Apache Spark is a popular open-source distributed data processing framework that can efficiently process massive amounts of data. It provides more than 180 configuration parameters for users to manually select the appropriate parameter values according to their own experience. However, due to the large number of parameters and the inherent correlation between them, manual tuning is very tedious. To solve the problem of tuning through personal experience, we designed and implemented a reinforcement-learning-based Spark configuration parameter optimizer. First, we trained a Spark application performance prediction model with deep neural networks, and verified the accuracy and effectiveness of the model from multiple perspectives. Second, in order to improve the search efficiency of better configuration parameters, we improved the Q-learning algorithm, and automatically set start and end states in each iteration of training, which effectively improves the agent’s poor performance in exploring better configuration parameters. Lastly, comparing our proposed configuration with the default configuration as the baseline, experimental results show that the optimized configuration gained an average performance improvement of 47%, 43%, 31%, and 45% for four different types of Spark applications, which indicates that our Spark configuration parameter optimizer could efficiently find the better configuration parameters and improve the performance of various Spark applications. Apache Spark  is a widApache Spark has more than 180 configuration parameters that users must manually adjust according to their application and cluster environment. Choosing the appropriate configuration parameters can not only significantly improve the performance of Spark applications and speed up the running time of Spark applications, but also improve the utilization of cluster resources. Unfortunately, some Spark’s configuration parameters are numerous, and their inter-relationship are also very complex . TherefoManually tuning Spark configuration parameters is cumbersome and time-consuming, and requires developers to have a deep understanding of the Spark framework, which inspired our interest in the automatic tuning of Spark configuration parameters. Generally speaking, automatic Spark configuration parameter tuning consists of two components: the Spark application performance prediction model and the configuration-parameter-searching algorithm. Performance prediction models render the evaluation process efficient and practical instead of methods of repeatedly executing the application.Gao et al.  employedWe designed and implemented a Spark configuration parameter optimizer that accurately predicts the execution time of Spark applications and gives the recommended configuration parameters. Experiments demonstrate that the performance of Spark applications using the recommended configuration parameters is significantly improved compared to the default configuration.From more than 180 Spark configuration parameters, we screened out key configuration parameters that had greater impact on the application to reduce the complexity of the model.We built a Spark application performance prediction model on the basis of a deep neural network, and verified the accuracy and usability of the model from multiple perspectives through experiments.On the basis of the Q-learning algorithm, we improved the optimal searching algorithm to explore unknown areas of the configuration parameter space, avoid local optimal solutions, and find a suitable parameter configuration for Spark applications in a short period of time.Recently, some methods based on reinforcement learning to find the optimal neural network model structure have been proposed. MetaQNN  applied The rest of this paper is organized as follows: In recent years, the performance optimization of large data processing systems has been a hot-spot academic issue. Big data processing systems contain a large number of configuration parameters such as controlling parallelism, memory settings, and I/O behavior. Inappropriate parameter settings can lead to severe performance degradation . GounariResearch on Spark performance prediction models has always been a hot topic. Singhal et al.  proposedParameter search mainly aims to design a search algorithm to quickly find better configuration parameters from a larger configuration space. Wang et al.  adopted In this part, we introduce the Spark application configuration parameter optimizer in detail, which mainly consists of three phases: the collection and preprocessing of historical Spark application data; the training of a Spark application performance prediction model; and the search for better configuration parameters, corresponding to In Spark configuration parameter optimization, it is very important to choose an appropriate parameter space. A large number of parameter features increase the searching time, while few parameter features may lead to sub-optimal configuration. In the Spark parameter list, some parameters (such as spark.app.name) have no influence on the performance of the application, which are removed directly. Lastly, on the basis of theories ,25 and eM represents the number of parameter combinations, N denotes the number of parameter types in each parameter combination, i-th parameter set obtained by sampling, and j-th parameter value in the i-th parameter set.In the data collection and preprocessing phase, we applied Hibench  to generi-th parameter set. Since the structure and format of each Spark configuration parameter are inconsistent, the value of each configuration parameter is normalized before model training by Equation T=t1Equation : (4)cij=After standardizing the base dataset for the Spark application, we generated a dataset for model training. To improve the efficiency of searching for optimal parameters, we used a performance model to evaluate the actual performance of the Spark application. In general, the performance of a Spark application  can be eIn this paper, we optimized the execution time of Spark applications by adjusting the Spark configuration parameters, which means that Equation , config n-th layer is connected with any neuron in the n+1-th layer. The connection relationship is expressed in Formula ym=fS is the finite state set, A is the finite motion set, R is the reward function [s. Under this configuration parameter, the Spark application prediction time is used as the value of the state s, and each execution of an action is converted into another state.The optimization problem of Spark configuration parameters in a discrete, finite search space can be modeled as a Markov decision process (MDP). An MDP consists of a quadruple function . After cs,a), uses S and A to construct a Q value table to store the value of Q, and selects the action that can obtain the maximal profit in the current state according to the Q value table in each step by adopting the r is the immediate reward by taking action a from state s to state Q-learning is a value-based reinforcement-learning algorithm that can solve the above MDP problem. The decision-making agent seeks the optimal strategy by maximizing the state action value function Q(Equation .(7)Qi+1a) are initialized to 0. Algorithm 1 The process of the improved Q-learning algorithm.Input:  S = [        State List Q = [        Qtable List Output:          bestConf;S, Q)  1: initQtable(  2: bestState = for round from 1 to rounds do  3: s = bestState //Store the best state  4:     while ratio > 0.1 do  5:     a = chooseAction( s ) //use the   6:         s,   7:         t, s,   8:         r = t −   9:         10:         updateQtable // update Qtable according to Formula if then11:         12:            bestState = t − t //compute the ratio of performance improvement13:         ratio = , evaluation metrics for Spark performance prediction models, and the analysis of experimental results.In order to evaluate the effectiveness of our proposed optimizer, we implemented the experiments on a cluster of six computing machines, each with Intel(R) Core(TM) i9-10900K CPU @ 3.70 GHz processor, 20 cores, 64 GB main memory, and 1 GigE Ethernet network. Our Spark cluster was based on CentOS Linux release 8.2.2004, JDK version 1.8, Apache Hadoop version 2.7.7, and Apache Spark 3.1.3.To test the effectiveness of our method on different Spark application types, we chose WordCount, PageRank, KMeans, and TeraSort, provided by Hibench , as our After the collecting training data, we measured the execution time of each Spark application under a given configuration 9 times, and used the median as the real execution time of each application to eliminate bias caused by other factors such as the computer hardware and network.In order to evaluate the quality of the Spark performance prediction model, we used the following evaluation metrics:Mean absolute error (MAE): The average value of the absolute error can reflect the actual situation of the predicted value error. The smaller the MAE value is, the higher the model accuracy is. Assuming that the real values in the test set were Equation .(8)MAE=Root mean square error (RMSE): The standard deviation of the residuals (prediction errors). The smaller the RMSE value is, the higher the accuracy of the Spark performance prediction model is. The representation of RMSE is in Equation (Equation .(9)RMSER-squared : The deformation  of the MAE. The smaller the value of MAPE is, the better the accuracy of the Spark performance prediction model is. The representation of MAPE is shown in Equation (Equation : (11)MAPRelationship between the performance prediction model and the number of training data: As with most machine-learning tasks, using more training data leads to better predictive models. However, in practice, since each training datum is collected by the Spark application execution in a real cluster environment, collecting too many training data increases the time cost. Therefore, we need to find a balance between various model evaluation indicators and the number of training data, that is, reduce the number of training data as much as possible under certain conditions. Performance comparison of deep neural network models and other regression models: We compared Spark performance prediction models built with linear regression (LR), support vector machine regression (SVR), external tree regression (ETR), random forest regression (RFR), decision trees (DTR), and deep neural networks (DNNs). To fairly compare these methods, we used the same training environment, training dataset, and testing set as those of our proposed DNN method. Performance comparison between the improved Q-learning algorithm and other parameter search algorithms:Time performance overhead evaluation: Our scheme consists of a performance prediction model and an improved Q-learning search algorithm. We applied the scheme proposed in this paper to four different types of Spark applications. Each type of application was conducted 9 times to search for the better configuration parameters and calculate the exact time cost, which is shown in Performance comparison between recommended configuration parameters and default configuration parameters: By using our method, the recommended configuration parameters for four different types of Spark applications were obtained, as shown in For Spark’s configuration parameter optimization problem, we employed a deep neural network to build a Spark performance prediction model, and designed an improved Q-learning algorithm as our Spark configuration parameter optimizer to search for better configuration parameters. The experimental results show that the Spark performance prediction model based on deep neural network is more accurate, and the improved Q-learning algorithm could greatly reduce the time cost of parameter searching while obtaining better results compared with the default configuration parameters for four different types of Spark applications.Not only can the configuration parameters of the Spark application affect the performance, but the state of the Spark cluster  can also influence the performance of the application. When CPU utilization is too high, it is better to allocate a small number of CPU cores to the application to reduce waiting time.Our current work can only solve the optimizer problem for a single Spark application on a given Spark cluster. In future work, we aim to simultaneously optimize multiple Spark applications with the resource constraints of a Spark cluster.


