The spatiotemporal control of DSB formation occurs within a specialized chromosomal structure characterized by sister chromatids organized into linear arrays of chromatin loops that are anchored to a proteinaceous axis. Although SPO11 and its partner proteins required for DSB formation are bound to the axis, DSBs occur preferentially within the chromatin loops, which supports the “tethered-loop/axis model” for meiotic recombination. In this mini review, we discuss insights gained from recent efforts to define and profile DSB hotspots at high resolution in eukaryotic genomes. These advances are deepening our understanding of how meiotic recombination shapes genetic diversity and genome evolution in diverse species.Homologous chromosomes must pair and recombine to ensure faithful chromosome segregation during meiosis, a specialized type of cell division that occurs in sexually reproducing eukaryotes. Meiotic recombination initiates by programmed induction of DNA double-strand breaks (DSBs) by the conserved type II topoisomerase-like enzyme SPO11. A subset of meiotic DSBs are resolved as crossovers, whereby reciprocal exchange of DNA occurs between homologous chromosomes. Importantly, DSBs are non-randomly distributed along eukaryotic chromosomes, forming preferentially in permissive regions known as hotspots. In many species, including plants, DSB hotspots are located within nucleosome-depleted regions. DSB localization is governed by interconnected factors, including  Meiosis is a specialized cell division program that is essential for sexual reproduction in eukaryotes. During this program, replication of chromosomal DNA to form sister chromatids is followed by two rounds of cell division. Maternal and paternal chromosomes (homologs) segregate at the first division and sister chromatids segregate at the second division. Chromosome number is thereby halved and, in diploid organisms, meiosis culminates in the production of haploid progeny cells (gametes). Chromosome segregation during meiosis is imperative for the continuation of the species, as it enables the formation of a zygote that inherits the full chromosome complement in the next generation by fusion of a male and a female gamete  and mouse, DSB hotspots have been defined as loci meeting or exceeding given thresholds for Spo11-oligo density and physical size  -based peak-calling approach  in gene promoters   than in wild-type budding yeast cells, indicative of a negative feedback loop in which homolog engagement following DSB formation suppresses Spo11 activity and prevents further breaks  group participate in the assembly of the synaptonemal complex and promote crossing over    targeted to meiotic hotspots suppresses crossover recombination , Copia LTR and LINE-1 classes less than expected (Choi et al., Arabidopsis and mouse, however, fewer-than-expected maize DSB hotspots occur in Copia LTR and LINE retrotransposons (He et al., Despite SPO11-1-oligo depletion in Arabidopsis, significant overlap occurs between comparable classes of DNA elements and DSB hotspots, many of which are located within functionally conserved sequences (Choi et al., Arabidopsis hotspots may be more evolutionarily stable. Nonetheless, comparisons between eukaryotes indicate that repeated sequences may influence meiotic recombination initiation landscapes in related ways.Citing the hotspot paradox hypothesis, Yamada et al.  speculatATM in budding yeast, suppresses the formation of clustered DSBs in cis over distances of ~70–100 kb (Garcia et al., ATM activity allows DSBs to form independently of one another over ±20–100-kb distances, giving rise to DSB formation in neighboring regions at frequencies comparable to those expected by chance. Over distances of ± ~7.5 kb, by contrast, Tel1ATM inactivation permits the formation of adjacent DSBs significantly more frequently than expected, generating localized regions of “negative DSB interference” (Garcia et al., cis-interference suggests that hotspots within the same loop domain are “primed” for cleavage. Cooper et al. (ATM-dependent cis-interference, which restricts DSB formation to only one of the primed intra-loop hotspots. Spatial regulation of meiotic DSB formation also occurs in trans via a mechanism involving Tel1ATM and Mec1ATR, another DDR signal transduction kinase (Zhang et al., trans-interference inhibits DSB formation at the corresponding locus on its sister, its homolog or frequently both. This mechanism is thought to ensure that an intact template is available for DSB repair, and to prevent DSB formation at allelic loci on both homologs (Zhang et al., Meiotic DSB hotspots are identified by mapping the DSB landscape in a population of cells. This landscape reveals a continuum of variation within which loci with high probabilities of DSB formation may be detected (Pan et al., r et al.  speculatGenome-wide DSB mapping in different eukaryotes has revealed diversity with regard to the hierarchical combinations of factors that shape meiotic recombination landscapes and hotspots. These distinctions highlight the importance of studying DSB landscapes in diverse eukaryotes and beyond model organisms. Efforts to elucidate the mechanisms that determine DSB hotspot designation may inform genetic or epigenetic manipulations intended to reshape naturally constrained meiotic recombination landscapes. For example, the presence of hotspots in conserved genomic elements, such as nucleosome-depleted promoters, has relevance for targeting crossover recombination to specific loci in plants (Sarno et al., AT and IH wrote and edited the manuscript. AT created Table 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.


Hepatitis E virus (HEV) can lead to high mortality during pregnancy. This study was to investigate the adverse pregnancy outcomes caused by different HEV genotypes and their prevention by HEV 239 vaccine in rabbits. Forty-two female rabbits were randomly and equally divided into 7 groups (A-G). HEV 239 vaccine and a placebo were administered to groups E (10 μg×2), F (5 μg×2) and G (1 mL of PBS×2) before copulation. After pregnancy, 1 mL of 1.5×106 copies/mL rabbit HEV3 was inoculated to groups A, E, F and G, swine HEV4/human HEV3 to groups B/C, and group D was a negative control. Anti-HEV antibody, HEV RNA, and alanine aminotransferase (ALT)/aspartate aminotransferase (AST) levels were monitored. Pregnant rabbits infected by HEV manifested HEV infection symptoms including fecal virus shedding, ALT/AST elevation, and histopathological changes, and adverse pregnancy outcomes. Immunized pregnant rabbits in groups E and F showed no HEV infection symptoms and adverse outcomes. The newborn rabbits delivered by pregnant rabbits with/without immunization showed without/with HEV infection symptoms. This study demonstrated that multiple genotypes of HEV infection can cause adverse outcomes and HEV 239 vaccine can prevent HEV-related adverse outcomes in pregnant rabbits. Hepatitis E virus (HEV) is the main cause of acute viral hepatitis in developing countries, which has aroused wide public concern . Each yeHEV often causes acute self-limited disease, with a mortality of approximately 0.2–1% . HoweverThe animal experiments were approved by the Committee of Laboratory Animal Welfare and Ethics, Peking University Health Science Center, and were in strict accordance with the protocol for the review of the Laboratory Animal Welfare and Ethics, Peking University Health Science Center (IACUC number: LA2017047).Forty-two 7-month-old female Japanese white rabbits weighing between 5.0 and 6.0 kg were obtained from the Department of Laboratory Animal Science of Peking University Health Science Center. Serum and feces specimens from selected rabbits were collected once a week for 2 weeks to test ALT and AST levels and to establish a baseline. Enzyme-linked immunosorbent assay (ELISA) and reverse transcription-nested PCR (RT-nPCR) were performed to ensure that all specimens were negative for HEV RNA and antibodies against HEV.Escherichia coli system derived from the 368–606 aa segment of the HEV1 (GenBank D11092) ORF2 protein. It is currently the only commercially available HEV vaccine globally. To date, the HEV 239 vaccine is only available in the Chinese market and has been approved for use in people older than 16 years, which is indicated for vaccinating individuals at high risk of HEV infection [The HEV 239 vaccine  is a 26 kDa recombinant polypeptide expressed by the GenBank D092 ORF2 6 copies/mL by using a one-step real-time quantitative PCR assay [The rabbit HEV strain , swine HEV strain  and human HEV strain  used in this study were all isolated from feces. Inocula were prepared as described previously . The virCR assay n = 42) were randomly divided into 7 groups (A-G), with 6 rabbits per group . Before copulation, rabbits in groups E and F were inoculated intramuscularly with a 10 and 5 μg dose of HEV 239 vaccine on weeks 0 and 4 respectively, while rabbits in group G as a vaccine control group were inoculated with a sterile PBS solution. On week 7, all female rabbits (n = 42) copulated with healthy male rabbits. After their pregnancy was confirmed [6 copies/mL rabbit HEV strain (rHEV3). Rabbits in groups B and C were inoculated intravenously with 1 mL of 1.5×106 copies/mL swine HEV4 strain (sHEV4) and human HEV3 strain (hHEV3), respectively, and rabbits in group D were inoculated intravenously with a rabbit feces suspension without HEV infection as a negative control.All selected female rabbits  by being immediately fixed in 10% neutral buffered formalin .To confirm the female rabbits’ pregnancy, their serum  and postpartum serum were all tested for progesterone and estrogen by the chemiluminescent enzyme immunoassay method on a Siemens Immulite 2000 Immunoassay System according to the manufacturer’s instructions .All serum samples were tested immediately for ALT and AST concentrations using standard methods on a Hitachi Automatic Clinical Analyzer 7180. When the ALT or AST level became more than 2 times the preinfection baseline value, the rabbit was considered to have liver inflammation .All rabbit serum samples were detected by ELISA for total anti-HEV antibodies according to the manufacturer’s instructions . When the S/CO value > 1, it was considered to be positive . The geoTotal RNA in serum and fecal suspensions was extracted according to the RNAgents Total RNA Isolation System Kit  instructions, and HEV RNA fragments were amplified by RT-nPCR . RT-nPCRAccording to the literature , real-ti+ and CD8+ cells were visualized with anti-CD4+  and -CD8+ polyclonal antibodies .For histopathology, tissue samples were prepared for hematoxylin and eosin (HE) staining, IHC  and immunofluorescence  by first being fixed in 10% neutral buffered formalin immediately following sampling according to the literature ,19. HEV http://www.ibm.com/cn/). Data were compared using Student’s t-test or chi-squared test. A P-value of <0.05 was considered significant.Statistical analysis was performed using the SPSS PASW Statistics v18.0 statistical software package  infection in pregnant rabbits, we inoculated pregnant rabbits in groups A, B and C with rabbit HEV3 (rHEV3), swine HEV4 (sHEV4), and human HEV3 (hHEV3) respectively after the pregnancy was confirmed. Group D was set up as a negative control group without HEV infection.P > 0.05)  was 83.3% (5/6), with 16.7% (1/6) maternal death, 50% (3/6) stillbirth, and 16.7% (1/6) miscarriage; in group B (sHEV4) was 83.3% (5/6), with 66.7% (4/6) stillbirth and 16.7% (1/6) miscarriage; and in group C (hHEV3) was 66.7% (4/6), with 16.7% (1/6) maternal death and 50% (3/6) stillbirth. All rabbits in group D had normal delivery and no adverse pregnancy outcomes occurred. While maternal death only occurred in groups A (16.7%) and C (16.7%) and miscarriage was only observed in groups