Osteosarcoma is the most common primary malignant bone tumor, occurring mainly in children and adolescents, and the limbs are the main affected sites. At present, limb‐salvage treatment is considered as an effective basic standard treatment for osteosarcoma of the limb. China has a vast territory, but the development of technology is not balanced,which requires sufficient theoretical coverage, strong technical guidance and the application of limb‐salvage treatment guidelines to the treatment of osteosarcoma. Therefore, to standardize and promote the development of limb‐salvage surgery technology and improve the success rate of limb‐salvage treatment, this guide systematically introduces limb‐salvage techniques for the treatment of patients with limb osteosarcoma through definition of limb‐salvage treatment, surgical methods, efficacy evaluation, postoperative treatment and prevention of complications, rehabilitation guidance, and follow‐up advice. Osteosarcoma is the most common malignant bone tumor, which occurs with a high degree of malignancy and rate of disability in adolescents. The 5‐year survival rate of the natural course of the disease is only 10%–20%. Classic osteosarcoma, or conventional osteosarcoma, is the most common subtype of osteosarcoma, accounting for 80% of all osteosarcomas. It usually occurs at the distal end of the femur and proximal end of the tibiaIn the past 30 years, significant progress has been made in China in the diagnosis and treatment techniques for osteosarcoma. Surgery is recognized as an effective basic treatment for primary and recurrent metastatic osteosarcoma. The 5‐year survival rate of conventional osteosarcoma has been significantly improved, with the tumor‐free survival rate reaching 60%–70% and the overall survival rate reaching 60%–80%All patients with suspected osteosarcoma should be referred to the osteosarcoma diagnosis and treatment center or institutions with a specialized osteosarcoma diagnosis and treatment system before biopsy, and the hospitals that implement the limb‐salvage treatment should have a team of experts in imaging, pathology, intervention, and other disciplines for the diagnosis of bone tumors, as well as a specialized department for bone tumor and an oncology department with experience in chemotherapy for osteosarcoma. The attending doctor is responsible for implementing the overall treatment plan of the patient and multidisciplinary team consultationChina is a vast territory, but access to technological advances is not equitable across all regions of the country, so it is necessary to provide sufficient theoretical coverage and strong technical guidance as well as apply limb‐salvage treatment guidelines to the treatment of osteosarcoma. Accordingly, in April 2018, members of the bone oncology group of the Chinese Orthopaedic Association jointly discussed the relevant information and developed this guide in accordance with the principles of science, practicability, and progressivenessLimb‐salvage therapy refers to a series of treatments, such as neo adjuvant chemotherapy, limb‐salvage surgery, and adjuvant chemotherapy, which are completed through the joint efforts of a multidisciplinary team of doctors. The purposes of limb‐salvage treatment are to reduce local recurrence as much as possible to retain good limb function and improve the survival rate of patientsLimb‐salvage surgery refers to the surgical procedure to restore bone and joint function after extensive resection of malignant bone tumors of the limbs.Neoadjuvant chemotherapy refers to the preoperative chemotherapy after the definite diagnosis of malignant bone tumorsTumor margin refers to the continuous integrity of the normal tissue adjacent to the tumor and the reaction area. The design of the surgical margin depends on the tumor margin, which has a barrier effect to prevent local invasion of the tumor and is a key factor in determining the prognosisThe surgical margin of the tumor refers to the actual extent of the tumor specimen to be removed. Extensive resection refers to the removal of the area of normal tissue surrounding the tumor response layer. After extensive resection, the local control rate of the tumor can reach more than 90%. The aim of surgical excision for limb‐salvage is to minimize the loss of normal tissue while obtaining a safe and tumor‐free surgical margin, so as to retain the best function of the limbSafety margin refers to the normal bone tissue in the longitudinal and/or transverse membranous and medullary cavity 3–5 cm away from the margin of the tumor, as well as the fascia, tendon, joint capsule, articular cartilage, blood vessel, nerve sheath, and outer membrane that are not invaded by the tumor, and where no tumor tissue was detected under the microscopeThe diagnosis of osteosarcoma of limb bones mainly relies on a combination of clinical, imaging, and pathological examination. The relatively constant cooperation of the multidisciplinary team participating in the diagnostic process can make the diagnosis and differential diagnosis more accurate. The composition of multidisciplinary teams and the division of tasks are shown in Table X‐ray: AP and lateral X‐ray of the primary lesion.Computed tomography : lesions and chest; chest examination require thin layer + coronal position.Magnetic resonance imaging (MRI) examination: weighted T1 and T2 and enhanced MRI.Bone scan: whole‐body + radioactive concentration region in TomoScan.It should be noted that positron emission tomography (PET), CT, and MRI examinations are optionalBiopsy is necessary for definitive diagnosis before treatment, and coarse needle biopsy is recommendedTypical limb‐salvage treatment methods for patients with osteosarcoma include neoadjuvant chemotherapy, limb‐salvage surgery, and adjuvant chemotherapy. The specific treatment process is shown in Fig. Besides controlling the primary lesion and killing distant micro metastases as soon as possible, the purpose of neoadjuvant chemotherapy is to reduce the tumor and surrounding inflammatory edema reaction area, in order to facilitate subsequent limb‐salvage surgery. The sensitivity of the tumor to chemotherapy was found to lay the foundation for further development of individualized postoperative chemotherapy regimensNeoadjuvant chemotherapy also has risks. For example, some patients have more lesions and/or reduced physical health after receiving neoadjuvant chemotherapy, making radical tumor resection impossible.In sequential or combined use, each patient should choose at least two or more drugs to be administered intravenously or intravenously according to the instructions. The initial dose is calculated according to the dose of the standard regimen, and the total dose intensity is maintained as long as possible. Under the premise of closely observing the effect of chemotherapy, it is recommended to use each drug for at least two cycles. Interval medication is required according to the selected standard regimen.CF, IFO.Tetracycline , Platinum , HDMTX‐Common non‐adjacent chemotherapy regimens:2 d1 + Cisplatin [DDP] 75 mg/m2,Q3W).AP regimen .MAP regimen DIA regimen .APIM regimen .IEP regimen  >60.There is a strong desire to retain limbs and limb functions.Good soft tissue coverageEn bloc resection and complete resection of bone and soft tissue containing the tumor, that is, complete sleeves of normal muscle and soft tissue, and interpretative visual thickness not less than 1 cm (based on MRI) are, albeit, still controversial. The safe margin of bone resection is 3 cm away from the tumor margin as shown by MRIAvoiding local recurrence is the standard for successful limb‐salvage surgeryBone and joint reconstruction surgery is a surgical treatment based on the safe edge, allowing the combined application of a variety of reconstruction techniques. When the tumor enters epiphysis or an adjacent joint, the joint and joint capsule need to be excised. Local muscle flap reconstruction and adequate coverage of the wound with normal soft tissue should be considered.The lower limbs of children can be extended 1–1.5 cm at a time, as far as possible to take into account the growth and development potential of childrenIt is recommended to apply the pre‐surgical design based on digital technology, which is conducive to the accurate resection of the tumorLimb‐salvage surgery allows the use of a variety of reconstruction techniques in combination or on their own, including tumor endoprostheses, autologous or extended allograft bone reconstruction, and soft tissue reconstruction at the site of tumor resection.