Of the 342 screws placed, 4% (14/342) were placed manually without the robot, due to surgeon discretion. Of the 328 screws placed with the robot, 2% (7/328) were repositioned based on the surgeon’s discretion, resulting in a 98% navigated robot-assisted pedicle screw placement success rate. In this cohort there were no revisions due to malpositioned screws. No complications due to screw placement were reported. This study demonstrates a high level (98%) of successful surgeon-assessed pedicle screw placement in minimally invasive navigated robot-assisted SP-LLIF, with no malpositions requiring a return to the OR.Minimally invasive lateral interbody fusion has distinct advantages over traditional posterior approaches. When posterior stabilization is needed, percutaneous placement of pedicle screws from the lateral decubitus position may potentially increase safety and improve operative efficiency by precluding the need for repositioning. However, safe placement of pedicle screws in the lateral position remains technically challenging. This study describes the pedicle screw placement of single-position lateral lumbar interbody fusion (SP-LLIF) cases in which navigated robotic assistance was used. A single-surgeon, single-site, retrospective Institutional Review Board-exempt review of the first 55 SP-LLIF navigated robot-assisted spine surgery cases performed by the lead author was conducted. An orthopaedic surgeon evaluated screw placement using plain film radiographs. In addition, pedicle screw malposition, reposition, and return to operating room (OR) rates were collected. In the first 55 SP-LLIF cases, 342 pedicle screws were placed. The average patient age and body mass index were 67 years and 29.5 kg/m Open lumbar interbody fusion is a popular method for treating patients with spinal back and leg pain who have failed conservative management. However, this technique is associated with decreased functional outcomes due to greater muscle and tissue disruptions, which may lead to a higher complication rate –3. MinimMinimally invasive lateral lumbar interbody fusion is a retroperitoneal approach that allows the placement of a larger implant, allowing for a larger fusion bed. When posterior stabilization is needed, percutaneous placement of pedicle screws from the lateral decubitus position precludes the need for repositioning, potentially increasing safety and reducing OR time, cost, and radiation. However, achieving safe placement of pedicle screws in the lateral position remains technically challenging.There is of high interest in the emerging field of robot-assisted spine surgery with navigation; however, studies evaluating the safety and accuracy of this technique are needed. This study describes the pedicle screw placement in single-position lateral lumbar interbody fusion (SP-LLIF) cases with navigated robotic assistance.®, Globus Medical, Inc. Audubon, PA, USA) were reviewed. An orthopaedic surgeon evaluated screw placement using plain film radiographs. Data on pedicle screw malposition, reposition, and return to OR rates were collected.This is a single-surgeon, single-site, retrospective Institutional Review Board-exempt review of the first 55 consecutive SP-LLIF navigated robot-assisted spine surgery cases. The demographic, intraoperative, and perioperative data of 55 patients who underwent lumbosacral pedicle screw placement with a minimally invasive navigated robot-assisted pedicle screw positioning system (ExcelsiusGPSA computed tomography (CT) scan of the spinal levels in the operative field was taken prior to the patient entering the OR, and screw placement planning was completed. The CT data set was transferred into the robotic positioning system, and then registration was completed for vertebral levels.In intraoperative CT mode, the image coordinate system was obtained from a portable intraoperative CT  or standard CT scan was taken at the time of surgery, with the patient already in position on the OR table. Spinal levels were identified and a CT scan was taken. Pedicle screw trajectories were planned and saved.The patient was positioned in the lateral decubitus position Fig. . A dynamFollowing pedicle screw placement, lumbar interbody fusion was performed using the lateral approach. The endplates were prepared and an interbody spacer of appropriate size was manually inserted. Rods were then placed in a standard fashion, with the patient remaining in the lateral position. Locking caps were set once the rods were in their proper position.Intraoperative fluoroscopy images were taken to verify the pedicle screw, interbody spacer, and rod position. Pedicle screw placement was qualitatively assessed using postoperative X-rays Fig. .Fig. 4Po® SPSS® Version 25 software . Data were presented as mean ± standard deviation. Statistical significance is shown at P < 0.05.The statistical analysis was performed using IBM2. Fifteen patients (27%) were either former or current smokers. The majority of patients were either retired (53%) or worked full time (31%) (Table The average patient age was 67 years and 49% (27/55) were female. The average BMI was 29.5 kg/mll time 3%  were treated at L4. Thirty-five percent (19/55) of patients underwent a two-level SP-LLIF. The average estimated blood loss was 117 cc. The average overall operative time was 155 min. The average length of hospital stay was 2.9 days. The most common workflow with the minimally invasive navigated robot-assisted pedicle screw positioning system was intraoperative CT (75%)  were placed manually, without the robot due to surgeon discretion. Of the 328 screws placed with the robot, 2% (7/328) were repositioned based on the surgeon’s discretion, resulting in a 98% screw placement success rate for navigated robot-assisted pedicle screw placement. In this cohort there were no revisions due to malpositioned screws. No complications due to screw placement were reported.This study demonstrates a high screw placement success rate in SP-LLIF with navigated robot-assisted spine surgery in the lumbosacral spine. High revision rates or neurological injuries with conventional freehand and two-dimensional fluoroscopy-guided pedicle screw placement has led to the development of multiple approaches to reduce complications and improve the accuracy of pedicle screw placement , 9. The The lateral approach facilitates thorough intervertebral disc preparation, creates a large surface area for fusion, and allows placement of a large elongated interbody implant while maintaining the annulus and the anterior longitudinal ligament –12. MiniThe advantages of minimally invasive surgery include less disruption of the paraspinal muscles and sparing of the posterior spinal column, as well as reduction of postoperative pain, operative time, and length of hospital stay. Wide exposure of the disc space in any anterolateral approach allows for the insertion of large interbody spacers, which is not possible with posterior or transforaminal lumbar interbody fusion due to proximity to the thecal sac and neural elements. An LLIF allows for the insertion of a long and wide interbody spacer that reaches the endplate diaphysis on both sides, allowing for a larger surface area for indirect decompression , 12, 13.Literature on pedicle screw placement success rate is variable. Santos et al.  studied The SP-LLIF approach has not gained widespread approval. Reports of neurological complications to the lumbar plexus range from 0.7-30% in the literature, which suggests inconsistent reporting . To reduInserting screws and interbody spacers using the SP-LLIF approach can be challenging for inexperienced surgeons due to the patient’s position. The robot used in this study is well-suited for the SP-LLIF approach, providing rigidity and stability to maintain the optimal trajectory during pedicle screw placement .Although this is a single-surgeon, single-site, retrospective study without a comparative control, the results are consistent with findings from the literature. This study forms the foundation for future studies with a higher level of evidence. Comparative studies with larger sample sizes and longer follow-up are needed to determine the effectiveness of SP-LLIF versus repositioning after LLIF.


Overexpression of members of the HER/erbB transmembrane tyrosine kinase family like HER2/erbB2/neu is associated with various cancers. Some heterodimers, especially HER2/HER3 heterodimers, are particularly potent inducers of oncogenic signaling. Still, from a clinical viewpoint their inhibition has yielded only moderate success so far, despite promising data from cell cultures. This suggests acquired resistance upon inhibitor therapy as one putative issue, requiring further studies in cell culture also aiming at rational combination therapies. In this paper, we demonstrate in ovarian carcinoma cells that the RNAi-mediated single knockdown of HER2 or HER3 leads to the rapid counter-upregulation of the respective other HER family member, thus providing a rational basis for combinatorial inhibition. Concomitantly, combined knockdown of HER2/HER3 exerts stronger anti-tumor effects as compared to single inhibition. In a tumor cell line xenograft mouse model, therapeutic intervention with nanoscale complexes based on polyethylenimine (PEI) for siRNA delivery, again reveals HER3 upregulation upon HER2 single knockdown and a therapeutic benefit from combination therapy. On the mechanistic side, we demonstrate that HER2 knockdown or inhibition reduces miR-143 levels with subsequent de-repression of HER3 expression, and validates HER3 as a direct target of miR-143. HER3 knockdown or inhibition, in turn, increases HER2 expression through the upregulation of the transcriptional regulator SATB1. These counter-upregulation processes of HER family members are thus based on distinct molecular mechanisms and may provide the basis for the rational combination of inhibitors. The HER/erbB transmembrane tyrosine kinase family comprises four members, HER1–HER4 (erbB1–erbB4). Receptor signalling is based on ligand-mediated receptor dimerization and subsequent phosphorylation, leading to the activation of downstream signal transduction. This includes the MAPK and PI3K-Akt pathways that promote cell growth, survival, invasion and angiogenesis and thus play pivotal roles in tumor cells . HoMechanisms of inherent or acquired resistance of tumors to HER-inhibiting therapies include somatic HER receptor mutations, compensatory signalling through activation of downstream signalling effectors or through receptors outside of the HER family, or increased expression of HER ligands  has been identified as a particularly potent mechanism of gene silencing that relies on small interfering RNAs (siRNAs) for sequence-specific mRNA cleavage and degradation . HoweverThe high potency of HER2-containing heterodimers and the emerging relevance of HER3 in solid tumors prompted us to analyse the effects of a double inhibition approach of HER family members through RNA-mediated dual knockdown. Interestingly, beyond the enhancement of anti-tumor effects upon the simultaneous inhibition of HER2 + HER3, we also found that this double knockdown counter-acts autocrine cellular adaptation processes that are based on the rapid counter-upregulation of the respective other HER family members after HER2 or HER3 knockdown, respectively. These effects, which rely on distinct molecular pathways, are also observed upon HER2 or HER3 inhibition through treatment with the inhibitors . As a mechanism of secondary resistance, it must be expected that therapeutic effects of the inhibition of a single HER receptor are limited by the counter-upregulation of other family members. Thus, our data establish a cellular and molecular basis for a combined inhibition/knockdown strategy to achieve more profound therapeutic effects and avoid rapid cellular adaptation processes.2) in IMDM  supplemented with 10% fetal calf serum (FCS). Athymic nude mice (Hsd:Athymic Nude-Foxn1nu) (nu/nu) were