A (16.7%) and B (16.7%), stillbirth was observed in groups A , B  and C . All HEV genotypes could induce adverse pregnancy outcomes in pregnant rabbits, and there was no statistically significant difference in the incidence of adverse outcomes among groups A, B, and C ( > 0.05) .Table 1P < 0.05)  began fecal virus shedding at 1 week post inoculation (wpi) that lasted for an average of 13.5 ± 0.4 weeks; rabbits in group B (sHEV4) began shedding at 2wpi that lasted for 10.2 ± 0.5 weeks on average; and rabbits in group C (hHEV3) began shedding at 2wpi that lasted for 4.8 ± 0.7 weeks on average. The durations of fecal virus shedding were statistically different among the three groups, with group A shedding (13.5 ± 0.4 w) being significantly longer than that of group B (10.2 ± 0.5 w) and group C (4.8 ± 0.7 w) ( < 0.05) . The seqP < 0.05)  of pregnant rabbits had viremia, while the rabbits in the other groups did not. Positive and negative HEV RNA strands in the livers and placentas were detected in 100% (6/6) and 50% (3/6) of group A’s rabbits respectively; positive and negative RNA strands were detected in the livers of 100% (6/6) and 50% (3/6) of the rabbits in groups B and C respectively. In groups A, B and C, 66.7% (4/6), 50% (3/6) and 16.7% (1/6) of rabbits showed a significant increase in ALT levels that was more than 2 times the baseline value respectively, while AST levels of 50% (3/6), 16.7% (1/6) and 0% (0/6) of rabbits in groups A, B and C significantly increased more than 2 times the baseline value respectively. In addition, seroconversion of anti-HEV antibodies in groups A (6/6), B (6/6), and C (4/6) was observed, and S/CO values in group A (20.3 ± 3.3) were significantly higher than those in groups B (10.7 ± 2.1) and C (6.1 ± 1.9) ( < 0.05) .To clarify the histopathological changes in pregnant rabbits’ tissues, we performed HE staining on rabbit livers and other organs . In grouTo assess the replication efficiency of different genotypes of HEV in the liver, ovary and placenta, the expression of HEV ORF2 and ORF3 was detected by immunofluorescence staining. HEV ORF2 and ORF3 antigens were positive in the liver, ovary, and placenta of rabbits in groups A, B, and C but not in the organs of group D .r2 = 0.4339) was observed, which indicated that estrogen levels were correlated with HEV infection and the higher the estrogen level, the higher the fecal viral load in pregnant rabbits was .By regression analysis of serum estrogen levels and fecal viral load in group A after virus inoculation, a positive correlation between serum estradiol levels and fecal viral load in pregnant rabbits .To investigate the relationship between HEV infection and cellular immunity in pregnant rabbits, we investigated the cellular immune response associated with HEV infection in the liver, ovary, and placenta through IHC. IHC experiments on CD4P < 0.05) (Supplementary Table 1).By analyzing HEV infection in surviving newborn rabbits delivered by pregnant rabbits infected with different genotypes of HEV, 50% (2/4) of surviving newborn rabbits of group A had fecal virus shedding after birth, 25% (1/4) were positive for anti-HEV antibody without HEV infection and 25% (1/4) did not have HEV infection; in surviving newborn rabbits of group B, 20% (1/5) had viremia after birth and 80% (4/5) did not have HEV infection; and in group C’s surviving newborn rabbits, 100% (7/7) were without HEV infection. The HEV infection rate of surviving newborn rabbits in group A was significantly higher than that of surviving newborn rabbits in group C (One newborn rabbit of group A (Aa) shed virus in its feces for 12 weeks, and the other (Ab) shed for 23 weeks. Both ALT and AST levels of these two rabbits significantly increased more than 2 times the baseline value and seroconversion of anti-HEV antibodies was also observed A and 3B.The liver tissues of euthanized newborn rabbits were analyzed by histopathology and immunofluorescence. Structural disorder, hepatocyte necrosis and inflammatory cell infiltration were observed in the liver tissue of Aa and extensive hepatocyte edema, balloon-like degeneration and hepatocyte necrosis were observed in the liver tissue of Ab. Immunofluorescence analysis showed positive signals for HEV ORF2 and ORF3 in the liver tissues of Aa and Ab D.Different doses of the HEV 239 vaccine were administered to pregnant rabbits to evaluate the protective efficacy. Four weeks after the first immunization in groups E and F, the serum anti-HEV antibody-positive rates of rabbits inoculated with the 10 and 5 μg dose HEV 239 vaccine were 83.3% and 66.7%, respectively. After the whole immunization process, all vaccinated rabbits were positive for anti-HEV antibodies, whereas the rabbits of the placebo group, G, were all negative for anti-HEV antibodies .Table 3To evaluate the dynamics of anti-HEV antibodies induced by different doses of the HEV 239 vaccine throughout the whole immunization process, the GMT was measured weekly. The GMT induced by the 10 and 5 μg dose vaccines gradually increased after the first immunization and increased rapidly after the second immunization A.FigureP > 0.05) and that the average anti-HEV antibody concentrations in groups E and F were higher than that in group G (P < 0.05)  B.After pregnancy was confirmed, rabbits in groups E, F and G were inoculated with the rHEV3 virus on the 10th day post copulation. None of the rabbits in groups E and F developed HEV infection symptoms, such as viremia, fecal virus shedding and elevation of ALT and AST levels, with only one rabbit in group F having a stillbirth. In contrast, all rabbits in group G began seroconversion to anti-HEV antibodies and fecal virus shedding at 2 wpi. Adverse pregnancy outcomes, including miscarriage, stillbirth, and death, occurred at 4 wpi, indicating that HEV can successfully infect rabbits without HEV 239 vaccination.P < 0.05). Two doses of the 5 μg HEV 239 vaccines could protect pregnant rabbits from rabbit HEV infection, with only one case of stillbirth occurring, which also had a significant difference compared with placebo administration (P < 0.05)  .Table 4Serum anti-HEV antibody levels of newborn rabbits in groups E and F were followed. From the 3rd to 8th week after birth, the levels of anti-HEV antibodies continued to decrease and then remained at the same level, while the anti-HEV antibody levels in the pregnant rabbits of groups E and F decreased slightly and remained at the same level. The initial level of anti-HEV antibody in newborn rabbits in group E was higher than that in group F but remained at the same level eventually C.Although studies have suggested that mainly HEV1 infection is associated with high mortality in pregnant women , there iThe vertical transmission of HEV has always attracted much attention , and sev+ and CD8+ T cells in the liver, ovary, and placenta and that the proportion of CD8+ T cells was higher than that of CD4+ T cells.The alteration in hormone secretion during pregnancy may be associated with hepatitis E-related poor pregnancy outcomes, and estrogen has been proven to be able to promote HEV replication in vitro . Our resIn this study, similar to previous studies ,29, our In summary, our results showed that different genotypes of HEV can infect pregnant rabbits and lead to adverse pregnancy outcomes and vertical transmission. The HEV 239 vaccine can effectively protect pregnant rabbits from rabbit HEV infection and adverse pregnancy outcomes. Although the animal numbers in the present study are limited, our results still suggest that to avoid hepatitis E-related adverse pregnancy outcomes and vertical transmission, childbearing-age women should be immunized with the HEV vaccine before pregnancy.Click here for additional data file.


BEST1, encoding a Ca2+-activated Cl− channel (hBest1), cause macular degenerative disorders. Best1 homolog structures reveal an evolutionarily conserved channel architecture highlighted by two landmark restrictions  in the ion conducting pathway, suggesting a unique dual-switch gating mechanism, which, however, has not been characterized well. Using patch clamp and crystallography, we demonstrate that both the neck and aperture in hBest1 are Ca2+-dependent gates essential for preventing channel leakage resulting from Ca2+-independent, spontaneous gate opening. Importantly, three patient-derived mutations  lead to Ca2+-independent leakage and elevated Ca2+-dependent anion currents due to enhanced opening of the gates. Moreover, we identify a network of residues critically involved in gate operation. Together, our results suggest an indispensable role of the neck and aperture of hBest1 for channel gating, and uncover disease-causing mechanisms of hBest1 gain-of-function mutations.Mutations of human  2+-activated Cl- channel, are both Ca2+-dependent gates. By showing hyper-activity of Bestrophin1, which is associated with its enhanced gate opening in the presence of three patient-derived mutations, this study provides mechanistic insights into the gain-of-function mutations that cause macular degenerative disease.Ji and Kittredge et al. demonstrate that the neck and aperture of human Bestrophin1, a Ca Over 250 patient-specific mutations in the BEST1 gene, which encodes hBest1, have been documented to cause retinal degenerative diseases10, underlying the most direct biomedical relevance known for the channel. Best1 is also distributed in the brain and spinal cord, where its physiological role remains elusive9. There are three other human bestrophin paralogs (hBest2–4) that share highly conserved transmembrane regions with hBest1 and were all originally identified as Ca2+-activated Cl− channels12. Therefore, deciphering the structure and function of Best1 has critical biological significance.Human bestrophin1 (hBest1) is a CaKlebsiella pneumoniae (KpBest) and the other from chicken (cBest1), have been previously reported14. The KpBest and cBest1 structures are very similar (root-mean-square deviation (RMSD) 2.4 Å) despite the relatively low sequence identity (17%) shared between the two proteins15, highlighting the evolutionarily conserved architecture of bestrophins. Overall, the channel is a pentameric assembly with a single ion-conducting pathway, which varies in diameter along its length and is constrained at two narrow points: a “neck” contributed by three residues (I62/I66/F70 in KpBest and I76/F80/F84 in hBest1/cBest1) on a transmembrane helix, and an “aperture” formed by a single residue  on a cytosolic helix. The neck and aperture are both apparent candidates for controlling Ca2+-dependent activation and ion permeability of the channel, but their regulatory mechanisms and individual contributions to the channel function are still unclear.Although the structure of hBest1 is still unsolved, two Best1 homolog structures, one from bacteria 2+-bound cBest1 structure are 1.1–1.3 Å, barely sufficient or insufficient for Cl− even in the dehydrated form  to pass through. As bigger anions such as Br−, I−, and SCN−  are also permeable to cBest116, the cBest1 structure, like the KpBest counterpart, is in a closed or inactivated state. Consistent with this idea, cBest1 structures containing an opened neck are recently obtained by removing a conserved inhibitory sequence at the C-terminus18. However, the open state conformation of the aperture still remains to be defined.The radii of the neck and aperture in the original Ca16, such that the neck is no longer a constraint for the passing ions and the aperture represents the only restriction in the ion-conducting pathway, functionally mimicking a state where the neck has been constantly opened/activated. With the same idea, alanine substitution at the aperture functionally mimics constant opening/activation of the aperture, leaving the neck as the sole constraint16.Gain-of-function