(i) Tumor endoprosthesisTumor endoprosthesis is the most commonly used technique for limb‐salvage reconstruction(ii) Autograft bone or large segment allograft bone reconstructionLong segment allograft: the limb‐salvage rate can reach 90%. The reconstruction success rate is related to the reconstruction site, and segment reconstruction success rate can reach 82%–84%in vitro or in vivo and then replanted in situ. It is generally considered to be an autologous bone graft. There are many inactivation methods, and there is no evidence of high‐level efficacy for most of them. Relatively, there is more clinical evidence for radio‐inactivation and freeze inactivation. The rate of bone end healing after treatment is 88%, which is higher than that of allograft, and has advantages in anatomical matching and soft tissue attachmentInactivated reconstruction: tumor segment is inactivated Allograft bone or inactivated bone combined with artificial joint: this approach can reduce the complications of articular cartilage degeneration caused by osteoarticular allograft, and is beneficial to soft tissue adhesion. It has advantages in limb‐salvage reconstruction of the proximal humerus, femur, and tibia. The complication rate is 23%Fibula transplantation: it is recommended that free fibula with blood vessels be combined with other repair materials to reconstruct long segmental bone defects, especially for lower limb long bone resection with a length of more than 15cm, in patients who are over the age of 18. Using allograft bone, or other biomaterial segment grafts, combined with free‐fibula grafts with blood vessels can significantly reduce the incidence of complicationsBone transport: For children who need to preserve epiphyses or joints can benefit, but indications are limited. Long segment bone transport may lead to complications, such as pin tract infection, limited movement of adjacent joints, and non‐union of bonesReconstruction of bone defects after tumor resection using autograft and/or allograft bone, with long‐term reliability bone reconstruction relying on bone‐to‐bone healing, including joint preserving reconstruction and joint fusion.(iii) Soft tissue reconstructionThis method should be performed simultaneously with bone and joint reconstruction, including the reconstruction of patellar ligament and attachment, joint capsule repair and other tissues related to joint stability, and it is recommended to complete soft tissue coverage in one phase as far as possible.Adjuvant chemotherapy after limb‐salvage surgery is an important component of the treatment of osteosarcoma. Surgery combined with neoadjuvant and adjuvant chemotherapy can improve the clinical efficacy of patients with conventional osteosarcomaThe aim of adjuvant chemotherapy is to eliminate subclinical lesions, reduce or delay distant metastasis and recurrence, and improve the efficacy of limb‐salvage therapy. The rate of tumor necrosis should be determined according to the pathological samples after the operation, and the efficacy of neoadjuvant chemotherapy should be evaluated to determine the adjuvant chemotherapy. To be sure the tumor necrosis rate and imaging results of the assessment is not exactly the same. The tumor necrosis rate for III‐IV level (necrosis area of ≥90%) patients with postoperative adjuvant chemotherapy can continue to use the neoadjuvant chemotherapy regimens, while the necrosis rate of I‐II level (necrosis area of < 90%), postoperative adjuvant chemotherapy need to adjust the chemotherapy regimen, but no decisions need to be made about whether to help improve overall survival. Postoperative adjuvant chemotherapy is generally no less than three cycles.The 10‐year limb‐salvage rate of epiphyseal preservation was 90%–97%This technique is suitable for children with osteosarcoma of the diaphysis or epiphysis, and neoadjuvant chemotherapy is effective. Preoperative evaluation of the relationship between tumor edge and epiphysis plate and epiphysis should be performed based on MRI. At present, the San Julian imaging method is widely used to determine the invasion of epiphyseal bone tumors in children. Type I is the absolute indication when the tumor is adjacent to the epiphyseal plate and the distance between the tumor edge and epiphyseal plate is more than 2 cm. Type II: when the distance between tumor and epiphyseal plate is less than 2 cm or adjacent. Type III is a part of the contact between the tumor and epiphyseal plate, which is more than 2 cm away from subchondral bone of the joint end. Types II and III are relative indicationsThis method is applicable to the bone defect after the resection of an osteosarcoma of the distal femur or the proximal tibia in children in the developmental stage, and the residual growth capacity is expected to be <4cm.The expected growth capacity of the limbs is calculated by referring to the methods of Anderson and PaleyLong‐term retrospective studies showed that this method had a higher incidence of complications, with the most common one being soft tissue complication (46%), followed by structural failure of the prosthesis (28%), infection (17%), and aseptic loosening (8%). The average extension was 4.4 times, and the average treatment of related complications was 2.5 timesThis method is suitable for reconstruction of defects after resection of osteosarcoma of the distal femur and proximal tibia in children younger than 11 years old. A semi‐knee prosthesis with biaxial motion trajectory can theoretically reduce the wear of the metal prosthesis on tibial articular cartilage in childrenComplications of any type of limb‐salvage reconstruction are common, with an overall incidence of 20%–30%Allograft bone: with an infection rate from 9% to 25%. Recently reported long‐term clinical studies showed an effective rate of 18% after debridement and antibiotic treatment. In 72% of the cases, allograft bone was removed and artificial body weight was used, and the reinfection rate was 12%Artificial joint prosthesis: the infection rate of lower limb tumor endoprostheses is 8%–10%, with most of the infections occurring within 2 years post operation, 70% of the deep infections occurring within 12 months post operation. Once infection occurred, the amputation rate was 23.5%–87%The risk of local infection after limb‐salvage surgery exists for a long time, the postoperative infection rate is 8%–15%, with the most common being staphylococcal infectionIn the case of neoadjuvant chemotherapy, extensive resection and long‐segment tumor metal prosthesis implantation are the high risk factors for postoperative limb‐salvage infection. The use of antibiotics is recommended according to the class of wound. The drug is selected according to the guiding principles for the clinical application of antibiotics (2015 edition). It is suggested to refer to the wound drainage time when calculating the usual time of antibiotics.The incidence of non‐union and fracture of allograft bone is 12%–63% Aseptic loosening of the prosthesis intramedullary needle is the main complication of distal femur tumor endoprosthesis replacement, and the incidence rate is 5%–11%The mechanical failure rate of the prosthesis is low, about 3%‐6%. The component fracture of the prosthesis, dislocated hinge device, and damage of the gasket are all defined as mechanical failure of the prosthesisLimb‐salvage treatment has the risk of local tumor recurrence, and the local recurrence rate is about 5.4%~10%. Local recurrence of osteosarcoma after limb salvage has an impact on the overall survival rate of the patients. The 5‐year tumor‐free survival rate is 10%–40%. The prognosis of patients with recurrence within 2 years after operation of conventional high‐grade osteosarcoma is poor33 beyond the normal tumor margins. Recurrent lesions >5 cm with metastases were independent factors for poor prognosisMulti‐factor analysis showed that the risk factors for local recurrence of osteosarcoma were failure to achieve safe surgical margin, poor histological response to chemotherapy and tumor growth during chemotherapyThe Musculoskeletal Tumor Society (MSTS) efficacy scoring system for limb‐salvage surgery is recommendedIncluding local and systemic control, it is recommended to use the efficacy evaluation standard of solid tumor version 1.1, and there is no high‐level evidence at present.Take the active exercise as the main activity, the passive exercise as the auxiliary. In addition to local fixation for tendon reconstruction, functional exercise was feasible 24 h after surgery. The specific method of functional exercise should be determined according to the surgical site and reconstruction methodAdjuvant chemotherapy after limb‐salvage surgery is an important part of the treatment of conventional osteosarcoma. Adjuvant chemotherapy can be administered after wound healing, and chemotherapy is recommended to be administered within 3 weeks after surgery. Studies have shown that delay in postoperative chemotherapy, especially in patients with poor histologic response to non‐adjuvant chemotherapy, increases the risk of local recurrenceth year follow‐up periodThe patients were followed up every 3 months for the first 2 years after limb‐salvage treatment. In the third year, the patients were followed up every 4 months. In the fourth and fifth years, the follow up was every 6 months. Eventually, the patients were followed up once a year from the fifth year of the 10Jing‐ping Bai, Wen‐zhiBi, Lin Cai, Zheng‐dong Cai, Tong‐wei Chu, Yang Dong, Wei Guo, Zheng Guo, Yong‐qiang Hao, Hong‐bo He, Yong‐cheng Hu, Ling Jiang, Zhi‐hong Li, Hao‐miao Li, Jia‐zhen Li, Jian‐min Li, Ya‐ping Li, Jian‐hua Lin, Xiao‐hui Niu, Guo‐fan Qu, Guan‐ning Shang, Zeng‐wu Shao, Jing‐nan Shen, Xiao‐dong Tang, Chong‐qi Tu, Guo‐wen Wang, Jin Wang, Zhen Wang, Su‐jia Wu, Jian‐ru Xiao, Zhi‐ping Yang, Zhao‐ming Ye, Zong‐sheng Yin, Xiu‐chun Yu, Chun‐lin Zhang, Guo‐chuan Zhang, Qing Zhang, Wei‐bin Zhang, Yong Zhou.