The basic surgical steps in the Goel technique of atlantoaxial fixation involve exposure of the atlantoaxial articulation, denuding of the articular cartilage, stuffing of bone graft pieces within the articular cavity, and subsequent instrumentation.“High-riding” vertebral artery in relationship to the pedicle-facet of C2 has been widely recognized to be a factor that makes insertion of the C2 pedicle-facet screw difficult or impossible. In this video, a technique of exposure and mobilization of the high-riding vertebral artery to permit safe C2 screw insertion is shown. An alternative option in the presence of such a high-riding vertebral artery is to use either C2 laminar or inferior facetal screw insertion.https://youtu.be/LjxxINmzph0The video can be found here:  I wish to demonstrate to you the technique of mobilization of high-riding vertebral artery into the C2 pedicle and facet to facilitate C2 screw insertion in the Goel’s technique of atlantoaxial lateral mass plate and screw stabilization.0:40 Clinical summary. This is a case of a 10-year-old girl who sustained blunt injury on the head 6 weeks prior to admission.0:48 Preoperative investigations. Investigations showed os odontoideum and mobile atlantoaxial instability. CT scan showed high-riding vertebral artery that deeply indented into the pedicle facet of C2 vertebra, more on the right side .1:08 Position. The patient is placed in head high position under cervical traction as shown in the figure.1:15 Surgical technique. Midline posterior incision is taken that is centered on the C2 spinous process. Dissection is done to expose subperiosteally the lamina and pedicle of C2. The technique of atlantoaxial fixation is shown on the left side where the vertebral artery is not so high. Atlantoaxial facetal articulation is exposed by subperiosteal dissection. The facet of C1 is widely exposed. C2 ganglion is displaced rostrally. Part of the pars and pedicle of C2 vertebra are drilled to facilitate positioning of the plate for plate and screw fixation technique. The joint cavity is exposed widely as seen here and power-driven drill is used to make a hole in the facet of atlas. Bone graft is packed into the atlantoaxial facetal articulation. Screw is first placed over the plate into the C1 facet. The vertebral artery in the region of the pedicle is identified, its course is delineated, and a screw insertion point on the pedicle is identified that is away from the loop of the vertebral artery. Navigation can be used in this instance if there is significant doubt of the course of the vertebral artery. After making a guide hole, the screws are inserted and later tightened .The dissection is now done on the right side where the vertebral artery is significantly high riding. The lamina is first exposed subperiosteally. The pedicles are identified. The course of the vertebral artery is identified and exposed. The vertebral artery is seen under the suction just below the atlantoaxial facetal articulation. A hole is made into the C1 facet. Bone graft is packed into the articulation as on the left side. Bone graft is packed as much as it is possible. This packing of bone graft is absolutely crucial for bone healing and also distraction and reduction of the atlantoaxial instability. Bone is removed on the posterior surface of the vertebral artery as much as its medial extension to facilitate its mobilization. As you can see, the vertebral artery is pulsating under the hole of the C2 portion of the plate. After protecting the vertebral artery, a hole is made into the C2 facet. C2 screw is then inserted after protecting the vertebral artery away from the site of screw insertion . The screws are then tightened .The arch of atlas and the C2 spinous process are denuded off all their muscle attachment and are drilled to make them suitable as a host bone for bone graft. Bone graft is harvested from the iliac crest.10:13 Postoperative imaging. Postoperative imaging shows reduction of the atlantoaxial instability and lateral mass plate and screw fixation. The C2 screw avoids the vertebral artery.


N2O is a greenhouse gas that has caused a sharp increase in global temperature. Biological nitrification inhibition refers to releasing natural compounds that inhibit nitrification from plant roots. The natural compounds released are called biological nitrification inhibitors (BNIs), which specifically inhibit the activity of microorganisms in soil nitrification. Biological nitrification inhibitors can significantly improve rice (Oryza sativa), corn (Zea mays) and other crops by 5–10%, which can increase the nitrogen utilisation rate of corn by 3.1%, and reduce greenhouse gas N2O emissions. Compared with plants that do not produce BNI, the amount of N2O released can be reduced by up to 90%. The BNI released by Brachialactone (Brachiaria humidicola) accounted for 60–90% of the total inhibition of nitrification. In summary, biological nitrification inhibitors that inhibit nitrification, improve nitrogen utilisation and crop yield, and reduce greenhouse gas emissions play an important role. This paper reviews the plants known to release BNIs, reviews the plants known to inhibit soil nitrification but with unknown BNIs and further discusses the important role of bio nitrification inhibition in agricultural systems.Based on an analysis of the current situation of nitrogen fertiliser application, it is suggested that improving the nitrogen utilisation efficiency of crops is an important means of promoting the sustainable development of agriculture and realises the zero increase in chemical fertiliser application. Nitrate loss and nitrous oxide (N Common industrial nitrification inhibitors are CP (2-chloro-6-(trichloromethyl) pyridine), DMPP , dicyanamide (DCD), etc.  secretion is the most significant, although the relevant mechanism of its inhibition has not been confirmed  and ammonia-oxidising archaea (AOA) and their related enzymes in nitrification, thereby inhibiting nitrification and the process of converting NHD), etc. , but theD), etc. ,b, certaD), etc.  found inD), etc. . The fute effect . It is pe effect . It was onfirmed . Studiesonfirmed , the cononfirmed ,b.2O gas emissions, and is of greatly affect the earth’s biochemical nitrogen cycle  and hydroxylamine oxidoreductase (HAO). The AMO pathway requires the participation of two nitrifying microorganisms, AOA and AOB, but the HAO pathway only requires one ammonia-oxidising microorganism, AOB. Whether its pathway has an impact on AOA remains to be confirmed. Therefore, BNI inhibits nitrification by inhibiting the AMO pathway .Brachiaria humidicola exhibits a lower abundance of AOB and AOA  is the first tropical grass found to have strong adaptability to a low nitrogen environment, and it can inhibit nitrification. One of the bionitrifying inhibitors it releases is named ‘Brachialactone’, a cycloditerpenoid with a unique 5-8-5-metacyclic system and alactone ring.N applied was 1.49 g/kg; soil the pH value is 6.2), the nitrification rate of the rhizosphere soil before and after planting was measured and the copy number of AOA and AOB was detected by quantitative PCR method; the results showed that the planting of Brachiaria humidicola cv. The N2O emission of the soil was 80 mg/m2, while the soil in which Brachiaria humidicola cv. Tully (BT) was planted with higher inhibition ability was only 32 mg/m2. The nitrification and denitrification in the BT rhizosphere soil the effect and abundance of AOA were significantly lower than those of BM, indicating that the forage with strong inhibitory ability played a leading role in inhibiting AOA activity  of the nitrogen in the soil was in the form of ammonia nitrogen. Significant inhibition of nitrification was detected in the soil of Lolium perenne Linn, where ammonium nitrogen was the source of nitrogen. Still no nitrate inhibition was detected in soil with a nitrate-nitrogen source , which mainly act by changing the activity of biological nitrification inhibitors  catechols (C6H6O2) biological nitrification inhibitor active substances during leaf litter decomposition, and they have a certain inhibitory effect on nitrification. The content of catechins and catechols in the soil of the roots of bayberry of the same volume is less than that released in leaf litter constitutes, so the main action of inhibition  had the highest nitrification potential, while perennial ryegrass (L. perenne) had the lowest. Eleven kinds of grasses, three kinds of non-grasses and two kinds of legume crops were further studied. Italian ryegrass had the lowest release of N2O gas, and mountain sparrow wheat (Bromus stamineus) had the highest release, but there was no significant difference (Raphanus raphanistrum), bromegrass (B. diandrus), wild oats (Avena fatua) and perennial ryegrass on nitrification under hydroponic conditions were studied. The results showed that radish root exudates inhibited nitrification most significantly, and its nitrification activity intensity was higher than that of Brachiaria wetland. Other plants also had a certain inhibitory ability −1, which was 10.3 t·(hm2 a)−1 higher than that of the control (application of urea), and the application of BNI significantly increased agricultural production . Compared to urea treatment, DCD treatment had no significant effect on N2O or agricultural NUE, while CP and BNI significantly reduced annual N2O emissions by16.5 and 18.1%, respectively; NUE increased by 12.6 and 6.7%, respectively, showing that BNI improves yield in vegetable ecosystem and reduces N2O emissions thus playing an important role .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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Here, we have prepared a protein kinase‐focused library by chemically modifying helix‐loop‐helix (HLH) peptides displayed on phage and subsequently tethered to adenosine. The library was screened against aurora kinase A (AurA). The selected HLH peptide Bip‐3 retained the α‐helical structure and bound to AurA with a KD value of 13.7 μM. Bip‐3 and the adenosine‐tethered peptide Bip‐3‐Adc provided IC50 values of 103 μM and 7.7 μM, respectively, suggesting that Bip‐3‐Adc bivalently inhibited AurA. In addition, the selectivity of Bip‐3‐Adc to several protein kinases was tested, and was highest against AurA. These results demonstrate that chemical modification can enable the construction of a kinase‐focused library of phage‐displayed HLH peptides.Conformationally constrained peptides hold promise as molecular tools in chemical biology and as a new modality in drug discovery. The construction and screening of a target‐focused library could be a promising approach for the generation of  Expanding helix‐loop‐helix (HLH) peptide libraries: A phage‐displayed HLH peptide library was chemically modified with adenosine as an anchor molecule for binding to the ATP binding site of protein kinase. This approach to the construction of target‐focused HLH peptide libraries would enable the efficient generation of selective inhibitors not only for protein kinases, but also for other proteins of interest. The adenosine derivative  was synthesized from 2’,3′‐isopropylidene adenosine‐5′‐(2′‐aminoethyl)‐amide via reaction with maleimide‐(CH2)2‐NHS and subsequent deprotection of the isopropylidene group , used as a model, binds to G‐CSF‐R with a KD value of 214 nM.P8‐2KA to G‐CSF‐R demonstrated its specificity; no binding was observed to the two other proteins examined, BSA and KDR (VEGF receptor). Phage clones (1011 cfu) of P8‐2KA were treated with 200 μL of serially diluted maleimide‐PEG2‐biotin (0.5–500 μM) in PBS at room temperature for 2 h . Higher concentrations of maleimide‐PEG2‐biotin led to a small loss of phage infectivity of the host TG1 E. coli cells and thus in further experiments we chemically modified the phage clones with 5 μM maleimide‐PEG2‐biotin. In phage ELISA experiments Mal‐Adc using the conditions described above. The chemically modified phage library was screened against AurA, a serine/threonine protein kinase involved in regulating mitosis and a common target of anti‐cancer drug discovery.Bip‐3 (CDGGSGGGS‐AELAALEAELAALE‐G7‐KLEYLKWKLWPLKG W) was most frequently observed. Interestingly, Bip‐3 showed no sequence homology with known ligands and inhibitors for AurA.Bip‐3 using a standard solid‐phase method and examined it in detail.We used the Bip‐3 has a high α‐helical content comparable with that of the original peptide YT1  were measured using various concentrations of ATP.[VX‐680 exhibited competitive inhibition for ATP whereas Bip‐3 showed mixed inhibition . Mixed‐type inhibition occurs, by definition, when an inhibitor binds the enzyme both before and after substrate binding and the affinities for the free enzyme and ES complex are different.CD spectrometry revealed that Bip‐3 was chemically modified with Mal‐Adc to provide the adenosine‐tethering peptide Bip‐3‐Adc, which was subjected to an IMAP® TR‐FRET inhibition assay for AurA . This result suggested that Bip‐3‐Adc interacted with the ATP‐binding pocket and its peripheral region, and the peptide moiety Bip‐3 appeared to dominantly contributed to the inhibitory activity for AurA. Lineweaver‐Burk plot analysis showed that Bip‐3‐Adc exhibited uncompetitive inhibition .Bip‐3‐Adc and obtain structural information. Finally, we evaluated the selectivity of Bip‐3‐Adc for AurA by comparing its inhibition activity against the aurora protein kinases AurA, AurB, and AurC, the serine/threonine‐protein kinases PAK4, Erk2, and PKAcα, and the tyrosine‐protein kinase Src. As shown in Figure Bip‐3‐Adc exhibited the highest inhibitory activity and remarkable selectivity for AurA.Next, peptide 7) and 12‐mer (X12) peptides, 7‐mer cyclic peptides with a disulfide bond (CX7C), and our conventional ‘α‐helical’ library (AELAALEAELAALE‐G7‐KLXXLKXKLXXLKA). Unfortunately, we observed no enrichment of phage clones binding to AurA. Screening of our 9‐mer ‘loop’ library L‐lib11 (AELAALEAELAALE‐GX9G‐KLAALKAKLAALKA) gave an HLH peptide inhibitor against AurA, but it had only weak inhibitory activity, showing 35 % inhibition at a concentration of 100 μM.de novo kinase inhibitors.In addition to using the kinase‐focused HLH peptide library, we attempted to generate AurA‐kinase inhibitors by screening several phage libraries: commercially available libraries of linear 7‐  should be addressed to the authors.Supporting InformationClick here for additional data file.


