser power of ~7 mW (measured before the sample). All animal experiments were performed in compliance with the Guide for the Care and Use of Laboratory Animals and the protocols approved by the Animal Study Committee of the School of Biomedical Engineering at Shanghai Jiao Tong University and the School of Life Science, at the University of Science and Technology of China.For in vivo experiments, 6- to 9-week-old C57BL/6 mice were anesthetized with 1% pentobarbital. After the surgical level of anesthesia was reached, the skin and skull (~1 mm diameter) above the desired brain area were removed in sequence. Then, dental cement was used to adhere the brain to the custom-made stage of the microscope and to form a recording chamber above the imaging window in order to keep the brain moist with ACSF. To introduce the calcium indicator to the targeted area of the brain, a glass pipette with a 20-μm diameter open tip filled with 1 μL Cal-520/AM (~0.6 mM) was inserted into the cortex . The fluorophore was injected at a speed of 40 nL/min. 1.5 h after the injection, the brain was observed by the upright two-photon confocal microscope with a 25×, 0.95 NA, water immersion objective lens . To activate a randomly selected neuron, the femtosecond laser light was scanned in a small area  and analyzed with GraphPad Prism 7 software throught the one-tailed paired t-test except where stated otherwise. The confidence interval for t-tests was set as 95%.As a cautious and strict standard, the CaSupplementary information, Fig. S1Supplementary information, Fig. S2Supplementary information, Fig. S3Supplementary information, Fig. S4Supplementary information, Fig. S5Supplementary information, Fig. S6Supplementary information, Fig. S7Supplementary information, Fig. S8Supplementary information, Fig. S9Supplementary information, Fig. S10Supplementary information, Fig. S11Supplementary information, Fig. S12Supplementary information, Fig. S13Supplementary information, Video legendSupplementary information, Video. S1Supplementary information, Video. S2


Interhemispheric connectivity of the two cerebral hemispheres is crucial for a broad repertoire of cognitive functions including music and language. Congenital amusia has been reported as a neurodevelopment disorder characterized by impaired music perception and production. However, little is known about the characteristics of the interhemispheric functional connectivity (FC) in amusia. In the present study, we used a newly developed voxel-mirrored homotopic connectivity (VMHC) method to investigate the interhemispheric FC of the whole brain in amusia at resting-state. Thirty amusics and 29 matched participants underwent a resting-state functional magnetic resonance imaging (fMRI) scanning. An automated VMHC approach was used to analyze the fMRI data. Compared to the control group, amusics showed increased VMHC within the posterior part of the default mode network (DMN) mainly in the posterior superior temporal gyrus (pSTG) and posterior cingulate cortex (PCC). Correlation analyses revealed negative correlations between the VMHC value in pSTG/PCC and the music perception ability among amusics. Further ROC analyses showed that the VMHC value of pSTG/PCC showed a good sensibility/specificity to differentiate the amusics from the controls. These findings provide a new perspective for understanding the neural basis of congenital amusia and imply the immature state of DMN may be a credible neural marker of amusia. Interhemispheric connectivity is a prominent feature of macroscopic brain organization characterized by a high level of collaboration between bilateral brain regions . Proper Congenital amusia is a neurodevelopmental disorder, which has been characterized by impaired music perception and production by several seminal studies . The doma priori selection of a voxel, cluster, or anatomical brain atlas region  and whether the character can be a neural marker of amusia, which may help understand the neural basis of congenital amusia. Accordingly, the first aim of the present study was to test the hypothesis that individuals with amusia may have abnormal interhemispheric FC in several different functional brain regions compared to the control group by the VMHC. Secondly, we tested the hypothesis that the abnormal interhemispheric FC was associated with the music perception ability by correlation analyses of the VMHC value with the musical test. Thirdly, we tested the hypothesis that whether the VNHC value of potential abnormal clusters in amusia can be credible neural markers of amusia evaluated by Receiver Operator Characteristic (ROC) curves.Native Mandarin-speaking participants were recruited from advertisements and campus screening. All participants reported right-handed, with no professional musical training, and no neurological or audiological deficit. All participants signed the informed consent form, and the study design was approved by the ethics committee of the Second Xiangya Hospital of Central South University. All participants could withdraw from the study if they experienced discomfort.All participants (age range: 18–24) were confirmed to have normal binaural hearing by pure tone audiometry. Amusia was defined by the Montreal Battery Evaluation of Amusia (MBEA). The MBEA includes three melodic subtests based on the pitch , two temporal subtests based on the time (rhythm and meter), and one musical memory subtest, and has been an effective tool to screen for amusia in western and eastern cultures . AccordiMagnetic resonance imaging scans were acquired using a 3T Siemens Skyra scanner . All participants were instructed to lie in a comfortable position, with eyes closed, and remaining awake. Resting-state functional magnetic resonance images (fMRI) time series were acquired with an echo-planar imaging sequence with the following parameters: repetition time, 2000 ms; echo time, 30 ms; flip angle, 80°; voxel size, 4.0 mm × 4.0 mm × 4.0 mm; the field of view, 256 mm × 256 mm; consisting of 32 slices covering the whole brain, and 216 volumes were obtained per participant. Simultaneously, high- resolution three-dimensional T1-weighted images were acquired using a gradient-echo sequence with the following parameters: repetition time, 1900 ms; echo time, 2.03 ms; flip angle, 9°; voxel size, 1.0 mm × 1.0 mm × 1.0 mm; slice thickness, 1 mm, the field of