In this study, using recombinantly expressed and purified proteins, various analytical methods, in vitro and in situ kinase assays, and mechanical measurements in isolated ventricular trabeculae, we demonstrate that human cMLCK is not a dedicated kinase for RLC but can phosphorylate other sarcomeric proteins with well-characterized regulatory functions. We show that cMLCK specifically monophosphorylates Ser23 of human cardiac troponin I (cTnI) in isolation and in the trimeric troponin complex in vitro and in situ in the native environment of the muscle myofilament lattice. Moreover, we observed that human cMLCK phosphorylates rodent cTnI to a much smaller extent in vitro and in situ, suggesting species-specific adaptation of cMLCK. Although cMLCK treatment of ventricular trabeculae exchanged with rat or human troponin increased their cross-bridge kinetics, the increase in sensitivity of myofilaments to calcium was significantly blunted by human TnI, suggesting that human cTnI phosphorylation by cMLCK modifies the functional consequences of RLC phosphorylation. We propose that cMLCK-mediated phosphorylation of TnI is functionally significant and represents a critical signaling pathway that coordinates the regulatory states of thick and thin filaments in both physiological and potentially pathophysiological conditions of the heart.Heart muscle contractility and performance are controlled by posttranslational modifications of sarcomeric proteins. Although myosin regulatory light chain (RLC) phosphorylation has been studied extensively  In co models –. RLC pho models –, 7 and h models –. Moreove models –.2+ and calmodulin-dependent protein kinase family; it has a C-terminal canonical calmodulin-binding site and Ca2+/calmodulin-dependent activity  have been associated with development of dilated cardiomyopathy in humans . In contrast, rodent cardiac troponin I differs considerably in its primary sequence surrounding the phosphorylatable serines 22/23 in its NTE, suggesting that rodent (including rat and mouse) cTnI might not be a substrate for cMLCK. The phosphorylatable serines 22/23 are followed by an asparagine residue (P+1 position) in human cTnI (A), which is similar to the consensus sequence found in rodent and human cardiac RLC. In contrast, in rodent cTnI, the phosphorylatable serines are followed by an alanine in the P+1 and an asparagine in the P+2 position (A), which might interfere with substrate recognition by cMLCK.We compared the primary sequence of known myofilament phosphoproteins from different species with the cMLCK consensus sequence in RLC and identified serines 22/23 in the cardiac specific N-terminal extension (NTE) of human cardiac troponin I (hcTnI) as potential substrates A, red. Iman cTnI A, which position A, which in vitro kinase assays and analyzed their phosphorylation profiles by Phos-tagTM SDS-PAGE. As shown in Fig. 1B, cMLCK specifically phosphorylated hcTnI to ∼0.6 mol Pi/mol cTnI within 30 min of incubation. No phosphorylation of cardiac troponin T or C was observed under the same conditions, suggesting that cMLCK has a high specificity toward hcTnI in vitro. In contrast, rat cardiac troponin I (rcTnI) was phosphorylated to less than 0.02 mol Pi/mol cTnI under the same conditions, suggesting that it is a poor substrate for cMLCK in vitro. Note that unphosphorylated rat cTnT and cTnI migrate faster and slower in SDS-PAGE, respectively, than the corresponding human proteins. Prolonged incubation of both troponin complexes for more than 60 min resulted in almost complete monophosphorylation of hcTnI (>0.9 mol Pi/mol cTnI) but less than 0.15 mol Pi/mol cTnI phosphate incorporation for the rat troponin complex . In contrast to cTnI, cMLCK monophosphorylates isolated human and rat cRLC under the same conditions, with a similar time course to ∼1 mol Pi/mol cRLC within 60 min  for 1 h and analyzed the phosphorylation profiles of cardiac troponin T, C, and I by Phos-tagTM SDS-PAGE and electron spray ionization (ESI) MS . Only hcTnI was specifically monophosphorylated to ∼1 mol Pi/mol cTnI under all conditions tested, supporting the hypothesis that cMLCK specifically phosphorylates a single residue in cTnI, i.e. either serine 22 or 23. We confirmed this result by phosphorylating isolated hcTnI with cMLCK (F), followed by Phos-tagTM SDS-PAGE and Western blotting using a cTnI pSer22/pSer23-specific antibody (G). The phosphospecific antibody clearly recognized the monophosphorylated species, supporting the above conclusion that Ser22, Ser23, or both are substrates for cMLCK in vitro. To identify the phosphorylated serine residue, we incubated isolated WT hcTnI or hcTnI with Ser22 or Ser23 substituted by alanine with Ca2+/calmodulin-activated cMLCK (H). Only WT and S22A-substituted cTnI were phosphorylated by cMLCK, localizing the phosphorylation site to Ser23, in good agreement with the sequence similarity to RLC (A). A minor bis-phosphorylated human cardiac troponin I species was observed when the protein was used in isolation in kinase assays (F and H), suggesting that cMLCK phosphorylated a secondary unspecific site that is, however, not accessible in the trimeric troponin complex.Next we tested the specificity of cMLCK for hcTnI by incubating human cardiac troponin complex with increasing concentrations of cMLCK  and an alanine in other vertebrates, suggesting that cTnI phosphorylation by cMLCK might be a specific evolutionary adaptation.A sequence alignment of the N-terminal extension of cTnI from different species revealed that the P+1 position is an asparagine in all Old and New World primate species and humans . Myofibrillar proteins after λPP treatment or sequential treatment with λPP followed by incubation with Ca2+/CaM/cMLCK for 1 h were separated by SDS-PAGE, and phosphorylated proteins were identified by phospho-protein staining with Pro-Q Diamond and total protein staining with Coomassie or SYPRO Ruby. As described previously , in good agreement with the calculated mass of cRLC. An additional band in the Pro-Q staining with an apparent molecular mass of ∼37 kDa  corresponds to cMLCK, which is endogenously phosphorylated . As expected, cRLC was highly phosphorylated after cMLCK treatment (∼0.7 mol Pi/mol cRLC). In contrast, Phos-tagTM Western blotting against cTnI with the same samples showed that, although λPP treatment resulted in efficient dephosphorylation of cTnI, the rat variant was not phosphorylated by cMLCK in situ.Next we confirmed the results for the isolated proteins described above in experiments with isolated cardiac myofibrils (CMFs), in which intact thin and thick filaments are organized into the native myofilament lattice. CMFs isolated from rat ventricular tissue were pretreated with λ protein phosphatase (λPP) to homogenously reduce protein phosphorylation levels. λPP treatment resulted in efficient dephosphorylation of cRLC, cTnI, cardiac troponin T, desmin, and cMyBP-C, as assessed by SDS-PAGE and Pro-Q Diamond phospho-protein staining  and human left ventricles, which exhibit a significantly higher cTnI primary sequence identity , including an asparagine in the P+1 position following serine 23 in the NTE. Pro-Q phosphoprotein staining of λPP/cMLCK-treated marmoset and human CMFs showed three bands with apparent molecular masses of ∼18, ∼24. and ∼37 kDa , consistent with the expected molecular masses of cRLC, cTnI, and cMLCK, respectively. As before, we used Phos-tagTM SDS-PAGE and Western blotting to confirm that cRLCs in both sets of CMFs were phosphorylated after cMLCK treatment (∼0.8 mol Pi/mol cRLC). However, in contrast to rat CMF, cTnI was phosphorylated in the same samples to about 0.3–0.6 mol Pi/mol cTnI , confirming that cMLCK phosphorylates native marmoset and human cTnI in the myofilament lattice in situ. Specific phosphorylation of marmoset and human cTnI at serine 23 in situ was further confirmed by Western blotting using a pSer22/pSer23-specific antibody . Incubation of human CMFs with smooth muscle MLCK did not result in phosphorylation of cTnI, suggesting that phosphorylation of cTnI is specific to the cardiac isoform of MLCK (D).We repeated the mol cTnI B, right, of MLCK D.A). Endogenous cTnI in demembranated ventricular trabeculae is highly phosphorylated because of residual kinase activity in the quiescent preparations (B), and overnight exchange with the unphosphorylated recombinant troponins reduced cTnI phosphorylation from ∼1.8 mol Pi/mol cTnI to ∼0.9 mol Pi/mol cTnI in both groups (C and D). As described previously, the level of RLC phosphorylation was low in demembranated trabeculae (<0.05 mol Pi/mol RLC) . Incubation in activating solution (pCa 4.5) containing 25 μmol/liter blebbistatin and 1 μmol/liter cMLCK/CaM had no effect on the cTnI phosphorylation level of rat troponin-exchanged trabeculae but significantly increased cTnI phosphorylation in the presence of human cardiac troponin to ∼1.3 mol Pi/mol cTnI . In contrast, RLC was highly phosphorylated in both groups of trabeculae after cMLCK treatment . In summary, these results show that cMLCK phosphorylates hcTnI in situ in the native environment of the myofilament lattice.To assess the functional consequences of hcTnI phosphorylation by cMLCK, we exchanged unphosphorylated rat or human cardiac troponin complex into demembranated rat right ventricular trabeculae with a roughly 70% exchange efficiency A. Endogearations B, and ovh groups , C and Dmol RLC) , and rec2+/CaM. However, exchange of ∼70% endogenous with recombinant troponin had only a moderate effect on the cTnI phosphorylation level observed after exchange, with about 30% still present as the monophosphorylated and 30% as the bis-phosphorylated form (C and D), suggesting that recombinant unphosphorylated cTnI was partially phosphorylated during the protein exchange by kinases still present in the freshly prepared ventricular trabeculae (B). Because of the high basal level of phosphorylation, the effects of kinase treatment on cTnI phosphorylation and myofilament function could not be unambiguously interpreted. We therefore employed an additional dephosphorylation step, using λPP as described previously for mouse ventricular trabeculae , but decreased the steepness of the force–calcium relation  (A). Moreover, λPP treatment decreased the rate of force redevelopment at intermediate and high levels of activation (B and C) but had little or no effect on passive or maximal calcium-activated isometric force. After the experiments, trabeculae were dismounted and dissolved in SDS-PAGE loading buffer, and cTnI phosphorylation levels were determined by Phos-tagTM SDS-PAGE followed by Western blotting against cTnI (D). Trabeculae from the same hearts that did not undergo λPP treatment were used as negative controls. As before, endogenous cTnI phosphorylation levels in untreated control trabeculae were high (∼1.8 mol Pi/mol cTnI), and incubation with λPP efficiently reduced cTnI phosphorylation to less than 0.1 mol Pi/mol cTnI, in good agreement with the CMF experiments described above. λPP treatment also resulted in efficient dephosphorylation of cMyBP-C, which was confirmed by Western blotting using a phospho-specific antibody . Comparison of the protein phosphorylation levels with the functional effects suggests that λPP treatment increases calcium sensitivity and decreases cross-bridge kinetics of ventricular trabeculae via dephosphorylation of cTnI and cMyBP-C, respectively. Therefore, to achieve a homogeneously low cTnI phosphorylation level, we first exchanged trabeculae overnight with unphosphorylated human or rat cardiac troponin, followed by λPP treatment directly before the mechanical experiments and subsequent incubation with Ca2+/CaM/cMLCK.First we tested the effects of λPP treatment on cardiac muscle mechanics. Incubation of rat ventricular trabeculae with λPP increased the calcium sensitivity of force, as indexed by an increase in , n = 4) A. Moreovtivation , B and Cnst cTnI D. Trabec2+/CaM-activated cMLCK increased the calcium sensitivity of force by 0.07 ± 0.01 pCa   but had no effect on the steepness of the force calcium relation , in good agreement with results obtained previously from native rat ventricular trabeculae  , suggesting that hcTnI phosphorylation by cMLCK in the same preparations mitigated the increase in calcium sensitivity associated with