The modified biochar MTBC was further used to remediate the fluoride-contaminated water. The fluoride adsorption testing was conducted using the batch method at 298, 308, and 318 K. The maximum fluoride removal efficiency (E%) using MTBC was 98% when the adsorbent dosage was 0.5 g/L and the fluoride concentration was 50 mg/L. The experiment data for fluoride adsorption on MTBC best fit the pseudo 2nd order, rather than the pseudo 1st order. In addition, the intraparticle diffusion model predicts the boundary diffusion. Langmuir, Freundlich, Temkin, and Dubnin–Radushkevich isotherm models were fitted to explain the fluoride adsorption on MTBC. The Langmuir adsorption capacity of MTBC = 18.78 mg/g was recorded at 298 K and decreased as the temperature increased. The MTBC biochar was reused in ten cycles, and the E% was still 85%. The obtained biochar with a large pore size and high removal efficiency may be an effective and low-cost adsorbent for treating fluoride-containing water.Fluoride contamination in water is a key problem facing the world, leading to health problems such as dental and skeletal fluorosis. So, we used low-cost multifunctional tea biochar (TBC) and magnetic tea biochar (MTBC) prepared by facile one-step pyrolysis of waste tea leaves. The TBC and MTBC were characterized by XRD, SEM, FTIR, and VSM. Both TBC and MTBC contain high carbon contents of 63.45 and 63.75%, respectively. The surface area of MTBC (115.65 m Fluoride is a naturally occurring element that can be exposed to humans through food and drinking water. The health effects of fluoride are mainly concentration dependent, as both low and high concentrations cause serious harm . The WHOBET, functional groups, and is suitable for adsorbing various environmental pollutants .Fe (NOPyrolysis = 0.516 USD (6 Units × 0.086 cost per unit).Total cost per gram = 0.0719 USD (0.719 ÷ 10).Total cost per Kg = 71.9 USD.The importance of this sorbent is based on its low-cost values, which helpfully remove fluoride from water. So, the cost analysis during this study was investigated. A stepwise cost analysis (in USD) is described as follows:Many countries contain high fluoride levels in their drinking water, especially Pakistan, India, China, USA, Italy, Ghana, Mexico, Sudan, Nigeria, etc. . Develop3)3 to form MTBC and successfully applied in groundwater defluoridation. The surface area of TBC (81.64 m2/g) was smaller than MTBC (115.65 m2/g). TBC and MTBC were characterized by their main components, carbon and oxygen along with other trace elements found in MTBC. The oxygen content of MTBC (25.56 wt %) was lower than that of TBC (26.18 wt %). MTBC was used to remove fluoride, and its removal efficiency reached 98% for the initial fluoride concentration of 50 mg/L at optimized conditions . The adsorption kinetics data explained the adsorption carried out by the chemical process and best fit the pseudo 2nd order model. The adsorption data are better fitted to the Freundlich model rather than the Langmuir model, which described the fluoride adsorption process controlled by multilayered process. The competitive ions Cl− and HCO3− have a slight effect, while other competitive ions  have a more significant impact on fluoride adsorption, due to their high charge density. Ion exchange, inner-sphere complex, electrostatic force of attraction, and hydrogen bonding probably take part in the defluoridation. Regeneration study and actual water treatment reveal that this adsorbent is helpful and supportable in fluoride removal in commercial and industrial uses because of its low cost and high removal efficiency.The one-step pyrolysis process was used to prepare the TBC, which was impregnated by Fe (NO


The nuclear matrix protein lamin A is a multifunctional protein with roles in DNA replication and repair, gene activation, transcriptional regulation, and maintenance of higher-order chromatin structure. Phosphorylation is the main determinant of lamin A mobility in the nucleus and nuclear membrane dissolution during mitosis. However, little is known about the regulation of lamin A phosphorylation during interphase. Interestingly, C-terminal lamin A mutations trigger cellular senescence. Recently, we showed that the C-terminal region of lamin A interacts with casein kinase II (CK2). In the present study, we have expanded on our previous research to further investigate lamin A phosphorylation and elucidate the mechanisms underlying the effect of C-terminal mutations on cellular senescence. Our results indicate that glycogen synthase kinase 3β (GSK3β) and CK2 jointly mediate the phosphorylation of lamin A at C-terminal Ser628 and Ser636 residues. Furthermore, a loss of phosphorylation at either of these two sites affects the nuclear distribution of lamin A, leading to an impaired DNA damage response as well as cellular senescence. Thus, phosphorylation at C-terminal sites in lamin A appears to be important for maintaining genomic stability and preventing cellular senescence. These findings provide insight into how loss of the C-terminal region of lamin A may induce premature aging. Furthermore, enhancement of GSK3β and CK2 activity may represent a possible therapeutic approach for the treatment of aging-related diseases. The nuclear lamina has roles in nuclear integrity, mitosis, mechano-sensation, DNA replication, signaling, regulation of transcription, and genome organization ,2. LaminStudies have shown that the depolymerization and nuclear localization of lamin A are closely related to its phosphorylation level ,13,14,15Through binding to a variety of nuclear proteins, lamin A acts as a signal integration platform. As such, phosphorylation of lamin A may have a direct impact on the binding sites of several proteins, affecting the transport of a series of important proteins in the nucleus and leading to changes in downstream signaling pathways. Studies have shown that phosphokinases that catalyze phosphorylation at specific lamin A sites are likely to directly participate in the regulation of activities such as the cell cycle and cellular senescence via the lamin A molecular platform. For example, the phosphokinase Cdk1 catalyzes the phosphorylation of N-terminal Ser22 and Ser392 sites in lamin A/C—an early event in cell mitosis . AdditioPro/+LMNA allele (LmnaG609G mutation flanked by two loxP sites) was generated by Cyagen Biosciences Inc., China. The G609G (GGC to GGT) mutation was introduced into exon 11 in the 3′homology arm. C57BL/6 embryonic stem cells were used for gene targeting [+/−LMNAintercrossed mice (C57BL background) were obtained from Z. Zhongjun (Hong Kong University).The argeting . LMNA+/−Pro/ProLMNAand LMNA−/− MEFs were isolated separately from 13.5-day-old embryos produced by Pro/+LMNAand +/−LMNA intercrossed mice, respectively (on a C57BL background). Littermate-matched Pro/Pro, LMNALMNA+/+, and LMNA−/− MEFs were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS). Cells were cultured at 37 °C in 5% CO2 and atmospheric oxygen conditions.HEK 293 cell lines (CRL-1573) were purchased from ATCC. LMNA-S625A-F: 5′-CCACACTGCGGTAGGCGCGAGTGACCGTGA-3′LMNA-S625A-R: 5′-TCACGGTCACTCGCGCCTACCGCAGTGTGG-3′LMNA-S628A-F: 5′-ACTGCCCCCCACAGCGCGGTAGCTGCGA-3LMNA-S628A-R: 5′-TCGCAGCTACCGCGCTGTGGGGGGCAGT-3′LMNA-S632A-F: 5′-GCTGCCACCCCCAGCGCCCCCCACACTG-3LMNA-S632A-R: 5′-CAGTGTGGGGGGCGCTGGGGGTGGCAGC-3′LMNA-S636A-F: 5′-GCAGTGGGGGTGGCGCCTTCGGGGACAATC-3′LMNA-S636A-R: 5′-GATTGTCCCCGAAGGCGCCACCCCCACTGC-3′LMNA-S652A-F: 5′-CTGGGTTCGGGGGGCGGAGTTGCCCAGG-3′LMNA-S652A-R: 5′-CCTGGGCAACTCCGCCCCCCGAACCCAG-3′Lamin A and progerin sequences were amplified and cloned into p3xFLAG-CMV or pGEX-4T-3 vectors. PCR-based deletion of the FLAG-Lamin A plasmid template was used to generate truncated forms of lamin A protein. Lamin A and point-mutated lamin A sequences were cloned into the lentiviral vector pLVX-mCherry-C1 by PCR. Transient transfections of these plasmids were performed using Lipofectamine 3000  according to the manufacturer’s protocol. Primer sequences for the mutation of lamin A were as follows:INK4a (sc-1661), and anti- p21WAF1/CIP1 (sc-6246) antibodies were purchased from Santa Cruz Biotechnology ; anti-γH2AX (05-636) was sourced from EMD Millipore ; and anti-KAP-1 (ab10484), anti-p-KAP-1 Ser824 (ab70369), anti-p-S/T (ab17464), anti-GSK3β (ab93926), and anti-GST (ab108524) antibodies were obtained from Abcam . Mouse anti-HA and anti-FLAG antibodies were obtained from Sigma-Aldrich, while mouse anti-actin and anti-GAPDH antibodies were obtained from Beyotime . Anti-pS628 and anti-pS632 lamin A polyclonal antibodies were generated by GenScript  using a specific phosphorylated peptide (Anti-lamin A/C (sc-20681), anti-p16 peptide .Cell extracts for Western blotting were prepared using modified RIPA buffer , boiled in sodium dodecyl sulfate (SDS) sample loading buffer, and resolved on SDS polyacrylamide gels before being transferred to PVDF membranes (Millipore). The membranes were blocked in 3% BSA in Tris-buffered saline with Tween 20  for 1 h at room temperature and incubated with primary antibodies (the antibody concentration was 1 µg/mL) overnight at 4 °C. The membranes were then probed with the respective HRP-linked secondary antibodies for 1 h at room temperature. Immunoreactive products were visualized using an enhanced chemiluminescence kit (Pierce 34095) and a Bio-Rad imaging system .For immunoprecipitation, cells exposed to the indicated treatments were harvested in lysis buffer  plus phosphatase inhibitors and protease inhibitors (Roche Complete). Cell lysates were incubated with 1 µg of the respective antibodies or control IgGs at 4 °C overnight. Then, bead-bound immunoprecipitates were washed with lysis buffer, after which the beads were boiled in SDS sample loading buffer and analyzed by Western blotting.GST-fusion proteins (bait and prey) were expressed in BL21 bacterial cells  and purified using glutathione-Sepharose 4B beads , with two washes in TEN buffer . For GST pull-downs, 1 µg GST, GST-lamin A, or GST-lamin A mutant protein was immobilized on Glutathione Sepharose 4B beads (GE) and incubated with 6× His-tagged GSK3β  at 4 °C overnight in GST-binding buffer . The beads were washed four times with GST-binding buffer and then analyzed by Western blotting with the indicated antibodies.2, and 0.2 mM ATP; kinase buffer for GSK3β: 25 mM Tris-HCl, 2 mM β-mercaptoethanol, 1 mM MgCl2, 0.5 mM ATP, and 5% glycerol). Each reaction