view, 256 mm × 256 mm; the number of slices, 176. All images were checked by a radiologist, who reported no anomalous findings.1 , and MBEA scores  between the amusia group and control group. FD values were calculated for each participant in data preprocessing  method (Voxel-based Two-sample ) method .To further examine potential core regions that could account for amusia, the mean VMHC values were extracted from these abnormal brain regions and conducted Pearson correlations with MBEA scores in all participants, amusia group, and control group. ROC analysis was conducted to examine whether the mean VMHC values of potential abnormal brain regions can be credible neural markers to identify the amusia from the controls using SPSS19.0 software. Notably, this method of ROC analysis has been used to assess whether the abnormal VMHC values obtained from group analysis could differentiate the disorder individuals from control individuals, such as conduct disorder , major dp > 0.05) and no significant differences in age, years of education, intelligence quotient (IQ), and FD between the amusia and control group . For the MBEA scores, amusia had a significantly lower global musical score (averaged over the six subtests of the MBEA) comparing to controls, as well as melodic subtest, temporal subtest, and memory subtest .As seen in t-test analyses on the VMHC maps between amusia group and control group. Compared to control group participants, amusics showed significantly increased VMHC  in the posterior superior temporal gyrus , and posterior cingulate cortex   .r < –0.5, p < 0.001). Among amusia group, there were significant negative correlations of the pSTG with global musical score , temporal subtest , and memory subtest , and melodic subtest , as well as no significant correlation of the PCC with temporal subtest  and memory subtest . Furthermore, there were no significant correlations between pSTG/PCC and MBEA scores among the control group , and 0.896 with PCC (p < 0.001). Results also showed that the VMHC value of the pSTG could correctly classify 28 of 30 amusics and 23 of 29 controls, resulting in a sensitivity of 93.33% and a specificity of 79.31%; the VMHC value of the PCC could correctly classify 23 of 30 amusics and 28 of 29 controls, resulting in a sensitivity of 76.67% and a specificity of 96.55%  can be credible neural markers to identify the amusia from the controls, we conducted the ROC analysis. The results showed that the areas under the ROC curve (AUCs) were 0.894 with pSTG  and posterior cingulate cortex (PCC). Correlation analyses revealed a negative correlation between the VMHC value in pSTG/PCC and the music perception ability among amusics. ROC analyses also showed that the VMHC value of pSTG/PCC showed a good sensibility/specificity to differentiate the amusics from the controls.The present study found increased VMHC in pSTG of amusia, which suggested the amusia had enhanced functional interactions between bilateral pSTG. The pSTG has been proposed to involve top–down feedback (IFG-pSTG) during vocal production in amusia in early life . The DTI2 , which includes medial prefrontal cortex, PCC, and temporoparietal junctions/inferior parietal lobule2 . The inc2 . A syste2 . The abn2 .Correlation analyses revealed both the VMHC value of pSTG and PCC were negatively related to the global music score in amusics. The abnormal increased interhemispheric FC may suggest a natural deficit in amusics. One explanation was that amusia was a neurodevelopment disorder, which may be related to neurodevelopmental immaturity due to a malformation in brain development . Prior fCorrelation analyses and ROC results also indicated the pSTG was negatively related to rhythm discrimination ability  in amusics, and higher sensibility in amusics and related lower specificity in controls according to the VMHC value of pSTG, while the PCC was negatively related to pitch discrimination ability (melodic subtest) in amusics but higher specificity in controls and related lower sensibility in amusics according to the VMHC value of PCC. One explanation was that there were two types of amusia, which were tone deafness (pitch deficit) associated with PCC and beat deafness (rhythm deficit) associated with pSTG. Prior studies have also shown beat deafness showed deficits in beat processing and could occur in isolation with tone deafness , but mosThe present study has some limitations. Firstly, gray/white matter abnormalities were not assessed, which has been reported to be associated with the VMHC deficits in psychiatric disorders such as schizophrenia . TherefoIn summary, the present study was firstly, directly to explore the interhemispheric functional interaction characteristics in congenital amusia. Results indicated amusics showed abnormally increased VMHC within the posterior part of the DMN mainly in the posterior superior temporal gyrus (pSTG) and PCC. Correlation analyses among amusics revealed a negative correlation between the VMHC value in pSTG/PCC and the music perception ability. Further ROC analyses showed that the VMHC has a good sensibility/specificity to differentiate the amusics from the controls. These findings provide a new perspective for understanding the neural basis of congenital amusia and imply the immature state of DMN in amusia may be a credible neural marker.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the ethics committee of the Second Xiangya Hospital of Central South University. The patients/participants provided their written informed consent to participate in this study.DW and ZJ designed the study. ZJ, SH, LJ, YY, MX, JW, and QL performed the research. ZJ analyzed the data. ZJ and DW wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Leukemia and treatment of male patients with anticancer therapy (aggressive chemotherapy and/or radiotherapy) may lead to infertility or even permanent male sterility. Their mechanisms of spermatogenesis impairment and the decrease in male fertility are not yet clear. We showed that under acute myeloid leukemia (AML) conditions, alone and in combination with cytarabine (CYT), there was significant damage in the histology of seminiferous tubules, a significant increase in apoptotic cells of the seminiferous tubules, and a reduction in spermatogonial cells (SALL and PLZF) and in meiotic (CREM) and post-meiotic (ACROSIN) cells. In