All treated subjects were clinically evaluated according to Flynn’s criteria and Mayo Elbow Performance Score, and radiographically, including the measurement of the Baumann angle. Results: Clinically, Group 1, according Flynn’s criteria, had excellent cosmetic outcome in 32 subjects (94.1%). Mean MAYO Score was 96.0 ± 3.8. Group 2, according Flynn’s criteria, had excellent cosmetic outcomes in 23 subjects (92.0%). Mean MAYO Score was 97.8 ± 3.3. Radiographically, mean difference of Baumann’s angle between the injured limb and the normal limb was 5.5° ± 1.0° in Group 1 and 5.1° ± 1.1° in Group 2. Conclusion: Both supine and prone positioning achieved a satisfying outcome with similar results in joint function recovery and complications.Background: Supracondylar humerus fracture (SCHF) is a frequent injury in pediatric ages. Closed reduction and percutaneous pin fixation is a common treatment of displaced SCHF. Surgery is usually performed in the supine position; otherwise the prone position allows an easier fracture reduction and a safe placement of pins. The aim of study is to compare the clinical and radiographic results of the treatment of displaced SCHF, comparing two different intra-operative positionings. Methods: 59 SCHF affected children were retrospectively divided into supine (Group 1;  Supracondylar humerus fractures (SCHF) represent 3% of all pediatric fracture, the most common elbow fracture ,2,3 and The purpose of the study is to analyze and compare the clinical and radiological outcomes in SCHF affected patients treated with CRPP in supine or prone position.A retrospective medical record review (1st May 2005 to 31st July 2014) was performed, including children younger than 12 years of age who underwent CRPP for displaced SCHFs at our institution. The study was conducted according to the declaration of Helsinki.The inclusion criteria were as follows: (1) confirmed diagnosis of Gartland type III; (2) patient age under 12 years; (3) trauma in extension resulting from a fall on the palm of the hand with a hyperextended elbow; (4) CPRR treatment within 8 h according the “treatment protocol” described below; (5) at least 6 months of follow-up; (6) complete clinical and radiographic data.The exclusion criteria were: (1) poly-traumatized patients with other associated fractures; (2) open or pathological fracture and (3) No complete clinical or radiographic data. According to inclusion and exclusion criteria, 59 subjects were eligible for the study. Informed consent was obtained by all patients.All patients were admitted through the emergency department with the following demographic and clinical data captured: gender, age at the time of trauma, mechanism of accident, involved side, presence or absence of associated neurovascular injury, and whether the fracture was closed or open.” In addition, the intraoperatively patient position was obtained from the medical records .Intra-operative positioning choice depended upon surgeon experience and fracture pattern .n = 34) and prone  (p > 0.05)Based on the patient position during the surgery, the samples were divided into groups: supine  . The cohAll subjects were treated within eight hours from presentation with a closed reduction and percutaneous pin fixation. After inducing general anesthesia, the arm was positioned at a “C” on the brightness amplifier plate. Intra-operative positioning could be supine or prone. According to the age of patients, closed reduction and fixation with 1.6 to 2 mm K-wire were used. The adopted configurations were cross-pins or lateral pins .After stabilization with wires, the children were immobilized with a simple posterior splint at 90 degrees of flexion. A clinical and radiographic evaluation was performed at 7 days from surgery. The wires were removed after 4 weeks, to start a rehabilitation protocol to restore the full range of motion of the elbow. All patients were assessed at 1, 3, 6, and 12 months and then every year.All treated subjects were evaluated clinically according to Flynn’s criteria  Table 2Table 2 at-test and Fisher’s exact test were used to compare age, follow-up, MEPS, range of motion for flexion-extension, supination-pronation, and modification of Baumann’s Angle means between the groups. The χ2-test was used to verify the homogeneity of the three groups based on gender and laterality—Flynn’s criteria. The selected threshold for statistical significance was p < 0.05. All statistical analyses were performed using the 2016 GraphPad Software . Continuous data are presented as means and standard deviations, as appropriate. The independent sample Student’s Group 1 was composed of 34 patients, 22 (64.7%) males and 12 (35.3%) females. The right side was involved in 18 (52.9%) cases, and the left side in 16 (47.1%). Mean age at time of surgery was 6.1 ± 2.8 (range 1.0–12.8), mean follow-up was 59.8 ± 15.8 months (range 24–79). Group 2 was composed of 25 patients, 12 (48%) male and 13 (52%) female. The right side was involved in 14 (56%) cases, the left side in 11 (44%). Mean age at time of surgery was 5.9 ± 2.3 (range 1.5–10.5). Mean follow-up was 59.9 ± 12.8 months (range 27–78) .Group 1, according Flynn’s criteria, had an excellent cosmetic outcome in 32 subjects (94.1%) and good in 2 (5.9%). The functional factor was satisfactory in 33 (97%) patients. Mean MEPS was 96.0 ± 3.8 (range 86–100). On examination at final follow-up, the range of motion in the treated arm resulted in values for flexion of 110.9° ± 14.3° (range 90°–135°), extension of 3.4° ± 1.8° (range 0°–10°), and supination to pronation of 84.9° ± 3.1° (range 81°–90°). Group 2, according Flynn’s criteria, had excellent cosmetic outcome in 23 subjects (92.0%) and good in 2 (8.0%). Functional factor was satisfactory in 100% of patients. Mean MEPS was 97.8 ± 3.3 (range 91–100). At the final follow-up, the range of motion gave a flexion range of about 113.6° ± 11.2° (range 94°–137°), extension of 2.9° ± 2.2° (range 0°–10°), and supination to pronation of about 86.2° ± 2.2° (range 84°–90°). Radiographically, mean difference of Baumann’s angle between the injured limb and the normal limb was 5.5° ± 1.0° (range 2.7°–6.7°) in Group 1 and 5.1° ± 1.1° (range 2.8°–6.6°) in Group 2. Minor complications encountered. Group 1: asymmetry of 0.8 cm in one (2.9%) case, ulnar nerve paresthesia in 2 cases (5.9%), which resolved spontaneously in about 2–3 months. Six degrees of varus deviation and 7.5° deficit of extension were reported in 1 case (2.9%). Group 2: mild hyperextension in one case (4%), local infection treated with antibiotic therapy in two cases (8%), spontaneous removal of K-wires in one case (4%). No other major complications were reported.Daily activities and sport participation were restored for all patients. Statistical analysis showed no significant differences between groups .According our data, supine and prone intraoperative positions are similar for functional and radiological outcomes. At the same time, no differences between both approaches were found, considering complication rate and anesthesia management. SCHFs were widely investigated in literature, but the management and the treatment are still debated; there is no common consensus among the pediatric orthopedics. Despite several societies and expert research committees ,20,21,22Timing of treatment, pin configuration and intraoperative patient position are the most controversial issues. In fact, to postpone surgery could significantly influence the necessity of open reduction approach, complication, or overall outcome . At the In 2018, the Italian Pediatric Orthopedic and Traumatology Society (SITOP) guidelines did not highlight the superiority of supine or prone position, considering radiation exposure, surgery length, reduction attempts, clinical and radiological outcome ,30, and Despite the SITOP recommendations, a multicenter study of the main Italian national pediatric trauma centers noted that only 53 out of 529 (10%) patients were surgically treated in prone position . In our p = 0.15) radiographic outcomes, according to modified Baumann’s angle. Hsuan-Kai Kao’s study [In our study, as in the series of Gurler et al. , both in’s study  showed tConsidering the equal clinical and radiological results, different authors have listed the advantages of prone intraoperative positioning, such as a simpler reduction, a more comfortable use of C-arm and a reduction in nerve injuries. In case of posterior displacement, the gravity force correctly positions the distal humerus in the frontal plane . MoreoveHowever, as well as the patient position, other parameters can influence the outcome. The fixation with elastic stable intramedullary nail (ESIN) after close reduction was described ,37,38. ESurgeon experience is another influencing factor for SCHFs-treated patient outcome ; TuomileThe limitationss of the study are its retrospective nature, small size, and the lack of objective measurements.In conclusion, regarding the surgical pinning of SCHFs, the supine intraoperative positioning is not superior to prone. In both groups, satisfying outcomes were achieved, with similar results in joint function recovery and complications. Surgeon experience is crucial in positioning choice.