required 6 µg substrate protein and 60 ng kinase in a total volume of 30 µL (supplemented with ddH2O); the samples were then mixed well and incubated at 30 °C for 2 h. The reactions were blocked by the addition of SDS loading buffer and the samples were analyzed by Western blotting; antibodies specific for phosphorylated lamin A were used to probe the membranes.Kinase reactions were initiated by incubating purified CK2α or GSK3β with GST-lamin A C-terminal protein and mutants in kinase buffer  according to manufacturer’s instructions. Briefly, cells were seeded in 6-well plates, fixed at room temperature for 15 min with 2 mL of 1× fixing solution, washed three times with 1× PBS, and stained with 1 mL of freshly prepared 1× β-galactosidase detection solution at 37 °C for 16 h in the dark. The cells were then washed twice with 1× PBS, overlaid with 70% glycerol/PBS, and photographed. The number of stained cells among more than 300 randomly chosen cells were counted. The data were analyzed using the two-tailed Student’s t-test to show differences between two groups, and the following p values were considered statistically significant: * p < 0.05, ** p < 0.01, and *** p < 0.001. Statistical analyses were performed using Graphpad Prism software v.5.03.At least three replicates were carried out for all experiments. Data are presented as means ± SD or means ± SEM (as indicated). Statistical analyses were conducted using the two-tailed Student’s pro/pro mice , NetPhos3.1 , and PhosphoSitePlus  to identify phosphorylation sites within the 50-amino acid region VGGSGGC-NH2 peptides. However, when the peptides were incubated together in vitro, phosphorylation at Ser628 was not apparent in the presence of GSK3β alone . As GSK3According to the online phosphorylation prediction tools used previously, we found that the lamin A Ser636 site was likely to be a substrate for phosphorylation by CK2α. CK2α catalyzes the phosphorylation of substrates with negatively charged amino acids such as aspartic acid (D), glutamic acid (E), or phosphoserine (pS) located at +1 or +3 residues on the carboxyl side D. PrevioTo further examine phosphorylation of the lamin A Ser636 site by CK2α, we generated an antibody against Ser636-phosphorylated lamin A and verified its specificity . In vitrThe above results demonstrated phosphorylation of lamin A at the Ser636 site by CK2α both in vivo and in vitro. We then investigated the role of Ser636 in activating phosphorylation at Ser628 through a subsequent series of in vitro kinase experiments using GST tag-purified lamin A C-terminal protein and mutants. In the presence of either CK2α or GSK3β, phosphorylation at Ser628 did not change. In contrast, when both CK2α and GSK3β were added, phosphorylation at Ser628 was markedly increased C,D. Thes−/− mouse model and used them to generate cell lines expressing wild type lamin A or mutant proteins with phosphorylation site-specific mutations tagged with mCherry , and the levels of lamin A protein and mutant proteins were determined by fluorescence and Western blot experiments. The results showed that cell lines expressing lamin A or point-mutated lamin A (LAS628A and LAS636A) were successfully constructed  on the phosphorylation of lamin A and confirmed that the kinases GSK3β and CK2 phosphorylated Ser628 and Ser636, respectively. We also found that phosphorylation at Ser636 influenced the phosphorylation of Ser628.The study of protein kinases involved in the regulation of aging has been a hot topic in recent years. Our previous studies confirmed that most tissues in premature and normal aging mouse models exhibited a decreased expression of CK2 and weakened CK2 enzymatic activity is an important cause of aging . This st−/− MEF cells supplemented with lamin A cell lines, during interphase, we observed that lamin A was diffusely distributed in the nucleus and exhibited punctate aggregation; however, punctate aggregation was not observed in LAS628A and LAS636A mutant cells. Moreover, foci in the lamin A-expressing cells were clustered around the perinuclear heterochromatin  have been observed in vivo [Nuclear lamins exhibit a high affinity to DNA and chromatin, which participate in various cellular processes including chromosome organization, transcriptional regulation, DNA damage response, cell cycle regulation, and mechanosensing . With re in vivo . Signifi in vivo . We willOverall, the present findings indicate that a loss of phosphorylation at the C-terminal of lamin A may lead to cellular senescence. Our results highlight the role of lamin A in intranuclear modification and communication and suggest a new mechanism to attenuate cellular senescence by activating the phosphorylation of lamin A using specific protein kinases. These findings may be beneficial for the future development of small-molecule drugs for use in anti-aging treatments.


In the present study we evaluated the effective phosphorylation of human AQP4ex. A small scale bioinformatic analysis indicated that only Ser335 is conserved in human, mouse and rat AQP4ex. The phosphorylation site of Ser335 was assessed through generation of phospho-specific antibodies in rabbits. Antibody specificity was first evaluated in binding phosphorylated peptide versus its unphosphorylated analog by ELISA, which was further confirmed by site-directed mutagenesis experiments. Western blot and immunofluorescence experiments revealed strong expression of phosphorylated AQP4ex (p-AQP4ex) in human brain and localization at the perivascular astrocyte endfeet in supramolecular assemblies identified by BN/PAGE experiments. All together, these data reveal, for the first time, the existence of a phosphorylated form of AQP4, at Ser335 in the extended sequence exclusive of AQP4ex. Therefore, we anticipate an important physiological role of p-AQP4ex in human brain water homeostasis.AQP4ex is a recently discovered isoform of AQP4 generated by a translational readthrough mechanism. It is strongly expressed at the astrocyte perivascular endfeet as a component of the supramolecular membrane complex, commonly called orthogonal array of particles (OAP), together with the canonical isoforms M1 and M23 of AQP4. Previous site-directed mutagenesis experiments suggested the potential role of serine AQP4 isxtension .335 to produce a phospho-specific antibody against human AQP4ex. We then employed this antibody to assess its specificity and the existence, localization, supramolecular aggregation and level of phosphorylation of AQP4ex in the human brain. Our results show the expression of highly phosphorylated AQP4 at Ser335 of the extended portion of AQP4ex.Phosphorylation is a common post-translational modification that can contribute to the regulation of protein function by inducing structural changes and regulating protein-protein interactions ,16. The The Entrez Gene database at the National Center for Biotechnology Information (NCBI)  was usedg for 10 min. The pellet was resuspended and plated on T25 flasks in DMEM-Glutamax , 1% penicillin/streptomycin, 10% fetal bovine serum (FBS) and maintained at 37 °C in a 5% CO2 incubator. After 7–10 days in culture, flasks were shaken to remove microglia and oligodendrocytes, and astrocytes were seeded on 12 mm round glass coverslips for immunofluorescence and in 12 multiwell plates for immunoblot analysis. The HEK293 cell line was cultured in Dulbecco’s high glucose medium  supplemented with 10% FBS  and penicillin/streptomycin . The day before transfection, cells at 60 to 70% confluency were plated using Optimem antibiotic-free medium . Transfection was carried out with 1 µg of pT-AQP4exS331A/S335A phosphonull, pT-AQP4exS331D/S335D phosphomimetic and pT-AQP4ex [Mouse astrocyte primary cultures were prepared as previously described . Briefly335, within the C-terminal extension at a concentration of 0.2 μg/mL for immunoblot and at 0.5 μg/mL for immunofluorescence. A custom rabbit polyclonal anti human AQP4ex [A custom affinity-purified peptide polyclonal antibody was generated by GenScript Biotech  against the human peptide containing the phosphorylated Sern AQP4ex  was usedn AQP4ex  was usedn AQP4ex  was usedTissues were obtained from neurosurgery. Specimens of human tissues were obtained from surgical intraoperatively resection of the following categories of patients with glioblastoma multiforme. (a) A 55-years-old patient with fronto-temporo-insular glioblastoma. (b) A 53-years-old-patient with frontal glioblastoma. (c) A 54-years-old patient with temporal glioblastoma. The brain region analyzed represents unaffected cortical brain areas supplied with glioblastoma multiforme, and defined as the zone of brain parenchyma surrounding tumor tissue, apparently normal under the microscope. Details on these biopsies have been recently reported . The stuImmunofluorescence experiments were performed as previously described . Brieflyp-AQP4ex antibody for 40 min at room temperature. After washing in PBS, cells were incubated with Alexa Fluor 488 anti-rabbit and Alexa Fluor 594 anti-human secondary antibodies for 30 min. Coverslips were mounted with Mowiol  added with DAPI . Images were obtained under an SP8 confocal automated inverted Lightning microscope (Leica TCS) using a 40X HC PL Apo oil CS2 objective.HEK293 cells were plated on round coverslips, fixed in 4% formaldehyde solution, washed with PBS, and permeabilized with 0.3% Triton X-100 in PBS. After blocking with 0.1% gelatin in PBS, the cells were incubated with primary antibodies for 40 min at room temperature. After washing in PBS, the cells were incubated with Alexa Fluor 488 anti-rabbit secondary antibody for 30 min. Finally, sections were viewed with a Leica DM2500 LED fluorescence microscope using 20×/0.55 and 40×/0.80 PL FLUOTAR objectives. Confocal images were obtained using an automated inverted Leica TCS SP8 confocal microscope with a 40× HC PL Apo oil CS2 objective for human brain slices, and on a ZEISS LSM900 with Airyscan 2–Super Resolution system using a Zeiss Plan-Apochromat 63×//1.4 Oil immersion objective for cell immunofluorescence. For AQP4-IgG (NMO) co-immunofluorescence, cells were seeded on round coverslips, washed with PBS and after blocking with 0.1% gelatin in PBS, were incubated with AQP4-IgG positive serum for 40 min at room temperature. Cells were washed, fixed, permeabilized and blocked as mentioned above and incubated with anti g for 40 min at 4 °C. The supernatants were