phosphorylation of cRLC. Moreover, cMLCK treatment reduced the steepness of the force calcium relation from Hn 5.51 ± 0.19 to 4.11 ± 0.25  in the presence of human cTn. cMLCK treatment slightly increased passive and active force of human and rat cTn-exchanged trabeculae.Incubation of rat cTn-exchanged and λPP-dephosphorylated trabeculae with Caktr) at different levels of activation by a rapid shortening–restretch maneuver. Rat and human cTn-exchanged trabeculae exhibited activation-dependent changes in ktr (D and E), ranging from ∼5 s−1 at low levels of activation to ∼15 s−1 at maximal activation. Data points were fitted to a quadratic equation to extract ktr at half-maximal levels of force (ktr 50%max). In human and rat cTn-exchanged trabeculae, cMLCK treatment increased ktr 50%max by ∼2 s−1 , suggesting increased cross-bridge kinetics at intermediate and high levels of activation.We determined the effect of cMLCK phosphorylation on cross-bridge kinetics by measuring the rate of force redevelopment . RLC was phosphorylated to 0.28 ± 0.08 mol Pi/mol RLC  and 0.36 ± 0.05 mol Pi/mol cRLC in rat and human cTn-exchanged trabeculae, respectively. cTnI was largely dephosphorylated in rat cTn-exchanged trabeculae. Because monophosphorylated hcTnI and unphosphorylated rcTnI comigrate in Phos-tagTM SDS-PAGE , we could not directly determine the level of troponin I phosphorylation, but it was estimated to be ∼0.3 mol Pi/mol, assuming a troponin exchange efficiency of 70%.After the experiments, trabeculae were dismounted, and phosphorylation levels were determined by Phos-tagsee also B, we coue.g. the skeletal isoform of MLCK has been shown to regulate skeletal muscle myogenesis via phosphorylation of the transcription factor MEF2c . cMLCK phosphorylated cTnI on serine 23 not only on the isolated cTnI subunit but also when it was incorporated within the trimeric troponin complex or the myofilament lattice  similar to that found in cMLCK's primary substrate RLC. Serine 23 in hcTnI is followed by an asparagine and an amphipathic amino acid residue (pSNY), which is similar to the conserved amino acid sequence found in the NTE of all RLC isoforms (pSNVF). Stepwise deletion of this conserved sequence in smooth muscle RLC decreased the maximal rate of phosphorylation, and it has been suggested that these residues contribute to substrate recognition by binding to a pocket found next to the catalytic cleft of smooth muscle MLCK (The NTE of hcTnI (amino acids 1–30) displays a sequence motif A similarcle MLCK , 23. Morcle MLCK . Specificle MLCK . Mutatiocle MLCK . The seqcle MLCK .in vitro . However, cMLCK did not phosphorylate N-terminal domains of cMyBP-C (C0C2), even at very high enzyme-to-substrate ratios , consistent with published results indicating that cMyBP-C is not a substrate for cMLCK , has been shown to monophosphorylate cTnI on serine 22 or 23 in response to α-adrenergic stimulation of cardiomyocytes . Moreoveor cMLCK .e.g. PKA activity is locally regulated within cells via interactions with so-called protein kinase A–anchoring proteins, which bind PKA with high specificity and affinity , and bis-phosphorylation has been proposed to be necessary for the downstream functional effects . The catalytic domains of human and rat cMLCK exhibit very high sequence identity and similarity (>98%), but the current results cannot exclude the possibility that rat cMLCK can phosphorylate rat cTnI.Our results suggest that there are likely fundamental differences between the physiological functions of cMLCK in rodent and human heart muscle. Although human and rodent cTnI have ∼92% sequence identity, human cMLCK specifically phosphorylates only the human but not the rodent protein isoform in situ , with onin vitro C. The caThe P+1 position following serine 23 is an alanine in rodent but an asparagine in human cTnI, and it has been shown previously that deletion of the analogous asparagine in RLC greatly reduces MLCK's activity toward RLC . The comThe concept of species-specific myofilament regulation has more fundamental implications for the study of heart failure, its etiology, and the development of pharmacological interventions using animal models. Loss-of-function mutations in cMLCK have been associated with dilated cardiomyopathy in humans , 16, whe2, 1 mmol/liter CaCl2, and 1 mmol/liter DTT. The salt concentration was reduced to 200 mmol/liter followed by 100 mmol/liter in the last two steps of dialysis. The complex was purified by ion exchange chromatography on a Resource-Q column with a two-step linear gradient of 0–200 mmol/liter NaCl over 5 column volumes and 200–450 mmol/liter NaCl over 30 column volumes. The final concentrated product was characterized by HPLC, ESI-MS, and size-exclusion multi-angle light scattering (SEC-MALS) . All measurements were consistent with a 1:1:1 complex of rat cardiac troponin T, I, and C. Troponin was dialyzed into a K2 EGTA buffer , 6.8 mmol/liter MgCl2, 10 mmol/liter K2 EGTA, and 1 mmol/liter DTT (pH 7.1)), and aliquots were stored at −80 °C. Na2 creatine phosphate (Na2 CrP) and Na2 ATP were supplemented before troponin reconstitution in trabeculae .The catalytic fragment of human cardiac myosin light chain kinase was prepared as described previously  based on2O  and HPLC-grade acetonitrile . Buffer A consisted of HPLC-grade H2O containing 0.1% (v/v) HPLC-grade TFA , and buffer B consisted of 80% (v/v) ACN in HPLC-grade H2O containing 0.1% (v/v) TFA.Analytical-scale HPLC was performed with an Agilent 1200 series system on octadecyl carbon chain (C18)–bonded silica columns . The solvent system was HPLC-grade H2O  containing 0.1% (v/v) formic acid , and buffer B consisted of 80% (v/v) ACN in MS-LC–grade H2O containing 0.1% (v/v) formic acid. Data analysis was performed with LC/MSD ChemStation software.Mass spectrometry analysis was performed on the Agilent Series 1100 LC-MS system in positive mode of ESI. The solvent system was as follows. Buffer A consisted of LC-MS grade Hm MgCl2, 1 mmol/liter CaCl2, 100 mm NaCl, and 100 mmol/liter tris(2-carboxyethyl)phosphine). Recombinant troponins were extensively dialyzed against SEC-MALS buffer before experiments. Light scattering and refractive index were measured on a Mini DAWN and OPTILAB DSP , respectively. Data were analyzed with custom-written software.SEC-MALS was performed on a Superdex 200 5/150 GL column  in SEC-MALS buffer (50 mmol/liter Tris-HCl (pH 7.4), 5 mAll animals were treated in accordance with the guidelines approved by the United Kingdom Animal Scientific Procedures Act (1986) and European Union Directive 2010/63/EU. Wistar rats  were sacrificed by cervical dislocation without anesthetics , and demembranated right ventricular trabeculae were prepared as described previously . Some trC. jacchus) ventricular tissue was kindly provided by the Biological Service Unit of King's College London. Cardiac myofibrils were prepared as described previously  Brij-35 (pH 7.5)). λPP was added to a final concentration of 4000 units/ml, and the mixture was incubated at 30 °C for 1 h. After incubation, CMFs were washed three times with relaxing buffer (20 mmol/liter MOPS (pH 7), 35 mmol/liter NaCl, 5 mmol/liter MgCl2, 1 mmol/liter EGTA, 1 mmol/liter CaCl2, 1 mmol/liter DTT, pCa 9) without ATP to remove Mn2+ and λPP.Human samples were obtained after informed consent and with approval of the institutional review board of the University of Kentucky  and the King's College London Research Ethics Panel . This investigation conformed with the principles of the Declaration of Helsinki (1997). Marmoset  via NAP5 columns . Concentrations were determined by UV absorbance at 280 nm using calculated extinction coefficients, and reactions were started by addition of calmodulin and cMLCK. Reactions were stopped by addition of SDS-PAGE loading buffer. Samples were denatured at 100 °C for 2 min, and phosphospecies were separated by Phos-tagTM SDS-PAGE (containing 50 μmol/liter Phos-tag reagent and 100 μmol/l MnCl2). Gels were subsequently stained with Coomassie, and band intensities were quantified using ImageJ. CMFs (2–4 mg/ml) were phosphorylated in situ by incubation in cardiac activating solution ) containing 25 μmol/liter blebbistatin and 1–2 μmol/liter cMLCK/CaM (0.5 nmol cMLCK/CaM per 1 mg of CMF protein) for 1 h at 30 °C. After incubation, CMFs were briefly washed in 10 mmol/liter Tris-HCl (pH 7) to remove EGTA. CMFs were dissolved in SDS-PAGE loading buffer, and samples were run on Phos-tagTM SDS-PAGE as described above. The assays were repeated at least twice to make sure that they were reproducible and at least three times for quantitation.Cardiac troponins were gel-filtered into cMLCK assay buffer , 192 mmol/liter glycine, 0.08% (v/v) SDS, and 15% (v/v) methanol) containing 5 mmol/liter EDTA, followed by three washes in buffer without EDTA. Proteins were blotted onto nitrocellulose or PVDF membranes for 1–2 h at 1 mA/cm2 in transfer buffer using a Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell (Bio-Rad). After blotting, membranes were blocked with TBS containing 0.05% (v/v) Tween 20 (TBS-T) and 5% (w/v) semiskimmed milk powder or 5% (w/v) BSA. Membranes were incubated with the following primary antibodies overnight in TBS-T containing 5% (w/v) semiskimmed milk powder or 5% (w/v) BSA: rabbit monoclonal anti-myosin light chain 2 , mouse monoclonal anti-cardiac troponin I , rabbit polyclonal anti-cardiac troponin I , rabbit polyclonal anti-cardiac troponin I , rabbit polyclonal anti-cardiac troponin T , and rabbit monoclonal anti-pSer282 cMyBP-C . Membranes were washed three times in TBS-T and incubated for 1 h at room temperature with secondary antibody  in TBS-T containing 1% (w/v) semiskimmed milk powder or 1% (w/v) BSA. Blots were washed in TBS-T and immersed in ECL Plus reagent , and bands were visualized on a Bio-Rad Gel-Doc Imager. SDS-PAGE gels were stained with Pro-Q Diamond and SYPRO total protein stain according to the manufacturer's instructions (Life Technologies) and imaged on a Bio-Rad Gel-Doc Imager using the appropriate excitation and emission filter settings.After Phos-tag2 CrP, 78.4 mmol/liter KPr, 5.65 mmol/liter Na2 ATP, 6.8 mmol/liter MgCl2, 10 mmol/liter K2EGTA, and 1 mmol/liter DTT (pH 7.1)) containing 1.5 mg/ml troponin complex and 30 mmol/liter 2,3-butanedione monoxime overnight at 4 °C. Trabeculae were mounted between a force transducer  and length controller . For dephosphorylation experiments, trabeculae were washed three times in MgCl2 rigor buffer , followed by three washes in Mn2+ rigor buffer  and incubated for 1 h at room temperature in Mn2+ rigor buffer containing 3000 units/ml λPP. Subsequently, trabeculae were washed three times for 10 min in relaxing buffer, and sarcomere length was adjusted to ∼2.1 μm by laser diffraction.Endogenous troponin was exchanged by incubation of trabeculae in relaxing solution ) containing 30 mmol/liter BDM, 1 μmol/liter calmodulin, and 1 μmol/liter cMLCK. Subsequently, trabeculae were washed three times for 10 min in relaxing buffer, and sarcomere length was adjusted to ∼2.1 μm by laser diffraction.Trabeculae were phosphorylated by incubation for 1 h at room temperature in activating solution ). Solutions with varying concentrations of free [Ca2+] were prepared by mixing relaxing and activating solutions using MAXCHELATOR software (RRID: SCR_000459). The dependence of force on free calcium concentration was fitted to data from individual trabeculae using nonlinear least-squares regression to the modified Hill equation:pCa = −log10 ([Ca2+]), pCa50 is the negative logarithm of [Ca2+] corresponding to half-maximal change in F, and Hn is the Hill coefficient. Trabeculae that showed a decline in maximal calcium activated force of more than 15% after the pCa titrations were discarded.Each activation was preceded by 2-min incubation in preactivating solution (25 mmol/liter imidazole, 15 mmol/liter Naktr.The rate of force redevelopment was measured by a fast release and restretch protocol . BrieflyI. R. S. and T. K. conceptualization; I. R. S. and T. K. data curation; I. R. S. and T. K. formal analysis; I. R. S., M. G., M. I., K. S. C., and T. K. writing-review and editing; B. B., S. P., M. G., M. I., K. S. C., Y.-B. S., and T. K. resources; B. B. and S. P. methodology; T. K. funding acquisition; T. K. writing-original draft.