addition, we showed a significant impairment in sperm parameters and fertilization rates and offspring compared to control. Our results showed a significant decrease in the expression of glial cell line-derived neurotrophic factor (GDNF), macrophage colony-stimulating factor (MCSF) and stem cell factor (SCF) under AML conditions, but not under cytarabine treatment compared to control. In addition, our results showed a significant increase in the pro-inflammatory cytokine interleukin-1 (IL-1) alpha in whole testis homogenates in all treatment groups compared to the control. Increase in IL-1 beta level was shown under AML conditions. We identified for the first time the expression of GCSF receptor (GCSFR) in sperm cells. We showed that GCSF injection in combination with AML and cytarabine (AML + CYT + GCSF) extended the survival of mice for a week (from 6.5 weeks to 7.5 weeks) compared to (AML + CYT). Injection of GCSF to all treated groups (post hoc), showed a significant impact on mice testis weight, improved testis histology, decreased apoptosis and increased expression of pre-meiotic, meiotic and post- meiotic markers, improved sperm parameters, fertility capacity and number of offspring compared to the controls (without GCSF). GCSF significantly improved the spermatogonial niche expressed by increased the expression levels of testicular GDNF, SCF and MCSF growth factors in AML-treated mice and (AML + CYT)-treated mice compared to those groups without GCSF. Furthermore, GCSF decreased the expression levels of the pro-inflammatory cytokine IL-12, but increased the expression of IL-10 in the interstitial compartment compared to the relevant groups without GCSF. Our results show for the first time the capacity of post injection of GCSF into AML- and CYT-treated mice to improve the cellular and biomolecular mechanisms that lead to improve/restore spermatogenesis and male fertility. Thus, post injection of GCSF may assist in the development of future therapeutic strategies to preserve/restore male fertility in cancer patients, specifically in AML patients under chemotherapy treatments.  Acute myeloid leukemia (AML) is a prevalent disease that appears at all ages, but is more frequent in adults. Recently, different studies have shown impairment in sperm parameters among leukemia patients even before anti-cancer treatments ,2,3,4,5.Leukemia may evoke a systemic response in the body. Cytokines such as interleukins, tumor necrosis factors, and other pro-inflammatory/anti-inflammatory factors secreted by tumor cells and immune system cells may mediate this systemic response ,22,23,24The aim of the present study is to evaluate and deepen our understanding of the possible role and mechanisms of granulocyte colony-stimulating factor in restoration of spermatogenesis and male fertility in AML-chemotherapy treated mice. Our results show that GCSF and GCSFR are presented in cells of the interstitial tissue and of the seminiferous tubules  A–C. Our Following the above results, we examined the effect of post injection of GCSF on the expression levels of GCSF and GCSF-R in the testes of AML- and CYT-treated mice. Our results show that injection of GCSF or CYT alone or in combination (GCSF + CYT), but not AML or AML + CYT, significantly increased the expression levels of testicular GCSF and GCSF-R compared to control mice (CT) E. HoweveWe injected GCSF at three different time points  in order to find the most effective time point of injection. Our results show that injection of the mice with PBS , GCSF (GCSF) or cytarabine (CYT) did not affect their survival A. InjectOur results did not show a significant effect on testicular weight of mice 3 and 5 weeks post injection of GCSF compared to CT , fertility capacity and number of offspring into untreated group (GCSF) compared to control group (CT) , (CYT (CYT + GCSF) and AML + CYT (AML + CYT + GCSF) significantly increased sperm concentration B, motiliOur results show that 3-week post-injection of GCSF had no significant effect on the percentages of seminiferous tubules with apoptotic cells compared to control (CT) A,B. On tWe also examined the expression levels of factors involved in the intrinsic (BAX) and extrinsic (FAS and CASP3) pathways of apoptosis in the different treated groups. Our results show that 3-week post-injection of GCSF did not significantly affect the expression levels of both pathways  in their testis compared to CT C. On theOur results show that 3-week post-injection of GCSF into control mice did not significantly affect the percentages and the expression levels of the pre-meiotic (SALL and PLZF), meiotic (CREM) (except the expression levels which increased its expression levels) and the meiotic/post-meiotic cell markers compared to CT . Our results show a significant decrease in the number/tubule of the pre-meiotic cells SALL4 and PLZF  that were positively stained to CREM (meiotic cells) , round spermatid (RSP) and sperm cells. Our results show that in the control group, almost 100% of tubules contained more than 10 cells/tubule (tens) that were positively stained for ACROSIN . Following these results and knowing that GCSF is used by clinicians after chemotherapy treatment , we deciWe also tested the expression of GCSF and GCSFR in the testes of mice from all treatment groups and found that after GCSF injection to all groups, GCSF and GCSFR expression was significantly increased in their testes. These findings suggest that GCSF regulates its own expression and its receptor in testicular cells. The injection of GCSF to all treated groups significantly increased their testes weight 3 and 5 weeks after the AML cell injection compared to AML alone. This was observed in parallel with a decrease in the percentage of the damaged seminiferous tubules . Additionally, we showed a significant decrease in apoptotic cells in all AML- and/or CYT-treated groups following GCSF injection. GCSF decreased expression of BAX and FAS apoptotic markers that activated both the intrinsic and the extrinsic apoptotic pathways. It is possible that the increase in testicular weight and the decrease in the percentage of tubules with apoptotic cells could be related to the protection against severe damage of the seminiferous tubules following AML disease and chemotherapy treatment. This finding is congruent with other reports demonstrating the