In their recent study, Garnett and colleagues (Gonçalves  CRISPR‐drug‐killing profiles in the Cancer Dependency Map.Deciphering the mechanism by which small molecules act in human cells is challenging. In their recent study, Garnett and colleagues present an approach to tackle this problem by correlating  Deciphering the mechanism(s)‐of‐action (MoA) by which small molecules act in human cells is key to identifying why patients do or do not respond to treatment, for developing next‐generation molecules with improved efficacy and selectivity and for identifying and preempting mechanisms of resistance. Yet, doing so is highly challenging.et al, For small molecules emerging from target‐based discovery campaigns, it is assumed that the answer revolves around the biochemical perturbation of the target. Despite this assumption, polypharmacological effects may play significant roles. Reciprocally, small molecules that emerge from phenotypic cellular‐based screens have historically required laborious biophysical approaches to unmask their MoA. A highly cited example is the elegant discovery of the MoA of the natural product rapamycin: the FKBP12 protein that disrupts mTOR signaling  in which hundreds of molecularly characterized cancer cell line models have been similarly subjected to genome‐wide RNAi or CRISPR (Tsherniak et al, et al, et al, To address this hypothesis, the authors leverage recently emerging data from the Cancer Dependency Map (The premise in this proof‐of‐concept manuscript that focuses on established cancer drugs with largely known MoAs is that the correlation in viability between one of ∼17,000 genetic knockouts and one of 397 established drugs across 484 diverse cell lines should rediscover the MoA. While examples of success have been reported, a broad‐scale study of this new use of the Cancer Dependency Map data has only become possible recently.Through an extensive series of supervised linear regression analyses, they demonstrate the merits of this approach. They find that in 26% of cases, the killing pattern of the drug is directly phenocopied by the CRISPR killing pattern of the known drug target. They investigate the 264 cases in which this is not the case and find, using protein–protein interactions, 76 additional examples in which the small molecule's killing pattern correlates with a first, second, or third order interactor. Thus, in aggregate, the authors conclude that either the target or pathway can be rediscovered in roughly 48% of cases.The authors next investigate some interesting examples. For instance, in the example of isoform‐specific PI3K inhibitors, the expected genetic knockdowns indeed correlate. In the case of MCL1 inhibition, they discover an exciting relationship between the MARCH5 E3 ligase and MCL1 inhibition. This finding has now been confirmed by other groups including reports of the MARCH5‐dependent degradation of the MCL1/NOXA complex. This discovery should prove very interesting for the therapeutic exploitation of the MCL1 dependency in many human cancers.et al, Finally, the authors explore concordant biomarkers that explain the sensitivity of both genetic and small molecule perturbations. They show how these biomarkers can inform MoA, along the lines of previous work focused on small molecules (Rees et al, This manuscript shares some similarities to a recent report (Corsello Additionally, the regression methods used in this paper may not fully account for the notion that most small molecules have diverse polypharmacological effects that together may conspire to explain the mechanism of killing across various cellular contexts. As the scale of the Dependency Map dataset grows, future computational approaches may be leveraged to produce consensus matches that aggregate individual targets in more complex ways.With these caveats aside, this report provides new insight into how valuable the Cancer Dependency Map is likely to be as a reference to guide MoA studies throughout the drug discovery process Fig . In the


Integral membrane proteins (IMPs) control countless fundamental biological processes and constitute the majority of drug targets. For this reason, uncovering their molecular mechanism of action has long been an intense field of research. They are, however, notoriously difficult to work with, mainly due to their localization within the heterogeneous of environment of the biological membrane and the instability once extracted from the lipid bilayer. High‐resolution structures have unveiled many mechanistic aspects of IMPs but also revealed that the elucidation of static pictures has limitations. Hydrogen–deuterium exchange coupled to mass spectrometry (HDX‐MS) has recently emerged as a powerful biophysical tool for interrogating the conformational dynamics of proteins and their interactions with ligands. Its versatility has proven particularly useful to reveal mechanistic aspects of challenging classes of proteins such as IMPs. This review recapitulates the accomplishments of HDX‐MS as it has matured into an essential tool for membrane protein structural biologists. Through the microscope or with the help of other biophysical tools, structural arrangements can be observed or deduced. This becomes a challenging task when the molecule of interest is embedded in a biological membrane. Structural biology of integral membrane proteins (IMPs) often implies extraction from the membrane, a process far from trivial.A detailed understanding of the molecular mechanism of any protein of interest is required to link structure to function. Deciphering the molecular mechanism requires the integration of structural information with dynamic information. In other words, one has to be able to monitor different motions the protein undergoes during a functional cycle, and to identify the allosteric networks associated with signal transmission. In that regard, HDX‐MS can provide invaluable information.There has been a recent surge in the application of HDX‐MS to IMPs. This is particularly timely as it correlates with the massive increase in structures resolved by high‐resolution methods, such as cryogenic electron microscopy (Cryo‐EM) and X‐ray crystallography.In this review, we will focus exclusively on efforts that employ HDX‐MS to the study of IMPs. Other recent reviews have wonderfully recapitulated the recent technical progresses made by the technique and the nuts and bolts of data interpretation, artefact detection, and prevention. These reviews also provide a great theoretical background of the fundamental principles of HDX which will not be covered here.A word of caution also accompanies this review. As the interest for the technique and its applications expands exponentially, we are constantly reminded that the fundamentals of HDX theory are still work in progress.2Detergent micelles remain the gold standard of membrane mimics. The hydrophobic tail of the detergent molecule shields the hydrophobic region of the IMP and the polar region faces the solvent, thus preventing aggregation in solution. Despite differing notably from the biological membrane, detergent micelles allow the isolation of many IMPs in a stable and functional state. The majority of biochemical and biophysical studies of IMPs have been done in detergent micelles and have unveiled the inner workings of many proteins. Specifically, most high‐resolution structures of IMPs have been obtained in detergent micelles.IMPs adopt different forms and functions but three major classes are overrepresented in terms of structural studies: transporters, channels and receptors. Two out of these three classes are also overrepresented in HDX‐MS research works: transporters and receptors. The average high‐molecular weight (~400 kDa or more) and oligomeric state of channels complicate data analysis and currently restricts their suitability for HDX‐MS studies, although it is likely that progresses in automatic data analysis will overcome this limitation.2.1Transporters is the class of IMPs that has been the most studied by HDX‐MS Figure . Their rTransporters can be broadly divided into two main classes: ABC transporters that rely on energy input from ATP hydrolysis to transport their substrate and secondary transporters that harness the energy potential contained in a transmembrane (TM) gradient to power substrate translocation. ABC transporters possess a cytoplasmic region comprising two nucleotide binding domains (NBDs) where ATP binds and is hydrolyzed. The presence of an important solvent accessible region on the transporter also facilitates HDX‐MS studies of this type of IMPs.2.1.1The multidrug exporter BmrA was the first ABC transporter to be studied by HDX‐MS.Similar strategies were used to follow the conformational transition of the eukaryotic ABC transporter P‐gP.It is interesting to note that all three studies highlighted asymmetry between the NBDs of the ABC transporters BmrA and P‐gP. These findings are in line with other spectroscopic studies using on P‐gP using DEER or single‐molecule FRET.2.1.2Aquifex Aeolicus and has long been used as a bacterial model of human homologs, before the recent structure resolution of the human NSS transporters DAT and SERT.+ and leucine) on the conformational dynamics. Furthermore, Rand et al. identified specific motif unfolding, supporting the notion that a transient local unfolding can be necessary for a global conformational transition to happen.LeuT is the prototypical protein from the neurotransmitter: sodium symporters (NSSs) family. This family of transporters has been under the spotlight for decades, because of their role in neurotransmission termination.+ and neurotransmitter dopamine. Specific locations were shown to behave differently upon Na+ and dopamine binding. Specifically, the intracellular loop (ICL) between TM8 and TM9 reports increased HDX upon sodium then dopamine binding. A thorough characterization of the conformational dynamics of SERT was carried out by the same research group. The effect of sodium, substrate serotonin (5‐HT), and inhibitors cocaine, citalopram, and ibogaine used as therapeutic or recreative drugs was systematically studied. This work revealed that EL4 and the T1a regions were consistent reporters of conformational changes, pointing them out as dynamic regions during the transport cycle. These regions present different patterns of HDX in the apo state, substrate (5‐HT)‐bound state, and inhibitor‐bound state. Interestingly, this study revealed opposite HDX patterns for the noncompetitive inhibitor ibogaine compared with competitive inhibitors cocaine and citalopram, indicating that a different mode of inhibition yields different structural dynamics.The human NSS transporters DAT2.1.3Proteins from the major facilitator superfamily (MFS) are ubiquitous—the overall architecture of 12 TM helices separated in two pseudo‐symmetric bundles is found in bacteria and humans, suggesting conserved mechanistic features.2.1.4To the best of our knowledge, the first transporter studied by HDX‐MS was the bovine ADP/ATP carrier bANC1p from the mitochondria.2.1.5A very interesting study showed how the power of HDX‐MS can be harnessed to go beyond structural information and confirm a specific mechanistic hypothesis. The authors solved the structure of the human excitatory amino acid transporter EAAT1 by crystallography.2.1.6+/H+ exchanger NhaA. The structure of NhaA has been obtained at acidic pH where the protein is inactive, and its mode of alternating‐access is not established. HDX‐MS studies at physiological pH 7.5 allowed to monitor the structural rearrangements upon ion binding.Another pioneering study examined the conformational changes of the Na2.1.72+ homeostasis is an elegant demonstration that obtaining a lot of sequence coverage is not always necessary to answer specific mechanistic questions.