IntroductionTM) that can be used for multi-level instrumentation with potential advantages for selected applications.The polyaxial head pedicle screw-rod system is a commonly used spinal instrumentation technique to achieve stabilization, deformity correction, and bony fusion. We present a novel plate-based pedicle screw system . The system and case examples are presented showing its potential advantages.ResultsThe quasi-static torsion, the mean for the angular displacement, torsional stiffness, and torsional ultimate strength was 2.5 degrees (SD ± 0.8), 5.3 N-m/mm (SD ± 0.7), and 21.6 N-m (SD ± 4.4). For the fatigue in axial compression, the closed ring construct failed when the applied load and bending moment were ≥ 1500 N and ≥ 60 N.m. This system maximizes the construct rigidity, allows easy extension to adjacent levels, and can be incorporated intuitively into practice. The ring construct with triangulation at each level, together with its biomechanical robustness, predicts a high pullout resistance. It requires an open posterior approach incompatible with minimally invasive techniques.ConclusionThis system may have advantages over the screw-rod systems in carefully selected situations requiring extra rigidity and high pull-out strength for complex reconstructions, sagittal and/or coronal correction, patients with poor bone quality, revisions, and/or extension to adjacent levels. Instrumented spinal fusion is a procedure that uses metal implants to minimize or eliminate the movement of the spinal column to promote bony fusion at the implanted levels . The incNotwithstanding the popularity of the polyaxial head screw-rod systems now in use, they have several drawbacks, including a less than optimal rigidity and a reduced bony surface available for fusion, which might necessitate the inclusion of additional spinal levels or anterior interbody fusion in the final construct. The screw-rod constructs have an appreciable failure rate in patients with a poor bony quality or multiple revisions -9. ConstThe system is titanium-based and was cleared by the Food and Drug Administration (FDA) 510(k) (No. K024313). It includes monoaxial pedicle screws, horizontal and vertical plates, and a multitude of washers, nuts, and bolts to connect these components .The concept behind the design is to create a ring construct forming rigid triangulation at each spinal level. This is done by interconnecting the screw heads at each level with a robust transversely oriented cross plate. The cross plates are then interconnected by sagittally oriented rigid plates situated medial to the screw heads Figure . These sBiomechanical testingThe construct was tested for quasi-static torsion, fatigue in axial compression, and quasi-static axial compression and bending. Operative techniqueThe system requires a standard open operative exposure, which includes a midline skin incision followed by subperiosteal dissection of the paraspinal muscles to expose the spinous processes, laminae, facets, and transverse processes. Then, a full laminectomy is required at each level to allow enough space for the construct placement. In cases requiring deformity correction, a facetectomy is typically performed as well. The pedicle screws are inserted using a standard technique . HorizonCase illustrationCase 1A patient presented with back pain because of pseudarthrosis after a lumbar fusion using the pedicle screw-rod system and interbody cage Figure . The imaCase 2A patient presented with lumbar coronal deformity (levoscoliosis) Figure , who undCase 3A patient presented with incomplete cauda equina syndrome. Imaging showed L4-5 anterolisthesis with severe canal stenosis Figure . The patCase 4A patient presented with multiple lumbar spine compression fractures because of osteoporosis. The UNIMAX system was used for stabilization and posterolateral fusion  using the UNIMAX system who presented with an L2 vertebral body fracture after a fall. An extension of the fusion to L1 was done by the partial exposure of the cranial aspect of the previous construct and adding hardware for the additional level without removing any previous material Figures -9C.The biomechanical testing showed the system was robust and met the requirements for FDA clearance. For the quasi-static torsion, the mean for the angular displacement, torsional stiffness, and torsional ultimate strength was 2.5 degrees (SD ± 0.8), 5.3 N-m/mm (SD ± 0.7), and 21.6 N-m (SD ± 4.4), respectively  and 5,000,000 (cycles to failure) with no construct failure Table . The conSpinal instrumentation continues to evolve to achieve better surgical outcomes. Spinal fusion procedures are commonly used to treat various spinal pathologies, including degenerative conditions, deformity, trauma, infection, and tumors ,4. The eThe UNIMAX pedicle screw-plate system has unique features that make it a potentially valuable addition to the spine surgeon’s armamentarium. A plating system is inherently more stable than rods, especially in axial rotation . One of A multilevel pedicle screw fixation requires a system that can accommodate different pedicle angles and inter-pedicular distances. The UNIMAX system achieves this by using the interlocking horizontal and vertical plates. The horizontal plates have a ball and socket type configuration to accommodate the varying pedicle screw angles, and the plates are available in different lengths with slots to accommodate any variability in the inter-pedicular/inter screw head distance. Another feature of this system is the ability to add additional vertical plates in a stacked and/or side-by-side configuration to provide additional posterior support to counteract both sagittal bending and torsional forces when needed. This feature can minimize the need for anterior column support, an especially useful advantage when dealing with osteoporosis and bone erosion from infection.The UNIMAX system's versatility allows the correction of sagittal, coronal, or rotational deformity. To correct a sagittal deformity, the use of the extra-long square bolts on the horizontal plate will allow the addition of vertical plates to reduce the anteriorly displaced vertebra into proper alignment. For the correction of coronal or rotational deformity, an adequate posterior facetectomy is performed, allowing the triangular constructs at each level to transform each vertebra into an independent unit that can be brought into alignment by using the vertical plates as a scaffold.The position of the vertical plates in the paramedian plane provides a larger bony surface area in the posterolateral area to contribute to the posterolateral boney fusion as compared to the traditional polyaxial screw-rod construct. This may enable a reduced need for simultaneous anterior interbody fusion or the use of fusion-promoting biologics with their inherent problems. The unique design and biomechanical characteristics of the UNIMAX system are predicted to increase the rate of bony fusion, which is important in all spinal fusion procedures and especially in clinical scenarios when robust fusion is of uttermost importance . Adjacent level disease is a known problem following instrumented spinal fusion. It sometimes requires the extension of a preexisting fusion, which can require the removal of the previously fused bone at multiple levels to allow for the release of the long rods. The UNIMAX system avoids this situation by simply adding sagittal extension plates that anchor the added level to the end of the existing construct.LimitationsDespite the several advantages of the UNIMAX system, it does have several acknowledged limitations. First, the system requires an open posterior approach, which prevents it from being used with minimally invasive techniques. Also, this system in its current design can only be used in the thoracolumbar spine. A formal comparison of the long-term outcomes of the UNIMAX system in comparison to currently used screw-rod systems in a clinical setting is outside the scope of this report but we believe the theoretical advantages shown here warrant further study. Also, the metallic components of the system can affect the radiological evaluation of bony and neural structures (on CT or MRI).The pedicle screw and plate-based UNIMAX system has unique design features that seek to maximize the pullout strength through rigid triangulation at each spinal level. This system also increases the available surface area for posterolateral bony fusion, and it can correct some types of sagittal, coronal, and rotational deformities. It also allows the easy extension of an existing fusion construct to adjacent levels and can be incorporated intuitively into practice. It does require an open posterior approach that makes it incompatible with minimally invasive techniques. This system may have advantages over currently popular screw-rod systems in carefully selected situations requiring extra rigidity and high pull-out strength for complex reconstructions involving infection, tumor, poor bone quality, and revisions. We suggest that it warrants further testing in comparison to existing pedicle screw-rod systems.


