Intramembrane proteases (IPs) function in numerous signaling pathways that impact health, but elucidating the regulation of membrane-embedded proteases is challenging. We examined inhibition of intramembrane metalloprotease SpoIVFB by proteins BofA and SpoIVFA. We found that SpoIVFB inhibition requires BofA residues in and near a predicted transmembrane segment (TMS). This segment of BofA occupies the SpoIVFB active site cleft based on cross-linking experiments. SpoIVFB inhibition also requires SpoIVFA. The inhibitory proteins block access of the substrate N-terminal region to the membrane-embedded SpoIVFB active site, based on additional cross-linking experiments; however, the inhibitory proteins did not prevent interaction between the substrate C-terminal region and the SpoIVFB soluble domain. We built a structural model of SpoIVFB in complex with BofA and parts of SpoIVFA and substrate, using partial homology and constraints from cross-linking and co-evolutionary analyses. The model predicts that conserved BofA residues interact to stabilize a TMS and a membrane-embedded C-terminal region. The model also predicts that SpoIVFA bridges the BofA C-terminal region and SpoIVFB, forming a membrane-embedded inhibition complex. Our results reveal a novel mechanism of IP inhibition with clear implications for relief from inhibition in vivo and design of inhibitors as potential therapeutics. Bacillus subtilis that is studied often as a model for these types of proteases.Proteases are a type of protein that work by cutting up other proteins. The part of the protease that does the cutting is called the active site. Intramembrane proteases are a specific group of proteases that cut up the proteins within cell membranes. There is a lot of interest in learning how to control intramembrane proteases because they are important in regulating the signaling processes that cells use to communicate. SpoIVFB is an intramembrane protease from the bacterium Bacillus subtilis uses SpoIVFB to produce spores, dormant reproductive cells that can survive extreme, harsh conditions for long periods with minimal energy. SpoIVFB is part of the system that allows spores to communicate with their ‘parent cells’, the cells they develop in. The activity of this protein is blocked by two other proteins called SpoIVFA and BofA. When these proteins are destroyed, SpoIVFB becomes active, but it is unclear exactly how SpoIVFA and BofA inhibit SpoIVFB. Understanding this relationship could help to reveal ways to regulate other intramembrane proteases.Bacillus subtilis. The results show that a region of BofA blocks the area of SpoIVFB that cuts a protein called Pro-σK, which stops SpoIVFB from releasing active σK into the ‘parent cell’. By making genetic variants of BofA, Olenic et al. identified three parts of BofA that are needed to fully inhibit SpoIVFB. A computer model predicts that these three parts give BofA the right shape to inhibit SpoIVFB, and that SpoIVFA helps by forming a bridge between BofA and SpoIVFB.To address this question, Olenic et al. used genetic, biochemical and computer modelling techniques to study how SpoIVFB activity is regulated in This investigation reveals how the intramembrane protease SpoIVFB is regulated by SpoIVFA and BofA. This information could be useful in developing inhibitors for other intramembrane proteases. The next stage will be to make and test artificial inhibitors based on the structures studied here. If successful, these could have applications in areas such as medicine, agriculture, industry and environmental protection. Intramembrane proteases (IPs) are membrane proteins containing a membrane-embedded active site. IPs cleave membrane-associated substrates within a transmembrane segment (TMS) or near the membrane surface during the process of regulated intramembrane proteolysis (RIP) . ReleaseK to σK, which directs RNA polymerase to transcribe genes necessary for endospore formation of Bacillus subtilis  and forespore (FS) compartments  of BofA occupies the SpoIVFB active site cleft. Together BofA and SpoIVFA block access of the N-terminal pro-sequence region (Proregion) of Pro-σ studies , partialK, we engineered Escherichia coli to synthesize variants of SpoIVFB and Pro-σK, and the inhibitory proteins BofA and SpoIVFA, in various combinations. The SpoIVFB variant contains the extra TMS cytTM (K(1–127) lacks the C-terminal half of Pro-σK, but SpoIVFB cleaves