mutations displaying elevated channel activity, best exemplified by alanine substitutions at the neck and aperture, have been a critical tool to study channel activation. As both restrictions are formed by side chains of the contributing hydrophobic residues, substitution with a shorter side-chained alanine will enlarge the size of the mutated restriction, resembling channel opening/activating. For instance, the structure of a cBest1 mutant with triple alanine substitution of the neck residues shows a much bigger opening at the neck14, suggesting an activation gate role for the aperture. In cBest1, triple alanine substitution of all three conserved hydrophobic residues at the neck  eliminated Ca2+ dependence without altering anion selectivity, while alanine substitution at the aperture (cBest1 V205A) abolished anion selectivity without affecting Ca2+ dependence16, suggesting an exclusive Ca2+-dependent activation gate role for the neck and a Ca2+-independent ion filter role for the aperture. These seemingly confusing results could be due to the different Best1 homologs and experimental methods involved in previous studies. In particular, the aperture-forming residue is a long side-chained isoleucine in KpBest (I180) and hBest1 (I205) but a less bulky valine in cBest1 (V205), resulting in a smaller aperture in KpBest/hBest1, which suggests a stronger gating capacity/effect. Consistent with this notion, KpBest I180 is the narrowest point in the channel ion-conducting pathway, but cBest1 V205 is not14. In Coot simulation, substituting cBest1 V205 with an isoleucine makes it the smallest restriction on cBest1, with the same radius (0.9 Å) as that at KpBest I180, predicting that I205 likely forms the narrowest restriction on hBest1 in the closed/inactivated state. Nevertheless, a careful examination of the neck and aperture in hBest1 is in urgent need.In KpBest and hBest1, alanine substitution at the aperture (KpBest I180A and hBest1 I205A) significantly enhanced channel activity2+-dependent gates, and their dysregulated opening by rationally designed or patient-derived mutations causes Ca2+-independent leak and elevates Ca2+-dependent channel activity. This illustrates, for the first time to our knowledge, gain-of-function disease-causing mechanisms of BEST1 mutations. Moreover, we identify a network of critical residues involved in operating the gates, a promotive effect of methanesulfonate− (CH3SO3−) on channel opening through the neck, and an estimated size of the opened aperture. Taken together, our results reveal fundamental gating mechanisms of bestrophin channels with direct pathological relevance.Here, by electrophysiological and structural analyses, we show that the neck and aperture in hBest1 are both indispensable Ca2+, all three mutants conducted robust Cl− current in patch clamp recordings, in sharp contrast to the tiny current in cells transfected with WT hBest1, which was indistinguishable from that of untransfected cells  at the aperture I205A), the neck , and both  13. This mechanism is possible, although unproven, considering the resolution of the structure and moderate oversize of the anions. However, the presence of a bulkier isoleucine at the hBest1 aperture (I205) poses a challenge to this hypothesis. To probe the hBest1 pore-opening capacity, we replaced Cl− in the external solution with CH3SO3−, which is an ideal choice for probing Ca2+-activated Cl− channels because of its spherical shape, big size (radius 2.6 Å in the dehydrated form), low relative permeability, non-association with Ca2+, and complete dissociation at physiological pH19. Strikingly, hBest1 conducted robust outward CH3SO3− current (CH3SO3− influx) in the presence of 1.2 μM Ca2+  has “breathing” to allow the passage of dehydrated anions such as Cla2+ Fig. , indicat3SO3− current in the absence of Ca2+  but also inward current (Cl− efflux) in WT hBest1 16. To characterize how alanine substitution affects the size of the aperture, we solved the crystal structure of the KpBest I180A mutant  at 2.9 Å  to pass through the aperture of hBest1 I205A in the absence of Ca2+.Consistent with the Clradius 3. Å16. To radius 3. Å16. To 6. As threonine has a shorter side chain than isoleucine, we speculated that I205T, like I205A, enlarges the opening at the aperture and enables Ca2+-independent leak activity. Notably, the threonine side chain possesses a hydrophilic hydroxyl group that is capable of forming H-bonds, which can further affect ion permeation. To test this idea, we solved the crystal structure of the equivalent KpBest I180T mutant at 3.2 Å , Y236C (a patient-derived mutation), and W287F , all of which showed normal overall expression and membrane trafficking in HEK293 cells after transient transfection  , respectively. Consistent with the functional results, no structural change was found at the aperture. Although the structure at the neck region was surprisingly unaltered, possibly due to the molecular packing effect in the crystal or the presence of Znly) Fig. , reaffirhBest1 D203A is a patient-derived mutation localized on an intracellular loop adjacent to the aperture Fig. . Structu2+-dependent Cl− current was bigger than that from WT hBest1, indicating a gain-of-function phenotype  all showed an overall similar structure to that of WT KpBest  upon channel activation and discovered a CH3SO3−-promoted trans effect through the neck. Importantly, using electrophysiological and crystallographic approaches, we identified three patient-derived mutations as gain of function due to enhanced opening at either one or both of the gates, directly linking impaired channel gating to disease-causing mechanisms. Moreover, in an alanine substitution screen, we identified multiple residues  critically involved in channel gating at the neck and/or aperture.A unique feature of Best1 channels, as indicated by the solved KpBest and cBest1 structures, is the presence of two restrictions, the neck and aperture, in the ion-conducting pathway. Given hBest1’s clear association with retinal diseases, it is of obvious importance to understand how these two restrictions on hBest1 contribute to channel gating. Here, by electrophysiological comparison of hBest1 mutants in which either one or both of the restrictions are removed, we demonstrated that the neck and aperture in hBest1 are both Ca2+-activated gates communicating through the I205–D203–P233–Y236/W287–F84 network. Notably, hBest1 D203/KpBest D179 is located on an intracellular loop essential for ATP-dependent activation of the channel15, potentiating the effect of ATP on both gates.While mutants with conformational changes at the aperture  or neck  do not have structural alteration at the other restriction, a mutation near the aperture  impacts both gates, suggesting both independent and cooperative regulations of the two channel gates. Based on these observations, we propose that the neck and aperture in hBest1 serve as two separate Ca2+-independent leak current in hBest1 3A was bigger compared to that in hBest1 I205A, suggesting a lower spontaneous open probability of the neck than the aperture, which is consistent with the formation of the neck by three rings of residues as opposed to only one ring of residues at the aperture. In the presence of Ca2+, the Cl− currents in hBest1 3A and I205A were enhanced to approximately two- and five-fold of those without Ca2+, respectively (compared to approximately nine-fold increase in WT), suggesting that Ca2+ has different stimulatory effects on the open probability of the aperture and neck. Consistent with this idea, KpBest I180A  displayed dramatically increased open probability but unchanged unitary current in lipid bilayer14.The Ca2+-activated Cl− channels, a wide range of Ca2+ dependence and sensitivity among them have been documented. For instance, hBest2 and Drosophila Best1 (dBest1) expressed in HEK293 cells exhibit Ca2+-independent activity23, while hBest1 and cBest1 are both Ca2+ dependent but display over nine-fold difference in Ca2+ sensitivity24. The existence of two gates, both of which allow Ca2+-dependent regulation, provides a versatile system for generating distinct Ca2+ responses in different bestrophin homologs, which conduct anion currents at various [Ca2+]i under physiological conditions. In particular, the aperture-forming residue is variable , in sharp contrast to the highly conserved neck-forming residues , underlining the aperture as a key regulatory element for Ca2+ response.Although bestrophins have been characterized as Ca16. Consistent with this idea, hBest1 I205A and I205T both displayed similar permeability to Cl− and CH3SO3−, which is significantly different in the WT. Importantly, the robust Ca2+-dependent CH3SO3− current in WT hBest1 and CH3SO3− leak in hBest1 3A indicate that the minimum size of the opened hBest1 aperture should be sufficient to accommodate a dehydrated CH3SO3− (radius 2.6 Å). On the other hand, to function as a size-selective filter, the opened aperture should not be bigger than hydrated anions (radius 3.3 Å for Cl−/Br−/I−). Therefore, we predict that the hBest1 aperture in its open state has a radius of 2.6–3.3 Å. Interestingly, asymmetry of the aperture is observed in the KpBest D179A and I180T mutants  results in a loss of the permeability sequence for anions, suggesting a “size-selective” filter role of the aperture: it represents a constraint to hydrated anions even when opened in WT channels, such that the permeability sequence of different anions is mainly determined by their dehydration energy, while in the mutant channel the opened aperture is sufficient to pass hydrated anions, bypassing the dehydration process and thus resulting in equalized permeabilityts Figs.  and 6b, 17. Opening of the cBest1 neck is mediated by a conformational change of the neck-forming S2b helices, which turns the side chains of I76, F80, and F84 away from the central axis of the channel pore. Importantly, F80 and F84 are stabilized in the open conformation by interactions with hydrophobic residues Y236 and W287. Moreover, consistent with our conclusion that hBest1 W287F is a gain-of-function mutation with increased open probability of the neck, a cBest1 W287F mutant structure shows a constantly opened neck17. However, the aperture remains unchanged in a presumably closed conformation in all solved cBest1 structures regardless of the status of the neck, possibly reflecting a lower open probability of the aperture and/or the requirement of additional activator(s), such as ATP15.The critical roles of hBest1 Y236 and W287 in gating of the neck have been confirmed by recently solved cBest1 structures with an open state neck−, respectively. As expected, the currents conducted by hBest1 4A in the absence of Ca2+ are significantly bigger than those from WT in the presence of Ca2+. Moreover, Ca2+ has no promotive effect on hBest1 4A, suggesting that the neck and aperture of the 4A mutant either cannot further dilate in response to Ca2+ or their further dilation does not contribute to the anion current amplitude (conductance). Interestingly, hBest1 I205A conducted significantly bigger currents compared to hBest1 4A in the presence of Ca2+ with CH3SO3− in the external solution  mutations at the neck/aperture, including I205A, I205T, 3A, and 4A, increase open probability by functionally mimicking a constant open state; and (3) mutations outside of the neck/aperture, including D203A, P233A, Y236A, Y236C, W287A, and W287F, alter open probability through the neck/aperture residues. The latter two groups of mutations may or may not affect channel unitary conductance, which requires further investigation such as single channel recordings.Here we identified 18 mutations that significantly alter hBest1 channel activity without affecting the whole cell and total membrane expression levels of