obtained from Harlan Winkelmann  and kept at 23 °C in a humidified atmosphere with food and water ad libidum. Animal studies were approved by the Regierungspräsidium Giessen, Germany.Chemically unmodified siRNAs were obtained from Thermo Scientific  or Dharmacon . A list of all siRNAs can be found in CP(target)/2CP(actin) with CP = cycle number at the crossing point (0.3).For the preparation of total RNA, the Tri reagent  was used according to the manufacturer’s protocol. Tissues were homogenized prior to RNA preparation in 200 μL Tri reagent. MiRNA was isolated using the miRVana Kit (Ambion) according to the manufacturer’s protocol. Reverse transcription (RT) was performed using the RevertAid H Minus First Strand cDNA Synthesis Kit from Fermentas  as described previously . Quantit6, 5 × 105 and 1 × 105 cells (6-well plate) for harvest after 72, 120 and 168 h after transfection, respectively, at 3 × 105 cells (12-well plate) for harvest at 48 h post transfection (NFκB reporter assay) and at 5 × 103 cells/well . Twenty-four hours after seeding, transfections were performed by the addition of the specific or non-specific siRNA , complexed with Interferin  according to the manufacturer’s protocol. Plasmid DNA was transfected at 1.5 μg or 0.5 μg/well of a 6-well or a 48-well plate, respectively, using PEI F25-LMW as described  fo fo41] foOur data, as well as previous findings on compensatory mechanisms that enhance HER2/HER3 signalling ,46, alsoAiming at combination therapies, alternative approaches of HER inhibition may provide advantages over antibodies or low molecular weight inhibitors. RNAi has been recognized as promising since it allows the highly specific knockdown of any given target gene, when using appropriate siRNAs, and their defined combination in one formulation. The major bottleneck so far, however, has been the efficient non-viral delivery of therapeutic siRNAs in vivo. Several strategies have been developed that may provide an avenue towards the therapeutic exploration of siRNAs (see e.g.,  for revi


Whereas transcriptional activation of kdpFABC expression is well studied, a mechanism for down-regulation when K+ levels are restored has not been described. Here, we show that KdpFABC is inhibited when cells return to a K+-rich environment. The mechanism of inhibition involves phosphorylation of Ser162 on KdpB, which can be reversed in vitro by treatment with serine phosphatase. Mutating Ser162 to Alanine produces constitutive activity, whereas the phosphomimetic Ser162Asp mutation inactivates the pump. Analyses of the transport cycle show that serine phosphorylation abolishes the K+-dependence of ATP hydrolysis and blocks the catalytic cycle after formation of the aspartyl phosphate intermediate (E1~P). This regulatory mechanism is unique amongst P-type pumps and this study furthers our understanding of how bacteria control potassium homeostasis to maintain cell volume and osmotic potential.KdpFABC is an ATP-dependent K The latter is responsible for sensing the environment and controlling the expression of the former  than the documented rate of protein turnover in bacteria  in the catalytic cycle of related P-type ATPases and the lack of precedent for an analogous post-translational modification in this well-studied superfamily of ATP-dependent cation pumps (307KTGT) in the centrally located cytoplasmic domain (P-domain). This phosphoenzyme intermediate (E1~P) transiently harnesses the energy of ATP, which is then used to drive conformational changes that lead to ion transport. The highly conserved TGES motif is found in a second cytoplasmic domain (A-domain) with Glu161 playing a crucial role in hydrolysis of the aspartyl phosphate in the second half of the catalytic cycle (E2-P → E2)  . Biochem-P → E2) . An X-ra-P → E2) . This cr-P → E2) , reveale+ concentrations, we found that moving cells to K+-rich media resulted in an inhibition of ATPase activity. We found that KdpFABC inhibition was time-dependent, was proportional to the extent of serine phosphorylation, and was restored by removing the phosphate with lambda protein phosphatase (LPP). To elucidate the mechanism of inhibition, we expressed a variety of mutants using the pBAD promoter in cells grown in K+-rich media and measured steady-state levels of the aspartyl phosphate intermediates (EP). We introduced the KdpB-S162D mutation to mimic the effect of serine phosphorylation and the KdpB-S162A mutation to prevent serine phosphorylation. Despite a lack of inhibition with KdpB-S162A, we found that EP levels for this mutation were much lower than for serine-phosphorylated, WT KdpFABC or for the KdpB-S162D mutation. Pulse chase studies with ADP indicated that both serine phosphorylation and the KdpB-S162D mutation trapped the pump in the E1~P state. Furthermore, although EP formation with the KdpB-S162A mutant was K+-dependent, serine phosphorylation as well as the phosphomimetic S162D mutation eliminated this K+-dependence. These observations suggest that serine phosphorylation disrupts allosteric coupling of the pump and provides secondary level of control over K+ homeostasis.To further explore the inhibitory effects of serine phosphorylation, we have established growth conditions that lead to phosphorylation of Ser162 on KdpB and have characterized its effect on individual steps in the reaction cycle. Consistent with previous observations , when wiE. coli cells, the promoter that controls transcription of kdpFABC genes is regulated by KdpE in response to ‘potassium need’ of the cell. In K+ replete conditions, KdpFABC is not expressed, but when K+ levels in the media fall into the micromolar range, the constitutive transport systems  fail to maintain the necessary chemo-osmotic gradient. KdpD senses this deficiency and activates KdpE such that transcription of kdpFABC is initiated. WT KdpFABC is a powerful pump, so the deficit is normally overcome by modest levels of expression. In order to obtain over expression, as required for structural or biochemical studies, a multicopy plasmid with kdpFABC driven by the native promoter is introduced into a cell line (TK2499) with Trk and Kup knocked out and cells are cultured with limiting concentrations of K+, which must remain in the low micromolar range for expression of WT kdpFABC . Inactive mutants cannot be expressed using this strategy, because they are incapable of rescuing cell growth in a K+-deficient environment. In those cases, the pBAD promoter can be used for expression with Top10 cells in K+ replete conditions (e.g. for previous cryo-EM structures), or a chromosomal copy of the kdpFABC gene can be used for rescue (TK2498 cells) and the mutant protein can be expressed from a plasmid carrying the native promoter. In this case, a His-tag is used to separate the mutant from the chromosomal copy by affinity chromotagraphy. In the following studies, we have used all three expression strategies. As we learned over the course of this work, the K+ concentration in the culture media is key to serine phosphorylation, making the expression protocol an important parameter to keep in mind.We used three different expression strains and a variety of different growth conditions to study the physiological stimulus for serine phosphorylation and to produce KdpFABC for in vitro experiments , Table 1 kdpFABC . Because+ transport. To this end, we setup a survey of alanine substitution mutants to assess the role of various residues implicated by the X-ray crystal structure. For expression of these mutants, we used E. coli strain TK2498 grown at 0.2 mM K+ (+ affinity and was therefore expressed at much lower levels of K+ (<10 μM) in the TK2499 strain. These results motivated us to examine whether phosphorylation of Ser162, and consequent inhibition of KdpFABC, was a cellular response to elevated K+ in the media.An overarching goal of our work is to understand the allosteric coupling between KdpA and KdpB that underlies ATP-dependent K.2 mM K+ , Table 1.2 mM K+ , causing.2 mM K+ , we used.2 mM K+ , which w.2 mM K+ . We then.2 mM K+ , thus su+ concentrations lead to serine phosphorylation, we first grew TK2499 cells with WT KdpFABC under K+ limiting (micromolar) conditions and then split the culture into two batches immediately prior to harvest. To one batch, we added 20 mM K+ - so called K+ shock - and growth was continued for 20 min. KdpFABC was then purified from both batches. ATPase assays documented a 40% inhibition for protein derived from the cells subjected to the K+ shock, which was fully restored after treatment with LPP  and LPP treatment largely relieved this inhibition  and confirmed Ser162 as the site of phosphorylation , which includes molecules in both E1~P or E2-P states  decay over time as the ATP in the solution is exhausted  and initial points for experiments with high turnover rates (i.e. at room temperature in the presence of K+) or pulse chase (see below). Normalization of data from each experiment was based on data from E161Q/S162A/Q116R, which consistently produced the highest EP levels and which were stable under all the experimental conditions that were tested. Thus, data in +. In contrast, EP levels for the Ser162A construct, which cannot be serine phosphorylated, are much lower and showed a significant dependence on K+. As expected, the D307A construct produced negligible levels of EP. These data suggest that formation of the high-energy aspartyl phosphate  and inactive (serine phosphorylated) molecules. At first glance, one might expect that the fraction of inactive molecules with serine phosphorylation would produce stable EP levels while unmodified, active molecules would turnover and very slowly exhaust the ATP in the solution. However, ADP is produced as the active molecules turnover which will then react with and dephosphorylate the molecules arrested in the E1 ~P state, as seen in the pulse chase experiments. This behavior would then account for the observed fall in overall EP levels of partially phosphorylated Q116R mutants over time.+ and ATP as well as inhibition by orthovanadate. Orthovanadate acts as a transition state analogue for inorganic phosphate and typically binds to the E2 state of P-type ATPases to form an inhibitory complex  and ATP (60 μM and 72 μM) were not statistically significant. Similarly, inhibition by orthovanadate under standard ATPase conditions (150 mM K+ and 2.4 mM ATP) indicate no significant change in IC50 (9.2 μM vs. 8.4 μM). Although not a comprehensive analysis of reaction cycle kinetics, these results reveal no significant changes in the affinity of these substrates and are therefore consistent with the interpretation that the KdpA-Q116R preparation consists of a fraction of molecules with normal binding affinities and a fraction of inactive molecules with serine phosphorylation. Although we consider it unlikely, it is possible that the Q116R mutation is masking an effect of serine phosphorylation on K+ affinity of the WT protein, which is expected to be in the micromolar range  was used to report electrogenic transport of K+ into the vesicles . This difference might reflect the much higher expression levels used for the current study. Specifically, we employed a multicopy plasmid in order to produce high levels of protein expression suitable for biochemical purification compared to the single chromosomal copy used in previous work. The higher expression level might affect the extent of the phosphorylation either by exceeding the capacity of the (still unknown) kinase system or by changing the ionic balance of cells and thus influencing the sensing mechanism. In any case, the inability to completely inhibit KdpFABC is likely to be detrimental to the cells, either by exhausting ATP levels or by creating an ionic imbalance. Either effect could interfere with progressive inhibition during long K+ shock experiments  (+-ATPases (2+-ATPase from sarcoplasmic reticulum (SERCA)  , yeast a-ATPases , and the-ATPases , the pum (SERCA)  and FxYD (SERCA) . In