collected, and the protein concentration was determined using the BCA protein assay . For dephosphorylation, 40 µg of lysates were treated with 200 U of Lambda protein phosphatase for 30 min at 30 °C  according to the manufacturer’s protocol.Harvested tissues were frozen in liquid nitrogen and stored at −80 °C for SDS-PAGE experiments [g for 30 min at 4 °C. A BCA Protein Assay Kit  was used to determine the total protein content of supernatants. The BN-PAGE experiments were performed as previously described [Human tissues were homogenized in 7 volumes of BN buffer  added with EDTA-free complete protease inhibitor cocktail, . After lysis on ice for 1 h, samples were centrifuged at 17,000× escribed  using 60Electrophoresis and immunoblotting were performed as previously described . BrieflyWe have previously provided evidence that serine phosphorylation in the extended C-portion may play a crucial role in AQP4ex water transport function .To evaluate whether phosphorylation is actually occurring in AQP4ex and to determine which amino acid is involved in the C-terminal extension of human AQP4ex, we first conducted a DNA and protein search of different genomes. The analysis showed that the C-terminus extended sequence is conserved among the vertebrate subgroups: fish, reptiles, birds, amphibians and mammals A.331 and Ser335) are only partially conserved among subgroups. In particular, among those analyzed, the wall lizard  and the mouse have Ser335 with the consensus motif conserved. Instead, among the analyzed species, Ser331 was not present or included in the consensus motif. Thus, to investigate the role of phosphorylation within the extended sequence, a multiple alignment was limited to that between species among some mammals and primates. Interestingly, we found that only one phosphorylation motif is conserved, which relates to Ser335  motif  are pres335 in mouse, rat and human, we decided to generate antibodies using a phosphopeptide (DRTESRQD-PSer-LELSSC) specific for the human form of p-AQP4ex. Antibodies were purified by affinity chromatography using the phosphorylated peptide, and specificity evaluated by indirect ELISA against the phosphopeptide and unphosphorylated peptides  and with aspartates  in an attempt to change the phosphorylation of serines in the AQP4ex C-terminal extension.For the phosphopeptide, the limiting dilution was 1:512,000, whereas it was 1:8000 for the unphosphorylated peptide. These data demonstrate that anti p-AQP4ex in cells expressing WT-AQP4ex, while both mutations affected p-AQP4ex antibody binding   . The pho in mice . The num111 in the cytoplasmic loop B [180 in loop D [111, which has been reported by other authors as not involved in AQP4 phosphorylation and water transport regulation [In the present study, we describe for the first time the identification of a functionally active phosphorylation site of AQP4 located in the extended portion of the human AQP4ex. Several phosphorylation-dependent pathways have been proposed for AQP4 water flux regulation, trafficking and subcellular localization ,26. In lc loop B  and Ser1n loop D , which hgulation . This cogulation ,17,29,30gulation ,32.331 and Ser335) located in the extended portion of human AQP4ex [335 appeared to be conserved in primates and rodents  with arginine (R). However, this modification belongs to a group of amino acids with similar properties, suggesting that in the course of evolution it was necessary to preserve the specific R-Q/R-DS sequence for an important physiological function. This phosphorylatable site could be modulated by a specific kinase whose selectivity is determined by residues close to the serine [335 is most likely to be the principal candidate for human AQP4ex phosphorylation. Thus, to verify this hypothesis we generated an antibody for the phosphorylated Ser335. Using several approaches we show here, for the first time, that AQP4 is constitutively phosphorylated in the human brain through the expression of AQP4ex at Ser335. Immunofluorescence experiments showed that p-AQP4ex is mainly expressed at the astrocyte processes surrounding brain vessels, overlapping the location of AQP4ex. The phosphorylation shown by immunoblot and immunofluorescence suggest an important role for post-translational modification in the basal condition. Mutational studies indicate that phosphorylation of human AQP4ex may decrease water channel activity [We recently reported the potential role in water channel activity of two serines  indicating that this modification could have a role in AQP4ex localization/anchoring at the perivascular pole and directly affect AQP4 complex stability. In the light of a recent study in which we reported that AQP4ex is critical in triggering the progressive mislocalization and downregulation of AQP4 in glioblastoma [p-AQP4ex is involved. Furthermore, it will be of interest to evaluate if p-AQP4ex is involved in gliosis, as recently reported for AQP4ex [BN-PAGE experiments showed the presence of blastoma , it willr AQP4ex .331 is not conserved in mouse and rat, it cannot be excluded that this second serine may play a role in human AQP4 as a second level of regulation by phosphorylation of this important region of AQP4. Further studies are required to answer this important point.Although Ser335 in the human brain. Similar to non-phosphorylated AQP4ex, p-AQP4ex is organized in supramolecular assemblies at the perivascular astrocytic domain abutting brain microvessels. Our data suggest that p-AQP4ex is part of the molecular functional unit of AQP4 at the blood-brain barrier level.In conclusion, we have shown that AQP4ex is highly phosphorylated at Ser


P. vivax. However, the highly polymorphic nature of this protein often misdirects immune responses, leading them to be strain-specific. Details of cross-reactive humoral immunity to DBPII variants have therefore become an important focus for the development of broadly protective vaccines. Here, cross-reactive humoral immunity against a panel of Thai DBPII variants (DBL-THs) was demonstrated in immunized BALB/c mice and P. vivax patients, by in vitro erythrocyte-binding inhibition assay. Sera from immunized animals showed both strain-transcending (anti-DBL-TH2 and -TH4) and strain-specific  binding to DBL-TH variants. Using anti-DBL-TH sera at 50% inhibitory concentration (IC50) of the homologous strain, anti-DBL-TH2 sera showed cross inhibition to heterologous DBL-TH strains, whereas anti-DBL-TH5 sera exhibited only strain-specific inhibition. In P. vivax patients, 6 of 15 subjects produced and maintained cross-reactive anti-DBL-TH inhibitory antibodies through the 1-year post-infection timepoint. Cross-reactive memory B cell (MBC) responses to DBL-TH variants were analyzed in subjects recovered from P. vivax infection (RC). The plasma samples from 5 RC subjects showed broad inhibition. However, MBC-derived antibodies of these patients did not reveal cross-inhibition. Altogether, broadly anti-DBP variant inhibitory antibodies developed and persisted in P. vivax infections. However, the presence of cross-reactive anti-DBL-TH inhibitory function post-infection was not related with MBC responses to these variants. More detailed investigation of long-lasting, broadly protective antibodies to DBPII will guide the design of vivax malaria vaccines.Duffy binding protein region II (DBPII) is considered a strong potential vaccine candidate of blood-stage  Plasmodium vivax is a major causative agent of malaria associated with high morbidity, particularly in tropical and subtropical regions  x 100. High inhibition (HI) was considered inhibition greater than or equal to 80% and less than 80% was defined as low inhibition (LI). Experiments were done in triplicate wells and were repeated two times.All pEGFP plasmids containing genes encoding different DBL-TH haplotypes were constructed by site-directed mutagenesis, following a previous protocol . To assein vitro binding inhibition. First, 50% inhibition concentrations (IC50) of anti-DBL-TH2 and -TH5 sera were determined by using undiluted and serially diluted antisera (1:1000–1:6000). Then, each antiserum at its IC50 was subsequently used for testing strain-transcending inhibition against a panel of DBL-TH variants by EBIA, as described above. The cross-reactive inhibition was defined when the inhibition of heterologous strain was not significantly different from that of homologous strain. All experiments were performed in triplicate and were independently repeated two times. Mouse anti-DBPII inhibitory monoclonal antibodies (2D10) characterized in a previous study were used as positive control [To demonstrate the immunogenicity of DBL-TH antigens in inducing cross-reactive antibody responses, anti-DBL-TH2 and -TH5 antisera representing broadly reactive and strain-specific antibodies, respectively, were assayed for  control  and pre-P. vivax plasma (diluted 1:200) at acute and 1-year post-infection in cohort study and RC subjects in cross-sectional study. Functional inhibition against a panel of DBL-TH antigens was classified as high inhibition (HI) if inhibition was greater than or equal to 80%, or lower inhibition (LI) if the assay result was less than 80%. Samples with HI to more than 3 strains of DBPII were classified as cross-inhibitory plasma. To analyze the inhibition end-point titers to a DBL-TH variant, broadly inhibitory plasma samples (n = 4) were titrated from 1:200 to 1:6400 and tested for inhibition. The end-point titer was defined as the last dilution that had greater than or equal to 80% inhibitory activity. The cross-inhibition between two variants was defined once the difference in end-point titers between those variants was less than or equal to two-fold. Plasma (n = 2) of subjects who displayed high inhibition against DBL-TH variants, as characterized in our previous study [EBIA was performed to assess the inhibitory activity of us study , were usP. vivax subjects who had recovered from infection for 1–3 months (RCs) and harbored cross-reactive inhibitory antibodies in circulating blood  were used to detect broad MBC responses to DBL-TH variants. PBMCs (1 x 106) from these RC patients (n = 5) and HCs (n = 4) were transferred into 24-well plates containing 1 mL of culture medium (RPMI + 10% FCS) and subsequently differentiated into antibody-secreting cells (ASCs) with stimulation of R848  and IL-2  at 37°C, 5% CO2 for 11 days, as previously described [Since