Greenland’s outlet glaciers have been a leading source of mass loss and accompanying sea‐level rise from the Greenland Ice Sheet (GrIS) over the last 25 years. The dynamic component of outlet glacier mass loss depends on both the ice flux through the terminus and the inland extent of glacier thinning, initiated at the ice‐ocean interface. Here, we find limits to the inland spread of thinning that initiates at glacier termini for 141 ocean‐terminating outlet glaciers around the GrIS. Inland diffusion of thinning is limited by steep reaches of bed topography that we call “knickpoints.” We show that knickpoints exist beneath the majority of outlet glaciers but they are less steep in regions of gentle bed topography, giving glaciers in gentle bed topography the potential to contribute to ongoing and future mass loss from the GrIS by allowing the diffusion of thinning far into the ice sheet interior. Greenland glaciers with high potential to contribute to mass loss identified using novel metricThe majority of thinning limits are set by a knickpoint, a steep drop in bed topographyGlaciers that transmit thinning far inland are preferentially in regions with gentle bed topography Negative values of Pe represent locations along glacier flow where thinning perturbations can advect upstream and, because of the particular flux‐geometry relationship that we use, this occurs where the bed slope is negative . Previous work has shown that using other typical formulations for the relationship between ice flux and geometry does not significantly affect the locations of our predicted flowline thinning limits . We then create one measure of the predicted inland thinning distance for each glacier, taking into consideration both (1) the furthest inland predicted flowline thinning limit, which accounts for the worst‐case, furthest‐inland distance over which thinning can diffuse, and (2) the spread in distances to predicted flowline thinning limits across glacier flow, which accounts for our uncertainty in the extent of thinning. We call this the “predicted glacier thinning limit,” calculated by subtracting one standard deviation of the distances to all predicted flowline thinning limits from the distance to the single furthest inland predicted flowline thinning limit. Herein, we discuss thinning limits along individual flowlines (predicted flowline thinning limits), which are used to examine spatial patterns of thinning within individual glaciers, as well as predicted glacier thinning limits, which are used to compare each glacier to one another.As an additional improvement on F17, we quantify uncertainties in both observed dynamic thinning and Pe and propagate these through our calculations of predicted glacier thinning limits. We shift values of dynamic thinning and Pe along each flowline using a systematic bias obtained from reported uncertainties in bed topography, surface DEMs, and RACMO‐estimated SMB to obtain worst‐case bounds on our calculated values of dynamic thinning and Pe (Text S9 and S10 in the supporting information). Using a Monte Carlo analysis, we randomly sample from these worst‐case calculations to examine uncertainty in our calibration of an empirical thinning limit and in the glacier thinning limits . We find that, even in this conservative uncertainty analysis, statistically, the majority of dynamic volume loss occurs downstream of Pe = 3 Figures . Thus, w3Examining predicted flowline thinning limits, we can categorize the results according to two end‐member glacier geometries: 1) those that permit thinning perturbations to diffuse very far inland and (2) those that set strong limits to the inland diffusion of thinning. We demonstrate the differences between these two geometry types through examination of two representative examples. Humboldt Gletscher (HUM) in Northwest Greenland flows over gentle bed topography from the ice sheet interior to its submarine terminus Figure . The Pe  those thKnickpoints are prevalent beneath GrIS outlet glaciers and play an important role in limiting the inland diffusion of thinning. Sixty‐eight percent of all surveyed flowlines have an identified knickpoint. To ensure that our sampling of glaciers with a variable number of flowlines does not skew this statistic, we survey one centerline per glacier and find that 65% of glacier centerlines have an identified knickpoint. Of those flowlines with an identified knickpoint, 88% have predicted flowline thinning limits within the extent of the knickpoints, indicating that knickpoints are important geometric features for mitigating the inland diffusion of terminus‐initiated thinning for the majority of outlet glaciers in Greenland. Knickpoints have a median height of hundreds of meters, relative to the median elevation of the trough, spatial extent of tens of kilometers , we find that the predicted flowline thinning limits that lie furthest inland are in regions with gentle bed topography Figure . Convers3/yr) but with thinning limits close to the ice sheet margin (<150 km). These glaciers include the well‐studied glaciers Jakobshavn Isbræ (JAK), Kangerlussuaq Gletscher (KAN), and Helheimgletscher (HEL), which are often examined in the literature due to their high flux. A second group of glaciers exists with relatively low ice flux (<10 km3/yr) but far inland thinning limits (>200 km). These glaciers do not receive much focused attention in the literature but they have the potential to be large contributors to dynamic ice mass loss as thinning perturbations diffuse hundreds of kilometers into the ice sheet interior within their catchments. The glaciers in this group are predominantly located in regions of gentle bed topography  and Cornell Gletscher (COR), where 9 of 12 neighboring glaciers have predicted glacier thinning limits >250 km from the ice sheet margin and, thus, they can collectively draw down a ∼140‐km‐wide region far into the ice sheet interior. By considering uncertainties in input datasets and our choice of Pe = 3 as the empirical thinning limit, we find that predicted glacier thinning limits are most uncertain in regions of gentle bed topography around the GrIS Figure  and we hOur results provide a mechanistic understanding for satellite observations that have measured the largest rates of dynamic thinning and mass loss along the southeast and northwest margins (Csatho et al., Although knickpoints may have a different initiation mechanism than their riverine counterparts, by drawing on ideas from fluvial geomorphology, subglacial knickpoints may also be used to reconstruct uplift rate histories (Roberts & White, 4Using a novel metric easily calculable from glacier geometry, we predict the inland extent of thinning for 141 GrIS outlet glaciers. This metric quantifies the ratio of the rate of advection to the rate of diffusion of a kinematic wave of thinning initiated at glacier termini. We find that the majority of limits on the diffusion of thinning are set by steep knickpoints in subglacial bed topography at the heads of troughs where the bed transitions from below to above sea level. Knickpoints at the heads of subglacial troughs are the locations where we would expect a large decrease in ice discharge when, sometime in the future, glacier retreat causes marine‐terminating outlets to become land‐terminating. We have shown that these same locations are already mitigating the impact of marine‐terminating outlet glaciers on dynamic ice sheet mass loss by limiting the diffusion of thinning further inland. Consequently, we find that glaciers that allow thinning to diffuse far into the interior of the GrIS are preferentially located in regions of gentle bed topography because, although knickpoints are prevalent around the entire ice sheet, they are less steep in regions of gentle bed topography. When we consider both inland thinning limits and ice flux as measures of each glacier’s potential for dynamic mass loss, we find that glaciers with low ice flux may be significant contributors because of their ability to transmit thinning far inland. We hypothesize that the low ice flux glaciers with far inland thinning limits will contribute to dynamic ice sheet mass loss over a longer timescale than high ice flux glaciers, as the rate of diffusion of thinning slows in the ice sheet interior. Of particular concern is a group of glaciers along the northwest coast of the GrIS, between Upernavik Isstrøm C and Cornell Gletscher, where 9 of 12 neighboring glaciers have the potential to lead to long‐term, diffusive thinning >250 km into the interior of the ice sheet over a ∼140‐km‐wide region. This provides a mechanistic explanation for why the northwest sector of the GrIS is the only region experiencing an ongoing increase in observed discharge.Supporting Information S1Click here for additional data file.Table S2Click here for additional data file.