+ ions for one Ca2+ but the binding sequence of events is difficult to understand. The crystal structure of NCX_Mj revealed four putative ion binding sites, three for Na+ binding sites and one for Ca2+.2+ and Na+. They obtain only 12 peptides but those included 10 of the 12 ion‐coordinating residues. By performing ΔHDX measurements, the authors could confirm that two ion binding sites are binding Na+, one is binding Ca2+ and another one is occupied by a water molecule, a finding in line with computational studies.A study on the exchanger NCX_Mj responsible for Ca2.1.8The conformational dynamics of the multidrug transporter NorM_PS from the multidrug and toxin extrusion (MATE) superfamily were studied using HDX‐MS by Eisinger et al.In conclusion, all the above‐mentioned studies have showcased the value of HDX‐MS as a sensitive tool to follow conformational transitions of transporters in the context of the alternating‐access model. This technique can easily identify how ligand binding and conserved structural motifs control these transitions.2.2One of the first families of IMPs to be studied by HDX‐MS was GPCRs, an interest likely sparked by their pharmacological relevance and the availability of structural data.2.2.1The first study that foresaw the value of HDX‐MS to capture the structural changes upon ligand activation dates back from 2010.3− and GDP, and finally the β2AR‐Gs complex with and without GDP. In agreement with crystallographic and biochemical data, the α5 helix of the Gα subunit undergoes a significant decrease in H/D upon complex formation  recruitment by β2ar.2.2.2CRD).CRD but unfortunately no peptides were detected at the interface region. Nevertheless, all the other peptides showing a change in HDX point toward the same direction: there is a global increase in structural dynamics upon Norrin binding that extends from the linker domain to the intracellular side of the TM region. The most important changes were observed on the linker domain, which corroborates the computational and biochemical findings of the study and pinpoint a role for this region in Norrin signaling.Frizzled receptors are a family of GPCRs involved in Wnt signaling, and most of them bind to WnT ligands. Norrin is a cysteine‐knot growth factor, distinct from WnT ligands, that activates the canonical Wnt pathway by interacting with receptor Frizzled4 cysteine‐rich domain .2.3Membrane proteins from less prominent drug target families were also studied by HDX‐MS, either to understand fundamental biological processes or as model systems for methodological developments. The following examples showcase the wealth of structural and dynamic information that HDX‐MS can provide to tackle more complex systems, such as multimers or multicomponent molecular machineries.2.3.1To the best of our knowledge, the first IMP studied by HDX‐MS was microsomal glutathione (GSH) transferase 1, a homotrimeric peroxidase that catalyzes addition of GSH to electrophilic species. In a seminal study from 2004, Busenlehner et al. used HDX‐MS to follow the conformational changes of the protein upon GSH binding and observed significant changes in HDX upon binding in several regions.2.3.2The second IMP studied by HDX‐MS is cytochrome c oxidase, a redox‐driven proton pump that acts as the terminal electron acceptor in the respiratory chain of aerobic organisms. Protons are distributed between the catalytic site and exit door for pumped protons using specific proton pathways, namely the K and D pathways. The authors carried out HDX‐MS experiments on the enzyme in different oxidation states: Oxidized (O), reduced (R), “peroxy” intermediate (Pm), and “ferryl” intermediate (f). HDX‐MS showed coordinated closing and opening of the K and D proton pathway, consistent with the oxidation state of the enzyme. For example, the enzyme in the R state shows clear HDX on peptides located near the K pathway but less HDX on peptides located on the D pathway.2.3.3Escherichia coli facilitates passive diffusion of water and glycerol into the cytoplasm. Each monomer is made of eight TMs arranged into a pore. The selectivity for water and glycerol is puzzling and cannot be rationalized by inspection of the high‐resolution structure.The glycerol facilitator (GF) was studied by Konermann et al. in 2012. The authors combined HDX‐MS and oxidative labeling to highlight the important role of TM7 in the structural rearrangements enabling function.2.3.4Another great example of the mechanistic insights afforded by HDX‐MS is the systematic investigation of the SecYEG‐SecA complex, involved in the translocation machinery of protein polypeptide chains. In order to understand how nucleotide binding was triggering the conformational changes enabling translocation, the authors performed ΔHDX experiments in the presence and absence of adenylyl‐imidodiphosphate , and ADP.3The biological membrane provides much more than a hydrophobic environment for IMPs. Both long‐range macromolecular effects and short‐range molecular interactions play an important role in the function of embedded proteins.Bicelles are well‐established membrane mimetics that have been extensively used for NMR studies.nanodiscs have become a very popular tool for IMPs biophysical studies. Nanodiscs allow to incorporate a purified IMP into a lipid bilayer of chosen composition surrounded by a protein belt consisting of an ApoA1 derivative.SMA‐based polymers.All these different biomimetics have pros and cons and their successful application will often be protein‐dependent. They offer the advantage to interrogate the structural dynamics of IMPs within an environment matching the native one more closely. They are also used to specifically examine the role of lipid‐protein interactions. However, they bring their own share of complications for their application for HDX‐MS. The presence of lipids implies a thorough clean‐up step prior to the peptides separation which has to be quick enough to reduce back‐exchange Table . Further3.13.1.1The use of HDX‐MS to study nanodisc embedded proteins has been reviewed in detail elsewhere.The first group that tackled the perilous task of performing HDX‐MS on an IMP reconstituted in nanodisc was that of John Engen. They used the human protein gamma‐glutamyl carboxylase as a model system to perform a proof‐of‐principle study in which they describe with great details the different aspects they optimized to obtain relevant structural information.The next protein to be studied in nanodisc was LeuT. Two independent groups reasoned that most of the available literature on LeuT dynamics used rather invasive techniques based on the covalent modification of cysteines which can have functional consequences. H/D labeling is comparatively mild, and, furthermore, gives a global overview of the structural dynamics. Adhikary et al. reconstituted LeuT into a POPG:POPC mix. They compared the conformational dynamics of the WT with a mutant known to favor the IF conformation  nanodiscs and studied in parallel with the protein in detergent micelles, to identify putative differences caused by the lipid environment.E. coli bacteria) or PC lipids (absent in E. coli).E for peptide identification, adequate (~30 μM minimum) concentration of the nanodisc samples and high‐pressure  on‐line pepsin digestion  in bicelles for HDX‐MS structural analysis and reported a major improvement in the sequence coverage, compared with detergent micelles3.2The goal of most structural biologists is to observe the molecular mechanism of their favorite membrane protein in an environment matching that of a cell as closely as possible. The use of detergent to extract proteins from the biological membrane is a step that can be detrimental, even if the protein is reinserted in a bilayer afterwards, as is the case for bicelles and nanodiscs. In that regard, the development of SMA‐based  polymers was a welcome addition to the existing toolbox. It allows to bypass the detergent‐based solubilization step by forming so‐called “native nanodiscs,” directly solubilizing the protein with its native lipid composition preserved.E. coli and growth temperatures) to alter the lipid composition of the biological membrane. The protein in three different environments were extracted from the biological membrane with SMALPs and analyzed by HDX‐MS. In parallel, the exact lipid composition of the SMALPs was determined by lipidomics. This work revealed clear differences in HDX patterns between the different lipid environments, illustrating once again the essential role of the lipids in modulating structural dynamics. Specifically, the decrease in temperature during the bacterial culture leads to increased structural dynamics in specific regions of the protease. This difference is likely caused by the increase in unsaturation in the fatty acid chains which in turn favors a more fluid bilayer.Reading et al. demonstrated the applicability of SMALPs nanodiscs for HDX‐MS analysis.E. coli.E. coli (Table The group of Lars Konermann did pioneering work on the Fo‐F1 ATP synthase from li Table . The autE. coli.Another study performed HDX‐MS experiments directly on outer membrane vesicles (OMVs) naturally released from 4This review provides an inventory of the work that has been done on IMPs by HDX‐MS. The evident conclusion is that the wealth of information afforded by the technique is directly proportional to the level of a priori structural and biochemical knowledge of the system under investigation. In that regard, HDX‐MS is ideally positioned to provide unique molecular insights into mechanistical questions and is likely to be used in a more systematic way in more structural biology studies. As integrative approaches combining biophysical, structural and biochemical data are more and more required to understand fundamental biological mechanisms, HDX‐MS is certain to shine in the set of biophysical tools accompanying the resolution revolution. A recent study on the Cop9 Signalosome—CRL2 supercomplex provided early evidence of how Cryo‐EM and HDX‐MS can go hand to hand for deciphering mechanistic insights otherwise inaccessible by each technique in isolation.The ability to look into complex and heterogeneous systems and extracting a signal unique to the protein of interest (at a peptide level of resolution) without invasive labeling and restriction in terms of size is an unmatched advantage. Improvement of automation and data analysis, peptide assignment, good practice, will hopefully facilitate the widespread use of this technique.Chloe Martens: Conceptualization; investigation; writing‐original draft; writing‐review and editing. Argyris Politis: Conceptualization; funding acquisition; investigation; writing‐original draft; writing‐review and editing.