prevention of testicular damage from chemotherapy and radiotherapy by GCSF injection ,40,41,42We also examined the possible involvement of GCSF in protecting the spermatogenesis process. After GCSF injection following AML conditions (with or without cytarabine treatment), the pre-meiotic marker PLZF and the post-meiotic marker acrosin quantity and expression were significantly increased compared to the control group. Additionally, the pre-meiotic marker Sall4 and the meiotic marker CREM were significantly increased (quantity and expression levels) following GCSF treatment of the AML- and/or CYT-treated groups. Our results are in agreement with the results of another group that tested the effect of GCSF on spermatogenic regeneration from surviving spermatogonia after busulfan chemotherapy. Normal mice treated with GCSF before or after busulfan treatment exhibited an increase in the numbers of PLZF cells . Our resParacrine/autocrine control plays a key role in the regulation of the spermatogenesis process. We demonstrated a decrease in SCF, MCSF, GDNF following AML condition. However, GCSF injection significantly increased the expression levels of SCF, MCSF and GDNF compared to groups without GCSF injection. These findings may suggest that GCSF improved/balanced the testis microenvironment of the study groups, and thus improved spermatogenesis.In addition, we measured inflammatory conditions (inflammatory cytokines) in the testes all examined groups. We showed that under AML conditions (with or without cytarabine) GCSF injection decreased IL-1 alpha and IL-1 beta expression. These findings may suggest that GCSF may be involved in the regulation of the inflammatory factors in the testes. This is supported by other studies that show the anti-inflammatory abilities of GCSF ,51,52 Due to the protective effect of GCSF in spermatogenesis, we evaluated its effect on the quality of sperm cells in all treated groups. We found that GCSF injection in all treatment groups significantly improved sperm concentration and motility compared to no treatment with GCSF. There was no significant difference in sperm viability between treatment groups. We also showed that GCSF injection improved the fertility capacity of the mice, which was shown by the numbers of offspring. These findings may suggest that GCSF could protect against AML and cytarabine testicular damage, affect sperm production and activity, and, as a result, improve the fertility capacity and eventual number of offspring. We showed for the first time the presence and the expression of GCSFR by sperm cells. This may suggest a direct paracrine effect of GCSF in the testes on the functionality of the sperm. Thus, our results may suggest that GCSF may affect spermatogenesis as a paracrine/autocrine factor, while also having a direct effect on the functionality of the developed sperm in the testes under normal and pathological conditions. Injection of GCSF under pathological conditions may restore these functions by increasing the production of GCSF and other paracrine/autocrine growth factors that affect spermatogenic cell development and niches. In conclusion, our study used for the first time the AML/CYT-mouse model to demonstrate that AML and CYT may lead to subfertility/infertility. Treatment of these AML/CYT-mice with GCSF enhanced the functionality of the cells in the testes and improved the development of spermatogenesis and fertility capacity. Our study suggests cellular and biomolecular mechanisms for the effectivity of GSCF in restoration and protection of spermatogenesis and fertility capacity in AML- and CYT-treated mice. These results may encourage the development of future therapeutic strategies to preserve male fertility in cancer patients, specifically in AML patients. This study was performed in accordance with the Guiding Principles for the Care and Use of Research Animals Promulgated by the Society for the Study of Reproduction. It was confirmed by the Ben-Gurion University Ethics Committee for Animal Use in Research (IL-70-11-2016). Six-week-old C57/BLACK mice were purchased from Envigo Laboratories, Jerusalem, Israel. 5 cells/100 µL were injected intravenously per mouse. Cytarabine  (100 µL of CYT (3 mg/kg)) was injected intraperitoneally into each mouse. The injections were performed 24 h after the injection of AML cells, 3 times every 12 h  injection of the murine C1498 (TIB-49) AML cells were performed according to our previous study . In genel., 2008 ; with adSurvival of mice was examined every week for 8 weeks.Mice were sacrificed by using Isoflurane  and testes were removed from the body and weighed 3 and 5 weeks post treatment. Testes were removed and Bouin fixed and paraffin embedded . Hematoxylin and Eosin Staining for Histological Evaluation The diameter of the testicular tubules was determined by using the mean of vertical and horizontal diameter of each seminiferous tubule (ST). On average, 17 ST were measured from each mouse.Sperm cells were extracted from the tail of the epididymis by squeezing in a Petri dish plate. Collected sperm from all examined groups of treated mice were examined for concentration and motility using a Makler counting chamber, as described previously . EvaluatPrimary and secondary antibodies used are described in Supplement, Double immunofluorescence staining was performed according to our the method described in our previous study ,54. For TUNEL assay was carried out using a commercial kit . The assay was conducted according to the manufacturer’s instructions. TUNEL positive cells were counted using the cell counter and images were acquired using charged coupled device (CCD) camera. The tubules with TUNEL positive cells (more than 5 cells/tubule) were counted and the results are expressed as % of total tubules.g for 5 min). After centrifugation, the media were removed and the pellet of the cells in the bottom of the tube was suspended in 1 mL of PBS.The interstitial cells were isolated from the testes of the all-treated groups of mice (3 weeks after injection). Testes were decapsulated and mechanically separated by few accurately aspirations through pipette tips and a 1-mL syringe containing phosphate-buffered saline [PBS, Beit HaEmek Biological Industries  and washed with 5 mL PBS (centrifugation at 100× This process was performed according to the method in our previous study  by usingt-test and was shown as: p-value < 0.05—*, p-value < 0.01—**, p-value < 0.001—***.The statistics values were calculated according to Av. ±2 S.D. the statistical significance was examined by two-tailed