Glycine max) was found to exhibit the strongest transport inhibition. From this extract, via a fractionation approach, we successfully isolated and identified genistein, a soy isoflavone, as a novel ABCC11 inhibitor with a half-maximal inhibitory concentration value of 61.5 μM. Furthermore, we examined the effects of other dietary flavonoids on the ABCC11-mediated DHEA-S transport to uncover the effects of these phytochemicals on ABCC11 function. While further human studies are needed, our findings here about the natural compounds will help develop a non-surgical therapy for AO.Axillary osmidrosis (AO) is a common chronic skin condition characterized by unpleasant body odors emanating from the armpits, and its aetiology is not fully understood. AO can seriously impair the psychosocial well-being of the affected individuals; however, no causal therapy has been established for it other than surgical treatment. Recent studies have revealed that human ATP-binding cassette transporter C11 (ABCC11) is an AO risk factor when it is expressed in the axillary apocrine glands—the sources of the offensive odors. Hence, identifying safe ways to inhibit ABCC11 may offer a breakthrough in treating AO. We herein screened for ABCC11-inhibitory activities in 34 natural products derived from plants cultivated for human consumption using an in vitro assay system to measure the ABCC11-mediated transport of radiolabeled dehydroepiandrosterone sulfate (DHEA-S—an ABCC11 substrate). The water extract of soybean ( Offensive or strong body odors can be a source of social embarrassment. Axillary osmidrosis (AO) is a chronic skin condition characterized by such body odors and excessive sweating from the armpits . In AsiaABCC11 gene, which codes a functionally null variant with a high allele frequency in East Asians -dehydroepiandrosterone sulfate (DHEA-S) , which is an ABCC11 substrate -DHEA-S (100 nM or indicated concentrations) in a reaction mixture  for 5 min at 37 °C, either without  or with the individual target fractions/authentic chemicals at the indicated concentrations. As the vehicle control, 1% water was used for plant extracts; 1% methanol  or 1% dimethyl sulfoxide  was used for the individual target fractions, as described below. Since stock solutions of authentic chemicals were prepared with DMSO at 10 mM, 1% DMSO was employed as the vehicle control for them. After incubation, the reaction mixture was mixed with 980 μL of an ice-cold stop buffer  and rapidly filtered on a membrane filter  for extract screening or Whatman™ Grade GF/F Glass Microfiber Filter Paper  for the other experiments). After washing with 5 mL of the ice-cold stop buffer three times, the plasma membrane vesicles trapped on the membrane filter were dissolved in Clear-sol II . Then, the radioactivity incorporated into the plasma membrane vesicles was measured with a liquid scintillator .In brief, the plasma membrane vesicles (0.25 mg/mL or indicated concentrations) were incubated with + and [M+Na]+ of the constituent, respectively . The three major soy isoflavones are genistein, daidzein, and glycitein, with the respective formulas (monoisotopic mass) of C15H10O5 (270.0528), C15H10O4 (254.0579), and C16H10O4 (284.0685) [We next conducted a series of spectrometric analyses  to obtai84.0685) . Additio50 of 61.5 μM , as well as their metabolites  on the ABCC11 function a,b. As e 61.5 μM c. If the 61.5 μM b, and th 61.5 μM c. After  61.5 μM , we concS)-equol, which is a daidzein-derived metabolite produced by the intestinal bacterial flora in human intestines [Daidzein and glycitein only exhibited weak and minimal ABCC11-inhibitory activity, respectively. , qualitative evaluation with accurate mass chromatograms revealed that daidzein and genistein were separately fractionated into the Fr.#10 and Fr.#11, respectively. Besides Fr.#11, Fr.#10 and Fr.#12 also showed noticeable ABCC11-inhibitory activity b. Thus, In conclusion, we found that the soybean extract inhibits the transport activity of ABCC11. From this extract, we successfully identified genistein, a compound known to be fairly safe for humans, as an active ingredient. Additionally, to the best of our knowledge, the present study is the first one demonstrating that some dietary flavonoids can inhibit ABCC11, at least in vitro. While human studies are needed to examine the effects of ABCC11-inhibitory phytochemicals on the AO phenotype, our findings here may provide a new clue for treating AO.


Cytochrome c oxidase (COX), the rate-limiting enzyme of mitochondrial respiration, is regulated by various mechanisms. Its regulation by ATP (adenosine triphosphate) appears of particular importance, since it evolved early during evolution and is still found in cyanobacteria, but not in other bacteria. Therefore the “allosteric ATP inhibition of COX” is described here in more detail. Most regulatory properties of COX are related to “supernumerary” subunits, which are largely absent in bacterial COX. The “allosteric ATP inhibition of COX” was also recently described in intact isolated rat heart mitochondria. There exists a basic difference in the regulation of ATP synthesis by oxidative phosphorylation (OxPhos) between bacteria (except cyanobacteria) and eukaryotic organisms. Aerobic bacteria grow and divide continuously under constant conditions. Higher organisms change the rates of ATP synthesis and consumption up to a factor of 10 depending on various inner and outer signals. This regulation is based on acquisition of additional regulatory protein subunits (supernumerary subunits) during evolution for the enzyme complexes of OxPhos, which in eukaryotes are located in special organelles, the mitochondria.1 complex), IV , and V (ATP synthase). According to the Mitchell theory [2), consisting of a membrane potential ΔΨm and a pH gradient across the inner mitochondrial membrane (Δp = ΔΨm − 59·ΔpH (mV)). Δp consists mostly of ΔΨm and is generated in the respiratory chain at enzyme complexes I, III, and IV via the translocation of protons from the matrix into the intermembrane space. Degradation of ΔΨm occurs predominantly via ATP synthase and “uncouplers” of OxPhos or uncoupling proteins [OxPhos includes the respiratory chain containing the enzyme complexes I (NADH: ubiquinone oxidoreductase), II (succinate dehydrogenase), III .3-type enzyme [Synechocystis contains four protein subunits corresponding to subunits I, II, III, and IV of vertebrate COX. The deduced amino-acid sequence of “subunit IV” from Synechocystis sp. PCC6803 showed approximately 50% and 20% sequence identity to COX subunit IV from Saccharomyces cerevisiae and beef heart, respectively. After reconstitution in liposomes, intraliposomal ADP stimulated and ATP inhibited the oxidation of ferrocytochrome c by cyanobacteria [Regulation of COX activity by adenine nucleotides evolved early during evolution, since it occurs already in COX of cyanobacteria containing an aae enzyme . Purifiebacteria .2+) [2+-activated protein phosphatase. It was suggested that the allosteric ATP inhibition of COX keeps the mitochondrial membrane potential (ΔΨm) at low values (120–140 mV) and prevents formation of high amounts of ROS (reactive oxygen species) [The allosteric ATP inhibition of COX was recently also shown with intact isolated rat heart mitochondria . In a pr2+) ,28 and dspecies) ,30. ROS species) ,32,33,34The relationship between stress factors and the generation of ROS was unknown until recently ,32. We pm, include high glucose [Stress factors are known to increase the cytosolic calcium concentration ,32,33, w glucose ,36, stre glucose ,39,40,41 glucose ,44,45,46 glucose .“resting state” of mitochondrial respiration . The high efficiency of dimeric COX is apparently due to allosteric ATP inhibition, which keeps ΔΨm at low values [m [+/e− stoichiometry of 2, instead of 1, which is thermodynamically possible [+/e− stoichiometry of COX is constant and always 1 [+/e− stoichiometry of 2 in COX was in fact measured by the Lehninger group in isolated rat liver mitochondria [The tion see  with higw values , thus pralues [m , which ialues [m  and causalues [m . In addialways 1  . The adalways 1 ,55,56, walways 1 . An H+/echondria ,59,60,61“active state” of mitochondrial respiration   contains only monomeric COX, the ordered cluster structure of the complete OxPhos system could allow a fast monomeric/dimeric configuration change. Thus, the establishment of “allosteric ATP control” mirrors the molecular composition [The ion) see  is chara)    (see m . These r)   is deleterious for life and has been shown to be involved in the generation of numerous diseases [In isolated mitochondria, supplemented with succinate, pyruvate, or glutamate, ADP stimulates oxygen consumption (state 3), whereas, after its conversion into ATP, respiration decreases (state 4) and ΔΨo 225 mV . The rat mV [2O2 . This hidiseases ,68,69,70diseases ,72, havediseases ,73.3, CuB, and a tyrosyl-group, allowing simultaneous transfer of four electrons to O2, thus preventing the formation of intermediate ROS compounds [m values above 140 mV [+ ratios lead to the formation of O2− [2O2 by superoxide dismutases in the matrix and in the intermembrane space [m values above 140 mV are not necessary for the synthesis of ATP, because the ATP synthase is already saturated and maximal at 100–120 mV [This high amount of ROS is produced in mitochondria mainly at complexes I–III ,75, but ompounds . It was e 140 mV ,77,78. Fn of O2− , which ine space . High an0–120 mV .m values are normally below 140 mV . Howevepoptosis ,83.m by ATP was also measured directly in isolated rat liver mitochondria using a tetraphenyl phosphonium electrode [Inhibition of ΔΨlectrode . Above 1lectrode ,76,77. Wlectrode ,73,75, hlectrode , are knolectrode ,67,68,69lectrode ,85,86.Allosteric ATP inhibition requires dimeric COX, based on its cooperativity of substrate action . Monomer2+-activated dephosphorylation of COX. The phosphorylation site is located at the cytosolic side of COX subunit I [The feedback inhibition of COX by ATP, discovered 23 years ago, was not recognized by the scientific community, because it was generally not found in isolated mitochondria for two reasons: (i) measurements in mitochondria were usually not performed in the presence of ATP and high ATP/ADP ratios; (ii) the mechanism is switched off under stress conditions via Caubunit I , and it 2+-activated protein phosphatase, allosteric ATP inhibition is switched off. Re-phosphorylation by a cAMP-dependent protein kinase switches it on again [2+ concentration and activate a Ca2+-dependent protein phosphatase, located at the intermembrane space, leading to dephosphorylation of COX with subsequent loss of allosteric ATP inhibition, increase in ΔΨm, and formation of ROS [After dephosphorylation of this site by a Caon again . These oon again ,90,93. Ton again . In thisn of ROS   generation in eukaryotic cells according to the actual requirements see .A further regulation of COX activity occurs via reversible phosphorylation of protein subunits ,92,94,95Regulation of COX activity via expressing isoforms of supernumerary subunits in the complex of 13 total subunits was first described in 1982 for subunits VIa, VIIa, and VIII , as showMeanwhile, six different isoforms were identified for supernumerary COX subunits . These iInterestingly, in the other complexes of OxPhos , no isoforms of supernumerary subunits were found.A specific expression of supernumerary COX isoforms concerns developmental-specific isoforms. During fetal development of mammalian embryos, the liver-type COX subunits VIa-L and VIIa-L are expressed, before switching to expression of the respective heart-type subunits during birth ,111.Copper (Cu) is an essential trace element required for the normal development of living organisms. Copper-dependent enzymes such as ceruloplasmin, superoxide dismutase SOD1 and SOD3, the group of metallothionein proteins, and COX are present at all stages of gametogenesis, as well as in the somatic cells of the testis. Due to its redox potential, copper is a cofactor in many enzymes responsible for important processes in cells. Copper is a very reactive element and, in its free state, it can trigger the production of large amounts of free radicals, which consequently lead to the damage of proteins and DNA [magnesium/manganese (Mg/Mn) site in cytochrome c oxidase has a possible role in proton pumping. Due to its close proximity and a shared ligand, oxidized Cu(A) is spin-coupled to the Mn(II) ion. This new observation of anion binding at the Mg/Mn site is of interest in terms of accessibility of the buried site and its potential role in redox-dependent proton pumping [hydrogen sulfide (2SH). However, studies conducted over the last two decades unveiled multiple biological regulatory roles of H2S as an endogenously produced mammalian gaseous transmitter. H2S serves as a stimulator of electron transport in mammalian mitochondria by acting as an electron donor, with sulfide/quinone oxidoreductase (SQR) being the immediate electron acceptor, and it stimulates mitochondrial ATP production [bile acid-binding site (BABS) was crystallographically defined in the membrane domain of mammalian COX and COX from Rhodobacter sphaeroides. Diverse amphipathic ligands were shown previously to bind to this site and affect the electron transfer equilibrium between heme a and a3 cofactors by blocking the K proton uptake path. Identified candidate ligands include steroids, nicotinamides, flavins, nucleotides, retinoic acid, and thyroid hormones, which are predicted to make key protein contacts with the residues involved in bile acid binding [Deoxycholate binds with its carboxyl group at the entrance of the K path; thus, this conserved steroid binding site could reveal a regulatory site for steroids or structurally related molecules that act on the essential K proton path and enzyme activity [3-CuB site, as well as the variation in the occupancy of this binding site with cholate or nucleotides, may modify the reactivity of the oxidized binuclear center toward cyanide [+/e− stoichiometry of proton pumping of non-muscle COX [epidermal growth factor receptor (EGFR), HIG1 domain family member 1A (Higd1a), B-cell lymphoma 2 protein (BCL-2), a hepatitis B viral protein (HBx), mitochondrial nuclear retrograde regulator 1 (MNRR1), Coiled-coil-helix-coiled-coil-helix domain containing 2 protein (CHCHD2), amyloid-β protein, Nitric oxide synthase 1 (NOS-1), NDUFA4, and NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 4-like 2 (NDUFA4L2), was discussed in former publications [Various metabolites have been described to bind to COX and change its activity.  and DNA . Hepatic and DNA . the ani pumping . Moreove pumping . Inhibitoduction . A conse binding . Deoxychactivity . Cholate cyanide . 3,5-Dii cyanide . Anotherscle COX . The binications ,107.Mitochondrial complexes are prone to sirtuin (Sirt)3-mediated deacetylation modification. COX subunit I was discovered as a new deacetylation target of Sirt3, indicating that the Sirt3/MT-CO1 axis is a promising therapy target of stress-related diseases .m maintained by the electron transport chain (ETC) was decreased [Upregulating the expression levels of mitochondrially encoded NADH dehydrogenase 1 (MT-ND1) and COX subunit I (MT-CO1) in tachypacing cardiomyocytes resulted in increased ATP content and superior cell viability, whereas the expression levels of NADH ubiquinone oxidoreductase core subunit 1 (NDUFS1) and COX subunit VIc (COXVIc) had no effect . Cytochrecreased . A systeecreased . Overexpecreased ,130. Oveecreased . In someecreased ,137,138.1III2IV1). Their properties were described in review articles [2 and CIV (SC I1+III2+IV1), known as the respirasome), as well as with CIII2 alone (SC I + III2). CIII2 forms a supercomplex with CIV (SC III2 + IV1), and CV alone forms dimers (CV2).Similar to the association of two COX monomers to the dimer, the complexes of the respiratory chain (CI–CIV) can associate to multiple complexes called “supercomplexes”. A well-known supercomplex is the “respirasome” (Iarticles ,141,142.