By using a general linear model for behavioral and fMRI data, dynamic causal modeling for effective connectivity, and other predictive approaches, we found strong statistical evidence for a reorientation effect for horizontal and vertical runs. However, neither neural nor behavioral measures differed between vertical and horizontal reorienting. Moreover, models from one run successfully predicted the cueing condition in the respective other run. Our results suggest that activations in the dorsal and ventral attention networks represent higher‐order cognitive processes related to spatial attentional (re‐)orientating that are independent of directional tuning and that unilateral attention deficits after brain damage are based on disrupted interactions between higher‐level attention networks and sensory areas.Hemispatial neglect, after unilateral lesions to parietal brain areas, is characterized by an inability to respond to unexpected stimuli in contralesional space. As the visual field's horizontal meridian is most severely affected, the brain networks controlling visuospatial processes might be tuned explicitly to this axis. We investigated such a potential directional tuning in the dorsal and ventral frontoparietal attention networks, with a particular focus on attentional reorientation. We used an orientation‐discrimination task where a spatial precue indicated the target position with 80% validity. Healthy participants orientation of spatial attention. In this study we find that attentional (re‐)orientation along both axes rely on very similar processes. However, our brain's capacities to process new data are limited. An effective selection of important over unimportant information is therefore critical to ensure efficient information processing. This selection process, in which salient features of the sensory environment, as well as our internal goals and preferences, are considered, is commonly referred to as selective attention.The allocation of attentional resources is controlled by neural structures that are thought to be organized in two distinct but interacting frontoparietal networks  orienting of attention involves a bilaterally organized dorsal frontoparietal network, encompassing the intraparietal sulcus (IPS) and the frontal eye‐fields (FEF). Converging evidence from functional imaging and transcranial magnetic stimulation (TMS) suggests that these regions may modulate the activity in sensory  cortices to prioritize the processing of stimuli at specific locations in space . Several attempts have already been made to disentangle the neural mechanisms underlying vertical as compared to horizontal attentional orienting. The evidence coming from different neuroimaging studies, however, is inconclusive about the brain regions involved. On the one hand, orienting attention along a horizontal relative to a vertical axis activated the lingual and right precentral gyrus, whereas orienting attention in a vertical dimension involved more pronounced activation in the precuneus, medial frontal cortex, anterior cingulate, and cerebellum  amplitudes and measures of effective connectivity were employed. We used a variant of Posner's spatial cueing paradigm Posner,  in which22.1SD = 0.14) with normal or corrected to normal vision, who gave written informed consent. One participant had to be excluded from both behavioral and fMRI analysis due to noncompliance with the task. Another participant was excluded only from further fMRI analysis due to excessive head movements (translation >3 mm); however, the participant's behavioral data were included in further analysis. The remaining 28 participants  were between 21 to 39 years  old. The ethics board of the German Psychological Association had approved the study. Participants were compensated with 15€ per hour.We recruited 29 right‐handed participants . Stimuli were displayed on a screen that was mounted at the end of the scanner's bore and could be seen by the participant via a mirror (245 cm distance). The mirror was mounted on top of a 32 channel head coil. Participants' task throughout the experiment was to report the orientation  of a target stimulus  using button presses of their left and right index fingers. Participants were instructed to continually fixate a diamond in the screen's center (0.5° wide). Next to the central diamond, empty boxes (1° wide) were presented in all four cardinalities throughout the experiment with their centers at 4° eccentricity. Each trial began with an alerting signal, a 500 ms brightening of the diamond's center, followed by a spatial cue (duration: 200 ms) after 1,000 ms. Brightening and widening of one of the central diamond's corners served as a symbolic cue (arrowhead), indicating the most likely upcoming target location with 80% probability. We informed the participants about the cue validity during the task instructions. After a variable interval of 400 or 600 ms, the target stimulus appeared (duration: 250 ms) in the cued box  or in the box opposite to the cue . Distractor stimuli were presented in the remaining three boxes for the same duration as the stimulus. Distractors were created by superimposing two Gabor patches, which were rotated by 45° and 135°. The resulting pattern matched the target stimulus in intensity and contrast  was calculated by dividing the model's posterior probability by the posterior probability of a null model . Additionally, we compared the model with the highest BF10 against all the other models (main effects and interaction). Following standard conventions, a BF10 > 3 is regarded as positive evidence and a BF10 > 10 as strong evidence in favor of H1. A BF10 < 0.33 is then seen as positive evidence and a BF10 < 0.1 as strong evidence in favor of the null hypothesis .We used a two‐step procedure to test for differences in reaction times and error rates between the vertical and horizontal runs and the effects of valid and invalid cueing. The resulting 2  x 2  design was subjected to a Bayesian implementation of an analysis of variance (BF_ANOVA), treating participants as random factors  sequence (time of repetition (TR) 2.2 s; echo time (TE) 30 ms; flip angle 90°). Each image consisted of 36 transverse slices (recorded in an interleaved and ascending manner), with a voxel size of 3.1 mm x 3.1 mm x 3.3 mm and 3 mm slice thickness (field of view 200 mm). We manually discarded the first 5 images of each run to account for T1 equilibrium artifacts. In addition to the BOLD images, we obtained a structural T1 anatomical image for each participant.The functional and anatomical data were preprocessed using fMRIPrep (version 1.1.1), a standardized and robust preprocessing pipeline  toolbox  The main effect of all invalid versus valid trials ((iL + iR + iD + iU) – (vL + vR + vD + vU)), (b and c) two contrasts for direction‐specific cueing effects: horizontal reorientation (iL + iR) – (vL + vR) and vertical reorientation (iD + iU) – (vD + vU), (d) a contrast for the main effect of direction ((iL + iR + vL + vR) – (iD + iU + vD + vU)), and (e) a contrast for the interaction of cueing and direction ((iL + iR – vL – vR) – (iD + iU – vD – vU)). Additional four tests were performed to show the effects of attentional and perceptual modulation in the visual areas by valid targets. These tests were performed separately for each visual field .p < .001 uncorrected (SnPM: fast option). We report the results of thresholded t‐maps, using a significance cut‐off of p < .05 (FWE corrected at the predefined cluster level). An overview of global and local maxima was created using the function “get_clusters_table” implemented in the Python package Nistats  with a predefined cluster forming threshold of 2.6As we did not find any significant differences in BOLD amplitudes between horizontal and vertical directions, and no significant activations for the interaction of direction and cueing , we conducted a more sensitive post hoc VOI based analyses. Here, we probed bilateral TPJ, FEF, and IPS, which are key regions of the ventral and dorsal attention networks. While these regions do not include the whole network , they are the ones we considered to be most likely influenced by the direction of attention reorienting due to their proximity to the visual areas. Furthermore, we limited the regions included in the network, also with regards to the computational complexity that would arise in the following DCM analyses.The global or local maxima corresponding to the six regions of the condition main effect Table  defined The mean beta values were averaged over visual fields to obtain values for the horizontal and vertical directions. For example, the beta value used for left IPS during invalid horizontal trials consisted of the average extracted beta values from iL and iR. For each VOI, we analyzed whether direction or interaction effects were present using BF_ANOVAs, with the participant as the random factor. We followed the rationale described for the analysis of the behavioral data.−4, 10−3 …, 103, 104]. The regularization parameter that achieved the highest average accuracy in the inner cross‐validation loop was used to refit the logistic regression on all of the training data. The run‐based model performance was then defined as the average accuracy over the splits. A similar approach was used to estimate generalized performance, where five‐fold cross‐validation was used on one run to find the best parameter C, and the accuracy was calculated for the predictions made on the other run.In addition to the BF_ANOVAs, we used logistic regression to test whether brain activity differences between cueing‐conditions of one direction were predictive for the cueing effect in the respective other direction. We again used the mean betas of each participant in the six VOIs for each of the eight regressors , this time not collapsing along meridians. Then, we tested whether BOLD amplitude patterns in the six VOIs of the horizontal run  were similar enough to differentiate valid and invalid trials of the vertical run , and vice versa. This was done using logistic regression implemented in scikit‐learn , refitting the logistic regression and then recalculating the accuracies . The permutation 2.7In addition to differences in BOLD amplitudes, we were interested in the cueing‐dependent effective connectivity patterns in the horizontal and vertical runs. To estimate effective connectivity, we used bilinear DCM . DCM is a state‐space model used to infer the cortical dynamics in time between brain regions. The approach leads to a generative model that, once inverted, can be used to simulate neural activity in the network. The state‐change equation of neural states in DCM is described by Equation z˙ is described by the fixed connectivity matrix A, which represents the coupling between brain regions in the absence of exogenous modulations (u). The coupling can be modulated by the j exogenous inputs (u), which are represented by the parameters in the matrix B (the connections in B are a subset of A). Lastly, the driving input regions, which represent the direct changes of hidden states, is defined by the matrix C. As we were interested in how connection strength differs between invalid trials in the horizontal (u1) as compared to the vertical run (u3), we restricted our analysis to the parameters in the matrices 1B and 3B. Matrices 2B and 4B were left empty, which means that connections were not modulated by valid trials . Since we were interested in investigating potential differences in reorienting of attention , we assumed that connectivity between