These observations suggest that the interplay between PTP1B and Grb14 can regulate PIP metabolism. Protein tyrosine kinases and protein phosphatases play a critical role in cellular regulation. The length of a cellular response depends on the interplay between activating protein kinases and deactivating protein phosphatases. Protein tyrosine phosphatase 1B (PTP1B) and growth factor receptor-bound protein 14 (Grb14) are negative regulators of receptor tyrosine kinases. However, in the retina, we have previously shown that PTP1B inactivates insulin receptor signaling, whereas phosphorylated Grb14 inhibits PTP1B activity. In silico docking of phosphorylated Grb14 and PTP1B indicate critical residues in PTP1B that may mediate the interaction. Phosphoinositides (PIPs) are acidic lipids and minor constituents in the cell that play an important role in cellular processes. Their levels are regulated by growth factor signaling. Using phosphoinositide binding protein probes, we observed increased levels of PI(3)P, PI(4)P, PIP Protein tyrosine phosphatase 1B (PTP1B) and growth factor receptor-bound protein Grb14) are two negative regulators of insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF-1R) . We prev are two Grb14 is a pseudosubstrate inhibitor of the IR, which interacts with IR’s substrate binding site and selectively inhibits the IR kinase activity . Tissue-2, PIP2, PIP2, and PIP3  are minor constitutes of phospholipids . Phospho3,4,5)P3 . These PPolyclonal Grb14 antibody was generated as described . Polyclo2 asphyxiation and were used for immunoblot and phosphoinositide analyses. We followed the National Institute of Health (NIH) Guide for the Care and Use of Laboratory Animals and ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The Institutional Animal Care and Use Committee (IACUC) at the University of Oklahoma Health Sciences Center approved all protocols (Protocol #13-027). PTP1B KO mice were obtained from Dr. Benjamin Neel . We characterized these mice in our previous publication . Grb14 K2, PIP2, and PIP3 were measured by ELISA using PIP-binding protein probes [Mouse retinas were homogenized in phosphate-buffered saline (PBS) and the retinal phosphoinositides were extracted as described . Lipid pn probes . The obtn probes , calculan probes  in the eImmunoblot analysis was carried out as described . The bloFASTA sequences for mouse Grb14 (Q9JLM9) and mouse PTP1B P35821) were isolated from the UniProt Repository. Sequences were then processed using the SWISS-Model ,17, and  were isoTarget Preparation: The modeled structure of PTP1B from SWISS-Model was opened in AutoDock Tools. Polar hydrogens were added to PTP1B. The structure was then saved as a pdbqt file using AutoDock Tools. Ligand Preparation: The Grb14 structure from SWISS-Model was opened in PyMOL and the C-terminal tail of Grb14 was saved as a separate molecule. The PyMOL plugin PyTMs was used to add phosphates to Tyr ; the resulting pGrb14 and Grb14 peptides were saved as PDB files. The pGrb14 and Grb14 peptides were individually opened in AutoDock Tools; the torsion count was modified so that the peptide backbone bonds in the Grb14 ligand were non-rotatable, but pGrb14 was rotatable. This was due to desolvation penalties associated with the presence of phosphate groups on alpha-helices . Therefo2, and PIP3; class III PI3K product PI(3)P; PI4-kinase product PI(4)P; and PIPK1 type I and type II product PIP2. We found significantly increased levels of PI(3)P, PI(4)P, PIP2, PIP2, and PIP3 in PTP1B knockout mouse retinas compared with control littermates P2, and PIP3 compared with wild-type littermates  interacts with PTP1B, which results in the inhibition of its activity . The docWe previously reported that PTP1B deleted mice exhibited enhanced retinal neuroprotection through constitutive activation of retinal insulin receptors . In the An RMSD value of less than 2 Å may indicate the accuracy of the software. In AutoDock Vina, RMSD values are given by comparing multiple docking poses. However, the use of a macromolecule peptide generated only a few docking poses as opposed to a small ligand. Docking the unphosphorylated-Grb14 peptide gave only one docking pose , whereas the phosphorylated-Grb14 peptide gave two docking poses . The RMSD values between the docking pose for the phosphorylated Grb14 peptide was 3.9 Å. It has been well documented in the literature that peptides are more flexible than proteins and tend to adopt numerous conformations . Our in 2, PIP2, and PIP3, whereas loss of Grb14 decreased these PIPs in the retina. Together, these observations suggest that the interplay between PTP1B and Grb14 modulates the key enzymes involved in PIP metabolism. Insulin and IGF1 receptor activation are modulated by PTP1B and Grb14, which in turn regulate class I PI3K, which generates PIP2 and PIP3. The observed increase in PIP2 and PIP3 in PTP1B KO mouse retina and decreased levels of the PIPs in Grb14 KO mouse retinas suggest the negative and positive regulatory roles of PTP1B and Grb14 on insulin and IGF1 receptors or other tyrosine kinase receptors in the retina. Interestingly, we observed increased levels of PI(3)P, PI(4)P, and PIP2 in PTP1B KO mouse retina. Decreased levels of these PIPs in the Grb14 KO mouse retina suggest that the key enzymes involved in the synthesis and degradation of PIPs may also be regulated by the interplay between PTP1B and Grb14. Further studies are needed to confirm these observations. The Grb14 has a PH domain, and we previously reported that it can bind to PIPs in vitro [In line with our earlier observations, loss of PTP1B resulted in increased PI(3)P, PI(4)P, PIPin vitro . We spec3 in cones, resulted in age-related cone degeneration [Akt, a canonical pro-survival molecule downstream of PI3K, is constitutively active in cone photoreceptors . The addneration . Disruptneration .In the retina, there is an active PIP cycle that responds to light ,34. Both2 and PI P3. In the current study, we showed that two regulators of the receptor tyrosine kinase signaling, PTP1B and Grb14, modulate the levels of PIPs in the retina. Our studies also showed modulations in the levels of PI(3)P, PI(4)P, and PIP2 in PTP1B and Grb14 KO mouse retina, suggesting that the enzymes involved in the generation of these PIPs may also be influenced by the interplay between PTP1B and Grb14. Furthermore, our in silico docking of phosphorylated Grb14 and PTP1B indicate critical residues in PTP1B that may mediate the interaction.Phosphoinositide lipids play an important role in cellular processes. In cells, PIPs are formed by the action of phosphoinositide kinases and phosphoinositide phosphatases. Class I phosphoinositide 3-kinase activation via growth factor signaling enhances PIP


Icing is a hazard which is important for the aerospace industry and which has grown over the last few years. Developing sensors that can detect the existence not only of standard icing conditions with typically small droplet size, but also of Supercooled Large Droplet (SLD) conditions is one of the most important aims in order to minimize icing hazards in the near future. In the present paper a study of the Fiber Bragg Grating Sensors’ (FBGSs) performance as a flight icing detection system that predicts the conditions of an icing cloud is carried out. The test matrix was performed in the INTA Icing Wind Tunnel (IWT) with several icing conditions including SLD. Two optic fibers with 16 FBGS in total were integrated in the lower and upper surface of an airfoil to measure the temperature all over the chord. The results are compared with a Messinger heat and mass balance model and the measurements of the FBGS are used to predict the Liquid Water Content (LWC) and Ice Accretion Rate (IAR). Finally, the results are evaluated and a sensor assessment is made. A good correlation was observed between theoretical calculations and test results obtained with the FBGS in the IWT tests. FBGS proved to detect the beginning and end of ice accretion, LWC and IAR quickly and with good precision. Atmospheric icing occurs when aircraft fly through a cloud with supercooled droplets. Currently, there is a need for sensors that can detect and evaluate the hazard in a short time and in a feasible way. Actual sensors used in aircrafts (A/C) such as the magnetostrictive sort  have proSeveral Flight Icing Detection Systems (FIDSs) have been developed (Jackson ) but in Many heat transfer studies have been carried out in order to predict the ice accretion in airfoils. Messinger  did a heAn important issue of modelling heat transfer is the convective heat transfer coefficient calculation. On turbulent flow, the heat transfer coefficient depends on the equivalent sand-grain roughness height ,12. The In this work a model for the prediction of the icing cloud conditions, heat transfer coefficient and ice accretion rate is presented based on the models from the LEWICE software  and G. Fn) [Firstly, it is important to describe briefly all the components involved in the tests. One of them is the Fiber Bragg Grating Sensors (FBGSs) from the company FBGS INTERNATIONAL, manufactured in the so-called draw tower process coated with ORMOCER  and withEquation  shows a ndex (n) :(1)λBqevapThe evaporative term is only taken into account in case of glaze ice, because in rime ice there is not a water film that flows on the airfoil surface. The water vapour densities at the surface tin 2006 ).(8)ρvsThe convective mass transfer coefficient hg  is expreThe third term represents the sensible heat of water and ice. In this term, the sensible heat of water and of ice on the surface of the impinging  which iThe frictional gained heat flux is proportional to the difference between the recovery temperature For solving the energy balance equation, the collection efficiency and convective heat transfer coefficient must be calculated. Both parameters depend on the fluid field which is calculated with a CFD model . Once thThe air stream around the airfoil was modelled using OpenFoam. Normally, in ice accretion codes, a potential flow model is used for solving the airflow. Due to the anomalous airfoil used for the tests  a turbuleference . All theOne of the most important parameters in predicting ice accretion behaviour is the convection. Many efforts have been made to determine the values of the convective heat transfer coefficient Samad, A . For calEquation ). The retion (Ve :(20)hl=2s):The thermal boundary layer thickness can be expressed as a function of the surface distance from the stagnation point . The log et al.  and usinajectory .