HSP10 (1 µmol·L−1) was infused into isolated hearts before I/R without blockers. At 10-min reperfusion, the mitochondria were isolated and mitochondrial function was assessed. In a subset of experiments, freshly isolated mitochondria were directly incubated with HSP10 or 5–10-sP with or without 5HD or chelerythrine before in vitro hypoxia/reoxygenation. Infusion of 5–10-sP (n = 5) and HSP10 (n = 5) into isolated hearts before I/R improved mitochondrial ADP-stimulated respiration, ATP production and prevented mitochondrial ROS formation compared to the I/R group (n = 5); this effect was abrogated by 5HD and chelerythrine. In freshly isolated mitochondria with in vitro hypoxia/reoxygenation, HSP10 (n = 16) and 5–10-sP (n = 16) incubation prevented reductions of mitochondrial ADP-stimulated respiration (91.5 ± 5.1 nmol O2/min/mg PTN), ATP production (250.1 ± 9.3 μmol ATP/200μg PTN), and prevented mitochondrial ROS production (219.7 ± 9.0 nmol H2O2/200μg PTN) induced by hypoxia/reoxygenation . 5HD reduced the ADP-stimulated respiration in the HSP10 group (65.84 ± 3.3 nmol O2/min/mg PTN), ATP production (193.7 ± 12.1 μmol ATP/200μg PTN) and increased ROS in the 5–10-sP group (274.4 ± 21.7 nmol H2O2/200 μg PTN). Mitochondria are a target of the cardioprotection induced by 5–10-sP and HSP10. This protection is dependent of PKC and mKATP activation. HSP10 can act directly on mitochondria and protects against hypoxia/reoxygenation injury by mKATP activation.Humoral factors released during ischemic preconditioning (IPC) protect the myocardium against ischemia/reperfusion (I/R) injury. We have recently identified 10 kDa-heat shock protein (HSP10) and a fraction of small 5–10 kDa peptides (5–10-sP) in the coronary effluent of IPC-treated hearts and demonstrated their cardioprotective potential. We here used our isolated mitochondria model to characterize the impact of exogenous HSP10 and 5–10-sP on mitochondria function from myocardium subjected to I/R injury. Isolated perfused rat hearts were submitted to 30-min global ischemia and 10-min reperfusion. Before ischemia, isolated hearts were infused with saline or 5–10-sP, with or without a mitochondrial ATP-sensitive-K IPC cannsfusion . Thus, ansfusion .Mitochondria are listed as end-effectors of many cardioprotective maneuvers . Indeed,ATP) by 5–10-sP (ATP channels (KATP), being this MPTP inhibition and KATP activation related to cell survival  among the humoral factors released on coronary effluent from hearts submitted to IPC, as having cardioprotective effects . Further 5–10-sP . The actsurvival . Moreovesurvival . HSP10 isurvival . Regardisurvival . In addisurvival . Indeed,survival .Thus, the aims of the present study were  to evaluAll chemicals  were obtained from Sigma-Aldrich (USA) if not otherwise specified. All solutions were freshly prepared and filtrated . Wistar rats  were used following the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health  and the local Institutional Animal Care and Use Committee (100/16).−1: NaCl 118.0, NaHCO3 25.0, KCl 4.7, KH2PO4 1.2, MgSO4 1.2, CaCl2 1.25, and glucose 11.0) at 37°C and equilibrated with a gas mixture of 95% O2 and 5% CO2 (pH = 7.4). The perfusate temperature was held constant by a heat exchanger located next to the aortic cannula. A fluid-filled latex balloon was inserted through the left atrium into the left ventricle and connected to a pressure transducer and the PowerLab/400 System  for continuous measurement of the left ventricular pressure (LVP). Initial left ventricular end-diastolic pressure was set to 10 mmHg by gradual balloon inflation. Hearts were continuously immersed in 37°C Krebs–Henseleit buffer to avoid hypothermia and allowed to stabilize for 20 min before being subjected to the following protocols .Isolated perfused hearts were perfused with coronary effluent from IPC hearts for 15 min before 30 min of global ischemia followed by 10 min of reperfusion.The coronary effluent was filtered using an ultrafiltration membrane  with a cut-off for 10 kDa. The retained fraction was collected and resuspended in 150 ml of Krebs–Henseleit buffer solution. The ultrafiltrated coronary effluent was perfused on the same day of the collection. Isolated perfused hearts were perfused with the >10 kDa peptides fraction for 15 min before 30 min of global ischemia followed by 10 min of reperfusion. The fraction was perfused within 2 h, including ultrafiltration and pH stabilization.The coronary effluent was filtered using ultrafiltration membranes  with cut-offs for 5 and 10 kDa. respectively. The IPC coronary effluent was initially ultrafiltrated using the 10 kDa cut-off membrane. The permeated fluid was collected and again ultrafiltrated using the 5 kDa cut-off membrane. The retained was resuspended in 150 ml of Krebs–Henseleit buffer solution. Isolated perfused hearts were perfused with the 5–10 kDa small peptides fraction (5–10-sP) for 15 min before 30 min of global ischemia followed by 10 min of reperfusion. The fraction was perfused within 2 h, including ultrafiltration and pH stabilization.−1. Isolated perfused hearts were treated with 5HD for 5 min before 5–10-sP infusion and together with 5–10-sP infusion. The protocol was otherwise identical to that for 5–10-sP infusion.Hydroxydecanoate (5HD) was added to the perfusate at a final concentration of 10 μmol·L−1. Isolated perfused hearts were treated with Chel for 5 min before 5–10-sP infusion and together with 5–10-sP infusion. The protocol was otherwise identical to that for 5–10-sP infusion.Chelerythrine (Chel) was added to the perfusate at a final concentration of 10 μmol·L−1 (Isolated perfused hearts were perfused with HSP10 1 μmol·L−1  for 10 m−1): 250 sucrose, 10 HEPES, 1 ethylene glycol tetra-acetic acid (EGTA), pH 7.4 with 0.5% w/v bovine serum albumin (BSA), minced thoroughly using scissors, and then homogenized with a tissue homogenizer (Ultra-Turrax) using two 10 s cycles at a shaft rotation rate of 6,500 rounds per min. This homogenate was further homogenized with proteinase type XXIV (8 IU/mg tissue weight) using a Teflon pestle. The homogenate was centrifuged at 700 g for 10 min at 4°C. The supernatant was collected and centrifuged at 14,000 g for 10 min. The resulting pellet was resuspended in isolation buffer without BSA and centrifuged at 10,000 g for 5 min at 4°C. This procedure was repeated, and the pellet was resuspended in isolation buffer. The protein concentration of the isolated pellet was determined using a protein assay  by comparison to a BSA standard . This methodology was used to isolate the total fraction of mitochondria from cardiac tissue.Mitochondria isolation was performed as described previously . After 3−1: 125 KCl, 10 MOPS, 2 MgCl2, 5 KH2PO4, 0.2 EGTA with pyruvate (5 mmol·L−1), and malate (5 mmol·L−1) as substrates for complex I. The oxygen electrode was calibrated using a solubility coefficient of 217 nmol O2/ml at 37°C. For the measurement of complex I respiration, the mitochondria  were added to 1 ml incubation buffer. After 2 min of incubation, 1 mmol·L−1 ADP was added, and ADP-stimulated respiration was measured for 2 min. Afterward, the mitochondria were used to either measure complex IV respiration, and maximal uncoupled oxygen uptake in the respiration chamber or incubation buffer containing the mitochondria was taken from the respiration chamber to measure ATP production or extramitochondrial ROS concentration, respectively. Complex IV respiration was stimulated by adding N,N,N,N′-tetramethyl-p-phenylenediamine  plus ascorbate (3 μmol·L−1), which donates electrons to cytochrome oxidase via the reduction of cytochrome c. Maximal uncoupled oxygen uptake was measured in the presence of 30 nmol·L−1 carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazone (FCCP)  at 37°C during magnetic stirring in incubation buffer containing in mmol·Le (FCCP) .2 ml−1. Mitochondria (200 μg) were added to 0.5 ml hypoxic buffer. After 8 min of hypoxia, the O2-saturated incubation buffer (0.5 ml) was added to achieve reoxygenation for 2 min. After simulated hypoxia/reoxygenation or the respective normoxic time control (10 min), were added pyruvate (5 mmol·L−1) and malate (5 mmol·L−1) as substrates for complex I or succinate (5 mmol·L−1) as substrates for complex II. Mitochondria were stimulated with ADP and respiration was measured for 2 min. For the measurement of complex II respiration, after hypoxia/reoxygenation, Rotenone (1 µmol·L−1) was added. After 2 min of incubation, 1 mmol·L−1 ADP was added. Finally, the mitochondria were used to either measure complex IV respiration and maximal uncoupled oxygen uptake, or extramitochondrial ROS concentration, or ATP production. Simulated Hypoxia/Reoxygenation protocols on isolated mitochondria were performed as follows and shown in The buffer  was made hypoxic by the introduction of purified nitrogen until the oxygen concentration was <15 nmol O2-saturated incubation buffer (0.5 ml) was added to achieve reoxygenation for 2 min. After simulated hypoxia/reoxygenation, the substrates for complex I were given. Finally, the mitochondria were used to either measure complex IV respiration and maximal uncoupled oxygen uptake, or extramitochondrial ROS concentration, or ATP production.Mitochondria were added to 0.5 ml hypoxic buffer. After 8 min of hypoxia, the O2-saturated incubation buffer (0.5 ml) was added for 2 min (time control 10 min), and substrates for complex I were given. Finally, the mitochondria were used to either measure complex IV respiration and maximal uncoupled oxygen uptake, or extramitochondrial ROS concentration, or ATP production.Mitochondria were added to 0.5 ml oxygenated buffer. After 8 min, O−1 HSP10. After 8 min of hypoxia, O2-saturated incubation buffer (0.5 ml), again supplemented with 1 μmol·L−1 HSP10, was added to achieve reoxygenation for 2 min. After simulated hypoxia/reoxygenation substrates for complex I or Rotenone and substrates for complex II were given. Finally, the mitochondria were used to either measure complex IV respiration and maximal uncoupled oxygen uptake, or extramitochondrial ROS concentration, or ATP production.Mitochondrial proteins were added to 0.5 ml hypoxic buffer supplemented with 1 μmol·L−1 Chelerythrine. After 1 min, was added 1 μmol·L−1 HSP10. After 8 min of hypoxia, O2-saturated incubation buffer (0.5 ml), again supplemented with 1 μmol·L−1 HSP10, was added to achieve reoxygenation for 2 min. After simulated hypoxia/reoxygenation substrates for complex I were given. Finally, the mitochondria were used to either measure complex IV respiration and maximal uncoupled oxygen uptake, or extramitochondrial ROS concentration, or ATP production.Mitochondrial proteins were added to 0.5 ml hypoxic buffer supplemented with 10 μmol·L−1 5HD. After 1 min, was added 1 μmol·L−1 HSP10. After 8 min of hypoxia, O2-saturated incubation buffer (0.5 ml), again supplemented with 1 μmol·L−1 HSP10, was added to achieve re-oxygenation for 2 min. After simulated hypoxia/reoxygenation substrates for complex I were given. Finally, the mitochondria were used to either measure complex IV respiration and maximal uncoupled oxygen uptake, or extramitochondrial ROS concentration, or ATP production.Mitochondrial proteins were added to 0.5 ml hypoxic buffer supplemented with 10 μmol·L2-saturated incubation buffer (0.5 ml), again supplemented with 5–10-sP, was added to achieve reoxygenation for 2 min. After simulated hypoxia/reoxygenation substrates for complex I or Rotenone and substrates for complex II were given. Finally, the mitochondria were used to either measure complex IV respiration and maximal uncoupled oxygen uptake, or extramitochondrial ROS concentration, or ATP production.Mitochondrial proteins were added to 0.5 ml hypoxic buffer supplemented with 5–10-sP. After 8 min of hypoxia, O−1 Chelerythrine. After 1 min 5–10-sP was