cross-reactive inhibition against DBL-TH variants was observed at 1-year post-infection, escribed . In thisP. vivax-exposed, or plasma (diluted 1:200) of healthy donors for 1 h at RT. After washing, horseradish peroxidase-conjugated goat anti-human IgG secondary antibodies  at 1:2000 dilution were added into the wells and incubated at RT for 1 hour before addition of TMB substrate. Absorbance was measured at 405 nm. All the samples were assessed in duplicate and mean values were used in the analyses. Acute-phase plasma samples (n = 3) of the vivax malaria patients who showed high OD values to DBL-TH variants, as characterized in previous study [Antibody responses to DBPII antigens were measured by indirect ELISA, as previously described . Brieflyus study , were usP. vivax-infected patients (n = 2) were used as positive control. Culture supernatant of non-stimulated wells were used as background control.To detect total IgG levels in culture supernatant, 1μg/ml of anti-human IgG MT91/145  was coated on 96-well plates and held overnight at 4°C. After blocking, culture supernatant (undiluted) was added into coated wells followed by detection with horseradish peroxidase-conjugated goat anti-human IgG secondary antibodies. Signal was developed with TMB and read at 405 nm. Plasma samples from To assess the inhibitory effect of MBC-secreted immunoglobulins, culture supernatants of 3 patients , of the 5 whose plasma showed cross inhibition, were chosen as representative of inhibition against 5, 4 and 3 DBL-TH strains, respectively. Undiluted culture supernatants were added into the wells to test inhibition against DBL-TH-2 -TH4, -TH5 and -TH9, and reference strain Sal I, following the above described EBIA protocol. Plasma (n = 2) of RC subjects who displayed high inhibition against DBL-TH variants were used as positive control. Only culture medium was used as negative control.P. vivax-infected subjects were cultured with R848 and recombinant human IL-2  at 37°C in a 5% CO2 incubator for 96 h. Then, the ELISPOT assay was performed by coating with 15 μg/mL of anti-human IgG MT91/145 (Mabtech), 5 μg/mL of DBL-TH and Sal I antigens, or 1 μg/mL of tetanus toxoid (TT) antigen  onto Multi-Screen IP plates (Merck Millipore). Cells were seeded in duplicate to yield 5 × 104 cells per anti-human IgG-coated well and 1 × 106 cells per antigen-coated well. After overnight incubation, 1 μg/mL of detection antibodies MT78/145 (Mabtech) was added and the mixture incubated for 2 h at RT. After washing, streptavidin-HRP at 1:1000 dilution (Mabtech) was added; plates were incubated for 1 h, then TMB substrate added. Plates were then rinsed with deionized water and analyzed with a Bioreader 5000 ELISPOT Reader. A positive ELISPOT response was defined as detectable spots in duplicate wells with the total spots in the specific antigen-coated wells being at least twice the number of spots detected with the negative control samples. The antigen-uncoated wells were used as negative controls for DBL-TH-specific ASC analysis.The presence of DBL-TH-specific MBCs was determined with an ELISPOT assay, performed as previously described . In brieStatistical analysis and figure presentation were performed using GraphPad Prism version 8.4.3 . One-way analysis of variance and multiple comparison analysis by Bonferroni test were used to analyze and compare the differences in antibody reactivity and percent inhibition. Differences were considered statistically significant when p≤ 0.05.The antisera from mice immunized with DBL-TH2, -TH4, -TH5, -TH6 or -TH9 were used to evaluate the presence of cross-reactive and/or variant-specific antibodies against a panel of DBL-TH variants after antibodies were depleted out by homologous antigen. Compared to homologous strain after complete depletion, the recognition of anti-DBL-TH2 antibodies to each heterologous strain was comparable without differing significantly . Anti-DBTo further analyze the presence of strain-transcending inhibitory antibodies against a panel of DBL-TH antigens, mouse anti-DBL-TH2 and -TH5 antisera  were used to test for inhibitory function. First, IC50 values of anti-DBL-TH2 and -TH5 antisera against erythrocyte binding were determined and found to be 10 μg/ml and 11.49 μg/ml, respectively . FurtherP. vivax infection, antibody titers and functional inhibition to DBL-TH variants and reference Sal I were tested in plasma collected during acute infection. As expected, all P. vivax-infected patients had higher antibody reactivity to tested antigens than did healthy controls  and three subjects in group B (B1-B2). From group A, 4 samples  maintained cross inhibition against at least three DBL-TH strains. Whereas, 5 patients  showed only short-lived broad inhibition  in the cohort study (groups A and B) after they recovered from their hibition . In grouhibition . CollectNext, to support the persistence of cross inhibition of anti-DBL-TH2, -TH4, -TH5 and TH9 antibodies, plasma samples from subjects with broad inhibition patterns  were titrated to compare the strength of inhibitory activity. The results clearly revealed that the plasma from A1 (PV04) showed no cross-reactivity to a panel of DBL-TH variants, while plasma from A2 (PV02), A3 (PV08) and B1(PV05) showed broad inhibition. Subject A2 (PV02) showed broad inhibition against DBL-TH2, -TH4 and -TH5, while subjects A3 (PV08) and B1 (PV05) displayed broad inhibition to DBL-TH2, -TH4 and -TH9 .P. vivax infection 1–3 months previous were studied, plasma samples from 5 patients showed broad inhibition to a panel of DBL-TH antigens  recovered from antigens . As abovin vitro stimulation cultures. The ASCs were assessed to determine the frequency of DBL-TH-specific ASCs and total IgG levels in culture supernatants. Our results showed that five RC patients harbored MBCs which produced antibodies with specific activity to a panel of DBL-TH variants and the reference strain Sal I  were differentiated into ASCs by in Sal I . Moreovemulation . By usinmulation .Plasmodium vivax [P. vivax (DBL-TH) were taken to interrogate the presence of broadly reactive antibodies in immunized mice. In parallel, long-term responses of cross-reactive anti-DBL-TH inhibitory antibodies were assessed in malaria-experienced patients. Few of the immunized mice produced anti-DBL-TH antisera with broad reactivity against DBL-TH variants. The monitoring broadly anti-DBL-TH inhibitory antibodies in P. vivax patients found that some individuals maintained cross-reactivity after parasite clearance. Although cross-reactive anti-DBL-TH inhibitory antibodies were present in plasma, MBC-derived antibodies from these P. vivax patients showed no broad inhibition against DBL-TH variants. These data could greatly assist the development and the use of DBPII-based vaccines.The highly polymorphic nature of DBPII impedes the development of a broadly effective vaccine against um vivax . Thus, mum vivax , 26, 27.P. vivax infections.An ultimate goal of an effective PvDBPII-based vaccine is to induce immune responses which inhibit all PvDBPII variants present in malaria-endemic areas . A previP. vivax [P. vivax infection was demonstrated. P. vivax-infected subjects  who were followed up for at least 12 months, maintained cross-inhibition to DBL-TH variants but not to the reference Sal I. Remarkably, DBL-TH2 and -TH4 were the most common variants shared in the cross-inhibition profile of studied subjects. This finding indicated that certain P. vivax-infected individuals were capable of mounting and maintaining inhibitory antibodies specific to multiple Thai DBPII haplotypes widespread in studied endemic area, but not to the reference Salvador 1 strain. Analyses of functional inhibitory antibodies to naturally occurring DBPII alleles are required for best design of efficacious and broadly-protective DBPII-based vaccine strategies. DBP-based vaccines containing multiple DBL-TH2 variants might be essential for protection against the multiple strains of P. vivax in Thailand and Southeast Asian.Naturally occurring anti-DBPII inhibitory antibodies that are cross-reactive to DBPII variants are documented in individuals who live in areas with low and seasonal transmission of P. vivax , 14. A sP. vivax . Here, pP. vivax infection. Our findings revealed that patients who developed strain-transcending inhibitory antibodies did not produce broadly reactive MBC-derived antibodies against DBL-TH variants. Possible explanations of the absence of concordance between circulating cross-reactive inhibitory antibodies and MBC responses to DBP variants include: (i) primary P. vivax infection triggered cross-reactive naïve B cells to activate and generate ASCs specific to DBL-TH variants with cross-reactive inhibitory anti-DBL-TH antibodies [Memory B cells are important for sustaining humoral immunity to malaria . Better tibodies ; (ii) trtibodies ; (iii) stibodies . It has tibodies –33. ThusP. vivax infection and the time of sample collection. Since strain-transcending immunity to DBPII develops only after repeated exposure to P. vivax [P. vivax patients or immunized animals for the production of monoclonal antibodies are warranted to analyze broad inhibition against a panel of DBPII variants.Limitations of this study include the relatively small number of patients in the cohort study of long-term broad inhibitory antibodies generated by vivax malaria. The lacking of association between broadly anti-DBL-TH inhibitory antibodies and cross-reactive MBC response to DBL-TH variants could be influenced by the period of P. vivax , 35, theP. vivax-infected human. Some patients maintained cross-reactive inhibitory antibodies through the convalescence phase for at least a year, whereas MBC-derived IgG exhibited only a limited breadth of inhibition towards DBL-TH variants. The insights into cross-reactive immunity will allow improvements in the design strategies of conserved epitope-targeting vaccines that are protective against diverse strains of P. vivax in the future.In summary, our study demonstrated the presence of broadly inhibitory anti-DBL-TH antibodies in both mice immunized by DBL-TH variants and S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 TablePolymorphic residues within DBPII and positions with reference to DBPII-Sal I (bold) are indicated. Conserved residues are represented by a dot (.).(DOCX)Click here for additional data file.S1 File(DOCX)Click here for additional data file.