Introduction: At a professional level, pianists have a high prevalence of playing-related musculoskeletal disorders. This exploratory crossover study was carried out to assess and compare quantitatively [using high density surface electromyography (HDsEMG)], and qualitatively  the activity of the lumbar erector spinae muscles (ESM) and the comfort/discomfort in 16 pianists sitting on a standard piano stool (SS) and on an alternative chair (A-chair) with lumbar support and a trunk-thigh angle between 105° and 135°.Materials and Methods: The subjects played for 55 min and HDsEMG was recorded for 20 s every 5 min. For the quantitative assessment of the muscle activity, the spatial mean of the root mean square (RMSROA) and the centroid of the region of activity (ROA) of the ESM were compared between the two chairs. For the qualitative assessment, musculoskeletal questionnaire-based scales were used: General Comfort Rating (GCR); Helander and Zhang’s comfort (HZc) and discomfort (HZd); and Body Part Discomfort (BPD).Results: When using the A-chair, 14 out of 16 pianists (87.5%) showed a significantly lower RMSROA on the left and right side (p < 0.05). The mixed effects model revealed that both chairs  and sides  contributed to the mean RMSROA variation by subject . GCR comfort indicated that participants found the A-Chair to be “quite comfortable,” and the SS to be “uncomfortable.” GCR discomfort indicated that the SS caused more numbness than the A-Chair (p = 0.05) and indicated the A-Chair to cause more feeling of cramps (p = 0.034). No difference was found on HZc (p = 0.091) or HZd (p = 0.31) between chairs. Female participants (n = 9) reported greater comfort when using the A-Chair than the SS  with respect to males. No differences between chairs were indicated by the BPD assessment.Conclusion: It is concluded that using a chair with lumbar support, such as the A-chair, will provide greater comfort, less exertion of the ESM and less discomfort than the standard piano stool. Pianists are a small professional category with a high prevalence of playing-related musculoskeletal disorders PRMD; . They arPlaying the piano at a professional level implies efforts due to the intensity of the practice which requires great precision, speed, accuracy and associated psychological stress due to the highly competitive environment . In partSeveral studies discuss PRMD risk factors, however, the results of the interventions to reduce these factors have been quantified in only a few cases  using siPrevious studies investigated the ESM of sitting workers and their pain mechanism using individual surface electromyography (sEMG) electrode pairs  whilst oComfort and discomfort can be assessed through subjective evaluations using scale-ranked questionnaires. These scales allow for a qualitative description of what is considered comfortable and uncomfortable for each individual . These eThe purpose of this exploratory research was to assess and compare quantitative measurements and qualitative evaluations obtained from 16 pianists continuously playing for 55 min while sitting on a SS in day 1 and on the A-chair in day 2. This exploratory crossover study is the first using a grid of 128 sEMG electrodes on the ESM on each side of the spine of pianists as well as using qualitative outcome measures and the interaction/correlation between them.Sixteen pianists , all self-reported as right dominant, participated in the study. The participants had at least 5 years of professional piano playing and played, on average, (15.7 ± 7.63) hours per week. None of them reported low back pain symptoms. All musicians provided informed consent prior to the tests. All the procedures used in this study were performed in accordance with the Helsinki Declaration of 1975, as revised in 2000 and 2008, and approved by the Italian National Health Service. Two chairs were tested on two different days, at least one week apart: the standard piano stool (SS) and the alternative chair (A-Chair). The A-Chair was a modified version of the Varier Move model with an added adjustable lumbar support providing a trunk-thigh angle of 115° ± 10°. The height of the SS and the lumbar support of the A-Chair were adjusted by the researchers to guarantee optimal position and comfort, for each pianist at the start of each session . The truThe pianists played continuously during 1 h a set of standard exercises. Firstly, the E major scale, moto retto, four octaves, three velocities followed by the E major scale, moto contrario, two octaves, average velocity, two modalities (low intensity increasing in the ascending phase and high intensity decreasing in the ascending phase). These exercises were repeated in an alternating order for the 55 min.Every 5 min the pianists switched to a standard music piece, well-known by pianists of all ages and experience . This piece was played for 20 s before switching back to the standard exercises. As such, a total of twelve 20-s recordings of HDsEMG were acquired. At the end of each session, the pianists completed questionnaires exploring their comfort/discomfort.The response of each subject to the two chairs was assessed in two ways:(1)Quantitative assessment of HDsEMG of the ESM during each of the twelve 20 s recordings; and(2)Qualitative assessment using different musculoskeletal questionnaires exploring comfort and discomfort in different ways.Prior to positioning of the HDsEMG grids, the skin was cleaned with abrasive paste  and cleaned with a wet cloth to avoid paste bridges between electrodes. The grids were then placed as indicated in RMS and input resolution of 0.5 μV). The differential signals were further digitally filtered with a fourth order Butterworth bidirectional  band-pass filter with high pass cut off at 20 Hz and low pass cutoff at 400 Hz.Monopolar HDsEMG signals were collected and differentiated along the column direction, approximately in the ESM fiber direction, with respect to a reference electrode placed on the knee. An amplifier of up to 400 channels  was used, .Background noise was estimated by performing a separate test with the same protocol on five additional subjects lying prone