added. After 8 min of hypoxia, O2-saturated incubation buffer (0.5 ml), again supplemented with 5–10-sP, was added to achieve reoxygenation for 2 min. After simulated hypoxia/reoxygenation substrates for complex I were given. Finally, the mitochondria were used to either measure complex IV respiration and maximal uncoupled oxygen uptake, or extramitochondrial ROS concentration, or ATP production.Mitochondrial proteins were added to 0.5 ml hypoxic buffer supplemented with 10 μmol·L−1 5HD. After 1 min, 1 μmol·L−1 5–10-sP was added. After 8 min of hypoxia, O2-saturated incubation buffer (0.5 ml), again supplemented with 5–10-sP, was added to achieve reoxygenation for 2 min. After simulated hypoxia/reoxygenation substrates for complex I were given. Finally, the mitochondria were used to either measure complex IV respiration and maximal uncoupled oxygen uptake, or extramitochondrial ROS concentration, or ATP production.Mitochondrial proteins were added to 0.5 ml hypoxic buffer supplemented with 10 μmol·L−1 5HD or 10 μmol·L−1 Chelerythrine. After 8 min of hypoxia, O2-saturated incubation buffer (0.5 ml), again supplemented with 1 μmol·L−1 HSP10, was added to achieve reoxygenation for 2 min. After simulated hypoxia/reoxygenation substrates for complex I were given. Finally, the mitochondria were used to either measure complex IV respiration and maximal uncoupled oxygen uptake, or extramitochondrial ROS concentration, or ATP production.Mitochondrial proteins were added to 0.5 ml hypoxic buffer supplemented with 1 μmol·L® M3, Molecular Devices, EUA) at 560 nm emission wavelength.After the measurement of ADP-stimulated respiration, the incubation buffer containing mitochondria was taken from the respiration chamber and immediately supplemented with the ATP assay mix (diluted 1:5). Mitochondrial ATP production after each respiration measurement was determined immediately and compared with ATP standards using a 96-well white plate and a spectrofluorometer . The integrity of the mitochondrial membrane was assessed by osmotically induced volume changes of the mitochondria and spectrophotometric determination of the apparent absorption of the suspension at 540 nm. A mitochondrial suspension (100 μg/ml) was added to the respiration medium in the absence of respiratory substrates, at 37°C and under constant stirring. The mitochondrial turgor was stimulated with 100 nmol·L−1 calcium. The swelling was expressed as a percentage of the absorption of the solution containing mitochondria in the presence of cyclosporin A , in relation to that absorbed after the addition of FCCP . For mitochondrial transmembrane potential (Δψm) determination the probe TMRM  was added in incubation solution containing 100 μg/ml of mitochondria for 1 h. The Δψm was estimated by fluorescence emitted by TMRM under 580 nm excitation. The Δψm was expressed as the percentage of fluorescence emitted by TMRM labeled mitochondria in the presence of cyclosporin A , relative to that emitted after the addition of FCCP to fully depolarize the mitochondria .The mitochondrial swelling and transmembrane potential were evaluated using a spectrofluorometer  at 540 nm emission and 580 nm extinction wavelengths.The Amplex Red Hydrogen Peroxide Assay Kit  was used to determine extramitochondrial ROS concentration. Amplex Red reacts at 1:1 stoichiometry with peroxides under catalysis by horseradish peroxidase (HRP) and produces highly fluorescent resorufin. The incubation buffer containing mitochondria was removed from the respiration chamber and immediately supplemented with 50 µmol·Lpost hoc tests. P < 0.05 was considered statistically significant.Data are presented as the mean ± standard error of the mean (SEM). Data were analyzed by Prism 6.0 software  using two-way ANOVA for repeated measures (Left ventricular developed pressure and coronary flow) and one-way ANOVA . When a significant difference was detected, one-way ANOVA was followed by Bonferroni The baseline left ventricular developed pressure was not different among the groups. Control hearts did not show LVDP oscillations during the 40-min normal perfusion. During the preischemic preconditioning, the LVDP values were similar among the groups. The preconditioned groups IPC, coronary effluent, 5–10-sP, and HSP10 presented a greater post-ischemic recovery of LVDP than I/R, > 10 kDa, 5–10-sP + 5HD and 5–10-sP + Chel  and indirect (in perfused isolated rat hearts) exposure. Although, it remains unclear how HSP10 directly protects the mitochondria against hypoxia/reoxygenation injury, our data suggested that this effect could be mediated by Kas BCL-2 . Furtheras BCL-2  and mitoas BCL-2 . Mitochoas BCL-2 . Furtheras BCL-2 . This isas BCL-2  and direas BCL-2 . A previas BCL-2 . Howeveras BCL-2 , consequnerators .ATP channels in the mechanisms by which the HSP10 can protect the mitochondria against injuries by hypoxia and reoxygenation. The activation of these channels creates an influx of potassium (positive charges) into the mitochondrial matrix  generated an increase in the production of mitochondrial ROS, however, this increase seems to be unrelated to ROS formation in protective amounts, but in an excessive manner that contributes to irreversible injury  in an isolated heart model treated with HSP10 represents a limitation in clarifying the pathways involved in mitochondrial protection conferred by HSP10. In fact, these experiments were not performed due to a large amount of HSP10 required for the execution of each experiment in an isolated heart model, since the synthesis, purification, and quantification of this amount of protein would take several months. The authors recognize a statistical limitation due to the repetition of some groups in different graphs regarding the assessment of mitochondrial function in hearts undergoing I/R.The lack of data using the PKC inhibitor (chelerythrine) and the blocker for KATP channel activation. Finally, this study shows the direct and indirect actions of HS P10 on mitochondrial function maintenance against I/R or hypoxia/reoxygenation injuries.In conclusion, our findings provide evidence that the improvement of mitochondrial function by preconditioned coronary effluent transfer is mediated by 5–10-sP humoral factors. This study also supports the mitochondria as an intracellular target of protection by 5–10-sP, and this protection is dependent on the PKC pathway and mitochondrial KThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by the Comissão de Ética no Uso de Animais (CEUA) in scientific experimentation of the Centro de Ciências da Saúde of the Federal University of Rio de Janeiro (100/16) and followed the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health .LM, AC, and JN: conception and design, acquisition of data, analysis and interpretation of data, drafting or revising the article, contributed unpublished essential data or reagents. DO and GM: acquisition of data, analysis, and interpretation of data. JN is the principal investigator. All authors discussed the results and commented on the manuscript.This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (grant 483639/2013-3 and grant INCT regenera 465656/2014-05) and the Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ) (grant E-26/112.085/2012).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.


Here, we report the draft genome sequences of nine bacterial isolates obtained after laboratory incubation of seawater, soil, and wastewater samples with polylactic acid, polyethylene, or polyethylene terephthalate film for 2 weeks. Assuming colonization as a prerequisite of degradation, these strains could contribute to a solution to the global plastic waste problem. Here, we report the draft genome sequences of nine bacterial isolates obtained after laboratory incubation of seawater, soil, and wastewater samples with polylactic acid, polyethylene, or polyethylene terephthalate film for 2 weeks. Assuming colonization as a prerequisite of degradation, these strains could contribute to a solution to the global plastic waste problem. OECD301 and OECD306 media contain 8.5 mg/liter KH2PO4, 217.5 mg/liter K2HPO4, 334 mg/liter Na2HPO4·2H2O, 5 mg/liter NH4Cl, 36.4 mg/liter CaCl2·2H2O, 22.5 mg/liter MgSO4·7H2O, and 0.25 mg/liter FeCl3·6H2O in either distilled water (OECD301) or seawater (OECD306) prepared with 2.5% sea salts (Sigma). Cultures were incubated in the dark at 16°C without shaking. After 14 days, the plastic pieces were transferred to fresh OECD301 or OECD306 medium and sonicated for 5 min. The samples were diluted 10-fold and plated onto OECD301 or OECD306 medium with 1.5% agar, 1% polypeptone, and 0.2% yeast extract. After incubation at 16°C for 7 days, colonies that were unique to each plastic type and different from the control samples were isolated by restreaking onto OECD301 or OECD306 1.5% agar plates with 1% polypeptone and 0.2% yeast and incubation for 4 days at 16°C.Plastic is omnipresent in our environment, and there is no sustainable recycling solution. Many pollutants can be degraded by microorganisms, and degradation is often induced by colonization of the material by the organism. Aiming to contribute a biological solution to the plastic problem, we isolated nine bacterial strains colonizing plastic. Environmental samples  were obtained from a wastewater treatment plant  on 14 September 2018, from a harbor  on 17 September 2018, and from a site near the university campus  on 11 October 2018. On the marine and terrestrial sides, plastic pieces (seawater plastic [SWP] and soil plastic [SP]) were also collected. Microbial communities were removed from plastic by sonication for 5 min, and all environmental samples were inoculated at a starting concentration of 10Genomic DNA was extracted using the NucleoSpin tissue kit . Five hundred nanograms of DNA per strain was submitted for sequencing at the Novo Nordisk Center for Biosustainability  using the NextSeq 500/550 midoutput kit v2 (Illumina) for 150-bp paired-end sequencing on a MiSeq Illumina platform. The sequence data were analyzed with KBase v1.8.9 . The reaPRJNA666993, and detailed information is listed in The genome assemblies have been deposited in GenBank under BioProject number