Pro-σK(1–127) accurately and the cleavage product is easily separated from the substrate by SDS-PAGE (K(1–127) upon production from a single plasmid  cleavage (To improve inhibition of cytTM-SpoIVFB in K(1–127), cytTM-SpoIVFB, and either GFPΔ27BofA, full-length BofA, or SpoIVFA. pET Triplets containing GFPΔ27BofA (K(1–127), and cleavage product accumulated, suggesting that BofA inhibits synthesis and/or enhances degradation of the other proteins when SpoIVFA is absent. pET Triplet SpoIVFA also exhibited cleavage (K(1–127), and cleavage product accumulated normally, but less SpoIVFA accumulated, suggesting that SpoIVFA synthesis decreases and/or degradation increases when BofA is absent. We conclude that inhibition of Pro-σK(1–127) cleavage by cytTM-SpoIVFB requires both inhibitory proteins in E. coli. Inhibition of full-length Pro-σK cleavage by cytTM-SpoIVFB also requires both inhibitory proteins in E. coli , cytTM-SpoIVFB, and SpoIVFA from pET Quartet plasmids in E. coli.To identify residues of BofA that may play a role in SpoIVFB inhibition, we analyzed an alignment of 70 BofA orthologs . Thirteesubtilis . We madeK(1–127), indicating that these substitutions strongly impaired inhibition of cytTM-SpoIVFB  cleavage, indicating less effect on cytTM-SpoIVFB inhibition in E. coli  prevents SpoIVFA cleavage, so a spoIVB165 single mutant exhibits little or no RIP of Pro-σK, but the double mutant exhibits unregulated Pro-σK cleavage because the additional null mutation in bofA (bofA::erm) relieves inhibition of SpoIVFB. We produced GFPΔ27BofA and the variants in the double mutant under control of the native bofA promoter from the non-native amyE chromosomal locus. We examined the levels of GFPΔ27BofA and the variants, and SpoIVFA, SpoIVFB, Pro-σK, and σK  during sporulation, and we included wild type, the spoIVB165 single mutant, and the spoIVB165 bofA::erm double mutant as controls.To test the effects of the three GFPΔ27BofA variants  during sporulation, we produced each in a  SpoIVFB . The nulK cleavage primarily between 4 and 5 hr poststarvation (PS) in wild type, very little cleavage in the spoIVB165 single mutant, and premature cleavage at 4 hr in the spoIVB165 bofA::erm double mutant (K cleavage at 5 hr (lane 8) than BofA in the spoIVB165 single mutant (lane 4), suggesting that full-length BofA is a slightly better inhibitor of SpoIVFB during B. subtilis sporulation, as observed in E. coli , suggesting that the T64A variant inhibits SpoIVFB, although not as well as GFPΔ27BofA (lane 7). We conclude that the three conserved residues of BofA are important for SpoIVFB inhibition during sporulation.Production of the T64A variant in the double mutant  than the strain producing GFPΔ27BofA T64A (lane 13), so the N48A substitution more severely impairs the ability of GFPΔ27BofA to inhibit SpoIVFB.Since GFPΔ27BofA co-localizes with SpoIVFA and SpoIVFB to the outer FS membrane during sporulation , we exam 3 hr PS . The N48 3 hr PS . Inabili at 4 hr , lane 11 at 4 hr  does not 4 hr (K . The strK(1–127), whose Proregion appears to occupy the SpoIVFB active site cleft  (which can be cleaved by active SpoIVFB) have been used previously (E. coli to coproduce combinations of four proteins and treated cells with the oxidant Cu2+(phenanthroline)3 to promote disulfide bond formation.We took advantage of the sole Cys residue of BofA, C46, near N48 and the middle of predicted TMS2 , to testeviously . We creaeviously . As a neFor MBPΔ27BofA C46 , lane 2,K(1–127) (K(1–127)  . TherefoK(1–127) , even thK(1–127) . MBPΔ27BBased on our initial cross-linking results, we modeled BofA TMS2 in the SpoIVFB active site cleft, and tested predictions of the model using additional disulfide cross-linking experiments . The resK(1–127) from interacting with SpoIVFB in E. coli. We coproduced a catalytically inactive E44C cytTM-SpoIVFB variant with a FLAG2 epitope tag with Pro-σK(1–127), SpoIVFA, and GFPΔ27BofA. We then prepared cell lysates and detergent-solubilized proteins, which we co-immunoprecipitated in pull-down assays with anti-FLAG antibody beads. We observed all four proteins in the bound sample (K(1–127) from interacting with the cytTM-SpoIVFB variant. However, we detected only the cytTM-SpoIVFB variant when the bound sample was diluted tenfold (lane 3) to match the input sample concentration (lane 1), and we observed portions of the other three proteins in the unbound sample (lane 2), indicating that