and relaxed on a bed for 1 h. HDsEMG signals were recorded with the same setup and procedure used for the pianists. The 12 spatial means of the background noise maps ranged from 1.90 μVRMS), a Region of Activity (ROA) was defined using the Active Contour Segmentation algorithm  of the entire map was higher than the noise level  and power line interference was evident. Power line interference was removed using the spectral interpolation technique  provided by each grid were obtained and the ROA was computed using EMG measurements were performed on the RMS maps over the twelve trials (20 s every 5 min during 55 min) and used to investigate and compare chair-specific spatial and time patterns of the images. The quantitative outcome measures used were:(1)Mean RMS values: As indicated in section “Electrode Placement, HDsEMG Recording and Processing,” these values were computed as the spatial means over the ROAs and defined, on the differential RMS maps, as RMSROA. The Mann–Whitney test was used to investigate differences between RMSROA associated to sides and chairs, and Mixed Effects Models were used to explore the RMSROA variations for each subject using the two chairs as well as for the entire group of 16 subjects. Mixed effects models allowed to better evaluate the RMSROA variations than conventional ANOVA with the correlated error and have previously been used on values obtained from sEMG signals  separately from the random effects (pianist and time). Interaction effects were also investigated to understand the population’s trend over time regardless of statistical significance. Finally, RMSROA change over time [defined as: 100 × /] were quantified and compared by participant using Wilcoxon signed-rank test (with chair as a factor). When statistically significant differences were found, post hoc Dunn’s Multiple Comparison tests were carried out. signals . The choROA, chairs, trials and pianists to understand how much each chair affected the activity of the muscles, and how this relation was influenced by the other variables.Finally, an interaction effect analysis was performed between RMS(2)CM, YCM coordinates)Centroid of the ROA compared by chair, side and time  were defined as:x and y are the discrete coordinates of pixel  in mm defined with respect to the origin located in the top left corner of the map. I  is the intensity of pixel ; and M and N are the total number of rows and columns, respectively. This computation only includes the pixels within the ROA; the pixels outside of the ROA were not computed in the summation.where The difference in displacement of the centroid of the A-Chair with respect to the SS, for the left (L) and right (R) sides was calculated as:andCM, YCM was Gaussian according to the Kolmogorov–Smirnov test. An additional ANOVA multivariate analysis was applied to explore the effects of chair, side, and time on the centroid to (a) identify significant changes in the location of the ROA centroid in time and by side of the ESM, and (b) test if the coordinates of the centroid were significantly affected by the chairs on each side of the back. An image interpolation by a factor of 15 was applied (1 pixel = 0.6 mm).The distribution of the XThe qualitative assessment was performed using different musculoskeletal questionnaire-based evaluations that were compared between the two chairs. The statistical assessment was performed treating the evaluation outcomes as continuous variables . These o(1)General Comfort Rating (GCR): This index explores the current musician status by a 1–10 scoring scale where 1: relaxed, 2: comfortable, 3: quite comfortable, 4: not very comfortable, 5: uncomfortable, 6: restless, 7: tight, 8: stiff, 9: numb, 10: in pain. The values reported were Gaussian distributed according to Kolmogorov–Smirnov test treated as continuous variables and comparisons were carried out through one-way ANOVA with chair as factor. When statistically significant differences were found, post hoc Tukey tests were carried out.(2)Helander and Zhang’s measures of comfort (HZc) and discomfort (HZd): These indicators have seven and nine statements, respectively. Each statement was ranked from 1 to 10, where 1 was “do not agree” and 10 was “totally agree”. This non-linear assessment of comfort/discomfort was analyzed by individual statements between chairs as a percentage of comfort and discomfort (t-tests.scomfort . The 1–1(3)Body Part Discomfort (BPD) rating: This index explores if there is pain or discomfort on 10 different body parts individually, ranking them from 0 to 10, where 0 was “no pain or discomfort” and 10 was “extreme pain or agony.”post hoc was applied to allow multiple comparison adjustments.Body part discomfort comparisons were performed between chairs (across pianists) using General Linear Mixed Models (GLMM). Dunn’s ROA changes in time between chairs (across subjects), with each qualitative measure obtained . This representation is only indicative since the 10 grades of each scale cannot be assumed to represent equally spaced values of comfort/discomfort.The relationship between quantitative and qualitative measurements was explored using Spearman’s correlation. This was done by correlating the RMSAll the statistical analyses were carried out using Minitab v19 .Subcutaneous adipose tissue thickness is known to reduce the sEMG RMS values . SAT thipost hoc tests were used to allow for multiple comparisons adjustments.The research question addressed in this work aims at assessing changes of sEMG RMS values and of subjective perception of comfort and discomfort induced by using the A-Chair compared to using the SS in 16 subjects. Twelve 20 s measurements were performed during 55 min of play, 5 min apart, on each subject for both chairs. Statistical tests concerning sEMG RMS values were the same as adopted in previous work  and baseThe qualitative assessment was carried out through questionnaire-based Likert-type evaluation which contained more than five categories. As such, an ordinal approximation of a continuous variable was used, and the evaluation of the outcomes of these scales was treated as a continuous variable .RMS. An example of the signal quality is shown on a 4 s recording window (out of the 20 s) in The spatial mean of RMS computed over the ROA , or over the entire map  ranged from 3.39 to 20.39 μVIn seven subjects (44%) out of 16, the