Three-dimensional (3D) organoid culture holds great promises in cancer precision medicine. However, Matrigel and stem cell-stimulating supplements are necessary for culturing 3D organoid cells. It costs a lot of money and consumes more time and effort compared with 2D cultured cells. Therefore, the establishment of cheaper and Matrigel-free organoid culture that can maintain the characteristics of a part of 3D organoids is demanded. In the previous study, we established a dog bladder cancer (BC) 3D organoid culture system by using their urine samples. Here, we successfully isolated cells named “2.5D organoid” from multiple strains of dog BC 3D organoids using 2.5 organoid media. The cell proliferation speed of 2.5D organoids was faster than parental 3D organoid cells. The expression pattern of stem cell markers was close to 3D organoids. Injection of 2.5D organoid cells into immunodeficient mice formed tumors and showed the histopathological characteristics of urothelial carcinoma similar to the injection of dog BC 3D organoids. The 2.5D organoids had a similar sensitivity profile for anti-cancer drug treatment to their parental 3D organoids. These data suggest that our established 2.5D organoid culture method might become a reasonable and useful tool instead of 3D organoids in dog BC research and therapy. It constitutes approximately 1–2% of all naturally occurring cancers in dogs, a nearly similar rate to humans3. The majority of dog BC is high grade invasive urothelial carcinoma5. In addition to the late diagnosis of dog BC, it is very difficult to treat them7. Although several experimental models of BC by using mouse and rat were developed10, they hardly reflect the characteristics of invasive or metastatic human BC11 and the mechanism of its arising and development still remains obscure. Interestingly, the naturally arising dog BC closely mimics invasive BC in human in the cellular and molecular features like histopathological architecture, local invasion, distant metastases, chemotherapy response, and prognosis7, suggesting that dog BC might be a pertinent model of invasive BC in human.Bladder cancer (BC), also known as transitional cell carcinoma (TCC) or urothelial carcinoma, is the most frequent form of urinary BC in dogs, affecting thousands of dogs annually worldwidein vivo architecture, functions, and genetic and molecular imprints of their original tissues. It holds great promise for use in medical research for establishing novel personalized therapies, especially cancer13.Three-dimensional (3D) organoid culture is derived from self-renewing stem cells that typically recapitulate the 14. The organoids recapitulated the tumor microenvironment of their parental tumor tissues and showed tumorigenesis in vivo. Furthermore, it could be used to investigate the sensitivity of anti-cancer drugs and their combinations. However, using the 3D organoid culture method for experiments costs a lot of money due to the expensive gel and the stem cell-stimulating supplements contained in the culture media. It also needs a long time because of the complicated handling (dissolving and solidifying gel) and the slow growth speed of the organoid cells compared with 2D cell lines. Although there is a need to establish a cheaper and easier-to-use organoid culture method, an alternative to the 3D organoid culture method has not been fully developed.In the previous study, we established the 3D culture method of dog BC organoids using the urine samples from BC diseased dogsIn the present study, we hypothesized that some media components enable patient-derived 3D organoid cells to culture in Matrigel-free 2D culture conditions without losing their characteristics such as marker expression, stemness, and drug sensitivity of their original 3D organoids (we named the cells 2.5D organoids). Here, we identified the suitable media components for culturing 2.5D organoids and established a new culture method of dog BC 2.5D organoids from different strains of dog BC 3D organoids. Since these 2.5D organoids partially exhibited similar characteristics to their parental 3D organoids, we expect that they could be useful as a new experimental tool not only in dogs but also in human BC research.To isolate 2.5D organoids from 3D organoids, we used a special 2.5D organoid media. The components were as follows: Advanced Dulbecco’s Modified Eagle’s Medium (DMEM)/F12  supplemented with 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid , 1% GlutaMax (Thermo Fisher Scientific Inc), 10 mM Nicotinamide , 1 mM N-acetyl-L-cysteine (Sigma-Aldrich), 0.5 μM A83-01 , 1% penicillin-streptomycin , and 5% fetal bovine serum . The difference of culture supplements between 2.5D and 3D organoid was shown in Table 14. Using our established 3 strains of BC organoids .In the previous study, we generated urine sample-derived dog BC 3D organoidsds Table , we gene2 incubator at 37 °C for 10 min. Thereafter, the EDTA solution was collected. Subsequently, 1 mL TrypLE Express solution (Thermo Fisher Scientific Inc.) was added to the cell pellets and they were incubated at 37 °C for 3–5 min. The trypsinized cell suspension was vigorously pipetted and collected into a 15 mL tube containing 100 μl FBS and then centrifuged at 600 g for 3 min. Cell pellets were mixed with new 2.5D organoid media and seeded into a 6 cm dish. We defined the passaged cells as early (4–8) and late passage (15–20) cells.After 70–80% confluent conditions, 2.5D organoid cells were passaged into a new 6 cm dish at a ratio of 1:3–4. To collect cells, 1 mL of 5 mM EDTA/PBS was added, and the culture dish was placed at COAfter the isolated 2.5D organoid cells were seeded into 6 cm dishes, they were cultured with 2.5D organoid media or 2D cell line media (DMEM containing 10% FBS and 1% PS) to compare the efficiency of cell attachment and proliferation after passaging. The bright-field images were obtained using a light microscope .15. Briefly, the 2.5D cells (2 × 105/well) were seeded on a coverslip in a 6-well plate. After 70% confluent conditions, the cells were fixed with 4% paraformaldehyde (PFA) solution for 10 min. Later, the cells were exposed to 0.2% Triton X/PBS for a few seconds. Blocking was done using 3% skimmed milk/PBS for 1 h. Subsequently, cells were treated with the primary antibodies  and kept at 4 °C overnight. After they were treated with the secondary antibodies and DAPI solution (1:1000) for 1 h at room temperature, the expressions were examined using a confocal microscope .Immunofluorescence staining of BC 2.5D organoids was performed as described before3 cells/well, each cell was cultured for 5 days. The number of living cells at day 1, 3 and 5 was evaluated by using a Prestoblue kit (Thermo Fisher Scientific Inc.). The fluorescence intensity was measured by a microplate reader  at an emission wavelength of 585 nm.After an equal number of 2.5D organoid cells at early and late passages and 3D organoid cells were seeded in 96-well plate at a density of 2×1014. Total RNA was extracted from 2 × 105 of 2.5D organoid cells at early and late passage, 3D organoids, and urothelial carcinoma cell lines by using a NucleoSpin kit  following the manufacturer’s protocol. First-strand cDNA was prepared using a QuantiTect Reverse Transcription Kit . Quantitative real-time PCR was done using a QuantiTect SYBR I Kit  and a StepOnePlus Real-Time PCR System . The ΔΔCq method was used for quantification. Specific primers used for dog BC stem cell markers, SOX2, CD44, and GAPDH were designed and are shown in Table Quantitative real-time PCR was performed as described previously15. Four NSG male immunodeficient mice (6 weeks old) were obtained from Japan SLC . They were housed under strict pathogen-free conditions. Here, 1×106 2.5D organoid cells were subcutaneously injected into the back of mice. Six weeks later, the 2.5D organoid-derived tumors were dissected under isoflurane anesthesia and used for H&E and immunofluorescence staining. All animal experiments in the present study were performed following the Guide to Animal Use and Care of Tokyo University of Agriculture and Technology and approved by the ethics committee .Mouse xenograft assay of 2.5D organoids was carried out as previously described15. After the isolated tumor tissues were fixed with 4% PFA for 24 h, they were embedded in paraffin. After deparaffinization, 4 µm-thick sections were stained with H&E. The images were obtained using a light microscope .H&E staining of 2.5D organoid-derived tumor tissues was performed as described previously14. Briefly, 1 × 103 cells of each 2.5D cells or 2 × 103 of their 3D organoid single cells were seeded in 96-well culture plates and incubated in a CO2 incubator at 37 °C for 24 h. The cells were treated with anti-cancer drugs including a microtubule inhibitor, vinblastine (0.01–10 nM), a topoisomerase inhibitor, mitoxantrone (0.1–100 ng/mL), or a DNA-damaging agent, carboplatin (0.1–100 µg/mL) for 72 h. Cell viabilities of the 3D and 2.5D organoid cells were examined in the same way as in the cell proliferation assay.Cell viability assay of the 2.5D organoid cells at early and late passages compared with their original 3D organoid ones was carried out as described beforeP-values ≤ 0.05.The obtained data were presented as means ± SEM. Statistical assessments were carried out using the one-way analysis of variance (ANOVA) method followed by Bonferroni’s test. Statistical significance was considered when 14. Our established BC 3D organoids expressed urothelial markers , showed the response to anti-cancer drugs, and exhibited tumorigenesis. In the present study, we hypothesized that some media components enable 3D organoid cells to culture in gel-free 2D culture conditions without losing their characteristics such as marker expression, stemness, and drug sensitivity. To prove the hypothesis, we made original media and checked whether the media efficiently generates 3D organoid-derived 2D cells (2.5D organoid) and can keep the characteristics . The 2.5D organoid cells (both early and late passages) expressed urothelial cell markers, CK7, CK20, and UPK3A Fig.  similar After seeding equal numbers of 2.5D organoid cells at early and late passages as well as their original 3D organoids, living cell number was evaluated by Prestoblue assay. The 2.5D organoid cells at early and late passages showed a significantly accelerated proliferation rate at day 5 compared with their original 3D organoids Fig. .To check whether a basal cell marker was expressed in 2.5D organoids, we investigated the expression of a basal marker, CK5. The 2.5D organoid cells expressed CK5 similarly to the parental 3D organoids Fig. .To check whether 2.5D organoids can revert to 3D organoids, we collected the generated 2.5D organoid cells and embedded them into Matrigel again. The 2.5D organoid cells were successfully reverted to 3D organoids Fig. , which wSince 3D organoids contain many stem cells compared with 2D cultured cells, we investigated whether BC 2.5D organoids maintain the expression level of stem cell markers as an indicator of stemness. Among 3D organoid, 2.5D early passage organoid, 2.5D late passage organoid, and urothelial carcinoma cell line, mRNA expression level of SOX2 was the highest in 3D organoid Fig. . In bothin vivo14. We therefore checked the tumorigenesis of the 2.5D organoids in vivo. After subcutaneous injection of the 2.5D cells into the back of immunodeficient mice, tumors of less than 1 cm in diameter were successfully formed after 6 weeks  2.5D organoid cells were successfully isolated from the different strains of 3D organoids by using our identified 2.5D organoid media  and UPK3A antibodies in humanlls Fig.  and xenolls Fig. , indicat45. CSCs are heterogeneous and composed of several phenotypes with different markers, which renders it extremely difficult to target in an individual patient. Therefore, identifying CSC-specific markers in each tumor is required for the establishment of tailored therapies46. Several CSC markers such as CD44, CD133, ALDH1, SOX2, EZH1, PD-L1, CD67LR, BCMab1, BMI1, MAGE-A3, and YAP149 were known to be expressed in BC. The transcription factor, SOX2 was found to be a CSC marker for both human and mouse BC and was absent in normal urothelial cells50. Additionally, SOX2 is associated with tumor progression and prognosis in urothelial carcinoma52. On the other hand, Chan et al. showed that 40% of more than 300 BC samples contained CD44+ cells and were able to form tumors in vivo 10–200 times than CD44- cells in mice53. On the contrary, the CD44+/CD24– cells were associated with a better prognosis, indicating that this phenotype is not suitable for detecting CSCs in dog mammary carcinomas54. Nevertheless, there is no data showing the expression of CD44 or SOX2 mediates the tumor progression in dog urothelial carcinoma. In the present study, SOX2 was upregulated while CD44 was downregulated in dog BC 3D and 2.5D organoids compared with the 2D urothelial carcinoma cell lines , the self-renewing and differentiating cells that mediate resistance to chemotherapy56, probably due to higher stemness and expression of multidrug resistance (MDR) proteins in the 3D conditions. In breast cancer, Imamura et al. assessed the difference in drug response between 2D and 3D culture of several breast cancer cell lines and primary cultured cells from a patient-derived xenograft and the patient’s original tumor tissues. They found higher resistance to paclitaxel and doxorubicin in the 3D cultured cells compared with the 2D cultured ones, probably due to the decreased expression of cleaved-PARP, cleaved-caspase-3, and Ki-67, greater G0-dormant subpopulation, and hypoxic condition57. In BC, Myeong et al. showed that the effect of rapamycin and Bacillus Calmette-Guérin (BCG) was more exaggerated in the 2D cell culture than that in the 3D cell culture environment58. Additionally, RT4 cells, a BC cell line cultured under 3D conditions also showed higher resistance to doxorubicin compared with 2D cultures59. On the other hand, our established 2.5D organoids showed similar responses to anti-cancer drug treatment compared with 3D organoids. Since the trend of stem cell marker expressions in 2.5D organoid cells is more close to 3D organoid ones compared with 2D cell lines as shown in Fig. The difference in the drug sensitivities between 2D and 3D culture conditions has been reported in different cancersin vivo and maintained the expression pattern of urothelial and bladder CSC markers. Furthermore, the 2.5D organoids showed a similar response to anti-cancer drugs with their parental 3D organoids. These results suggest that our established gel-free 2.5D culture method can be used as a cheaper and less time-consumed research model instead of 3D organoid studies to investigate the mechanisms of BC diseased dogs. It also provides new insights into the development of dog BC therapy.We for the first-time generated BC 2.5D organoids from 3D organoids. These 2.5D organoids showed constant passages and higher proliferation speed than their parental 3D organoids. The 2.5D organoids demonstrated the tumorigenesis