Aquaporin-4 (AQP4) is the main water channel protein expressed in the central nervous system (CNS). AQP4 is densely expressed in astrocyte end-feet, and is an important factor in CNS water and potassium homeostasis. Changes in AQP4 activity and expression have been implicated in several CNS disorders, including (but not limited to) epilepsy, edema, stroke, and glioblastoma. For this reason, many studies have been done to understand the various ways in which AQP4 is regulated endogenously, and could be regulated pharmaceutically. In particular, four regulatory methods have been thoroughly studied; regulation of gene expression via microRNAs, regulation of AQP4 channel gating/trafficking via phosphorylation, regulation of water permeability using heavy metal ions, and regulation of water permeability using small molecule inhibitors. A major challenge when studying AQP4 regulation is inter-method variability. A compound or phosphorylation which shows an inhibitory effect in vitro may show no effect in a different in vitro method, or even show an increase in AQP4 expression in vivo. Although a large amount of variability exists between in vitro methods, some microRNAs, heavy metal ions, and two small molecule inhibitors, acetazolamide and TGN-020, have shown promise in the field of AQP4 regulation. While+ , suggest+ ion homeostasis [AQP4 expression and activity in various neurological disorders has been examined, and several intriguing discoveries have been made. In cerebral edema and ischemia, AQP4 expression is increased ,16, and eostasis ,41,42,43eostasis ,16,19, aeostasis ,34,42,43Regulation of AQP4 protein activity starts with regulation of gene expression. A developing field in AQP4 regulation is the application of microRNAs which target AQP4 . MicroRNPhosphorylation is a relatively well-studied post-translational protein modification, which can change the activity or expression of a protein by modulating protein structure, protein–protein interactions, and marking proteins for trafficking ,57. PhosThe subcellular localization of AQP4 can be controlled by phosphorylation at several serine residues in the protein ,59. ProtA similarly complicated area of study is whether or not phosphorylation driven gating exists for AQP4 ,67,68,69Phosphorylation of AQP4 remains a complex topic. While most current knowledge focuses on S111, S180, and S276, there remain many more proposed phosphorylation sites, including S285, S315, S316, S321, and S322 , the fun2+ [2+ on AQP4 is intracellular, meaning that in the case of oocytes and most cell models, Hg2+ cannot reach its direct binding site on AQP4 and can therefore not modulate its activity  enhanced the inhibitory effect, decreasing the concentration needed for 30% inhibition to 20 μM [2 when expressing AQP4 and AQP7 [2+ inhibiting AQP4 via the same mechanism as mercury and zinc, by binding to the C178 residue of AQP4, which is not accessible in oocyte or cell models where Cu2+ is unable to cross the cell membrane [A second metal ion which appears to bind to the same C178 residue is zinc . Experimto 20 μM . In addito 20 μM . This into 20 μM . In the to 20 μM . Prior tand AQP7 . This ismembrane ,82.2+) [2+ significantly increased mAQP4 expression, and this change was eliminated by MAPK inhibitors and reduced by treatment with antioxidants [2+-induced cell death, as astrocytes treated with Fe2+ and control siRNA showed significant levels of cell death in comparison, which suggests that AQP4 plays a key role in Fe2+-induced apoptosis [2+ [2+, it is instead suggested that the increase of mAQP4 and mAQP9 expression is a result of an increased expression and nuclear translocation of Nuclear Factor κB (NF-κB) p65, a regulator of transcription of several genes involved in immune and inflammatory responses [2+ and treatment with either curcumin or NF-κB inhibitors interfered with the NF-κB pathway and inhibited the increase in protein concentration of mAQP4 and mAQP9 [2+ overload.Not all metal ions which regulate AQP4 activity act via direct binding, some act via indirect means, triggering a phosphorylation pathway which leads to changes in AQP4 expression or water permeability. As briefly mentioned above, lead exposure increased water permeability of cultured rat astrocytes by 40% via activating a CaMKII pathway leading to the phosphorylation of S111 , althoug2+) . Treatmeoxidants . Interespoptosis . Experimpoptosis ,88. In tpoptosis . Treatmeosis [2+ . In a moosis [2+ . Via tesesponses , as the nd mAQP9 . It is pIn general, it appears that metal ions which are able to bind directly to AQP4 often cause an inhibitory effect, decreasing the water permeability of the water channel itself ,80,82,8350) of AZA in proteoliposomes was approximately 1.1 mM and max inhibition was 53.3%, which is in stark contrast to the IC50 reported in oocytes of 0.86 μM and max inhibition of 80% [+/+ and mAQP4−/− mice, and a stopped flow assay using mouse erythrocytes [A small molecule inhibitor is a compound which may act as an inhibitor of AQP4 without the inclusion of a metal ion in the compound. The identification of small molecule AQP4 antagonists could be extremely valuable, as AQP4 activity or expression is implicated in several pathological conditions ,36,92,93n of 80% ,97. The n of 80% ,96,97. An of 80% ,95,96) an of 80% . The eighrocytes . These rhrocytes . It is whrocytes . An in shrocytes . Of thesWhile in vitro assessments of some of these compounds have shown conflicting results ,96,97,9850 of 3 μM) that has been evaluated in vivo is 2-(Nicotinamide)-1,3,4-thiadiazole, or TGN-020 [+/+ and mAQP4−/− mice were treated with TGN-020, and live imaging was used to determine the relative cerebral blood flow. TGN-020 induced an increased blood flow in mAQP4+/+ mice, but no increased flow in mAQP4−/− mice, implying that the TGN-020 is acting on mAQP4 [Another compound identified as a potent hAQP4 M23 inhibitor in oocytes (73% max inhibition, IC TGN-020 ,96. In s TGN-020 . TGN-020 TGN-020 . Both mAon mAQP4 . Furtheron mAQP4 . A singlon mAQP4 .While the in vitro screening of small molecule inhibitors remains a controversial field, the increasing number of publications which indicate an effect of some of these molecules in vivo shows that there is a possibility that some of these compounds may actually inhibit AQP4. With that being said, it is entirely possible that treatment of in vivo models with these small molecule inhibitors may cause inhibition or loss of AQP4 expression due to indirect methods, rather than via direct binding, as in vitro methods tend to attempt to detect. Regardless, the potential of a non-toxic AQP4 specific inhibitor in several neurological conditions makes this an intriguing field not only in theoretical research but also in the medical field.Regulation of AQP4 in the central nervous system remains a complicated and intricate topic, with many potential interactions and a large amount of model based variability. AQP4 appears to be regulated via many pathways, from gene silencing/activation to protein modification, and via direct binding of ions or compounds. Recently more and more studies are revealing different aspects of a larger picture in AQP4 regulation, although it is necessary to continue to develop this field of research, as many questions remain unanswered. A clear understanding of AQP4 regulation is required, as AQP4 has an important role both in the normal function and dysfunction of the CNS. The role of AQP4 in several CNS disorders has heightened the importance of identifying a non-toxic AQP4 activity modulator which could be used for therapeutic applications. The main challenges facing development of AQP4 targeting therapeutic agents are selectivity, toxicity, functional inhibition, and delivery into the CNS. Metal ions, while effective, are often too toxic to be used. The conflicting results shown when using small molecule inhibitors showcase the difficulty in identifying an effective activity modulator. Both a phosphorylation based approach and a microRNA based approach show promise, but with such complex pathways, disruption of the status quo could lead to unforeseen consequences. While several antibody based therapies have been developed for application in NMO, only one specifically targets AQP4, Aquaporimab, and this drug has not been tested in humans, nor is it designed to have an effect on water permeability through AQP4. At this point in time, the most promising AQP4 specific activity modulator appears to be TGN-020, as it has demonstrated selectivity, functional inhibition, and successful delivery to the CNS in vivo, though only during studies with one single dose just prior to analysis, so whether or not sustained therapeutic concentration is possible has not been established. Even though a vast amount of information has recently been revealed regarding AQP4, there remains a need to continue to study the regulatory mechanisms of this molecule, and a need to standardize the analysis methods used in order to combat the inter-method variability present in this field.


Rat allergen-specific IgG antibodies sustained upon treatment discontinuation. Sera of rats immunized with BM4 were able to significantly suppress binding of human IgE to the wild-type allergens and CD23-mediated human IgE-facilitated Bet v 1 binding on B cells. By contrast, treatment-induced IgE antibody levels were low or undetectable. In summary, BM4 induced a robust IgG immune response that efficiently blocked human IgE-binding to wild-type allergens, underscoring its potential therapeutic value in AIT.Allergic sensitization to the major allergen Bet v 1 represents the dominating factor inducing a vast variety of allergic symptoms in birch pollen allergic patients worldwide, including the pollen food allergy syndrome. In order to overcome the huge socio-economic burden associated with allergic diseases, allergen-specific immunotherapy (AIT) as a curative strategy to manage the disease was introduced. Still, many hurdles related to this treatment exist making AIT not the patients’ first choice. To improve the current situation, the development of hypoallergen-based drug products has raised attention in the last decade. Herein, we investigated the efficacy of the novel AIT candidate BM4, a hypoallergenic variant of Bet v 1, to induce treatment-relevant cross-reactive Bet v 1-specific IgG antibodies in two different mammals, Wistar rats and New Zealand White rabbits. We further analyzed the cross-reactivity of BM4-induced Wistar rat antibodies with the birch pollen-associated food allergens Mal d 1 and Cor a 1, and the functional capability of the induced antibodies to act as IgE-blocking IgG antibodies. Enzyme-linked immunosorbent assay (ELISA) was used to determine the titers of rat IgG1, IgG2a, IgG2b, and IgE, as well as rabbit IgG and IgE antibodies. To address the functional relevance of the induced IgG antibodies, the capacity of rat sera to suppress binding of human IgE to Bet v 1 was investigated by using an inhibition ELISA and an IgE-facilitated allergen-binding inhibition assay. We found that the treatment with BM4 induced elevated Bet v 1-specific IgG antibody titers in both mammalian species. In Wistar rats, high BM4-specific IgG1, IgG2a, and IgG2b titers (10 Betula verrucosa) represents the major elicitor of tree pollen-associated allergic symptoms in Europe, with sensitization patterns ranging from 54 up to 92% among patients allergic to tree pollen  and Cor a 1 . Although the adverse health effects caused by birch pollen are efficiently treatable by allergen-specific immunotherapy (AIT), the concomitant treatment of PFAS symptoms induced by Bet v 1-associated food sources remains debatable, highlighting strengths, and possibilities for therapeutic improvements  and ameliorated immunogenicity (enhanced T cell-activating capacity and proliferation) –20. BM4 Herein, we show that immunizations of Wistar rats and NZW rabbits with BM4 resulted in high levels of IgG antibodies cross-reactive to wild-type Bet v 1. In Wistar rats, functional Mal d 1- and Cor a 1-cross-reactive IgG antibodies were induced, however, to a lesser extent compared to the Bet v 1. In rats receiving repeated immunizations with BM4, sustained IgG antibody titers remained even upon treatment discontinuation. In addition, BM4-induced IgG antibodies displayed a functional inhibitory activity toward the binding of human IgE to the wild-type pollen allergen (Bet v 1) and the associated food allergens .BM4  was produced recombinantly and endotoxin-free under GMP conditions based on the described protocol . Both BM–1) were recorded at a constant temperature (25°C) for each protein at concentrations of 1.5–2.0 mg/ml using an AquaSpec transmission cell adapted to a Tensor II FTIR system . The data were analyzed using the OPUS spectroscopy software 6.0 . The second derivative of the amide 1 spectra was