Robotic assistance with integrated navigation is an area of high interest for improving the accuracy of minimally invasive pedicle screw placement. This study analyzes the accuracy of pedicle screw placement between an attending spine surgeon and a resident by comparing the left and right sides of the first 101 consecutive cases using navigated robotic assistance in a private practice clinical setting. A retrospective, Institutional Review Board-exempt review of the first 106 navigated robot-assisted spine surgery cases was performed. One attending spine surgeon and one resident performed pedicle screw placement consistently on either the left or right side (researchers were blinded). A CT-based Gertzbein and Robbins system (GRS) was used to classify pedicle screw accuracy, with grade A or B considered accurate. There were 630 consecutive lumbosacral pedicle screws placed. Thirty screws (5 patients) were placed without the robot due to surgeon discretion. Of the 600 pedicle screws inserted by navigated robotic guidance (101 patients), only 1.5% (9/600) were repositioned intraoperatively. Based on the GRS CT-based grading of pedicle breach, 98.67% (296/300) of left-side screws were graded A or B, 1.3% (4/300) were graded C, and 0% (0/300) were graded D. For the right-side screws, 97.67% (293/300) were graded A or B, 1.67% (5/300) were graded C, and 0.66% (2/300) were graded D. This study demonstrated a high level of accuracy (based on GRS) with no significant differences between the left- and right-side pedicle screw placements  in the clinical use of navigated, robot-assisted surgery. The traditional teaching method of orthopedic residents for spine surgery is through a preceptorship or apprenticeship approach to orthopedic surgical training with 1:1 training with an attending surgeon, rotating through orthopedic subspecialties. The residents work towards improving comprehensive patient care, along with surgical and clinical skills.Safe pedicle screw placement may be a factor in achieving positive clinical outcomes as well as avoiding catastrophic complications. Therefore, orthopedic surgical resident education in proper pedicle screw placement is essential. Advances in medical imaging have improved the accuracy of pedicle screw placement, from fluoroscopic-guided to computer-aided navigation. Auode et al. have shown that computer-aided navigation is an effective tool for training orthopedic surgery residents in pedicle screw placement . Free-haIn a recent study analyzing the accuracy of thoracic pedicle screw placement placed by neurosurgery residents, it was found that six postgraduate year (PGY-6) residents were associated with the highest rate of cortex violations compared to PGY-2 and PGY-3 residents [The robot affords a stable platform for guiding pedicle screw placement to pre-planned trajectories, which facilitates accurate pedicle screw placement. Evaluation of pedicle screw accuracy is necessary to determine the efficacy of navigated robotic guidance in spine surgery. To the best of the author's knowledge, pedicle screw accuracy based on the surgeon’s level of training using robotic guidance has not been studied. This study analyzes the accuracy of pedicle screw placement between an attending spine surgeon and a resident by comparing the left and right sides of the first 101 consecutive cases using navigated robotic assistance in a private practice clinical setting.This is a retrospective, Institutional Review Board exempt study of the first 101 navigated robot-assisted spine surgery cases at a single site. Per case, one attending spine surgeon and one resident performed pedicle screw placement consistently on either the left or right side (researchers were blinded). The attending surgeon is a supervising physician in a Committee on Advanced Subspecialty Training accredited spine fellowship, who has been in practice for 24 years. The residents participated in minimally-invasive spinal surgery during their PGY-6 of neurosurgery residency. Radiographic evaluation of preoperative plan and postoperative CT scans was performed and complication rates were collected.®; Globus Medical, Inc., Audubon, PA, USA) uses radiological patient images , along with a dynamic reference base and positioning camera to guide pedicle screw placement in real time. Robotic assistance can help guide the surgeon’s planning and approach prior to and during surgery, and is designed to improve pedicle screw accuracy.The robotic positioning system  or a standard CT scan taken at the time of surgery with the patient already in surgical position (prone). After a CT scan was taken and the spinal levels identified, pedicle screw trajectories were planned and saved. Reference frames were installed and fixated to the pelvis, and instruments and arrays with reflective markers were registered. A surgeon-controlled foot pedal activated and positioned the robot arm to the planned pedicle trajectory. Stab incisions were made on the skin using a scalpel. Pedicle screws were inserted percutaneously using navigated instruments guided by the robotic arm. Per case, one attending spine surgeon and one resident performed pedicle screw placement consistently on either the left or right side. This sequence was repeated until all pedicle screws were placed. Rods were then placed and locking caps were set once the rods were in the proper position. Intraoperative CT images were taken to verify screw and rod position. Interbody devices when used were inserted manually. Surgical incisions were cleaned and closed in the standard fashion.A CT-based Gertzbein and Robbins System (GRS) was used to classify pedicle screw accuracy, in which screws were graded as A (screw is completely within the pedicle), B , C , D , and E  . The nump < 0.05 for all statistical analysis.Statistical analysis was performed using SPSS Statistics Version 25 software . Data were presented as mean ± standard deviation. The level of statistical significance was set to 2. A majority of the indications were degenerative disc disease (79) and adjacent segment disease (19)  were placed without the robot due to surgeon discretion. Of the 600 pedicle screws inserted by navigated robotic guidance (101 patients), 300 screws were placed on the left and 300 screws were placed on the right. Only 1.5% (9/600) were repositioned intraoperatively. The average age was 64.8 years, and 55% were female. Average body mass index was 31 kg/mOf the 600 screws inserted with navigated robotic guidance, 26.0% (156/600) were performed at L4 and 25.7% (154/600) were performed at L5. The most common screw used was 25% of the time, 7.5 × 50 mm. Average estimated blood loss was 165 cc. Mean operative time was 142 min. The mean length of hospital stay was 4.6 days. The most common disposition was home (32%) and rehabilitation (31%) (Table p > 0.05).Based on the GRS CT-based grading for the left side screws, 98.67% 296/300) were graded A or B, 1.33% (4/300) screws were graded C, and 0% (0/300) screws were graded D   screws was 1.74 ± 1.31 mm from tip, 1.75 ± 1.24 mm from tail, and 1.98 ± 1.43° of angulation. The average offset from preoperative plan to actual final placement of right-side (attending) screws was 1.75 ± 1.39 mm from tip, 1.82 ± 1.14 mm from tail, and 2.11 ± 1.75° of angulation. Using an independent samples Two complications—interbody removal and wound vacuum-assisted closure—were reported as requiring a return to the OR, but these were not related to robotic guidance or pedicle screws.The accuracy of pedicle screw placement is of the utmost importance for the safety of patients undergoing instrumented spinal fusion. Two- and three-dimensional fluoroscopy along with intraoperative CT for freehand real time navigation based on actual patient anatomy has greatly improved pedicle screw accuracy compared to freehand manual guidance . In a syThe current literature is not consistent on whether surgical education has an effect on pedicle screw placement and/or accuracy. Baird et al. compared the surgical skills of a PGY-2, PGY-4 orthopedics resident, a spine surgery fellow, and a board-certified attending spine surgeon . It was The arrival of advanced minimally invasive imaging technology may allow residents and fellows to place pedicle screws as accurately as experienced surgeons. In a retrospective radiological study by Laudato et al., 569 thoracolumbar pedicle screws were inserted in 84 patients using three different insertion methods: robotic assistance, O-arm navigation, and freehand lateral fluoroscopy. Screw position was evaluated using Rampersaud criteria, similar to the Gertzbein-Robbins scale used in our study . It was Although this is a retrospective study, the results are consistent with findings from the literature. The number of pedicle screws inserted by residents and attending prior to using the robot is not quantified. Also, pedicle screw accuracy prior to using the robot is unknown. This study forms the foundation for future studies with a higher level of evidence. Comparative studies with larger sample sizes are needed.This study demonstrated a high level of accuracy (based on GRS) with no significant differences between the left- and right-side pedicle screw placements  in the clinical use of navigated, robot-assisted surgery. In this cohort, both resident and attending surgeon placed pedicle screws successfully under navigated robot guidance.