Schizosaccharomyces pombe cells, especially during S-phase in synchronized cultures. By visualizing individual DNA molecules, we observed shorter DNA fragments of newly replicated DNA in the PhenDC3-treated cells, suggesting that PhenDC3 impedes replication fork progression. Furthermore, a novel single DNA molecule damage assay revealed increased single-strand DNA lesions in the PhenDC3-treated cells. Moreover, chromatin immunoprecipitation showed enrichment of the leading-strand DNA polymerase at sites of predicted G4 structures, suggesting that these structures impede DNA replication. We tested a subset of these sites and showed that they form G4 structures, that they stall DNA synthesis in vitro and that they can be resolved by the breast cancer-associated Pif1 family helicases. Our results thus suggest that G4 structures occur in S. pombe and that stabilized/unresolved G4 structures are obstacles for the replication machinery. The increased levels of DNA damage might further highlight the association of the human Pif1 helicase with familial breast cancer and the onset of other human diseases connected to unresolved G4 structures.G-quadruplex (G4) structures are stable non-canonical DNA structures that are implicated in the regulation of many cellular pathways. We show here that the G4-stabilizing compound PhenDC3 causes growth defects in  G4 structures have high thermodynamic stability under physiological conditions  . These snditions , and thenditions ,4. Bioins . Thesenditions . Notablyreatment . The forreatment ,16, but reatment .in vitro polymerase stop assay performed in the presence of a G4-stabilizing small molecule combined with whole-genome sequencing, suggested the presence of >700 000 predicted G4 structures in the human genome , nucleolin is reported to bind to G4 structures in the c-MYC promoter of HeLa cells  experiments to determine the presence of G4 structures in vivo. Results from our ChIP-seq experiments performed at the optimal PhenDC3 concentration, where we used the catalytic subunit of the leading-strand DNA polymerase as a marker for slowed replication, identified genomic G4 sites that could impede replication fork progression. In vitro G4 assays confirmed that these sequences form G4 structures in the presence of KCl and that these predicted G4 structures cause stalling of DNA synthesis. In particular, the use of PhenDC3 improved our detection of G4 structures that are less stable. Furthermore, recombinant Pfh1  and YNS219 (MDRΔ) strains were grown overnight in liquid EMM2 media (Formedium). Starting with 2.4 × 106 cells/ml, 5-fold serial dilutions of the cells were made, and 5 μl of these dilutions were spotted on EMM2 agar plates containing 1.3% (v/v) DMSO or 50 or 100 μM PhenDC3. Plates were incubated at 30°C for 5 days. The determination of the optimal PhenDC3 concentration was performed by growing YJJ21 cells in liquid EMM2 media containing 0.03% (v/v) DMSO and 10, 20, 50 or 100 μM PhenDC3. Five-fold serial dilutions of the cells were spotted on EMM2 plates, and these were incubated at 30°C for 5 days. Each experiment was performed in triplicate. To determine the doubling time, exponentially growing YJJ21 cells were inoculated at 1 million cells/ml in EMM2 media containing 0.13% DMSO (v/v), 20 or 50 μM PhenDC3. Cultures were grown at 30°C at 180 rpm and counted after 12 h using a Bürker chamber. Next, the cells were diluted to 1 million cells/ml in the presence of fresh DMSO, or 20 or 50 μM PhenDC3, grown for additional 12 h, and counted to calculate the doubling time. Three independent experiments were performed to calculate the doubling time using the following formula: Doubling time = t / log2 (x /x0), where t is time in hours; x is counted cell number at 12 h and x0 is counted cell number at 0 h.PhenDC3 was synthesized as previously described . To testS. pombe cells (YIO4) were grown to 107 cells/ml in the presence of 20 μM PhenDC3 or 0.03% (v/v) DMSO at 25°C in liquid EMM2 media. Subsequently, the cultures were diluted twice and incubated at 36.5°C for 5 h to arrest the cells at the G2-phase. These cells were then released from the G2-phase by shifting the temperature to 25°C. At 30 min after release from the G2-phase, 66 μM final concentration of bromodeoxyuridine (BrdU) was added and the cells were allowed to incorporate the BrdU into their DNA for 35 min. A stop solution (250 mM EDTA (pH 8.0) and 0.16% sodium azide) was added, and cells were harvested by centrifugation and resuspended in cold 70% ethanol. The ethanol was removed by centrifugation, and the cells were washed in PBS and resuspended in digestion buffer ) for cell-wall digestion using 200 U/ml lyticase from Arthrobacter luteus (Sigma-Aldrich). Cells were pelleted and resuspended in 100 μl PBS. Spreading of DNA fibers on glass slides was performed as previously described , and 5 mM β-mercaptoethanol), their DNA was stretched along the slide by tilting the slide to 15° and fixing with 4% paraformaldehyde. The slides were incubated in 0.5 M NaOH for 25 min to denature the DNA. Immunostaining of BrdU incorporated into DNA was detected using rat anti-BrdU clone [BU1/75 (ICRI)] primary antibody (ABD Serotec) and goat anti-rat IgG Alexa Fluor 568 secondary antibody (Life Technologies), while ssDNA was detected using anti-DNA antibody, single-stranded, clone 16–19 primary antibody (Sigma Aldrich) and goat anti-mouse IgG2a (γ2a) Alexa Fluor 488 secondary antibody (Life Technologies). An Axio Imager Z1 microscope (Zeiss) was used to visualize the stained DNA fibers, and images of untangled DNA fibers were obtained at randomly selected fields of view. Only DNA fibers with BrdU labeling at intact ssDNA ends or DNA fibers with BrdU labels measuring >70 μm were selected for analysis using the Zen 2.6 blue edition (Zeiss) and ImageJ software packages. The experiments were repeated two times independently, and at least 17 images were taken for each condition in each experiment. DNA fibers were measured in micrometers and converted to kilobases using a conversion factor of 1 μm BrdU label corresponding to roughly 2 kb  DMSO. Cells were pelleted, washed with 1 ml distilled water, resuspended in 0.3 ml distilled water, and 0.3 ml 0.6 M NaOH was added and the cells were incubated for 10 min. After centrifugation, the supernatant was carefully removed and the cells were gently resuspended in 70 μl 2× sample buffer (100 mM Tris-HCl (pH 6.8), 4% (w/v) SDS, 0.2% (w/v) bromophenol blue and 200 mM β-mercaptoethanol). Proteins were separated on a 9% SDS-polyacrylamide gel and blotted onto a PVDF membrane. Cdc20-HA and Cdk1 were detected using anti-HA (Santa Cruz Biotechnology) and anti-Cdk1 (Abcam) antibodies, respectively. Pfh1–13Myc in YJJ16 cells was detected using anti-Myc (Takara Bio), while Pfh1 in YJJ21 cells was detected with anti-Pfh1. For the cellular fractionation experiments, the S. pombe strain (YJJ21) was grown in liquid EMM2 media for 12 h in the presence of 20 μM PhenDC3 or 0.03% (v/v) DMSO. Harvested cells were resuspended in ChIP lysis buffer (50 mM Hepes/KOH (pH 7.5), 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, and 0.1% Na-deoxycholate) and lysed using glass beads in a FastPrep-24™ homogenizer . Lysed cells were separated into the soluble fraction (supernatant) and insoluble fraction (pellet) by centrifugation at 20,900 × g for 15 min at 4°C. Fractions were treated with SDS sample buffer prior to SDS-PAGE and Western blot analysis.An alkaline extraction method described in  was usedS. pombe cells were grown in liquid PMG medium (Formedium) at 25°C. Cells were diluted to 1.5 × 106 cells/ml and grown for 30 min at 25°C prior to arresting the cells at late G2-phase by incubating for 5 h at 36.5°C. After 5 h, the cells were rapidly cooled down to 25°C in an ice bath to synchronously release them from the late G2-phase. The cultures were divided into different flasks and supplemented with 0.05% DMSO, or 20 or 50 μM PhenDC3. Samples for flow cytometry analysis were taken immediately after the cells were cooled down to 25°C (time 0), after 40 min, and then every 20 min until 240 min. For cell fixation, 2 ml of cell suspension (approximately 3 × 106 cells) was transferred into a 15 ml tube with 1 ml of stop solution (50 mM EDTA and 0.1% sodium azide) and incubated for 10 min. After centrifugation at 1000 × g for 5 min, the cells were resuspended in 700 μl of 70% EtOH and stored at 4°C. A total volume of 300 μl cells (1.5 × 106 cells) was washed in 3 ml of 50 mM sodium citrate (pH 8) and incubated overnight in 500 μl of 50 mM sodium citrate (pH 8) and 0.2 mg/ml of RNAseA (Thermo Fisher Scientific). The cells were then separated by sonication and mixed with 500 μl of staining solution (50 mM sodium citrate (pH 8) and 1 × Sybr Green I (Thermo Fisher Scientific)). The cells were incubated at room temperature for at least 30 min before analysis on a Beckman Coulter Cytomics FC500 flow cytometer. Data were smoothed in OriginLab software for better visualization.YJJ32 S. pombe cells (strain YJJ21) were grown in liquid EMM2 media for 12 h in the presence of 20 μM PhenDC3 or 0.03% (v/v) DMSO. Subsequent procedures for the ChIP experiments were performed as previously described , and the quality was assessed using FastQC (v.0.11.5) (REF: https://github.com/s-andrews/FastQC). Reads were mapped against the S. pombe reference genome ASM294v2 by Bowtie2 v.2.3.2 (https://github.com/taoliu/MACS/. The IDR (irreproducible discovery rate) pipeline from ENCODE was used to generate high-confidence peak lists from each pair of biological replicates, as well as to evaluate replicate reproducibility  using IDR (IDR < 0.01). To evaluate reproducibility, reads from each pair of replicates were merged and randomly subsampled into two pseudo replicates (pRep) using SAMtools. Reproducible peaks for the pseudo replicates (Np) were again detected using MACS2 and IDR and compared to the number of peaks in the original replicates (Np/Nt). An Np within a factor of 2 from Nt (Np/Nt <|2|) indicates reliable replicates according to ENCODE, and this condition was fulfilled for all of our replicates (Table www.ebi.ac.uk/ena) under accession number PRJEB37862.ChIP-seq data were trimmed using Trim Galore