the protein. Notably, the short extracellular domains of hBest1 represent a challenge for quantitative measurement of the surface expression by labeling techniques . They can be classified into three groups by different molecular mechanisms: (1) mutations on residues involved in CaBEST1 patient mutations. To our knowledge, for the first time, we identified gain-of-function disease-causing mutations  and revealed the underlying molecular mechanisms. As BEST1 patients clinically display decreased light peak, which is presumably generated by hBest1-mediated Cl− current in RPE, gain of function represents a disease-causing mechanism fundamentally distinct from loss of function. Therefore, it will be very interesting to examine how these gain-of-function mutations affect the cellular behavior of endogenous hBest1 and the electrophysiological property of endogenous Cl− in RPE25. From a therapeutic perspective, rather than gene supplementation for the rescue of hBest1 loss of function, a different strategy such as gene suppression may be required for the treatment of gain-of-function mutations26.Previously, only loss-of-function phenotypes have been recognized for 14, disqualifying KpBest for the investigation of the non-conserved residues in hBest1. Second, as KpBest is a Na+ channel that does not require Ca2+ while hBest1 is a Ca2+-activated Cl− channel14, KpBest is not suitable for functional studies on the Ca2+ dependence or ion selectivity of hBest1. In regard to these concerns, we only utilized KpBest for structural modeling of mutations on the highly conserved residues in this work.Although the highly conserved architecture of Best1 homologs potentiates KpBest as a simple structural model to study hBest1, one should be aware of two major limitations. First, KpBest and hBest1 share only 14% sequence identity−1 glucose, Corning 10013CV) supplemented with 10% fetal bovine serum and 100 μg ml−1 penicillin–streptomycin. 4,6-Diamidino-2-phenylindole staining revealed no mycoplasma contamination.HEK293 cells authenticated by short tandem repeat DNA profiling were kindly gifted from Dr. David Yule at the University of Rochester. HEK293 cell cultures were maintained in Dulbecco’s modified Eagle’s medium  was used to transfect HEK293 cells with plasmids bearing the indicated hBest1 WT or mutant (1 μg). After 6–8 h, the transfection mix was removed and cells were washed with phosphate-buffered saline and cultured in supplemented DMEM. Twenty-four hours after transfection, cells were again detached from the cell culture dishes by trypsin and split onto fibronectin-coated glass coverslips for patch clampHEK293 cells transiently expressing yellow fluorescent protein-tagged hBest1 WT and mutant channels were harvested 48 h post transfection. The M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific 78501) and Mem-PER Plus Membrane Protein Extraction Kit (Thermo Fisher Scientific 89842) were used to prepare the total cell lysates and membrane-associated proteins, respectively. The samples were mixed with Laemmli buffer and denatured at 95 °C for 5 min, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and wet transfer to nitrocellulose membranes. Immunoblotting was performed with green fluorescent protein primary antibody  and fluorophore-conjugated secondary antibody , and subsequently detected by infrared imaging.27 was used to perform whole-cell patch clamp recordings. Micropipettes were pulled and fashioned from filamented 1.5 mm thin-walled glass (WPI Instruments) and subsequently filled with internal solution containing (in mM): 130 CsCl, 1 MgCl2, 10 EGTA, 2 MgATP (added fresh), and 10 HEPES . The desired free Ca2+ concentration (0 and 1.2 μM) was obtained by adding CaCl2 (http://maxchelator.stanford.edu/CaMgATPEGTA-TS.htm). Series resistance was typically in the range of 1.5–2.5 MΩ, with no electronic series resistance compensation. The external solution consisted of (in mM): 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES , and 15 glucose. When replacing Cl− with CH3SO3−, the external solution (in mM) was 120 NaCH3SO3 and 10 HEPES . Solution osmolarity was 310–315 mOsm. A repetition interval of 4 s was used to acquire traces28. Sampled at 25 kHz, currents were filtered at 5 or 10 kHz. To generate I–V curves, a group of step potentials  were used. The experiments were conducted at room temperature (23 ± 2 °C). Liquid junction potentials were measured and corrected using HEKA built-in functions. Solution changes were performed manually.Electrophysiological analyses were conducted 24–72 h after transfection. An EPC10 patch clamp amplifier (HEKA Electronics) controlled by Patchmaster (HEKA)All KpBest constructs have 11 residues truncated from the C-terminus of WT. Point mutations of hBest1 and KpBest were made by site-directed mutagenesis PCR with the In-fusion Cloning Kit (Clontech). All constructs were verified by sequencing.15, BL21 plysS cells containing KpBest expression vectors were cultured in Terrific Broth medium overnight, inoculated 1:100 (v/v) into fresh Terrific Broth medium, and grown at 37 °C to OD 0.6–0.8 over 2–3 h. The culture was henceforth induced with 0.4 mM IPTG and grown at 20 °C overnight.BL21 plysS cells were kindly gifted by Dr. Wayne Hendrickson. For protein production18. For protein purification29, cell pellets were re-suspended in a buffer containing 50 mM HEPES (pH 7.8), 300 mM NaCl, 5% glycerol, 20 mM imidazole, and 0.5 mM TCEP. Cells were then lysed using an emulsiflex-C5 high pressure homogenizer (Avestin). The cell lysate was incubated with 2% DDM (w/v) for 1 h at 20 °C. The non-dissolved matter was pelleted by ultracentrifugation at 150,000 × g at 4 °C for 30 min. The supernatant was carefully collected and immediately loaded to a pre-equilibrated 5 mL HisTrap Ni2+-NTA affinity column . A 13-column volume buffer wash was performed, and the protein was eluted with a buffer containing 25 mM HEPES (pH 7.8), 200 mM NaCl, 5% glycerol, 500 mM imidazole, 0.1 mM TCEP, and 0.05% (w/v) DDM. To remove the 10×His tags, TEV proteinase was added at 1:1 mass ratio, followed by incubation at 4 °C for 30 min. The resulting samples were concentrated with 100 kDa centrifugal filter units  to a final volume of 400–500 μL. Samples were purified via size-exclusion chromatography with a Superdex-200 column by HPLC .After centrifugation at 4 °C, cell pellets were immediately processed (for purification) or stored at −80 °C until use−1. The sitting-drop vapor diffusion method was used to grow crystals at 20 °C. The growing conditions for the best diffracted crystals are summarized in Supplementary Table Purified protein was concentrated to ~5 mg mLt test was used to determine statistically significant differences (P < 0.05) between means of two groups. Data are presented as means ± s.e.m.30.Data from whole-cell patch clamp recordings were processed in Patchmaster off-line. Built-in functions in Origin were used to perform statistical analyses. According to the specific method utilized in a given experiment, we examined a sufficient number of samples to reach statistical conclusion. Student’s 3SO3− to Cl− relative permeability (PCH3SO3/PCl) was calculated according to the Goldman–Hodgkin–Katz equation: ΔErev = 59 log(150PCl/120PCH3SO3), as the internal Cl− concentration is 150 mM and the external CH3SO3− is 120 mM in the presence of Ca2+. The relative CH3SO3−/Cl−  inward movement (outward current) conductance (GCH3SO3 with ex-CH3SO3/GCl with ex-Cl) was measured as slope conductance at the reversal potential plus 50 mV, and CH3SO3− current was multiplied by 1.2 to compensate for the concentration difference. The trans effect, representing the relative Cl−/Cl−  outward movement (inward current) conductance (GCl with ex-CH3SO3/GCl with ex-Cl) was measured as slope conductance at the reversal potential minus 50 mV.The CH31 via the RAPD system of APS NE-CAT and AIMLESS32. To solve the mutant structures, the WT KpBest structure (PDB code: 4WD8) was used as a search model during molecular replacement, which was carried out using Phaser33 implemented in Phenix suite34 and MOLREP35 implemented in the CCP4 suite36. Coot37, phenix.refine in the Phenix suite34, and Refmac5 in the CCP4 suite38 were utilized to carry out model building and refinement. Table 39. All figures were made in PyMOL.The X-ray data sets on KpBest mutants were processed and scaled using XDSFurther information on research design is available in the Description of Additional Supplementary FilesSupplementary InformationReporting SummarySupplementary Data 1


Sisymbrium officinale (L.) Scop. is a wild common plant of the Brassicaceae family. It is known as “the singers’ plant” for its traditional use in treating aphonia and vocal disability. Despite its wide use in herbal preparations, the molecular mechanism of action of S. officinale extracts is not known. The plant is rich in glucosinolates and isothiocyanates, which are supposed to be its active compounds. Some members of this family, in particular allylisothiocyanate, are strong agonists of the transient receptor potential ankyrin 1 (TRPA1) channel, which is involved in the somatosensory perception of pungency as well as in the nociception pathway of inflammatory pain. This study aims to isolate the glucosinolates and isothiocianates from fresh S. officinale to identify the major components and test their activity in in vitro assays with a cloned TRPA1 channel. Samples of cultivated S. officinale have been extracted and the active compounds isolated by column chromatography, HPLC and PTLC. The main components glucoputranjivin, isopropylisothiocyanate and 2-buthylisothiocianate have been tested on TRPA1. The glucosinolates glucoputranjivin and sinigrin turned out to be inactive, while isopropylisothiocyanate and 2-buthylisothiocyanate are potent agonists of TRPA1, with an EC50 in the range of the high potency natural agonists identified so far for this somatosensory channel. Sisymbrium officinale (L.) Scop. (Brassicaceae) (SO) is an annual plant which is spread mostly in the Eurasiatic Region and North Africa. It is very common in bare ground, on roadsides, dumps and on the edges of fields. The plant is the object of a multidisciplinary project named “Erisimo a Milano” studying its diffusion and ecology in North Italy, agronomic potential, phytochemistry and food applications i) were determined using the selective intracellular fluorescent probe Fluo-4. Assays of the rat TRPA1-mediated elevation of intracellular Ca2+ in transfected HEK-293 cells were performed as described i in not-transfected HEK-293 cells were used as a baseline and subtracted from the values obtained from transfected cells. Agonist efficacy was expressed as a percentage of the effect on [Ca2+]i observed with 100 μM allylisothiocyanate (AITC). The potency of the compounds  was determined as the concentration required to produce half-maximal increases in [Ca2+]i. Antagonist/desensitizing behavior as evaluated against TRPA1 agonist AITC 100 μM by adding the compound directly into the quartz cuvette 5 min before the stimulation of cells with the agonist. IC50 is expressed as the concentration exerting a half-maximal inhibition of an agonist effect, taking as 100% the effect on [Ca2+]i exerted by the agonist alone. Dose–response curves are fitted by a sigmoidal regression with a variable slope. Curve fitting and parameter estimation were performed with a GraphPad Prism . Three independent experiments were done for each determination; all determinations were performed at least in triplicate. The time-course spectra showing the increase of intracellular Ca2+ upon application of the compounds are available in Compound effects on intracellular Caescribed . Briefly