Mutations in the p62/SQSTM1 gene have been known to be associated with patients with amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Parkinson disease (PD). The aim of the present study was to create a novel model of human neurogenerative disease in Drosophila melanogaster by altering the expression of Ref(2)P, the Drosophila orthologue of the human p62/SQSTM1 gene. Ref(2)P expression was altered in all neurons, the dopaminergic neurons and in the motor neurons, with longevity and locomotor function assessed over time.Sequestosome 1 (p62/SQSTM1) is a multifunctional scaffold/adaptor protein encoded by the Ref(2)P resulted in a significantly increased median lifespan in the motor neurons, followed by a severe decline in motor skills. Inhibition of Ref(2)P in the dopaminergic neurons resulted in a significant, but minimal increase in median lifespan, accompanied by a drastic decline in locomotor function. Inhibition of Ref(2)P in the ddc-Gal4-expressing neurons resulted in a significant increase in median lifespan, while dramatically reducing motor function.Inhibition of  UAS-LacZ has been used as a benign control in a number of experiments produced in our lab as other controls investigated to date do not have a benign response and may reduce median lifespan. Drosophila melanogaster was maintained on a standard media comprised of 65 g/L cornmeal, 50 ml/L fancy grade molasses, 10 g/L yeast and 5.5 g/L agar which was then treated with 2.5 ml propionic acid and 5 ml of 0.1 g/ml methylparaben. This mixture was then allowed to solidify at the bottom of vials and stored in 4 to 6 °C until use. Stocks were stored at room temperature (~ 21 °C), while crosses and experiments were performed at 25 °C. Quantification of inhibition efficiency was not conducted in this study, however, the UAS-Ref(2)P-RNAi HMS00551 has been quantified by Liu et al. (2018).The D42-Gal4, TH-Gal4, ddc-Gal4HL4.3D lines were bred with male flies which expressed Ref(2)P inhibition. The consistent material and paternal sources allow for minimal genetic differences. Two Ref(2)P inhibition lines UAS-Ref(2)P-RNAi HMS00551, UAS-Ref(2)P-RNAiHMS00938 and the control line UAS-lacZ were used. Critical class male progeny from these crosses were assessed for longevity and locomotor ability through ageing and climbing assays. While UAS-LacZ does not produce a non-silencing RNAi control it is still a valid control for the GAL4 and responding transgenes. Future experiments may include the addition of a RNAi-silencing control if shown to be reasonable.Virgin female flies from the P-value less than or equal to 0.05 with Bonferroni correction being considered statistically significant. Longevity data is a representation of the summation of independent replicates.The survival of Drosophila was analyzed to examine the lifespan of experimental flies in comparison to control flies. Critical class male progeny were collected daily and placed in vials with fresh medium. A sample size of approximately three hundred males was collected in total and stored at 25 °C for the duration of the experiment. The flies were scored every two days to examine if any death had occurred. A fly was considered dead when no movement was observed. Males were transferred onto fresh media every 4 days to obtain a healthy environment. Graphpad Prism 8 (Graphpad Software Inc.) was used to analyze longevity data, and survival curves were analyzed and compared using the Log-rank (Mantel-Cox) test, with a D42-Gal4, TH-Gal4, and ddc-Gal4HL4.3D flies and male UAS-Ref(2)P-RNAi HMS00551, UAS-Ref(2)P-RNAiHMS00938 and UAS-lacZ flies. This assay was used to measure the ability of flies to climb up a narrow glass tube over the course of their lifespan, with 50 male flies from each genotype being evaluated once every 7 days, beginning at the 7th day post-eclosion. Climbing analysis was conducted until flies had a minimum climbing score for 2 consecutive weeks, or less than 10 flies remained alive for that genotype. Class males were maintained in vials with ten flies per vial, stored at 25 °C, and placed on new medium once per week throughout the experiment. Climbing analysis followed the standard protocol outlined by our laboratory. Graphpad Prism 8 (Graphpad Software Inc.) was used to analyze the data, and to generate climbing curves fitted using non-linear regression. 95% confidence intervals were used to test for significance with the curves considered to be significantly different if P < 0.05. Locomotive data is a representation of the summation of independent replicates.Approximately 70 critical class male progeny to be collected from crosses between female Ref(2)P via the UAS-Ref(2)P-RNAi HMS00551 transgene through D42-Gal4 resulted in a significant increase in median lifespan by approximately 22%. Compared to the control UAS-lacZ median lifespan of 70 days, inhibition of Ref(2)P via Ref(2)P-RNAi HMS00551 lead to a median lifespan of 86 days P-RNAiHMS00551 flies lost their ability to climb at week 4 P via the UAS-Ref(2)P-RNAiHMS00938 transgene through D42-Gal4 resulted in a significant increase in median lifespan by approximately 17%. Compared to the control UAS-lacZ median lifespan of 70 days, inhibition of Ref(2)P via Ref(2)P-RNAiHMS00938 lead to a median lifespan of 82 days P-RNAiHMS00938 flies lost their ability to climb at week 3 P via the UAS-Ref(2)P-RNAi HMS00551 transgene through TH-Gal4 resulted in a significant increase in median lifespan compared to the control UAS-lacZ median lifespan of 82 days, inhibition of Ref(2)P via Ref(2)P-RNAi HMS00551 lead to a median lifespan of 88 days P-RNAi HMS00551 flies lost their ability to climb at week 4 P via the UAS-Ref(2)P-RNAiHMS00938 transgene through TH-Gal4 resulted in a significant increase in median lifespan compared to the control UAS-lacZ median lifespan of 82 days, inhibition of Ref(2)P via Ref(2)P-RNAiHMS00938 lead to a median lifespan of 86 days P-RNAiHMS00938 flies lost their ability to climb at week 4 P via the UAS-Ref(2)P-RNAi HMS00551 transgene through ddc-Gal4HL4.3D resulted in a significant increase in median lifespan compared to the control UAS-lacZ median lifespan of 70 days, inhibition of Ref(2)P via Ref(2)P-RNAi HMS00551 lead to a median lifespan of 74 days P-RNAi HMS00551 flies lost their ability to climb at week 4 P via the UAS-Ref(2)P-RNAiHMS00938 transgene through ddc-Gal4HL4.3D resulted in a significant increase in median lifespan by approximately 34%. Compared to the control UAS-lacZ median lifespan of 70 days, inhibition of Ref(2)P via Ref(2)P-RNAiHMS00938 lead to a median lifespan of 94 days P-RNAiHMS00938 flies lost their ability to climb at week 4 P gene results in a significant increase in median lifespan, accompanied by a severe reduction in locomotor function over time. The inhibition of Ref(2)P through the expression of UAS-Ref(2)P-RNAi HMS00551 in the motor neurons gave a significant increase in median lifespan by approximately 22%, while resulting in a drastic decrease in locomotor function over time. Similarly, the inhibition of Ref(2)P through the expression of UAS-Ref(2)P-RNAiHMS00938 in the motor neurons gave a significant increase in median lifespan by approximately 17%, while severely reducing locomotor ability over time. The increase in lifespan accompanied by a sharp decrease in motor function over time may be interpreted as trade-off, where the slight increase in longevity is a type of compensation for a severe decline in motor skills. Inhibiting Ref(2)P in the motor neuron was conducted as both ALS and Parkinson Disease are known to result in a loss of motor function, with ALS being a motor-neuron disease. This significant reduction in motor ability and increase in lifespan when Ref(2)P expression is altered corresponds with the characteristic loss of motor neurons associated with ALS, making the inhibition of Ref(2)P in the motor neuron an imperfect model of neurodegenerative disease.The Ref(2)P through the expression of UAS-Ref(2)P-RNAi HMS00551 in the dopaminergic neurons gave a significant increase in median lifespan, while resulting in a drastic decrease in locomotor function over time. Similarly, the inhibition of Ref(2)P through the expression of UAS-Ref(2)P-RNAiHMS00938 in the dopaminergic neurons gave a significant increase in median lifespan, while severely reducing locomotor ability over time. Similar to the inhibition of Ref(2)P in the motor neurons, the minimal increase in lifespan and drastic reduction in motor skills seen under the inhibition of Ref(2)P in the dopaminergic neurons can be interpreted as a type of compensation. As dopaminergic transmission is known to be altered during Parkinson Disease pathology, investigating the consequences of Ref(2)P in the dopaminergic neurons would be of great interest. This significant reduction in motor ability and minimal increase in lifespan seen when Ref(2)P expression is altered, corresponds with the characteristic loss of dopaminergic neurons associated with Parkinson Disease, making the inhibition of Ref(2)P in the motor neuron an promising model of neurodegenerative disease.The inhibition of Ref(2)P through the expression of UAS-Ref(2)P-RNAi HMS00551 in the ddc-Gal4HL4.3D-expressing neurons gave a significant increase in median lifespan, while dramatically reducing locomotor ability over time. The ddc-GAL4-expressing neurons include both dopaminergic and serotonergic neurons, as dopaminergic transmission is known to play a role in Parkinson Disease investigating the consequences of Ref(2)P inhibition in these neurons was conducted. This significant reduction in motor ability and minimal increase in lifespan seen when Ref(2)P expression is altered suggest that the inhibition of Ref(2)P in the ddc-Gal4HL4.3D-expressing neurons to be a promising model of neurodegenerative disease. On the other hand, the inhibition of Ref(2)P through the expression of UAS-Ref(2)P-RNAiHMS00938 in the ddc-Gal4HL4.3D-expressing neurons gave a significant increase in median lifespan by approximately 34%, accompanied by a severe reduction locomotor ability over time. This significant reduction in motor ability and increase in lifespan seen when Ref(2)P expression is altered may also be interpreted as a type of compensation, and seen as an imperfect model of neurodegenerative disease.The inhibition of Ref(2)P expression in a tissue-specific manner demonstrates the phenotypic consequences to the organism in a more direct manner. By inhibiting expression in the motor neuron, dopaminergic neuron and ddc-GAL4-expressing neurons, rather than the generating mutant Ref(2)P critical class which express Ref(2)P inhibition throughout the whole body, less restrictive results are produced that show the consequences of tissue-specific loss-of-function. While the loss-of-function of Ref(2)P has shown a reduction in climbing ability, which can be a promising model of neurodegenerative disease, but yet increase in median lifespan, this cannot be explained through this study, and instead can be seen as a compensational trade-off.Investigating the inhibition of Ref(2)P would be highly desirable and essential for a “gene replacement” study of Ref(2)P. Although several Gal-4 transgenes were employed in this set of experiments, a number of others could prove to informative. Our future plans include combining the inhibition of Ref(2)P with other gene manipulations to identify interactions including synergies that further increase lifespan and improve locomotor activity over time. Although UAS-LacZ is not the best control it has been used as benign control in our lab, we aim in the future to explore other controls that may be better. Finally, as critical class males were analysed, broadening the study to critical females might be very provide useful data.To significantly add to our experimental design, the directed overexpression of