mostE. coli: ubiB and yegI . In contrast, inactive constructs such as S162D, E161Q, and the serine phosphorylated species are essentially stuck in one of these phosphoenzyme states. ADP-sensitivity seen in the pulse chase experiments show that E1~P is the prevalent species and that serine phosphorylation prevents, or at least greatly slows down, the E1~~P to E2-P transition. In contrast, the E161Q mutation typically interferes with hydrolysis of the aspartyl phosphate . It seems plausible that a phosphate moiety on Ser162 would sterically interfere with this interaction, thus preventing formation of E2-P. For the Q116R/S162A/E161Q, the lack of serine phosphorylation allows this construct to achieve the E2-P state, but the isosteric E161Q mutation prevents hydrolysis of the aspartyl phosphate that is normally orchestrated by this residue. Finally, although serine phosphorylation eliminates the K+-dependence of EP formation, the complex is not truly uncoupled since transportof K+ across the membrane, both active and passive, is also inhibited.In addition to this inhibition of catalytic activity, serine phosphorylation of KdpFABC also eliminates the Kncertain , the canby SERCA  and acceby SERCA . The unu4 in samples used for the cryo-EM studies.This mechanism of inhibition differs from our earlier speculation that salt bridges between the serine phosphate and the N-domain simply held the protein in the inactive conformation seen in the X-ray structure . Indeed,+ homeostasis in bacteria. Fundamental questions remain regarding the molecular mechanism of KdpFABC, including the role of a unique tunnel that runs between the subunits and the precise path taken by K+ as it crosses the membrane. Given the effects that we have documented on the enzymatic cycle, efforts should be made to ensure that serine phosphorylation is not a factor in future structural and biochemical work on this intriguing membrane pump.In conclusion, our work has characterized a novel mechanism for regulating P-type ATPases and maintaining KE. coli strain TK2499 , which lacks all other K+ transport systems with genotype F−, thi, rha, lacZ, nagA, trkA405, trkD1, Δ(kdpFABC)5, Δ(ompT) 2SO4, 0.4 mM MgSO4, 6 μM FeSO4, 1 mM sodium citrate, 0.2% glucose, 1 μg/mL thiamine, 50 μg/mL carbenicillin) supplemented with additions of 10 μM KCl every 90 min after culture density reached an OD600 of ~0.1. Total culture volumes ranged between 4 and 9 L. Cells were harvested at an OD600 of ~0.3–0.4 by centrifugation at 3500 g for 20 min; the resulting cell pellets were frozen at −80°C for storage. For K+ shock experiments, the culture was divided into equal portions and 20 mM KCl was added from a 2M stock solution followed by further incubation for defined periods of time prior to harvest.For studies of serine phosphorylation, the pSD107 plasmid was transformed into  Δ(ompT) . This plE. coli strain TK2498 with genotype F−, thi, rha, lacZ, nagA, trkA405, trkD1, kdpA-Q116R, Δ(ompT), also from W. Epstein . This culture was transferred to 500 mL K1-medium (K0-medium supplemented with 1 mM KCl) and incubated at 37°C for 8 hr. The 500 mL cell culture was transferred again to 18 L K0.2-medium (K0-medium supplemented with 0.2 mM KCl) and incubated at 31°C to induce expression of kdpFABC. Cells were harvested at a culture density of OD600 = ~0.8 by centrifugation at 3500 g for 20 min at 4°C and cell pellets were either frozen or used immediately for purification.To generate site-specific Ala mutants, site-directed PCR mutagenesis was used to alter codons in the WT operon on the plasmid described above. These plasmids were transformed into  Epstein . Unlike  Epstein . This taE. coli strain Top10 (Invitrogen). Cells were grown in 4L flasks at 37°C in LB media supplemented with 100 mg/mL ampicillin to an OD600 of 0.5. Expression was then induced by addition of 0.02% arabinose and the culture continued to grow at 37°C for 4 hr. Cells were then harvested by centrifugation as above and frozen for storage. Total culture volumes were typically 2 L.For studies of EP formation, the kdpFABC operon with the 8x histidine tag was cloned into the pBAD vector  and introduced into 2, 10% glycerol, protease inhibitor tablets  and 1 mM DTT, and lysed with an Emulsiflex C3 high-pressure homogenizer . Whole cells and debris were removed by centrifugation for 15 min at 12,000 g, and membranes were pelleted by centrifugation for 2.5 hr at 90,140 g. Membranes were solubilized by overnight incubation in 50 mM Tris pH 7.5, 600 mM NaCl, 10 mM MgCl2, 10% glycerol, 1 mM TCEP and 1.2% n-decyl-β-maltoside (DM) at 4°C using 20 mL per gram of membrane. After centrifugation for 30 min at 90,140 g, the solution was loaded on a 5 mL Ni-NTA HiTrap chelating column  that was equilibrated in Ni-NTA base buffer  supplemented with 20 mM imidazole. The column was washed with 20 mL of this same buffer, followed by 20 mL of Ni-NTA base buffer supplemented with 50 mM imidazole. Stepwise 5 mL elutions (collected in 1.5 mL fractions) were then performed as the imidazole concentration was incremented in steps of 50 mM up to a final concentration of 300 mM imidazole. Alternatively, elution was done with a continuous gradient running from 50 to 500 mM imidazole. The fractions containing KdpFABC were pooled, concentrated to ~0.7 mL using a 100 kDa cut-off Amicon centrifugal filter  and introduced onto a Superdex 200 size exclusion column  pre-equilibrated with 25 mM Tris pH 7.5, 100 mM KCl, 10% glycerol, 1 mM TCEP, and 0.15% DM. For measurements of EP formation, the NaCl was substituted for KCl during this purification. Fractions were either stored for short periods (days) at 4°C or frozen in liquid nitrogen and stored at −80°C.For purification of the KdpFABC complex, cells were resuspended in 50 mM Tris pH 7.5, 1.2 M NaCl, 10 mM MgClThe Phospho-Tag phosphoprotein gel stain kit  was used to evaluate levels of serine phosphorylation. Specifically, 20 μg of precipitated protein was resuspended in sodium dodecylsulfate (SDS) buffer and run on a 10% SDS-PAGE gel. After staining according to manufacturer’s instructions, gels were imaged in a Chemidoc imaging system  using the epi-green illumination for Cy3 fluorescence. KdpB-S162A was used as a negative control and as background in quantifying band intensities.4HCO3 and incubated for 4 hr at 37°C, followed by 500 ng GluC or AspN protease (Promega) overnight. Digestion was stopped with 10% formic acid and peptides extracted with 5% formic acid in 50% acetonitrile, followed by final extraction with acetonitrile. Combined peptide pools were concentrated to a small droplet by centrifugation under vacuum. The resulting peptides were desalted using a Stage Tip manually packed with Empora C18 High-Performance Extraction Disks (3M) . Slices from SDS gels were digested with 500 ng trypsin solution (10 ng/μL)  in 50 mM NHsks (3M) . Desalte6 ions. Precursors were isolated with a window of 1.6 Th, survey scans were acquired with a resolution of 120,000 at m/z 200 and HCD spectra with 15,000 at m/z 200.LC-MS/MS was performed with a ThermoEasy nLC 1000 ultra-high-pressure UPLC system  coupled online to a Q Exactive HF mass spectrometer with NanoFlex source (ThermoFisher Scientific). Analytical columns (25–30 cm long and 75 μm inner diameter with 8 μm spray needle) were packed in-house with ReproSil-Pur C18 AQ 3 μm reversed-phase resin . The analytical column was placed in a column heater  regulated to a temperature of 45°C. The peptide pool was loaded onto the analytical column with buffer A (0.1% formic acid) at a maximum back-pressure of 300 bar; peptides were eluted with a 2-step gradient of 3% to 40% buffer B (100% ACN and 0.1% formic acid) over 40 min and 40% to 90% B over 5 min, at a flow rate of 250 nL/min over 60 min before direct infusion into the mass spectrometer. Mass spectrometric data were acquired using a data-dependent (DDA) top-15 method, dynamically choosing the most abundant, not-yet-sequenced precursor ions from the survey scans (300–1750 Th). Peptide fragmentation was performed via higher energy collisional dissociation with a target value of 10Mass spectra were first processed with Proteome Discoverer 1.4 and then compared with a SwissProt Database with a Taxonomy filter for E coli  from 072014 using Mascot . Decoy protein sequences with a reversed order were included to estimate false discovery rates, which averaged ~1% at protein and peptide levels. Peptide spectral matches were further analyzed with Thermo Scientific Xcalibur software. Phosphoserine peptide ion intensities  were manually extracted, and intensity ratios of singly phosphorylated peptides relative to total (phosphorylated and nonphosphorylated) peptide intensities were calculated for each sample.2, 9.6 U/mL lactate dehydrogenase, 9.6 U/mL pyruvate kinase, 2.4 mM ATP, 0.5 mM phosphoenol-pyruvate, 0.36 mM NADH, and 0.15% DM. Concentrations of KCl and ATP were varied for determination of KM. Orthovanadate was prepared by adjusting the pH of a 200 mM solution of sodium othovanadate to 10 using NaOH, boiling until the yellow color cleared, cooling to room temperature, and repeating this cycle until the pH stabilized. Assays were run in triplicate and data from titrations with K+, ATP and VO4 were fit using Prism8 software .Rates for ATP hydrolysis were measured with a coupled enzyme assay  with 5 μ+ transport, KdpFABC was reconstituted into proteoliposomes using the method of E. coli polar extract and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine)  was prepared by evaporating a chloroform stock solution (25 mg/ml) with Ar gas followed by 2 hr in a vacuum chamber. This dry film was resuspended in transport buffer  to make a 10 mg/mL lipid stock solution, which was subjected to five cycles of freezing and thawing using liquid N2. The stock solution was then extruded 13 times through a 400 nm nucleopore filter  to create homogenous, unilamellar liposomes. Proteoliposomes were made at a lipid:protein weight ratio of 20:1 by combining 1.25 mg liposomes, 62.5 μg of KdpFABC, and 375 μg Triton X-100 (Sigma Aldrich) in a total volume of 250 μL. This solution was stirred at room temperature for 30 min followed by addition of 7.5 mg BioBeads (BioRad) and a further 90 min incubation. Finally, 15 mg BioBeads were added before overnight incubation with stirring at 4°C. Reconstituted proteoliposomes were harvested and bath-sonicated three times for 10 s each before use in the transport assay.For measurement of KThe transport assay was performed as described by 2, 0.15% DM and 10 mM of either KCl or NaCl. The reaction was started by addition of 100 μM [γ32P]-ATP and 200 μL aliquots containing 25 μg of protein and 2.7 μCi were taken at intervals of 0, 15, 30, 60, 120 s and quenched with 300 μl of 35% ice cold trichloroacetic acid (TCA). For the pulse-chase experiments, samples were incubated for 15 s at 4°C, an aliquot was recovered followed by addition of 10 mM ADP and 10 mM EDTA with additional aliquots taken various time intervals. After completing the assay, aliquots were centrifuged for 10 min at 14,000 g and washed twice with either 5% ice-cold TCA (for filtration) or water (for SDS-PAGE). For filtration, pellets were resuspended in 500 μL 5% TCA, filtered with 0.22 mm MCE filters  and washed three times with 5% TCA. Radioactivity was then quantified with a scintillation counter. For SDS-PAGE, the pellets were resuspended in 30 μL of 5 mM TRIS-phosphate pH 5.8, 6.7 M urea, 0.4 M DTT, 5% SDS, 0.014% bromophenol blue. The stacking gel contained 4% polyacrylamide, 65 mM TRIS-phosphate pH 5.5, 0.1% SDS. The resolving gel contained 7.5% polyacrylamide, 65 mM TRIS-phosphate pH 6.5, 0.1% SDS. The running buffer was 0.17 M MOPS pH 6 and 0.1% SDS. Gels were dried and exposed to a storage phosphor screen overnight which was then imaged with a Typhoon Trio+ . For each construct, data was obtained from three to four independent cell expression/purification procedures and individual samples were measured in triplicate.For measurements of EP formation, aliquots of purified KdpFABC were diluted to 0.25 mg/ml in 50 mM HEPES pH 7.8, 2 mM MgCl In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:This paper provides convincing evidence that post-translational modification by phosphorylation is a key feature of the bacterial potassium uptake system KdpFABC, an unusual complex of a P-type ATPase (subunit KdpB) and a potassium channel. Specifically, the results show that phosphorylation serves to inhibit the complex once cellular potassium levels have been restored, a novel regulatory strategy that may present another unique feature of KdpFABC systems, but that also provides a new lens through which other P-type ATPases should be viewed.Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Olga Boudker as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Howard Young (Reviewer #3).Thank you for submitting your article "Serine Phosphorylation Regulates the P-type Potassium pump KdpFABC" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission. Please aim to submit the revised version within two months.Summary:+ into the cell thereby restoring the intracellular potassium concentration. Surprisingly, recent structural data for KdpFABC complexes revealed the phosphorylation of a conserved TGES motif in the A domain of KdpB, and subsequent experiments demonstrated that phosphorylation reduces the ATPase activity of KdgFABC. Using activity and phosphorylation assays, and mass spectrometry data, Sweet et al. demonstrate that this post-translation regulation occurs in response to increased K+ levels. Phosphorylation therefore serves to inhibit the complex once the cellular potassium levels have been restored. This novel regulatory strategy may present another unique feature of KdpFABC systems, but also provides a new lens through which other P-type ATPase should be viewed.The bacterial potassium uptake system KdpFABC is an unusual complex of a P-type ATPase (subunit KdpB) and a potassium channel. Its expression levels are increased in response to low-potassium stress conditions, at which point it utilizes ATP to pump KEssential revisions:While the observations regarding the role of Ser162 in phosphorylation are clear and robust, more conclusive data must be provided regarding the molecular basis of inhibition, according to the following:1) The results describing the experiments identifying the step in the Post-Alber cycle affected by phosphorylation are confusing, in part because of the various mutants and growth conditions. This section requires clarification. It may also help to rename E161Q to reflect the additional mutations that accompany it.2) Based on Figure 4 it is concluded that KdpFABC is inhibited in the E1~P state. There are two issues with the experiment.- First, all variants were produced at high K, and thus in the Q116R mutant, Ser162 would be mostly phosphorylated. The authors claim that the phosphorylation of Asp307 is uncoupled from the presence of potassium, which is why high phosphorylation is found for the inhibited variants Q116R and S162D . But why does the EP level of Q116R decrease over time, if it is inhibited? The "rate" of dephosphorylation is at least as fast as for S162A, which is not inhibited and the dephosphorylation is nearly 100%. Considering that only a fraction of the protein (e.g. 75%) is inactive, the dephosphorylation should only apply to a small portion of the phosphorylated protein. How does this experiment look for Q116R expressed at low K? Is the initial EP level much lower due to fast turnover as suggested for S162A? And is the dequenching even faster?- Second, from the pulsed-chase experiment the authors claim that the protein is trapped in an E1~P state. However, from the inhibition with orthovanadate the authors concluded that the equilibrium between E1/E2 is not affected by the phosphorylation. In the best case this statement is only a bit misleading as it only refers to the non-phosphorylated fraction. If something else was meant, this statement should be clarified. Further, the pulsed-chase experiment can be explained by two scenarios: the inhibition of the protein in one state and the E1-E2 transition as rate-limiting step. For S162A the authors claim the experiment reflects the latter, while S162D is inhibited in E1-~P.3) The plots in 2G and 2H are misleading as they are based on the assumption that upon 100% phosphorylation the ATPase activity is completely abolished. However, there is no formal proof of this. In fact, the maximal inhibition seen is approx. 60% after 90 min K shock when compared to the dephosphorylated sample. On the other hand, the level of phosphorylation cannot be reliably estimated, neither from mass spec nor from the phos-tag signal. One could thus speculate that the rate of phosphorylation is even higher, particularly because no significant increase was detected upon K shock after 20 minutes , and that the phosphorylation does not completely inhibit KdpFABC but instead slows down the state transitions (e.g. E1 to E2) such that 100% inhibition would never be seen. This scenario may also explain the experiments performed in Figure 4.4) The molecular dynamics simulations presented rely on the deletion of the phosphate group from Ser162 as a proxy for the dephosphorylated state. This assumption is flawed as it involves artifactual, high-energy states, and does not consider the metastable conformations that are likely to be involved immediately before or after kinase/phosphatase activity. Moreover, the simulations of the phosphorylated structure were initiated using a system in which the cavities in the structure have not been hydrated , with the exception of one highly-ordered water molecule observed in the structure. Such cavities are unlikely to become hydrated on the timescale of the equilibration, but could have a significant effect on the dynamics of the protein system. Therefore, these simulation data should be removed. In case the authors chose to provide new computational data in its place, substantially more detail must be provided to describe the analysis, e.g. which atoms are included in RMSD calculations. Please also note that radial distribution functions are not typically appropriate for describing residue-residue interactions; distance distributions are preferable.5) A central theme of the proposed mechanism is that Ser162-Pi interacts with K357+R363, as observed in the available crystal structure (PDB code 5MRW) and that dephosphorylation would abolish this interaction leading to an allosteric effect on the transmembrane domains. However, other structures of KdpFABC  contain Ser162-Pi and yet do not show this interaction, despite representing, at least in one case, a similar state in the transport cycle. Moreover, one of those structures is an E2 state of Ser-phosphorylated KdpB, which questions the hypothesis that the phosphorylated state is truly inhibited in an E1 conformation. Thus, any discussion of a molecular mechanism must carefully and convincingly consider those structures and their implications. eLife. Your article has been reviewed by Olga Boudker as the Senior Editor, a Reviewing Editor, and two reviewers. The reviewers have opted to remain anonymous. The Reviewing Editor has also reviewed the article, and has drafted this decision to help you prepare a revised submission.Thank you for submitting your article "Serine Phosphorylation Regulates the P-type Potassium pump KdpFABC" for consideration by Summary:+ into the cell thereby restoring the intracellular potassium concentration. Recently, structural data for KdpFABC complexes revealed a surprising observation, namely phosphorylation of a serine in a conserved TGES motif in the A domain of KdpB, based on which it was shown that phosphorylation reduces the ATPase activity of KdgFABC. In the present work, Sweet et al., demonstrate that this post-translation regulation occurs in response to increased K+ levels, using activity and phosphorylation assays, and mass spectrometry data. Phosphorylation therefore serves to inhibit the complex once the cellular potassium levels have been restored. This novel regulatory strategy may present another unique feature of KdpFABC systems, but also provides a new lens through which other P-type ATPases should be viewed.The bacterial potassium uptake system KdpFABC is an unusual complex of a P-type ATPase (subunit KdpB) and a potassium channel. Its expression levels are increased in response to low-potassium stress conditions, at which point it utilizes ATP to pump KThe authors satisfactorily addressed the major concerns regarding the biochemical aspects of the work, and the experimental systems and major conclusions of the work are more clearly presented. They also provided additional experimentation that test a foundational assumption regarding the simulation data. Unfortunately, the new experiments illustrate that key residues interacting by the phosphorylserine group in the starting structure of the MD simulations are not required for inhibition. Therefore, the simulations of this structure are unlikely to be relevant for the  question at hand. Moreover, the authors use, as their control, a modified (dephosphorylated) version of that structure, which is bound to have unfavorable interactions that most probably do not reflect the true dephosphorylated protein.  Based on these concerns, if the authors wish to resubmit a revised version of the manuscript, the MD simulations must be removed.Clarification:In referring to a high-energy, artifactual state, we referred not to the crystal structures themselves, but rather to the molecular system created by the authors when the phosphate group was deleted from the serine in the crystal structure prior to running the MD simulations, i.e., the SER system. The concern is that introducing this dramatic artificial change into a structure  creates an system with a set of new high-energy interactions; these could be the cause of the "uncoupling of the A- and N-domains" . In other words, the SER system does not reflect a true dephosphorylated protein. The true dephosphorylated protein may or may not ever visit the exact same conformation that is present in this starting structure. In an ideal world, one would initiate the simulations with an experimental structure of a dephosphorylated protein in the same state of the transport cycle. Obviously, such a structure is not available, but the alternative adopted here is insufficiently robust to address the question at hand. Essential revisions:While the observations regarding the role of Ser162 in phosphorylation are clear and robust, more conclusive data must be provided regarding the molecular basis of inhibition, according to the following:1) The results describing the experiments identifying the step in the Post-Alber cycle affected by phosphorylation are confusing, in part because of the various mutants and growth conditions. This section requires clarification. It may also help to rename E161Q to reflect the additional mutations that accompany it.+ concentrations used for growth, as well as Table 1, which summarizes information for each construct. We have also changed the nomenclature for the constructs in the text to explicitly acknowledge double and triple mutations.We agree that the diversity of samples used for this work could cause confusion. Keeping track of the wide array of mutations and varying growth conditions is critical for understanding the implications of individual experiments and for the overall functioning of KdpFABC. To assist in this task, we have added a new paragraph at the beginning of the Results section to enumerate the mutations and to explain differences in the growth conditions used to produce them. This explanation is accompanied by a newly added Figure 2, which diagrammatically illustrates the bacterial strains as well as K2) Based on Figure 4 it is concluded that KdpFABC is inhibited in the E1~P state. There are two issues with the experiment.Note that data for these experiments are now shown in Figure 5 of the revised manuscript.