v.0.4.1)  sequences. Extension of the annealed primer was performed at 37°C for 1 min in the presence of either 1% (v/v) DMSO and 100 mM KCl or 0.1 μM PhenDC3 and 100 mM KCl using 0.063 U of an exonuclease-deficient Klenow fragment of Escherichia coli DNA polymerase (Thermo Fisher Scientific). Reaction products were separated on 10% polyacrylamide gels containing 8 M urea, 25% formamide, and 1 × Tris/borate/ethylenediaminetetraacetic acid (TBE). Bands were visualized and quantified using a Typhoon Scanner 9400  and the ImageQuant 5.2 software .The DNA primer extension assay was performed as described previously . BrieflyS. pombe genomic DNA, and 1 × SyGreen mix (PCR Biosystems) in the presence of either 0.65% (v/v) DMSO, 25 mM KCl, or 25 mM KCl and 0.5 μM PhenDC3. The reactions were performed on a LightCycler 96 thermocycler (Roche) using the following program: 95°C for 5 min (1 cycle) followed by a 2-step reaction of 85°C for 10 s and 60°C for 20 s (33 cycles) in one-point acquisition mode. ΔΔCq values were determined to express the relative DNA amplification.The qPCR stop assay was performed as described previously . BrieflyG4 oligonucleotides (50 μM) in water or buffer  were heated at 95°C for 5 min and allowed to fold at room temperature for 3 h, and 5 μM folded G4 oligonucleotides were incubated with 10 μM PhenDC3 or 1.3% (v/v) DMSO. CD spectra were recorded between 205 and 350 nm at 25°C using a JASCO-720 spectrometer with a Peltier temperature control in a quartz cuvette (0.1 cm path length). Each spectrum was the accumulation of four measurements. A buffer (10 mM Tris-HCl (pH 7.5) and 100 mM KCl) containing 1.25% (v/v) DMSO or 10 μM PhenDC3 was used as a blank for baseline corrections. All data were normalized to molar ellipticity by using the formula below:m° is CD signal in millidegrees; M is molecular weight of oligonucleotides in g/mol; L is path length of cell in cm; C is concentration of oligonucleotides in g/l.Where 32P-ATP were used to 5′ end label 0.5 μM 10A-G4 oligonucleotides or their mutated variants at 37°C for 75 min. Subsequently, 1 μl 0.5 M ethylenediaminetetraacetic acid (EDTA) was added and the reaction was incubated at 78°C for 1 min to inactivate the T4 PNK. Labeled DNA was purified on a G50 column .T4 polynucleotide kinase (PNK) (Thermo Fisher Scientific) and γ-To fold the G4 oligonucleotides, 20 μl of labeled 0.2 μM 10A-G4 oligonucleotides, or their mutated variants, was mixed with an equal volume 2 × folding buffer (20 mM Tris (pH 7.5) and 200 mM KCl). The reaction was incubated at 95°C for 5 min and allowed to cool down to room temperature for 3 h. The oligonucleotides were loaded on a 10% native polyacrylamide gel containing 50 mM KCl and separated in a cold ice box run at 100 V for 80 min.2 and 100 mM KCl were incubated for 10 min at 30°C with various concentrations of Pfh1 . For the helicase trap assay, 10 nM oligonucleotide complementary to the sequence of the G4 motifs was included in the reaction. The reaction was stopped by adding 4 μl 6 × stop solution (60 mM EDTA (pH 8.0), 40% (w/v) sucrose, 0.6% SDS, 0.2% bromophenol blue, and 0.5 mg/ml proteinase K) on ice. Reaction products were separated by electrophoresis using a 10% polyacrylamide gel containing 50 mM KCl. Bands were visualized and quantified using a Typhoon Scanner 9400 and ImageQuant 5.2 software. The amount of unwound DNA was calculated using the formula [% unwound = 100 × (P/(P + S))], where P is the pixel intensity of unwound DNA band and S is the background minus pixel intensity of the corrected intact DNA band.The Pfh1 G4 unwinding assay was performed as reported previously . BrieflyS. pombe cells were grown in 50 ml EMM2 media containing 0.066% (v/v) DMSO or 20 or 50 μM PhenDC3 for 16 h until reaching 10 million cells/ml. Genomic DNA was isolated using CHEF Genomic DNA Plug Kits (Bio-Rad) following the manufacturer’s protocol.YJJ21 Agarose plugs containing DNA isolated from either untreated or PhenDC3-treated cells were washed in 500 μl of Milli-Q for 1 h at room temperature with gentle shaking. The supernatant was discarded, and each plug was resuspended in 200 μl of Milli-Q. The agarose plugs were first melted at 55°C for 5 min and then incubated at 42°C for 5 min. After thermal equilibration of the plugs, 1 U of agarase (Thermo Fisher Scientific) was added and the samples were incubated at 42°C for 1 h. The agarase-digested plugs were stored at 4°C until further use. Before the labeling of DNA damage, the samples were heated at 55°C for 5 min.DNA samples (100 ng) were incubated with 2.5 U each of apurinic/apyrimidinic endonuclease 1 (APE1), formamidopyrimidine glycosylase (FpG), Endonuclease III (Endo III), endonuclease IV (Endo IV), and endonuclease VIII (Endo VIII) in 1× CutSmart Buffer (New England BioLabs (NEB)) for 1 h at 37°C. These enzymes are collectively referred to as the ‘enzyme cocktail’. All enzymes were procured from NEB. The PhenDC3 damage sites were labeled with dNTPs (Sigma-Aldrich) ) and DNA polymerase 1 (DNA pol 1) (1.25 U) at 20°C for 1 h in 1 × NEBuffer 2 (NEB). Finally, the reaction was terminated with 2.5 μl of 0.25 M EDTA (Sigma-Aldrich) and the samples were stored at 4°C until analysis.A total of 5 μl of the fluorescently labeled DNA was stained with 320 nM YOYO-1 (Invitrogen) in a final volume of 50 μl of 0.5 × TBE, and the samples were heated at 55°C for 15 min. One microliter of β-mercaptoethanol (Sigma-Aldrich) was added to the samples just before stretching to avoid photo bleaching of the samples while imaging. A total of 3.5 μl of each sample was then stretched on the silanized coverslips by placing the labeled DNA samples at the interface of the coverslip and a glass slide  . No. 1 cThe DNA molecules were stretched on the silanized coverslips and then imaged with a fluorescence microscope (Zeiss Observer.Z1) equipped with an Andor iXON Ultra EMCCD camera and a Colibri 7 LED illumination system. This set up has band-pass excitation filters (475/40 and 640/30) and bandpass emission filters (530/50 and 690/50) for YOYO-1 and Aminoallyl-dUTP-ATTO-647N, respectively. An EM gain setting of 100 and exposure times of 30 and 500 ms for YOYO-1 and Aminoallyl-dUTP-ATTO-647N, respectively, were used.A custom-made software that reports the damage as dots/μm was used to analyze the data. The data was then converted to dots/MBp using a conversion factor of 3000 bp/μm. The damage caused by PhenDC3 is reported in terms of damage detected (DD), which is the total number of sites detected by the enzyme cocktail.in vitro. Wild-type (wt) S. pombe cells are resistant to multiple drugs due to efficient efflux pumps that expel toxins out of the cell  strain. This strain is sensitive to a number of drugs  strain. At the restrictive temperature (36.5°C), cdc25–22 cells arrest at the G2-phase, and can then be released at the permissive temperature (25°C) and followed during the cell cycle in a synchronous manner. We therefore arrested the cdc25–22 MDRΔ cells at 36.5°C, released them in the presence of DMSO or 20 or 50 μM PhenDC3 at 25°C, and collected samples every 20 min for up to 3 h. Flow cytometry was used to monitor the cell cycle phase of these samples. While the cells treated with DMSO or 20 μM PhenDC3 entered S-phase at about 60 min after release and completed the cell cycle by returning to G2-phase after 180 min, the 50 μM PhenDC3-treated cells showed a slightly delayed and significantly prolonged S-phase, which was still not completed even at 180 min after release  strain with the MDRΔ cdc25–22, hENT1 and hsv‐tk background that was engineered to incorporate extracellular thymidine or its analogues into DNA through the nucleotide salvage pathway. These cells were grown in the presence of DMSO or 20 μM PhenDC3, arrested at G2-phase, and released at the permissive temperature. At 30 min after release, the thymidine analogue BrdU was added and allowed to be incorporated into the newly replicated DNA during early S-phase. The DNA from these cells was spread on microscope slides, immunostained with anti-BrdU and anti-ssDNA antibodies, and visualized by fluorescence microscopy. Cells treated with 20 μM PhenDC3 showed significantly reduced lengths of newly replicated DNA compared to cells without PhenDC3 treatment 3 G≥3  growth of Cdc20-HA MDRΔ was impaired at 50 and 100 μM PhenDC3, while PhenDC3 concentrations of 20 μM or lower showed no adverse effect on growth (P = 0.002). Based on these experiments, we grew the strain in the presence of 20 μM PhenDC3 (to reduce the possibility of secondary defects of PhenDC3-treatment) or DMSO (as the control) for the ChIP-seq experiments and then immunoprecipitated the DNA associated with Cdc20 and performed whole-genome sequencing. The input DNA used for ChIP was also sequenced and used as a control.To perform ChIP with Cdc20, we created a yeast strain (YJJ21) expressing Cdc20-HA  in the Mn growth . Furtherr = 0.97 and r = 0.99 in untreated and PhenDC3-treated samples, respectively) (Table P < 0.005). Next, we compared the overlap of these peaks with the 446 previously identified G4 motifs (Supporting File 2) (Each ChIP-seq experiment (PhenDC3-treated and untreated) was performed in duplicate. A high Pearson correlation coefficient between the duplicate samples indicated good concordance between the two replicates for both experimental conditions (y) Table . Using M File 2) . The num File 2) .S. pombe, both tDNA and 5S rDNA genes are dispersed throughout the genome  Table . Thus, iNext, we performed a more stringent discrimination of the identified peaks and focused on the 100 most-consistent Cdc20 peaks in the untreated and treated samples according to IDR (Supporting File 1). The number of Cdc20 peaks in the untreated and PhenDC3-treated samples that overlapped with G4 motifs were 13 and 22, respectively  identified in both treated and untreated ChIP-seq analyses that were not close to any tDNA or 5S rDNA genes, and performed several different o Figure . Twelve o Figure ,65,66. Ho Figure . To detein vitro.We first used circular dichroism (CD), which is a versatile and common technique for studying G4 structure formation and topology . DNA oliNext, we used native polyacrylamide gel electrophoresis (PAGE) to determine if the oligonucleotides formed intra- or intermolecular G4 structures. On a native gel, an intramolecular G4 structure that forms within a DNA molecule migrates faster due to its compact nature compared to its ssDNA counterpart, while an intermolecular G4 structure formed between two or more DNA molecules migrates slower due to its larger size ,71. Becain vitro and in vivo data suggest that the tested G4 motifs form G4 structures and that replication is slowed down in the presence of PhenDC3. To determine if the tested G4 structures slow down replication in vitro, we utilized the qPCR stop assay  sequence showed almost no inhibition of amplification of its DNA under any of the conditions . Compared to the non-G4 DNA, the templates containing G4 motifs showed reduced amounts of full-length products in the presence of KCl  . This coS. pombe, we measured the formation of DNA lesions in PhenDC3-treated MDRΔ cells using a DNA damage detection assay based on imaging of single DNA molecules (MDRΔ (YJJ21) cells . This can explain our surprising findings that PhenDC3-treated cells showed enhanced Cdc20-association to 5S rDNA genes and suggests that PhenDC3 binds and stabilizes these two stack-layered G4 structures, which results in slower replication fork movement. Formation of both canonical and non-canonical RNA G4 structures in human cells are reported, and these structures are implicated in either transcriptional or translational regulation  under accession number PRJEB37862.The sequencing data have been deposited at the European Nucleotide Archive (ENA, gkaa820_Supplemental_FilesClick here for additional data file.