brain regions in valid trials was the same for both runs .The change in the hidden neural states p < .05 uncorrected), and the time series were adjusted for the nuisance regressors and mean activity. We used the contrast of valid trials against baseline to select the VOIs for bilateral FEF and IPS, and the contrast invalid trials against baseline for the TPJ VOIs.We limited the network analysis to the most representative regions of the classic models of visual spatial attention and extracted the time series in the regions of the VOI analyses. The time series of both runs were concatenated (spm_concat). For the estimation of our DCMs, we defined a new design matrix for each participant. The target onsets of all trials served as driving inputs to the DCM. As in the GLM analysis above, we included the seven motion parameters as nuisance regressors and added run specific intercepts (to center the time series of each run). The VOI coordinates (see Table A) was defined by fully connected intra‐hemispheric regions and inter‐hemispheric connections of homologous regions. All nodes received all driving‐inputs because visual input was carefully matched across conditions and visual areas were comparably activated.The underlying network structure describing the intrinsic coupling during the task B were the same. Hence, while the connectivity parameters could differ, the overall modulation structure by invalid trials stayed the same. Finally, we used BMA on the winning model‐family on a participant level, to get more reliable point estimates for the different connections.Hierarchical family‐wise Bayesian model selection (BMS) implemented in the MATLAB VBA‐toolbox , we simulated the BOLD signal by swapping the inputs (u) between the horizontal and the vertical runs  ↔ iV (3u), vH (2u) ↔ vV (4u)). This approach allowed us to evaluate the specificity/generality of the parameters for horizontal and vertical reorienting of attention. If the model performance with the parameters of the respective other run is comparable to the original data, we can conclude that, regardless of specific parameter values, the neural processes of invalid trials are similar across runs.We tested whether the modulation by invalid trials differed between the vertical and horizontal session by calculating the BFu were kept, but the input streams  were assigned randomly. We report the proportion of participants with permutation P‐values lower than p < 0.05 in the original and swapped conditions. The permutation P‐values were calculated as the proportion of models where the root mean squared error  and in the vertical run . A similar pattern was observed for error rates , similarly so in the vertical run . The BF_ANOVA for reaction times yielded strong evidence only for the main effect of cueing‐condition with a BF10 of 17,275.39 against the baseline model. This model was also superior to the other possible combinations of the 2x2 design . The analyses of the error rates yielded similar results. The model including only a cueing main effect had the highest BF10 against the intercept model (BF10 = 16.8), and also stood out against all other possible combinations of factors . In sum, these analyses show that the main manipulation of the experiment—the reorientation of attention in invalid trials—induced the expected reaction time costs and increased difficulty, as seen in the error rates. Moreover, they provided positive to strong evidence that neither the overall level of reaction times nor the reorienting costs after invalid cueing differed between the horizontal and vertical runs.Participants' reaction times in invalid trials were higher than reaction times in valid trials, both in the horizontal  yielded a cluster forming threshold of k ≥ 58 voxels. Cluster size in cubic millimeter, global maxima, up to four local maxima, and their respective t‐statistics are provided in Table Figure The run‐specific activation maps of reorienting‐related activity are depicted in Figure Tests for main effects of direction (k ≥ 57) and the interaction of direction and cueing (k ≥ 47) did not yield any significant voxels surviving the cluster‐based FWE correction.Our analysis of the attentional modulation in valid trials in relation to the visual fields Figure  revealedhttps://identifiers.org/neurovault.collection:5622).The statistical t‐maps of the GLM analysis can be found on Neurovault in a thresholded and un‐thresholded form . Comparing the cueing‐only effect against the main effect of direction, both main effects, and main effects plus interaction (Table 10 > 3) in most of the VOIs. In the right IPS VOI, however, there was only anecdotal evidence (BF10 = 1.07) favoring the cueing‐only model against the model including both main effects, meaning that we cannot convincingly exclude an additional effect of direction for this region.As a potentially more sensitive approach, we extracted the regression (beta) weights of the main GLM analysis in six regions that showed significant reorienting related activity (see Table p = .021) for the horizontal and with an accuracy of 62.3% (p = .021) for the vertical run. More importantly, the model trained on the horizontal run generalized to the vertical run with an accuracy of 59.3% (p = .016), and the model trained on the vertical run generalized to the horizontal run with an accuracy of 62.0% (p = .006). These results support the observation that the activation patterns in the six VOIs were highly similar, so that those predictive models generalized well across the two runs.Using logistic regression, we tested whether the average beta weights of the eight regressors  could predict the cueing‐condition  in the respective other run. The prediction was significant for each run with an accuracy of 62.2% (3.4ep] = .54) when compared to the other two families . This family was then further subdivided into three families consisting of models describing the direction of the interhemispheric IPS connections. The model family with a modulation from right IPS to left IPS had the highest evidence with an ep of .76 . Finally, the models in this winning family were subjected to BMA. The participant‐specific DCM models with averaged parameter estimates had a good to moderate fit to the data, with a mean coefficient of determination (R2) of 33.74 .The DCM analysis was carried out using data from 26 of the remaining 27 participants, as for one participant, the coordinates for the left TPJ VOI could not be established. To select the DCM with the highest evidence of generating the network activity in our data, we applied a hierarchical familywise model selection. The family with modulations in both hemispheres was slightly superior (exceedance probability [1B) and invalid vertical (3B) trials were compared using Bayesian paired t‐tests. Most modulations provided positive evidence for an absence of differences between both runs .The parameters for invalid horizontal (ns Table . For theSD = 0.08) performed in general better than the random models . Swapping the vertical and horizontal inputs (iH (1u) ↔ iV (3u), vH (2u) ↔ vV (4u)) led to slightly worse performance in each model , when compared to the original data. Still, the swapped model was superior to a random input model for most participants . Using a cutoff of p < .05 , the model with swapped inputs performed better than the random input model in 18 out of 26 participants (69%).Further interrogating the DCMs for each participant revealed that DCMs based on the BMA  of the effects of interest. In the case of Bayes factor ANOVAs, this provided us with the possibility to compare the main effect of cueing against other possible interactions and main effects. For the behavioral measures, this analysis revealed evidence in favor of a model including an effect of attentional reorienting only, without additional interactions. Similar results were observed in the fMRI analyses suggesting a similar neural mechanism of attentional reorienting in different spatial directions.p‐values, strongly suggests that the higher‐order neural mechanisms underlying attentional reorienting are insensitive to different spatial directions.In addition to statistical analysis, we were also able to show that predictive models trained on BOLD data related to attentional reorienting along one meridian generalized well to the other. In other words, the effect of direction was not only statistically insignificant but also had no impact on the generalizability of statistical models—so that the cueing condition in one run could be successfully predicted by the model from the respective other run. This novel analysis approach, which does not rely on classical inferential statistics based on Along the same lines, we also demonstrate that the network dynamics of a DCM between runs were so similar that they could be used to reproduce the BOLD activity patterns induced by attentional reorienting in the respective other spatial direction.Our results replicate the findings of Macaluso and Patria , who alsStill, other studies contrasting vertical and horizontal stimulus layouts have shown direction‐sensitive effects for behavioral and neuroimaging data. For example, differential activity in superior parietal and frontal areas was found in fMRI studies using an attentional cueing paradigm (Mao et al., One reason for these discrepancies might be that horizontal and vertical asymmetries critically depend on the basic perceptual properties of the visual system. For example, it has been argued that horizontal and vertical asymmetries (Rizzolatti, Riggio, Dascola, & Umiltá, However, despite carefully controlling for bottom‐up influences and using an attentional cueing task, Mao et al.  reportedSimilarly, higher activity in the ventral network correlates with worse behavioral performance in valid trials (Wen, Yao, Liu, & Ding, While the previous studies focused on the right TPJ, we observed that invalid cueing heightens TPJ activation in both hemispheres. Similar bilateral involvement of the TPJ has been described previously (Beume et al., The spatial independence of attention networks observed in the present study seems to contradict clinical data: Patients with ventral parietal lesions to one hemisphere are not able to reorient attention to an invalidly or neutrally cued target in the visual field contralateral to the lesion (Posner, Walker, Friedrich, & Rafal, Unilateral lesions to the ventral system may, therefore, lead to dysfunction and imbalance in the reallocation of attention in the dorsal system, resulting in attentional deficits in the horizontal spatial dimension in patients with neglect (Corbetta & Shulman, Further experiments will be necessary to investigate whether the dorsal and ventral attention network interact in the hypothesized way. Despite extensive work using fMRI, for example on the direction coding in IPS (Molenberghs et al., In conclusion, we observed that reorienting visuospatial attention along the horizontal and vertical meridians relies on very similar neural processes in frontoparietal areas of the dorsal and ventral attention network. The absence of direction‐specific effects in the ventral attention network, together with the bilateral involvement of the TPJ, corroborates the notion that this network is involved in higher‐order cognitive processes such as violations of prior expectations, rather than being dependent on stimulus properties, such as its spatial location (Geng & Vossel, The authors declare no competing financial interests.AppendixS1 Supporting InformationClick here for additional data file.