- First, all variants were produced at high K, and thus in the Q116R mutant, Ser162 would be mostly phosphorylated. The authors claim that the phosphorylation of Asp307 is uncoupled from the presence of potassium, which is why high phosphorylation is found for the inhibited variants Q116R and S162D . But why does the EP level of Q116R decrease over time, if it is inhibited? The "rate" of dephosphorylation is at least as fast as for S162A, which is not inhibited and the dephosphorylation is nearly 100%. Considering that only a fraction of the protein (e.g. 75%) is inactive, the dephosphorylation should only apply to a small portion of the phosphorylated protein.We do have an explanation for the decrease in EP levels for the Q116R mutant. As the reviewers acknowledge, this preparation has a mixture of serine phosphorylated (inhibited) and unmodified (fully active) protein. We believe that the fraction that is serine phosphorylated is blocked in the E1~P conformation, whereas the active fraction will turnover. As a result of this enzymatic turnover, ADP will be produced and, as demonstrated by the pulse-chase experiments , ADP rapidly reacts with the E1~P species to reverse the phosphorylation of Asp307 . Thus, as time goes on and more ADP is produced, the total steady-state levels of EP will fall to zero. This effect only occurs in the presence of K, because otherwise there is no turnover and no ADP is produced. We previously thought that this detail might be confusing to readers, but we are pleased that the reviewers brought it up and we have included an explanation in subsection “Effect of serine phosphorylation on partial reactions” of the revised manuscript.How does this experiment look for Q116R expressed at low K? Is the initial EP level much lower due to fast turnover as suggested for S162A? And is the dequenching even faster?With our current expression strategies, we are not able to produce the Q116R mutant at low K. Because of the lower K affinity produced by this mutation, it cannot rescue cell growth at μM levels of K, but requires at least 0.2 mM at which concentration there is a already a substantial amount of serine phosphorylation. In principle, one could try to express a His-tagged Q116R construct in a background of WT KdpFABC. Although we do not have the strain necessary to attempt this experiment, it would be interesting to pursue this approach in follow-up studies.- Second, from the pulsed-chase experiment the authors claim that the protein is trapped in an E1~P state. However, from the inhibition with orthovanadate the authors concluded that the equilibrium between E1/E2 is not affected by the phosphorylation. In the best case this statement is only a bit misleading as it only refers to the non-phosphorylated fraction. If something else was meant, this statement should be clarified. Further, the pulsed-chase experiment can be explained by two scenarios: the inhibition of the protein in one state and the E1-E2 transition as rate-limiting step. For S162A the authors claim the experiment reflects the latter, while S162D is inhibited in E1-~P.The titrations with orthovandate, K and ATP address the possibility that serine phosphorylation alters the rate of a step in the reaction cycle rather than blocking it altogether. If rates were affected, the fraction of unphosphorylated molecules would behave normally, but the serine-phosphorylated molecules – comprising the majority of the population – would behave differently and thus shift one of these titration curves. The fact that all three titration curves, now shown in Figure 6, were not significantly affected by levels of serine phosphorylation is consistent with (though not necessarily proof of) our hypothesis that serine phosphorylation completely blocks turnover and that the observed activity comes only from unphosphorylated molecules. In order to make this reasoning clearer, we have moved this section to a later point in the manuscript so that there is better context for these experiments. We have also rewritten the relevant paragraphs in subsection “Effect of serine phosphorylation on ligand binding”.3) The plots in 2G and 2H are misleading as they are based on the assumption that upon 100% phosphorylation the ATPase activity is completely abolished. However, there is no formal proof of this. In fact, the maximal inhibition seen is approx. 60% after 90 min K shock when compared to the dephosphorylated sample. On the other hand, the level of phosphorylation cannot be reliably estimated, neither from mass spec nor from the phos-tag signal. One could thus speculate that the rate of phosphorylation is even higher, particularly because no significant increase was detected upon K shock after 20 minutes , and that the phosphorylation does not completely inhibit KdpFABC but instead slows down the state transitions (e.g. E1 to E2) such that 100% inhibition would never be seen. This scenario may also explain the experiments performed in Figure 4.As discussed above for point 2, the data are consistent with the idea that serine phosphorylation completely inhibits turnover. This conclusion is further supported by results from the S162D mutation, which is completely inactive and produces a steady level of EP in both the presence and absence of K. However, the reviewers are correct that we have no formal proof that serine phosphorylation has this same effect and perhaps S162D is a misleading – though commonly used – mimetic of phosphorylation. The right-hand axes are not essential to our story so we have removed them from the revised figures and will simply report phosphorylation in arbitrary units, since this will convey the point without unnecessary confusion or controversy.The data after 90 min of K shock is somewhat puzzling, as one would logically expect continued increases in inhibition over time. After reconsidering this matter, however, we believe that cell metabolism is likely to be compromised under extended conditions of K shock. Unlike the complete inhibition observed in the previous publication by Roe et al., (2000), the inhibitory system (as yet uncharacterized) is not 100% effective in shutting down KdpFABC activity in our expression system. In the original manuscript, we speculated that this outcome is due to forced overexpression of KdpFABC from a multicopy plasmid that has overwhelmed this regulatory system. As a result of this deficiency, we expect that K shock will be detrimental to ionic homeostasis and potentially compromise survival of cells after they are returned to K replete conditions. If so, K shock would cause unexpected results during long incubations This effect may be reflected in the lower activity seen in LPP treated samples from the longer periods of K shock. This issue is discussed in more detail in the Discussion section in the revised manuscript.4) The molecular dynamics simulations presented rely on the deletion of the phosphate group from Ser162 as a proxy for the dephosphorylated state. This assumption is flawed as it involves artifactual, high-energy states, and does not consider the metastable conformations that are likely to be involved immediately before or after kinase/phosphatase activity.The reviewers raise important points about our analysis of MD simulations that have caused us to reconsider their contribution. The bottom line is that simulations show a dramatic difference in the behavior of serine phosphorylated and unphosphorylated KdpFABC and, although they do not offer definitive answers, we believe that they make a valuable contribution to our story. For the revised manuscript, we have refocused the analysis on the dynamic properties of cytoplasmic domains of KdpB and on the linkers connecting the A-domain to the membrane helices. We have downplayed the issue of hydration and have presented a testable hypothesis to explain the functional effects seen in our biochemical experiments.We are not certain what is meant by "artifactual high-energy states" and the "metastable conformations […] immediately before or after kinase/phosphatase activity". In our minds, this statement could refer to either (1) states related to the conformational cycle of the P-type pump and phosphorylation/hydrolysis of Asp307, (2) the regulatory process involving phosphorylation/dephosphorylation of the Serine162 or (3) the influence of crystal environment that could force flexible regions to adopt a high-energy conformation. For our response, we have assumed that the "artifactual high-energy state" refers to the packed environment in the crystals that produced the starting model for our MD simulations (#3 above) and that kinase/phosphatase activity refers to the unknown agent that is acting on Ser162 (#2 above). We sincerely hope that this is as intended.We agree that the X-ray structure is possibly in a high-energy conformation, stabilized by the chemical environment of the crystallization experiment . We nevertheless used the X-ray structure because it is based on data of considerable higher quality compared to the EM models. This is exemplified by the higher resolution of the X-ray data (2.9 Å) compared to the cryo-EM structures (3.7 and 4.0 Å), and is especially evident in the definition of the cytoplasmic domains . We note that crystal packing is likely not the primary determinant of the observed conformation, given that the asymmetric unit contained three copies of KdpFABC, each having unique interactions with neighboring molecules but all adopting the same conformation . In general, the use of crystal structures as a starting point of Molecular Dynamics is standard practice, and any "high-energy" strain imposed by the crystallographic experiment is normally relieved either by the initial equilibration procedure or during the restraint-free production simulations. It is the undeniable that the choice of the starting conformation can potentially influence the average thermodynamic quantities calculated from the simulations, but we feel that our choice is well justified and was effective not only in revealing distinct character for the two systems but also in highlighting a region of the molecule that may be responsible for allosteric coupling. For the revised manuscript, we added a brief explanation for the basis of selecting the X-ray structure over the cryo-EM structures in subsection “Effects of serine phosphorylation on structural dynamics”.With regard to the metastable intermediate states associated with phosphorylation, it was not our goal to study this process, but rather to compare the properties of the protein in the presence and absence of serine phosphorylation.Moreover, the simulations of the phosphorylated structure were initiated using a system in which the cavities in the structure have not been hydrated , with the exception of one highly-ordered water molecule observed in the structure. Such cavities are unlikely to become hydrated on the timescale of the equilibration, but could have a significant effect on the dynamics of the protein system. Therefore, these simulation data should be removed. In case the authors chose to provide new computational data in its place, substantially more detail must be provided to describe the analysis, e.g. which atoms are included in RMSD calculations.There is ample evidence in literature that the free water movement in MD simulations is able to accurately hydrate protein cavities. For example, Tajkhorshid and colleages showed that cavities of amino acid transporters were hydrated on a timescale comparable to our simulations (~100 to 200 ns) . In another example, water molecules spontaneously hydrate or dehydrate a key enzymatic active site in the cytochrome c oxidase . With regard to ion pumps, we have not been able to find any report on MD simulations in which a hydrating procedure was used . These examples illustrate the broad acceptance of MD simulations of transmembrane protein systems without using cavity-hydrating programs such as Dowser. As a result, we believe that the distribution of water seen during our simulations has good precedent in the field as a measure of hydration for KdpFABC.We did nevertheless use Dowser++ as proposed on the KdpFABC as well as on the Na/K-ATPase system that we have previously examined in a number of publications. During this process, we communicated with the authors of Dowser++ to ensure that our protocol was optimal . Despite our best effort, we obtained gross over-hydration of the membrane domain for both protein systems. As a result of this over-hydration, the ensuing MD runs produced anomalous results. In reviewing the use of Dowser++ in other publications , we noted that the number of water molecules inserted by Dowser++ often increases significantly as the resolution of the structure decreases and that very high-resolution (sub-Å) crystal structures are typically used for benchmarking Dowser++ performance.