Proteasome activator 28γ (PA28γ/REGγ) is a member of the 11S family of proteasomal regulators that is constitutively expressed in the nucleus and implicated in various diseases, including certain cancers and systemic lupus erythematosus. Despite years of investigation, how PA28γ functions to stimulate proteasomal protein degradation remains unclear. Alternative hypotheses have been proposed for the molecular mechanism of PA28γ, including the following: (1) substrate selection, (2) allosteric upregulation of the trypsin-like (T-L) site, (3) allosteric inhibition of the chymotrypsin-like (CT-L) and caspase-like (C-L) sites, (4) conversion of the CT-L or C-L sites to new T-L sites, and (5) gate opening alone or in combination with a previous hypothesis. Here, by mechanistically decoupling gating effects from active site effects, we unambiguously demonstrate that WT PA28γ allosterically activates the T-L site. We show PA28γ binding increases the Kcat/Km by 13-fold for T-L peptide substrates while having little-to-no effect on hydrolysis kinetics for CT-L or C-L substrates. Furthermore, mutagenesis and domain swaps of PA28γ reveal that it does not select for T-L peptide substrates through either the substrate entry pore or the distal intrinsically disordered region. We also show that a previously reported point mutation can functionally switch PA28γ from a T-L activating to a gate-opening activator in a mutually exclusive fashion. Finally, using cryogenic electron microscopy, we visualized the PA28γ-proteasome complex at 4.3 Å and confirmed its expected quaternary structure. The results of this study provide unambiguous evidence that PA28γ can function by binding the 20S proteasome to allosterically activate the T-L proteolytic site. DespiteTrypanosoma brucei (PA26) 13, ticks   . Regulati.e., regulatory particle or 19S) utilize ATP to recognize, unfold, and translocate substrates into the degradation chamber . Our recombinant human PA28γ in the presence of human 20S proteasome (H20S) induces an almost 20-fold increase in proteolytic activity for the T-L proteolytic site-specific peptide substrate (RLR-AMC) when compared to H20S only controls , as was similarly reported in Gao et al . These results replicate past observations that the PA28γ-K188E mutation alters PA28γ activity and likely switches it to a gate-opening form (similar to PA28αβ). This is because gate opening is expected to stimulate the degradation of all peptides regardless of their proteolytic site specificity, as it should freely allow the entry of all peptide substrates. These results clearly demonstrate that PA28γ stimulates the proteasome differently than PA28αβ and that the K188E mutation alters how it stimulates 20S function. These results can be explained by several possible mechanisms used by PA28γ that are listed in the introduction. It is also not clear from these results alone if WT PA28γ can function via both gate-opening and T-L–like activation or if these mechanisms could function mutually exclusively in WT PA28γ.Before specifically asking which hypothesis PA28γ uses to activate 20S function, we first sought to validate the activity of our purified recombinant PA28γ, as previous groups indicated different PA28γ functions that were deemed to be dependent on the specific purification strategy employed , 34. As Fig. 1B). Importantly, WT yeast 20S respond similarly to activation by PA28αβ and PA28γ as do human 20S . However, if the activator’s regulatory mechanism included allosteric changes to the proteolytic sites, this could be clearly observed enzymatically in the absence of a functioning gate. Our results demonstrate that there were no changes to the fold activation of α3ΔN-Y20S in the presence of PA28αβ and PA28γ-K188E , which demonstrates and confirms that PA28αβ and PA28γ-K188E indeed function to open the proteasomal gate allowing peptide to diffuse in and do not themselves affect the activity of proteolytic sites. However, PA28γ induced ∼15-fold activation of the α3ΔN-Y20S in the presence of the T-L substrate RLR-AMC, when compared to the α3ΔN-Y20S controls and did not inhibit or alter the degradation of CT-L or C-L substrates (LLVY-AMC and nLPnLD-AMC) . It is important to note that the cleavage rates of the peptide substrates vary between each peptide and that the T-L peptide, RLR-AMC, has the lowest Vmax when compared to the CT-L and C-L peptides . α3ΔN-Y20S has an N-terminal truncation mutation to the α3 subunit of the Y20S that alleviates the closed gate conformation induced by the N termini of the WT Y20S α-subunits  Fig. 1BBFig. 1B.uman 20S . The α3Δpeptides . These dA). When tested in a proteasome activity assay using WT H20S or α3ΔN-Y20S, the results reveal that PA28γ2XCys has similar activity patterns to that of PA28γ-K188E or PA28αβ and opens the proteasomal gate without inducing T-L activation (E and F). As cysteine residues can form disulfide bonds, we tested these mutants under both reducing, noncrosslinked and oxidizing, crosslinked conditions. Under both conditions, PA28γ2XCys stimulated all three activities in the WT 20S but did not stimulate the open channel 20S indicating that it induces only gate opening. One caveat is that PA28γ2XCys did not stimulate gate-opening as well as did PA28γK1888E, presumably due to a less efficient conformational state induced by changing hydrophilic pore residues to hydrophobic ones. Nevertheless, these results further demonstrate that pore stabilizing mutations in PA28γ, whether salt-bridge, hydrophobic, or covalent in nature, can switch PA28γ from an allosteric activating state to a gate-opening state.Disulfide crosslinking has been established to be a useful tool in studying proteasomal activators’ structure and function, particularly for homomers, such as the archaeal proteasomal AAA+ ATPase, PAN . BrieflyFig. 1A). However, PA28γ stimulation of the T-L substrate, RLR-AMC, was essentially unaffected in the presence of epoxomicin. Alternatively, in the presence of the β2 inhibitor, leupeptin, our data demonstrated that T-L activity was diminished 94 to 100% in the absence or presence of all the indicated 11S activators, including PA28γ where activity went from 30-fold stimulation  to no stimulation . Our results definitively show that PA28γ′s ability to activate T-L activity does not require a functional CT-L site and cannot simulate T-L activity if the T-L–like site is inhibited. Therefore, PA28γ does not function by altering CT-L catalytic activity, as has been suggested for the PA200 activator.While intuitively one would think that an increase in T-L like activity is likely due to an increase in the proteolytic activity of the T-L proteolytic site, this may not necessarily be the case. A recent cryo- EM study of the human PA200-20S complex determined that PA200’s upregulation of T-L activity is the result of conformational changes to the S1 pocket of the β5 subunit to change from CT-L activity to T-L activity . TherefoFig. 2m or Vmax for any of the three peptide substrates  of the T-L site by PA28γ. These results demonstrate that binding of PA28γ to the 20S increases both the catalytic affinity and the maximum proteolytic rate of the T-L site, without affecting the CT-L– or C-L–like activities. Therefore, PA28γ can use a nongating activation mechanism, which is mechanistically distinct from PA28αβ. While it is tempting to speculate that these results strongly suggest allosteric activation of the T-L site, similar results could possibly be obtained if PA28γ allowed the passage of some peptide substrates differently than others to function as a substrate filter or sieve based on peptide charge. We will investigate this last alternative mechanism next.The results from our inhibitor data led us to ask the question of how PA28γ or PA28αβ changes proteolytic activity for each active site. To answer this question, we wanted to monitor the kinetic changes of each proteolytic site in the presence of either PA28γ or PA28αβ. Using the α3ΔN-Y20S, we were able to effectively answer this question as we can directly decouple the effects of activator binding and gate-opening from changes to the proteolytic sites. We performed dose responses of CT-L (LLVY-AMC), T-L (RLR-AMC), and C-L (nLPnLD-AMC) peptides to monitor kinetic changes of α3ΔN-Y20S proteolysis in the presence of either PA28γ or PA28αβ. Compared to the α3ΔN-Y20S controls, PA28αβ demonstrated negligible changes to the Kbstrates . This obi.e., it swaps IDR domains). A PA28α-γ swap was also created but did not heptamerize under our conditions and so could not be analyzed. The PA28γΔIDR is a deletion of PA28γ′s IDR but includes an 8-residue serine–glycine linker to limit the introduction of steric hinderance that a complete loss of the IDR would likely cause. When we tested the ability of these two PA28γ variants to activate 20S function, we found that both the PA28γ-α and PA28γΔIDR upregulated T-L substrate degradation, similar to WT PA28γ, for both H20S and α3ΔN-Y20S. In addition, these two IDR variants showed no change in activity for the C-L or CT-L substrates in the presence of either H20S or α3ΔN-Y20S, also similar to WT PA28γ (B and C). Based on these results, we can conclude that PA28γ′s T-L activation mechanism does not require its IDR domain, and thus this domain must also not confer any peptide “filtering” capacity. In addition, the IDR domain from PA28α does not confer any gate opening activity to PA28γ when swapped.The above results indicate that PA28γ can upregulate T-L activity by a nongating mechanism, but what remains unclear is whether PA28γ functions to exclude entry of some but not other substrates from the proteasome whereby, it could specifically allow entry of T-L peptide substrates . All of WT PA28γ , B and CA). We therefore asked the question if the charges in this pore region could affect which types of peptides are able to pass through PA28αβ. To ask this question, we introduced point mutations into PA28αβ that would mimic the charges in the substrate entry pore of PA28γ. We then asked if these mutations would be sufficient to switch PA28αβ′s activity to that of PA28γ. This experiment was performed for two reasons: (1) to test whether PA28αβ and PA28γ can filter peptide substrates through their substrate entry pores and (2) to implicate helix 3 in the unique functional activity of PA28αβ and PA28γ on the 20S proteasome. As PA28αβ has two separate subunits, we created this mutant by inducing point mutations in helix 3 of PA28α-D183K and PA28β-D173K, which will further be called PA28αβ2XLys (C). A proteasome activity assay using WT H20S revealed that PA28αβ2XLys still stimulates the degradation of all three types of substrates though at a reduced capacity: approximately 2- to 10-fold less activity amongst all peptide substrates when compared to WT PA28αβ (D). Nevertheless, these results demonstrate that PA28αβ2XLys is functional to induce gate opening but importantly does not selectively prevent the passage of some types of peptides, despite its very different pore charges. To confirm this conclusion, we also found that PA28αβ2XLys was unable to stimulate peptide hydrolysis in the α3ΔN-Y20S at any capacity (similar to WT PA28αβ), which also indicates that PA28αβ2XLys maintains its gate opening ability (E) but does not “filter” substrates based on their charge. This result reveals that the generation of a completely positively charged pore in PA28αβ was not able to switch it to a T-L activating complex (like PA28γ), thus demonstrating that differences in pore charge are unable to selectively exclude some types of peptides from entering the degradation chamber. One caveat of this experiment is that mutagenesis of helix 3 in PA28αβ diminished the activator’s ability to induce gate-opening, but it did not change its general function. Based on the structure of PA28γ, there are three different regions where peptide passage through PA28γ and entry into the 20S could be affected: (1) the IDR, (2) the substrate entry pore, and (3) the proteasome gate. We have generated mutants in all three of these locations and found that none of them could change the effect that PA28γ has on the proteasome. Therefore, these results clearly demonstrate that PA28γ does not select for peptide substrates through its IDR or its substrate entry pore and does not specifically open the proteasomal gate for T-L peptide substrates, but in fact, allosterically upregulates the catalytic capacity of the β2 subunits, thus increasing T-L–specific peptide proteolysis.As discussed previously, PA28γ-K188E has a substrate entry pore that mimics the substrate entry pore of PA28αβ and changes the functional mechanism of PA28γ from allosteric activation to gate-opening A. We the8αβ2XLys C. A protT PA28αβ D. Nevert ability E but doeA). This electron density reveals that PA28γ binds to the 20S proteasome using its C termini to dock into the proteasomal intersubunit pockets of the α-ring, like PA26 . The tertiary and secondary structures of PA28αβ and PA28γ also align similarly relative to one another (D and E). Figure 5E also shows a slice through the PA28γ map that includes the PA28αβ model, and it is clear the helix 3 (center most helix) occupies similar space in both homologs. Interestingly, our structure reveals a density in the pore of PA28γ that does not appear in the model of PA28αβ (B and