Malaria during pregnancy results in intrauterine growth restriction, fetal anemia, and infant mortality. Women are more susceptible to malaria during pregnancy due to malaria‐induced inflammation and the sequestration of infected red blood cells in the placenta, which bind to the chondroitin sulfate portion of syndecan‐1 on the syncytiotrophoblast and in the intervillous space. Syndecan‐1 is a dimeric proteoglycan with an extracellular ectodomain that is cleaved from the transmembrane domain (referred to as “shedding”) by matrix metalloproteinases (MMPs), likely the secreted MMP‐9. The ectodomain includes four binding sites for chondroitin sulfate, which are proximal to the transmembrane domain, and six distal binding sites primarily for heparan sulfate. This “shedding” of syndecan‐1 is inhibited by the presence of the heparan sulfate chains, which can be removed by heparanase. The intervillous space contains fibrin strands and syndecan‐1 ectodomains free of heparan sulfate. The following is proposed as the sequence of events that leads to and is primarily responsible for sequestration in the intervillous space of the placenta. Inflammation associated with malaria triggers increased heparanase activity that degrades the heparan sulfate on the membrane‐bound syndecan‐1. Inflammation also upregulates MMP‐9 and the removal of heparan sulfate gives MMP‐9 access to cleave syndecan‐1, thereby releasing dimeric syndecan‐1 ectodomains with at least four chondroitin sulfate chains attached. These multivalent ectodomains bind infected RBCs together leading to their aggregation and entrapment in intervillous fibrin. This mechanism suggests possible new targets for anti‐placental malaria drugs such as the inhibition of MMP‐9. Doxycycline is an antimalarial drug which inhibits MMP‐9. CSAchondroitin sulfate ACSCchondroitin sulfate CCSPGchondroitin sulfate proteoglycanDBLDuffy binding likekDakilodaltonsMMPmatrix metalloproteinaseMT‐MMPsmembrane‐type matrix metalloproteinasePfEMP1Plasmodium falciparum erythrocyte membrane protein 1RBCred blood cell1Plasmodium falciparum due to inflammation and cell sequestration in the placenta . P. falciparum causes the most severe disease and is the most deadly among several species of the unicellular protozoan genus Plasmodium  whereas P. vivax infects only reticulocytes and relatively young erythrocytes  but not to the pronounced sequestration of infected RBCs that occur in placental malaria.The virulence of 1.1Anopheles  receptors, that become embedded in the plasma membrane of the RBC γ domains (referred to as VAR2CSA receptors) that can bind to the chondroitin sulfate A (CSA) portion of chondroitin sulfate A proteoglycans which is the target receptor in the placenta. These infected RBCs with VAR2CSA receptors start out as a small portion of the total infected RBCS but are selected for and develop rapidly  association between peripheral malaria and placental malaria , the percentage of 10,032 term pregnancies that were infected with placental malaria was 29.9%  and that the affinity of the infected RBCs for chondroitin sulfate was similar for the three stages of gestation evaluated.Other studies, though, have used methods to detect the presence of placental infected RBCs in peripheral blood prior to delivery. For example, isolates of infected RBCs collected from peripheral blood can be evaluated for transcripts of the 3.3Active infection: Parasites and pigments in maternal RBCs in the intervillous spaces but no pigment or cells within fibrin.Active‐on‐past infection: Parasites and pigment in maternal RBCs and pigment and/or cells within fibrin.Past infection: Parasites not present and pigment and/or cells confined to fibrin.The stages of infection within the placenta have been identified histologically and one system of classification originally developed by Bulmer, Rasheed, Francis, et al. , Bulmer,In Bulmer, Rasheed, Francis, et al. , 121 pla3.4Maternal anemia Brabin, ;Pre‐eclampsia  or the 6‐carbon position (referred to as chondroitin‐6‐sulfate or chondroitin sulfate C [CSC]). In some cases, both chondroitin sulfate A disaccharides and chondroitin sulfate C disaccharides can be included within the same chain. Alternatively, the disaccharide can consist of l‐iduronic acid (instead of glucuronic acid) and galactosamine  with the 4‐carbon position of the galactosamine being sulfated (referred to as chondroitin sulfate B or dermatan sulfate). Chondroitin sulfate proteoglycans (CSPGs) are cell surface and extracellular matrix components.Chondroitin sulfate is actually the name of a family of GAGs. The disaccharide repeating units of chondroitin sulfate can consist of 5.2P. falciparum can bind to chondroitin sulfate A attached to a plastic Petri dish. Fried and Duffy  to a CSPG, decorin, which is a small proteoglycan (MW ~40 kDa) with a single chondroitin sulfate chain. Larger VAR2CSA fragments which contained this 62 kDa segment had somewhat greater affinity including FV2  for which the KD was 5.2 nM.Clausen et al., 5.4Several studies have contributed to the characterization of the binding site(s) on the chondroitin sulfate A ligand. Both Beeson et al.  and PouvAlkhalil, Achur, Valiyaveettil, Ockenhouse, and Gowda  measuredThus, it seems likely that the binding site for VAR2CSA on placental chondroitin sulfate A consists of between 6 and 19 chondroitin disaccharide units.5.55.5.1N‐acetylgalactosamine groups in the GAG chains were sulfated in the 4‐position. In the less common CSPG from the intervillous space, about 1 in 10 of the N‐acetylgalactosamine groups in the GAG chains were sulfated in the 4‐position. There were also two primary CSPGs identified as being cell associated and these were also not fully sulfated with about one‐half of the N‐acetylgalactosamine groups being sulfated.Achur et al.  studied Achur et al., Achur et al.  also obsBeaudet et al.  washed eKD = 37 nM) than that obtained for the binding to the chondroitin sulfate A‐binding region (ID1‐DBL2Xb) by the CSPG decorin (KD = 21.8 nM).Beaudet et al. also evaluated the binding of the GAG preparations from the three regions to the 62‐kDa chondroitin sulfate A binding fragment from VAR2CSA (ID1‐DBL2Xb) studied by Clausen et al. . The aff5.5.2One of the proteoglycans of the endothelial glycocalyx of the human syncytiotrophoblast is syndecan‐1  that is found in human plasma at concentrations normally in the range of 150–400 mg/dL  and serum concentrations of syndecan‐1 ectodomain progressively increase during gestation.Syndecan‐1 in the intervillous space must be the syndecan‐1 ectodomain as intact syndecan‐1 is a membrane protein.Both the cell‐associated and intervillous proteoglycans have substantial chondroitin sulfate but there is substantial heparan sulfate only in the cell‐associated fraction.The lack of heparan sulfate on the proteoglycans in the intervillous space could imply that the syndecan‐1 is not shed until the heparan sulfate on the syndecan‐1 ectodomain has been removed, presumably by heparanase.Heparan sulfate on syndecan‐1 inhibits the shedding of the syndecan‐1 ectodomain, perhaps by forming a canopy that blocks the access of secreted MMPs to the base of the ectodomain.Heparanase degrades the heparan sulfate on syndecan‐1 and is upregulated by inflammation.Heparanase expression also leads to the upregulation of MMP‐9.Inflammation is known to increase syndecan shedding and the upregulation of heparanase and MMP‐9 could be the primary mechanism.8Since syndecan‐1  and the estimate provided here for the prevalence of placental malaria in sub‐Saharan Africa (30%), a high transmission area, could be more than a coincidence. It seems possible that the fibrin that occurs spontaneously in the placentas of some women provides the initial lattice for the trapping of aggregated infected RBCs  and leads to placental malaria. Thus, the presence of intervillous fibrin mesh could predispose women to placental malaria.The finding of pigment or cells (without parasites) within perivillous fibrinoids on the surface of the syncytiotrophoblast in cases of past placental malaria could indicate that these fibrinoids are the product of the intervillous mesh after it has collapsed.The endothelial glycocalyx is complex. Nevertheless, it is proposed that the following cascade of events plays a significant role in the genesis of the sequestration of infected RBCs in the intervillous space in placental malaria  could be effective against placental malaria.P. falciparum malaria in pregnant women (Gaillard, Boxberger, Madamet, & Pradines, Doxycycline is a tetracycline antibiotic that has been used to treat and prevent malaria (Cross, Ling, Day, McGready, & Paris,