+ independence of EP formation caused by serine phosphorylation, we have de-emphasized this point by moving it to the Discussion section with the relevant data moved to the supplemental material . We believe that this observation provides a potentially important lead for future studies into the allosteric effects of serine phosphorylation and therefore makes a valuable contribution to the manuscript.Given the uncertainties of using Dowser++ with low resolution structures, and the poor behavior of the resulting hydrated complexes of KdpFABC and Na/K-ATPase during simulations, we believe that this issue requires further experimentation which is outside of the scope of the current manuscript. We have therefore refocused the revised manuscript on the dynamics of the cytoplasmic domains and the identification of a potential allosteric control site in the linker between the A-domain and the membrane domain. Although our observation of increased hydration is noted as a potential explanation for the KPlease also note that radial distribution functions are not typically appropriate for describing residue-residue interactions; distance distributions are preferable.We agree that distance distributions may be more suited to represent residue-residue interactions. We have therefore included RMSD distance distributions alongside the radial distribution functions. In general, RMSD was calculated from the last 100 ns of the simulations after correcting individual structures for bulk translational motion; the calculations include all atoms from the relevant residues. The radial distribution functions and RMSD values both show comparable effects and support the striking difference in behavior after phosphorylation of Ser162. The details of the RMSD calculation have been included in legend for Figure 7—figure supplement 1.5) A central theme of the proposed mechanism is that Ser162-Pi interacts with K357+R363, as observed in the available crystal structure (PDB code 5MRW) and that dephosphorylation would abolish this interaction leading to an allosteric effect on the transmembrane domains. However, other structures of KdpFABC  contain Ser162-Pi and yet do not show this interaction, despite representing, at least in one case, a similar state in the transport cycle. Moreover, one of those structures is an E2 state of Ser-phosphorylated KdpB, which questions the hypothesis that the phosphorylated state is truly inhibited in an E1 conformation. Thus, any discussion of a molecular mechanism must carefully and convincingly consider those structures and their implications. These are very valid observations that indicate our failure to express clearly our point of view about the interaction between Ser162-Pi and K357-R363. Although this interaction was highlighted in the X-ray structure and proposed as a potential mechanism for inhibition, the paper also noted lack of conservation for K357 and R363 in KdpB from other species. Given the cryo-EM structures from the Hänelt group and the MD studies presented here, our view on this matter has shifted. We now believe that the observed interaction with K357 and R363 in the crystal structure is not essential for the inhibition to occur. We have brought this issue front-and-center in the Introduction of the revised manuscript (Introduction) and return to the lack of sequence conservation in the Discussion section. In addition, we have added a key experiment to show that mutation of Lys357 and Arg363 does not prevent inhibition . In particular, we expressed the K357A/R363A/Q116R mutant in the TK2498 strain in 0.2 mM KCl  and used the Phos-tag stain to show that serine phosphorylation had occurred. We then compared ATPase activity of this preparation before and after LPP treatment. The ~4-fold stimulation produced by the phosphatase is analogous to the effect on WT protein, providing clear evidence that Lys357 and Arg363 are not essential for the inhibitory mechanism. These new data are referenced together with the MD results .4. Given our evidence that serine-phosphorylation eliminates K-dependence of the reaction cycle, this combination of ligands could plausibly stabilize E1, E1-ATP, E1~P, or E2-P states. Although we conclude that serine-phosphorylation blocks the reaction cycle in the E1~P state, the E2-P state would still be accessible via back-door phosphorylation, especially given the uncoupling of the Post-Albers reaction cycle with respect to K. These thoughts about the cryo-EM structures are included in the penultimate paragraph of the Discussion section, which also dismisses roles for Lys357 and Arg363 and offers an alternative explanation for stabilization of the E1~P state.With regard to the cryo-EM structures from the Hänelt group, these two structures were derived from a single sample prepared in the presence of AMP-PCP, K, and AlF


Since 2009, unconventional natural gas development (UNGD) has significantly increased in Appalachia’s Marcellus Shale formation. Elevations of fine particulate matter <2.5 µm (PM2.5), have been documented in areas surrounding drilling operations during well stimulation. Furthermore, many communities are experiencing increased industrial activities and probable UNGD air pollutant exposures. Recent studies have associated UNGD emissions with health effects based on distances from well pads. In this study, PM2.5 filter samples were collected on an active gas well pad in Morgantown, West Virginia, and three locations downwind during hydraulic stimulation. Fine particulate samples were analyzed for major and trace elements. An experimental source identification model was developed to determine which elements appeared to be traceable downwind of the UNGD site and whether these elements corresponded to PM2.5 measurements. Results suggest that 1) magnesium may be useful for detecting the reach of UNGD point source emissions, 2) complex surface topographic and meteorological conditions in the Marcellus Shale region could be modeled and confounding sources discounted, and 3) well pad emissions may be measurable at distances of at least 7 km. If shown to be more widely applicable, future tracer studies could enhance epidemiological studies showing health effects of UNGD-associated emissions at ≥15 km. Particulate air pollution (PM) is known as a significant contributor to the global burden of disease and mortality. PM is a physicochemically diverse mixture, with a variety of amounts, shapes, sizes and origins—all of which contribute to PM toxicity. PM can be delineated into three sizes PM <10 µm , <2.5 µm , and PM <0.1 µm . Smaller-sized particles deposit deep in the lungs, which has been shown to invoke an inflammatory immune response and produce oxidative stress ,4, asthmSince 2009, unconventional natural gas development (UNGD) has significantly increased ,20. ElevThe scientific literature currently lacks methodologically rigorous studies that link UNGD-related particulate matter with health outcomes ,32,33. OWe hypothesized that well pad air emissions could be determined by elemental analysis of collected PM2.5 downwind. To determine how far away from the well pad air emissions can be detected, we tested the hypothesis that tracer elements unique to the UNGD well pad can be identified downwind and off site of the gas well. We also sought to determine whether these tracer elements corresponded to PM2.5 measurements simultaneously collected in multiple stations. In this study, we collected PM2.5 on polytetrafluoroethylene (PTFE) filters at three points downwind of a UNGD well pad in West Virginia during hydraulic fracturing. High-resolution inductively coupled plasma mass spectrometry (HR-ICP-MS) was applied to analyze the filters for major and trace elements . Further analysis was conducted using an experimental source identification model incorporating wind patterns to determine which elements, if any, could be traced downwind of the UNGD site.No humans or animals were subjected to testing in this study. Permission to conduct environmental sampling was obtained for each site. Researchers obtained International Association of Drilling Contractors (IADC)/SafeLand USA RigPass certification through West Virginia University Safety and Health Extension office, a requirement for conducting sampling on the well pad.In this study, we collected 48 hour samples of PM2.5 at three points predominantly downwind  of an active Marcellus Shale gas well pad in Morgantown, West Virginia. Samples were obtained during an 8 day hydraulic fracturing stimulation process that occurred on October 28–November 5, 2015 . SamplinSamples were collected every two days at each of the four operational sampling sites  using a Gast Compressor Vacuum Pump DOA-V191-AA  during the 8 day stimulation process. Materials were captured on a Teflon Membrane Disc Filter  utilizing pre-sterilized URG-2000-30FG 47 mm filter holder and the URG-2000-30EH 16.7 Lpm, 2.5 μm Teflon Coated Aluminum Cyclone . Daily flow calibration was conducted using the Sierra 822 Top-Trak Mass Flow Meter  and Key Instruments adjustable flowmeter (10-100 SCFH) . Total flow (m3) was tracked utilizing the Kimmon SK25EX  dry gas meter.Teflon forceps were used to handle the filters which were then stored in plastic petri dishes and transported immediately from each site in a cooler. Within 60 minutes of collection, samples were transferred for storage at 277.15 K at the West Virginia University Occupational and Environmental Health Sciences laboratory. Samples were shipped overnight on dry ice to the Lamont Doherty Earth Observatory in Palisades, New York, at Columbia University for HR-ICP-MS analysis.https://www.wunderground.com/personal-weather-station/dashboard?ID=KWVMORGA25#history/tgraphs/s20151031/e20151031/mdaily last accessed: 8-8-2017) for Morgantown, West Virginia, using the Brooks green roof (KWVMORGA25) sampling station that averaged temperature, humidity, pressure, UV, and wind direction approximately every 9 minutes. Wind data were synchronized with sample collection data using time of the first sample taken at the first site and time of the last sample taken at the last site during the sampling timeframe. For each sampling period, we calculated the percentage of time in which the wind derived from a specific direction, and the speed  at which the wind was blowing. Our primary wind patterns of interest were periods when the wind was blowing greater than 1 mph and derived from Southeast (SE), South–Southeast (SSE), South (S), South–Southwest (SSW), and Southwest (SW). Wind direction data for sampling periods where the wind was not blowing greater than 1 mile per hour and/or in the direction of our sampling stations for more than 5% of the time were excluded.Wind data were obtained on the Weather Underground online historical database  Thermo Element XR, a magnetic sector high-resolution inductively-coupled plasma mass spectrometer (HR-ICP-MS). The study utilized “Summer New York” analysis methods previously published [Elemental analysis of the digested air filters was conducted at the Columbia