The majority of acute myeloid leukemia (AML) patients suffer from relapse and the exact etiology of AML remains unclear. The aim of this study was to gain comprehensive insights into the activity of signaling pathways in AML. In this study, using a high-throughput PepChip™ Kinomics microarray system, pediatric AML samples were analyzed to gain insights of active signal transduction pathway. Unsupervised hierarchical cluster analysis separated the AML blast profiles into two clusters. These two clusters were independent of patient characteristics, whereas the cumulative incidence of relapse (CIR) was significantly higher in the patients belonging to cluster-2. In addition, cluster-2 samples showed to be significantly less sensitive to various chemotherapeutic drugs. The activated peptides in cluster-1 and cluster-2 reflected the activity of cell cycle regulation, cell proliferation, cell differentiation, apoptosis, PI3K/AKT, MAPK, metabolism regulation, transcription factors and GPCRs signaling pathways. The difference between two clusters might be explained by the higher cell cycle arrest response in cluster-1 patients and higher DNA repair mechanism in cluster-2 patients. In conclusion, our study identifies different signaling profiles in pediatric AML in relation with CIR involving DNA damage response and repair. Therefore, improved therapy of AML will require therapeutic agents directed against the perturbed pathways downstream of these events.Acute myeloid leukemia (AML) forms a spectrum of diseases that share clinical and pathological features. Improvements in chemotherapeutic regimens and supportive care have increased overall survival rates in younger patients (<15 years of age) to more than 60%, however, 40% ultimately relapse requiring salvage therapy . ExtensiTo address this, we recognized the need to develop a global map of the activation patterns going on in all of the signal transduction pathways within a cell. Therefore, we utilized a high-throughput peptide microarray array and investigated the peptide activation of 976 peptides simultaneously and quickly to generate a global map of pathway activation in pediatric AML. Unsupervised clustering clearly divided the AML samples into two different profiles, named cluster-1 and cluster-2 of which cluster-2 was strongly related to a higher cumulative incidence of relapse (CIR). In addition, cluster-2 samples showed to be significantly less sensitive to various chemotherapeutic drugs than cluster-1 samples. The identified activated peptide patterns of two clusters were independent of patient characteristics such as FAB, WHO classification with recurrent genetic abnormalities and mutations. GeneGO pathway analysis revealed that cell cycle arrest response was highly activated in cluster-1 and DNA repair process in cluster-2.max) using average linkage algorithms. That resulted in 192/976 recorded peptides which significantly separated the samples into two main clusters (cluster-1 and cluster-2) based on gap statistics  compared to cluster-1 (5 × 109/L)  with multiple testing corrections was observed. The patient karyotypes of two clusters are shown in × 109/L) . Additio× 109/L) . Only FL× 109/L)  whereas  2 test, .(B)To understand the different signaling dynamics activated in the two clusters, we performed pathway analysis of highly activated peptides for both cluster-1 and cluster-2 separately. Metacore GeneGO pathway analysis was used to identify the top ranked most significantly altered pathways using enrichment analysis. Top 20 pathways for proteins of corresponding peptides upregulated in both cluster-1 and cluster-2 in GeneGo are presented in This network analysis suggested that the cell cycle axis within DNA damage response was highly activated in cluster-1 patients as p53 and p21 peptides were intense on the microarray . In clusIn cluster-2, higher intensities of BRCA1, Chk1 and Chk2 were found. BRCA1 is known to be phosphorylated by Chk1 and Chk2 phosphorylation. Highly phosphorylated BRCA1 could participate in DNA repair mechanism and probably facilitate higher DNA replication process of cluster-2 AML cells. In addition, it could be hypothesized that faster cell cycle progression is promoted by higher phosphorylation of Rb and lower E2F1 phosphorylation in cluster-2 patients. These data suggest that the AML samples in cluster-1 have elevated p53 signaling possibly leading to apoptosis while AML samples in cluster-2 have elevated signaling reflected in DNA damage tolerance and repair.To confirm peptide microarray results of the cell cycle regulatory process, western blot analyses were conducted for phosphorylation status and total amounts of JNK1, p53, p21, Chk1, Chk2 using leukemic blasts of three leukemic patients for each cluster , 4C. It This study has demonstrated that peptide microarray profiling of AML samples provides significant details about AML signaling and identified two clusters associated with DNA damage response and repair system, resulting in significantly different CIR. The present study shows the power of high throughput peptide microarrays for the first time in a larger number of pediatric AML patients.In vitro peptide microarray profiling may reveal phosphorylation of specific peptides which may not occur in vivo since the tertiary structure of phosphorylation sites and the spatiotemporal regulatory mechanisms are lost [in vitro culture and organoids [Our previous studies have shown the feasibility of peptide microarray technology to develop treatment possibilities with specific kinase inhibitors for pediatric brain tumor, acute lymphoblastic leukemia and subsets of AML patients , 19, 20.are lost . Therefoare lost . With MErganoids . TherefoAt first, this study characterized the deregulated signaling pathways in pediatric AML. The peptide microarray profiles provided a comprehensive insight into signaling pathways active in pediatric AML patients. As expected, we found activation of proteins belonging to cell cycle regulation, cell proliferation, cell differentiation, apoptosis, PI3K/AKT signaling, MAPK signaling along with the regulators of metabolism, transcription factors and GPCRs signaling regulators in pediatric AML. Moreover, AML-associated peptides  were found to be activated in our array. This finding underscores our previous observation in leukemia that CREB, NF-ĸβ, E2F1 and EP300 were highly phosphorylated in MLL-rearranged AML . Other sin vitro, cluster-2 samples were more resistant to chemotherapeutic drugs than cluster-1 samples. Dissecting the peptides activation by bioinformatics analyses and subjecting the activated peptides to pathway analysis yields direct insights into signaling differences into the two clusters. It is tempting to hypothesize that the lower CIR found in cluster-1 AML samples is related to some remaining functionality of p53 and p21 in driving the cell towards cell cycle arrest, followed by apoptosis as shown in the low Bcl2 and high BAX protein levels. Mutations in p53 are rare in pediatric AML (approximately 1%) and correlate with worse outcome [Secondly, by unsupervised hierarchical clustering, two clusters of AML samples were identified, without the knowledge of prior class definitions and independent of patient characteristics e.g. blast percentage, WBC counts, age, sex and karyotypes but related to CIR. Moreover,  outcome . Previou outcome . Activat outcome . It is k outcome , 33.In cluster-2 samples, high BRCA1 activation may facilitate DNA repair mechanism as well as the phosphorylation of Rb. BRCA1 plays a pivotal role in the repair of DNA damage, especially induced following chemotherapy and ionizing radiation. There are only limited data on the role of BRCA1 activation in leukemia, higher BRCA1 expression was found in resistant AML sample , 35. PhoIn conclusion, this study demonstrates the feasibility and strength of this peptide microarray array platform in a large group of AML samples. Results elucidated deeper insights in the active signaling pathways, its high interconnections, as well as the essential role cell cycle regulation plays in the observed difference in the cumulative incidence of relapse. And so it is tempting to explore whether the use of cell cycle inhibitors focusing on DNA damage response and DNA repair pathways might be beneficial new additives to AML treatment schedules.This study used a total of 100 primary blood or bone marrow samples of newly diagnosed AML patients. Patients were treated according to Dutch Childhood Oncology Group (DCOG). After obtaining written informed consent, primary AML samples at diagnosis were collected from pediatric patients in accordance with the declaration of Helsinki and the study was approved by all participating review boards. Mononuclear cells were separated by Lymphoprep density gradient , and cryopreserved in liquid nitrogen until use. The cryopreserved leukemia cells were thawed rapidly at 37° C and diluted in a 6 ml volume of newborn calf serum, as described previously . The remn = 96) and CD34+ normal bone marrow (NBM) samples (n = 4) were determined using the PepChipTM Kinomics microarray system  as described previously [6 AML cells) were lysed in 100 μl of cell lysis buffer . Peptide array incubation mix was produced by adding 10 ml of filter-cleared activation mix ). Next, the peptide array mix and sample lysate were added onto the chip and incubated at 37° C in a humidified stove for 90 min. Subsequently, the peptide chip was washed twice with Tris-buffered saline with Tween, twice in 2M NaCl, and twice in demineralized H2O and then air-dried at 37° C.The peptide microarray profiles of AML samples (eviously . Brieflyr2) of triplicates was below 0.6. For further analysis medians from the triplicates were used. Quantile normalized data were analyzed using Qlucore Qmics Explorer version 3 . In order to identify differentially activated peptides and patients clusters, peptides with variance 0.15 (σ/σmax) were selected for further analysis [The chips were exposed to a phosphoimager plate for 72 hours, and the density of the spots was measured and analyzed with array software . Using grid tools from ScanAlyze software, spot density and individual background intensities were analyzed. An overview of the data analysis procedure is provided in analysis , 41. Thaanalysis .5 cells per 100 μl/well in a 96 wells plate in RPMI medium supplemented with 10% fetal calf serum with and without chemotherapeutic drugs (range of concentration), mitoxantrone , etoposide  and amsacrine . These concentration ranges were determined empirically to obtain the largest number of evaluable dose-response curves in AML and allowing the calculation of LC50 values. The mitochondrial activity of AML cell was measured after 48 h using a microplate reader at 450 nm . Cell survival percentages (viability) are determined relative to non-treated cells.The WST-1 assay was performed to assess the leukemic cell viability of primary leukemic samples after treated with chemotherapeutic drugs as described previously . BrieflyThe cell viability data is presented by calculating the LC50 for leukemic cells with the following formula:U test. P< 0.05 was considered statistically significant (2-tailed test). The difference in median LC50 values for a specific drug between 2 groups of patients was presented in the graph.Differences in the distribution of LC50 values were analyzed using nonparametric Mann-Whitney TM , based on a manually curated database of known molecular interactions, pathways, and processes, was used to explore the signaling pathways of the 192 differentially activated peptides. The activated peptides of the two different clusters were compared to score and rank entities in pathways most relevant for these 192 peptides. In MetaCoreTM, the statistical significance  is calculated using a hypergeometric distribution. Additionally, the GeneGo pathway analysis platform was used to generate a protein-protein interactional network, based on evidence in literature [A system biology pathway analysis tool, MetaCoreterature .6 cells were lysed in laemmli sample buffer . Proteins were separated by SDS-polyacrylamide gel electrophoresis, and transported to nitrocellulose membranes as described previously [1 × 10eviously . First, p-values were presented with Bonferroni corrections. The Kaplan-Meier method was used to generate the survival curves between two clusters . Cumulative incidence of relapse (CIR) which is the measure of relapse frequency during the follow-up time between two clusters was tested using Pearson Chi-Square test. Results were considered statistically significant when p-values were < 0.05. Associations between two clusters and patient characteristics were assessed in SPSS . Mann-Whitney U test (for continuous variables) and Pearson Chi-Square test  were used to assess the difference between the two clusters.One way ANOVA was used for multiple comparisons of peptide activation between AML patients of cluster-1 and cluster-2 and CD34+ NBM samples and