Appropriate personal protective equipment should be worn in all clinical settings during the COVID‐19 pandemic because anyone could be carrying SARS‐CoV‐2. Peroral endoscopy is the procedure potentially generating large volumes of aerosols through stimulation of patient coughing. The present study investigated the efficacy of a specially designed acrylic box (Endo‐Splash Protective [ESP] box) for preventing droplet transmission as part of droplet precautions for endoscopists and assistants during routine endoscopy for potential asymptomatic carriers or emergent endoscopy for confirmed or suspected COVID‐19 patients.P = 0.239) or assistant (P = 0.576) positions. We could block the droplets by using ESP box.ESP box was fabricated for use in peroral endoscopy performed with the patient in either a left lateral or prone position. Circular ports were created, one for scope insertion and one for each of the assistant's hands. Simulated droplets were released inside ESP box, and the number of droplets was counted every 100/3 ms for 5 s pre‐release and post‐release at the positions of the endoscopist and assistant. The experiment was repeated eight times at the endoscopist and assistant positions, and the median numbers of droplets were calculated and compared. No significant differences were observed between the median number of droplets counted for 5 s prerelease and postrelease at either the endoscopist (The present findings suggest that use of ESP box during peroral endoscopy may reduce endoscopist and assistant droplet exposure, potentially reducing the risk of droplet transmission to healthcare professionals performing peroral endoscopy during the COVID‐19 pandemic. The present findings suggest that use of ESP box during peroral endoscopy may reduce endoscopist and assistant droplet exposure, potentially reducing the risk of droplet transmission to healthcare professionals performing peroral endoscopy during the COVID‐19 pandemic. Coronavirus disease 2019 (COVID‐19), caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), a novel coronavirus, has spread globally since the initial outbreak in Wuhan City, Hubei Province, China, in December 2019. The World Health Organization declared COVID‐19 a Public Health Emergency of International Concern on 30 January 2020 and subsequently labeled the outbreak a pandemic on 11 March. In Japan, the first patient was reported on 16 January, COVID‐19 was classified as a Designated Infectious Disease on 1 February, and a state of emergency was declared on 7 April.The causative agent of COVID‐19, SARS‐CoV‐2, mainly causes respiratory illness, but it is thought to be less pathogenic than Middle East respiratory syndrome (MERS) or severe acute respiratory syndrome (SARS). The incubation period for COVID‐19 ranges from 1 to 14 days, with symptom onset around 5 days after exposure. Unlike SARS and MERS, COVID‐19 is highly infectious after onset, causing community transmission.Therefore, because anyone could be carrying SARS‐CoV‐2, appropriate personal protective equipment (PPE) should be worn in all clinical settings during the COVID‐19 pandemic.According to the Japanese Society for Infection Prevention and Control guidance for exposure risk assessment and countermeasures for healthcare providers (HCPs),et al.The Gastrointestinal Endoscopy in the Era of the Acute Pandemic of COVID‐19: Recommendations by the Japan Gastroenterological Endoscopy SocietyThe present study investigated the efficacy of a specially designed acrylic box (Endo‐Splash Protective box [ESP box]) for the prevention of droplet transmission as part of droplet precautions for operators and assistants during routine endoscopy for potential asymptomatic carriers or emergent endoscopy for confirmed or suspected COVID‐19 patients.An acrylic box measuring 500 mm (height) × 500 mm (width) × 600 mm (depth) was fabricated from 5‐mm acrylic sheet for use in peroral endoscopy performed with the patient in either a left lateral or prone position . Circular ports were created, one for scope insertion on one side wall  and one for each of the assistant's hands on the opposite side wall and end wall nearest the top of the patient's head . We made the hole for inserting the endoscope into a circular shape in order to improve the operability of the endoscope. A latex glove was taped to the scope insertion port, and a hole was opened in the tip of the middle finger of the glove through which the scope could be inserted into the box. Vinyl arm covers were taped to the two ports for the assistant's hands to access the box. A vinyl curtain was attached to the open side of the box positioned over the patient's shoulders Peroral upper gastroscopy was also performed on healthy volunteers with 1 g of corn starch  placed in front of the mouth, and photos and video images were recorded to visualize and compare simulated droplet dispersion with and without the acrylic box. Images were recorded using a light source  and camera .P‐values <0.05 were considered significant in two‐sided tests. SPSS  for Windows was used for the statistical processing.The Wilcoxon signed‐rank test was used for continuous parameters with a non‐normal distribution. P = 0.239; Fig. P = 0.576; Fig. No significant differences were observed between the median number of simulated droplets counted every 100/3 ms for 5 s pre‐release and post‐release at either the endoscopist  pre‐release to 10.5 (1–25) post‐release.Static images to visualize simulated droplet dispersion during peroral endoscopy performed with and without ESP box demonstrated that droplet exposure occurred without Fig. , but notThe primary route of infection of SARS‐CoV‐2, the causative agent of COVID‐19, is droplet transmission, but contact transmission is also possible. Though symptomatic individuals are the primary agent for transmitting infection, asymptomatic carriers also pose an infection risk.With droplet transmission, infection occurs via direct inhalation of droplets released by patients within a very close range. Comprising aqueous particles, these respiratory droplets are larger and fall much more rapidly than droplet nuclei, remaining in the air for only a short period of time. Droplets released into the air fall onto surfaces and objects within 1 to 2 m of the source. Therefore, for prevention of droplet transmission, precautions for shared air close to the patient are more important than those for the entire room.No significant differences were observed between the number of simulated droplets counted pre‐ and post‐release at either the endoscopist or assistant positions outside ESP box. Particles were visible in the post‐release images; however, dust and other particles were also present within the imaging range before release of the simulated particles; thus, it was possible to demonstrate the preventive efficacy of ESP box by comparing the number of simulated particles counted pre‐ and post‐release. Since this verification was done in an environment with dust and other particles, it was impossible to reduce the number of particles to zero before release of the simulated particles, and the only way to do so was to compare pre‐ and post‐release the droplets. Visual observation also suggested that HCPs exposure to droplets during endoscopy was prevented.In experiments when a small gap opened up between the latex glove and the scope insertion port, the number of simulated droplets clearly increased after release, suggesting that droplet exposure can occur with even small gaps. Based on these findings, the latex glove was attached to ESP box using a commercial drainage channel gasket through which the latex glove was tucked into the scope insertion port, rather than using tape to prevent gaps between the box and the glove Fig. . FurtherIn conclusion, the present findings suggested that use of ESP box during peroral endoscopy may reduce endoscopist and assistant droplet exposure, reducing the risk of droplet transmission to HCPs performing peroral endoscopy during the COVID‐19 pandemic.Video S1. Video to visualize the simulated droplet dispersion during peroral endoscopy.Click here for additional data file.