No studies have compared monoaxial and polyaxial pedicle screws with regard to the von Mises stress of the instrumentation, intradiscal pressures of the adjacent segment and adjacent segment degeneration.Short-segment monoaxial/polyaxial pedicle screw fixation techniques were compared using finite element methods, and the redistributed T11-L1 segment range of motion, largest maximal von Mises stress of the instrumentation, and intradiscal pressures of the adjacent segment under displacement loading were evaluated. Radiographic results of 230 patients with traumatic thoracolumbar fractures treated with these fixations were reviewed, and the sagittal Cobb’s angle, vertebral body angle, anterior vertebral body height of the fractured vertebrae and adjacent segment degeneration were calculated and evaluated.The largest maximal values of the von Mises stress were 376.8 MPa for the pedicle screws in the short-segment monoaxial pedicle screw fixation model and 439.9 MPa for the rods in the intermediate monoaxial pedicle screw fixation model. The maximal intradiscal pressures of the upper adjacent segments were all greater than those of the lower adjacent segments. The maximal intradiscal pressures of the monoaxial pedicle screw fixation model were larger than those in the corresponding segments of the normal model. The radiographic results at the final follow-up evaluation showed that the mean loss of correction of the sagittal Cobb’s angle, vertebral body angle and anterior vertebral body height were smallest in the intermediate monoaxial pedicle screw fixation group. Adjacent segment degeneration was less likely to be observed in the intermediate polyaxial pedicle screw fixation group but more likely to be observed in the intermediate monoaxial pedicle screw fixation group.Smaller von Mises stress in the pedicle screws and lower intradiscal pressure in the adjacent segment were observed in the polyaxial screw model than in the monoaxial pedicle screw fixation spine models. Fracture-level fixation could significantly correct kyphosis and reduce correction loss, and adjacent segment degeneration was less likely to be observed in the intermediate polyaxial pedicle screw fixation group. The posterior transpedicular pedicle screw fixation technique has been widely adopted for the management of an unstable spine, mainly due to trauma, and approximately 10-20% of such injuries are burst fractures In the current study, the biomechanical characteristics of fixation techniques  were compared, using finite element methods, with regard to the redistributed ROM, the VMS of internal fixation devices, and the IDPs of the adjacent segment under displacement loading. Two hundred thirty patients presenting with traumatic thoracolumbar fractures and treated with these types of fixation techniques were reviewed, and ASD was evaluated.A finite element model including seven vertebrae and six discs between T9 and L3 of the spine was reconstructed and analyzed using finite element analysis software Two hundred thirty patients presenting with a single traumatic thoracolumbar fracture with a fractured vertebral body between T11 and L3 underwent surgery in our hospitals using posterior short segment fixation techniques. The inclusion criteria consisted of a traumatic T11-L3 fracture, a type A fracture according to the AO classification p<0.05 was considered significant (two-tailed). Independent samples t-tests and one-way ANOVA were used to compare group means. The chi-square test was conducted to assess frequency data when appropriate.We used SPSS 15.0 software  to perform all statistical analyses, and a value of The SM4+2, IM6+2 and IP6+2 fixation models presented a decreased ROM compared to the intact normal spine model in all states of motion, and these results are illustrated in The LMS of the pedicle screws was observed in flexion and axial rotation in the monoaxial pedicle screw fixation models (MPSF) and in flexion and extension in the polyaxial pedicle screw fixation models (PPSF). The upper pedicle screw presented the LMS in the SM4+2, SP4+2 and IP6+2 models, and the lower pedicle screw presented the LMS in the IM6+2 model . The LMSMaximal IDPs of the adjacent segment were observed during lateral bending. The maximal IDPs of the upper adjacent segments were all larger than those of the lower adjacent segments. In the MPSF models, the maximal IDPs were larger than those in the corresponding segment of the normal model. In the PPSF models, some IDPs of the adjacent segments were less than those of the normal model .The postoperative SCA, VBA and AVBH were significantly improved compared to the preoperative values in all four groups. The postoperative SCA in the SP4+2 group and the AVBH in the IM6+2 group were significantly larger than the preoperative values. The final follow-up evaluation showed that the mean loss of correction of the SCA, VBA and AVBH were smallest in the IM6+2 group . In the Reinforcement with fixation at the fracture level can help to improve kyphosis correction and biomechanical stability The LMS of the upper pedicle screw was larger than that of the lower pedicle screw in the SM4+2, SP4+2 and IP6+2 models. The LMS values of the instrumentation in the MPSF group were larger than those in the PPSF models. These results may suggest that the PPSF technique can decrease the VMS of the instrumentation. Upon suspecting that a pedicle screw is broken, the upper pedicle screw must be the focus of attention for patients subjected to SM4+2, SP4+2 and IP6+2 fixation techniques, and the lower pedicle screw is the focus for the IM6+2 fixation technique. Clinically, 12 patients presented with fixation failure, including instrument breakage and screw loosening. Six patients presented with pedicle screw breakage, all of which occurred at the root of the pedicle screw because the results of stress distribution within the instruments demonstrated that the largest stress occurred at the pedicle screw root.In the SP4+2 group, pedicle screw breakage was observed in 2 patients , and screw loosening occurred in 1 patient (upper pedicle screw). In the SM 4+2 group, screw loosening occurred in 4 patients . We suggest that the phenomenon may be related to the fact that the upper pedicle screw presented the LMS in the SP4+2 and SM4+2 groups, and the redistributed ROM was largest in these two fixation models. The rate of screw loosening in the SM 4+2 group (7.5%) was significantly higher than in the SP 4+2 group (3.7%). It is difficult to precisely align the side opening of two monoaxial pedicle screw heads for insertion of the rod, and small discrepancies in the fixation of monoaxial pedicle screws may increase the rotational forces and intervertebral translational forces with negative effects at the screw-to-vertebral-body interface, which may lead to screw cut-out or loosening In the current study, the maximal IDPs of the adjacent segment were observed during lateral bending. The maximal IDPs of the upper adjacent segments were all larger than those of the lower adjacent segments in the MPSF models. These results were consistent with those of previous studies This study has many limitations. First, the number of patients included in the study was small, and this was a retrospective study. Second, a selection bias may exist because this study included patients referred to our teaching hospitals. Third, it is necessary to discuss several factors, including different patient conditions, muscle forces, ribs, and the length and diameter of pedicle screws, for a more clinically feasible conclusion because these factors can influence finite element analysis results. Fourth, the stress of the screw-to-vertebral-body interface was not analyzed in the current study; we will conduct further research specifically considering spinal models with different bone quality in the near future.The use of additional intermediate monoaxial pedicle screws may result in a stiffer construct and reduced VMS of the pedicle screws than in SPSF models. In comparison, use of additional intermediate polyaxial pedicle screws may result in a stiffer construct than the normal spine model and lower VMS of the pedicle screws and IDPs of the adjacent segment than MPSF models. Fracture-level fixation could significantly correct kyphosis and reduce correction loss, and ASD was less likely to occur in the IP6+2 group. Our results provided a theoretical basis for the treatment of spinal fractures using minimally invasive intermediate polyaxial pedicle screw fixation.Wang H, Zhao Y and Mo Z conceived of the study, collected the data, participated in the data analysis, drafted the manuscript and performed the statistical analysis. Han J conceived the study and participated in its design, coordination and drafting. Chen Y, Yu H, Wang Q, Liu J and Li C participated in the data analysis and interpretation. Zhou Y and Xiang L participated in the review, coordination and drafting of the manuscript and contributed to the analysis via constructive discussions.


There is no study concerning safety and accuracy of pedicle screw placement in Marfan syndrome. The objective of this study is to investigate accuracy and safety of pedicle screw placement in scoliosis associated with Marfan syndrome.CT scanning was performed to analyze accuracy of pedicle screw placement. Pedicle perforations were classified as medial, lateral or anterior and categorized to four grades: ≤ 2 mm as Grade 1, 2.1–4.0 mm as Grade 2, 4.1–6.0 mm as Grade 3, ≥6.1 mm as Grade 4. Fully contained screws or with medial wall perforation ≤ 2 mm or with lateral wall perforation ≤ 6 mm and without injury of visceral organs were considered acceptable, otherwise were unacceptable.P < 0.05), higher in lumbar region than in thoracic region  and higher in concave side than in convex side . No injury of visceral organs especially aorta erosion was noted in the series. 7 cases of dural tear caused by misplaced screws occurred, and 4 cases developed cerebro-spinal fluid leak. Drainage and pressure dressings were applied for these patients, and no infection was observed. Leg pain was observed in 7 cases, and 2 cases simultaneously complained of leg weakness. Revision surgery was conducted to remove the misplaced screws for these 2 patients. Conservative treatment was applied for the 5 patients without leg weakness. Symptoms of leg weakness and pain resolved in all patients.976 pedicle screws were placed, 713 screws (73.1%) were fully contained within the cortical boundaries of the pedicle. 924 (94.7%) screws were considered as acceptable, and 52 (5.3%) as unacceptable. The perforation rate was higher using free-hand technique than O-arm navigation technique (30.8% VS. 11.4%, Placement of pedicle screw in Marfan syndrome is accuracy and safe. O-arm navigation was an effective modality to ensure the safety and accuracy of screw placement. Special attention should be paid when screws were placed at the lumber spine and the concave side of spine deformity to avoid the higher rate of complications. Marfan syndrome, a disease caused by an autosomal dominant mutation of the fibrillin-l gene occurred in around two to three in 10,000 people  Many sysThe incidence of scoliosis ranges from 52% to 100% in patients with Marfan syndrome –8. BracePatients with scoliosis associated with Marfan syndrome receiving posterior fixation from January 2007 to March 2016 were included in the study. The diagnoses were made by two spinal surgeons and a genetic specialist according to the Ghent nosology. Pre-operative and post-operative CT scans were performed on all patients. The medical records including gender, age at the time of surgery, magnitude of pre and post-operative curves, curve pattern, number of inserted pedicle screws and complications were collected for all patients. The magnitude of the curve was evaluated by using the Cobb method. Pedicle screw placements were performed with the help of fluoroscopy or O-arm navigation system.Free-hand pedicle screw placement technique Entry point for the thoracic spine was selected as the junction of the outer third and inner two thirds of the superior facet joint. For the lumbar spine, entry point was the junction of the mammillary process, inferior aspect of the transverse process and pars interarticularis. The entry was made with a probe into the pedicle. Pathfinder was used to make further passage. After removing pathfinder, the intact of cortices were checked by a ball-tipped probe. If any perforation was detected, the entry would be remade in an adjusted direction. Fluoroscopy was used to verify the position of each screw and to revise screw position .A dynamic reference frame  was installed on a spinous process. Then, the first 3D scan was acquired, and images of each section were recorded. The 3D images were reconstructed and then transferred to the navigation station . Under the guiding of navigation, the entry point and trajectory were designed. An awl was used to make the entry and the pedicle tract was made with a probe. By a navigated screwdriver, the pedicle screw was placed to a proper depth. The position and screw trajectory were displayed on the monitor  Fig.1)..1).Post-operative CT-scanning was performed for all the patients on instrumented segments. The assessment was performed on a PACS image system. An attending surgeon assessed all CT scanning to analyze screw accuracy. Pedicle perforations were classified as medial, lateral or anterior and categorized into one of four grades: ≤ 2 mm as Grade 1, 2.1–4.0 mm as Grade 2, 4.1–6.0 mm as Grade 3, ≥6.1 mm as Grade 4. Fully contained screws or with medial wall perforation ≤ 2 mm or with lateral wall perforation ≤ 6 mm and without injury of visceral organs were considered acceptable, otherwise were unacceptable  .The variables of pedicle screw as defined by fully contained screw accuracy and acceptably positioned screw accuracy were compared using Fisher (two-tailed) exact test as to: 1) convex side or concave side; 2) thoracic spine or lumbar spine; 3) free-hand or O-arm navigation.A series of 75 patients were included. There were 35 males and 40 females whose ages ranged from 10 to 28 years old, with an average age of 16.4 years old. The pre-operative Cobb angles were 45° to 165°, with an average angle of 74.8°. The post-operative Cobb angles ranged from 16° to 85°, with an average angle of 30.8°. Free-hand pedicle screw insertion technique was used for 63 patients and O-arm navigation technique for 12 patients.976 screws were placed, 713 screws (73.1%) were fully contained within the cortical boundaries of the pedicle. 