(23)β.The model algorithm resolution begins in the stagnation point node , so theSeveral tests at different temperatures and ordered in a test matrix were carried out in order to obtain repetitive results. The tests were planned to have outcomes in two types of ice—glaze and rime. All tests were recorded with the same time sequence for having a visual explanation of the sensor signals and for giving sense to the signal events recorded.The test matrix was designed with the goal of comparing the results with a constant parameter in different conditions of LWC and MVD. Each test matrix has constant ambient temperature and a fixed air speed of 70 m/s. Two matrices were tested, one at −5 The airfoil used in the tests is a NACA 0012, cut for not having a too large chord for testing in the icing tunnel. The dimensions of the airfoil are shown in In order to detect ice formation and to obtain information about the temperatures in different locations all over the chord, eight sensors were placed in the upper surface and another eight in the lower surface. Of those, four were placed in the leading edge, two in the upper and another two in the lower surface. Leading edge sensors have a big temperature step in the conditions of The temperature is stabilised in an equilibrium temperature before the fogging.In the 40th second the temperature rises abruptly due to the beginning of the icing cycle. It stabilizes in a new equilibrium temperature.In the 130th second the fogging stops and the temperatures drop abruptly.Every test consists of one icing cycle of approximately 90 s. An example of the signals of the tests can be seen in Several tests were carried out in order to validate the thermal model (Equation ). The fi. Fortin ), but tha), (b) and (c), the impinging limits are between −20 and 20 mm and in the test cases (g), (h) and (i) they are between −40 and 40 mm. This fact occurs because there are differences between the MVD of both (test cases (a), (b) and (c) have an MVD of 70 g), (h) and (i) cases have a MVD of 20 Another aspect to take into account is the similarity between There is a high accuracy of the model in rime ice with different angles of attack as well. For example, in Initially, it was observed that there is more water fluency in the surface of the airfoil than in the end of the test. This could produce a more intense water freezing in the areas further away from the leading edge in the beginning of the test and less water freezing in the area close to it. This fact can be corroborated in After a certain time, less water flowing was observed in the test, so there is more ice accretion in the area close to the leading edge. In Glaze ice results are less accurate because in the applied model the evaporative term causes different solutions depending on the relative humidity of the air. Another problem of glaze ice tests is a temperature drop during the nebulization . This coThe freezing fraction is the magnitude that measures the fraction of water that freezes as a function of the position. In the i as the nodes of the energy balance equation, it could be expressed as a function of the mass flux of impinging water, recovery temperature, specific heat of ice in the surface One of the advantages of using FBGSs in rime ice is the easiness of the convective heat transfer coefficient calculation. Considering the sensors’ positions The It is shown that FBGS can be a very useful tool in order to predict where the transition between laminar and turbulent flow is located. In this case, as can be seen in Once the test temperature has reached the equilibrium temperature , the temFor certain conditions, collection efficiency is higher than 0.8 so high LWC accuracy could be achieved. The conditions with high collection efficiencies were calculated using the method laid out in reference  and reprThe LWC prediction procedure consists in calculating the impinging mass flow in the stagnation point sensor using the impinging water mass flow Equation  and simpThe collection efficiency of the problem is unknown so a The prediction results can be seen in In terms of modeling, glaze ice has the problem that the stagnation point temperature is 0 essinger ), so usiquations  and 28)∘C, for wThe accretion rate was calculated using the heat balance Equation . In rimeCombining Equations  and 28)28), the This variable does not depend on the liquid water content, the stagnation collection efficiency or the freezing fraction, so the value is expected to be more accurate. The total temperature of the test is the leading edge sensor temperature before nebulization. In Using the ice accretion rate, it is possible to know approximately the ice geometry in the airfoil, applying Equation  in all sAn optic fiber with eight FBGSs was integrated in each of the upper and lower surfaces of a cut NACA 0012 aerodynamic profile in order to measure the temperature in sixteen points along its chord. An icing test campaign was performed in the INTA IWT, some of them in hazardous SLD conditions. Analytical and experimental results, using a Messinger heat and mass balance model and the FBGS measurements show a good correlation of the theoretical and empiric data. The icing prediction temperatures obtained with the integrated FBGSs along the chord are quite accurate near the leading edge, demonstrating that these optical sensors enable fast and precise detection of the beginning and the end of the icing hazard, the evaluation of the liquid water content and the ice accretion rate. Downstream from the leading edge an increment of errors occur that are caused by the aerodynamic turbulence. The LWC predictions obtained in the test campaign show a maximum error of 20% for Finally, it is important to note that the integrated fiber optic sensors can be used not only for the ice detection and icing condition evaluation in aircrafts, which is what they were initially developed for, but can help also to know and properly interpret some physical events during IWT tests. Knowing the surface temperature during an icing test can give much information about the heat transfer events and, therefore, the ice accretion in the airfoil surface in the first moments of the flogging cycles. An integration of the tiny fiber optic sensors in these IWT test models can be carried out normally without a significant influence in its aerodynamic performance.


In the kidney, a major site of ion transport, NKA consumes ~ 50% of ATP, indicating a tight coordination of NKA and energy metabolism. AMP-activated protein kinase (AMPK), a cellular energy sensor, regulates NKA by modulating serine phosphorylation of the α1-subunit, but whether it modulates other important regulatory phosphosites, such as Tyr10, is unknown. Using human kidney (HK-2) cells, we determined that the phosphorylation of Tyr10 was stimulated by the epidermal growth factor (EGF), which was opposed by inhibitors of Src kinases (PP2), tyrosine kinases (genistein), and EGF receptor . AMPK activators AICAR and A-769662 suppressed the EGF-stimulated phosphorylation of EGFR (Tyr1173) and NKAα1 at Tyr10. The phosphorylation of Src (Tyr416) was unaltered by AICAR and increased by A-769662. Conversely, ouabain (100 nM), a pharmacological NKA inhibitor and a putative adrenocortical hormone, enhanced the EGF-stimulated Tyr10 phosphorylation without altering the phosphorylation of EGFR (Tyr1173) or Src (Tyr416). Ouabain (100–1000 nM) increased the ADP:ATP ratio, while it suppressed the lactate production and the oxygen consumption rate in a dose-dependent manner. Treatment with ouabain or gene silencing of NKAα1 or NKAα3 subunit did not activate AMPK. In summary, AMPK activators and ouabain had antagonistic effects on the phosphorylation of NKAα1 at Tyr10 in cultured HK-2 cells, which implicates a role for Tyr10 in coordinated regulation of NKA-mediated ion transport and energy metabolism.Na PCR plates and PCR plate adhesive films were from Bio-Rad or Nippon Genetics. Amersham ECL Full-Range Rainbow Molecular Weight Markers were from GE Healthcare Life Sciences. Polyvinylidene difluoride (PVDF) membranes and all other reagents, unless specified otherwise, were from Merck.2 up until the passage 10. A day prior experiment the cells were seeded on 6-well cell culture plates at density of 2 × 105 cells/well or on 12-well cell culture plates at density of 1 × 105 cells/well (unless specified otherwise) to obtain 100% confluency at the time of experiment. For the last 4 h the cells were serum-starved in DMEM without serum and antibiotics.HK-2 cell line was grown in DMEM GlutaMAX  supplemented with 10% FBS and 1% Pen Strep at 37 °C in humidified air with 5% COd-ribofuranoside  and A-769662 , mitochondrial oxidative phosphorylation uncoupler carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone , inhibitor of Src kinases 3-(4-chlorophenyl)-1--1H-pyrazolopyrimidin-4-amine , inhibitor of ATP synthase oligomycin , insulin , Na+,K+-ATPase inhibitor ouabain , epidermal growth factor , EGFR tyrosine kinase inhibitor gefitinib , tyrosine kinase inhibitor genistein , electron transport chain complex I inhibitor rotenone , and complex III inhibitor antimycin A .The following substances were used for the treatment of the cells: activators of AMPK 5-aminoimidazole-4-carboxamide 1-β-2HPO4, 1.8 mM KH2PO4, pH 7.4). Cells were lysed with Laemmli buffer (62.5 mM Tris–HCl (pH 6.8), 2% (wt/vol) sodium dodecyl sulphate (SDS), 10% (wt/vol) glycerol, 5% (vol/vol) 2-mercaptoethanol, 0.002% (wt/vol) bromophenol blue) and were subsequently sonicated and heated at 56 °C for 20 min. Proteins were separated by SDS polyacrylamide gel electrophoresis with 4–12% gels and transferred to the PVDF membranes with wet electrotransfer. After the transfer, membranes were stained with Ponceau S [0.1% (wt/vol) in 5% (vol/vol) acetic acid] to evaluate uniformity of sample protein concentration. Membranes were then blocked in 7.5% (wt/vol) dry skimmed milk in Tris-buffered saline with Tween  Tween 20, pH 7.6) for 1 h at room temperature, washed several times in TBST and then incubated with appropriate primary antibody  or human Na+,K+-ATPase α3 mRNA  (both ON-TARGETplus–SMARTpool from Horizon Discovery) using Invitrogen Lipofectamine 2000 Transfection Reagent (Thermo Fisher Scientific) according to the manufacturer’s guidelines with siRNA in the final concentration of 5 nM. To test the off-target effects of siRNA, a non-targeting siRNA pool  was used. After 24 h of transfection the medium was replaced with fresh DMEM  and the cells were incubated for another 24 h. To enable measurement of lactate secretion into medium, the cells were grown in serum-free DMEM for the last 8 h. The cells were washed with PBS and frozen at − 80 °C until further analysis. Gene knock-down was assayed using qPCR and immunoblot.HK-2 cells were seeded on 6-well cell culture plates at density of 1 × 10+,K+-ATPase (No. Hs00167556_m1) and human α3-subunit of Na+,K+-ATPase (No. Hs00958036_m1). Expression of target gene was normalized to expression of cyclophilin , which was used as an endogenous control. Efficiency of PCR was estimated with LinRegPCR software (2018.0)  or E.Z.N.A. HP Total RNA Kit (Omega Bio-tek) and reverse transcribed to cDNA with High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). Quantitative real-time polymerase chain reaction (qPCR) was performed on 7500 Real-Time PCR System  using TaqMan Universal MasterMix and TaqMan gene expression assays for human α1-subunit of Na3 cells/well. 24 h after seeding, growth medium was replaced with Seahorse XF DMEM medium (Agilent) supplemented with 10 mM glucose, 1 mM pyruvate and 2 mM glutamine. Cells were incubated for 1 h at 37 °C in normal atmosphere  and then transferred into Seahorse. In Seahorse, cells were treated with ouabain or vehicle for ~ 3½ h, which was followed by successive treatment with 1 μM oligomycin, 0.75 μM FCCP, and a combination of 1 μM rotenone and 1 μM antimycin A.ECAR and OCR were analysed with Seahorse XFe24 Analyzer (Agilent). Cells were seeded on Seahorse XF24 cell culture microplates (Agilent) at density of 50 × 10At the end of the experiment cells were detached with trypsinization and stained with FITC-conjugated annexin V (for detection of phosphatidylserine on the outer membrane of apoptotic cells) and propidium iodide  using Annexin V FITC Assay Kit . Cells were analysed with Cell Lab Quanta SC flow cytometer (Beckman Coulter) and divided into four populations based on the intensity of FITC and PI fluorescence: live , early apoptotic , late apoptotic , and necrotic .3H)-labelled 2-deoxy-glucose (2-DG), as described  and incubated in HBS with 10 μM 2-DG and 1 μCi/mL 2--DG (PerkinElmer) for 10 min at 37 °C. Cells were subsequently washed with PBS supplemented with 25 mM glucose and lysed with 0.04% (wt/vol) SDS in water. Cell lysates were then analysed for protein content with Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) or mixed with liquid scintillation cocktail Aquasol 2 (PerkinElmer) and analysed for radioactivity with MicroBeta TriLux scintillation counter (PerkinElmer). Amount of 2-DG taken up by the cells was determined with the help of a standard  and expressed in pmol/min/mg of protein. Control cells were treated with inhibitors of glucose transporters (phlorizin (100 μM) or cytochalasin B (10 μM)) during 2-DG uptake to confirm that 2-DG was taken up via glucose transporters.Glucose uptake was determined by measuring the uptake of tritium (Lactate dehydrogenase (LDH) and lactate in cell medium were measured with Cytotoxicity Detection Kit (LDH) (Roche) and Lactate Assay Kit (Merck), respectively. The total protein content