calculated after vector-normalization (25 smoothing points) using the Savitzky–Golay algorithm.The structural composition of the recombinant proteins was determined using circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy. The CD spectra were recorded at 20°C between 190 and 260 nm using a JASCO J-815 spectropolarimeter . All proteins were diluted in a 10 mM potassium phosphate buffer to a final concentration of 0.1 mg/ml. For the structural analysis using FTIR, the spectra in the range of the amide 1 and amide 2 peaks . Since in human allergen-specific subcutaneous immunotherapy (SCIT) AIT vaccine is administered via the s.c. route of administration, BM4 was administered likewise. Treated animals were daily monitored in a specific pathogen-free (SPF) environment on a frequent basis for 14 days post-injection for clinical signs of test item effects, such as changes in skin and fur, respiration, eyes and mucous membranes, circulation and behavior patterns, and then sacrificed. Blood samples were collected pre- and 14 days post-injection and stored at −20°C until analysis. Sera of the single immunization model were only used for an initial screening of BM4-specific and Bet v 1-cross-reactive antibodies, whereas a further functional characterization of these sera was not pursued.Both species, NZW rabbits  and Wistar rats , received a single subcutaneous (s.c.) injection (hereinafter “single immunization model”) of either 320 μg of BM4, formulated as 320 μg BM4/2 mg aluminum hydroxide/0.9% NaCl/ml, or the respective amount of adjuvant without antigen, termed “placebo” in the following (n = 10 per gender per treatment group) were sacrificed 1 week after the last injection and those in the recovery group (5 per gender per treatment group) 6 weeks after the last injection. The dose level used in the study was based on the doses employed in human AIT protocols.According to the OECD Guidelines for the testing of chemicals no 420, the rat is the preferred species. Therefore, the repeated toxicity study analyzing potential toxic effects of the hypoallergen was only conducted in Wistar rats . Injections containing either 160, 80, 40, or 20 μg of BM4  were administered s.c. on a bi-weekly schedule over a period of 6 months ; termed “repeated immunization model” in the following. This model was conducted in order to provide a immunization schedule relatable to a human AIT protocol. The animals of the main group 17) and the standard operating procedures (SOPs) of UTUCAL. All procedures and protocols were approved by the National Animal Experiment Board of Finland in accordance with the EU Directive 2010/63/EU on the protection of animals used for scientific purposes under the license numbers ESAVI/7217/04.10.03/2012, and ESAVI/8528/04.10.07/2015. UTUCAL animal facilities operate in compliance with the OECD Principles of Good Laboratory Practice (GLP) and have Animal Welfare Assurance by the Office of Laboratory Animal Welfare (OLAW), PHS, NIH. Assurance identification number is A5040-01.® flat-bottom 96 well plates . Serial dilutions of rat sera were incubated with the respective antigen overnight at 4°C. Horseradish peroxidase (HRP)-conjugated mouse anti-rat IgG1, IgG2a, IgG2b , and IgE  antibodies diluted 1:5000 were used as detection antibodies. The SureBlue TMB  Microwell peroxidase substrate  was used for detection. Since NZW rabbits only possess a single IgG subclass, the BM4- and Bet v 1-specific rabbit IgG ELISAs were performed using a HRP-conjugated goat anti-rabbit IgG antibody, Fc Fragment  for detection . Either BM4, Bet v 1, Mal d 1, or Cor a 1 were coated in a concentration of 2 μg/ml diluted in 1× phosphate-buffered saline on Nunc MaxiSorpetection . Plates etection . For the® flat-bottom 96 half-area well plates . After blocking with 0.5% BSA, the immobilized antigen was incubated with rat sera in dilutions of either 1:32 for Bet v 1 or 1:2 for Mal d 1, and Cor a 1 for 2h at room temperature. To exclude any potential interference of IgE antibodies, the rat sera were heat-inactivated at 56°C for 1h prior to the experimental procedure. After the inhibition step, the samples were incubated with different human reference serum pools per antigen , diluted 1:2 for Bet v 1 and Mal d 1, and 1:8 for Cor a 1 (depending on the presence of specific IgE in the serum pools), overnight at 4°C. An alkaline phosphatase-conjugated mouse anti-human IgE antibody , diluted 1:1000, and the alkaline phosphatase substrate p-nitrophenyl phosphate  were used for detection. The absorbance was recorded at 405 nm after 3h of incubation at room temperature. The data were normalized to the uninhibited control (100%). The percentage of inhibitory activity of each individual serum was calculated by inverting the normalized absorbance value  after background subtraction (detection antibody control).For the inhibition ELISA, 2 μg/ml of either Bet v 1, Mal d 1, or Cor a 1 were coated on Nunc MaxiSorpn = 20, recovery group: n = 10). A total of 15 μl of each rat serum was incubated with 10 ng/ml Bet v 1 for 1h at 37°C, followed by the addition of 1 μl of the human serum pool and further incubation for 1h at 37°C. Subsequently, 1 × 105 Epstein–Barr virus (EBV)-transformed B cells expressing CD23, the low IgE-binding receptor , were added to the antibody allergen reaction mix. The cells were stained using a PE-labeled anti-human CD23  and a FITC-labeled anti-human IgE  in the dilutions 1:40 and 1:100, respectively. The experimental conditions, including antigen concentration, amount of human reference serum and the dilution of the FITC-labeled anti-human IgE antibody, were defined prior to the experiment  in a dilution 1:100 was used for live/dead (L/D) discrimination. As positive control, the uninhibited human serum pool  was used for IgE-Bet v 1 complex formation. The cells were analyzed using a Cytoflex S  and FlowJo v 10 . Doublet discrimination was performed by gating the side scatter area (SSC-A) versus the SSC height (SSC-H). Only living EBV-transformed B cells were considered for the data analysis. The detailed gating strategy is shown in We performed inhibition FAB assays to determine the capacity of BM4-induced rat antibodies to prevent the formation of Bet v 1-IgE complexes using a defined human indicator serum pool obtained from six individual birch pollen allergic patients. The indicator serum pool contained high levels of Bet v 1-specific IgE antibodies, as determined by ImmunoCAP assays . For thiperiment . To avoit-test was used to compare pre- and post-immunization sera . Statistics were calculated on transformed data [Y = Log(Y)]. A paired ion sera , 3A–B anion sera , whereasion sera . For comion sera , 4, 6, 7p < 0.0001) of the mean antigen-specific IgG level post-immunization compared to the levels of the placebo control that remained unaltered (p < 0.0001). The mean titers increased 24,721-fold for IgG1, 1,560-fold for IgG2a, and 402-fold for IgG2b . In comparison, the IgG titers of the placebo group, pre- and post-immunization, showed no change. Additionally to the IgG antibody titers, we sought to monitor the antigen-specific IgE levels in order to evaluate possible IgE-mediated side effects caused by the treatment. The treatment did not affect the total IgE level within rabbit sera . In contrast to the single immunization model, the highest doses of BM4 (80 and 160 μg) in the repeated immunization model also caused a slight elevation of BM4-specific IgE antibodies compared to the placebo group . On average, IgG1 and IgG2a levels were still 500 to 1,000-fold higher than IgE. Even the IgG2b titers, which increased less compared to IgG1 and IgG2a, were still 63.4-fold higher than the IgE levels. Interestingly, it appears that there are two types of responses among animals, one with increased IgE and the other with no alterations in the IgE levels.In order to mimic a standard AIT protocol using BM4 and to evaluate its consequences on the humoral immune response, we immunized naïve Wistar rats with BM4 bi-weekly over a period of 6 months . The determined mean IgG2a antibody titers were 17,969, 24,764, 40,608, and 28,456 for 20, 40, 80, and 160 μg BM4, respectively, and, thus, exceeded the placebo group (16.8) on average 1,663-fold. The IgG2b levels induced by the treatment with BM4 were approximately 30-times lower than the corresponding IgG1 and IgG2a titers. The highest antigen-specific mean IgG2b titer compared to placebo (11.6) was observed for the 40 μg BM4  treatment group, followed by 20 μg , 160 μg , and 80 μg . A statistical comparison of the main and the recovery group of BM4-specific antibody titers, as well as of the following Bet v 1-, Mal d 1-, and Cor a 1-cross-reactive antibodies can be found in Regarding the recovery group, the mean of BM4-specific IgE levels of each treatment group dropped below 1.5 resembling the placebo group. Of note, the 40 μg BM4 group was even significantly lower than the placebo group (p < 0.0001). Side-by-side comparison of BM4-specific and Bet v 1-cross-reactive titers of the post-immunization sera revealed that the Bet v 1-cross-reactive IgG1, IgG2a, and IgG2b levels were marginally but significantly lower than the corresponding BM4-specific values . In contrast, no difference was observed in NZW rabbit post-sera (p = 0.0011), 40 (p < 0.0001), and 160 μg BM4 (p = 0.0271), were actually significantly higher than the corresponding BM4-specific titers. In all three investigated IgG subclasses the repeated immunizations with 40 μg BM4 resulted in the highest mean Bet v 1-cross-reactive titers , followed by 20 μg BM4 . The mean titers induced by 80 and 160 μg BM4 were comparatively lower. A similar pattern was observed for IgG1 and IgG2a titers of the recovery group . These food allergens share a sequence similarity of 56.6%  and 67.3% (Cor a 1) with Bet v 1 and exhibit the common PR-10 fold. The structural integrity and similarity of the recombinant proteins used in this study with wild-type Bet v 1 was assessed by determining the secondary structural elements of each protein by CD and FTIR . A high In direct comparison with the induced mean Bet v 1-specific IgG titers, it was apparent that all investigated Cor a 1- as well as Mal d 1-specific IgG subclasses were markedly lower . DependiSummarizing the specificity of the BM4-induced antibodies of the repeated immunization model, we can conclude that the highest IgG antibody levels were induced toward the wild-type Bet v 1 allergen rather than its hypoallergenic variant . IgG antThe induction of blocking antibodies (IgG4/IgG1) is a hallmark of successful AIT, thus, a sole characterization of the antibody pattern as well as of their specificity is not sufficient enough to draw conclusions of the functional relevance of the induced antibodies regarding the IgE-blocking activity of IgG antibodies. Therefore, we decided to investigate to which extent rat sera of the repeated immunization model (20 and 40 μg BM4) containing BM4-induced antibodies are able to compete with human IgE antibodies for binding of wild-type Bet v 1, Mal d 1, and Cor a 1. For this purpose, we used an inhibition ELISA . In rat r = 0.8153, p < 0.0001, and r = 0.6972, p < 0.0001, respectively), whereas the IgG2b titers showed a relatively low correlation with the inhibition of human IgE binding , 30.8% (Cor a 1), 15% , and 66.4% (Bet v 1), 32.3% (Cor a 1), 11.65%  was observable in the main and recovery group, respectively, compared to placebo. For Bet v 1, the inhibition results positively correlated with the respective cross-reactive IgG1 and IgG2a titers (p < 0.0001) compared to placebo in the main as well as in the recovery group  by inverting the percentage of allergen-IgE complexes . An efficient mean inhibitory activity of 95% was found for sera of rats receiving BM4 of the main group (p < 0.0001), and 74%  to 82%  for those of the recovery group , whereas there was a relatively low correlation with the Bet v 1-cross-reactive IgG2b titers  assay protocol by Shamji et al.  was adapry group . A mean ry group . A posit4 to 106) were induced even upon receiving just a single immunization. Since neither Wistar rats nor NZW rabbits possess the IgG4 subclass, we sought to cover the complete IgG repertoire among these species, except for rat IgG2c that is primarily recognizing carbohydrate epitopes .in vivo models. FS provided the formulated GMP drug products for the in vivo experiments and BM4 for the ELISAs. LJ and RV devised the experiments and interpreted the data. All authors read the manuscript. All authors contributed to the article and approved the submitted version.LA devised and performed most experiments, wrote the manuscript, and created the figures. AB and MG conducted the experiments. PC, U-MJ, and EY designed, organized, and performed the FS is an employee of Biomay AG. The remaining 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.