263 (26.2%) screws were in perforation of the pedicle wall. 136 were lateral perforations, 102 medial and 25 anterior. 924 (94.7%) screws were acceptably positioned, and 52 (5.3%) were unacceptably positioned. Of the 263 screws in perforation, 173 were rated as Grade 1, 52 as Grade 2, 32 as Grade 3, and 6 as Grade 4.783 screws were placed by free-hand technique and 193 by O-arm navigation technique. 241 perforations occurred by free-hand technique and 22 by O-arm navigation technique. The perforation rate was higher using free-hand technique than O-arm navigation technique .P < 0.05).In the thoracic spine, 592 screws were placed and 384 in the lumbar spine. The perforation rate was higher in the lumbar region than in the thoracic region .507 screws were placed in the convex side of the spine and 469 in the concave side. 106 perforations occurred in the convex side and 157 in the concave side. The perforation rate was higher in the concave side than in the convex side , and 2 cases (1 at L1 and 1 at L2) simultaneously complained of leg weakness. Revision surgery was conducted to remove the misplaced screws for these 2 patients. Conservative treatment was applied for the other 5 patients without leg weakness. All symptoms resolved at final follow-up. For O-arm navigation group, only 1 case of dural tear caused by misplaced screws was observed, and no cerebro-spinal fluid leak for this patient.Numerous studies have investigated the safety and efficacy of pedicle screw placement in spinal surgery. However, the perforation rates vary from 1% to 43% in literature, and the variation seems to be attributed to the postoperative imaging technique, the grading scheme used, and the threshold for classifying a screw as misplaced –19. CT hThe grading system evaluating perforation with 2 mm increments was utilized in this study. This method is widely considered the standard method to evaluate the accuracy of pedicle screw placement in spine surgery. The system is based on anatomic measurements featuring good reliability and readiness , 18. In Belmont et al.  reportedUnlike AIS, the perforations occurred more frequently in lumbar spine than in thoracic spine (57/193 VS. 24/102). Double major curves and lumbar curves were more frequently seen in Marfan syndrome, and pedicles of lumbar spines were always extremely thin; thus, even when pedicle screws were placed in the right directions, perforations would inevitably occur (Fig.Many methods to enhance safety and accuracy of pedicle screw placement have been developed, such as funnel technique, intraoperative C-arm image intensifier, CT-assisted technique and computer-assisted technique –31. WithThere is a significant learning curve associated with pedicle screw placement , and allPlacement-related complications include neurologic impingement, aorta injury, dural tear, vertebral fracture , 17, 33.In conclusion, placement of pedicle screw in Marfan syndrome is accuracy and safe. O-arm navigation was an effective modality to ensure the safety and accuracy of screw placement. Special attention should be paid when screws were placed at the lumber spine and the concave side of spine deformity to avoid the higher rate of complications.


Phosphorylation under plausible prebiotic conditions continues to be one of the defining issues for the role of phosphorus in the origins of life processes. In this review, we cover the reactions of alternative forms of phosphate, specifically the nitrogenous versions of phosphate (and other forms of reduced phosphorus species) from a prebiotic, synthetic organic and biochemistry perspective. The ease with which such amidophosphates or phosphoramidate derivatives phosphorylate a wide variety of substrates suggests that alternative forms of phosphate could have played a role in overcoming the “phosphorylation in water problem”. We submit that serious consideration should be given to the search for primordial sources of nitrogenous versions of phosphate and other versions of phosphorus. One of the most important elements for life on the earth is phosphorus . As a crHowever, there is a parallel approach where “alternative forms” of phosphate have been considered inspired by the discovery of phosphonic acids in Murchison meteorite by Schwartz ,6. The oWe begin with the prebiotically plausible amidophosphate derivatives which have been shown to be excellent phosphorylating reagents in aqueous medium, of biogenic and prebiological organic molecules, in a prebiotic context. Then, we describe other nitrogenous analogs (P–N derivatives) of various oxidized states of phosphorus that have been discovered and used in other contexts (such as organic synthesis) along with their useful applications in origins of life chemistry research. In the next section, biochemically relevant P–N derivatives that are used as phosphorylating (phosphoryl transfer) agents are discussed. We then present a survey of plausible prebiotic P–N based species and end with a perspective that the reactivity of the plausible prebiotic, synthetic and biological P–N variants may provide inspiration and insights for finding a library of solutions to the ‘prebiotic phosphorylation in water problem’.4O10 at elevated temperature . Kr. Kr3 as n yields  37]. Al. Al3 as n-alkylamines 16 with trimetaphosphate 1 generated the corresponding N-triphosphate 17, which underwent an intramolecular phosphorylation to form potentially prebiotic amphiphiles such as O-monophosphate 19 in the absence of divalent metals at pH 10 (18 (not observed). Depending on the length of the alkyl chain, varieties of different products were obtained, and the longest chain compound, where R=C8H17 produced O-monophosphate product 19 . Ho. Ho3, it3 for the potential prebiotic synthesis of phosphoenol pyruvate 34 which is the highest-energy molecule involved in extant metabolic pathways of glycolysis . Su. Su1 to 44  was highlighted in another publication by Zhao et al. ..98].115  was discovered in skeletal muscle [70 to regenerate ATP 67 and creatine 118 as shown in the following reaction in Another important phosphorylating reagent in biology, phosphocreatine l muscle  and servEscherichia coli Histidine Triad Nucleotide Binding Proteins (Hint) using phosphoramidate pronucleotides and have shown that the therapeutic utility of nucleoside phosphoramidates could be expanded by improving their cellular uptake and also by incorporating an additional tissue targeting compound . However, there seems to have been no necessity (from a prebiotic perspective) to consider corresponding P–N analogs, even though P–N chemistries have been invoked in origins of life studies as exemplified by the central role played by the development of phosphoroimidazolides of nucleotides necessary for the RNA world hypothesis. And the reason seems to be solely based on the focus on the role of phosphates in extant biological pathways. However, as will be argued below, there may be other compelling reasons to reconsider this approach.J = 5–4 transition during the analysis of Orion-KL spectral line [J = 2–1, 3–2, 5–4 and 6–5 rotational transitions of this species in molecular sources and found all the four lines toward Orion-KL [The first reason is that primordial P–N sources are indeed available. That it is not unreasonable to propose primordial P–N containing compounds gains some credibility based on the fact that the first series of phosphorus compounds that were detected in the interstellar medium (ISM) diffuse clouds and several star-forming regions were in fact P–N species , whose oral line . To veriOrion-KL . Their aOrion-KL . FurtherOrion-KL . HoweverOrion-KL . And in The challenges concerning the prebiotic phosphorylation by phosphate containing minerals ,109,110 3P), an analogue of schreibersite, in water containing acetate gave diverse water soluble reduced phosphate species such as orthophosphate, hypophosphate, phosphites, and even organic phosphate compounds such as cyclic organic phosphate, phosphonoacetate and others [3P were proposed to be responsible for the formation of such reduced phosphate from the minerals.The formation of reduced state P compounds from the meteorite mineral schreibersite, a common mineral in iron meteorites was demonstrated by Lauretta and Pasek ,9 and Ked others  nt et al . They fuper year , which sper year .. As opposed to the suggestions made in the paper [An alternate route to synthesize a wide range of organophosphates of biological significance in a deep eutectic solvent (2:1 urea and choline chloride), utilizing various orthophosphate sources was again demonstrated by Gull et al. . As oppohe paper , the acthe paper  and alsohe paper .3P (major component of schreibersite) with glycerol under aqueous condition demonstrates the ability of such minerals in the prebiotic phosphorylation of biological molecules on early Earth [Again, the synthesis of membrane making glycerol phosphate along with other reduced oxidation state phosphorus species by the reaction of Fely Earth .5(PO4)3OH, one of the most common phosphorus minerals in Haden Earth, is insoluble in water but in the presence of this eutectic solvent is able to take part in the phosphorylation reaction with increased rate [As discussed elsewhere ,119 minesed rate .4PO4·6H2O). Pasek and Gull demonstrated the effectiveness of struvite in phosphorylating the prebiotic molecule like glycerol and choline chloride although they raised the question on the prebiotic relevance of struvite [43–) was added to seawater containing more than 0.01 M ammonium [4+ ions [The more efficient mineral for prebiotic phosphorylation consideration is struvite  that have been invoked to enable phosphorylation in a prebiotic context. Extending this trend backwards in time, it is worthwhile to speculate about the existence and availability of the nitrogenous versions of the reduced phosphate species  were used with the sole purpose of driving the reaction towards complete formation of AmTP and DAP quickly. Such short-time conversions need not be necessary in the case of prebiotic geochemical scenarios, and could allow for lower milli- or micro-molar concentrations of compounds to react over longer time scales. Whether such concentrations of ammonia would have been available on early earth has been a matter of debate [In this context, it is once again important to note that the only known formation of AmTP f debate  in termsf debate ,130,131.f debate ,133. Whaf debate  which caf debate ,136,137 The various chemistries of P–N derivatives remain largely unexplored and underutilized in the context of prebiotic phosphorylation chemistry even though they have been repeatedly demonstrated to function effectively in aqueous medium with respect to the important process of phosphorylation, overcoming the “water problem”. The examples reviewed above, drawn from synthetic organic chemistry and biochemistry show that P–N derivatives do have the capacity to phosphorylate a wide variety of substrates, and in many cases also have been demonstrated to work under prebiotically relevant aqueous conditions. Furthermore, some of these examples show the potential to go beyond the phosphorylation reactions. For example, amino acids once phosphorylated and having formed P–N derivatives have been shown to form peptides. These reinforce the possibility that there is not only potential for overcoming the phosphorylation problem, but also the potential to connect the various classes of compounds to their respective higher order structures (polymers). This could be extended to a) the nucleosides giving rise to oligonucleotides, and b) the formation of phospholipids giving rise to self-assembled enclosed structures such as vesicles. As more and more chemistries of the P–N derivatives are demonstrated in a prebiotically relevant context, the search for prebiotically plausible sources and existence of P–N derivatives on the early earth will become critical, if these findings are to have any meaningful impact on, and be pertinent to, the origins of life research.