University Lamont Doherty Earth Observatory using the lab’s Axiomublished . This meublished . Sample ublished . ResultsAn analysis of the correlation between the elements determined was conducted using the raw HR-ICP-MS analysis of 34 elements from digested filters during each sampling period. To determine the most appropriate trace elements associated with emissions from the well site and to discriminate emissions from intervening sources, a logic tree model was established. While there may be elements that are uniquely associated with the well pad emissions, if the concentration of those elements is not sufficient to allow measurement of them, they cannot be considered. For exclusion criterion 1 (detectable mass), therefore, elements were excluded when total concentration on the filter did not rise above the quantification limit at any of the downwind locations.3 = ng trace element/m3 of air flow)  . Using t2 > 0.6) for the power trendline was used to decide whether the decreasing concentrations were a consistent trend with distance downwind from the well site. It was assumed that the introduction of other sources of the tracers at intervening distances downwind would result in the increases in concentration and diminution of the correlation coefficient. It is, of course, still possible that the actual dispersion of the elemental tracers is not following the power function relationship exactly. However, this was still considered a conservative assumption for exclusion. Those elements with r2 < 0.6 were therefore excluded under exclusion criterion 3 (inconsistent diminution of concentration with distance)  a–d. For istance) e–h.2 value between elements was greater than 0.6 and the acceptable slope of the comparison of the selected element to three or more other elements during the sampling period was nominally zero but at least between values of plus and minus one. This served as exclusion criteria 4 . We assumed that the wind would need to be blowing at least 1 mile per hour in the direction of the sampling sites at least five percent of the time in order to detect trace elements  were performed at each site using TSI DustTrak™ II 8530 Aerosol Monitors during the sampling periods. Measurements were taken every minute at all sites except for the well site which was taken every two minutes. DustTrak™ monitors were situated beside other monitoring devices downwind of UNGD operations. Raw data from these experiments were saved in a Microsoft ExcelThese PM2.5 mass measurements were corrected against a factory calibrated DustTrak device, and the corrected DustTrak values were then averaged daily and compared to the elemental data.We selected the October 30–November 1, 2015 sampling period because it had the most consistent dust generation from the well site along with appropriate windspeed and direction. Distance was inserted into the elemental power trendline calculation for the October 30–November 1, 2015 sampling period using a distance of 0.1 km for the well site. This established the elemental value to daily DustTrak average by distance. We then compared these values using by Microsoft Excel® power trendline calculation. This was repeated for all elements deemed significant during the October 30–November 1, 2015 sampling period to determine whether we could use the DustTrak alone to follow the plume profile.2 > 0.6, slope 1) to at least three other elements during the sampling period; and Mo (n = 1) was excluded due to lack of proportionality to at least three other elements across other sampling periods. Wind pattern data demonstrated that the wind was blowing greater than 1 mile per hour (mph) from the directions of interest  for approximately 42% of the overall sampling time. Results of the wind analysis for Oct. 28–30 are listed in 2 = 0.91, slope = 0.008), Co , Sr , U , and V , was the only remaining element to match the selection criteria.The 34 trace elements initially considered for inclusion in the analysis for each sampling period are listed in 2 > 0.6, slope 1) to at least three other elements during the sampling period. Wind pattern data demonstrated that the wind was blowing greater than 1 mile per hour (mph) from the directions of interest  for approximately 38% of the overall sampling time. Cr, Fe, La, Ti, U (n = 5) were excluded due to lack of proportionality to at least three other elements across other sampling periods. Similarly with the previous sampling period, Mg was the only remaining element to meet all of the selection criteria, and demonstrated proportionality to Ba , Co , Sr , U , V , in addition to Cs , Fe , La , Mn , Ni , Rb , and Ti . For the October 30–November 1 2015 sampling period, all elements met the detection limit. Ag, As, Bi, Cd, Cu, Pb, S, Sb, Se, Tl (n = 10) were excluded because their concentration did not decrease overall with distance, while Be, Li, Na, Sn, Zn (n = 5) did not have a strong association for proportional concentration decrease in distance. Al, Ba, Ca, Co, Cs, K, Mn, Mo, Ni, P, Rb, Sr, V (n = 13) did not demonstrate a strong proportional fit with  to at least three other elements including Ba , which is consistent to the October 28–30 and October 30–November 1 sampling periods, Fe , and Ni . Wind patterns indicate that the wind was blowing in the directions of interest for 11% of the total sample period.During the November 1–3, 2015 sampling period, Cr (n = 1) was the only element excluded for not meeting the detection limit. Additionally, Ag, As, Bi, Cd, Na, Pb, S, Sb, Se, Tl, and Zn (n = 11) were excluded because their concentration did not decrease overall with distance, and Al, Be, Ca, Co, Cs, Cu, K, La, Li, Mn, P, Rb, Sn, Sr, Ti, U, and V (n = 17) did not have a strong association for proportional concentration decrease in distance. Ba, Fe, Mo and Ni (n = 4) were excluded for not having a strong proportional fit to at least three other elements. This left Mg (n = 1) as the only remaining element that demonstrated a strong proportional fit  were excluded for concentrations not meeting the detection limits, followed by exclusion of Be, Bi, Cd, Cu, Na, P, Pb, S, Sb, Se, Sn, Tl, and Zn (n = 13) for concentration not decreasing overall with distance. All remaining elements Ag, Al, Ba, Ca, Co, Cs, Fe, K, La, Li, Mg, Mn, Mo, Ni, Rb, Sr, Ti, U, V (n = 19) were excluded for lack of strong association for proportional concentration decrease in distance prior to even considering the role of wind patterns, which only met the criteria of interest approximately 4.5% of the time.Results of the trace elemental analysis are summarized in In this hypothesis generation study, we collected PM2.5 samples at three points downwind of a UNGD well pad in West Virginia during the stimulation process and used HR-ICP-MS to analyze trace elemental content. We sought to determine 1) what trace elements unique to the UNGD well pad could be identified at distances downwind, and 2) whether these elements corresponded to PM2.5 measurements. With this information, we developed an experimental source identification model for the wind patterns and topography of the surrounding area to test our hypotheses.Limited information about the source of Mg is available for UNGD. Mg is a major element in the earth’s crust and is present in a variety of minerals, especially mafic minerals . It is kWhile Mg may not serve as a generalizable trace element, this source identification model could be used to identify localized trace elements in different shale plays and for different point sources. It should be noted that although air emissions could be detected at our furthest sampling point at 7 km, the highest emission levels were detected on site at the gas well. It appears that when the wind is blowing at speeds greater than 1 mph towards the sampling devices, more often, there is a greater number of proportional correlations.Our model for identifying tracers is a more sensitive way to distinguish a PM2.5 derived source from background for the particular topography, meteorology, shale play at depth, and UNGD process unique to the location and timeframe being sampled. While these results should not be immediately extrapolated to other sources, we hypothesize that this method of elemental identification can be used to characterize the distance that PM2.5 air emissions travel from any given point source. Further studies using this model could assist in developing an exposure matrix for epidemiological studies assessing off-site exposures to PM2.5.The trace elements from the UNGD well pad we analyzed decline after 1 km, consistent with the 2012 study by McKenzie et al. . Other pThere were other limitations of our study. Disturbances in the flow sampling at Site 0 resulted in suboptimal vacuum pressure as described in 2 > 0.6 may be too conservative an approach. For the purposes of this study, this likely excluded a number of elements simply because there was not enough dust detected at 7 km out from the well due to the reduced concentration of PM2.5 at this sampling site. Using exclusion criteria based on a coefficient of determination of rFuture studies should work to confirm our initial findings at other wells in the Marcellus Shale region. Furthermore, these techniques could be applicable to other UNGD in the Barnett, Utica, and other reservoirs.2 = 0.278), Cr (r2 = 0.253), La (r2 = 0.444), Fe (r2 = 0.477), Ti (r2 = 0.352), and U (r2 = 0.355)  .2 > 0.6. These results provide strong evidence that Mg can be used as a tracer to detect off-site PM2.5 emissions generated during the stimulation process at the UNGD well site. Findings indicate that Mg is consistently proportional to multiple other elements at sampling sites 7 km downwind of the Morgantown UNGD well site, with a correlation of r


Three-dimensional intracardiac echocardiography (3D ICE) with wide azimuthal elevation is a novel technique performed for assessment of cardiac anatomy and guidance of intracardiac procedures, being able to provide unique views with good spatial and temporal resolution. Complications arising from this invasive procedure and the value of 3D ICE in the detection and diagnosis of acute cardiovascular pathology are not comprehensively described. This case illustrates a previously unreported iatrogenic complication of clot displacement from the intra-vascular sheath upon insertion of a 3D ICE catheter and the value of 3D ICE in immediate diagnosis of clot in transit through the heart with pulmonary embolism.We conducted a translational study of 3D ICE with wide azimuthal elevation to guide implantation of a left ventricular assist device (Impella CP®) in eight adult sheep. A large-bore 14 Fr central venous sheath was used to enable right atrial and right ventricular access for the intracardiac catheter. Insertion of the 3D ICE catheter was accompanied by a sudden severe cardiorespiratory deterioration in one animal. 3D ICE revealed a large highly mobile mass within the right heart chambers, determined to be a clot-in-transit. The diagnosis of pulmonary clot embolism resulting from the retrograde blood entry into the large-bore sheath introducer, rapid clot formation and consequent displacement into venous circulation by the ICE catheter was made. The sheep survived this life-threatening event following institution of cardiovascular support allowing completion of the primary research protocol.This report serves as a serious warning to the researchers and clinicians utilizing long large-bore sheath introducers for 3D ICE and illustrates the value of 3D ICE in detecting clot-in-transit within right heart chambers. Intracardiac echocardiography (ICE) is increasingly used for diagnostic procedures and imaging guidance during interventions. It offers unique high-resolution views of cardiac anatomy and intracardiac devices. The intracardiac echocardiographic catheter is inserted through a vascular sheath following central venous catheterization . The tipContemporary two-dimensional (2D) ICE catheters have a diameter of 8Fr and a length of 90 cm. Three-dimensional (3D) ICE catheters have a diameter up to 12.5 Fr and are 90 cm in length. 