Channa pleurophthalmus Blkr) and identify its antiallergenic properties.The study about the antiallergenic properties of inedible fish body parts is still limited. Therefore, this study aimed to characterize the charcoal from the body parts of Kerandang fish  using a maceration method with different solvents . The identification of active compounds in the extract was carried out using liquid chromatography–high-resolution mass spectrometry (LC-HRMS) analysis, while the antihyaluronidase activity was determined using the antihyaluronidase test. The highest charcoal antihyaluronidase activity-extract was applied to ovalbumin-induced mice for 7 days with various doses . The specific immunoglobulin E (IgE) was measured using enzyme-linked immunosorbent assay on day 8.50) of hyaluronidase was found in the ethyl acetate extract of fish caudal fins at a concentration of 4 mg/mL. We found that 15 mg/kg body weight of charcoal of fish caudal fins suppressed IgE expression in male mice.Our LC-HRMS analysis showed that the active compound of charcoal in the caudal fins of Kerandang fish was hexadecanamide. The highest inhibition (ICOur findings indicate that the charcoal of non-edible body parts of Kerandang and one of its constituent, hexadecanamide, may have strong antiallergic effects. Many natural resources in Indonesia that are used in traditional medicine are now being studied for their potential application to the development of modern public health. However, despite the advancements of modern medicine and technology, traditional medicine remains a key component of public health in Indonesia, one that the government is still actively promoting . The useAllergens can be in the form of dust particles, plant dust, drugs, or food. The allergic mechanism is controlled by mast cells: When exposed to the allergen, they release the IgE antibody. IgE is also produced in large quantities when allergens attach to B lymphocyte cells ,8. The rThe Kerandang fish has recently gained significant attention in Central Kalimantan, as charcoal can be made from several of the inedible body parts of the fish, then used as a traditional medicine to treat hereditary allergic reactions. The body parts of the fish are burned into charcoal, and then smeared on itchy skin or on the bumps that arise on the skin after an allergic reaction. Many fish that inhabit peatlands possess various bioactive materials, which can be used for several medicinal purposes .Channa pleurophthalmus Blkr) and identify its antiallergenic properties.Thus far, the antiallergenic properties of charcoal from the Kerandang fish have not been studied. In this study, we attempt to characterize charcoal from the body parts of Kerandang fish .This study was conducted from September 2018 to September 2019. We obtained inedible parts of Kerandang fish, including the scalp, scales, dorsal fins, pectoral fins, ventral fins, anal fins, and caudal fins, from fishermen in Sebangau Kereng Bengkirai Lake, Central Kalimantan, Indonesia.The fish samples were cleaned and dried for 2-3 days, then burned to charcoal using a normal oven with a maximum temperature of 200°C, until the resulting charcoal attained a mass of 500 g per sample. The extraction was done at a temperature of <4°C, using ethanol, ethyl acetate, and chloroform solvent, under the following protocol: 100 g of dry samples were added 500 mL of solvent, incubated for 24 h, and filtered through a vacuum filter. The filtrate was dried using a vacuum rotary evaporator. The crude extract was stored until further analysis .Each extract was diluted in a solvent until reaching a volume of 1300 μL. All extracts were spun for 2 min and filtered using a 0.22 μm syringe filter. The samples were then inserted into the autosampler and injected into the LC-HRMS. The data were converted into a NetCDF format to ease data processing using mzCloud™ . MzCloud data processing consists of several steps, i.e., creating a chromatogram, reducing noise, identification based on molecular weight, and compiling data .2+ was reacted with 250 μL sodium hyaluronate (1.2 mg/mL) dissolved in 0.1 M acetate buffer (pH 3.5), then incubated at 37°C for 40 min. Next, 100 μL of 0.4 M sodium hydroxide and 100 μL of 0.4 M potassium borate were added to the reaction mixture and incubated in a bath of boiling water for 3 min. After cooling at room temperature, 1.5 μL of dimethylaminobenzaldehyde (DMAB) (4 g DMAB dissolved in 350 μL of 100% acetic acid and 50 μL of 10 M hydrochloric acid) was added to the reaction mixture, then incubated at 37°C for 20 min. Optical density (OD) in the reaction mixture was measured using a spectrophotometer at 585 nm. The percentage of inhibition was calculated using the following equation:The antihyaluronidase activity was examined with a slight modification from the original protocol ,19. The % Inhibitors = [(ODc − ODs) / ODc] × 10050 values causing 50% inhibition were determined using a linear regression analysis.Note: ODc is the OD of the control, ODs is the OD of the sample being tested. ICThe highest charcoal antihyaluronidase activity was applied topically as a cream to ovalbumin (OVA)-induced allergy mouse for 7 days at three different treatment doses ; hydrocortisone cream was used as a positive control. The specific IgE was measured on day 8 after the application by collecting the blood serum using the Mouse OVA sIgE enzyme-linked immunosorbent assay marker kit.Data from the antihyaluronidase test and specific IgE levels were analyzed using a one-way analysis of variance using SPSS 23.0. LC-HRMS qualitative analyzed according to database mzCloud and compared with PubChem, Lipinski, and SwissADME bioinformatic method.50 value for the seven types of charcoal tested with three different solvents . Data of IC50 of hyaluronidase activity were obtained from a linear regression analysis. IC50 values represent the extract concentration of samples needed to inhibit 50% of hyaluronidase activity, which determined by linear regression analysis [We measure the inhibition of hyaluronidase using charcoal extracts from inedible parts of the Kerandang fish body . The higanalysis . The higanalysis -22. Thisanalysis -25. A pranalysis ,27.The IgE levels in male mice were increased after sensitization OVA , which iThe average level of IgE in control mice was 404.63±4.57 ng/mL . In the Histamine plays a key role in the pathogenesis of allergies through the regulation of differentiation of CD4+ Th-cell lymphocytes . The mecTopical nasal AHs such as azelastine are also available and recommended forrunny nose due to allergic effects. To increase the efficacy of oral AHs in allergic rhinitis for those who continue to exhibit symptoms, the preferred topical therapy is a corticosteroid nasal spray. These sprays should be considered a first-line treatment in moderate to severe allergic rhinitis ,34.Treatment for allergic reactions takes several forms: One can increase IgE levels so incoming antigens can be destroyed through the complement system; others provide the patients with AHs, reduce IgE levels to inhibit the binding between antigens with IgE, prevent the entry of antigens into the body, or again inhibit mastocyte degranulation to prevent the release of chemical mediators, which stimulate the allergic reaction -37. In o16H33NO [3mol−1 [The identification of charcoal ethyl acetate extract from Kerandang fish caudal fins using LC-HRMS is shown in 16H33NO . Accordi [3mol−1 . Compari [3mol−1 -43. BaseOur findings indicate that the charcoal of non-edible body parts of Kerandang and one of its constituent, hexadecanamide, may have strong antiallergic effects. More methodological work is needed on how to determine the effect inedible Kerandang fish body part on other parameter related to skin allergy.AA designed the research and wrote the manuscript. ES helped in experimental method. BBS collected and analyzed the data and HH revised the manuscript. All authors read and approved the final manuscript.