signals from most electrode pairs, presented visually evident burst-like activity patterns as observed previously  on the sROA was different between chairs. The effect of the chair on the RMSROA of each map was quantified by the mean (over 12 measurements) percentage change of the RMSROA for each subject sitting on the A-Chair with respect to the SS, defined as –100 [RMSROA(A-Chair)–RMSROA (SS)]/RMSROA (SS). Wilcoxon ranked sign tests with Dunn’s post hoc were used to compare the median differences for each pianist (N = 16), computed across trials (N = 12) between chairs (N = 2), for each side. These comparisons  and end  obtained from both chairs are shown in parisons  showed tolinists .ROA amplitude throughout the trials when compared to the SS. This lower amplitude reflects less involvement of the ESM whilst using the A-Chair. This observation was further confirmed when changes of the RMSROA were assessed for each pianist by chair and side of the ESM  % on the right side and (29.69 ± 23.01) % on the left side. This difference between sides was significantly different from zero (Wilcoxon signed-rank test with post hoc Dunn’s test).The A-Chair was generally associated to lower RMS the ESM . The meaROA change in time individually and across the sample population to compute the differences taking fixed and random factors into consideration . The statistical tests revealed that both chairs  and sides contributed to the RMSROA variation with the right side presenting greater RMS amplitude than the left side . When these factors were assessed for each subject, the individual RMSROA differences were confirmed to be influenced by the chairs and side . A global decreasing trend in time of RMSROA was observed on both chairs, with a mean difference over time between chairs of 2.80 μV with (RMSROA of SS) > (RMSROA of A-Chair). The model goodness-of-fit explained 96.97% (R2) of the RMSROA with a st. dev. of 0.70. This highlights that the changes on the RMSROA were directly influenced by the chairs and sides of the ESM.The mixed effects model was applied to assess the RMSThe averaged displacement difference (A-Chair – SS) of the CM over 55 min  for the right side was ΔR :  mm; and for the left side ΔL :  mm. In All the results from the questionnaires were Gaussian-distributed as confirmed by the Kolmogorov–Smirnov test unless otherwise explicitly mentioned.p < 0.001).The A-Chair was considered most comfortable (mode = 3: “quite comfortable”) when compared to SS, which was found to be mostly uncomfortable (mode = 5: “uncomfortable”)  was obtained. For the rest of the comfort statements, no statistically significant differences were found, however, there was an overall positive trend across statements in favor of the A-Chair, with respect to the SS. These results highlight a slightly greater positive subjective perception of the A-Chair, with respect to the SS.Paired-sample s the SS . From tht-tests were run on the nine statements for the 16 pianists to determine whether there were statistically significant mean differences in discomfort when using the A-Chair versus the SS  was obtained. In contrast, pianists agreed more to feeling more “numbed” when using the SS (2.88 ± 1.86), than with the A-Chair (2.00 ± 1.37); a statistically significant mean decrease of 0.88 ,  was obtained.Paired-sample s the SS . Out of t-tests, HZc: p = 0.091; t-tests, HZd: p = 0.31).When comfort and discomfort were assessed using the percentages of comfort (HZc) and discomfort (HZd), there was no association between these and the chairs  were used to explore the extent of pain experienced on the different body parts and the chairs. No statistical difference was found between the results associated to the A-chair and those associated to SS; however, the SS was found to cause slightly more pain  across body parts than with the A-Chair.ROA and the GCR, HZc, and HZd. A correlation was found on the left side of the ESM between the change of the RMSROA on the A-Chair with respect to the SS and the GCR scores , which highlights that at lower sEMG amplitude, the A-Chair is perceived to be more comfortable.Spearman’s correlation was used to assess the relationship between the time change of the RMSROA differences between R and L sEMG amplitudes and ROAs as well as between their percent changes are due to the neural drive, postural factors and chairs.The mean SAT on the right (R) and left (L) side was 8 mm and 9 mm, respectively. No significant differences were found between 12 measurements of the SAT on the R and L side of each subject at any of the three anatomical levels. The lack of significant differences between sides of the SAT thicknesses indicates that the RMSROA decreased as the SAT thickness increased ; this non-significant correlation was also observed on the SS, with RMSROA decreasing as SAT thickness increased . On the R side, the A-Chair was associated to a significant positive correlation between RMSROA and SAT thickness, with RMSROA increasing as SAT thickness increased ; on the SS, a non-significant positive correlation was found with RMS values increasing as SAT thickness increased  .Finally, on the L side, the A-Chair was associated to a non-significant negative correlation, implying that the RMSROA of sEMG detected over the ESM, in performing pianists, by (35.47 ± 21.46) % on the right side and (29.69 ± 23.01) % on the left side (Wilcoxon signed-rank test with post hoc Dunn’s p < 0.01).A novelty of this work is the use of a large electrode grid on each side of the spine and the definition of a ROA for each electrode grid to test the ESM of pianists. This eliminates the confounding factor due to the location of a single electrode pair, whose signal may be quite different depending on electrode location as evident from Qualitative comfort changes indicate that the A-Chair provided greater comfort (when assessed using the GCR) than the SS. Additionally, the A-Chair was associated to more positive feelings with “likeliness” being significantly greater when compared to the SS. These qualitative results are found to be in line with the quantitative measurements of RMS and findings reported .Two discomfort assessments were found to be of significance with the A-Chair causing greater feeling of “cramping” than the SS, and for the SS to cause a greater perception of “numbness” when compared to the A-Chair. No