The superfamily 2 helicase XPB is an integral part of the general transcription factor TFIIH and assumes essential catalytic functions in transcription initiation and nucleotide excision repair. The ATPase activity of XPB is required in both processes. We investigated the interaction network that regulates XPB via the p52 and p8 subunits with functional mutagenesis based on our crystal structure of the p52/p8 complex and current cryo-EM structures. Importantly, we show that XPB’s ATPase can be activated either by DNA or by the interaction with the p52/p8 proteins. Intriguingly, we observe that the ATPase activation by p52/p8 is significantly weaker than the activation by DNA and when both p52/p8 and DNA are present, p52/p8 dominates the maximum activation. We therefore define p52/p8 as the master regulator of XPB acting as an activator and speed limiter at the same time. A correlative analysis of the ATPase and translocase activities of XPB shows that XPB only acts as a translocase within the context of complete core TFIIH and that XPA increases the processivity of the translocase complex without altering XPB’s ATPase activity. Our data define an intricate network that tightly controls the activity of XPB during transcription and nucleotide excision repair. ERCC3 (XPB) or ERCC2 (XPD) genes can cause xeroderma pigmentosum (XP), trichothiodystrophy (TTD) and sometimes combined features of XP and Cockayne syndrome (CS)  gene, which are known to cause TTD (The ATPase activity of the superfamily 2 (SF2) helicase XPB is indispensable for two main cellular processes, transcription and nucleotide excision repair (NER) . XPB is ome (CS) . No diseause TTD .ERCC3 gene are located in domains other than its two RecA like domains (HD1 and HD2), which harbor all seven conserved helicase motifs  and two unique motifs named the thumb like motif (ThM) and the RED motif . CtXPB was inserted into both, the pBADM-11 vector (EMBL) containing an N-terminal hexa-histidine tag with a TEV cleavage site, and the pFastBac vector (Invitrogen) containing a C-terminal Twin-Strep tag and a 10×-histidine tag (The latter ctXPB was only used for the pull-down assays). Ctp44, ctp34, ctp8 and ctXPA were inserted individually into the pBADM-11 vector containing an N-terminal hexa-histidine tag with a TEV cleavage site. CtXPD was inserted into the pBADM-11 vector with or without an N-terminal hexa-histidine tag. Ctp62 and ctp52 were each inserted into the pETM-11 vector (EMBL) without tag. The constructs ctp52_1–321, ctp52_121–514 and ctXPB_60–345 were cloned utilizing the full length cDNA sequences via sequence and ligation independent cloning (6 cells/ml was reached followed by transfection with 10% (v/v) virus containing medium. Protein expression was allowed for 72 h at 27°C. CtXPB from the pBADM-11 vector was expressed in Rosetta™ 2 (DE3) cells (Merck Millipore). CtXPD was expressed in ArcticExpress (DE3) RIL cells (Agilent). Ctp62/ctp44, ctp52/ctp8 as well as ctp52/ctp34 were co-expressed in BL21 CodonPlus (DE3) RIL cells (Agilent). Ctp8, ctp52_1–321, ctp52_121–514 and ctXPB_60–345 were separately expressed in BL21 CodonPlus (DE3) RIL cells. CtXPA was expressed in LOBSTR BL21 (DE3) RIL cells (Kerafast). The E. coli cells were grown at 37°C in either Lennox broth  or Terrific broth  medium (Carl Roth) to reach a final OD600 of 0.6 or 1.2, respectively. For the seleno-methionine containing variants of ctp52_1–321 and ctp52_121–514 the cells were grown in M9 minimal medium supplemented with L-seleno-methionine at 37°C to a final OD600 of 0.6  was purified using a 5 ml StrepTrap HP column (Cytiva) followed by SEC using a HiLoad 16/600 Superdex 200 prep grade column. For SEC, the buffer contained 20 mM Hepes pH 8.0, 200 mM NaCl and 1 mM TCEP. CtXPB (expressed in E. coli), ctXPB_60–345, ctp52/ctp34, ctp52/ctp8, ctp52_1–321, ctp52_121–514 and ctp8 were purified via IMAC  and SEC using a HiLoad 16/600 Superdex 200 prep grade column (Cytiva). For ctXPB and ctp52/ctp34, the SEC buffer contained 20 mM HEPES pH 7.5, 250 mM NaCl, and 1 mM TCEP. For ctXPB_60–345, ctp52_1–321 and ctp52_121–514, the SEC buffer contained 20 mM Tris–HCl pH 7.5 and 250 mM NaCl. For ctp52/ctp8 and ctp8 the SEC buffer contained 20 mM Hepes pH 8 and 375 mM NaCl. All variants were expressed and purified as described for the wild type proteins. In case of ctp8, two versions were used. For the co-crystallization with ctp52_121–514, ctp8 containing an N-terminal hexa-histidine tag and a TEV cleavage site was used. For the other studies, the tag was removed by incubation with TEV protease after IMAC and the protein solution was subjected to another IMAC to remove the TEV protease and uncut protein prior to SEC. CtXPA was purified via IMAC and AEC using a MonoQ 5/50 GL column with buffers containing 20 mM Tris–HCl pH 8, 50/1000 mM NaCl and 2 mM TCEP.For purification of the ctXPD/ctp44/ctp62 complex, ctp44 and ctp62 were co-purified via immobilized metal affinity chromatography (IMAC) using Ni IDA beads (Macherey-Nagel) followed by size exclusion chromatography (SEC) via a HiLoad 16/600 Superdex 200 prep grade column (Cytiva) with 20 mM Hepes pH 7.5, 250 mM NaCl, and 1 mM TCEP. The elution fractions containing ctp44/ctp62 were pooled and directly added to the cleared lysate of ctXPD. This mixture was subjected to another IMAC, followed by SEC and anion exchange chromatography (AEC). For SEC, the same column and buffer as above was used. For AEC the mixture was applied to a MonoQ 5/50 GL column (Cytiva), with buffers containing 20 mM Hepes pH 7.5, 50/1000 mM NaCl, and 1 mM TCEP. To obtain the ctp44/ctp62 complex in the absence of ctXPD, ctp44 and ctp62 were purified via IMAC and SEC as described above, followed by AEC using a MonoQ 5/50 GL column with the same buffers as for the ctXPD/ctp44/ctp62 complex. To obtain ctXPD, the protein was purified via IMAC, followed by SEC and AEC. The SEC buffer contained 20 mM Hepes pH 7.5, 150 mM NaCl, 5 mM MgClAll proteins were concentrated to 50–1000 μM, flash frozen in liquid nitrogen, and stored at –80°C.Crystallization was performed via the vapour diffusion method at 20°C. For ctp52_1–321, a seleno-methionine derivative was crystallized at concentrations of 4–7 mg/ml. The reservoir solution consisted of 100 mM calcium acetate, 12.5% (w/v) PEG 8000, and 100 mM HEPES pH 7.5. For ctp52_121–514, a seleno-methionine derivative was crystallized at protein concentrations of 5–6 mg/ml. The reservoir solution consisted of 7.5% (w/v) PEG 4000 and 100 mM HEPES pH 7.0. For the ctp52_121–514/ctp8 complex, ctp52_121–514 at concentrations of 5–6 mg/ml was incubated for 1 h at 4°C with a twofold excess of ctp8 prior to crystallization. The reservoir solution consisted of 150 mM ammonium chloride, 15% (w/v) PEG 4000, and 100 mM Tris–HCl pH 8.5.X-ray diffraction data were processed with XDS . Diffracin vitro ATPase assay in which ATP consumption is coupled to the oxidation of NADH via pyruvate kinase and lactate dehydrogenase activities. Activities were measured at 30°C in 100 μl solution containing 1.3 U pyruvate kinase, 1.9 U lactate dehydrogenase , 1.6 mM phosphoenolpyruvate, 0.24 mM NADH, 10 mM KCl, 2.5 mM MgCl2, 1 mM TCEP, and 20 mM Tris–HCl pH 8. The assay was carried out under saturating concentrations of ATP using ctXPB wild type and ctXPB_K392R at a concentration of 250 nM. Ctp52, ctp52 variants, ctp8 and ctp34 were added in a twofold molar excess to ctXPB. When core ctTFIIH was assembled, all subunits were present at an equimolar ratio and a final concentration of 250 nM. CtXPA was added in a 4:1 stoichiometric ratio to ctXPB or core ctTFIIH. If not stated otherwise, double stranded (dsDNA)  was used at a final concentration of 125 nM for ctXPB or 1 μM for core ctTFIIH. The sample was preincubated until a stable base line was achieved. Enzyme catalysis was initiated by the addition of 2.5 mM ATP. The activity profiles were measured at 340 nm using a Clariostar plate reader (BMG Labtech) and 384-well F-bottom μClear™ microplates (Greiner Bio-One). Initial velocities were recorded and ATP consumption was determined using the molar extinction coefficient of NADH. Curves were fitted with GraphPad Prism. All measurements were carried out at least in triplicates, using at least two independently purified batches of the two helicases XPB and XPD as well as their Walker A variants. Example time courses are shown in CtXPB’s ATPase activity was quantified utilizing an 2, 16 mM HEPES–KOH pH 8, 4% (v/v) glycerol, 0.8 mM TCEP, 2.2 mM phosphoenolpyruvate, 1.8 U pyruvate kinase (Sigma-Aldrich) and 150 nM triplex DNA. The triplex DNA consists of a 52 bp dsDNA strand with a 5′ dabcyl label  and a 5′ Cy3-tagged TFO (5′-Cy3-TTCTTTTCTTTCTTCTTTCTTT-3′). The triplex DNA was annealed as previously described  were equillibrated with 100 μl buffer containing 150 mM NaCl, 1 mM TCEP and 20 mM HEPES pH 7.5. 