D). This cryo-EM structural reveals that PA28γ adopts a similar tertiary conformation to the fellow mammalian homolog, PA28αβ, even though PA28γ only has 42.1% sequence identity to PA28α and 33.6% identity with PA28β, and even though both activators activate proteasome function in very different ways.Recently, the structures of various 11S family members have been solved using cryo-EM and X-ray crystallography , 40, 41.Fig. 5ike PA26  and PA28ike PA26 , 41 actiike PA26 . Furthere.g., T-L peptides). Further, if PA28γ functioned as a “sieve”, it would also limit CT-L and C-L peptides from entering the proteasome, which would appear as inhibition in the open channel 20S. Our results from Figure 1D clearly demonstrate that PA28γ upregulates the degradation of the T-L peptide but did not inhibit the degradation of CT-L or C-L peptides in the constitutively active proteasome. Moreover, it is reasonable to assume that if PA28γ was using a sieve model, that the IDR surrounding the substrate entry pore may play a role in substrate selection. C–E) demonstrated that this 2XLys mutation did not confer any “sieving-like” properties to PA28αβ and did not change how it activated 20S function. These results clearly rule out contributions from mechanism 1 for PA28γ function. None of our experiments could rule out mechanism 2—allosteric activation of the T-L like site. Mechanism 3 or allosterically inhibition of CT-L or C-L activity (combine with gate-opening—mechanism 5) to increase T-L degradation is ruled out by A and E show that the equilibrium kinetics of CT-L and C-L peptides remain unchanged when PA28γ is bound to open channel protesasome complex demonstrating that PA28γ cannot inhibit these active sites . Together, m) and increased the Vmax of the T-L like site for RLR-AMC , inducing a 13-fold increase in catalytic efficiency of the T-L site. These results suggest that PA28γ can allosterically activate the T-L proteolytic site. Mechanism 4 is ruled out by results shown in C and D) demonstrates that it does not need to induce gate opening to stimulate T-L activity (ruling out mechanism 5). Taken together, our results clearly demonstrate that PA28γ, through long-range interactions (A), can allosterically affect the β2 subunits of the 20S proteasome to upregulate its T-L enzymatic activity. These results suggest that one biological function of PA28γ in the nucleus could be to upregulate the T-L catalytic activity of β2, as it has been shown that even with the highest affinity peptide substrates catalysis of T-L substrates is far lower than that of C-L or CT-L substrates  specific substrate selection, (2) allosteric upregulation of the T-L catalytic site, 3) allosteric inhibition of the CT-L and C-L catalytic sites, (4) conversion of the CT-L or C-L sites to new T-L catalytic sites, and (5) gate-opening in combination with a previous hypothesis. Our results effectively ruled out mechanism 1 that the PA28γ could function as a substrate selective “sieve” and allow only entry of only peptide substrates with positive charges. The “sieve” model would require that PA28γ is able to open the 20S gate, but only allow entry of select peptides . This indicates that there must be other differences between PA28αβ and PA28γ that compensate for their mechanistic differences besides pore stability. Although mutations that increase helix 3 interactions in the pore of PA28γ can cause it to switch activity, it is not known if WT PA28γ, under physiological conditions, can similarly switch states as part of its normal function to degrade proteins, though it is tempting to speculate that this switching mechanism could be a component of how it regulates protein degradation, which is currently not understood.WT PA28γ has two basic residues on helix 3 (K188 and K192), while WT PA28αβ has a basic (lysine) and acidic (aspartate) residue in the homologous positions of PA28α (D183 and K187) and PA28β . Further, the overall structure of the PA28γ-20S proteasome complex revealed that PA28γ docks its C termini into the intersubunit pockets of the proteasome’s α-ring, a mechanism paralleled by other 11S regulators, and does not bind the β ring directly to upregulate T-L proteolysis. We also revealed a density unique to PA28γ that occupies the substrate entry pore. It is expected that this density is its IDR, and although we could not resolve this region, it supports the idea that this region is highly disordered. Our structure allows us to visually conclude that PA28γ in general has many overall qualities that align with the 11S family of regulators. While the application of C7 symmetry to the PA28γ-20S complex is reasonable and useful since PA28γ is a homoheptamer, the same symmetry application to the heteroheptomeric 20S averages out its pseudosymmetry, thus causing loss of asymmetric structural information, which would be required to observe structural changes in the β2 subunits. Unfortunately, the particle number obtained was too low for 3D reconstruction without symmetry application. Lesne et al.’s hydrogen-deuterium exchange coupled to mass spectrometry study complements our biochemical results and supports our hypothesis that PA28γ uses a distinct proteasomal activation mechanism from other 11S regulators, while structurally preserving similar tertiary and quaternary protein structures.Based on our observations, we hypothesize that PA28γ′s positively charged substrate entry pore allows it to adopt a conformational that can generate the long-range allosteric effects that allow it to activate the 20S T-L site. In support of this notion, a recent study by Lesne to PA28γ . Our strThe PA28γ-20S complex has recently been demonstrated to degrade unfolded proteins  and is k4 gradient. Eluted fractions with significant proteolytic activity were pooled and dialyzed, and B20S purity (>95%) was determined using SDS-PAGE and densitometry . Concentration was determined using a Bradford assay with bovine serum albumin as the reference protein. Eukaryotic α3ΔN-Y20S were expressed and purified from yeast using anion exchange chromatography, as described with minor modifications (Bovine 20S (B20S) proteasomes were purified from bovine liver as described . Liver wications  Briefly,ications .E. coli and purified using strong anion exchange , followed by hydroxyapatite , and finished with SEC . Recombinant PA28γ was expressed in Rosetta E. coli and purified using Ni-NTA affinity resin (Qiagen) and followed with SEC , as previously described , Y20S (1 nM), H20S (1 nM), and α3ΔN-Y20S (1 nM) were all assayed using fluorogenic peptides, as previously described , using aAssays were performed under reaction conditions and protein concentrations similar to proteasome activity assays. B20S proteasomes were incubated in reaction buffer with either PA28αβ, PA28γ, or PA28γ-K188E, and either epoximicin (100 nM) or leupeptin (40 μM) was subsequently added . Assays were controlled for using replicates without inhibitors read simultaneously.CAT/KM) was calculated as FC in VMAX/KM because KCAT = VMAX/[E], and the [E] was the same in both conditions used to calculate FC.Dose response assays were done following a modified proteasome activity assay protocol. Briefly, α3ΔN-Y20S (1 nM) was put into a proteasome activity assay buffer alone or with either PA28αβ (50 nM) or PA28γ (62.5 nM). Fluorogenic substrate was added in a dose-dependent manner . Assays were analyzed for the MaxV at each concentration, and dose responses were analyzed using GraphPad Prism9. The fold change (FC) in catalytic efficiency (KWT PA28γ and PA28γ2XCys were assayed using a modified protocol . Briefly2 was saved as a movie with each movie being dose fractioned into 40 frames within a target defocus range of -2.5 to -1.25. All data were collected using cryoSPARC software (Thermo Fisher).Copper Quantifoil R 1.2/1.3300 mesh (EMS) grids were cleaned and treated with amylamine using a PELCO easiGlow Glow Discharge cleaning system. PA28γ and H20S were mixed at a 1:1 M ratio, and 3 uL of the sample mixture was placed onto a grid. Grid was subsequently blotted by hand using blotting paper and immediately flash frozen in liquid ethane using a manual plunge freeze apparatus. Data collection was done using a Titan Krios transmission electron microscope (Thermo Fisher) operated at 300 kW and a magnification of x81,000, which resulted in 1.08 Å/px. Images were collected using a Falcon IIIEC direct electron detector camera equipped with a K3/GIF system operating in counting and super resolution modes. Electron dose per pixel of 50 e-/ÅB). Three thousand three hundred fifty eight particles primarily side view orientation  from single and double cap in PA28 years-20S 2D classes were used to generate five ab-initio models to further remove junk particles . One of the resulting ab-initio models produced a PA28 years-20S complex from 876 particles, which were used for homogeneous regiment for 3D classification with C7 symmetry applied using the map from prior determined ab-into model (C and D). The gold standard (0.143) Fourier shell correlation resolution was calculated form 2 half maps (Cryosparc) to be 4.3 Å . All representations of the PA28γ-20S proteasome complex were created using UCSF ChimeraX .This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Storstrømmen and L. Bistrup Bræ are 20‐ and 10‐km wide, surge type glaciers in North Greenland in quiescent phase that terminate in the southernmost floating ice tongue in East Greenland. Novel multi‐beam echo sounding data collected in August 2020 indicate a seabed at 350–400 m depth along a relatively uniform ice shelf front, 100 m deeper than expected, but surrounded by shallower terrain (<100 m) over a 30‐km wide region that blocks the access of warm, salty, subsurface Atlantic Intermediate Water (AIW) at +1.6°C. Conductivity temperature depth data reveal waters in front of the glaciers at −1.8°C not connected to AIW in the outer fjord, Dove Bugt. The recent grounding line retreat of the glaciers is attributed to glacier thinning at its ablation rate, with little influence of ocean waters, which illustrates the fundamental importance of knowing the bathymetry of glacial fjords. Storstrømmen Glacier, Greenland  stands in cold waters behind shallow bathymetryThe frontal regions are 100 m deeper than expected but access of warm Atlantic Intermediate Water (AIW) is limited by a wide and shallow ridgeRetreat of Storstrømmen and L. Bistrup Bræ glaciers has operated relatively independent of changes in AIW temperature in Dove Bugt We combine the AXCTD data with historical CTD data from the Hadley center (bodc.ac.uk) spanning from 1960 to 2017 to document temperature changes in Dove Bugt. We analyze the CTD data in front of the glaciers   GEOMAR Helmholtz Centre for Ocean Research Kiel's Elac SeaBeam 1050, 50 kHz multibeam echo sounder (MBES) which we combined with an interferometric swath sonar for the shallower water (<350 m), the ITER‐SYSTEM’s Bathyswath‐2 117 kHz. The Elac sonar was mounted on the stern of the boat 0.8 m below the water level whereas the Bathyswath‐2 was mounted on the hull of the M/V Wave at an angle of 42° from the vertical. Correction for sound velocity  is performed using real‐time sound velocity sensor mounted on the head of the transducers and CTD data collected at regular intervals  is found below 250 m depth . The ocean is too cold to yield much glacier undercutting.As the glacier does not terminate in a calving cliff, but as a floating ice shelf, it is difficult to assess the impact of the ocean waters on ice melt. Assuming that the undercutting model developed for a vertical face is also applicable at the glacier grounding line of Storstrømmen, we calculate the rate of undercutting as in Rignot et al. . The ratu Figure , thermalThe ocean warming in Dove Bugt of 0.7°C ± 0.2°C is less than the 1.3°C ± 0.5°C warming for Zachariae Isstrøm and Nioghalvfjerdsfjorden between 240 and 500 m depth, but comparable to the 0.85°C ± 0.2°C warming at 300–400 m depth for Humboldt. Due to the presence of broad sills.4A wide region of shallow ground, with numerous islands, protects Storstrømmen and L. Bistrup Bræ from the influence of warm AIW waters at present. The bathymetry reveals an over‐deepening in Borgfjorden, where the two glaciers merged in earlier times and most likely yielded enhanced basal erosion, but the overdeepening does not extend more than 46 km past the current ice front, hence does not form a natural, deep passage for AIW to reach the glaciers from Dove Bugt. Although a number of deep passages are detected in between the islands, we find no natural passage for AIW to enter the inner fjord, as confirmed by the CTD data.The grounding line retreat is well explained by ice thinning at its ablation rate. The same conclusion applies to Soranerbren Glacier, a glacier immediately southeast of L. Bistrup Bræ  outer fjord (Dove Bugt) to the  glacier fronts. At the glacier fronts, the ocean temperature is only −1.8°C, which does not yield much bottom melt of the ice shelf. For Storstrømmen and L. Bistrup Bræ glacier, we are also able to interpret their grounding line retreat as almost entirely caused by ice thinning. In contrast to most other large outlet glaciers in Greenland, the ocean waters do not play a strong role in the evolution of Storstrømmen and L. Bistrup Bræ glaciers in a warming climate.