was determined with Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). All assays were performed according to the manufacturer’s instructions. The DNA content was determined with a DNA assay based on the fluorescent DNA dye Hoechst 33342 (Thermo Fisher Scientific) as described  according to the manufacturer’s instructions. Cells were seeded in white 96-well microplates with clear bottom (PerkinElmer). Luminescence was measured with VICTOR microplate reader.P ≤ 0.05 was considered as statistically significant.Results are presented as means with standard error of the mean (± SEM). Results were statistically analysed with GraphPad Prism 6 software using one-way ANOVA followed by Bonferroni’s or Dunnett's post hoc test to compare different treatments and control samples or Student's t-test to compare only two samples. NKAα1 is the predominant NKAα isoform in HK-2 cells; however, they also express NKAα3  at all time points  or 120 nM insulin (5–60 min) , HK-2 cells were treated with 1 μg/mL EGF (10 min) in the presence of EGFR inhibitor gefitinib (10 μM) or tyrosine kinase inhibitor genistein (100 μM)  did not alter the phosphorylation of NKAα1 at Tyr10  in the absence or presence of 100 nM ouabain or 300 μM A-769662 , and cytochalasin B (a glucose transporter inhibitor)  Fig. I. ExceptTo determine whether ouabain altered mitochondrial function, the oxygen consumption rate (OCR) Fig. J and extA major finding of our study is that the EGF-stimulated phosphorylation of NKAα1 at Tyr10 was suppressed by two different AMPK activators and enhanced by the cardiotonic steroid ouabain. This finding highlights the phosphorylation of Tyr10 as a focal point of regulation by multiple signalling pathways. Moreover, it implicates a role for AMPK in regulation of tyrosine phosphorylation of NKA.EGF was previously shown to increase tyrosine phosphorylation of NKAα1 in the proximal tubules   and an increased phosphorylation of the inhibitory site Tyr527 (aka Tyr529). In direct contrast, PP2 (in concentrations that abolish Src activity) may enhance the phosphorylation of Src at Tyr416, induced by exogenous stimuli, such as bombesin and angiotensin II . Despite these differences both AMPK activators decreased the phosphorylation of EGFR as well as NKAα1 at Tyr10, which strengthens the argument that AMPK plays a role in regulation of Tyr10 phosphorylation via inhibition of the EGFR signalling.Ouabain enhanced the EGF-stimulated phosphorylation of Tyr10 although it had no effect when used alone. Importantly, EGF action on Tyr10 was enhanced although phosphorylation of EGFR and Src were similar between the EGF and the combined (EGF and ouabain) treatment. While the underlying mechanism was not dissected it seems clear that ouabain did not promote the phosphorylation of Tyr10 by augmenting the activation of EGFR-Src pathway. We can therefore speculate that ouabain might have induced conformational changes in NKAα1 that made Tyr10 more accessible to phosphorylation by Src. Alternatively, ouabain might have enhanced the association between NKA and Src. If ouabain increased the interaction between NKA and Src via either of these two mechanisms, the pool of activated Src molecules may have been more efficient at phosphorylating Tyr10. This hypothesis is consistent with the above stated idea that A-769662 activated a pool of Src that was not close to NKA. Notably, ouabain increased interaction between Src and NKAα1 and NKAα2 in isolated skeletal muscle (Kotova et al. + and K+ (Pirkmajer et al. The EGFR/Src/ERK1/2 signalling pathway was traditionally considered to be a particularly important link between ouabain and its intracellular effects. However, its universal significance as well as the underlying molecular mechanisms that link NKA to this pathway have recently been questioned Askari , b. WhilOuabain had no effect on the ATP production in isolated mitochondria (Kajikawa et al. In addition, ouabain was previously shown to increase glycogen synthesis in isolated skeletal muscles as well as cultured skeletal muscle cells Clausen , 2006b. Here we did not examine the functional consequences of the Tyr10 phosphorylation, but these may theoretically include modulation of NKA kinetics, protein–protein interactions with signalling proteins or NKA regulators, such as FXYD proteins, or NKA trafficking between the plasma membrane and the intracellular compartment. The ultimate effect of the tyrosine phosphorylation of NKAα1 is unclear since the studies which examined the functional significance of unspecific tyrosine phosphorylation or phosphorylation of Tyr10 did not produce consistent results. For instance, insulin and EGF increased tyrosine phosphorylation of NKAα1 and activity of NKA in rat proximal tubules (Feraille et al. In conclusion, AMPK activation suppressed the EGF-stimulated phosphorylation of Tyr10 in HK-2 cells, while NKA inhibitor ouabain promoted it. AMPK likely exerted this effect by suppressing the EGFR-Src pathway, but activation of tyrosine phosphatases, which dephosphorylate Tyr10 cannot be excluded. The mechanism by which ouabain enhanced the EGF-stimulated Tyr10 phosphorylation did not seem to involve altered signalling via EGFR or Src. Collectively, our results suggest a new mechanism by which AMPK regulates NKA, while highlighting a link between regulation of energy metabolism and NKA-mediated ion transport.


Our recent extensive global proteome and phosphoproteome analysis revealed that SmKIN3 is a target of the striatin-interacting phosphatase and kinase (STRIPAK) multisubunit complex. Here, using protein samples from the wild type and three STRIPAK mutants, we applied absolute quantification by parallel-reaction monitoring (PRM) to analyze phosphorylation site occupancy in SmKIN3 and other septation initiation network (SIN) components, such as CDC7 and DBF2, as well as BUD4, acting downstream of SIN. For SmKIN3, we show that phosphorylation of S668 and S686 is decreased in mutants lacking distinct subunits of STRIPAK, while a third phosphorylation site, S589, was not affected. We constructed SmKIN3 mutants carrying phospho-mimetic and phospho-deficient codons for phosphorylation sites S589, S668, and S686. Investigation of hyphae in a ΔSmkin3 strain complemented by the S668 and S686 mutants showed a hyper-septation phenotype, which was absent in the wild type, the ΔSmkin3 strain complemented with the wild-type gene, and the S589 mutant. Furthermore, localization studies with SmKIN3 phosphorylation variants and STRIPAK mutants showed that SmKIN3 preferentially localizes at the terminal septa, which is distinctly different from the localization of the wild-type strains. We conclude that STRIPAK-dependent phosphorylation of SmKIN3 has an impact on controlled septum formation and on the time-dependent localization of SmKIN3 on septa at the hyphal tip. Thus, STRIPAK seems to regulate SmKIN3, as well as DBF2 and BUD4 phosphorylation, affecting septum formation.We showed recently that the  STRIPAK is involved in a broad variety of developmental processes in higher and lower eukaryotes. For example, proliferation of several mammalian cancer cells is correlated with dysfunctional STRIPAK subunits  and label-based approaches (isobaric tags for relative and absolute quantitation [iTRAQ] and tandem mass tags [TMTs]), are indispensable for large-scale detection of changes in the phosphorylation of peptides and the identification of potential molecular targets of kinases and phosphatases  approaches. These can be employed as a means to validate a subset of the results obtained by shotgun experiments. While targeted MS is widely used for accurate protein quantification, the high variability, increased experimental effort, and need for validation has limited the implementation of targeted approaches in phosphoproteomics analysis  SmKIN3, which, however, was absent in the global proteome and thus did not permit quantitative measurement of phosphorylation. Therefore, SmKIN3 phosphorylation was determined by applying absolute quantification by synthesis of stable isotope-labeled standard (SIS) peptides combined with parallel-reaction monitoring (PRM), a method which has not yet been applied to a fungal organism to identify phosphorylation sites.Among the 781 regulated proteins in septation initiation network (SIN) complex (sterile protein (STE) kinase, a GCK, and a nuclear DBF2-related (NDR) kinase  kinase . For exaes DBF-2 . The funes DBF-2 , 16, 17.es DBF-2 , 19.Here, we show that STRIPAK-directed phosphorylation of SmKIN3 has a significant impact on proper hyphal septation and septal localization. This is the first report about STRIPAK-dependent phosphorylation analyzed by targeted quantification of phosphorylation sites, and it will have an impact on understanding the function of mammalian homologs.N. crassa, but similarity with other homologues from ascomycetes is low. Using the eukaryotic linear motif (ELM) database (large tumor suppressor (LATS) kinase recognition motifs, and a binding motif for forkhead-associated (FHA) domains. The COILS sequence analysis program (http://www.ch.embnet.org/software/COILS_form.html) revealed two predicted putative coiled-coil domains located next to each other in a region between amino acids (aa) 688 and 788. At the C-terminal end of SmKIN3 (aa 805 to 811), we detected a conserved sequence motif, previously called the T-motif -PRM-based workflow was established to quantify the site-specific phosphorylation states of putative STRIPAK targets. First, 70 phosphorylated peptides and their corresponding nonphosphorylated counterparts were selected, and SIS were synthesized. The median coefficients of variation (CV) of the biological replicates were calculated as 16.3% for all nonphosphorylated peptides and 16.1% for all phosphorylated peptides. To further prioritize our approach, we selected 15 pairs of peptides, representing phospho sites from proteins belonging to the SIN signaling pathway. Dilution series were prepared to verify a linear response and determine the limit of detection (LOD) of each SIS peptide, as described in Materials and Methods. LODs ranged from 1.7 amol to 19.3 fmol per injection for phosphorylated SIS peptides and from 1.43 amol to 32.3 fmol per injection for their nonphosphorylated counterparts. The average lower limit of quantification (LLOQ) was 375 amol or 135 amol on column for phosphorylated or nonphosphorylated peptides. The median CV throughout all dilution steps was calculated as 6.7% for phosphorylated peptides and 5.0% for nonphosphorylated peptides. These results are summarized in A targeted bipartite TiO10.1128/mBio.00658-21.8DATA SET S1Data Set S1, XLSX file, 0.3 MB.Data from the PRM analysis. Download Copyright © 2021 Stein et al.2021Stein et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the S. macrospora wild type and the three STRIPAK deletion strains, the Δpro11, Δpro22, and Δpp2Ac1 mutants, were grown in triplicates prior to being subjected to lysis and protein extraction  in the SmKIN3 protein.Finally, as a third option, we found seven phosphorylation sites on five proteins, where only the phosphorylated peptides were detected and no site occupancy value could be calculated . Howevert-test P value < 0.05; the results are summarized in Overall, 19 phosphorylation sites showed significantly different site occupancy between the wild type and at least one of the STRPIAK mutant strains (Student's 10.1128/mBio.00658-21.4FIG S1y axis on the left side gives the amount of the peptide SPDKPVLSAsPTK in attomoles as a nonphosphorylated variant (green) and phosphorylated variant (orange). The y axis on the right side shows the quantity of phosphosite occupancy in percentages as red dots. Lowercase letter in the peptide indicates the phosphosite. Error bars indicate standard deviations. Download FIG S1, TIF file, 0.6 MB.Quantitative analysis of the nonphosphorylated and phosphorylated peptides containing phosphorylation site S589 of SmKIN3 in three different STRIPAK deletion mutants. The Copyright © 2021 Stein et al.2021Stein et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the in vitro mutagenesis, replacing the serine triplets with either alanine (no phosphorylation) or glutamic acid (mimics phosphorylation) triplets, as described in Materials and Methods. The phosphorylation of S589 is apparently STRIPAK independent -labeled SmKIN3 is fully functional. We then investigated three homokaryotic ascospore isolates of each phospho-mimetic strain, i.e., those with S589E, S668E, and S686E, and phospho-deficient strains, i.e., those with S589A, S668A, and S686A. Growth rates of mutant strains, grown at different temperatures and under different light conditions, resemble that of the wild type  staining. We investigated at least 200 single hyphal branches for each strain in three technical triplicates. In these cases, at least 600 hyphal branches were counted for each recombinant strain  Southern blot analysis of the Smkin3 copy number in SmKIN3-phospho variants. Genomic DNAs of the wild type, ΔSmkin3::Smkin3-gfp strain, and S589A, S589E, S668A, S668E, S686A, S686E, S668A S686A, and S668E S686E phospho-mutants were digested with the restriction enzymes ApaI and PstI for hybridization with Smkin3. Autoradiograph of Southern blot hybridization with radioactively labeled probe specific for Smkin3. The wild-type Smkin3 band is visible at 7241 bp and the recombinant Smkin3 gene is visible at 5757 bp. The wild type and a complemented ΔSmkin3 strain were used as controls and are shown only once, indicated by the black lines. Download FIG S2, TIF file, 1.2 MB.