This nanogenerator could be used to convert the ambient mechanical vibrations into electrical energy and supply low-power consumption devices.Piezoelectric nanogenerators can convert energy from ambient vibrations into electrical energy. In the future, these nanogenerators could substitute conventional electrochemical batteries to supply electrical energy to consumer electronics. The optimal design of nanogenerators is fundamental in order to achieve their best electromechanical behavior. We present the analytical electromechanical modeling of a vibration-based piezoelectric nanogenerator composed of a double-clamped beam with five multilayered cross-sections. This nanogenerator design has a central seismic mass (910 μm thickness) and substrate (125 μm thickness) of polyethylene terephthalate (PET) as well as a zinc oxide film (100 nm thickness) at the bottom of each end. The zinc oxide (ZnO) films have two aluminum electrodes (100 nm thickness) through which the generated electrical energy is extracted. The analytical electromechanical modeling is based on the Rayleigh method, Euler–Bernoulli beam theory and Macaulay method. In addition, finite element method (FEM) models are developed to estimate the electromechanical behavior of the nanogenerator. These FEM models consider air damping at atmospheric pressure and optimum load resistance. The analytical modeling results agree well with respect to those of FEM models. For applications under accelerations in  The world population growth and technological development have increased the energy consumption through electronic components and devices. For instance, the world population will use approximately 25 billion devices in the following years . These dy-direction of 2.50 m/s2, and an optimal load resistance of 32,458 Ω. An array of these nanogenerators with diode-bridge rectifiers can be used to charge capacitors to DC voltage. This electrical charge could supply low-power consumption electronic devices.Commonly, the piezoelectric micro and nanogenerators have been investigated to convert kinetic energy from vibrations or mechanical motions into electrical energy ,32,33,34This work is organized as follows: In this section, we present the modeling to estimate the electromechanical behavior of the piezoelectric nanogenerator. In addition, the quality factor and air damping at atmospheric pressure of the nanogenerator are obtained.A piezoelectric nanogenerator  is desigmth, nth, pth, qth and rth layers, which have symmetry on the x-y plane. jth cross-section of the nanogenerator. In order to simplify the analytical model, the layers of the nanogenerator are assumed as homogeneous and isotropic.The Rayleigh method ,48 is usSja) of the jth section of the piezoelectric nanogenerator can be calculated as [iSjE is Young’s modulus of the ith layer in the jth section, hi−1)Sj( is the distance from bottom side of the first layer to the top side of the (i − 1)th layer of the jth section, hiSj is the distance from the bottom side of the first layer to the top face of the ith layer of the jth section, iSjb and iSjt = iSjh − hi−1)Sj and SjA is the area of the jth section.The elastic centroid (lated as :(1)aSj=(zEI)Sj as [The elastic centroid of each section is used to calculate its bending stiffness (Iz)Sj as :(2)(EIz)mK) and potential (mP) energies of the five sections are determined as:L12 = L1 + L2, L123 = L1 + L2 + L3, L1234 = L1 + L2 + L3 + L4 and L12345 = L1 + L2 + L3 + L4 + L5. Furthermore, Sjy is the static deflection of the jth section and iSjρ is the density of the ith layer in the jth section.Based on the Rayleigh method, the maximum kinetic (mP = mK), we obtain the resonance frequency as:Applying the conservation of energy (Sjy) of the five sections of nanogenerator. We consider an initial deflection of the nanogenerator equal to zero. After, the deflections of the nanogenerator can be calculated using the Euler–Bernoulli beam theory [SjM is the bending moment of the jth section.The Equations (3) and (4) need the values of deflections (m theory :(6)(EIz)The boundary conditions for each one of the sections of the nanogenerator are given by:F (x)) and applying the Macaulay method [The bending moment is determined by integrating twice the load function (y method .(16)F(xV(x) is obtained by integrating Equation (16):Considering the integration rules of the Macaulay functions, the shear load function M(x) is calculated by integrating the Equation (17):Next, bending moment function C1 = 0 and C2 = 0) of Equations (17) and (18) are defined with the boundary conditions V(0) = R0 and M(0) = M0.The integration constants (y-z plane (M0 = M1 and R0 = R1). Moreover, the weight per unit length of sections 1 and 2 are equal to those of sections 5 and 4 (Sω1 = Sω5 and Sω2 = Sω4), respectively. To determine the reaction force (R0) on a clamped support and the weight per unit length (Sjω) of all the sections of the nanogenerator, we use the following Equations:g is the gravitational acceleration.Due to the symmetry of the nanogenerator with respect to -z plane , their bThe bending moments of the five multilayered sections of the nanogenerator are obtained through Equation (21):x < L1For 0 < L1 < x < L12For L12 < x < L123For L123< x < L1234For L1234< x < L12345For Sjy) in each section of the nanogenerator, Equations (21)–(25) are substituted into Equations (6)–(10)and integrated using Macaulay’s function integration rules [To obtain the static deflections  of the nanogenerator is calculated using the following boundary condition of deflection y5(x) in the right clamped support:The bending moment on the left support (The first bending resonant frequency of the nanogenerator is obtained by substituting Equations (3) and (4) into Equation (5). FEM models are developed to predict the first resonant frequencies and vibration modes  of the nanogenerator. The deflection and mechanical stress of the nanogenerator are obtained through static structural analysis with the FEM models. Finally, the dynamic deflection, normal stress and output power are estimated by means of a harmonic response analysis. In the modal analysis, the first four vibration modes and resonant frequencies of the nanogenerator are obtained. aQ is the quality factor of the nanogenerator and it can be estimated using [b, h, and ρ are the width, thickness, and density of the double-clamped beam, respectively, µ0 and ρ0 are the viscosity and density of air, respectively, and rf is the resonant frequency of the double-clamped beam.The piezoelectric nanogenerator is designed to operate at atmospheric pressure and its damping ratio is given by:ed using ,54,55: is calculated by [pC is the capacitance of the ZnO layer.Based on Equation (33), the quality factor and damping ratio of the nanogenerator have values of 99.98 and 5 × 10lated by :(35)RoptV is the generate voltage.The optimal resistance is required to determine the maximum power generated by the nanogenerator. This optimal resistance is defined as :(37)P=V2This section presents the results and discussion of the electromechanical performance of the piezoelectric nanogenerator determined by the analytical and FEM models.y-axis. First, we determined the maximum deflections of the nanogenerator due to the static loads. An initial deflection of the nanogenerator equal to zero is assumed. For this, both analytical and FEM models considered the acceleration of Earth’s gravity along the y-axis on the office desk of 0.0879 m/s2 [−3 W/m3. x, y and z of the nanogenerator. The normal stress in the x-direction registers the maximum value of 14.31 MPa when the nanogenerator operates at resonance. This normal stress does not exceed the yield stress of ZnO (412 MPa) and PET (54.5 MPa) [Next, a harmonic response analysis of the FEM model of the nanogenerator is studied considering an acceleration of vibration along the 879 m/s2 . Figure 4.5 MPa) ,58.y-directions, four magnitudes of acceleration  along y-axis are considered , PET (54.5 MPa) and aluminum (280 MPa) layers. The maximum normal stresses occur on the ZnO layer close to the ends of the double-clamped beam. x, y and z-directions of the ZnO layer. However, the maximum normal stress in x-direction of the ZnO layer is close to its yield stress. To avoid the increase in this maximum normal stress, we propone an over range protection of the maximum deflection of the nanogenerator (see y-direction of the nanogenerator to 448 μm. These two rigid structures are composed by two double-clamped beams, in which the upper beam will have a gap of 448 μm with respect to the outer surface of the seismic mass. On the other hand, the lower beam will have a gap of 448 μm with respect to the bottom surface of the PET. Thus, the maximum deflection of the nanogenerator is limited to 448 μm, although the accelerations in y-direction overcome 2.5 m/s2. This can constrain the maximum normal stress of the ZnO layer of the nanogenerator, keeping its operation safe.In order to predict the output power of the nanogenerator under different acceleration amplitudes in nsidered . Figure ator see . This ovThe nanogenerator can use diode-bridge rectifiers to transform the alternating current to direct current. Thus, one array of the designed nanogenerators can include diode-bridge rectifiers to charge capacitors to DC voltage. Finally, this electrical charge could be applied to low-power consumption electronic devices. Moreover, the designers can enhance the nanogenerator behavior for different applications through the modification of its geometrical dimensions and materials. Additionally, the output voltages and power of nanogenerators can be increased when they operate at resonance with similar frequencies to those of ambient vibrations or mechanical motions. Therefore, the magnitudes of main design parameters of the nanogenerators can be adjusted by employing electromechanical models to increase their output voltage and power.Analytical models to predict the first bending resonant frequency and deflections of a piezoelectric nanogenerator with multilayered cross-sections were presented. This nanogenerator was formed by a double-clamped beam composed of five sections with different layers. The nanogenerator was designed with a flexible PET substrate, ZnO layer, aluminum electrodes, and a seismic mass. The mechanical behavior of the nanogenerator was estimated using analytical models based on the Rayleigh method, the Euler–Bernoulli beam theory and the Macaulay method. Additionally, FEM models of the nanogenerator were used to predict its electromechanical behavior. The results of the mechanical behavior of the nanogenerator determined by the analytical models agreed well with respect to those of the FEM models. The first bending resonant frequency of the nanogenerator calculated with the analytical model had a relative difference of −8.07% in comparison to the FEM model. The nanogenerator can be used to convert the ambient mechanical vibrations under different accelerations into electrical energy. This electrical energy can be used to supply low-power consumption electronic devices. In addition, several nanogenerators can be used with diode-bridge rectifiers to charge capacitors to DC voltage. Furthermore, an over range protection of the maximum deflection of the nanogenerator was proposed to keep its safe operation during high accelerations.Future research work will include the fabrication and characterization of piezoelectric nanogenerators and their implementation in potential applications.