Reperfusion therapy is the most important method for treating acute myocardial infarction. However, myocardial ischemia reperfusion injury (MIRI) can offset the benefit of reperfusion therapy and worsen the outcome. In both ischemia and reperfusion, autophagy remains problematic. Activating molecule in Beclin1-regulated autophagy (Ambra1) is an important protein in autophagy regulation, and its function in MIRI remains unclear. Thus, we used H9C2 cells to investigate the function of Ambra1 in MIRI and the underlying mechanisms involved. Hypoxia and reoxygenation of H9C2 cells were used to mimic MIRI in vitro. During hypoxia, autophagy flux was blocked, then recovered in reoxygenation. Ambra1 overexpression increased autophagy in the H9C2 cells, as the LC3B II/I ratio increased, and alleviated cellular necrosis and apoptosis during hypoxia and reoxygenation. This effect was counteracted by an autophagy inhibitor. Knocking down Ambra1 can block autophagy which P62 sediment/supernatant ratio increased while the ratio of LC3B II/I decreased, and worsen outcomes. Ambra1 enhances autophagy in H9C2 cells by improving the stability and activity of the ULK1 complex. Reactive oxygen species (ROS) are an important cause of MIRI. ROS were reduced when Ambra1 was overexpressed and increased when Ambra1 was knocked down, indicating that Ambra1 can protect against hypoxia and reoxygenation injury in H9C2 cells by promoting autophagy and reducing ROS. As morbacidosis , 11. Aut in MIRI –15.Autophagy is the cellular process of digesting damaged proteins and organelles and maintaining cellular environmental homeostasis and is very important under both physiological and pathological conditions . AutophaAutophagy is very important during both ischemia and reperfusion; however, the effect of autophagy in MIRI remains controversial. Some studies have shown that enhancing autophagy can protect the heart from ischemia and reperfusion –28, whilμg/ml streptomycin  at 37°C in a 5% CO2 incubator. Cells were passaged when they reached 90% confluence.H9C2 cells were obtained from the American Type Culture Collection  and cultured in Dulbecco's modified Eagle's medium  supplemented with 10% fetal bovine serum , 100 U/ml penicillin, and 100 H9C2 cells were cultured in complete medium, and control adenovirus, Ambra1-overexpression adenovirus, and shAmbra1 adenovirus with GFP flag  were added, respectively, when the cell density reached 50–70%. After 8 h, the medium was changed to fresh complete medium. At 72 h after adenovirus transfection, GFP and Western blot were analyzed to confirm that the adenovirus was transfected successfully; then, downstream experiments were performed.2 for 6 h. Next, the medium was replaced with normal complete medium and cultured in a standard cell culture incubator for 4 h .To mimic ischemia and reperfusion, the primary culture medium was discarded and washed with phosphate-buffered saline . The H9C2 cells culture medium was replaced with DMEM without glucose or FBS, then subsequently placed in a hypoxia incubator with 1% Oβ-actin antibody was purchased from Solarbio (China). After washing with TBST , the secondary antibody  was incubated. Finally, the membranes were visualized using the Gel Doc XR+ System .H9C2 cells were lysed with RIPA buffer mixed with proteinase inhibitor cocktail  and PMSF . The cell extract was centrifuged at 12000 × g for 30 min at 4°C. Supernatant concentrations were measured using a BCA Protein Quantitation Kit . The concentration of the protein precipitate was proportional to the supernatant. After denaturation, both the supernatants and precipitates were separated via SDS-PAGE, then transferred to a polyvinylidene fluoride (PVDF) membrane . Membranes incubated in the corresponding antibodies and stored at 4°C overnight. Anti-LC3B antibody and anti-P62 antibody were purchased from Abcam (Britain). Anti-Ambra1 antibody was purchased from Cell Signaling Technology (USA). Anti-H9C2 cells cultured on slides were washed with PBS, then fixed with 4% paraformaldehyde  for 1 hour. After fixation, PBS was used to wash the redundant paraformaldehyde three times for 10 min each time. Subsequently, cells were permeabilized with 0.5% Triton X-100  for 10 min, then washed again with PBS. After blocking in 1% BSA , cells were incubated with primary antibodies overnight at 4°C, then visualized using secondary antibodies under a confocal microscope .μl of cell suspension was transferred to a cell-counting chamber, then inserted into a cell-counting machine  to analyze cell viability.H9C2 cells digested in prewarmed trypsin solution  were mixed in the trypan blue solution . After mixing for three minutes, 20 μl Annexin-APC  and PI  were added to 100 μl cell suspension, then incubated in the dark for 15 minutes at room temperature. After adding 200 μl of 1× binding buffer, the cell suspension was analyzed via flow cytometry .Culture media was removed from the dishes, and H9C2 cells were washed once with PBS. Prewarmed trypsin solution without EDTA  was added to digest the cells from the dishes. Culture media with 10% FBS was added to neutralize the extra trypsin solution. Cell suspensions were transferred to tubes and centrifuged at 1000 rpm for 3 minutes. The supernatant was then discarded, and the cells were resuspended with 1× binding buffer. Next, 5 μl working solution as above, then incubated in the dark for 30 min at 37°C. After incubation, the cells were washed with complete medium, then analyzed via flow cytometry.Dihydroethidium  was dissolved in DMSO, then diluted with complete medium into working solution. H9C2 cells were made into a cell suspension with 200 t-test was used to evaluate the differences between two groups. All data are presented as the means ± S.D. of at least three independent experiments. P < 0.05 was considered statistically significant.The SPSS 21 software was used to perform the statistical analyses. One-way analysis of variance was used to analyze the differences between multiple groups, and Student's To determine the function of Ambra1 in autophagy in H9C2 cells, we constructed control adenovirus (Ad-Ctrl), Ambra1-overexpression adenovirus (Ad-Ambra1), and shAmbra1 adenovirus (Ad-shAmbra1), then transinfected these adenoviruses into H9C2 cells. At 72 h after transfection, Western blot was used to examine the Ambra1 expression and autophagy flux. After Ad-Ambra1 was transinfected, Ambra1 was successfully overexpressed, and LC3B detection showed that the LC3B type I conversion to type II was increased, and the P62 supernatant was increased, while the P62 sediment was decreased . This chTo determine the optimum time for hypoxia and reoxygenation in H9C2 cells, several conditions were set, which revealed 6 hours of hypoxia, and then 4 hours of reoxygenation was the best. After 6 h of hypoxia and 4 h of reoxygenation, trypan blue staining was used to detect the cell viability. Cell viability was decreased during both hypoxia and reoxygenation . WesternTo investigate the role of autophagy during hypoxia and reoxygenation, the autophagy inhibitor, chloroquine (CQ), was used to block autophagy by blocking autophagosome and lysosome fusion. Western blot results showed that when CQ was added, the P62 sediment increased as the supernatant decreased during hypoxia and reoxygenation compared with the no-CQ group, indicating that CQ successfully blocked autophagy flux . We thenBecause blocking autophagy resulted in poor outcomes for H9C2 cells during both hypoxia and reoxygenation and Ambra1 increased autophagy in H9C2 cells under basal condition, we decided to examine the role that Ambra1 plays in hypoxia and reoxygenation in H9C2 cells. We overexpressed Ambra1 by transfecting an Ambra1-overexpression adenovirus and used CQ to block autophagy flux by stopping the autophagosomes combining with lysosomes. We also knocked down Ambra1 to determine what occurs in H9C2 cells during hypoxia and reoxygenation. So we set up several groups: Ad-Ctrl group, Ad-Ambra1 group, Ad-Ambra1+CQ group, and Ad-shAmbra1 group. Western blotting showed that Ambra1 overexpression contributed to enhancing autophagy in both conditions, while CQ blocked autophagy flux successfully . ConversThe exact causes of MIRI remain unclear, but extra ROS generation is an important reason that causes cell injury. As Ambra1 overexpression yielded better outcomes in H9C2 cell hypoxia and reperfusion model, and Ambra1 has been reported that it can reduce ROS through mitophagy, whether Ambra1 affects ROS in H9C2 cell hypoxia and reoxygenation model should be further explored. We used DHE to detect the superoxide anion. After living cells uptake DHE, it can be dehydrogenated by superoxide anions to produce ethidium. Ethidium produces red fluorescence when combined with RNA or DNA. The red fluorescence intensity parallels the ethidium level; thus, when the superoxide anion levels in cells are higher, more ethidium is produced. After transfecting the control, Ambra1-knockdown, and Ambra1-overexpression adenoviruses for 72 h, cells were incubated with DHE for 20 min in the dark; then, the red fluorescence was detected via flow cytometry and the mean fluorescence intensity was calculated. We found that overexpression of Ambra1 alleviated the superoxide anion regardless of hypoxia or reoxygenation, while decreasing Ambra1 aggravated superoxide anion production Figures . This inIn order to explore the mechanism by which Ambra1 promotes autophagy in H9C2 cells, we overexpressed Ambra1 by transfecting an Ambra1-overexpression adenovirus or reduced Ambra1 by transfecting an Ambra1-knockdown adenoviruses. As shown in Here, we demonstrated for the first time the function of Ambra1 during hypoxia and reoxygenation in H9C2 cells, which mimics MIRI. Our results indicated that overexpressing Ambra1 enhanced autophagy in H9C2 cells in both physiological and pathological conditions, which benefits cell survival during both hypoxia and reoxygenation, and this outcome can be reversed by an autophagy inhibitor. Correspondingly, Ambra1 knockdown blocked autophagy flux and worsened the results in H9C2 cell hypoxia and reoxygenation. Moreover, Ambra1 reduced the superoxide anion during hypoxia and reoxygenation. Thus, Ambra1 may play several important roles in alleviating MIRI.Ambra1 is a novel BH3-like protein that has been studied over the last decade  and playAutophagy in myocardial ischemia and reperfusion remains controversial, both in function and change. Our research showed that autophagy flux was blocked during hypoxia, then restored during reoxygenation, as the P62 sediment/supernatant ratio was increased with LC3B II/I ratio decreased during hypoxia. After reoxygenation, the LC3B II/I ratio was increased without increasing the P62 sediment/supernatant ratio, indicating that autophagy flux was restored. Our study showed that blocking autophagy by autophagy inhibitor, CQ, aggravated cell damage in both hypoxia and reoxygenation. Marek-Iannucci et al. also showed that increased autophagy plays a protective role in MIRI . ConversAs ULK1 complex plays an important role in activating autophagy, and we demonstrated that in H9C2 cells, an increase in Ambra1 results in an increase in ULK1. Further, P-ATG13 was also increased in this situation while there are no difference in ATG12 and Beclin1. All of this indicates that Ambra1 improves the stability and activity of the ULK1 complex, and this effect is exclusive. Bartolomeo et al. has shown that ULK1 kinase can phosphorylate Ambra1 thus release the autophagy core complex from dynein , 37. TheMyocardial ischemia and reperfusion injury are severe problems in blood restoration and may counteract the benefit of reperfusion therapy . The exaOur research explored the function of Ambra1 in MIRI on H9C2 cells. Overexpression and knockout of Ambra1 in animal models are closer to the MIRI pathophysiology. Autophagy flux, ROS, cell necrosis, and apoptosis detected in vivo will better reveal the role that Ambra1 plays in MIRI. Our further research will focus on these issues.