(A) Expression control of phospho-mutated variants of SmKIN3 tagged with GFP. Strains were grown for 3 days in liquid medium  as a surface culture. For each strain, 10 μg of crude protein extract was subjected to SDS-PAGE. Western blot analysis was performed with an anti-GFP antibody and an anti-α-tubulin antibody as control. SmKIN3 tagged with GFP has a mass of 118 kDa, while α-tubulin has a mass of 55 kDa. SmKIN3-GFP was detected in all eight different phospho-mutants . The wild type and a complemented ΔCopyright © 2021 Stein et al.2021Stein et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.00658-21.6FIG S3Smkin3 strain and both the Δpro11::Smkin3-gfp and Δpp2Ac1::Smkin3-gfp strains. Download FIG S3, PDF file, 0.5 MB.Characterization of the wild type and mutant strains. (A) Phenotypic characterization of the growth rate and fertility of strains, as indicated, when grown at 27°C under light conditions. The phospho-mutants resemble the wild type when grown under different physiological conditions, as noted in Materials and Methods. (B) The SmKIN3-S686E protein localizes to septa of vegetative hyphae (white arrows) having different ages and close to the nucleus at spindle pole bodies (red arrows). In addition, SmKIN3 appears at septa of vegetative hyphae and localizes as well to septa of young and mature hyphae marked by a white arrow. Strains were grown for 1 day on a solid-BMM-coated glass. Samples were stained with calcofluor white M2R . The image serves as an example for all phospho-mutated strains, as well as for the complemented Copyright © 2021 Stein et al.2021Stein et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.00658-21.10DATA SET S3Data Set S3, XLSX file, 0.01 MB.Data for quantification of the hyper-septation phenotype. Download Copyright © 2021 Stein et al.2021Stein et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the S. macrospora wild type was 4.4% ± 1.6%. The hyper-septation value for the complemented strains and phospho-mutants were as follows: for the ΔSmkin3::Smkin3-gfp strain, 4.0% ± 2.7%; for the S589A phospho-mutant, 2.6% ± 1.9%; for the S589E phospho-mutant, 3.4% ± 1.9%; for the S668A phospho-mutant, 13.5% ± 4.8%; for the S668E phospho-mutant, 16.2% ± 6.0%; for the S686A phospho-mutant, 16.0% ± 7.0%; and for the S686E phospho-mutant, 13.7% ± 5.1%. The values  and phospho-deficient (S668A S686A) double mutants and analyzed derived ascospore isolates. The strains were fully fertile, and the phospho-mutated SmKIN3 proteins are still found at septa and at nuclei in young hyphae . We founpro11 strain), the catalytic subunit of the STRIPAK phosphatase (Δpp2Ac1 strain), or the STRIP1/2 homologue (Δpro22 strain). We found no hyper-septation in the Δpro11, Δpro22, or Δpp2Ac1 strain . We selected the four terminal septa at the hyphal tip for our detailed localization analysis. We counted the first terminal septa at hyphal tips, where GFP fluorescence indicated SmKIN3 localization  or CFW-stained septa. Inhibiting arrows point to septa where SmKIN3 is missing. This figure corresponds to Copyright © 2021 Stein et al.2021Stein et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the pro11 and Δpp2Ac1 strains. In both deletion strains, SmKIN3 localized mainly at the first or second septum, as with the S668 and S686 phospho-variants and unlike in the wild-type strain. This suggests that temporally controlled phosphorylation at sites S668 and S686 is required for the localization of SmKIN3 to early septation sites  complex, the STRIPAK homolog , followed by CR constriction. The CR protein complex includes structural proteins, molecular motors, and signaling enzymes  strains revealed a hyper-septation phenotype, which was also observed in both double phospho-mutants. In contrast, the S589 phospho-mutants showed a septation phenotype comparable to that of the wild type. Since phosphorylation at distinct SmKIN3 sites is regulated by STRIPAK, we hypothesize that the fine spatiotemporal tuning of the phosphorylation status of S668 and S686 mutants is important for the septation process. Thus, the coordinated formation of septa is impaired in both phospho-mimetic and phospho-deficient mutants. The outcome of our mutant analysis is reminiscent of a comparable analysis in yeast , where tAspergillus nidulans, SepH, the homolog of CDC7, has been identified as a central component for the initiation of septation prior to actin ring formation. SepH is the kinase upstream of SepL, which is the homolog of SmKIN3, followed by SidB (homologue of DBF2)  , 37. SINof DBF2) . Downregle septa . This innidulans . Comparaof septa . Phosphoguanine exchange factors (GEFs) and GTPases, which in turn phosphorylate the formin Cdc12p. The phosphorylation of Cdc12p is important for the assembly of actin to form the CR. The NDR kinase Sid2p is necessary to phosphorylate the septin Cdc12p and other proteins to form the CR , the SIN kinase SepH (CDC7), and on its own phosphorylation state.We hypothesize further that deletion of STRIPAK subunits results in higher phosphorylation of CDC7, which prevents assembly of SIN . This re STRIPAK . This isN. crassa, phosphorylation analysis revealed that the phosphorylation of DBF-2 is stimulated by SID-1. Interestingly, phospho-deficient and phospho-mimetic mutants of DBF-2 phosphorylation site S499 in N. crassa are nonfunctional in vivo and reduce the kinase activity of DBF-2 in vitro (S. macrospora) seems not to be regulated by STRIPAK but is essential for septum formation in N. crassa  media -coated glass. Isogenic and homokaryotic strains were generated by genetic crossing and ascospore isolation . The number of ectopically integrated recombinant DNA fragments was verified by Southern hybridization analysis  media . In all solation . To obtasolation  were traanalysis .10.1128/mBio.00658-21.2TABLE S2Table S2, DOCX file, 0.02 MB.Strains used in this work. Download Copyright © 2021 Stein et al.2021Stein et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Smkin3-gfp was created by restricting the plasmid pIG1783 with NcoI. Smkin3 was amplified via PCR with overhangs at the 5′ and 3′ ends containing recognition sites for NcoI. Ligation was performed with T4 DNA ligase. For phospho-mimetic and phospho-deficient strains, plasmid pIG1783-Smkin3-gfp was used for Q5 mutagenesis (New England Biolabs). With specific primers  coupled with a CoolSNAP HQ camera (Roper Scientific) and a Spectra-X LED lamp (Lumencor) at room temperature. Images were acquired and edited with MetaMorph . Strains were grown on glass slides covered with solid BMM and incubated for 24 to 48 h. GFP fluorescence was analyzed using filter set 49002 . Septa in vegetative hyphae and ascogonial coils were stained using calcofluor white M2R  with a concentration of 1 μg/ml CFW stock solution diluted 1:400 in distilled sterile water. CFW fluorescence was analyzed using Chroma filter set 31000v2 .pro11, Δpp2Ac1, and Δpro22 STRIPAK deletion mutants, we investigated at least 100 hyphal branches from three technical replicates. We counted all septa within 20 μm behind hyphal branches. Septa within a distance of maximum 12 μm were designated hyper-septation septa. Septal distances in hyphae were measured using MetaMorph .To analyze the distribution of SmKIN3 on septa, at least 50 hyphal tips of the growing front were observed in 10 to 15 independent samples per strain in three different independent strains. Septa were stained with CFW, and SmKIN3-GFP localization was considered only when detected with an exposure time between 100 and 1,000 ms. For analysis of hyper-septation, e.g., double, triple, quadruple, or quintuple septa, we investigated at least 200 single hyphal branches for each strain in three technical repeats. In the case of recombinant strains, three different single ascospore isolates were investigated to exclude side effects from random integration of recombinant DNA. In these cases, at least 200 hyphal branches were counted for each recombinant strain. For the ΔBased on the results of the global iTRAQ-based (phospho-)proteomic analyses previously performed , 9, as w13C615N2) and arginine (13C615N4) were incorporated at the C-terminus, and amino acid analysis was applied to determine peptide concentrations  and Fmoc chemistry. Synthesis and subsequent purification were performed as described previously . Heavilytrations . A five-Protein extraction, digestion, and normalization and phosphopeptide enrichment were performed as recently reported .rapid-separation liquid chromatography nano (RSLCnano) high-performance LC (HPLC) system coupled with a Q Exactive HF mass spectrometer . The HPLC was equipped with a trapping column  for preconcentration and an analytical column  for separation of the peptides. Preconcentration was performed for 5 min at a flow rate of 20 μl/min using 0.1% trifluoroacetic acid (TFA), and separation was performed at a flow rate of 250 nl/min. An optimized binary gradient of solvent A (0.1% formic acid [FA]) and solvent B  was used with the following steps: 0 min, 2% B; 5 min, 2% B; 10 min, 5% B; 50 min, 9% B; 73 min, 15% B; 100 min, 21% B; and 115 min, 45% B. This was followed by two washing steps for 5 min at 95% B and 20 min of equilibration at 2% B. The MS was operated in PRM mode at a resolution of 60,000 (at 200 m/z) with a fixed first mass of 150 m/z. The AGC target was set to 1 × 106 and a maximum injection time of 118 ms. Targeted precursors were isolated with a quadrupole isolation width of 0.4 m/z and fragmented with a normalized collision energy of 27. PRM acquisition was scheduled with a retention time window of 2 min per target.Samples were analyzed on an Ultimate 3000 Enriched phosphorylated peptides were analyzed with the same instrumentation and settings as described above. The LC gradient was optimized to suit the targeted phosphopeptides, and the steps were modified as follows: 0 min, 3% B; 5 min, 3% B; 15 min, 7% B; 37 min, 10% B; 90 min, 20% B; 110 min, 27% B; and 120 min, 45% B. This was followed by the same washing and equilibration steps as above.limit of blank (LOB), LOD, and LLOQ, response curves of both phosphorylated and nonphosphorylated SIS peptides were acquired. In both cases, a background matrix was generated by pooling aliquots of all individual samples. SIS peptides were spiked in at eight different concentrations, covering a range of 3 orders of magnitude. Based on prior determination of individual SIS response factors, the highest concentrations for nonphosphorylated SIS peptides varied between 54 fmol and 2.73 pmol on column, while the lowest concentrations were between 13 and 667 amol on column. For the analysis