statistical difference was found by body part.ROA differences between sides cannot not be attributed to the SAT. Theoretically, if two measurements of sEMG RMS are performed using the SS and A-Chair one week apart, then SAT thickness, muscle and SAT-skin conductivities remained the same, the % variation of sEMG RMS should be independent of these three parameters. In this study, a significant correlation was observed between RMSROA values and SAT thickness on the right side of the ESM for both chairs, with RMSROA values increasing as SAT thickness increased. In contrast, on the left side, a correlation was found with RMSROA values decreasing as SAT thickness increased, but this was not statistically significant. The relation between RMSROA and SAT values requires further investigations that exceed the purpose of this work.The lack of significant differences in the SAT values between sides of the ESM indicates that any RMSROA amplitude and PRMD, are not only related (or caused by) muscular activity: they can also be due to the mechanical conditions of the intervertebral disks and the curvature of the spine, clinically associated to back pain . Similar patterns have been studied in sitting violinists  and shouROA value measured from the ESM on both violinists and pianists. When using the A-chair rather than the standard orchestra chair, nine out of nine violinists showed a significantly lower RMSROA on both the R and L sides (Wilcoxon signed rank test). When using the A-chair rather than the piano stool, 14 out of 16 pianists showed a lower RMSROA on the L side and R side . The probability of this happening by chance, according to the binomial distribution, is less than 0.004 in each case. Other qualitative findings are similar in the two studies.Our results are in agreement with those obtained in previous work on sitting violinists  and showBurst-like ESM activation is more evident in the violin players (eight out of nine subjects)  than in This is the first work addressing the ESM of pianists with large electrode grids providing overall topographical maps. As shown for other muscles , the HDsThe relevance of HDsEMG is confirmed by ROA of the right and left side of the ESM. This difference may be due to hand dominance and deserves further investigation.There was a significant difference between the RMSIt is also concluded, from the relationship between quantitative and qualitative measures, that using a chair with lumbar support and a trunk-thigh angle of 115° ± 10° provides greater comfort, less exertion of the ESM, and less discomfort than the standard piano stool. This is in agreement with the findings of a previous work on sitting violinists .Finally, a decreasing trend of mean RMS was observed over time, but no significant change of the ROA’s centroid was observed possibly because the inter-subject variability was considerable.ROA due to chairs reported in Actual sEMG RMS values (expressed in μV) are used in this study to estimate differences between chairs and sides, expressed as percent changes. They have not been normalized with respect to a reference value . On the other hand, normalization would not change the relative variations of RMSROA value was taken as the noise level and RMSROA was considered reliable if >5 μVRMS.It is good practice to estimate the background noise level in every subject, in relaxed conditions, before testing. Because of the limited time availability of the subjects this was replaced by a separate estimate of background noise measured on the ESM on a test group of four subjects lying prone on a bed, as described in section “Electrode Placement, HDsEMG Recording and Processing.” Noise measurements were repeated 12 times (for 20 s each time) every 5 min. Twice the mean RMSThe same piece of music was played by all subjects during each 20 s test. The relationship between sEMG and the music type and speed were not investigated and no metronome was used.The number of subjects was limited by availability but significant differences in sEMG were found in 14 subjects in favor of the A-chair (see section “Results”). The sample of investigated subjects was not homogenous and was not large enough to allow investigation of the effects of sex, age, and experience. However, the fact that the chair effect is so clearly evident, suggests that it is present across sexes, ages and expertise/years of practice. Our results justify larger studies, focused on these confounding factors. In this work, the effect of the chair stands out of these factors.For organizational reasons, in all cases, the test on the SS chair was done first and the test on the A-chair was done one week later. It was not possible to blind the pianists to the two type of chairs. However, in crossover studies (such as this one), one new treatment (A-Chair) is tested against a standard treatment (SS) that has been used for a long time. With this type of studies, every participant serves as its own control. The order of testing cannot be randomized. If we take into account that each subject has played the piano a minimum of 7 h and up to 34 h a week using the SS, then the session on A-Chair could never be done before using the SS, which has been used for at least 5 years prior to the day these measurements took place. For this reason, the only factors that we could possibly change by changing the order of testing would have been any psychological effects . It is unlikely that these factors influenced the sEMG. Finally, it has been demonstrated in other studies that one week between tests provide a minimal memory effect, yet short enough to avoid changes in muscle strength .Surface EMG spectral features (mean or median frequency of the sEMG power spectrum) were investigated. In our work the signal/noise ratio appeared to be too small for a reliable estimate of spectral variables.The data will be available upon resonable request.The studies involving human participants were reviewed and approved by the Italian National Health Service. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.AR, FS, SD’E, and AA-G collected the data. AR and AA-G performed the analyses. AA-G prepared the manuscript. RM supervised the work. All authors read and provided feedback on the manuscript.FS was employed by company Montecatone Rehabilitation Institute S.p.A. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.