Direct repair of the pars defect in lumbar spondylolysis is an effective surgical procedure, but it is technically challenging. We assessed the feasibility of a new robotic system for intralaminar screw fixation of spondylolysis.A 26-year-old man complained about frequent low back pain after failed conservative treatments.The lumbar computed tomography images demonstrated the presence of bilateral spondylolysis at the L5 level, with no spondylolisthesis.We performed one surgery of direct intralaminar screw fixation under the guidance of the TiRobot system. The trajectory of the screw was planned based on intraoperative 3-dimensional radiographic images. Then, the robotic arm spontaneously moved to guide the guide wires and screw insertion.Bilateral L5 intralaminar screws were safely and accurately placed. No intraoperative complications occurred. Postoperative computed tomography showed good radiological results, without cortical perforation.We report the first case of robot-assisted direct intralaminar screw fixation for spondylolysis using the TiRobot system. Robotic guidance for direct repair of spondylolysis could be feasible. Direct repair of the pars defect has been proved to be an effective surgical procedure in lumbar spondylolysis.–4 This motion-preserving technique has also demonstrated better biomechanical results. In recent years, we applied a new robot system  in clinical orthopedic surgery. The accuracy of this novel system in pedicle screw insertion was above 95%. We present the first case of robot-assisted (RA) direct intralaminar screw fixation for spondylolysis performed under the guidance of the TiRobot system.Spondylolysis occurs in nearly 6% of the general population.2A 26-year-old male complained of frequent low back pain for 10 months without any significant radicular pain. He had failed conservative treatments after 8 months. On neurological examination, there was no sensory-motor deficit.The lumbar computed tomography images demonstrated the presence of bilateral spondylolysis at the L5 level Fig. , with noThe patient has provided informed consent for publication of the case. After approval by the ethics committee and informed consent, a surgery of RA direct intralaminar screw fixation was scheduled. The patient was placed in a prone position after general anesthesia. A standard posterior midline exposure of the involved vertebra was made. The fibrous tissue and the sclerotic bone in the pars defect were cleaned till bleeding bony surface. After the reference tracker was mounted at the spinous process of L4, a set of 3-dimensional images was acquired by an intraoperative C-arm scan. Based on these images, intraoperative planning of the intralaminar screws was performed using the TiRobot system. The robot could spontaneously move to the chosen trajectory. Then, we inserted the guide wires through the cannula on the robotic arm under real-time navigation monitoring and adjustment Fig. . Two 4.5Immediate postoperative recovery was achieved, with no neurological deficit and other complications. A soft brace patient was worn after ambulation, and no sports were allowed for 3 months. A postoperative computed tomography scan showed acceptable screw positions  returned to competitive sports with this technique. However, on the other hand, it is a technically demanding procedure.,14 In response, we applied this new surgical robot to reduce the operating difficulty, thus verify the feasibility of RA direct repair of pars defect.Several techniques have been applied for the surgical treatment of a pars defect. Direct repair of the defect in spondylolisthesis, by which screws are placed directly through the fractured pars, was first described by Buck in 1970.–17 Higher accuracy and minimal invasion were the advantages of RA technology when compared to traditional methods. Traditional methods require extensive exposure of paravertebral muscles and ligaments, to find the correct orientation. However, using the RA technique, the direction of screws is not tricky through a percutaneous approach. Decreased soft tissue dissection has been shown to decrease infection, muscle atrophy, length of stay, and blood loss.–20 Besides, the RA technique could decrease the operating duration by rapid planning and placement of the screws.This new surgical robot was reported that could provide accurate positioning, adequate steadiness, and repeatability in surgery.This study has some limitations. First, only one case of intralaminar screw fixation was completed. A much larger number of patients and a control group would be needed to assess the safety and accuracy of this RA technique. Second, long-term follow-up with patient-reported outcomes would be required to evaluate the postoperative outcomes and bone union.We report the first case of RA direct intralaminar screw fixation for spondylolysis using the TiRobot system based on intraoperative 3-dimensional images. The use of robotic guidance in the direct repair of spondylolysis could be feasible.Conceptualization: Wei Tian, Qi Zhang, Ya-Jun Liu.Data curation: Xiao-Guang Han, Qiang Yuan.Formal analysis: Qi Zhang, Xiao-Guang Han, Da He.Funding acquisition: Wei Tian, Ya-Jun Liu.Methodology: Qi Zhang, Xiao-Guang Han, Qiang Yuan.Project administration: Wei Tian, Da He, Ya-Jun Liu.Resources: Qiang Yuan.Software: Qi Zhang.Supervision: Wei Tian.Validation: Xiao-Guang Han, Qiang Yuan.Visualization: Ya-Jun Liu.Writing – original draft: Qi Zhang.Writing – review & editing: Wei Tian, Qiang Yuan, Da He, Ya-Jun Liu.


TARGET OF RAPAMYCIN (TOR) kinase has been recognised as a key developmental regulator in both plants and animals. Despite their distinct developmental programmes, all eukaryotes studied possess a functional TOR kinase, which integrates environmental and nutrient signals to direct growth and development. This is particularly important in plants, as they are sessile and must sense and respond to external signals to coordinate multicellular growth appropriately. Thus, the investigation of TOR is essential for plant developmental studies in the context of the resources available for growth. Recently, links have been shown between TOR and plant development from embryogenesis through to senescence, however more investigation is crucial to fully elucidate TOR function in each developmental process. Mounting evidence suggests that integrated signaling and metabolic networks play an instructive role in developmental programs and responses to environmental changes and stresses . RemarkaStreptomyces hygroscopicus  and RAPTOR2   . In siliin algae , it has ant copy , however defects , further defects . The pro defects . The cono leaves , howevero leaves . Future LST8 gene, however two LST8 genes (LST8-1 and LST8-2) have been found in Arabidopsis thaliana and A. lyrata as a result of a gene duplication event in their common ancestor  nuclei and cellularization does not occur  and EXPANSIN B1 (EXPB1) after AtTOR inhibition  relays  relays . Further) relays .raptor1 mutants (SALK_101990 and SALK_022096) had seeds with delayed germination and reduced stress resistance, resulting in reduced viability (Two independent iability . Furtheriability . There wvia either asTORis (active site ATP-competitive TOR inhibitors) or genetic suppression, reduces cotyledon greening, chloroplast development and seedling growth (via the regulation of BR INSENSITIVE 2 (BIN2) (See review: The transition from dark-grown (etiolated) to light-grown (de-etiolated) seedlings is accompanied by several morphological changes; elongation rate is reduced, the apical hook opens, true leaves undergo expansion and mature chloroplasts develop. The inhibition of AtTOR in seedlings, g growth . 40S ribvia Rho-like small GTPase 2 (ROP2) (An exciting link has been made between light and the activation of AtTOR-RPS6 (RIBOSOMAL PROTEIN S6) in de-etiolating seedlings. Light is first perceived by photoreceptors such as phytochrome A and cryptochromes, leading to the inactivation of the negative regulator CONSTITUTIVE PHOTOMORPHOGENESIS 1 (COP1), which triggers the activation of the auxin pathway and thus AtTOR-dependent phosphorylation of RPS6 . It has 2 (ROP2) . TOR mayAtTOR is known to be expressed in these tissues  and the Root Apical Meristem (RAM) maintain undifferentiated stem cells responsible for the formation of the above- and below-ground organs, and  tissues . Consistnt lines . Recent nt lines . AtTOR iyak1 loss-of-function mutants are resistant to the asTORis AZD-8055, whilst Arabidopsis overexpressing YAK1 are hypersensitive. Treatment of WT plants with AZD-8055 reduces the number of dividing cells in the meristematic zone (MZ) inducing early differentiation. In yak1 mutants MZ size was not reduced in the presence of the inhibitor. Furthermore, when treated with pINDY, an ATP-competitive inhibitor of the animal AtYAK1 homolog DYRK1A (DUAL SPECIFICITY TYROSINE PHOSPHORYLATION REGULATED KINASE 1A), MZ size was restored in WT plants treated with AZD-8055. These results indicate that YAK1 controls cell proliferation in the MZ in a TOR-dependent manner.YET ANOTHER KINASE 1 (YAK1) has recently been reported as a downstream target of the AtTOR pathway and a major regulator of RAM activity . YAK1 waSIM, SMR, and CYC genes revealed that, in the absence of AtTOR activity, YAK1 induces the expression of SMR genes, which in turn repress CDKs to promote differentiation (Cell proliferation is controlled by CYCLIN DEPENDENT KINASES (CDKs), which are regulated by the periodic activation of cyclins (CYC). CDK-CYC activity is regulated by plant-specific CDK inhibitors such as SIAMESE (SIM) and SIAMESE-RELATED (SMR)  and CLAVATA (CLV) genes has also been connected to AtTOR activity  were observed when treated with asTORis, suggesting that AtTOR may not regulate meristem patterning in the RAM    to targeAtTOR show hypersensitivity to high nitrate inhibition of roots (Nutrients such as nitrogen (N), phosphate (Pi), and sulfur (S) play crucial roles in the promotion of plant growth and recent studies suggest that AtTOR functions in these processes. For example, S availability coordinates glucose signaling to activate AtTOR . Furtherof roots . By sensAbscisic acid (ABA) signaling has been implicated as a critical player in the inhibition of plant growth under stress and recent studies propose TOR is the key mediator of this process . Upon stvia TOR include SnRK1, a conserved glucose/energy sensor protein kinase. Glucose can activate AtTOR indirectly via the inactivation of SnRK1   . Future lst8-1 produces flowers with smaller floral organs, but no changes in organ patterning or number have been reported  and BIGPETALp (BPEp)  and flanking floral organ primordia. Mutation of reported , suggested fully . Furtherp (BPEp) .S6K (LIS6K) in A. thaliana produces flowers with shortened petals and stamens, due to reduced cell expansion and normal cell division (AUXIN RESPONSE FACTORS (ARFs) and BASIC LEUCINE ZIPPER 11 (bZIP11) are encoded by uORF-mRNAs and therefore their translation reinititation may be under the control of TOR via S6K (MONOPTEROS (ARF5) and ARF2 (Interestingly, ectopic expression of Lily division . S6K is division . S6K1 indivision . Plant s via S6K . It wouland ARF2  are actiThe TOR signaling pathway is vital to integrate information about the nutrient and energy status of cells and tissues to direct the appropriate developmental and physiological response . Our undKM drafted the manuscript and all authors revised it.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.