of the individual samples, a total amount of nonphosphorylated SIS peptides ranging from 0.05 fmol to 60 fmol was spiked into 3 μg of the total protein digest, depending on the expected endogenous concentration  peptides were included in the assay, but no endogenous peptide was detected. These peptides could be further used to rule out the presence of these isomers through knowledge of their retention time and diagnostic transitions. The median CV of the biological replicates was calculated as 16.3% for all nonphosphorylated and as 16.1% for all phosphorylated peptides. The lowest average site occupancy was detected at 0.06% on the phosphorylation sites S369 in the HSP90 protein SMAC_04445. This site was included as a negative control, and its occupancy showed no significant difference between the wild type and any of the deletion strains. The phosphorylation site S2349 in the phosphatidylinositol 3-kinase TOR2 (SMAC_03322) exhibited the highest site occupancy, with 92.2% measured in the wild type.In addition, six phoshphoisomeric 2-based phosphopeptide enrichment and nano-LC-MS/MS measurement in technical triplicates (16.7% of eluate per replicate) as detailed above. Additionally, two replicates without the SIS spike-in (DS0) were processed in parallel to appropriately determine background signal levels. For the final analysis of individual samples, a total amount of 410 amol to 4.1 fmol was spiked into 450 μg of the protein digest, followed by phosphopeptide enrichment and nano-LC-MS/MS measurement of 25% of the eluates after enrichment.All calibration curve samples were subjected to TiOc) of phosphorylated and nonphosphorylated peptide per sample. The phosphorylation site occupancy was calculated using the following formula:t-test.Skyline software (version 4.1 ) was usehttps://panoramaweb.org/SmKIN3.url.All targeted proteomics data and raw files are available through the Panorama repository  under th


When a hip screw needs to be changed, choosing between the conventional (C-type) and helical blade (H-type) types is difficult. In this biomechanical study, we compared these two screw types relative to the type of the initial screw used.3; PCF 10, 160 kg/m3; and PCF 15, 240 kg/m3), and then successively or alternately inserted (following screw) after the leading screw removal. An original model  of a leading screw without removal was created as a control. The strengths of resistance to pullout (PO) and rotational stress were measured. For each experimental condition, there were 30 experimental models.C- or H-type screws were inserted (leading screw) in three types of polyurethane bone models  of different bone mineral densities  showed the greatest resistance to PO, and using the H-type screw as the following screw  showed superior resistance to rotational strength. However, the rotational strength of the C2 screw decreased by more than 50% compared with that of the original C screw. Moreover, the PO and rotational strengths of the H2 screw decreased to less than 30% overall compared with those of the original H screw.The H-type screw should be used for second-time screw insertion procedures in cases where it is difficult to choose between PO and rotational strengths. Femoral intertrochanteric fractures (IT Fx) are representative osteoporotic fractures in elderly patients, and their incidence has markedly increased in aging societies . For mos3), mild osteopenic , and osteopenic . Two types of hip screws were used: a third-generation gamma nail  as the C-type screw and a PFN antirotation  as the H-type screw. The length of the hip screws were uniform at 100 mm  which is widely used in biomechanical experiments. IT Fx occur not only in patients with osteoporosis but also in patients with an osteopenic bone mineral density (BMD). Thus, the experimental bone models reflected three types of BMD: severe osteoporotic  insertion of the original screw  as the leading screw, and without removal, (2) removal of the leading screw and insertion of a subsequent screw of the same type without reaming , (3) removal of the leading screw and insertion of a following screw of a different type  after reaming (RO), and (4) removal of the leading screw and insertion of a subsequent screw of a different type  without reaming (RX). By applying these four experimental conditions to the two different types of hip screws, eight experimental models were obtained: original H, H1-H2, H1-ROC2, and H1-RXC2; and original C, C1-C2, C1-ROH2, and C1-RXH2. For all eight models, the pullout (PO), clockwise rotation (CWR), and counterclockwise rotation (CCWR) strengths of the following and original screws were evaluated, considering the BMD. We further evaluated the change in strength reduction in the PO, CWR, and CCWR strengths, between the original and the following screw .A servohydraulic testing machine  was used to ensure that the same force was applied during insertion, removal, and reinsertion of the hip screws. Using an automated torque tester , an axial loading of 9.8 1-kgf) to 29.4 (3-kgf) N at a speed of 3 r/min was applied. The entire experimental process was supervised by one orthopedic surgeon with > 10 years’ experience in surgically treating hip fractures to reduce the difference in force applied to screw insertion and removal  for using a two-sided two sample t-test with an effect size of 0.8. Twenty-six samples were needed to achieve a power of 80% at 0.05 significance level. Owing to predicted test failures, we proceeded to 30 per each experimental model.The screws were evaluated for resistance to PO, CWR, and CCWR. Evaluations were performed using 30 lab blocks for each experimental model. Each lab block was fixed firmly with a custom-made jig in the frame of a universal material testing machine . Lag in the lab block was tested according to the international test standard, ASTM F543. PO stress was applied at a speed of 5 mm/min until the screw came out Fig. A, B. Aftp value < 0.05. All analyses were performed using R 4.0.1 .Descriptive data are presented as means and standard deviations. Comparisons between two groups were performed using an independent t-test when normality was satisfied, and the Mann–Whitney U test when normality was not satisfied. Normality tests were performed using the Shapiro–Wilk test. For comparisons among three groups, we used analysis of variance (ANOVA) with Tukey’s test or the Games–Howell post hoc test when normality was satisfied. The Kruskal–Wallis and Dunn tests when normality was not satisfied. If the results of each ANOVA and Kruskal–Wallis test were significant, the corresponding post hoc analysis was used. Statistical significance was defined as a When the C-type screw was the leading screw, using the C-type screw as the following screw C1-C2) gave the greatest resistance to PO with a significant difference compared with using the H screw as the following screw (C1-H2). When the H-type screw was the leading screw, using the C-type screw as the following screw (H1-C2) gave the greatest resistance to PO with a significant difference, compared to the H screw (H1-H2) in all bone models. When the leading screw and the following screw were different (C1-H2 or H1-C2), reaming did not affect the PO strength, except in the case of C1-RXH2, where the PO strength was significantly lower than PCF 10  gave superior resistance to rotational strength with a significant difference compared to using the C-type screw (C1-C2), regardless of the rotation direction and BMD. When the H-type screw was the leading screw, using the H-type screw as the following screw (H1-H2) gave superior resistance to rotational strength with a significant difference compared to using the C-type screw (H1-C2), regardless of the rotation direction and BMD. In particular, when the leading screw was an H-type screw and a C-type screw was the following screw after reaming (H1-ROC2), the rotational strength was significantly lower than when the reaming was not performed (CW on PCF 5 and 10 and CCW on PCF 10 and 15)  showed that the C-type screw was significantly superior to the H-type screw for all BMDs. In contrast, the H-type screw was significantly superior to the C-type screw in terms of rotational strength, regardless of the rotation direction and the BMD Table  Fig. 4AA4A, B, CWhen the following screw was a C-type screw , the PO strength for the C2 significantly decreased to < 50% overall compared with that for the original C. When the following screw was a C-type screw , the rotational strength for C2 significantly decreased compared with that for the original C in both CW and CCW. In particular, the rotation strength compared with that for the original C decreased by > 50% in all other cases except for C1-C2 CCW, 47.13%) in PCF 5 and C1-C2 , H1-ROC2 , and H1-RXC2  in PCF 15 (Table 7.13% in p = 0.358). When the following screw was used as an H-type screw , the rotational strength for H2 significantly decreased compared to that of the original H in both CW and CCW, except for H1-H2  and C1-RXH2 . In particular, the rotational strength compared to that of the original H decreased by < 30% in all cases , the PO strength for H2 decreased to < 30% overall compared to that of the original H, and the difference was significant except for H1-H2 , compared to that of the original C. In particular, in H1-ROC2 with osteoporosis (PCF 5 and 10), the rotation strength for CW and CCW significantly decreased by > 60%. Therefore, considering rotational strength, we recommend using the H-type screw as the following screw , regardless of the type of leading screw.The differences in fixation strength when the C- and H-type screws are inserted into the cancellous bone and the stability after use for treating IT Fx have been researched. However, there is still controversy on which type of screw is superior –24. In tThis study has several limitations. First, we used polyurethane bone models, which do not reflect cancellous bone impaction or autogenous cancellous bone grafting that occurs with normal bone. However, using a fresh bone model would have been inappropriate to reflect various BMDs, and to consistently reproduce the experiments under the same conditions. Our study aimed not to examine the stability of fixation for IT Fx, but to determine the fixation of the following screw in the cancellous bone after removing the leading screw. Therefore, a cancellous bone model with the same shape and BMD was more appropriate than a fourth-generation compact material or a fresh femoral bone model. Moreover, the authors did not attempt a finite element model test (FEM) as it was not possible to implement reaming and reinsertion after screw removal, and it was difficult to confirm the difference in mechanical characteristics according to the thread of the screw. Second, the study did not reflect the type of IT Fx, which may affect the stability of the hip screw, and did not reflect the time elapsed between inserting the leading and following screws, which may affect the strength of the cancellous bone. In addition to the motions of the fracture site, the loss of the lateral wall of the trochanter can affect the stability of the following screw. In particular, a comparative study on bone defects in the lateral wall of the femur after removal of PFNA-II and gamma 3 showed that gamma 3 using a C-type screw as a hip screw resulted in significantly more bone defects . TherefoIn conclusion, it would be more appropriate to select the C-type screw if the PO strength of the following screw is important and select the H-type screw if rotational strength is important. However, considering the differences in the unique design of the two screws and the important role of rotational strength, we believe that the H-type screw should be chosen as the subsequent screw, in cases in which it is difficult to choose between the PO and rotational strengths. Finally, the authors believe that the results of this study will act as an important reference for selecting reinsertion hip screws, specifically for patients whose screws need to be reinserted in the same position after surgical treatment with NSH.