Based on a plenty of different applications, intracardiac echocardiography (ICE) is now a well-established technology in complex electrophysiological procedures. Recently, ICE has become the most widely used ultrasound-based imaging tool to guide diagnostic endomyocardial biopsy (EMB). EMB of cardiac mass guided by ICE is an interesting application of ICE. Allowing a correct positioning of the bioptome, ICE reduce the procedure-related risks and the need of a diagnostic open-chest procedure reserving the more invasive approach to selected cases.Hereby we report a case series of right ventricular masses in which the EMB was safely and effectively performed under ICE guidance giving essential information for planning the therapeutic strategy.The diagnosis of both metastatic and primary cardiac tumors relies on the histopathological analyses. The endomyocardial biopsy is a valuable tool for preoperative diagnosis and surgical planning of intracardiac masses suspected for tumors. In our experience, the use of ICE for right ventricle EMB of an intracardiac mass is an attractive modality thanks to the precise localization of the cardiac structures and the ability to guide bioptic withdrawal in the target area.The online version of this article (10.1186/s12872-018-0749-9) contains supplementary material, which is available to authorized users. Intracardiac echocardiography (ICE) is a well-established imaging technology frequently used in complex electrophysiological procedures, such as atrial fibrillation and ventricular tachycardia ablation. As in other scenario, ICE can contribute in improving the efficacy and the safety of procedures. It allows a better definition of the anatomical structures involved in ablation procedures and permits a continuous monitoring of catheter-tissue contact avoiding procedure-related complications . For theWe describe three different cases of EMB of right ventricular masses performed under ICE guidance. In all cases, ICE imaging was performed using a 9F ViewFlex™ Xtra 64-element phased-array and 4-way steerable ultrasound catheter  inserted via the right femoral vein or 8F Acunav 4-way steerable ultrasound catheter . The technique used by our operators to perform EMB guided by ICE is described in Additional file The study was conducted according to our Institutional Review Board and in adherence to the Declaration of Helsinki. A written informed consent was obtained from all patients or their closest relatives.A 65-year-old woman smoker with a long history of malignancy was referred to our Center due to the evidence of an intracardiac mass at transthoracic echocardiography (TTE). Her past medical history included pulmonary adenocarcinoma treated with right lobectomy and lymphadenectomy, right nephrectomy due to clear cell renal carcinoma and total gastrectomy for adenocarcinoma. The TTE revealed a hyperechogenic thickening at the apex of the right ventricle (RV) without clear evidence of pathological mass. CMR showed a 20 × 28 mm intramyocardial mass localized at the apex of the RV Fig. a. Due toA 54-years-old male, with previous history of liver transplant related to hepatocellular carcinoma (HCC) and HBV-cirrhosis, presented to our center for severe dyspnea. TTE demonstrated a 23 × 27 mm multi-lobed mass in the right ventricular outflow tract (RVOT) involving the pulmonary trunk. CMR imaging and contrast-enhanced CT also confirmed the intracardiac mass detected by TTE  [–18.3% [3Although clinical diagnosis of intracardiac masses could be mainly done by echocardiography, CT, and/or CMR, histopathological analysis remains crucial for differential diagnosis. Histology provides critical information for treatment and prognosis, which are largely dependent upon tumor histotype and biological behavior. EMB is a valuable tool for diagnosis of intracardiac masses. It is mainly indicated for the investigation of right-sided masses showing an infiltrative or obstructive growth pattern and for the differential diagnosis of sarcomas, lymphomas, and metastatic tumors. Unresectable intracardiac masses may benefit from EMB to plan therapy or palliative treatment .We demonstrated the usefulness of ICE guidance to perform EMB in different sites and pathologies involving the RV. In all cases, the histopathological diagnosis was made on samples obtained by EMB ICE-guided. Our approach was helpful not only for the final diagnosis but also to plan the optimal therapeutic strategy, reserving more invasive procedure only for selected cases. Indeed, the use of ICE eliminates the risks of a diagnostic open-chest procedure. According to this strategy, surgery is reserved only for patients who would benefit the most in terms of prognosis. Another approach to perform transvenous EMB is under fluoroscopic or 2D- or 3D–transesophageal echocardiography (TEE). However, the use of fluoroscopic guidance to perform right ventricular EMB has some drawbacks, in particular the inability to accurately guide the bioptome, the longer radiation exposure and some possible complications including myocardial perforation, tricuspid valve apparatus disruption and arrhythmias. On the other hand, 2D or 3D–TEE can be cumbersome, it requires general anesthesia and a skilled echocardiographist in the operating room .Recently, the use of ICE has increased in many procedures in order to facilitate transseptal puncture, ablation of cardiac arrhythmias, percutaneous mitral valve intervention, interatrial septal abnormalities closure, and left atrial appendage closure. The capability to clearly show intracardiac structures makes ICE a safe and effective tool to guide transcatheter EMB of right side cardiac mass. Moreover, EMB can be performed under conscious sedation with direct visualization of the mass minimizing the risk for perforation. Of note, there are some drawbacks related to the use of ICE, such as the need of a venous approach, the risks of right chamber catheterization and some technical and economical aspects as the probe cost .To the best of our knowledge, there are few reports of ICE-guided EMB of right atrial cardiac tumor –9, in coAdditional file 1: Movie 1.The whole action of EMB is showed. Fluoroscopy image in RAO view: ICE probe is placed across the tricuspid valve and positioned in the right ventricular inflow tract to image the ventricles in the long axis view. Through a long-deflectable sheath  the bioptome is inserted and directed to the mass visualized by ICE. After checking carefully the placement of the bioptome, a sample is taken. (MOV 4285 kb)Additional file 2: Movie 2.ICE imaging of EMB taken in the right ventricular apex. (MOV 4960 kb)Additional file 3: Movie 3.ICE imaging of massive multi-lobed intracardiac mass in the right ventricular outflow tract. (MOV 4341 kb)Additional file 4: Movie 4.EMB guided by ICE. ICE visualizes a good contact between the bioptome and the mass. (MOV 4225 kb)


Here we evaluated by site-directed mutagenesis the contribution of Lys251/Asp252 pair  in electron transfers and associated with it proton uptake to the quinone reduction site (Qi site). We showed that the absence of protonable group at position 251 or 252 significantly changes the equilibrium levels of electronic reactions including the Qi-site mediated oxidation of heme bH, reverse reduction of heme bH by quinol and heme bH/Qi semiquinone equilibrium. This implicates the role of H-bonding network in binding of quinone/semiquinone and defining thermodynamic properties of Q/SQ/QH2 triad. The Lys251/Asp252 proton path is disabled only when both protonable groups are removed. With just one protonable residue from this pair, the entrance of protons to the catalytic site is sustained, albeit at lower rates, indicating that protons can travel through parallel routes, possibly involving water molecules. This shows that proton paths display engineering tolerance for change as long as all the elements available for functional cooperation secure efficient proton delivery to the catalytic site.Describing dynamics of proton transfers in proteins is challenging, but crucial for understanding processes which use them for biological functions. In cytochrome  •i catalysis of cyt bc1 is explored by mutagenesis.Contribution of D252 and K251 in Q•The proton path is disabled only when both protonable groups are eliminated.•2 triad in Qi site.H-bonding network influences thermodynamic properties of Q/SQ/QH•i site are implicated.Parallel routes for proton transfer to the Q In Peter Mitchell's redox loop mechanism, proton translocation is achieved by a functional coupling of two reactions: an oxidation of quinol with release of two protons at one side of the membrane and a reduction of quinone with uptake of two protons at the opposite side of the membrane bc1 is a functional dimer b subunit, which together with cytochrome c1 and iron-sulfur (ISP) subunit form the catalytic core of the monomer o and Qi sites, respectively. In the Qo site, the oxidation of quinol releases two protons to the intermembrane space. The electrons from this reaction are directed into two separate cofactor chains. The high potential c-chain transfers one electron to cytochrome c via iron-sulfur cluster [2Fe-2S], while the low potential b-chain delivers the second electron through hemes bL and bH to the Qi site. The sequential reduction of quinone to quinol through a semiquinone intermediate (SQi) is associated with an uptake of two protons from the mitochondrial matrix or cytoplasm i site requires oxidation of two quinol molecules at the Qo site. In addition, the electron transfer between two hemes bL is possible o and two Qi sites in the dimer.Cytochrome bc1 are well defined, the proton paths are much less known. This is in part due to the lack of methods that can directly monitor proton transfers. While uncertainties related with proton transfers concern both the Qo and Qi sites, here we focus just on the Qi site.While the electron paths within cytochrome bc1 were known, early site-directed mutagenesis successfully identified several key protonable residues associated with the operation of the Qi site i site i) and/or its protonation/deprotonation. Besides these amino acid side chains, cardiolipin (CL) was also postulated to facilitate proton transfers at the entry point from the protein exterior (dimer interface) to the Qi site. In this scenario, CL together with a neighboring lysine residue (Lys251) and water molecules can form the CL/K pathway delivering protons to the site Before X-ray structures of cytochrome i site to form a salt bridge with the deprotonated and negatively-charged Asp252 side chain. This fully bent Lys251 conformation, which is not seen in any substrate-bound X-ray crystal structures, results from semiquinone binding in the simulations, but pKa calculations indicate that the switch-like motion would be pH-dependent and possible even without a bound substrate at the Qi site.Our recent MD simulation study i site. In view of this new finding, we examined the consequences of replacements of Lys251 and Asp252 with non-protonable residues for the functioning of cytochrome bc1in vivo and for the kinetics of electron and proton transfers. Comparative analysis of separate replacements of either Lys251 or Asp252 side chains (single mutants) and simultaneous replacements of both side chains (double mutants) supports the idea that functional cooperation between Lys251 and Asp252 facilitates proton transfers to the Qi site. It also reveals a limited plasticity of this path to accommodate a lack of one, but not two of protonable groups from the Lys251/Asp252 pair.The rotation of the Lys251 side-chain implicates the possibility of functional connection between Lys251 and Asp252 for proton transfers to the Q22.1Rhodobacter (R.) capsulatus cells containing substitutions at 251 and 252 positions in cytochrome b subunit were obtained using a genetic system originally developed by Dr. F. Daldal b gene using QuikChange site-directed mutagenesis system (Stratagene) and the following PCR primers:CGG CTG TTC GCG CTG GCC-3′;D252A_F: 5′-TAT TTC GTG ATC AAG CGC CTT GAT CAC GAA ATA CGG-3′;D252A_R: 5′-CAG CGC GAA CAG AAC CTG TTC GCG CTG GC-3′;D252N_F: 5′-TTC GTG ATC AAG TGT CTT GAT CAC GAA ATA CGG-3′;D252N_R: 5′-AG CGC GAA CAG TGA GAC CTG TTC GCG C-3′;K251M_F: 5′-G TAT TTC GTG ATC ATC GAT CAC GAA ATA CGG C-3′;K251M_R: 5′-C GAA CAG GTC TG GCGA CTG TTC GCG CTG GCC C-3′;K251M/D252A_F: 5′-G TAT TTC GTG ATC CGC CAT GAT CAC GAA ATA CGG C-3′;K251M/D252A_R: 5′-GC GAA CAG TG AACA CTG TTC GCG CTG GCC C-3′;K251M/D252N_F: 5′-CCG TAT TTC GTG ATC T CATGT GAT CAC GAA ATA CGG C-3′.K251M/D252N_R: 5′-GGC CAG CGC GAA CAG petABC operon was used. The BstXI-XmaI fragment of the operon containing the desired mutations, and no other mutations, were inserted into pMTS1 vector and introduced into MT-RBC1 R. capsulatus strain using triparental crossing petB gene on plasmid isolated from mutated R. capsulatus strains. R. capsulatus bacteria were grown under semiaerobic or photoheterotrophic conditions as described previously + phenotype (photosynthetic competence) were isolated, and reversion mutations were identified by sequencing the entire petABC operon.As a template DNA pPET1 plasmid containing wild type (WT) 2.2R. capsulatus cells growing under semiaerobic conditions. After isolation, chromatophores were homogenized and suspended in MOPS pH 7.0 or Tris pH 9.0 buffer (for light-induced electron transfer measurements) or in 50 mM Tris pH 8.0 buffer containing 100 mM NaCl, 0.01% DDM and 20% glycerol (for protein purification). Cytochrome bc1 complexes were isolated from detergent-solubilized chromatophores using ion-exchange chromatography (DEAE-BioGel A) as described Procedure described previously in ref. 2.3bc1 in chromatophores. Transient kinetics of hemes b were measured at 560–570 nm after activation by single saturating flash (~ 10 μs). Measurements were performed at pH 7.0  or pH 9.0  under conditions of low (100 mV) or high  ambient redox potential. Experiments were performed under anaerobic conditions in the presence of redox mediators and valinomycin as described in bH reduction in the presence of antimycin, bH re-oxidation without inhibitors and to heme bH reduction from reverse reaction in the presence of myxothiazol  and subsequent addition of 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzohydroquinone (DBH2) as a substrate. The negative control was obtained by addition of antimycin (Qi site inhibitor) to samples treated previously with myxothiazol and DBH2. Both DBH2 and myxothiazol were used at final concentration of 200 μM while antimycin was used at 400 μM. Quantitative EPR analysis of the semiquinone was performed using 4-Hydroxy-TEMPO (TEMPOL) as a standard as described in i measurements.CW EPR spectra of semiquinone were obtained for isolated cytochrome 33.1Conclusions drawn from MD simulations described by Postila et al. bc1 with all three catalytic subunits . The difference optical spectra of all mutated complexes in the isolated form were similar to that of the native complex. The ability to grow under photosynthetic (Ps) conditions, which tests functionality of cytochrome bc1in vivobc1 is not functional in vivo  under ambient redox potential setting hemes b oxidized and the quinone pool half-reduced prior to flash activation. Under these conditions, heme bH in the native enzyme undergoes light-induced reduction followed by re-oxidation (o site. The re-oxidation phase occurs through the action of the Qi site (reduction of quinone to semiquinone and then semiquinone to quinol) and is blocked by antimycin, a potent inhibitor of this site . While the reduction rates in the presence of antimycin in all mutants are similar and comparable to WT  this level approaches approximately 70% of the maximum reduction level (seen in the presence of antimycin), in the double mutants, it reaches the maximum reduction level (the amplitude of black and red trace are comparable).Kinetic transients shown in le to WT  the leve2 to heme bH at the Qi site (reverse reaction) under conditions where the Qo site is blocked by myxothiazol and the reduction power of Q pool is increased (by increasing pH). Reduction of heme bH under these conditions is not observed on a millisecond time scale in D252A and in both double mutants. In D252N this reaction is 70 times slower than in WT .Kinetic transients shown in 3.3i site, we conducted a series of measurements of electrogenic reactions associated with the operation of cytochrome bc1 by following the antimycin-sensitive phase of carotenoid bandshift  clear SQi signal can be observed only in WT and D252N .Semiquinone in the Q – 2.004 . Typicalnd D252N A, C. Qua44.1i site without much influence on the Qo site.The roles of Lys251 and Asp252 in proton management of the Qi site, suggested by MD simulations o site was inferred from little influence of the mutations on the rates of Qo site-mediated heme bH reduction  for which the kinetics comparable to WT were reported bH re-oxidation in the light-induced kinetics in the absence of inhibitors Similar slowing of the re-oxidation of heme bH and Q or SQ was shifted in the mutants towards reduction of heme bH in comparison to WT (vs red traces), implicating that heme bH in mutants faces difficulty in delivering electron to quinone occupying the Qi site. This effect is apparently not a result of a changing in the redox midpoint potential (Em) of heme bH given the values of Em determined by redox potentiometry  upon generation of transmembrane electric field. The antimycin-sensitive phase of carotenoid bandshift is associated with the action of cytochrome bc1 complex. Concerning the previous studies i site after the full quinone reduction is completed. This concerns protonation of oxygen atoms at both the C-1 (through the K251/D252 path) and C-4 groups (through the H217 path) of reduced quinone.For all these mutants the process of proton uptake from bulk solutions to the Qi site, while the elimination of this phase in double mutants indicate much more severe blocking of this process. Single K251M does not influence much the proton uptake, as indicated by similar rate and amplitude of the carotenoid bandshift phase in this mutant (comparing to WT). The mutants of Asp252 in R. sphaeroides also affected this phase: D252N showed a slowing, with diminished amplitude while in D252A this phase was abolished. K251M showed a slower phase without amplitude change. In all three cases, changes in the carotenoid bandshift appear to be more severe in R. sphaeroides than the effects of respective mutants shown here i site.In light of this assumption, the diminished amplitude of the carotenoid bandshift phase in D252A and D252N, and additional slowing in D252A, reflect overall difficulty in uptake of protons to the Qin vivo but replacement to Leu deactivated the enzyme leading to loss of its functional competence in vivoThis, in view of electron transfer measurements, MD simulations and crystal structure data, is most likely associated with the hampered K251/D252 path affecting protonation of quinone C-1 carbonyl. The role of His217 in C-4 carbonyl protonation is inferred from previous studies which showed that replacing His217 to Asp or Arg yielded enzymatically active complexes functional bc1in vivo: only mutants that show this phase at measurable rates and amplitudes are able to grow photosynthetically. This is understandable, if one considers that the efficiency of proton transfers ultimately defines proton motive generating capacity of the enzyme in vivo. This further substantiates the notion that this phase reflects the protons uptake from aqueous phase into the Qi site.We note that, if this and other mutational works including Additional indication for involvement of D252 in proton transfer came from the observation that barely functional D252A and non-functional K251M/D252A or K251M/D252N mutants regained functionality by restoring protonable group (either E or D) at position 252 .4.2i  and Qi site E, F). Al2 and heme bH are generally similar for K251M, D252A and D252N we notice some differences that result from different effect of Lys and Asp on Q/SQ/QH2 binding and proton transfer between protein interior and exterior. The sharpest differences between Lys and Asp mutants become visible when analyzing traces in which only theoretically one-electron reactions are involved. It is clear that when Q is awaiting electron from heme bH in all three mutants K251M, D252A or D252N the electron is mostly retained at the level of heme bH as if the potential of Q/SQ couple was lowered. For K251M, it may reflect a higher degree of deprotonation of Asp carboxyl group that cannot be stabilized by interaction with amine group of Lys which leads to destabilization (weaker binding) of Q or SQ within the Qi site. This destabilization seems to be even more severe for mutant having Asp replaced with non-protonable residues (D252A and D252N) for which there is no direct partner for quinone or semiquinone that may deliver proton and stabilize the binding.The more complex effects are associated with single replacements of either K251 or D252 with non-protonable amino acids. Although the reactions associated with electron transfer between Q or QH2 oxidation by heme bH in the Qi site) the differences between the mutants shed light on the proton reactions associated with the SQ/QH2 couple. A lack of QH2 oxidation in D252A mutant indicates that deprotonation of QH2 is blocked when direct proton exchanger (Asp) is replaced by hydrophobic residue. As a result, the semiquinone at the Qi site cannot be effectively formed , as these mutants still retain much of the electron and proton transfer capabilities and remain functional in vivo. This could be result of another protonable group/groups, possibly water molecules, taking over the function of the original side chains that are missing in the mutants, or a reminiscence of natural existence of parallel (multiple) paths for protons in native protein B site of photosynthetic reaction center i site which yields mutants non-functional in vivo with fully inactive Qi site. This indicates that at least one of the protonable side chains at either position 251 or 252 must by present. In addition, in R. sphaeroides it was observed that the inversion of charges at positions 251 and 252 (double mutant K251D/D252K) had little effect on enzymatic activity and did not affect the function of enzyme in vivoWe emphasize, however, that in light of experimental results, any scenario assuming a sequential mechanism of transfer of protons involving Lys251 and Asp252 should be considered as a possible, but certainly not the unique path available for protons to enter the QTransparency document.Image 2