A set of collocated, in situ oceanographic and glaciological measurements from Petermann Gletscher Ice Shelf, Greenland, provides insights into the dynamics of under‐ice flow driving basal melting. At a site 16 km seaward of the grounding line within a longitudinal basal channel, two conductivity‐temperature (CT) sensors beneath the ice base and a phase‐sensitive radar on the ice surface were used to monitor the coupled ice shelf‐ocean system. A 6 month time series spanning 23 August 2015 to 12 February 2016 exhibited two distinct periods of ice‐ocean interactions. Between August and December, radar‐derived basal melt rates featured fortnightly peaks of  Concurrent measurements of melt rate and hydrography under Petermann Gletscher Ice Shelf reveal dynamics of ice shelf meltingMaximum melt rates occur during periods of elevated buoyant under‐ice flow rather than when ocean temperatures are greatestInitiation of buoyant flow is modulated at the grounding line by the spring‐neap tidal cycle and seasonality in subglacial runoff The mooring line freezes into the ice shelf, which prevents recovery of these instruments. Data acquisition hence relies on communications cables running from the ocean, through the ice shelf, to a data logger on the surface. This approach has been successfully used to collect multiyear ocean time series beneath various Antarctic ice shelves . We use Equation QOT: QOT on In Equation QIT: pi=2,009 J kgκiT=1.14×10−6 mδTiδzB using Equations 23, 26, and 31 of Holland and Jenkins u∗2U reduces the unknown parameter for these events to CD1/2ΓT, an expression referred to as the Thermal Stanton number. We use a range of Thermal Stanton numbers to compute melt rates during the six melt rate peaks and select an initial value that produces calculated melt rates closest to our measurements. Differences between calculated and measured melt rates are then attributed to variations in U. We address these variations by considering the Thermal Stanton number along with the instantaneous measured melt rate at the arrival time of the six ocean meltwater pulses, then choosing the Thermal Stanton number that produces a median U equal to the 0.23 m sD1/2ΓS of D1/2ΓT = 0.0011, CD1/2ΓS=3.1×10−5 ; however, the boundary layer was strongly stratified, with calculated interfacial salinities between 26 and 27 g kgTime series of basal melt rates and ocean mixed layer properties depict two distinct periods of ice‐ocean interactions during the 6 months of data Figure . The fir1 Figure , which vm Figure , when th Figures –4e. DuriΘ−ΘB=0.25°C and SA=33.50 g kgAs the tidal range declined toward neap conditions, large melt peaks of  Figures  and 4d,  Figures  analyzedΘ−ΘB=0.10 to 0.80°C, SA=33.20 to 34.20 g kgΘ−ΘB=1.00°C, SA=34.50 g kgAfter the final meltwater pulse, melt rates reduced to The above estimates of current speed and interfacial temperature and salinity hinge on the choice of constant Thermal and Haline Stanton numbers for the full data record. While the lack of measured current speeds and upper ocean hydrographic vertical resolution necessitated this assumption, the implementation of constant turbulent heat and salt transfer coefficients during variable melt and mixed layer conditions could have led to inconsistencies in the estimated variables. Stanton numbers are likely most accurate during the melt rate peaks at the arrival time of meltwater pulses, as these events were used to tune the boundary layer parameterization equations, but the efficiency of turbulent mixing of heat and salt across the boundary layer varies with the degree of upper ocean stratification. In periods of high thermal forcing and low melting such as Period 1 background conditions, increased stratification would potentially reduce the efficiency of turbulent mixing. This would lower the Stanton numbers relative to those estimated here, which would require a combination of higher current speeds and higher interfacial salinities (weaker stratification) to drive the observed melt rates. This could indicate that our estimated currents and interfacial salinities are biased low and our interfacial temperatures are biased high during this section of the record. In contrast, in periods of colder and fresher ocean conditions and low melting such as during Period 2 spring tides, decreased stratification would potentially increase the efficiency of turbulent mixing. This would raise the Stanton numbers relative to those estimated here, which would require a combination of lower current speeds and higher interfacial salinities (weaker stratification) to drive the observed melt rates. This could indicate that our estimated currents and interfacial temperatures are biased high and our interfacial salinities are biased low during this section of the record.K1=1.01 cpd, O1=0.93 cpd) and semidiurnal  tidal frequencies. While these frequencies dominate variability during Period 2, melt rate spectra in Period 1 register comparable diurnal levels and higher semidiurnal levels than Period 2. However, elevated Period 1 background melt rate variance masks these signals, which suggests different regimes between periods. The ocean spectra corroborate this, as diurnal and semidiurnal levels are much lower during Period 1 than Period 2.Autopower spectra of the data from these two periods reveal the distribution of variability at discrete time scales in the ice shelf basal melt rate, ocean mixed layer hydrographic properties, and estimated current speeds Figure . When coCross‐power spectra confirm that the basal melt rate did indeed covary with the mixed layer hydrographic properties Figure , as spec55.1The lack of semidiurnal and diurnal tidal variations in estimated ocean currents during Period 1 suggests that some other component dominated flow beneath the ice shelf during this time. The higher speeds in Period 1 compared with Period 2 further demonstrate that this other component contributed significantly to under‐ice currents and the resultant ocean‐forced melt Figure . Washam In each event, melt rates Figure  began toThis pattern of buoyancy‐enhanced melt held true for five of the six meltwater pulses; however, the sixth pulse exhibited a different structure, as thermal forcing was already low, meltwater concentrations were high, and the peak current speed preceded the peak melt rate. This event marked a maximum current speed of 0.40 m sΘ−ΘB=0.80 to 1.00°C, SA=34.20 to 34.60 g kgWasham et al.  noted th Figures  –4e. Eve Figures , and 7.In Period 2 the ocean mixed layer oscillations at our study site are diurnal Figures  even tho2πf  prevent linear Rossby wave theory from applying. Ignoring this limitation, we estimate the along‐slope phase velocity of a (short) diurnal topographic Rossby wave in a two‐layer ocean with an upper layer 5.2We interpret the behavior of estimated currents in Period 1 as a persistent, seaward flowing meltwater plume that periodically accelerated when a large pulse of SR discharged across the grounding line. This interpretation is supported by phase‐lagged cross correlations over this time period between three mooring sites, which showed that ocean mixed layer signals occurred first near the grounding line, then at the site of interest here, and finally at a location further seaward  along the upper section of the ice shelf basal channel between our site and the grounding line by dividing The impact of these swift meltwater pulses on ocean‐driven melting is clear in the e Figure ; howeverBulk upstream melt rates using this method Figure  vary conn Figure  instead n Figure . This isWe estimate that the total upstream portion of the channel melted by The coupled relationship between SR flux rates and bulk melt rates (from GMW fluxes) indicates that the discharge of SR across the grounding line placed a first order control on the strength of channelized melting downstream. Previous modeling studies derived an empirical relationship to relate the maximum melt rate at a tidewater glacier  melts at a location in its central basal channel. High melt rates occurred periodically at our study site over the 3 months following the 2015 summer season, but ended afterward. These high melt rates aligned with estimates of swift under‐ice currents that resulted from the discharge of buoyant subglacial runoff across the glacier's grounding line into the ocean. The flow initiated by this freshwater source efficiently mixed the relatively warm and saline seawater in the underlying ocean mixed layer across the stratified boundary layer, providing a source of heat to melt the ice base. When placed into the larger context of atmospheric warming in northern Greenland (Orsi et al.,


Significance: Infrared (IR) inhibition can selectively block peripheral sensory nerve fibers, a potential treatment for autonomic-dysfunction-related diseases . Lowering the IR inhibition threshold can increase its translational potentials.Aim: Infrared induces inhibition by enhancing potassium channel activation. We hypothesized that the IR dose threshold could be reduced by combining it with isotonic ion replacement.Approach: We tested the IR inhibition threshold on the pleural-abdominal connective of Aplysia californica. Using a customized chamber system, the IR inhibition was applied either in normal saline or in isotonic ion-replaced saline, which could be high glucose saline, high choline saline, or high glucose/high choline saline. Each modified saline was at a subthreshold concentration for inhibiting neural conduction.Results: We showed that isotonically replacing ions in saline with glucose and/or choline can reduce the IR threshold and temperature threshold of neural inhibition. Furthermore, the size selectivity of IR inhibition was preserved when combined with high glucose/high choline saline.Conclusions: The present work of IR inhibition combined with isotonic ion replacement will guide further development of a more effective size-selective IR inhibition modality for future research and translational applications. The ner2.2.2Aplysia saline , allowing the simultaneous application of both inhibitory modalities . The sol1.Aplysia saline, in which all the ion concentrations were diluted to 80% of the level in normal Aplysia saline. The elevated glucose concentration . In this modified saline, concentrations of the added glucose and choline were cut in half  to measure the temperature distribution of an approximated midplane solution as previously reported.2.4Aplysia saline. Control tests to generate CAPs in normal Aplysia saline before and after all the inhibition tests were conducted to assess the nerve’s health. More specifically, in each 105-second experimental trial, three 10-second applications of IR light were applied at the 10th, 45th, and 80th seconds of the trial. The IR power was carefully ramped up across trials to identify the IR threshold for inhibition.We used separate groups of nerves to test the hypothesis that the IR inhibition threshold can be lowered by combining each type of isotonic ion-replaced saline. Using high-glucose saline as an example, here are the testing steps: (1) verify that high-glucose saline could not induce inhibition; (2) identify the IR threshold  of the CAPs to verify that the IR thresholds under different testing solutions correspond to the same inhibition level. Since heat accumulates during the first 6 seconds, only the CAPs from the 7th to 10th second of each IR application were analyzed. For the initial and final trials without IR application, CAPs at the identical time points were used, which helped take account of the very gradual rundown of the CAP due to electrical stimulation over time. To make a comparison of inhibition strength across nerve samples, the average RAUC of each nerve’s response at the designated time points was normalized to the average RAUC of the first 10 CAPs of its first normal saline control test.For each group of nerves tested with given modified saline, we compared the IR thresholds in the modified saline versus in normal saline, once the normalized RAUC response confirmed the consistency of IR inhibition strength. A one-tailed We conducted a repeated measures of analysis of variance (ANOVA) test on those percentage changes of IR threshold under different types of modified saline to explore if there was a preferable method. For the quantification and statistical analysis of size selectivity, we used the same method as previously described.3Aplysia saline was higher [All three types of subthreshold ion-replaced saline were able to lower the IR inhibition threshold. For example, the CAPs . In contthe CAPs . The radThe RAUC result confirmed that IR combined with high-glucose saline achieved the same inhibitory effect as IR alone, but required lower IR levels. A paired hibition  did not hibition . The RAUhibition  did not Aplysia saline [Aplysia saline [Infrared radiant exposure thresholds for inhibition were significantly lower  the stability of the isotonic ion-replaced saline’s reduction effect on the IR inhibition threshold over the repeated test; (2) multiple ion-replacement compounds, in combination, could be used at concentrations that were suitable for potential translational applications; (3) the ratio between the inhibition threshold and the damage threshold in normal saline versus in isotonic ion-replaced saline.The present work is an acute study of IR inhibition combined with isotonic ion replacement. To accurately find the IR threshold for each condition, six tests were performed see , which tInfrared neuromodulation has been combined with other modalities to lower the IR threshold. For example, Duke et al.