The phosphatidylinositol 3′ kinase (PI3K)‐related kinase ATR is crucial for mammalian meiosis. ATR promotes meiotic progression by coordinating key events in DNA repair, meiotic sex chromosome inactivation (MSCI), and checkpoint-dependent quality control during meiotic prophase I. Despite its central roles in meiosis, the ATR-dependent meiotic signaling network remains largely unknown. Here, we used phosphoproteomics to define ATR signaling events in testes from mice following chemical and genetic ablation of ATR signaling. Quantitative analysis of phosphoproteomes obtained after germ cell-specific genetic ablation of the ATR activating 9-1-1 complex or treatment with ATR inhibitor identified over 14,000 phosphorylation sites from testes samples, of which 401 phosphorylation sites were found to be dependent on both the 9-1-1 complex and ATR. Our analyses identified ATR-dependent phosphorylation events in crucial DNA damage signaling and DNA repair proteins including TOPBP1, SMC3, MDC1, RAD50, and SLX4. Importantly, we identified ATR and RAD1-dependent phosphorylation events in proteins involved in mRNA regulatory processes, including SETX and RANBP3, whose localization to the sex body was lost upon ATR inhibition. In addition to identifying the expected ATR-targeted S/T-Q motif, we identified enrichment of an S/T-P-X-K motif in the set of ATR-dependent events, suggesting that ATR promotes signaling via proline-directed kinase(s) during meiosis. Indeed, we found that ATR signaling is important for the proper localization of CDK2 in spermatocytes. Overall, our analysis establishes a map of ATR signaling in mouse testes and highlights potential meiotic-specific actions of ATR during prophase I progression. Meiosis is a specialized cellular process whereby a single round of DNA replication is followed by two successive rounds of cell division to produce haploid gametes. To ensure proper chromosome segregation at the first meiotic division, meiotic cells must undergo a series of highly regulated processes, including programmed double-strand break (DSB) formation, recombination, and chromosome synapsis . The serATR activation is relatively well understood in somatic cells, and some of the molecular determinants of ATR activation are shared in meiosis. In mitotic cells, ATR is activated at sites of single-stranded DNA (ssDNA) that arise during replication or DNA repair . ATR is Stra8-Cre. Depletion of RAD1 in germ cells is anticipated to disrupt all potential 9-1-1 complexes, including those that could form with the testes-specific paralogs Hus1b and Rad9b , and therefore significantly impair ATR signaling  followed by mass spectrometry to generate phosphoproteomic datasets. By combining these datasets, and after data filtering, we were able to identify a set of 401 phosphorylation events that were dependent on both ATR and RAD1, including phosphorylation of crucial DNA damage signaling and DNA repair proteins at the preferential motif for ATR phosphorylation (S/T-Q). Interestingly, the localization of several of these targets to the sex body, such as Senataxin and RANBP3, was found to be disrupted after ATR inhibition. Finally, the set of ATR- and RAD1-dependent phosphorylation events included enrichment for phosphorylation at the S/T-P-X-K motif, suggesting that ATR promotes signaling of proline-directed kinase(s) in meiosis. Consistent with this model, ATR signaling was important for the proper localization of CDK2 in spermatocytes. Overall, our analysis establishes a comprehensive map of ATR signaling in spermatocytes and highlights potential meiotic-specific actions of ATR during prophase I progression.While hundreds of ATR targets have been characterized using quantitative phosphoproteomics mitotic cells , much leignaling . In paraRad1 conditional knockout model in which the RAD1 subunit of the 9-1-1 complex is depleted in germ cells under a Stra8-Cre promoter. By comparing the datasets generated from these two methods of impairing ATR signaling, we took advantage of the germ cell specificity of the conditional knockout model as well as the rapid dynamics of inhibition with the ATR inhibitor treatment. Our rationale was that phosphorylation impaired both by ATRi treatment and Rad1 cKO should reflect events that occur early in the ATR response and are specific to germ cells, therefore overcoming limitations intrinsic to each of these experimental setups if analyzed in isolation.Despite the central role of ATR in prophase I of mammalian meiosis, the meiotic ATR signaling network remains largely unknown. Here, we generated phosphoproteomic databases from two sets of mice with independent methods to impair ATR signaling . First, Rad1 cKO mice and littermate controls. Tissue samples were lysed and digested with trypsin, followed by enrichment of phosphopeptides and labeling with the 6-plex TMT reagent  treatment resulted in strong reduction of γH2AX at the sex body as well as a complete loss of the diplonema population . For our reagent . HILIC p reagent . After son sites .Rad1 cKO mice  and Rad1 cKO datasets , an RNA:DNA helicase with established roles in transcriptional regulation and genome maintenance  to be doeiosis I . Senataxeiosis I . Senataxeiosis I . To asse spreads . Strikin spreads . While iold mice . Previouold mice . Our resRad1 cKO pachytene-like spreads in both 8–12-week and 14-day-old mice (Another protein with a S/T-Q phosphorylation site downregulated upon RAD1 loss and ATR inhibition was RANBP3 (phosphorylation at serine 283) . RANBP3 old mice , suggestDespite phosphorylation at the S/T-Q motif being enriched in Q2, it still represented only a small portion of the RAD1- and ATR-dependent sites identified in both datasets for 4 hr and 2.5–3 days of ATRi . The finInterestingly, we noticed that several proteins involved in the biogenesis of piRNAs were phosphorylated at S/T-P sites in a RAD1- and ATR-dependent manner . piRNAs Rad1 cKO genetic mouse model and ATR inhibitor-treated mice, we enhanced our confidence in identifying meiosis-enriched ATR-dependent signaling events. Importantly, this dual approach allowed us to remove phosphorylation events changing as a result of loss of spermatocyte and sperm populations in Rad1 cKO testes. Importantly, we did not observe gross changes in cellular composition after 4 hr of ATRi treatment , the ATRi conditions showed more variability along principal component 2, which could reflect the difficulties in reproducibly delivering ATR inhibitors to the testis via oral/systemic administration  and found that ATR signaling is important for proper localization of CDK2 to autosomes. A simple model would predict that ATR somehow activates CDK2 during prophase I , which cOverall, our work represents an initial attempt to reveal the scope of targets and processes affected by meiotic ATR signaling. As expected, the ATR signaling network in meiosis is overwhelmingly complex and multifaceted. Major challenges remain, especially to untangle the functional relevance of most of the identified signaling events, and understand how the different modes of ATR signaling are coordinated for proper control of meiotic progression. Another key outstanding question is to understand how the ATR kinase, which imposes cell cycle checkpoints in most other cell types, is so highly activated in spermatocytes without inducing cell cycle arrest. A potential explanation may lay at the specificity of ATR’s action at the sex body, which may be devoted to the regulation of checkpoint-independent processes such as the control of RNA processing during meiotic prophase I , as suppAZ20 was reconstituted in 10% DMSO (Sigma), 40% propylene glycol (Sigma), and 50% water. Control mice were treated with 10% DMSO (Sigma), 40% propylene glycol (Sigma), and 50% water. Wild-type C57BL/6 male mice aged to 8–12 weeks old were gavaged with 50 mg/kg of AZ20 (Selleckchem) and euthanized at indicated time points. Specific time points examined in this study include collection after 2.5 or 3 days of 50 mg/kg of AZ20 per day or 4 hr after one dose of 50 mg/kg of AZ20.Rad1 cKO mice used in this study, +/-, Stra8-Cre+Rad1 mice were crossed with flox/floxRad1 mice to generate -/fl, Stra8-Cre+Rad1 animals. Whole, decapsulated testes from these Rad1 cKO and littermate controls on a 129Sv/Ev background were collected at 8 or 12 weeks  supplemented with 1 mM PMSF and PhosSTOP (sigma) and sonicated. 4 mg of protein  was collected, denatured with 1% SDS, and reduced with 5 mM DTT at 65°C for 10 min followed by alkylation with 60 mM iodoacetamide. Proteins were precipitated in a cold solution of 50% acetone, 49.9% ethanol, and 0.1% acetic acid, and protein pellet was washed once with a solution of 0.08 M urea in water. Protein pellet was resuspended at a concentration of 10 mg/ml in 8 M urea, Tris 0.05 M, pH 8.0, NaCl 0.15 M, and diluted fivefold prior digestion with 80 µg of trypsin  overnight at 37°C. Protein digests were acidified to a final concentration of 1% TFA and cleaned up in a solid-phase extraction (SPE) C18 cartridge pre-conditioned with 0.1% TFA solution. Peptides were eluted from the cartridges with 80% acetonitrile, 0.1% acetic acid aqueous solution, and dried in a speedvac. Phosphopeptide enrichment was performed using a High-Select Fe-NTA Phosphopeptide Enrichment Kit according to the manufacturer’s protocol . Phosphopeptide samples were split into four aliquots  and dried in salinized glass insert tubes. For each experiment, the three 30% aliquots from each control and AZ20-treated, or control and Rad1 cKO, samples were resuspended in 35 µL of 50 mM HEPES and labeled with 100 µg of each of the TMT sixplex Isobaric Label Reagents (Thermo Scientific), previously diluted in 15 µL of pure acetonitrile. TMT-labeling reaction was carried out at room temperature for 1 hr and quenched with 50 µL of 1 M glycine. All sixplex TMT-labeled aliquots were mixed, diluted with 200 µL of aqueous solution of formic acid 1% (v/v), and cleaned up in a SPE 1 cc C18 cartridge . Bound TMT-labeled phosphopeptides were eluted with 50% acetonitrile, 0.1% formic acid in water, and dried in a speedvac.Whole, decapsulated testes were collected and frozen at –80°C from 8- to 12-week-old AZ20 and vehicle-treated C57BL/6 mice 4 hr after treatment as indicated. To generate the 12 weeks . Rad1 cK18 reversed-phase resin (Reprosil Pur C18AQ 3 μm). Q-Exactive HF was operated in data-dependent mode with survey scans acquired in the Orbitrap mass analyzer over the range of 380–1800 m/z with a mass resolution of 120,000. MS/MS spectra were performed selecting the top 15 most abundant +2, +3, or +4 ions and precursor isolation window of 1.2 m/z. Selected ions were fragmented by Higher-energy Collisional Dissociation (HCD) with normalized collision energies of 28, and the mass spectra acquired in the Orbitrap mass analyzer with a monitored first mass of 100 m/z, mass resolution of 15,000, AGC target set to 1 × 105, and max injection time set to 100 ms. A dynamic exclusion window was set for 30 s.Dried TMT-labeled phosphopeptides were resuspended in 16.5 µL water, 10 µL formic acid 10% (v.v), and 60 µL of pure acetonitrile and submitted to HILIC fractionation prior to mass spectrometry analysis in a TSK gel Amide-80 column  using a three-solvent system: buffer A (90% acetonitrile), buffer B (75% acetonitrile and 