Here we identify the functionally relevant site for PKA phosphorylation on the human cardiac L-type Ca2+ channel pore forming α1 subunit using a novel approach. We used a cell free system where we could assess direct effects of PKA on human purified channel protein function reconstituted in proteoliposomes. In addition to assessing open probability of channel protein we used semi-quantitative fluorescent phosphoprotein detection and MS/MS mass spectrometry analysis to demonstrate the PKA specificity of the site. Robust increases in frequency of channel openings were recorded after phosphorylation of the long and short N terminal isoforms and the channel protein with C terminus truncated at aa1504. A protein kinase A anchoring protein (AKAP) was not required. We find the novel PKA phosphorylation site at Ser1458 is in close proximity to the Repeat IV S6 region and induces a conformational change in the channel protein that is necessary and sufficient for increased calcium influx through the channel.The “Fight or Flight” response is elicited by extrinsic stress and is necessary in many species for survival. The response involves activation of the β-adrenergic signalling pathway. Surprisingly the mechanisms have remained unresolved. Calcium influx through the cardiac L-type Ca S proteins. The locally increased cAMP level activates cAMP- dependent protein kinase A (PKA) that phosphorylates many target proteins3 ultimately increasing the intracellular calcium level and contractile force.It is well recognized that many species are able to evade a threat by way of activation of the “Fight or Flight” response. The sympathetic nervous system drives this through activation of the β-adrenergic receptor signaling pathway that results in positive chronotropic, inotropic and lusitropic effects in the heart. At the level of cardiomyocytes, β-adrenergic receptor (β-AR) stimulation leads to an increase in cyclic AMP (cAMP) production by adenylate cyclase coupled to Gv1.2 protein  is the pore forming and ion conducting subunit of the cardiac L-type Ca2+ channel and the main route for calcium influx into cardiac myocytes. Calcium influx through the channel initiates contraction and regulates excitation. Although β-AR-mediated stimulation of Cav1.2 has been studied for decades, and PKA is a recognized downstream effector of β-AR signaling, the molecular mechanism responsible for PKA regulation of the channel remains unresolved.The Cav1.2 is the target for PKA-dependent phosphorylation. The distal carboxyl terminus (DCT) has been demonstrated to impose an autoinhibitory effect on channel function4, contains the sites for calcium and calmodulin binding, and interaction with A kinase anchoring proteins (AKAPs)5. AKAP is responsible for proper compartmentalization of PKA in cardiac myocytes9, and binds to Cav1.2 channels via the leucine zipper motif in the DCT10 but in vivo studies have failed to prove a direct effect of AKAPs on cardiac contractility12. It has been suggested that the physiologically cleaved C terminal α1 C fragment remains functionally associated with the channel and is responsible for autoregulation14. Deletion of the Cav1.2 α1 DCT at G1796 reportedly caused a loss of β-adrenergic regulation when adenylyl cyclase was directly activated using forskolin in mouse embryonic ventricular myocytes15. Truncation of DCT also caused prenatal or neonatal death, due to incorrect membrane insertion of the channel, leading to proteasomal degradation of the truncated protein16.Many studies have suggested that the C terminus of the Ca17. Since then, it has been concluded that serine 1928 is not essential for PKA-mediated stimulation of Cav1.2 activity19. Serine 1700 and threonine 1704 in the proximal C terminal domain of cardiac Cav1.2 channels were also identified as phosphorylation sites21. Both serine 1700 and threonine 1704 are conserved in the skeletal muscle Cav1.1 channels and serine 1575 and threonine 1579 have been identified as substrates for cAMP-dependent protein kinase. Serine 1575 can be modified by calcium/calmodulin-dependent protein kinase II (CaMKII) in neurons22. Serine 1575 and threonine 1579 in Cav1.1 channels and their homologs in Cav1.2 channels are proposed to be located at a regulatory interface between the distal and proximal C terminal domains22. More recently it has been demonstrated that serine 1700 is not necessary for β-adrenergic responses in a mouse model23 but is necessary for physiological regulation of the channel24.Serine 1928 in the DCT was first identified as the main target for PKA-dependent phosphorylation in rabbit heart25, alternative splicing26, and post-translational proteolytic cleavage at alanine 1800 in the DCT that occurs in vivo14. Choosing the most appropriate model system is pivotal for comparative studies. However limited knowledge of the β-AR signaling pathway makes it difficult for its full reconstitution in heterologous expression systems27.Controversies in phosphorylation studies may originate as a result of the complexity of the β-AR signaling pathway, the heterogeneity of calcium channels in the models testedv1.2 channel by reconstituting the channel protein into artificial liposomes. This cell-free system allows for the measurement of changes in ion-conducting channel function as a result of direct modification of the channel protein measured as single channel currents using patch-clamp technique. The major benefit of this functional assessment compared with the use of expression systems is that channel function is studied in isolation from the auxiliary subunits of the channel complex and the regulatory pathways present in the cell28.In this study, we used a direct approach to study the human cardiac Cav1.2 channel (corresponding to serine 1535 in Q13936-1) is the target for PKA phosphorylation. Homologous serine 1517 in murine neurons29 and serine 1570 in rabbit heart30 mediate Ca2+ current facilitation. CaMKII shares the same consensus binding site with PKA, and can phosphorylate and modify the function of voltage-dependent calcium channels31. It has been suggested that simultaneous activation of these two intracellular signaling pathways contributes to the ‘Fight or Flight’ response32. Our results demonstrate that phosphorylation of serine 1458 by PKA is necessary and sufficient to increase open probability of Cav1.2. This is likely to be due to the close proximity of the serine to the ion pore forming region of the channel. CaMKII further potentiates the increase in open probability as a result of binding to the DCT in the full length channel protein.We find that serine 1458 in the truncated C terminal short-NT isoform of the human cardiac Cav1.2 channel is approximately 240 kDa  was used to promote channel openings. We confirmed the properties of the channel by voltage-stepping from −150 mV to +150 mV and measuring the magnitude of the current and its sensitivity to the L-type Ca2+ channel antagonist nisoldipine. The magnitude of the outward single channel current at 150 mV was approximately 2.5 pA, decreased as voltage approached 0 mV, reversed at 0 mV (consistent with symmetric solutions) with a slope conductance of approximately 21.3 pS  was 0.022 ± 0.002 (n = 7) and the mean open dwell time was 23.0 ± 3.4 ms  for the C terminal truncated channel  as well as the incidence of mode 2 openings of the channel (35.2 ± 8.3% vs 4.7 ± 2.4% long-NT isoform) in the absence of BayK. This supports a role for the intracellular C terminus in channel gating, supporting mode 0 and mode 1 openings.Previous nus Fig.  by truncnel Fig. . This isImmunoblot analysis did not detect a difference in phosphorylation between the full length or truncated isoforms of the channel  and trials of in silico phosphorylation site prediction programs  were used to predict PKA substrate sites. Considering direct enzyme-substrate interactions when a serine was preceded by an arginine at −3 position, serine 436 (homologous to S465 in Q13936-1) in the Repeat I-II linker, serine 754 (S783) and serine 834 (S863) in the Repeat II-III linker region, and serine 1458 (S1535) in the C terminal end before the truncation site were mutated to alanine in order to prevent potential phosphorylation of the serine in vitro.The long-NT isoform of Cao = 0.046 ± 0.01, n = 7), current amplitude (2.5 ± 0.2 pA) or dwell time  truncated C terminal Cav1.2 channel, when voltage was stepped to 150 mV , without altering the current-voltage relationship, amplitude or mean open dwell time  and 0.040 ± 0.007 to 0.105 ± 0.025 (n = 6) respectively, without altering other characteristics . Application of PKA had no significant effect on the Po or current-voltage relationship  of WT and mutant proteins was determined by taking the temperature at which the RFU (relative fluorescence unit) reached 50% of the maximum. The WT truncated C terminal short-NT isoform and the S1458A mutant isoform demonstrated similar melting peak profiles  in control samples and 1786.84 (596.62 3+ m/z) in PKA treated samples, showing a 79.98 mass difference, consistent with peptide phosphorylation. A neutral loss of 97.97 from the serine 7 residue was observed in MS/MS spectra of the PKA treated peptide and this was confirmed by in vitro phosphorylated YLTRDWSILGPHHL peptide and the synthetic phospho-peptide  on PKA phosphorylation of the channel, we used 10 μM St-Ht31 inhibitor peptide to specifically block the association of the regulatory subunit of PKA and AKAP proteins. Following addition of St-Ht31, PKA significantly increased the function of the long-NT isoform Fig. . We alsov1.2 channel protein (Table The effect of 5 μM PKI (PKA Inhibitor fragment 5–24 amide) was also examined Fig. . Incubatv1.2 channel protein to PKA resulted in a significant increase in Po to 0.109 ± 0.021, while subsequent exposure to PP2A decreased Po to 0.041 ± 0.013  is the target for PKA phosphorylation. The orthologous serine at 1517 in rabbit Cav1.2 is proposed to be a target for CaMKII phosphorylation30. We did not find any evidence for a further increase in channel function by CaMKII in the C truncated Cav1.2 protein  of the truncated C terminal short-NT protein were resistant to phospho-modification by cAMP dependent protein kinase. Quantitative fluorescent phosphoprotein analysis detected only one extra phosphate group after v1.1 was recently determined42, the structure of the Cav1.2 protein has not yet been fully resolved. Serine 1458 is located in the proximal carboxyl terminus in close proximity to the Repeat IV S6 membrane spanning region, which is part of the ion conducting pore of the α1 C subunit. Phosphorylation at this site on the proximal C terminus would be expected to induce a local conformational change and add a negative charge close to the ion conducting region, which remains in contact with the rest of the channel following proteolytic cleavage of the distal C terminal domain4. The proximal C terminus is also suggested to interact with the N terminus in the folded channel protein.Although the structure of Cav1.2 activity, but β1-AR stimulation activates PKA more globally and results in phosphorylation of non-sarcolemmal proteins (such as phospholamban and troponin I), while β2-AR stimulation is more local, limited to activation of PKA compartmentalized with Cav1.244. G protein coupled receptor activation can be further complicated as a result of activation of β3-AR’s that can antagonise β1 and β2-AR signalling via activation of nitric oxide45. Compartmentalization is facilitated by proteins such as AKAP’s that assist by directing PKA to target proteins41. AKAP’s are proposed to mediate specialised functional responses via specific isoforms. The targeted responses can be directed to compartments within the cell or from cell to cell as is the case with sympathetic innervation of the heart by neurons delivering catecholamines46. Recently it was identified that AKAP-mediated PKA responses can be constrained within close proximity of the target47. In failing heart, the chronically elevated adrenergic agonist down-regulates the dominant β1-AR and uncouples the β2-AR from downstream Ca2+ regulatory target proteins via AKAP signalling complexes48. A higher phosphorylation level of E-C coupling proteins is also well documented in heart failure. This results in less efficient excitation-contraction coupling and decreased contractile function49, leading to impaired adaptation to hemodynamic demands.In the heart adrenergic stimulation is mediated via activation of β1 and β2-AR’s. Both adrenergic receptors are capable of stimulating the adenylate cyclase-cAMP-PKA pathway enhancing Cav1.2 channel by PKA alters function and we confirm that direct activation is a requirement for increased open probability. The response does not require the presence of the carboxyl terminal domain and therefore suggests that in vivo the physiologically cleaved protein may participate in adrenergic responses. We demonstrate that auxiliary proteins or additional regulatory proteins such as AKAPs are not required for direct activation of the Cav1.2 channel. Our data suggest that in vivo auxiliary or regulatory proteins modulate channel function in addition to direct regulation. We