3D ICE catheters provide a 90° imaging sector and are commercially available in narrow-angle (22°)  and wideThere is limited world-wide clinical experience  and a paWe conducted a translational research project to assess the implantation of the left ventricular assist device Impella CP® guided by 3D ICE using an adult ovine model . The preThe study was approved by the University of Sydney  Animal Research Ethics Committee (2019/1650 amendment) and conducted at the Charles Perkins Centre for Research, The University of Sydney . It was carried out in accordance with the National Institutes of Health guide for the care and use of Laboratory animals  and in accordance with the relevant guidelines and regulations. Sheep number three of eight developed the reported complication.2O monitored by ventilatory pressures, continuous pulse oximetry and end tidal CO2. The sheep was in the right lateral position for a left thoracotomy. Invasive monitoring of arterial pressure, central venous pressure and cardiac output were continuously recorded. The anaesthetised sheep was euthanised on completion of the study.This adult female cross merino sheep was acclimatised for at least two weeks prior to the procedure and received routine preventative treatments prior to arrival. The sheep was anaesthetised and mechanically ventilated in synchronized intermittent positive pressure ventilation mode with positive end-expiratory pressure of 5 cm HA physician qualified in transthoracic echocardiography   performeA 14 Fr, 20 cm valved intra-vascular sheath  was used for the intracardiac catheter access. A cut-down technique was used to expose the left internal jugular vein and the sheath was inserted by a modified Seldinger technique with a standard dilator. The dilator was removed immediately after intravenous placement of the vascular sheath. The sheath was sutured to the skin.For the initial echocardiographic assessment, an AcuNav Volume 12.5 Fr, 90 cm four-ways steerable ICE catheter  was inserted through the haemostatic valve of the vascular sheath approximately 5 min following removal of the dilator during which time systemic heparinisation had not been administered until immediately prior to the ICE catheter insertion.Live three-dimensional volumes were acquired at 6 Mhz fundamental and 8 Mhz harmonic frequencies with the sector set at 90 × 50° as standard and adjusted together with the depth as required. Maximum achievable frame rate was 14–20 frames per second for 3D grey scale imaging. Images were subsequently cropped and processed using on-cart software.Sudden profound arterial oxygen desaturation was detected following insertion of the 3D ICE catheter via the sheath into the superior vena cava.A 3D ICE right atrial view in live full volume modality and in the default ICE catheter position initially revealed a large mobile mass (white arrows) within the right atrium Fig.  Panel a.Intracardiac echocardiography is mostly used by electrophysiologists and interventional cardiologists. Its proximity to the cardiac structures creates unique imaging capabilities for the assessment of cardiac anatomy, intracardiac masses and procedural guidance in interventional cardiology . However3D ICE with wide azimuthal elevation offers unique incremental value over 2D ICE by volumetric display and spatial imaging relationship between complete cardiac structures, intracardiac masses and devices. The trade-off is that 3D ICE catheters have larger diameters and require larger central venous sheath introducers. Premature removal of the dilator from the large-bore sheath following its insertion and inadequate flushing of the sheath with crystalloid solution may lead to the retrograde blood entry into the bore. In the absence of adequate systemic anticoagulation, this can predispose to the rapid formation of sizeable clots within the sheath and the potential for clot migration into pulmonary arterial tree. Clot displacement from the sheath will occur upon insertion of the ICE catheter. To our knowledge, such complication has not been previously described. Our report suggests that life-threatening pulmonary embolism should be considered among other diagnoses in patients undergoing ICE who develop sudden episodes of desaturation or significant haemodynamic compromise.Sheep have a clotting time similar to humans , 10, thuAn ability of ICE to identify formed thrombi in the left atrial cavity , the rigThis report highlights the newly identified risk of a potentially life-threatening complication during ICE resulting from rapid clot formation within a long large-bore central intra-venous sheath and consequent thrombus displacement into the pulmonary circulation during insertion of the ICE catheter. 3D ICE with wide azimuthal elevation demonstrated excellent diagnostic capabilities to image the clot in-transit within right heart chambers.Additional file 1.


Furthermore, we found that c-Jun, a known potent repressor of myogenesis, which is coincidently also a down-stream target of MEK/ERK signaling, was phosphorylated and accumulates in the nucleus in response to TGFβ activation. Taken together, these observations support a model in which TGFβ activates a MEK/ERK/c-Jun pathway to repress skeletal myogenesis, maintaining the pluripotent undifferentiated state in myogenic progenitors.Transforming growth factor β (TGFβ) is a pluripotent cytokine and regulates a myriad of biological processes. It has been established that TGFβ potently inhibits skeletal muscle differentiation; however, the molecular mechanism is not clearly defined. Previously, we reported that inhibition of the TGFβ canonical pathway by an inhibitory Smad, Smad7, does not reverse this effect on differentiation, suggesting that activation of receptor Smads (R-Smads) by TGFβ is not responsible for repression of myogenesis. In addition, pharmacological blockade of Smad3 activation by TGFβ did not reverse TGFβ’s inhibitory effect on myogenesis. In considering other pathways, we observed that TGFβ potently activates MEK/ERK, and a pharmacological inhibitor of MEK reversed TGFβ’s inhibitory effect on myogenesis, as indicated by a  TGFβ is the prototype of a large family of pluripotent cytokines with diverse effects on cellular proliferation, tumor growth, apoptosis, differentiation, fibrosis, anti-inflammation, and embryo development ,2,3,4,5.The general canonical view that has been established for TGFβ signaling is that it binds to its cognate type II receptor, which facilitates receptor complex formation and activation of the cytoplasmic serine/threonine kinase activity of the type I receptor leading to phosphorylation of Smad2/3 (receptor regulated Smads: R-Smads). Phosphorylation of R-Smads at the C-terminal SXS motif results in association with the common Smad, Smad4, and translocation into the nucleus to regulate target gene transcription through complex interactions with heterogeneous transcription complexes ,8. WhilePreviously, we observed that an inhibitory Smad (I-Smad), Smad7, potently counteracts Smad3 activation by TGFβ in a myogenic cell line. However, exogenous Smad7 was unexpectedly not able to prevent inhibition of muscle differentiation by TGFβ . These oWe previously found that although exogenous Smad7 strongly repressed Smad3 activation by TGFβ, the inhibition of muscle differentiation by TGFβ was not rescued by exogenous expression of Smad7 . These oWe hypothesized that if Smad3 activation is insufficient to inhibit muscle differentiation, TGFβ must activate a non-canonical pathway to repress myogenesis. We did note that TGFβ-treated C2C12 cells reached a high density and survived better in differentiation medium (DM) (unpublished observation). This observation warrants further investigation into the possible effects of TGFβ on myoblast proliferation and survival. In other cell models, TGFβ has been observed to activate the MEK/ERK pathway ,21, and Assessment of the MEK signaling pathway activation by immuno-blotting with antibodies recognizing the total and phosphorylated forms of MEK revealed that phosphorylated MEK was enhanced in C2C12 cells treated with TGFβ (2ng/mL) compared to that in solvent treated control cells. TGFβ was more potent than CT-1 (10ng/mL) in terms of MEK activation based on the ratio of P-MEK to total MEK . Since wWe next tested the possibility that prevention of MEK activation by a MEK-specific inhibitor might activate myotube formation and MyHC accumulation in the presence of TGFβ activation. As seen in Reversal of TGFβ mediated myogenic repression by MEK inhibition is not due to inhibition of Smad signaling since MEK/ERK signaling positively modulates R-Smad activity by phosphorylating the linker region of Smad2/3 ,27,28,29myog gene, which is a critical myogenic target gene in the hierarchical control of myogenesis pyridin-3-yl)-propenone hydrochloride  were resuspended with DMSO and added into the cell culture media for 30 min prior to adding TGFβ.C2C12 myoblast were obtained from the American Type Culture Collection; USA and cultured in growth medium (GM) consisting of 10% fetal bovine serum (FBS)  in high-glucose Dulbecco’s modified Eagle’s medium (DMEM)  supplemented with 1% penicillin-streptomycin  at 37°C and 5% COPhase contrast photomicrographs were obtained using an epifluorescence microscope , with appropriate phase and filter settings, and either 4X NA 0.10 or 10X NA 0.25 Achrostigmat objective lenses. Images were recorded with a digital camera .® kit (Pierce) according to the manufacturer’s protocol.Nuclear proteins were extracted from the cells by NE-PERTotal cellular protein extracts were prepared in NP-40 lysis buffer ). Protein concentrations were determined by a standard Bradford assay . Equivalent amounts of protein were resolved by SDS-PAGE gels, followed by electrophoretic transfer to an Immobilon-P membrane  as directed by the manufacturer (Millipore). Blots were incubated with the indicated primary antibody in 5% milk in PBS or Tris buffered saline (TBS)-T  or 5% Bovine serum albumin (BSA) in TBS-T according to the manufacturer’s protocol at 4°C overnight with gentle agitation. After washing briefly, the blots were incubated with the appropriate HRP-conjugated secondary antibodies in 5% milk in PBS or TBS-T at room temperature according to the manufacturer’s protocols . After being washed three times with 1XPBS or 1XTBS (depending on the primary antibody) at room temperature, the blots were treated with the enhanced chemiluminescence reagent  to detect immuno-reactive proteins. The immunoblots were exposed to Biomax film  for visual representation. The band intensity was measured using ImageJ and graphed.2O2 in PBS) to detect MyHC by immunocytochemistry. The nuclei were counter-stained with hematoxylin. Images were recorded with a microscope  with either 4X NA 0.10 or 10X NA 0.25 Achrostigmat objective lenses with a digital camera .C2C12 cells were washed with phosphate buffered saline (PBS) (pH7.4) and fixed with 90% methanol at −20 °C for 10 min. After fixation, the cells were incubated in 5% milk in PBS for 30 min at 37 °C for blocking. Cells were incubated at room temperature with MF-20 (primary antibody) diluted in blocking buffer (5% milk PBS) for 1 h. After incubation, the cells were washed three times with PBS and incubated for 60 min at room temperature with a horseradish peroxidase (HRP)-conjugated α-mouse secondary antibody. The cells were washed again three times with PBS and incubated in developer . Luciferase and β-galactosidase enzyme assays were performed according to the manufacturer’s protocol (Promega). Luciferase activity was quantified using a luminometer  and standardized according to β-galactosidase activity. Relative Luciferase units normalized for β-galactosidase activity  were determined and plotted as an average of triplicate determinations and error bars represent standard deviations of the triplicate values.