0.005% trifluoroacetic acid), and buffer C (0.025% trifluoroacetic acid). The chromatographic runs were carried out at 150 µL/min and gradient used was 100% buffer A at time = 0 min; 94% buffer B and 6% buffer C at t = 3 min; 65.6% buffer B and 34.4% buffer C at t = 30 min with a curve factor of 7; 5% buffer B and 95% buffer C at t = 32 min; isocratic hold until t = 37 min; 100% buffer A at t = 39–51 min. 1 min fractions were collected between minutes 8 and 10 of the gradient; 30 s fractions between minutes 10 and 26; and 2 min fractions between minutes 26 and 38 for a total of 40 fractions. Individual fractions were combined according to chromatographic features, dried in a speedvac, and individually submitted to LC-MS/MS analysis. Individual phosphopeptide fractions were resuspended in 0.1% trifluoroacetic acid and subjected to LC-MS/MS analysis in an UltiMate 3000 RSLC nano chromatographic system coupled to a Q-Exactive HF mass spectrometer (Thermo Fisher Scientific). The chromatographic separation was carried out in 35-cm-long 100 µm inner diameter column packed in-house with 3 µm Chttp://massive.ucsd.edu) and received the ID: MSV000086764, doi:10.25345/C57N54, and ProteomeXchange ID: PXD023803. Following processing by Comet, PeptideProphet, Libra, and PTMProphet, phosphoproteomic datasets were exported as .xls files for further processing via R scripts. A first script calculated fold changes between TMT reporter ion channels using the median of Libra-provided values for each condition. This script eliminated any peptides that featured missing reporter ion intensity values for channels in both conditions for a given experiment. A second script normalized the fold changes and procedurally clustered phosphorylation sites if PTMProphet localized them to adjacent phosphorylatable residues. These clustered sites’ localization probabilities were summed, and if this cumulative score remained above 0.85, the phosphorylation sites were designated a valid cluster and remained in the dataset for further analysis. After quantification, normalization, and clustering of each experimental dataset, AZ20-treated and Rad1 cKO experiments were compared with one another such that sites seen in both sets of experiments could have their average fold changes plotted against each other on Cartesian coordinates. Quadrants were designated as Q1–Q4 delimited by an interval of correlation correspondent to fivefold of the log2 scale. Any sites whose average ratios fell within a circle of radius of 0.7-fold change centered on the origin were designated as ‘Center’ sites and were considered unregulated. Phosphorylation sites placed into quadrants were subjected to a second filtering step to account for any inconsistent ratios between replicates of each experiment. This filter removed any site that featured at least one inverted (opposite of expected quadrant sign) experimental ratio with a magnitude greater than or equal to 0.25-fold change.The peptide identification and quantification pipeline relied on the Trans Proteomic Pipeline (v. 5.2.0) and the Comet search engine (v. 2019.01 revision 5) . The Mou2O for 10 min twice and allowed to dry before mounting with DAPI/antifade. Slides were imaged on a Leica DMi8 Microscope with a Leica DFC9000 GTC camera using the LAS X (Leica Application Suite X) software. For every condition, a minimum of 50 images from three independent mice were acquired. To quantify florescence intensity, the LAS X software quantification tool was used. Briefly, a region of interest (ROI) line was drawn over the sex body and mean intensity of the underlying pixels was recorded. Additionally, two ROI lines of equal length were placed over two autosome cores and the mean pixel intensity was also recorded to serve as an internal control for background florescence. The sex body ROI intensity was then normalized to the average of the two autosomal ROI intensities for each individual cell. For the autosomal signal CDK2, a ROI was drawn along each autosomal core as identified by SYCP3 staining. The signal intensity for each autosome was then averaged per cell. For the Rad1 cKO mice, where a true sex body does not form, a line was drawn over chromosomes that were best morphologically identified as the sex chromosomes by SYCP3 staining.Meiotic spreads were prepared as previously described . BrieflyAdult testes were dissected and incubated in Bouin’s fixative for 7 hr, washed 4× in 70% ethanol and embed in paraffin. 5 mM sections were mounted on slides and rehydrated in SafeCear Xylene Substitute followed by decreasing amounts of ethanol. Slides were then stained with hematoxylin followed by eosin and gradually dehydrated by incubation in increasing concentrations of ethanol before mounting using permount mounting medium. This paper nicely provides a valuable list of phosphorylation targets by the DNA damage checkpoint kinase, ATR, during mouse spermatogenesis. Importantly, by using the dataset, the authors now showed that ATR controls the localization of CDK2 on meiotic chromosomes, which is critical for meiotic crossover formation. public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, including Akira Shinohara as Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Jessica Tyler as the Senior Editor.Thank you for submitting your article "Phosphoproteomics of ATR Signaling in Prophase I of Mouse Meiosis" for consideration by eLife as a "Tools and Resource" paper. However, we all agree that you need a major revision of your paper, particularly in respect to the relationship with the co-submitted paper with Dr. Weiss.The Reviewing Editor and other two reviewers all acknowledge the importance of this work and recommend possible publication in Since using the same data sets in co-submitted papers is unusual, we ask you to integrate most of the ATR-dependent phospho-proteomic data described in Figure 6  from the accompanying paper by Weiss into your paper. Importantly, to strengthen your conclusion in the paper, we recommend you to re-analyze your data sets. Below are the list of experiments and additional analysis that we would like you to add in the revised version. We would like revisions to be returned within two months. If the revisions take a lot longer, you may want to resubmit as a new manuscript, and we will make sure that it goes to the same editors and reviewers.Essential points (experiments):1. One major issue is on the different cellular compositions between controls and Rad1-conditonal knockout (cKO) testes. The authors likely detected developmental changes in phosphorylation sites in spermatogenesis, and the authors are aware of this caveat (Line 160-165). To circumvent this issue, the authors may perform phosphoproteomics of first-wave Rad1-cKO testes , where the cellular composition of Rad1-cKO testes was not grossly changed from that of controls. They should also show the testicular section data and confirm the cellular composition of the testis they analyze. You may add these data set as Supplement; Duplicate would be preferred, but not essential.2. There are two different experiments for ATRi-treated mice (2 pairs after 2.5-3 days of treatment and 2 pairs 4 hours after a single dose). However, these results are not distinguished in the analysis, and there is no evaluation of testicular morphology in both cases after the ATRi treatment. The authors should analyze these data separately as the experiments were distinct. Also, they should show the testicular morphology after the ATRi treatment and discussed the difference a little bit more.3. It is very interesting hypothesis raised by the authors that ATR may control the activity of other kinases and phosphatases which will be responsible for most of the phosphorylation site changes observed in this study. To follow up on this, the author may check the localization of CDK1/2, MAK, NEK1, PKMYT1, PPM1G, and/or PP1R7 in control, ATRi-treated, and RAD1cKO spermatocytes? For some of these proteins, commercial antibodies are available, even for their phosphorylated forms.Other points (text editing etc);1. Consider changing the title of the manuscript since you are studying the ATR signaling in mouse testis, not only meiotic cells.2. The abstract was overstated. The study unbiasedly identified any phosphorylation sites. The abstract is misleading because "12,000 phosphorylation sites" are all phosphorylation sites detected in this study. This is not surprising, as previous studies have already detected many more phosphorylation sites . The sentence "Here we defined ATR signaling during prophase I in mice" may be an overstatement.3. The introduction section contains several errors:4. Ref #10 did not examine meiosis.5. Line 49: CHK1 is not required for MSCI, and CHK1 and BRCA1 do not spread from the axes to chromatin loops.6. Some references for ATR's function in meiosis are missing. Please cite Fedoriw, et al., Development 2015 and Menolfi, et al., Nat Commun 2018.7. A study showed that ETAA1 unlikely functions in meiosis . Discussion about TEAA1 can be adjusted.8. There are numerous errors in figure numbers, and these results are difficult to evaluate.9. Table S1 was referenced in many places, but it is impossible to interpret the described data. Table S1 is just a list of ~12,000 sites. Appropriate data should be shown for each description.10. Figure 2G is not described in the text.11. The entire section following "Connectivity analysis defines ATR-regulated sub-networks" does not make sense. Figure 3A is not STRING analysis but is described so in the text. All figure numbers for sup data are misaligned. Line 213: there is no data about "cell cycle" in the figure.12. The data pertaining to ATR-dependent SETX and RANBP3 are interesting, but the authors cannot conclude if this is phosphor-dependent. Similarly, they cannot conclude that "ATR-dependent phosphorylation promotes the recruitment or retention of Senataxin at the sex body" (Line 298).13. Table S1 Please provide the protein name also apart from protein ID.14. Revise supplemental figure citation because I think most of them are incorrect.15. Figure 3E. I would like the authors to either refer to the Phospho-S/T-D/E sites in the main text or simplified the figure by pooling these with the other phosphorylation sites.16. I have missed in the results, or the discussion, the mention of one of the best known ATR targets (CHK1). Was this protein found in the analysis (I could not find it in Table S1)? If not, how do the authors explain this result?17. Although the authors used spermatocytes to identify the ATR-dependent targets, they have not mentioned the phosphorylation of meiosis (spermatocytes)-specific proteins. Moreover, the authors described mainly targets which have been identified in mitotic cells such as MDC1, TOPBP1, and CTIP etc. It would be interesting to compare phospho-proteome of spermatocytes with that of mitotic cells with exogeneous DNA double-strand breaks (DSBs).18. Given that ATR belongs to the PI3 kinase family, in some senses, ATR is, in some cases, redundant to ATM kinase (and DNA-PK), it would be very nice to talk about the specificity of ATRi, AZ20. This is very important since there is only weak enrichment of ATM/ATR consensus sequence, S/TQ in the data set.19. It would be nice to show what kind of meiotic defects are induced after acute and chronic treatment of