have identified the critical serine for the response. We propose that direct phosphorylation of the Cav1.2 by cAMP-dependent protein kinase is sufficient to alter the conformation of the channel and increase calcium influx during sympathetic stimulation.We examined whether phosphorylation of the Ca2+ channel α1 subunit were used, the long N terminal , and the short N terminal (short-NT) isoform, which also lacked the exon 45 . Using the short-NT isoform cDNA as a template, S1928A mutant and leucine 1504 deletion mutant (Ctrunc) constructs were generated. The cDNA of the different Cav1.2 α1C forms was cloned into the pcDNA3.1 vector (Invitrogen), and modified to include a HIS6 tag at the N terminus.In this study two natural variants of the human cardiac L-type voltage-gated Ca2. Cell transfection with pcDNA3.1-h CAC1C was carried out using Lipofectamine2000 (Invitrogen) according to the manufacturer’s instructions. In brief, 80–90% confluent cells were incubated for 72 h in the presence of the lipofectamine–plasmid DNA complexes. Serum was added only after the first 6 h incubation. The cell pellet was lysed in lysis buffer , centrifuged at 4,200xg for 10 min at 4 °C then purified using Ni-NTA magnetic agarose beads (Qiagen). The purified protein was eluted and stored in −80 °C in elution buffer  then analyzed by SDS-PAGE and immunoblotting  were cultured in DMEM medium, supplemented with 5% fetal bovine serum and antibiotics (Invitrogen) at 37 °C and 5% CO38) were used to predict the most likely PKA substrate sites. Considering the probability of direct enzyme-substrate interaction, the intracellularly located serine 436 in the Repeat I-II, serine 754 and serine 834 in the Repeat II-III linker region and serine 1458 in the C terminal end before the truncation site were chosen to be mutated to alanine in order to prevent potential phosphorylation of the –OH group in vitro when preceded by an arginine at -3 position.To predict the most probable site for protein kinase A phosphorylation, a comparative study of available phosphorylation databases  and trials of in silico phosphorylation site prediction programs . Two oligonucleotide primers containing each desired mutation were extended by Pfu Ultra II DNA polymerase (Stratagene) during PCR. A standard PCR amplification protocol was performed. The quadruple mutant (S436A + S754A + S834A + S1458A) was also constructed from the appropriate mutant cassettes in a stepwise manner. The mutant sequence, orientation, and reading frame of all constructs were confirmed by automated DNA sequencing and restriction digests using XbaI and BamHI restriction enzymes (Promega). Mutant DNA was then purified from an overnight culture using low copy plasmid purification protocol (Nucleobond Xtra Midi plus).Site-directed mutagenesis was undertaken to construct point mutations at serine 436, serine 754, serine 834, serine 1458 using cDNA encoding the ΔL1504 deletion mutant of CaSince the Repeat I-II as well as the II-III linker region contains a further 7 and 9 serine residues aside from the mutated serines, two small GST-fusion peptide constructs corresponding to the linker regions were also generated and tested for protein kinase A modification.v1.2 channel was used as a template to generate the cDNA fragments corresponding to linker I-II and linker II-III region via PCR amplification using appropriate primers and KOD Hot Start DNA polymerase kit (Merck-Millipore). The linker I-II region sequence incorporating glycine 377 to asparagine 495 or the linker II-III region sequence from aspartic acid 725 to threonine 871 was cloned into BamHI and EcoRI sites in pGEX-2T vector with a glutathione-S-transferase (GST) tag. Vector and insert were digested and gel purified, then ligated at a 4:1 ratio using T4 DNA ligase (Invitrogen). Ligated product was transformed into XL-2 blue competent cells (Stratagene). Positive clones were confirmed by Colony PCR and Sanger DNA sequencing. The recombinant pGEX-2T vector was grown in E. coli on Luria Bertini-Ampicillin-Chloramphenicol agar plates overnight, and then amplified in liquid media. Once the OD 600 nm reached 0.6, 100 mM IPTG was added to the culture and then incubated for 5 h at 37 °C. The cell suspension was lysed by sonication. The GST fusion peptides corresponding to linker I-II and linker II-III regions of the Cav1.2 channel were then purified by affinity chromatography using glutathione agarose (Sigma), the product was eluted  then stored at −80 °C for later use. The purified protein was analyzed by SDS-PAGE.The cDNA of the truncated C terminal construct of the Cav1.2 channel protein (1:1000 protein/lipid ratio) following the dehydration/rehydration method as previously described28. Liposomes were added to the bath filled with solution  for 10 min at room temperature to promote the formation of blisters. External and pipette solution  contained recording solution with 2 μM BayK8644 (Sigma) to enhance channel openings. The back filled microelectrode had an average resistance of 16–17 MΩ. Single channel currents were filtered at 1 kHz, digitized at 100 kHz, and analyzed using pClamp software (Molecular Devices)28. The Cav1.2 channel was determined by the magnitude of the current, changes in open probability of the channel (Po) and sensitivity of the current to the L-type Ca2+ channel antagonist nisoldipine. Current traces from control conditions and after addition of 0.5 µM PKA  reduced with 1 mM dithiothreitol  and 5 mM N-ethyl-maleimide  substituted with 1 mM ATP-disodium salt, Sigma) treatments within the same patch were compared during analysis. To specifically block the association of the regulatory subunit of PKA and AKAP 10 μM InCELLect™ AKAP St-Ht31 inhibitor peptide (Promega) was used. FMP-API-1 (Sigma) non-specific PKA-AKAP inhibitor was also applied at 1 mM concentration. For direct inhibition of PKA 5 μM PKI  30 min incubation was used before PKA treatment. PP2A (Promega), a major phosphatase for L-type Ca2+ channels34 was used at the same concentration as PKA, to specifically remove the phosphate group from PKA phosphorylated substrate. For CaMKII phosphorylation studies we substituted the recording solution with 12.2 pM  recombinant human CaMKII δ (Abcam) in the presence of 47 nM (0.8 μg/ml) active recombinant human calmodulin protein (Abcam) activated with 200 μM CaCl2 and 1 mM ATP disodium salt.Single channel experiments were performed on liposome-reconstituted purified Cav1.2 protein samples and GST fusion cytoplasmic linker I-II and linker II-III peptides were phosphorylated in vitro using 1 Unit PKA catalytic subunit (Promega) per 0.5 μg protein for 2 hrs at 37 °C in kinase buffer , Phosphatase inhibitor cocktail IV and V (Merck Millipore)). Dephosphorylation of the protein samples was achieved by using PP2A (0.05 Unit/reaction Promega) for 2 hrs at 37 °C in phosphatase buffer containing 50 mM Tris-HCl, 150 mM NaCl, 1 mM MgCl2, 1 mM DTT, pH 8 with freshly added EDTA free cOmplete mini tablet (Roche). Native PAGE and Western blots were performed to examine protein kinase A phosphorylation over time during in vitro treatment (0–60 min), using SDS PAGE but excluding SDS from all solution  was used. To confirm the position of the channel protein on the membrane, the membrane was reprobed after stripping  with anti Cav1.2 channel primary antibody  and then goat anti-rabbit HRP conjugate secondary antibody (Abcam). The channel protein was finally detected by ChemiDoc MP Imaging System using Luminata Forte, Western HRP substrate (Millipore). Densitometry analysis was performed on 3 individual immunoblots, using the ImageJ software.Purified Ca51. The blots were visualized using ChemiDoc MP. Pro-Q Diamond phosphoprotein blot stain detects phosphoserine-, phosphothreonine- and phosphotyrosine-containing proteins, independent of the sequence context of the phosphorylated amino acid residue. This sensitive technique (detection limit 8–16 ng of phosphoprotein) and signal correlates with the number of phosphate groups, in order to perform quantitative analysis. Subsequently, densitometry was performed for each band on fluorescent images captured by Chemidoc on triplicates, using ImageJ software. Phosphoprotein fluorescent intensity was normalized to total protein fluorescent intensity and then the normalized values were compared with the following protein standards with a known number of phosphate groups (marked for each protein): bovine serum albumin (0), pepsin (1), ovalbumin (2), β-casein (5), α-casein (8) in a concentration range between 10–200 pmol/l52.In separate experiments, phosphoprotein specific fluorescent dye was used to detect phosphorylation. After performing staining with fluorescent ProQ Diamond Phosphoprotein Blot Stain (Molecular Probes) to detect phosphorylated molecules, SyproRuby Protein blot stain was used to detect total proteinv1.2 channel primary antibody, then the corresponding band was excised from the gel and was cut into fine pieces. The fine pieces of gel were immersed in 0.5 ml ddH2O and incubated overnight in a rotary shaker at room temperature. Following incubation, the suspension was centrifuged at 10,000xg for 20 min at 20 °C. The supernatant was transferred into a new tube. Four μg of the purified protein, treated, or non-treated, was mixed with 1:1000 dilution of 5000X Sypro Orange (Molecular Probes). All experiments were performed in triplicates. The samples were heated from 25 °C to 95 °C with a heating rate of 1 °C/min. Protein thermal unfolding curves were monitored by detection of changes in fluorescence of the Sypro Orange dye. The melting temperature (Tm) of the truncated C terminal short-NT isoform S1458A mutant channel proteins was determined by converting the raw fluorescence data to the first derivative of the fluorescence with respect to the temperature (dF/-dT).Truncated C terminal short-NT isoform S1458A mutant channel was purified from 8% native polyacrylamide gel. The protein band was identified on nitrocellulose membrane after electroblot, using specific anti Cav1.2 protein were synthesized by Philip Thompson’s laboratory at Monash Institute of Pharmaceutical Sciences, Faculty of Pharmacy and Pharmaceutical Sciences, Monash University using standard Fmoc Solid Phase Synthesis Protocols on 2-chlorotrityl chloride resin. Phosphoserine was coupled as its benzyl protected derivative Fmoc-Ser[PO(OBzl). The peptide-resin was cleaved with 95% TFA: 2.5% dimethoxybenzene: 2.5 triisopropylsilane; and purified by RP-HPLC to > 95% purity as measure by HPLC-MS.Synthetic peptides of amino acid sequence YLTRDWSILGPHHL, corresponding to aa 1453–1466 of the short N terminal form of Ca2, 1 mM ATP, 34 mM potassium phosphate, 100 μM DTT, 250 U (~5.35 μg) PKA catalytic subunit (Promega)/5 μg peptide in 25 μl reaction mixture. Synthetic peptides were dissolved in 20 μl of 5% acetonitrile (v/v), 0.1% formic acid (v/v), and 5 μl of this resuspension was loaded onto a C18 polaris high capacity nano LC chip (Agilent) in 95% Buffer A (0.1% (v/v) formic acid in Optima grade water (Fisher)) and 5% Buffer B (0.1% formic acid in Optima grade acetonitrile (Fisher)) using a 1200 series capillary pump (Agilent Technologies). Following loading, samples were eluted from the C18 column and into an inline 6550 Series QTOF mass spectrometer (Agilent Technologies) and separated according to the details outlined in Nelson et al. (2014). Agilent .d files were convert to MGF peak lists and were searched against the Human-Uniprot protein database using Mascot 2.3 running on an in-house server, allowing for variable phosphorylation of the protein, no enzymatic digestion, 2+, 3+ and 4+ charge state matches,100 ppm mass tolerance for the parent ion, and 0.5 Da tolerance on MS/MS peaks.Peptide phosphorylation was performed at 37 °C for 1 hour in 50 mM Tris, 10 mM MgClThe Mascot output of the search against the Human-Uniprot database was transformed and analyzed using R (v3.2.2). The data was filtered for the YLTRDWSILGPHHL peptides and grouped by their treatment. The relative phosphorylation was calculated by dividing the number of peptide counts showing a serine phosphorylation by the total number of peptide counts from triplicates of MS measurement analyzed by Mascot .In compliance with journal policy we have made data available and archived on a specialised public repository at The University of Western Australia.Supplementary information