FLEC suppresses spontaneous Ca2+ waves in Casq2-knockout (Casq2−/−) cardiomyocytes, a CPVT model. However, a report failed to find FLEC efficacy against Ca2+ waves in another CPVT model, RyR2-R4496C heterozygous mice (RyR2R4496C+/−), raising the possibility that FLEC efficacy may be mutation dependent.The dual Na−/− and RyR2R4496C+/− cardiomyocytes and mice under identical conditions.To address this controversy, we compared FLEC in Casq22+ waves were quantified during a 40 s pause after 1 Hz pacing train in the presence of isoproterenol . FLEC efficacy was also tested in vivo using a low dose (LOW: 3 mg/kg ISO + 60 mg/kg caffeine) or a high dose catecholamine challenge (HIGH: 3 mg/kg ISO + 120 mg/kg caffeine).After 30 min exposure to FLEC (6 μM) or vehicle (VEH), spontaneous Ca2+]. At 2 mM [Ca2+], only Casq2−/− myocytes exhibited Ca2+ waves, which were strongly suppressed by FLEC. At 3 mM [Ca2+] both groups exhibited Ca2+ waves that were suppressed by FLEC. At 4 mM [Ca2+], FLEC no longer suppressed Ca2+ waves in both groups. Analogous to the results in myocytes, RyR2R4496C+/− mice (n = 12) had significantly lower arrhythmia scores than Casq2−/− mice (n = 9), but the pattern of FLEC efficacy was similar in both groups .In cardiomyocytes, FLEC efficacy was dependent on extracellular [Ca2+ waves in RyR2R4496C+/− cardiomyocytes, indicating that RyR2 channel block by FLEC is not mutation-specific. However, FLEC efficacy is reduced by Ca2+ overload in vitro or by high dose catecholamine challenge in vivo, which could explain conflicting literature reports.FLEC inhibits Ca In appr (Casq2) , or calm (Casq2) . At the hycardia . Drug thtiveness .−/−) mice, a CPVT model  and were approved by the institutional Committee of Ethics in Animal Research at Vanderbilt University Medical Center.Adult Casq22 and placed on a heating pad. Loss of toe-pinch reflex and respiration rate was used to monitor levels of anesthesia -loaded cardiac myocytes were field-stimulated at 1 Hz for 20 s to reach a constant Ca2+ transient height. All single cell experiments were conducted at room temperature. After 30 min exposure of Fura-2 AM loaded, quiescent cardiomyocytes to FLEC (6 μM) or vehicle (VEH), spontaneous Ca2+ waves were quantified during a 40 s pause after the 1 Hz pacing train in the presence of isoproterenol . At the end of the pause, myocytes were exposed to 10 mM caffeine for 5 s using a rapid concentration clamp system. SR Ca2+ content was estimated by the amplitude of the Ca2+ transient induced by rapid Caffeine application. Spontaneous SR Ca2+ waves (SCWs) were defined as any spontaneous increase of 0.07 ratiometric units or more from the diastolic Fratio. To calculate incidence of spontaneous Ca2+ releases (SCRs), cells that displayed a specific event  during the 40 s pause period were counted as positive, and then expressed as a percentage of total cells analyzed. To estimate the inhibitory concentration at which flecainide achieved 50% of the maximal effect (IC50) of Ca wave suppression, we used 0 and 1 as asymptotes for maximum and minimum inhibition and fitted the mean and SEM of three flecainide concentration data sets  to a Boltzmann Function using the OriginPro 9.0 software.Ventricular myocytes were isolated by a modified collagenase/protease method as previously described . All exp2+ waves were compared by non-parametric Mann–Whitney U test. Results were considered statistically significant if the p-value was less than 0.05.Differences among the groups were assessed using one-way ANOVA. The incidence of arrhythmia and spontaneous Ca2+ waves (−/− mice) (R4496C+/– mice) (2+ waves due to increased RyR2 activity at diastolic [Ca2+] (2+ wave suppression by flecainide in single Casq2−/− cardiomyocytes (2+ releases (SCRs) events were defined as either spontaneous Ca2+ waves or spontaneously triggered beats. SCRs were elicited by applying a pacing train in the presence of isoproterenol   and mice/– mice) . Single c [Ca2+] . To ensumyocytes . Spontanoterenol . Suppresoterenol . We then± 0.2 μM . Higher 2+ waves of ventricular cardiomyocytes isolated from Casq2−/− and RyR2R4496C+/– mice under identical experimental conditions  that reliably induces ventricular arrhythmias in Casq2−/− mice  in combination with isoproterenol. The incidence of ventricular arrhythmias after high-dose challenge increased to 75% in RyR2R4496C+/– mice. Taken together, these results indicate that the Casq2−/− mice have a more severe CPVT phenotype compared to RyR2R4496C+/– mice, which mirrors the results from the single cell studies  and caffeine (120 mg/kg). However, in Casq2−/− mice, a much smaller dose of epinephrine (0.5 mg/kg) as a single agent was sufficient to induce PVC’s and non-sustained VT in 100% of mice tested  to achieve a robust level of arrhythmias in both animal models. Flecainide completely suppressed PVCs in RyR2R4496C+/– and strongly reduced PVCs in Casq2−/− mice. These findings further support the hypothesis that flecainide is effective in reducing arrhythmias independent of the causative mutation  in permeabilized cardiomyocytes under experimental conditions that render Na+ channels inactivated  may have contributed to the lack of flecainide activity on Ca2+ waves in some reports.Our group has attributed flecainide’s striking efficacy against spontaneous Cahibition . Althoug+ sparks . The hypctivated . In cont2+ waves . Hence, All datasets generated for this study are included in the manuscript and/or the supplementary files.All protocols were following the Guide for the Care and Use of Laboratory Animals  and approved by the Vanderbilt University Institutional Committee of Ethics in Animal Research.in vivo mouse ECG experiments. MB and HH wrote the initial draft of the manuscript. JR re-analyzed ECG recording and prepared the figures. HH and BK designed and supervised the overall project, and prepared the final manuscript and figures.HH conducted and analyzed the isolated myocyte experiments. MB and MF conducted and analyzed the 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.


The partner of activated epidermal growth factor receptor (EGFR), growth factor receptor bound protein-7 (Grb7), a functionally multidomain adaptor protein, has been demonstrated to be a pivotal regulator for varied physiological and pathological processes by interacting with phospho-tyrosine-related signaling molecules to affect the transmission through a number of signaling pathways. In particular, critical roles of Grb7 in erythroblastic leukemia viral oncogene homolog (ERBB) family-mediated cancer development and malignancy have been intensively evaluated. The overexpression of Grb7 or the coamplification/cooverexpression of Grb7 and members of the ERBB family play essential roles in advanced human cancers and are associated with decreased survival and recurrence of cancers, emphasizing Grb7′s value as a prognostic marker and a therapeutic target. Peptide inhibitors of Grb7 are being tested in preclinical trials for their possible therapeutic effects. Here, we review the molecular, functional, and clinical aspects of Grb7 in ERBB family-mediated cancer development and malignancy with the aim to reveal alternative and effective therapeutic strategies. GRB7 gene encodes a 532-amino acid protein [Through studies of binding targets of tyrosine phosphorylated EGFR, growth factor receptor bound protein 7 (Grb7), a 535-amino acid multidomain adaptor protein, was initially identified as an EGFR binding partner . Of note protein ,2. Funct protein . Through protein ,7.GRB7 gene is located on chromosome 17q12, in close proximity to the cancer-associated ERBB2 gene, which has been identified as an ERBB2 amplicon [GRB7 and ERBB2 are highly increased in many human cancers [ERBB2 gene amplification and ERBB2 oncoprotein expression in invasive cancers [Additionally, the human amplicon , suggest cancers ,10. More cancers .This review will provide an overview of the basic properties and regulatory mechanisms of Grb7, as summarized in The N-terminus of the Grb7 protein contains a proline-rich region with five minimal consensus PxxP motifs for Src homology region 3 (SH3)-mediated protein–protein interactions . HoweverCaenorhabditis elegans [Caenorhabditis elegans, suggesting an important role of Grb7 in cell migration [The GM region possesses approximately 300 amino acids with 50% amino acid identity between the Grb7 adaptor protein family and Mig-10 of  elegans . Functioigration ,14. Therigration ,15.Sequence analysis identifies the structural conservation of the RA domain in the Grb7 adaptor protein family , suggestThe PH domain has been shown to govern cellular signaling events, such as vesicle trafficking or cytoskeletal organization , and theThe BPS/PIR region of Grb7 has been shown to interact with the activated insulin receptor in the SH2 domain of Grb7 dependent and independent interactions , implyinFor the spatial and temporal effects of Grb7 on the regulation of multiple signal transduction cascades, the SH2 domain of Grb7 functions in a critical role for Grb7 by binding to activated receptor tyrosine kinases/nonreceptor tyrosine kinases or other tyrosine-phosphorylated signaling proteins . The SH2Grb7 can be tyrosine phosphorylated by various kinases, including EGFR , EphB1 , Tek/TieSimilarly, a specific tyrosine residue (Tyr-1100) of Tek receptor tyrosine kinase functions as a functional docking site for binding to Grb7, whereas the mutated Tyr-1100 of Tek abolishes the Grb7-binding ability and tyrosine phosphorylation of Grb7 by Tek . On the In addition to the functional effects of receptor tyrosine kinases on Grb7, nonreceptor tyrosine kinase FAK also endows a critical impact on the tyrosine phosphorylation of Grb7. FAK is a critical upstream factor in Grb7-mediated signaling pathways that enable activation by growth factor receptors or integrins during cancer development . The binSerine/threonine phosphorylation has also been reported to be involved in Grb7-mediated cellular functions. In response to EGF  or heregIn response to upstream signal activation and the recruitment of downstream signaling molecules, the subcellular localization of Grb7 has been investigated in the cytoplasm, focal adhesions, stress granules, and the nucleus ,38,43,44The dimerization or well-defined oligomerization of proteins is a common characteristic of signaling proteins. Although most SH2 domains exist mainly as a monomer, several studies have suggested that the SH2 domain of Grb7 can exist as a homodimer ,47,48. IGRB7 expression decreases the expression of the anti-apoptotic B-cell lymphoma-2 (BCL-2) and increases the expression of the pro-apoptotic BCL2-associated X, leading to a significant increase in apoptosis [Grb7- and ERBB family/Grb7-mediated signal transduction cascades have been