ATRi such as apoptosis or impaired progression of meiosis. Essential points (experiments):1. One major issue is on the different cellular compositions between controls and Rad1-conditonal knockout (cKO) testes. The authors likely detected developmental changes in phosphorylation sites in spermatogenesis, and the authors are aware of this caveat (Line 160-165). To circumvent this issue, the authors may perform phosphoproteomics of first-wave Rad1-cKO testes , where the cellular composition of Rad1-cKO testes was not grossly changed from that of controls. They should also show the testicular section data and confirm the cellular composition of the testis they analyze. You may add these data set as Supplement; Duplicate would be preferred, but not essential.Rad1 cKO and WT testes. We believe that our dual approach of only considering sites that are depleted in both the Rad1 cKO as well as the 4 hour ATRi treated mice (which do not have changes in cellular composition) (see point 2) addresses this concern. We do acknowledge that it would be ideal to perform the phosphoproteomic experiments in Rad1 cKO 14 day-old mice, before significant cell loss has occurred. Unfortunately, the amount of tissue required to perform phosphoproteomic analysis on first-wave Rad1 cKO testes would be prohibitive and we were not able to perform this experiment. However, with the limited number of Rad1 cKO mice available to us, we were able to confirm that SETX and RANBP3 localization in 14-day old Rad1 cKO testes was not different compared to 8-12 week Rad1 cKO animals from our initial analysis, supporting the validity of our approach .We thank the reviewers for this suggestion. We agree that the differences in cellular composition is a convoluting factor when comparing the 2. There are two different experiments for ATRi-treated mice (2 pairs after 2.5-3 days of treatment and 2 pairs 4 hours after a single dose). However, these results are not distinguished in the analysis, and there is no evaluation of testicular morphology in both cases after the ATRi treatment. The authors should analyze these data separately as the experiments were distinct. Also, they should show the testicular morphology after the ATRi treatment and discussed the difference a little bit more.We thank the reviewers for this important suggestion. We have now analyzed the 2.5-3 day ATRi treatment separately from the 4 hr ATRi datasets. Importantly, we repeated the 4 hr treatment condition (now five replicates) to enhance our confidence in this dataset. We have included the 2.5-3 day ATRi dataset analysis in separate supplemental figures and tables, and have used it for the analysis of the S/T-P-X-K motif and to compare the changes in this motif over the 4hr and 2.5-3 day inhibition period. We have confirmed that tubule morphology and cellular composition is not altered in the 4 hr ATRi treatment condition and have added this data to the supplement.3. It is very interesting hypothesis raised by the authors that ATR may control the activity of other kinases and phosphatases which will be responsible for most of the phosphorylation site changes observed in this study. To follow up on this, the author may check the localization of CDK1/2, MAK, NEK1, PKMYT1, PPM1G, and/or PP1R7 in control, ATRi-treated, and RAD1cKO spermatocytes? For some of these proteins, commercial antibodies are available, even for their phosphorylated forms.We thank the authors for the positive comment on the finding that ATR is likely regulating other downstream kinases and phosphatases. We thank the suggestion of monitoring potential localization changes of some of the kinases/phosphatases upon ATR inhibition. We performed a series of IF experiments to assess the localization of several kinases/phosphatases for which antibodies were available. Despite multiple attempts, we could not detect specific signal in meiotic spreads for several of the proteins in the vehicle or 4 hour ATRi treatment condition. Excitingly, we were able to observe specific signal for CDK2 and found that its localization in meiotic spreads was significantly reduced after 4 hours of ATRi treatment . We believe that this is an important finding that highlights the potential for ATR activity to regulate CDK localization and allow CDK-dependent phosphorylation of several targets, consistent with our finding that ATRi treatment results in the loss of several phosphorylation events at the S/T-P-X-K motif . We have included this new data to a remodeled Figure 5 and updated the discussion accordingly.Other points (text editing etc);1. Consider changing the title of the manuscript since you are studying the ATR signaling in mouse testis, not only meiotic cells.We agree with this reviewer comment since our analysis was done on whole testes and not purified meiotic cell populations. We have changed the title of the manuscript to “Phosphoproteomics of ATR signaling in Mouse Testes”.2. The abstract was overstated. The study unbiasedly identified any phosphorylation sites. The abstract is misleading because "12,000 phosphorylation sites" are all phosphorylation sites detected in this study. This is not surprising, as previous studies have already detected many more phosphorylation sites . The sentence "Here we defined ATR signaling during prophase I in mice" may be an overstatement.We thank the reviewer for this feedback and have revised the abstract to more accurately describe our approach and results. We now emphasize that while we identified more than 14,000 phosphorylation sites, with 401 of these sites being dependent on ATR and RAD1. Furthermore, we have cited the publications mentioned by the reviewer.3. The introduction section contains several errors:We have carefully revised all the text in the manuscript, including introduction.4. Ref #10 did not examine meiosis.Thank you for this correction, we have removed reference 10.5. Line 49: CHK1 is not required for MSCI, and CHK1 and BRCA1 do not spread from the axes to chromatin loops.We thank the reviewer for this correction and have updated the text to correct this error.6. Some references for ATR's function in meiosis are missing. Please cite Fedoriw, et al., Development 2015 and Menolfi, et al., Nat Commun 2018.We thank the reviewer for this correction and have added these references.7. A study showed that ETAA1 unlikely functions in meiosis . Discussion about TEAA1 can be adjusted.We agree that ETAA1 is unlikely to function in meiosis, although we cannot exclude that as a possibility in our system. We have updated the introduction and discussion to reflect this.8. There are numerous errors in figure numbers, and these results are difficult to evaluate.We apologize for the errors. We thank the reviewers for pointing this and have corrected figure numbers.9. Table S1 was referenced in many places, but it is impossible to interpret the described data. Table S1 is just a list of ~12,000 sites. Appropriate data should be shown for each description.We have added more specific referencing to the data. We have also added more comprehensive table descriptors to Supplementary Files 1 and 3, which contain extensive relevant data for the descriptors in the text, including sequence motif for each site, designated quadrant, etc.10. Figure 2G is not described in the text.We thank the authors for this correction and have updated the text to describe this data.11. The entire section following "Connectivity analysis defines ATR-regulated sub-networks" does not make sense. Figure 3A is not STRING analysis but is described so in the text. All figure numbers for sup data are misaligned. Line 213: there is no data about "cell cycle" in the figure.To improve clarity, we have completely remodeled Figure 3 and related text. Parts of it were moved to supplemental data.12. The data pertaining to ATR-dependent SETX and RANBP3 are interesting, but the authors cannot conclude if this is phosphor-dependent. Similarly, they cannot conclude that "ATR-dependent phosphorylation promotes the recruitment or retention of Senataxin at the sex body" (Line 298).Rad1 cKO mice. Moreover, both SETX and RANBP3 have identified S/T-Q sites that are also lost in ATRi treated mice and Rad1 cKO mice. For the sake of extra caution, we have modified our conclusions to use the words “strongly suggest”.We believe that our results strongly suggest that the localization of SETX and RANBP3 is dependent on ATR signaling since the localization is lost in both the ATRi treated mice and 13. Table S1 Please provide the protein name also apart from protein ID.We agree and have added this to Supplementary Files 1 and 3.14. Revise supplemental figure citation because I think most of them are incorrect.Thanks, we have corrected these errors.15. Figure 3E. I would like the authors to either refer to the Phospho-S/T-D/E sites in the main text or simplified the figure by pooling these with the other phosphorylation sites.Done. Of note, previous Figure 3E is now Figure 5F. Also, we have completely remodeled previous Figure 3, which is now Figure 5 focused mostly on S/T-X-P-K sites and the new finding that ATR inhibition alters localization of CDK2.16. I have missed in the results, or the discussion, the mention of one of the best known ATR targets (CHK1). Was this protein found in the analysis (I could not find it in Table S1)? If not, how do the authors explain this result?We appreciate the reviewer bringing this to our attention. We did not detect CHK1 in our analysis, which we believe reflects technical challenges in achieving complete coverage of the phosphoproteome and the potential low abundance of CHK1 in spermatocytes. We have updated the discussion to address this concern and have also reiterated that we did identify other known ATR targets such as MDC1 and yH2AX.17. Although the authors used spermatocytes to identify the ATR-dependent targets, they have not mentioned the phosphorylation of meiosis (spermatocytes)-specific proteins. Moreover, the authors described mainly targets which have been identified in mitotic cells such as MDC1, TOPBP1, and CTIP etc. It would be interesting to compare phospho-proteome of spermatocytes with that of mitotic cells with exogeneous DNA double-strand breaks (DSBs).We appreciate this suggestion but feel this is beyond the scope of this study. We believe future work will address this point.18. Given that ATR belongs to the PI3 kinase family, in some senses, ATR is, in some cases, redundant to ATM kinase (and DNA-PK), it would be very nice to talk about the specificity of ATRi, AZ20. This is very important since there is only weak enrichment of ATM/ATR consensus sequence, S/TQ in the data set.19. It would be nice to show what kind of meiotic defects are induced after acute and chronic treatment of ATRi such as apoptosis or impaired progression of meiosis.We thank the reviewer for bringing this point and have added additional citations to previous work where the consequences of ATRi treatment for 3-7 days was discussed, showing loss of spermatocyte populations arising from DNA repair, chromosome synapsis and sex body formation defects. We also discuss the point about specificity of AZ20, which is part of a new generation of highly specific inhibitors of ATR, therefore not expected to interfere with a range of S/T-Q sites targeted by ATM or DNA-PKcs, as pointed out by the reviewer.