P. asiatisca, and its representative four single compounds, acteoside, 1H-indolo-3-carbaldehyde, isoacteoside, and myristic acid, were evaluated by electron transfer signal blocking activities (ETSBA), which is based on the electron transfer signal of XOD enzymatic reaction. The blocking activities were detected using an electrochemical biosensing method. Compared with control, significant effects were observed after the addition of P. asiatica extract, acteoside, and 1H-indolo-3-carbaldehyde . The IC50 values of the extract and acteoside are 89.14 and 7.55 μg·mL−1, respectively. The IC20 values of the extract, acteoside, and 1H-indolo-3-carbaldehyde are 24.28, 3.88, and 16.16 μg·mL−1, respectively. Due to the relatively lower inhibitory potential of 1H-indolo-3-carbaldehyde, its IC50 was not obtained. In addition, isoacteoside and myristic acid did not show any XOD inhibitory effects. Our data demonstrated that the XOD inhibitory effects of the extract, acteoside, and 1H-indolo-3-carbaldehyde can be accurately evaluated by the ETSBA method. The results from this study indicated that Plantaginis Semen significantly inhibited XOD activities to reduce hyperuricemia and treat gout. The study also proves that measuring the electron transfer signal blocking activities is a simple, sensitive, and accurate method to evaluate the XOD inhibitory effects.The XOD inhibitory effects of Plantaginis Semen, that is, the seeds of Gout is medical condition caused by long-term hyperuricemia and has become increasingly common over the last 15 years. This disease can be a severely disabling disorder inducing significant pain and limiting daily activity and social functioning, leading to a poor health-related quality of life . XanthinRecently, XOD inhibitors from natural products have attracted a great deal of attention due to their nontoxic properties –7. For tPlantago asiatica L. is a perennial herbal plant frequently used as an ingredient in dietary supplements, and the dried ripe seeds of P. asiatica, that is, Plantaginis Semen, are one of the most popular folk herbal medicines in China and other Asian countries as plant-based diuretics  spectrophotometric method may easily give false results as certain compounds in a given botanical can absorb light [It has been shown that XOD inhibitors not only affect the reacting products, but also block the electron transfer activities of the enzymatic reactions –11. By qrb light , 13, whirb light , 15.The electrochemical biosensing method has been shown to have a number of advantages such as simplicity, rapidity, high sensitivity, and low costs. An electrochemical biosensing method was used to evaluate the XOD inhibitory effect, by quantifying enzymatic reaction product, hydrogen peroxide, detected at a single potential using chronoamperometry . However Plantaginis Semen extract and its four single compounds of acteoside, 1H-indole-3-carbaldehyde, isoacteoside, and myristic acid, which represent those four groups, was evaluated using the ETSBA. We observed that the extract has very significant inhibitory effects and that the variable activities of the four tested single compounds can also be accurately measured by this simple ETSBA testing. Our data supported the notion that the ETSBA successfully provided a simple and accurate evaluation of the XOD inhibition activities of natural products with their potential inhibition properties.In relation to XOD activity, the compounds in natural products could be classified into two groups: with or without the XOD inhibition effect. Each group could be further classified into two subgroups: with or without electrochemical activity. In this work, the XOD inhibitory activity of3 for 10 h [; the purity was found to be >95% [Plantaginis Semen was purchased from a cultivation base in Jiujiang city and identified by Professor Shouwen Zhang of Jiangxi University of Traditional Chinese Medicine. Xanthine oxidase (XOD)  was obtained from the Nanjing Jiancheng Bioengineering Institute . Shorted double-wall carbon nanotubes (DCNTs) with 95% purity were purchased from Shenzhen Nanotech Port Co.  and purified by refluxing the as-received DCNTs in 2.6 M HNOfor 10 h . Xanthin be >95% , 20 and Cyclic voltammetry measurements were performed with an Autolab Potentiostat/Galvanostat electrochemical station . A conventional three-electrode system with the modified glassy carbon electrode  as the working electrode, an Ag∣AgCl∣KCl  reference electrode, and a platinum wire electrode as the counter electrode  were used. All applied potentials were measured and reported versus AE and all experiments were carried out at room temperature. Scanning electron microscopy (SEM) images were obtained by using a Nova NanoSEM450 field emission SEM .μm) on a polishing cloth until a mirror finish was obtained. After 10 min of sonication, the electrodes were transferred to the electrochemical cell for cleaning by cyclic voltammetry between −0.5 V and +1.2 V at 50 mV/s in PBS , until a stable profile was obtained. The prepared electrodes were dried under a high purity nitrogen stream and used for modification immediately. 1.0 mg DCNTs was dispersed in 1.0 mL doubly distilled water with the aid of sonication. The DCNTs modified electrode was prepared by casting 3 μL of the dispersion on the surface of a GCE and air-dried at room temperature. The DCNTs modified GCE was further casted by 6 μL 0.147 mM XOD and air-dried at room temperature. The DCNTs and DCNTs/XOD modified electrodes were denoted as DCNTs/GCE and XOD/DCNTs/GCE hereafter, respectively. When not in use, the modified electrodes were stored in a 4°C refrigerator.The GCEs were carefully polished with emery paper and aqueous slurries of fine alumina powders .At first, the response current of xanthine in the absence of inhibitors was calculated by two steps measurements. Firstly, the electrode was immersed into the PBS solution without addition of xanthine and inhibitors, and then the current located at 610 mV was recorded. This current is the background current 1. Secondly, xanthine was added to the solution standing for 5 minutes; the peak current of xanthine located at 610 mV was recorded and noted as current 2. Hence, current 2 is subtracted from background current 1; the obtained result is the response current of xanthine (ΔIp1).Then, the response current of xanthine in the presence of inhibitor was measured via the following steps. Firstly, the inhibitor was added to the PBS solution standing for 10 minutes, and then xanthine was added to the solution standing for further 5 minutes; the peak current located at 610 mV is recorded and noted as current 3, which includes the response current of xanthine and inhibitors. Current 3 is subtracted from background current 1; the obtained result is the response current of xanthine and inhibitors. Here it is noted as current 4. Then, the current of the inhibitor at 610 mV was recorded when there is no xanthine under the same conditions and noted as current 5. Current 5 is subtracted from background current 1; the obtained result is the response current of inhibitors and noted as current 6. Hence, current 4 is subtracted from current 6; the obtained result is the response current of xanthine in the presence of inhibitors  using Sigmaplot 10.0. Significant differences among means of samples were evaluated using a one-way analysis of variance (ANOVA). Blank samples without the addition of XOD inhibitors were used as the control.Data were expressed as mean ± standard deviation , which are attributed to the excellent electron transfer ability of DCNTs. The anodic peak potential (Epa) and cathodic peak potential (Epc) are located at −350 and −375 mV, respectively. The peak-to-peak potential separation is about 25 mV. The redox peak couple is attributed to the conversion of the oxidized FAD cofactor into a reduced FADH2 cofactor of XOD, which is consistent with the literature [μg·mL−1 xanthine, or further added with 1.8 μg·mL−1 allopurinol. A sharp and sensitive peak current can be seen at about 610 mV. The current of electron transfer signal is confirmed to have exclusively resulted from the over-potential-oxidation of the FAD/FADH2 redox couple [μg·mL−1 allopurinol, the peak current decreased sharply, which proved that electron transfer signal blocking activities are affected by allopurinol with high sensitivity. Hence, ETSBA could be undoubtedly applied to evaluate the XOD inhibitory effect of natural products, which has been applied successfully in our recent work [Uric acid and hydrogen peroxide of XOD enzymatic reaction products are widely used to evaluate the inhibitory effect of XOD inhibitors –16, but terature , 11. Figx couple , 21, whient work .μg·mL−1 xanthine with the same XOD/DCNTs/GCE and 5 different XOD/DCNTs modified GCEs was 2.8% and 3.2%, respectively. These results suggested that there is excellent reproducibility and long-time stability for ETSBA, which is very facilitative for the evaluation of the XOD inhibitory effect.Stability and reproducibility of the ETSBA of the XOD/DCNTs/GCE stored in the 4°C refrigerator were also investigated using CVs in PBS . The response currents of the XOD/DCNTs modified electrodes almost did not change in 24 hours. The relative standard deviation (RSD) of 10 measurements of 28  Plantaginis Semen [The reported electrochemical biosensing method for screening XOD inhibitors is based on the enzymatic reaction products of hydrogen peroxide detected at a single potential using chronoamperometry, which may be easily disturbed by electrochemical active constituents . ETSBA iis Semen , 20 wereIt is very important to accurately evaluate the XOD inhibition effect of extract, as it is the prerequisite and basis for further active constituent research. However, the traditional ultraviolet and visible spectrophotometry (UV) method may give false results easily because the constituents in the extract are very complicated, while HPLC-UV/MS methods need complicated pretreatment procedures and expensive apparatus –15, whic Plantaginis Semen extract, which is caused by the reduction and oxidation of the electrochemical active constituents. The peak currents increased  to (7) represent the concentration of extract being 0, 36, 72, 108, 144, 180, and 216 μg·mL−1, respectively. The peak current of xanthine located at about 610 mV was accordingly decreased with addition of extract. The corresponding current was 52.36 ± 1.35 μA, 35.84 ± 1.12 μA (p < 0.05), 27.17 ± 1.18 μA (p < 0.01), 22.12 ± 1.05 μA (p < 0.001), 19.89 ± 1.08 μA (p < 0.01), 18.06 ± 0.84 μA (p < 0.01), and 14.04 ± 0.65 μA (p < 0.001), respectively. The inhibitory rates for the treatments were 0%, 31.55%, 48.11%, 57.75%, 62.01%, 65.51%, and 73.19%, respectively  of XOD were modulated by the extract of Plantaginis Semen.It can be seen from Figures  Figures  with the Figures . In this Plantaginis Semen extract.The above-mentioned ETSBA characteristics are very valuable. Firstly, it is very easy to eliminate false results as ETSBA not only showed the increased current of the electrochemical constituents, but also displayed the blocked electron transfer signal of XOD. Secondly, an established condition for the evaluation of an extract could be applied for the evaluation of all compounds from the extract. Lastly, the extract could be tested directly and did not need complicated sample pretreatment procedures using a cheap electrochemical station. Hence, ETSBA is very convenient to accurately evaluate the XOD inhibition effect ofμg·mL−1. The IC50 value of the