reported to facilitate cancer cell survival through the regulation of mitochondria-associated apoptotic pathways . In contpoptosis ,50. Addipoptosis . Upon expoptosis . When thpoptosis . Additiopoptosis , indicatpoptosis . Subsequpoptosis , suggestGrb7 together with lapatinib, a dual tyrosine kinase inhibitor that blocks EGFR and ERBB2 signals, contributes to the inhibition of protein kinase B  activity and decreased cell viability [Moreover, several ERBB family-mediated signals contribute to Grb7-mediated cell survival as well as anti-apoptosis in cancer cells. For instance, EGF signaling stimulates an interaction in mitochondria between Grb7 and HS1 associated protein X1 (Hax1) isoform 1, an anti-apoptotic protein, and subsequently regulates cancer apoptosis by affecting caspase3-mediated Hax1 cleavage . Moreoveiability . Moreoveiability .GRB7 expression reduces cancer proliferation and anchorage-independent growth in ERBB2/Grb7-overexpressing breast cancers [Upregulation of oncogenic ERBB family-mediated signals stimulates cancer proliferation. The expression of Grb7, one of the most important mediators in ERBB family-mediated signaling, is significantly correlated with cell cycle progression and anchorage-independent growth ,39,42,54 cancers . MoreoveGRB7 expression has been indicated to attenuate ERBB2 and AKT phosphorylation in breast cancer cells, affecting ERBB2-mediated cancer growth in vivo [ERBB2 amplicon, including GRB7, leads to an additive effect on the decreased cell-cycle progression and cancer proliferation [GRB7 coamplified with ERBB2 in their contribution to cancer proliferation.On the other hand, knockdown of  in vivo . Notablyferation , emphasiThe protein stability, protein binding ability, and phosphorylation of Grb7 are also critical for Grb7- or ERBB family/Grb7- mediated cancer proliferation. Indeed, Grb7 protein stability modulated by the peptidyl-prolyl cis/trans isomerase Pin1 has been implicated in the regulation of cell cycle progression at the G2-M phase . AdditioGRB7 expression reduces gap closure migration of cancers in response to the EGF treatment [Grb7- or ERBB family/Grb7- mediated signal transduction cascades have been implicated in the promotion of cancer migration. Indeed, it has been shown that high Grb7 expressing cancer cells exhibit a higher migratory ability than low Grb7 expressing cells ,39,58. Mreatment . Howeverreatment . Additioreatment . In ERBBreatment . Beyond reatment  or FAK [reatment  enhancesreatment ,32. Intrreatment . Additioreatment .GRB7, which ablates MMP-9 expression [The coexpression of Grb7 with the ERBB family, especially EGFR and ERBB2, is significantly correlated with the invasion of advanced esophageal carcinomas . Moreovepression , attenuapression . Consistpression . Moreovepression . In a mopression ,7 as welpression . These sGRB7 and ERBB2 are key genes that predict the recurrence of breast cancer in clinical trials [Cooverexpression or coamplification of Grb7 and the ERBB family has been clinically investigated in human breast cancers ,63, cervl trials ,67. BaseClinical studies have indicated that upregulation of Grb7 is significantly correlated with the recurrence and low survival of patients with cancer ,63,68,69ERBB2 amplicon, where GRB7 is located on the chromosome next to ERBB2, the copy number and expression levels of genes within the ERBB2 amplicon, such as GRB7 and ERBB2, were highly increased in human breast cancers [ERBB2 gene amplification as well as ERBB2 overexpression in invasive breast cancer [+ breast cancers [By using comparative genomic hybridization microarray analyses of human chromosome 17q12 in the  cancers , gastric cancers , high gr cancers . Moreovet cancer . Cooveret cancer , suggestt cancer  and facit cancer ,70. Of n cancers , reflectIn clinical prognosis, metastasis is one of major risk factors affecting mortality in patients with diagnosed malignant cancers. In agreement with the clinical relevance of the Grb7 expression level in cancer malignancy, overexpression of Grb7 was significantly correlated with the lymph node-positive status of cancer metastasis . AdditioGRB7 RNA expression groups, respectively [GBR7 expression was correlated with resistance to neoadjuvant doxorubicin and taxane therapy [GRB7 and ERBB2 have been indicated as two key genes in the 21 gene recurrence score assay to predict the recurrence of tamoxifen-treated breast cancer [GRB7 expression is a highly significant predictor for recurrence after cancer treatment.Predictive factors of cancer drug response and recurrence are also important indicators during cancer treatment. In triple-negative breast cancer (TNBC) patients treated with standard adjuvant anthracycline and taxane therapy, the five-year recurrence rates were 10.5% and 20.4% in the low and the high ectively . In addi therapy . The expt cancer ,67,71. TIn agreement with the importance of the SH2 phosphotyrosine binding site of Grb7 in Grb7-mediated signal transduction cascades and cancer malignancy, the specific Grb7 peptide targeting the SH2 phosphotyrosine binding site of Grb7 efficiently blocks Grb7-mediated signal transduction cascades. Using phage display random peptide libraries as ligands to the SH2 domain of Grb7, Pero et al. found that peptides containing a nonphosphorylated Tyr-X-Asn (YXN) motif exhibited high specificity and binding affinity to the SH2 domain of Grb7 . In contGRB7 in ovarian cancers [GRB7 [GRB7 knockdown ERBB2+ breast cancer, suggesting the effects of Grb7-mediated EGFR activation on the malignancy of ERBB2+ breast cancer [+ breast cancer [Understanding the detailed regulatory mechanisms of Grb7 in cancer development can provide alternative methods for cancer therapy. Recently, a study indicated a negative correlation between miR-193a-3p and  cancers . miR-193rs [GRB7 , suggestt cancer . Taking t cancer .+ breast cancer treated with two approved therapies targeting ERRB2, Herceptin and lapatinib, tend to experience relapses or malignant outcomes [GRB7 in ERRB2+ breast cancers [As numerous studies have illustrated, Grb7 amplifies oncogenic signals to promote cancer development, thereby highlighting that targeting both Grb7 and Grb7-amplified oncogenic signals provides a synergistic therapeutic effect on anti-cancer therapy. Clinically, patients with ERBB2outcomes , indicatoutcomes ,63,68,69 cancers . Additio cancers . TogetheGRB7 and ERBB in the prediction of the recurrence of breast cancer has been validated in a clinical trial. Additionally, shRNA or peptides targeting Grb7 expression or the SH2 phosphotyrosine binding site display promising anti-cancer effects in preclinical studies. Since the Grb7 protein interacts with several oncogenic protein tyrosine kinases, especially the ERBB family, combination therapy with peptides targeting Grb7 and protein tyrosine kinase-targeting agents/drugs can be viewed as an innovative therapeutic intervention. Of note, the detailed regulatory mechanisms, side effects, proper dose, tolerability, as well as treatment-induced resistant cancers against molecules targeting Grb7 or in combination with protein tyrosine kinase-targeting agents/drugs remains to be evaluated. Collectively, the molecular mechanisms and cellular functions of Grb7 in ERBB family-mediated cancer have provided valuable basic and clinical information for targeting Grb7 or Grb7-amplified oncogenic signals to improve the outcomes of human cancer cases.Grb7 as a functionally multidomain adaptor is essential for cancer development by binding to phospho-tyrosine-related oncogenic signaling molecules to amplify oncogenic signal transduction cascades. In particular, the multifunctional features of Grb7 have been emphasized as being involved in ERBB family-mediated cancer malignancy. However, the functional role and pathological significance of Grb7 in vivo, during the entire process of ERBB family-mediated cancer development, as well as tumor microenvironment formation need to be illustrated. Based on preclinical and clinical studies, the upregulation of Grb7 in numerous human cancers highlights Grb7 as a potential prognostic marker and a therapeutic target. Currently, the use of the expression of both


These mutations have previously been associated with elevated inositol trisphosphate receptor (IP3R) levels in skeletal muscle myotubes derived from patients. However, the functional relevance and the relationship of IP3R mediated Ca2+ signalling with the pathophysiology of the disease is unclear. It has also been suggested that mitochondrial dysfunction underlies the development of central and diffuse multi-mini-cores, devoid of mitochondrial activity, which is a key pathological consequence of RyR1 mutations. Here we used muscle biopsies of central core and multi-minicore disease patients with RyR1 mutations, as well as cellular and in vivo mouse models of the disease to characterize global cellular and mitochondrial Ca2+ signalling, mitochondrial function and gene expression associated with the disease. We show that RyR1 mutations that lead to the depletion of the channel are associated with increased IP3-mediated nuclear and mitochondrial Ca2+ signals and increased mitochondrial activity. Moreover, western blot and microarray analysis indicated enhanced mitochondrial biogenesis at the transcriptional and protein levels and was reflected in increased mitochondrial DNA content. The phenotype was recapitulated by RYR1 silencing in mouse cellular myotube models. Altogether, these data indicate that remodelling of skeletal muscle Ca2+ signalling following loss of functional RyR1 mediates bioenergetic adaptation.Core myopathies are a group of childhood muscle disorders caused by mutations of the ryanodine receptor (RyR1), the Ca Different mode of inheritance of mutations in the RYR1 gene and associated distinct clinico-pathological phenotypes are recognized: the dominantly inherited central core disease ; autosomal recessive multi-minicore disease , RYR1-related centronuclear myopathy (CNM) (Mutations of the type 1 ryanodine receptor (RyR1), a key sarcoplasmic reticulum (SR) Cahy (CNM) ,5. In sphy (CNM) . Accordi2+-mediated excitation-contraction (EC) coupling in skeletal muscle, significant research has focussed on the consequences of mutations on this process. The emerging picture divides the functional outcome into (i) defects in mechanical coupling in the triad structure and increased Ca2+ leak through RyR1 channels, mostly characterizing dominant mutations, and (ii) loss of RyR1 function, often associated with recessive mutations leading to reduced expression of RyR1 H redox state and increasing ATP production, long-term Ca2+ overload can lead to mitochondrial permeability transition  patient p7322 exhibiting CCD pathology due to a dominant mutation in the MH/CCD hotspot 3  and (ii) patient p3369 with MmD pathology with compound heterozygous mutations in regions outside of MH/CCD hotspots presumably resulting in truncated non-functional RyR1 protein (c.A4711G/p.I1571V + c.9407delT/p.L3136Rfs). For gene expression studies, in addition to these prototypic MmD and CCD patients, a further affected child and two adult CCD patients were used with dominant mutations in MH/CCD hotspots 1 and 3 . The localization of the mutations and the summary of the patient material is shown in In order to understand the consequences of RyR1 mutations in primary patient-derived tissues and differentiated myotubes, we have selected biopsies collected from patients with CCD and MmD with 2+ fluxes in patient cells carrying RyR1 mutations that underlie CCD and MmD pathologies, we first evaluated nuclear and cytosolic Ca2+ signals in primary differentiated myotubes obtained from muscle biopsies. Cells were loaded with the Ca2+-sensitive fluorescent ratiometric dye fura-2-AM. RyR1-mediated Ca2+ signals were induced by the nicotinic