2 μM Twin-Strep-tagged ctXPB was added and incubated for 45 minutes with the beads. 16 μM ctp52/ctp8 or ctp52_E359K/ctp8 were added and incubated for another 45 minutes. As control, 16 μM ctp52/ctp8 was incubated with the beads in the absence of ctXPB. Unbound protein was washed off with buffer and bound protein was eluted in 20 μl buffer supplemented with SDS loading dye and boiled for 5 min at 95°C. Elution fractions were analysed by SDS PAGE. The intensities of the ctp52/p8 gel bands were quantified using ImageJ and graphs were generated via GraphPad Prism. Three technical replicates were performed.2, 1 mM TCEP, and 6 nM DNA at room temperature. CtXPB was used at concentrations of 0.5–500 nM as indicated. Ctp52/ctp8 was added at a 2:1 stoichiometric ratio to ctXPB. Fluorescence polarization was detected at an excitation wavelength of 540 nM and an emission wavelength of 590 nM with a Clariostar plate reader (BMG LABTECH) using 384-well F-bottom Fluotrac high binding microplates (Greiner Bio-One). The gain was adjusted to a well containing buffer and DNA only. Curves were fitted with GraphPad Prism. Three technical replicates were performed.DNA binding was analysed by fluorescence polarization employing a duplex DNA with a Cy3 label (fw: 5′-Cy3-AGCTACCATGCCTGCACGAATTAAGCAATTCGTAATCATGGTCATAGC-3′ rv: 5′-GCTATGACCATGATTACGAATTGCTTAATTCGTGCAGGCATGGTAGCT-3′). Assays were carried out in 50 μl solution with 20 mM HEPES pH 7.5, 10 mM KCl, 2.5 mM MgClCircular dichroism (CD) spectra were recorded under constant nitrogen flush using a Jasco J-810 spectropolarimeter over 190–260 nm. Measurements were performed at 20°C in eight scans with a speed of 50 nm/min and a band width of 2 nm. The proteins were diluted to 4 μM in a buffer containing 19 mM dipotassium phosphate and 1.2 mM monopotassium phosphate at pH 8. All samples were centrifuged for 20 minutes at 30 000 g prior to the measurement. The buffer spectrum was used as a baseline and subtracted from all protein spectra.® Gel 30 (37.5:1) (Roth), 0.07% (w/v) APS, and 0.1% (v/v) TEMED. All samples contained 10 μM protein in 4 mM Tris, 16 mM HEPES pH 8, 350 mM NaCl. The samples were incubated on ice for 1 h, supplemented with 5× loading dye (0.08% (w/v) Ponceau S and 50% (v/v) glycerol) and loaded on the gel. Electrophoresis was performed at 4°C in a buffer containing 12.5 mM Tris and 96 mM glycine pH 8.9 for 90 min at 100 V followed by 3:45 h at 200 V. Protein bands were visualized by Coomassie G-250 staining.Native PAGE was performed using Tris/glycine gels consisting of 12.5 mM Tris, 96 mM glycine pH 8.9, 12% (v/v) ROTIPHORESE2, 8% (v/v) ROTIPHORESE® Gel 40 (29:1) (Roth), 0.1% (w/v) APS and 0.1% (v/v) TEMED. All samples contained 1 μM triplex DNA supplemented with 5 x loading dye. Electrophoresis was performed in a buffer containing 40 mM Tris pH 5.5, 40 mM acetate, 5 mM magnesium acetate, and 1 mM MgCl2 for 55 min at 100 V. DNA was visualized using a Pharos FX™ Plus imager (Bio-Rad).To confirm correct triplex DNA annealing, native PAGE was performed using Tris-acetate gels consisting of 40 mM Tris pH 5.5, 40 mM acetate, 5 mM magnesium acetate, 1 mM MgClChaetomium thermophilum (ctp52) using x-ray crystallography. Ctp52 shares 35% and 39% sequence identity with the human p52 and yeast Tfb2 sequences, respectively  led to phenotypic consequences when they are mutated . We thers ATPase . All ctps ATPase . Based os ATPase . The moss ATPase . All ctps ATPase , lane 2.s ATPase , lane 10s ATPase , shows as ATPase , lane 14s ATPase  were capSince the cryo-EM structure of holo-TFIIH suggests the presence of a second XPB/p52 interface located in the HD2 domain of XPB and the MD2 of p52 Figure  26) we  we 26) wVmax of 64.6 μmol ATP*l−1*min−1 , 140.0 μM (ctXPB/ctp52/ctp8), and 45.0 μM (ctXPB/ctp52/ctp8 with excess dsDNA). These values indicate a positive effect on the KM for ATP in the presence of dsDNA. However, the corresponding Vmax value of ctXPB with excess dsDNA (74.9 μmol ATP*l−1*min−1) is around three times higher than the Vmax of ctXPB/ctp52/ctp8/dsDNA (23.4 μmol ATP*l−1*min−1). The latter value fits well with the Vmax of ctXPB/ctp52/ctp8 without dsDNA (20.7 μmol ATP*l−1*min−1) indicating that ctp52/ctp8 predominantly affects the Vmax of the ctXPB/dsDNA complex. In addition, we calculated the kcat/KM ratios for ctXPB/ctp52/ctp8 and ctXPB/ctp52/ctp8/dsDNA with 0.7 μM ATP*min−1 and 1.8 μM ATP*min−1, respectively. Both are significantly lower than the kcat/KM ratio of the ctXPB/dsDNA complex with 4.8 μM ATP*min−1, indicating that also the catalytic efficiency is significantly decreased by p52/p8 when dsDNA is bound to XPB.We next aimed to investigate the influence of DNA binding on the ATPase activity of ctXPB. In the sole presence of double stranded DNA (dsDNA) the activity of ctXPB reached a max of the ctXPB/ctp52_E359K/ctp8 complex is not repressed and reaches XPB/dsDNA like activity. Ctp8 by itself also does not exert any effect . It seems that ctp8 in this case partially compensates for the presence of dsDNA since the maximum activity value of the ctXPB/ctp52/ctp34 complex with excess of dsDNA . These data indicate that no further activation of the ctXPB ATPase in the core ctTFIIH environment is achieved by the presence of the additional subunits. The ctXPD/ctp44/ctp62 complex displays an ATPase activity of 59.5 μmol ATP*l−1*min−1, rendering XPD the major ATPase in core ctTFIIH. Finally, we investigated core ctTFIIH lacking ctp8. Here, we observed an activity of 65.7 μmol ATP*l−1*min−1. This corresponds to a reduction of around 8 μmol ATP*l−1*min−1 when compared to core ctTFIIH containing ctp8. This reduction in activity is in line with the observed reduction in activation of XPB’s ATPase, which emphasizes the importance of ctp8 within core ctTFIIH  has no effect on the translocase activity .Interestingly, removal of ctp8 from the core ctTFIIH/ctXPA complex also decreases the translocase activity significantly to 1628 u rel. FC*min1 Figure . To our Finally, we addressed the question if the increase in translocase activity by ctXPA is due to the stimulation of XPB’s ATPase activity. Interestingly, ctXPA does not seem to influence ctXPB’s ATPase activity in any of the analysed conditions , thus exRecent structural data derived from cryo-EM studies have greatly improved our understanding towards the architecture of core TFIIH and its individual components . HoweverOur crystal structures of ctp52 show that p52 is a flexible protein that requires its binding partners XPB and p8 to assume a complete functional state. The high structural homology of the individual p52 domains but the different orientation of the domains relative to each other within our crystal structures compared to the cryo-EM TFIIH structures provides support for this hypothesis. We cannot exclude the possibility that the domain arrangement observed in our crystal structure is a crystallization artefact due to crystal packing. The modular composition of p52, however, may well be necessary to provide the flexibility to assume different conformational states thereby fulfilling TFIIH’s functions in transcription and NER ,44.C. thermophilum and the human proteins indicates a high functional homology of the two organisms that has been described previously  and is et al. . XPA intet al. . This coet al. . We thuset al. . Coin ett of XPA . Hence, t of XPA ,50.Our data show that XPB is a highly regulated protein. This regulation is embedded in several layers and could depend on the cellular process the protein is involved in. The maximum activation of XPB by DNA may only be important for other, less explored functions of XPB related to its localisation at the centrosome during mitosis or mRNA export ,52 and tXPB’s ATPase activity is crucial for NER and transcription initiation , howevermax of XPB’s ATPase activation by DNA and thus assumes the function of a speed limiter, which clearly dominates XPB’s regulation within TFIIH. Lastly, we show that despite its full ATPase capacity in isolation, XPB only acts as a translocase in the context of core TFIIH. Combined, our work redefines the regulatory network of the XPB ATPase and translocase shedding light on the inner regulatory mechanisms of core TFIIH in transcription and DNA repair.In conclusion, our analysis permitted us to decipher the tight regulatory network controlling XPB’s ATPase activity within TFIIH. We show in a stepwise approach how p52 and p8 activate XPB in an additive way. Importantly, this is independent from the activation of XPB by DNA. Despite being seen as an activator, the p52/p8 complex also limits the VAtomic coordinates and structure factors have been deposited in the Protein Data Bank under the accession codes 6TRS (ctp52_121–514/ctp8) and 6TRU (ctp52_1–321).gkaa911_Supplemental_FileClick here for additional data file.