extract has been calculated to be 89.14 μg·mL−1, which is close to Kong's observation [The IC20 value of the extract has been calculated to be 24.28 ervation , which m Plantaginis Semen. It was reported that acteoside has XOD inhibition activity [μg·mL−1 acteoside (for line (1) to line (5), respectively), while the peak current located at about 610 mV decreased accordingly , 43.53 ± 1.15 μA (p < 0.01), 27.87 ± 0.94 μA (p < 0.001), 17.06 ± 0.43 μA (p < 0.001), and 11.13 ± 0.22 μA (p < 0.001). The inhibition rates were 0%, 16.86%, 46.88%, 67.42%, and 78.30%, respectively. The relationships of the response current of ETSBA and inhibition rate of acteoside are summarized in μg·mL−1. The IC50 value of acteoside was calculated to be 7.55 μg·mL−1. This value is also smaller than the 12.5 μg·mL−1 that was reported in previously published data [The representative natural compounds with XOD inhibitory effects could be divided into two subgroups according to their electrochemical activity. Acteoside is the main active constituent inactivity . In this Figures . The corμg·mL−1, respectively), the currents located at 610 mV decreased accordingly , 45.39 ± 0.91 μA (p < 0.01), 38.68 ± 0.82 μA (p < 0.01), and 36.13 ± 0.79 μA (p < 0.001), respectively. The inhibition rate for the compound was 0%, 8.35%, 15.36%, 29.60%, and 30.99%, respectively. The inhibitory effect of 1H-indole-3-carbaldehyde could not exceed 50%. Hence the IC20 value was tested and calculated to be 16.16 μg·mL−1, which shows that it is a low activity XOD inhibitor. As the peak current located at about 610 mV came exclusively from the electron transfer signal of the enzyme catalytic reaction, the XOD inhibitory effects of compound group without any electrochemical activity could be evaluated by ETSBA and caused no false results even if it is a low XOD inhibitor.1H-indole-3-carbaldehyde has never been reported with XOD inhibition activity before. Its electrochemical characteristics and the inhibitory effect on XOD were studied by ETSBA and the results were listed in  Figures . The corμg·mL−1 of isoacteoside (for lines (1) to (4), respectively), which is similar to that of acteoside. This phenomenon is reasonable as the electrochemical active functional group of isoacteoside is the same as that of acteoside. The currents located at 610 mV remains constant, and there was no significant difference observed for isoacteoside. This means that isoacteoside could not affect the electron transfer signal blocking activities and it is not a XOD inhibitor. This result is very trusted as the oxidation-reduction current of isoacteoside increased with the increased concentration, while the current of electron transfer signal was kept constant with the successive addition of isoacteoside. The result is also consistent with previously published work [Isoacteoside is an isomer of acteoside. It can be seen from Figures hed work . It is rhed work  to exhibμg·mL−1, respectively). To clearly illustrate the inhibition effects of isoacteoside and myristic acid on XOD, the CVs were successively shifted to the left by every 50 mV, respectively. As seen from Figures Myristic acid is a long chain fatty acid and its inhibitory effect on XOD is shown in Figures The mechanism of electron transfer blocking activities (ETSBA) is completely different from other traditional methods. The XOD inhibitory effects of the extract and four representative compounds from Plantaginis Semen can be evaluated by ETSBA as a simple and sensitive and technique. Plantaginis Semen possesses significant XOD inhibitory effects and can be applied for treatment of gout and hyperuricemia as it contains XOD inhibitors. The IC50 value of the extract can be accurately detected, while the activity of acteoside is significantly higher. The IC50 values for the other three single compounds cannot be quantified. Thus, this ETSBA technique can be used to accurately measure XOD inhibitory effects using the electrochemical activity assessment. It appears that the ETSBA technique may be a novel way to effectively evaluate XOD inhibitory activities.


The objective of this study is to analyse the effectiveness of the surgical torque limiter during operative use. The study also investigates the potential differences in torque between hand and drill-based screw insertion into locking plates using a standardised torque limiter.Torque for both hand and power screw insertion was measured through a load cell, registering 6.66 points per second. This was performed in a controlled environment using synthetic bone, a locking plate and locking screws to simulate plate fixation. Screws were inserted by hand and by drill with torque values measured.The surgical torque limiter (1.5 Nm) was effective as the highest recorded reading in the study was 1.409 Nm. Comparatively, there is a statistically significant difference between screw insertion methods. Torque produced for manually driven screw insertion into locking plates was 1.289 Nm (95 % CI 1.269–1.308) with drill-powered screw insertion at 0.740 Nm (95 % CI 0.723–0.757).The surgical torque limiter proved to be effective as per product specifications. Screws inserted under power produce significantly less torque when compared to manual insertion by hand. This is likely related to the mechanism of the torque limiter when being used at higher speeds for which it was designed. We conclude that screws may be inserted using power to the plate with the addition of a torque limiter. It is recommended that all screws inserted by drill be hand tightened to achieve adequate torque values. Locking plates are widely used in orthopaedic surgery and are highly effective in the osteosynthesis of fractures , 2. The The increased time required for insertion of long screws by hand into locking plates raises questions around surgical efficiency and surgeon fatigue. Despite potential benefits through efficiency of drill-based screw insertion, there appears to be little evidence evaluating potential differences in applied torque between the two modes of insertion and on their relative impact on construct strength and risk of complications.A literature review found no studies comparing insertional torque differences between hand and drill power when using a standardised torque limiter, and a number of implant manufacturers could not supply this data to us readily. Our aim was to quantify and compare peak insertion torque values using a 1.5-Nm surgical torque limiter in an attempt to further define optimum method of screw insertion and better consider risk to a locking plate construct. Our hypothesis was that a locking screw inserted under drill power will generate higher insertional torque and consequently put the locking screw interface at risk of over tightening.Synbone) was used to simulate bone with a Synthes LCP Reconstruction Plate fixed with two screws equidistant from the central screw. The model was secured onto a rotating platform  with torque limiter attachment. A standardised number of five audible clicks, representing triggering of the torque limiter five times, were used for each screw insertion to ensure appropriate capture of the insertional torque forces generated. This process was repeated ten times to increase statistical power.The second round of testing analysed screw insertion under power, also with 50 clicks recorded from ten separate screw insertions. A trauma drill  was used with the torque limiter attached. The maximum RPM for the drill was measured using a tachometer at 1506 RPM. Care was taken to create a controlled speed of insertion of the screw; however, technically, this was more difficult. Audible clicks were again used as in surgical practice.From the data recorded, peak insertion torque values were calculated for the different groups. Analysis of the data demonstrated that the surgical torque limiter, rated at 1.5 Nm, was effective in our simulated operative setting. The highest recorded torque value was by hand at 1.409 Nm. Comparatively, the highest recorded value at insertion under power was 0.898 Nm.n = 50) was 1.289 Nm with a standard deviation (SD) of 0.069  1.269–1.308).For the screws inserted by hand, data points clustered in peaks for each separate screw insertion. As seen in Fig. n = 50) was significantly less at 0.740 Nm with a SD of 0.045 (95 % CI 0.723–0.757). The difference between the two groups was statistically significant (p = 0.0001).Torque values generated by screw insertion under power also relied on five audible clicks to capture the force produced for each screw insertion. As seen in Fig. Closer focus on the torque patterns of individual screw insertion showed that the maximum torque in the power group Fig.  was achiThroughout testing, we did not observe any evidence of head or thread stripping, evident wearing, cold welding, screw breakage or difficulty with screw removal.We acknowledge that our single user model was simplistic and this data is applicable only to the small fragment system torque limiter rated at 1.5 Nm (Synthes). Our study used a 3.5-mm-diameter screw with a locking plate system and a synthetic bone model to simulate surgical fixation. Results may vary for different sized screws and constructs.p < 0.001) than a screw inserted by drill (mean 0.7 Nm). This disproves our initial hypothesis and current belief that screws inserted by drill generate higher torque values and are at increased risk of over tightening or damage.Our results proved the effectiveness of the torque limiter as no value exceeded the manufacturer specified limit of 1.5 Nm in any group. The study also demonstrated that the mean torque applied to a manually inserted locking screw (mean 1.2 Nm) is significantly greater  likely facilitates an early triggering of the torque limiting mechanism, limiting the torque to a subpar 0.7 Nm rather than the specified maximum 1.5 Nm. This raises concerns over the longevity and effectiveness of the torque limiter when used at higher speeds for which they may not have been designed.Our results have shown significantly less insertional torque values when using the drill. This would suggest a lowered risk from increased torque and theorised damage to the involved construct. Supposed benefits could be reduced operating time and surgeon fatigue by inserting locking screws to a plate under power rather than hand.While locking plates do not require the same levels of torque to be generated as their non-locking counterparts , they arFurther testing using a cadaveric model may be useful in better replicating a true model. It is also not known what impact large fragment systems, variable angle technology, different drills, plate interfaces, and varying screw size or length would have on torque values and could potentially be the interest of further study. Due to differences in torque limiter internal mechanism design between manufacturers and instrument sets, these results are not generally applicable to all torque limiting devices and other devices may behave quite differently. We advise caution when extrapolating these results to equipment different to those considered in this study.Based on these findings, we suggest that locking screws can be inserted to the plate under power with a torque limiting attachment. As the insertional torque values produced are significantly less, it is therefore recommended that final screw tightening be performed by hand. The recommendation by companies not to use power for locking of screws may be based on risks of under-tightening or internal mechanism wear rather than over-tightening.In our model, we found that the surgical torque limiter is effective at preventing excess torque as per specifications. As less torque is generated by drill-powered insertion, we advise that screws can be powered directly into a plate without the risk of excessive torque. It is highly recommended that all screws inserted by drill be hand checked to ensure adequate torque and to decrease the risk of construct failure.