acetylcholine (ACh) receptor agonist carbachol  in the presence of the muscarinic ACh receptor blocker atropine  which activates the dihydropyridine receptor -RyR1 axis. IP3R-mediated Ca2+ release was triggered by the G-protein coupled receptor agonist ATP (100 μM). Myotubes were stimulated in the absence of extracellular (EC) Ca2+ (medium supplemented with 200 μM EGTA) (2+ (1 mM)   or in th+ (1 mM)  to discrchannels . Thus, i EC Ca2+ .2+ release in the cytosol which was reflected by a similar down-regulation when Ca2+ signals were quantified over the nuclear regions of interest (ROIs)  showed significantly increased nuclear Ca2+ signals compared to controls  , indicatpatients . The redmyotubes . While Rcontrols . The myo EC Ca2+ . To addrantibody . Importa2+ signalling results in increased mitochondrial Ca2+ uptake in myotubes from the patient with MmD (p3369), we transfected cells with 2mtGCaMP6m, a recombinant fluorescent Ca2+ probe specifically targeted to mitochondria  are shown in 2+ are shown in Since we have observed loss of RyR1 in both MmD and CCD patient-derived myotubes, in order to directly address the role of RyR1 in remodelling myotube CaRYR1 led to significantly enhanced IP3-mediated Ca2+ release, in both the cytosolic and nuclear compartments  are shown in 2+ are shown in 2+ uptake followed the changes seen in nuclear and cytosolic Ca2+ signals. In RYR1 siRNA-transfected mouse myotubes, ATP-induced, IP3R-mediated mitochondrial Ca2+ uptake was significantly increased compared with control cells, while the RyR1-mediated, CCh-induced signals were significantly reduced  in the CCD and MmD patient-derived myotube models. For these measurements, we used the membrane permeable cationic dye tetramethyl rhodamine methyl-ester at low concentration , and quantified average intensities in two distinct subcellular mitochondrial populations, in the perinuclear and sub-plasmalemmal (SP) regions, since in differentiated myofibres the proteomic composition and function of SP mitochondria differs from the interfibrillar population  following K+ stimulation  patient myotubes (data not shown). Altogether these data indicate that the rate of mitochondrial ATP synthesis is higher and make a greater contribution to cellular ATP demand in patient myotubes. Importantly, no differences between the recessive MmD and dominant CCD patient-derived myotubes were observed, suggesting that a shared feature, such as the loss of RyR1 is the underlying cause.In order to understand the functional consequences of the remodelled compartmental Capulation . In addimulation . Lower sprotocol . InteresRyR1 siRNA-transfected myotubes in the absence or presence of XeB, a specific inhibitor of IP3R-mediated Ca2+ release. As shown in RyR1 led to significantly increased basal respiration, which was prevented by treating cells with XeB. Increased respiration was specific differentiated myotubes  in C2C12 mouse myotubes using the Seahorse technology. We have compared OCR in control and myotubes , and wasmyotubes . No sign2+ signalling in at least two different ways. Direct IP3R-mediated Ca2+ transfer to mitochondria has been previously shown to contribute to maintenance and activation of the TCA cycle, respiratory activity and ATP production under both basal and stress conditions  or mitochondrial pathways  in C2C12 myotubes we co-transfected myoblast with 3 µM of the mitochondrial targeted probes 2mtGCaMP6m or Cepia3mt and 1 µM siRNA-RYR1 . Images acquisition rate was 1/s. Images were generated using a 475–415 excitation filter with exposure time set to 200 ms for 475 nm and 100 ms for 415 nm, emitted light was collected was collected through a 515/30 nm band-pass filter (Semrock). To measure mitochondrial Ca2+ levels in patient-derived differentiated myotubes we infected cells with an adenoviral 2mtGCaMP6m construct (1 ug/well in six-well plates) by adding the virus directly to the culture medium 36 h before performing the experiments. Imaging was performed using an Zeiss Axiovert 200 microscope equipped with a 63×/1.4 N.A. Plan Apochromat objective. Excitation was performed with a DeltaRAM V high-speed monochromator  equipped with a 75 W xenon arc lamp. Images were captured with a high-sensitivity Evolve 512 Delta EMCCD (Photometrics). The system was controlled by MetaMorph 7.5 (Molecular Devices) and was assembled by Crisel Instruments.Cells for imaging were seeded on 1% gelatin pre-coated 24 mm coverslips placed in six-well plates. Human-derived primary myoblasts were plated at a concentration of 2 × 104. 4 days after addition of differentiation medium, cells were infected with 1 µM adenoviral cytosolic-targeted aequorin (cytAEQ). The following day (5th day in differentiation medium) coverslips with infected cells were incubated with 2 μM coelenterazine for 2 h in KRB supplemented with 1 mM CaCl2, and then transferred to the perfusion chamber. All luminometer measurements were carried out in KRB and agonists were added to the same medium. Experiments were terminated by lysing the cells with 100 μM digitonin in a hypotonic high [Ca2+] solution (10 mM CaCl2 in water), to discharge the remaining aequorin pool. The light signal was collected and calibrated into [Ca2+] values by an algorithm based on the Ca2+ response curve of aequorin at physiological conditions of pH, [Mg2+] and ionic strength , 0.9% PMSF, 0.1% DTT, 9% phosphostop phosphatase inhibitor cocktail tablets, Roche 04906845001) supplemented with a cocktail of protease inhibitors (1%) (Sigma). Supernatants were collected after 25 min centrifugation at 12 000 rpm at 4°C. The total protein content was determined using the BCA protein assay . A total of 20 µg proteins were loaded on a Nu-page 3–8% tris-acetate precast gel (Lifetechnologies) immersed in tris-acetate SDS running buffer (Lifetechnologies). Running was set up as follows: for IP3R and RyR1 30 min at 50 V and 2 h at 70 V; for mitochondrial respiratory complexes and PGC1-α 1 h 30 min at 130 V. Wet transfer was performed 2 h onto nitrocellulose (IP3R and RyR1) or methanol pre-activated PVDF membrane (Biorad) . Membranes were blocked with 5% milk in TBS-Tween 1% for 1 h and incubated with primary antibodies overnight at 4°C in 1% milk in TBS-Tween 1%. We used the following antibodies: rabbit-IP3R , mouse RyR1 , mouse mitoprofile  rabbit PGC1-α , rabbit β-tubulin . Detection was carried out by incubation with secondary horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse IgG antibodies  for 1 h at RT. Proteins were visualized by the chemiluminescent reagent ‘super signal west pico chemioluminescent substrate’ . Densitometric analyses were performed by using ImageJ analysis software. We analysed by means of densitometric measurements of at least three independent experiments, normalized to β-tubulin.C2C12 siRNA 6 on 1% gelatin pre-coated 24 mm coverslips. After 5 days in differentiation medium, mitochondrial membrane potential (Δψm) was measured by confocal imaging. Cells were loaded with 4 nM tetramethyl-rhodamine-methyl-ester  for 30 min at 37°C in KRB (supplemented with 2 mM CaCl2) to measure Δψm at resting conditions (steady state) or loaded in a modified KRB with [NaCl] lowered to 70 mM and supplemented with 2 mM CaCl2 and 70 mM KCl. About 0.8 μM cyclosporine-H was also used in these experiments to reduce active TMRM extrusion. Z-stack images were acquired with a Leica Confocal SP5 microscope using a 100× immersion oil objective (Zeiss). TMRM was excited at 535 nm and emission was measured with a 560 nm LP filter. Eight bit images were taken every 20 s for 4 min with a fixed 1 s exposure time; laser power was set to 8%. Pinhole was set to 1.5 airy units and pixel size was set as 1024 × 1024 nm. ImageJ was used to quantify TMRM intensity on background-corrected images applying a constant threshold and quantifying the fluorescence intensity mean values in selected region of interests . After an equilibration step, basal OCR(pMoles/min) were recorded using 3 min mix, 2 min wait and 3 min measure (looped three times) cycles prior to the injection of 2 uM oligomycin to inhibit the ATP synthase. Three more measurement loops were recorded prior to injection of substrate plus 0.8 uM FCCP to induce maximal oxygen consumption. Following recording of three more measurement loops, rotenone 1 uM and 1 uM antimycin A  were injected to assess non-mitochondrial OCR. Stimuli prepared in MB (70 μl volumes) were pre-loaded and sequentially injected as indicated through ports in the XF24 calibration cartridge to final concentrations of 1 μg/ml oligomycin, 800 nM FCCP and 1 μM rotenone and antimycin-A. XeB (2 μM) was added to siRNA scr and siRNA RYR1-transfected cells 30 min prior to measurements, and was included in ports in the XF24 calibration cartridge to maintain final concentrations of 2 μM when injected. Since the number of differentiated cells in the culture was not feasible to determine, OCR values are expressed as pMoles/min/20 K seeded cells. As controls, non-differentiated myoblasts were treated and measured in the same way. Values were corrected to non-mitochondrial respiration, i.e. by subtracting OCR values after rotenone/antimycin-A addition.Low-passage C2C12 myoblast were seeded 20.000/well in a V7 XF 24-well cell culture microplate  in culture medium. Cells were transfected (as reported in the previous section) with 0.1 ug/well siRNA RyR1 (Sigma) and 0.5 ul lipofectamine 2000 (Invitrogen) per well and the following day culture medium was replaced with differentiation medium. After 5 days in differentiation medium, mature myotubes were gently rinsed in measurement buffer (MB) consisting of DMEM  8.3 g/l, sodium pyruvate 1 mM (1.1 g/l), glucose 25 mM (4.5 g/l), NaCl 33 mM (1.85 g/l), phenol red (15 mg/l), et al. . Enrichment analyses were performed using g: Profiler (https://www.broadinstitute.org/scientific-community/science/programs/metabolic-disease-program/publications/mitocarta/mitocarta-in-0) (http://bioinfogp.cnb.csic.es/tools/venny/index.html). Microarray data have been deposited in GEO (GSE103855).Total mRNA was extracted from 40 mg of frozen human or mouse muscle tissues using 1 ml trizol per sample using a tissue lyser . Gene expression studies were performed using Agilent technology with one-color (quick amp labelling) kit following the manufacturer’s instructions. Quantitative and qualitative analysis of total RNA were performed with NanoDrop spectrophotometers (Thermo Scientific) and Nanochip BioAnalyzer (Agilent). Human samples were analysed using the Human 4x44K V2 platform (Agilent) and data were normalized with the intra-array (Multiplicatively Detrended) and the inter-array (Quantile) methods. To evaluate the differences in transcript expression levels we used significance analysis of microarray (SAM) and we performed cluster analysis only on altered transcripts with false discovery rate (FDR) analysis of 0% using the complete linkage Euclidean method. EDL and soleus mouse samples were analysed using the Mus musculus 4x44K platform and data were normalized with the intra-array (Multiplicatively Detrended) and the inter-array (Quantile) methods. Differentially expressed transcripts were found using permutations of Profiler . To identa-in-0) , using Vhttp://biit.cs.ut.ee/gprofiler/help.cgi? help_id=21). Significant TFs were further analysed by listing all the genes ordered by how many TFs target them in the human patient and mouse model samples. In addition, we created heatmap plots of the genes ordered by this ranking and the TFs ordered by hierarchical clustering (see Putative TF-binding sites were determined using the TRANSFAC database (2015.3)  by a cuspost-hoc tests for multiple comparisons, or Student’s t-tests.Data are reported as mean ± S.E.M. Statistical differences were evaluated by ANOVA and HMG online.Supplementary DataClick here for additional data file.