Physical exercise elicits physiological metabolic perturbations such as energetic and oxidative stress; however, a diverse range of cellular processes are stimulated in response to combat these challenges and maintain cellular energy homeostasis. AMP-activated protein kinase (AMPK) is a highly conserved enzyme that acts as a metabolic fuel sensor and is central to this adaptive response to exercise. The complexity of AMPK’s role in modulating a range of cellular signalling cascades is well documented, yet aside from its well-characterised regulation by activation loop phosphorylation, AMPK is further subject to a multitude of additional regulatory stimuli. Therefore, in this review we comprehensively outline current knowledge around the post-translational modifications of AMPK, including novel phosphorylation sites, as well as underappreciated roles for ubiquitination, sumoylation, acetylation, methylation and oxidation. We provide insight into the physiological ramifications of these AMPK modifications, which not only affect its activity, but also subcellular localisation, nutrient interactions and protein stability. Lastly, we highlight the current knowledge gaps in this area of AMPK research and provide perspectives on how the field can apply greater rigour to the characterisation of novel AMPK regulatory modifications. The net result of repeated exercise-induced fluctuations in these pathways is an increase in the steady-state abundance of requisite signalling proteins  and energy availability  to better combat future metabolic disturbances . Ul. Ul2-terIn a physiological context, AMPK has a well-known ability to stimulate cellular glucose uptake. Although AMPK was originally thought to be responsible for augmenting insulin-independent glucose uptake into skeletal muscle during exercise, there is mounting evidence suggesting this is not the case and it instead plays a more pivotal role in post-exercise glucose uptake and subsequent glycogen resynthesis ,19,20. TAt the molecular level, AMPK is subject to a plethora of dynamic PTM events as expected of a major nutrient-sensing hub . FunctioPhosphorylation events on AMPK with known function are summarised in α1-T174/α2-T172 (henceforth α-T172), which align with the activation loop sites on numerous other typical eukaryotic protein kinases. α-T172 was discovered to be phosphorylated over 20 years ago and was shown to lead to >50-fold activation of AMPK [2-terminally myristoylated, while AMP and ADP binding can also protect α-T172 from phosphatase dephosphorylation independent of myristoylation status [in vitro, and was required for the phenformin-  and 5-aminoimadazole-4-carboxamide-1-β-D-ribofuranoside-  induced phosphorylation of α-T172 in multiple mammalian cell lines [2+ ionophores stimulated α-T172 phosphorylation in an LKB1-null background, an effect abolished by the CaMKK inhibitor STO-609 [The α-subunit kinase domain possesses an archetypal kinase domain structure consisting of two lobes, a small N-lobe and larger C-lobe. The C-lobe contains a series of essential residues collectively termed the activation loop . Phosphorylation of protein kinase activation loops is critically important for their activity, as the negatively charged phosphate group coordinates both the active site and substrate binding groove into a catalytically-favourable conformation . In AMPK of AMPK . Phosphon status ,65,66. Tll lines ,32,67. Ill lines ,69, has ll lines . Currentll lines ,71. The ll lines  in cell- STO-609 ,34,72. s STO-609 ,35,36.α1-S175/α2-S173, adjacent to α-T172, can be phosphorylated by protein kinase A (PKA) to inhibit α-T172 phosphorylation and reduce AMPK activity [Other kinases have been proposed to activate full-length AMPK in vitro by α-T172 phosphorylation, such as transforming growth factor-β-activated kinase (TAK1) ,36. Overactivity . This meactivity . Two subactivity  and insuactivity , and thiactivity .2+/Mn2+-dependent protein phosphatase 1E (PPM1E) [Inactivation of AMPK is an equally important and tightly controlled aspect of its regulation. This mechanism involves the dephosphorylation of α-T172 by the protein phosphatases; protein phosphatase 2A (PP2A), protein phosphatase 2C (PP2C) and Mg (PPM1E) ,78. Prot (PPM1E) . Whether (PPM1E) ,79,80. F (PPM1E) , while s (PPM1E) ,82. Thisα1-S347/α2-S345 (henceforth α-S345), which is phosphorylated in C2C12 myotubes, mouse liver and human vastus lateralis muscle [Immediately following the α-subunit kinase domain lies a tri-helical autoinhibitory domain  ,84 and ts muscle ,41, and s muscle ,87,88. Cs muscle . We reces muscle  and TSC2s muscle . As AMPKs muscle , an evens muscle  and to as muscle . Howevers muscle , a substs muscle , suggests muscle  expressis muscle  recentlys muscle . An α-sus muscle . Since ps muscle  to in tuα1-Y432/α2-Y436 (hereafter α-Y436) that was found to be phosphorylated in skeletal muscle by Fyn, a member of the Src family of protooncogene non-receptor tyrosine kinases with key roles in inflammation [At the extreme COOH-terminus of the α-subunit lies a β-subunit interacting domain  that is crucial for stabilising the entire AMPK complex . The majammation . TNFα cyammation , an effeα1-T481/α2-T485 (hereafter α-T481), which was shown to be phosphorylated in cellulo and in cell-free assays by glycogen synthase kinase 3 (GSK3)α and GSK3β [α1-S477 and α1-T473 to create a phosphorylation cluster dependent on prior “priming” phosphorylation on α-T481, characteristic of many GSK3 substrates [α1-S487/α2-T491 (hereafter α-S487), however this was not a typical GSK3 priming event, as the GSK3β R96A mutant which blocks GSK3 phosphorylation of primed but not non-primed substrates, is still capable of inducing α-T481 phosphorylation [At the start of the ST loop is nd GSK3β , while αnd GSK3β . Phosphobstrates . Howeverrylation ,101. InsPhosphorylation of α-S487 was first reported in 2003 on bacterially expressed α1β1γ1, however this site was originally labelled α1-S485/α2-S491 as the α1 sequence used was a noncanonical rat sequence missing α1-T3 and α1-A4 . α1β1γ1 Some studies have suggested that α-S487 is an autophosphorylation site, as bacterially expressed kinase active α1β1γ1 produced a α1-pS487 immunoblot signal that was abolished with substitution to Ala . This is2-terminal sequence.  that is highly divergent between β1 and β2 isoforms [S24 and S25 were originally shown to be phosphorylated in AMPK purified from rat liver in a mutually exclusive manner, with β1-S24 the most abundant [The β-subunit contains an unstructured NHisoforms . Within abundant . Phosphoabundant , and whiabundant  it is noabundant . Howeverβ1/2-S108, with β1-S108 identified in AMPK purified from rat liver as an autophosphorylation site. β2-S108 may also be an autophosphorylated residue [cis [Immediately following the NTD is a conserved carbohydrate binding module , that forms an interface with the N-lobe of the α kinase domain creating a hydrophobic cleft termed the allosteric drug and metabolite (ADaM) site . The ADa residue ,57,62. βdue [cis , whereasdue [cis ,56, wherdue [cis . Additiodue [cis .β-T148 lies within the carbohydrate binding pocket of the CBM, which was shown to be autophosphorylated on both α1β1γ1 and α1β2γ1 purified heterotrimer complexes, as well as in HEK293T and HepG2 cells [The more traditional function of the CBM is to target AMPK to glycogen, an interaction we recently showed is critical for glucose handling and maximal exercise capacity . The obsG2 cells . This waβ1-S182/β2-S184 (hereafter β-S182), where β1-S182 was originally shown to be stoichiometrically phosphorylated in AMPK purified from rat liver [Located at the extreme COOH-terminus of the β-subunit is a highly conserved α-γ-subunit binding sequence  that forms a three-stranded antiparallel β-sheet, where the last β-strand forms an inter-subunit β-sheet with a β-strand from the γ-subunit. The α-γ-SBS is absolutely required for AMPK complex formation . Locatedat liver , while βat liver . Early fat liver . Howeverγ1-S192 which is located on the linker between CBS2 and CBS3, and γ1-T284 which is located in CBS4 [32P-incorporated autoradiogram signal in the isolated γ1-subunit. These phosphorylation events reportedly work in concert to promote lysosomal localisation, which ultimately increases LKB1-mediated α-T172 phosphorylation, without having any effect on AMP stimulation. However, further validation of direct phosphorylation of the γ1-subunit is required using heterotrimeric AMPK, while it is unknown if the γ3-subunit is phosphorylated at the equivalent sites.Up until recently, phospho-regulation of the regulatory γ-subunit has been largely ignored, as the majority of studies have investigated AMPK PTM regulation through the α- and β-subunits. However, a new study has recently identified two phosphorylation sites on the γ1-subunit,  in CBS4 . These sRefer to S. cerevisiae AMPK ortholog Snf1 to destabilise the yeast complex and down-regulate signalling [AMPKα-subunit ubiquitination by makorin ring finger protein 1 (MKRN1) suppresses AMPK signalling through protein destablisation, leading to reduced glucose consumption and increased lipid accumulation [gid genes extended maximum lifespan of C. elegans by up to 35%. The E3 ligase cullin-RING 4A (CRL4A) polyubiquitinated, and decreased the stability of, AMPKα through non-canonical K29 and K63 linkages [γ1-subunit to promote its proteasomal degradation [Crbn knockout mice associated with high AMPK activity were restored following acute pharmacological AMPK inhibition [Ubiquitination is a reversible, highly regulated PTM central to cellular signalling . The prognalling . AMPKα-smulation . Consequmulation . Intrigulinkages . These mlinkages ,126,140.radation . CRBN alradation ,142. CRBradation . Finallyhibition . These sα2-K60 and K391, and analogous α1-K62 and K388, to increase association with LKB1. Other candidate substrates are not conserved (α2-K379/K470 and α1-K276/K476), hinting at isoform-specific regulation at the deubiquitination step. In this thorough study, AMPK was also shown to activate USP10 via phosphorylation of S76, indicating the presence of a positive feedforward loop important for maintenance of metabolic homeostasis in liver.The α-subunit has been reported to be degraded by UPS pathways associated with a PEDF/PEDFR/peroxisome proliferator activated receptor γ (PPARγ) axis, although the AMPK modifying ligase was not identified. PPARγ or proteasomal inhibition disrupted an AMPKα/PPARγ interaction, abolishing α-subunit degradation in cardiomyocytes . Loss ofα1-subunit by the TRIM27 ligase, directed by melanoma antigens (MAGE) A3 and A6 upregulated in many cancer states, has been demonstrated in HeLa cells and leads to AMPK degradation, reduced autophagic flux and hypersensitisation to metformin [Specific ubiquitination the etformin . RING fietformin . Besidesetformin . While ietformin .Ube2o gene deletion restored α2-subunit levels, or impaired tumor initiation, growth and metastasis, and switched off tumor cell metabolic reprogramming in mice. The second study defined UBE2O as an essential regulator of α2-dependent glucose and lipid metabolism programs in mouse skeletal muscle, extending the therapeutic potential of the axis to metabolic disorders.AMPK α2-subunit protein levels in C2C12 myotubes are reported to be regulated by UPS pathways. α2 ubiquitination and degradation were decreased following treatment with serum obtained from calorie-restricted mice, an effect possibly controlled by the ubiquitin-specific protease USP9X that was previously shown to interact with the AMPK-related kinase NUAK1 ,138. Conβ1-subunit isoform has been shown to be mediated through polyubiquitination by cell death-inducing DFF45-like effector A (CIDEA) [Cidea−/− mice, previously shown to be resistant to high fat diet-induced obesity and diabetes through increased energy expenditure [β2-subunit by a complex formed between laforin, a dual-specificity phosphatase, and malin, an E3 ubiquitin ligase, resulted in steady-state accumulation of β2, possibly due to subcellular re-localisation [Stability of the  (CIDEA) . AMPK leenditure . Similarenditure . E4BP40 lisation .Ubiquitination has also been detected by multiple (>3) HTPs at α1-K387/α2-K391 and K476, and γ1-K234, K264 and K329, although the functional consequences of these PTMs are unclear [β2 isoform, but not β1, as a substrate for the E3-SUMO ligase protein inhibitor of activated STAT PIAS4  in HEK293 cells [α1 and α2-subunits have both been shown to be sumoylated by PIAS4 in MEFs and HEK293 cells [Sumoylation has many similarities to ubiquitination, except in this case Small Ubiquitin-like Modifier  proteins are covalently attached to Lys residues on target proteins. Sumoylation does not usually target proteins for proteasomal degradation, but rather may compete for ubiquitination sites or act as a tag for subsequent UPS degradation. This reversible PTM also affects cellular processes such as apoptosis, protein stability, localisation and cell cycle progression. First evidence pointing to sumoylation of AMPK revealed the 93 cells . Attachm93 cells . The mod93 cells . Intriguγ1 NH2-terminus in AMPK isolated from rat liver, and a handful of proteomic studies identifying acetylation of α1-K31, K33, K71/α2-K69, γ1-K264 and γ2-K9/K10. The α1 residues K31 and K33 (conserved in α2) are of particular interest as structurally they stabilise the AMPK ADaM site through interactions with the negatively charged phosphate group of phosphorylated β1-S108, shown to mediate sensitivity to A769662 and lauroyl-/myristoyl-CoAs [S. cerevisiae AMPKβ homolog Sip2, regulated by acetyltransferase NuA4/Esa1 and deacetylase Rpd3 [2-terminal sites was sufficient to enhance an interaction between Sip2 and Snf1 (AMPKα) to decrease Snf1 activity. With no clear phylogenetic conservation, it remains unclear the extent to which NH2-terminal acetylation regulates mammalian AMPK, however it is perhaps worth noting that Sip2-K256 structurally aligns to β2-K167, found in the αC-interacting helix known to be important for AMPK activation by ADaM agonists but not AMP [Lysine acetylation is a reversible PTM involved in the regulation of a diverse array of cellular processes, of which the most intensely studied is gene expression through modification of core histones. Evidence for acetylation of mammalian AMPK is limited to an early study from Mitchelhill et al. , demonstoyl-CoAs ,56,106. ase Rpd3 . Sip2 ac not AMP .DNA methylation is an adaptive mechanism by which skeletal muscle and other tissues respond to repeated bouts of exercise associated with endurance training. These modifications lead to altered gene transcription profiles to e.g., increased oxidative capacity and substrate utilisation, and hypertrophy, that intriguingly may be inheritable to confer beneficial effects on offspring health ,150. Pro2O2 stimulated AMPK in HEK293 cells but inhibited in neonatal primary cardiomyocytes [α2-C130 and α2-C174 following treatment of COS7 cells with H2O2 has been reported to induce formation of an intermolecular disulphide bond, preventing association with AMPK activating kinases and suppressing signalling. The modification was negatively regulated by the oxidoreductase Trx1 (itself positively regulated by AMPK) to promote AMPK activity in the ischemic mouse heart [2O2 also reversibly inhibited activities of purified AMPK α1β1γ1 and AMPK-related kinases SIK1-3 [2O2 directly activated the isolated AMPK kinase domain, and glucose oxidase-generated H2O2 oxidation triggered an increase in α-T172 and AMPK activation in HEK293 cells without any detectable change in ADP:ATP ratio [α1-C304 and, to a lesser extent, α1-C299 in the AID, although regulatory roles for these residues were not detected in a subsequent report [2O2 or hypoxia/ischemia via indirect pathways. Incubation at 1.5% O2 up-regulated activities of LKB1 in MEFs [2+ influx and relocation of stromal interaction molecule 1- (STIM1) to the plasma membrane [2+ entry, hinting at negative feedback regulating AMPK signalling during exercise. Finally, AMPK was reported to be activated independently of changes to adenine nucleotide in mild ischemic rat hearts (39% reduced coronary flow for 30 min) [2O2-incubated rat epitrochlearis muscle to induce uptake of 3-O-methyl-D-glucose [2 (1.5–3%) [Reactive oxygen species (ROS) are generated in exercising skeletal muscle via a number of processes, most notably the mitochondrial electron transport chain coupled to a rise in oxygen consumption. Exercise-generated ROS regulate many protein classes, including metabolic signalling kinases, via a suite of oxidative modifications. We direct you to a recent and extensive review concerning dynamic regulation of AMPK following generation of ROS under glucose stress . In essemyocytes , which tse heart . H2O2 als SIK1-3 . The majs SIK1-3 ,153, altTP ratio . This prt report . Other s in MEFs , and CaMmembrane . Intrigumembrane . Phospho 30 min) , in H2O2-glucose , and in (1.5–3%) , althoug(1.5–3%) ,104.2O2 also induced elevated AMP:ATP ratios and AMPK signalling in cultured NIH3T3 or HEK293 cells, respectively [2O2-induced AMPK signalling was blunted in HEK293 cells stably expressing an AMP-insensitive γ1-R531G mutant [On the other hand, several studies have attributed redox stress-induced AMPK activation to fluctuations in intracellular adenine nucleotide pools. Evans et al. reported in vivo activation of α1 AMPK in rat pulmonary arterial smooth muscle following 1 h of hypoxia was accompanied by a 2-fold increase in AMP:ATP ratio . Exogenoectively ,162. AccG mutant . More coα1-C176 and α1-C416 were found to be s-nitrosylated in treated HEK or human pulmonary arterial endothelial cell (HPAEC) lysates, and a range of Cys residues in all AMPK isoforms, with the exception of γ3, were modified in mouse tissues and human cell lines, although functional consequences remain unknown [ unknown ,134,164.+2 was found to negatively regulate activity of human Aurora A (C290), supporting previous in vitro and in vivo studies examining PKA (C199) [+2 (α1-C176) to Ser prevented accumulation of autophosphorylated species following induction, without deleteriously affecting allosteric regulation [+2.AMPK is one of 102 human Ser/Thr kinases with a Cys residue at position +2 in relation to the activating phosphorylation site in the kinase domain activation loop . These kA (C199) . Using agulation . One expAMPK is an ancient energy sensor that calibrates cellular growth potential with nutrient availability. Given its standing in the governance of cellular metabolism, it is hardly surprising that AMPK succumbs to a multiplicity of PTMs whose diversity  is likely reconciled by the heterogeneity of AMPK expression profiles in distinct cells and tissues.2-terminal extensions, respectively. A total of 46 putative phospho-sites, conserved between mouse and human species, have been flagged in these in these two γ2 and γ3 regions in HTP studies. Notwithstanding that species conservation of only 2 sites exist in γ3, this region in humans still harbours a total of 34 Ser/Thr residues, suggesting some have evolved to cater for the differential metabolic requirements of human skeletal muscle [2-terminal extension is one such mechanism modulating activity. It is also noteworthy that of the two longer γ isoforms, γ2 contains 14 Ser-Pro sites, which is significant when one considers the critical role of proline-directed kinases in cell cycle regulation and their intense interest for the cancer and neurodegeneration fields [2-terminal extension contains nuclear localisation and export sequences, whereby γ2-containing complexes were found to phosphorylate and inhibit the nuclear transcription factor TIF-IA to prevent ribosome biogenesis [As discussed, the overwhelming majority of investigations of posttranslational regulation of AMPK to date have investigated α- and β-subunits, with independent studies of the γ-subunit dominated by its conventional nucleotide-sensing capacity. However, if we wish to fully understand how signalling events like phosphorylation cascades control AMPK throughout the body, and the implications in for health and disease, then it would be remiss to ignore that both γ2 and γ3 isoforms are characterised by unique, 241 and 168 amino acid NHl muscle , the soll muscle , it stann fields . In factogenesis , a proceogenesis . This alogenesis , these sFinally, if the field as a whole seeks to move forward in its collective understanding of AMPK regulation, greater rigour in regulatory site identification needs to be respected. For example, full-length heterotrimeric AMPK as a substrate, as opposed to protein fragments, are a must for phosphorylation reactions under cell-free conditions, preferably over a shorter duration more indicative of the rapidity of cellular signalling. Promiscuous kinases may hit multiple sites on purified enzyme substrates, with little-to-no applicability to complex biological systems in cells. Taken into account, we need more orthogonal approaches to PTM validation involving a variety of techniques, such as mass spectrometry, stringent phospho-antibody validation, and confirmation in heterogenous cell lines. Furthermore, conclusions should not be solely based on overexpression and/or knockdown effects in cells; while many inhibitors have off-target effects, and these need to be considered when analysing changes in site regulation. Without doubt this creates challenges for researchers in the field but will ultimately be advantageous to provide greater clarity on the regulation of AMPK biology.