co-purification was inefficient. A negative control with the cytTM-SpoIVFB variant lacking the FLAG2 epitope tag showed no Pro-σK(1–127) in the bound sample, but did show small amounts of GFPΔ27BofA and SpoIVFA (lane 8), indicative of weak, nonspecific binding to the beads.Since our cross-linking results show that BofA TMS2 occupies the SpoIVFB active site cleft, we tested whether BofA and SpoIVFA prevent Pro-σd sample , lane 4,6 tag on Pro-σK(1–127), and we performed both types of pull-down assays  on proteins from E. coli coproducing full-length BofA rather than GFPΔ27BofA with the other three proteins (K(1–127) from interacting with the cytTM-SpoIVFB variant. However, GFPΔ27BofA and SpoIVFA reduced co-purification of full-length Pro-σK-His6 with the cytTM-SpoIVFB variant in both types of pull-down assays (K(1–127)  , suggestK(1–127) predicts extensive interactions between the SpoIVFB CBS domain and the Pro-σK(1–127) C-terminal region (K(1–127) C-terminal region upon oxidant treatment of E. coli coproducing the proteins (K(1–127) susceptibility to cleavage (K(1–127) from interacting with the CBS domain of SpoIVFB in E. coli, consistent with the results of our pull-down assays  (K(1–127) formed a cross-linked complex with single-Cys  cytTM-SpoIVFB variants (SpoIVFB cleaves Pro-σK  and Pro-K(1–127)  between variants . The comK(1–127) variants coproduced in the absence of inhibitory proteins in E. coli. The V20C and K24C Pro-σK(1–127) variants formed abundant complex that increased over time, but the F18C and S21C variants formed much less complex, only slightly more than the Cys-less Pro-σK(1–127) negative control (K(1–127) variants formed much less complex and its abundance did not increase over time (K(1–127) variants. The effects of MBPΔ27BofA (lacking TMS1) and full-length BofA were indistinguishable. These results likely explain why BofA TMS1 is dispensable for most of the inhibitory function of BofA  variants in the absence of inhibitory proteins  in E. coli  (K(1–127) (K(1–127) susceptibility to cleavage (K(1–127)   to guide( 1–127)  can be cK(1–127) . We showcleavage . Finallycleavage , similarK(1–127) . The simK molecule (residues 1–114), referred to as ‘fb.sigk’; (2) full-length SpoIVFB, again modeled as a tetramer, with one molecule each of full-length BofA and parts of Pro-σK (residues 38–114) and SpoIVFA (residues 65–111), referred to as ‘fb.sigk.bofa.fa.’ We omitted residues of Pro-σK and SpoIVFA that we could not place with sufficient confidence. We generated computational models using a similar protocol as described previously , but incK(1–127) (K(1–127) (red). The enlarged view of the SpoIVFB active site cleft shows BofA TMS2 surrounded by SpoIVFB (TMSs labeled 1–6). The top view of the membrane domain emphasizes proximity between BofA TMS2, SpoIVFB TMSs 2–4, and the zinc ion (magenta) involved in catalysis.K(1–127) . The memK(1–127)  . Our modl region . All thrK(1–127) and the SpoIVFB CBS domain. The mechanism of SpoIVFB inhibition is novel in comparison with previously known mechanisms of IP regulation. Structural modeling predicts that conserved BofA residues interact to stabilize TMS2 and a membrane-embedded C-terminal region. The model also predicts that SpoIVFA contacts the BofA C-terminal region and SpoIVFB TMS4, bridging the two proteins to stabilize the inhibition complex. The model has clear implications for relief of SpoIVFB and its orthologs from inhibition during sporulation, as well as for IP inhibitor design.Our results provide evidence that BofA TMS2 occupies the SpoIVFB active site cleft. Both inhibitory proteins block access of the substrate N-terminal Proregion to the SpoIVFB active site, but do not prevent interaction between the C-terminal region of Pro-σPrevious work revealed three mechanisms of IP regulation—substrate localization, substrate extramembrane domain cleavage, and substrate interaction with an adapter protein. Substrate localization to a different organelle than its cognate IP is common in eukaryotic cells . An earlE. coli involves envelope stress regulating site-1 cleavage of the RseA extramembrane domain by DegS, allowing site-2 cleavage by RseP  cleavage and reduced the SpoIVFA and cytTM-SpoIVFB levels upon coproduction  more than MBPΔ27BofA  from interacting with cytTM-SpoIVFB  are well-advanced . TranslaE. coli in a recent study , selectively inhibited RseP in nt study . A lack nt study . The hydnt study  for actint study  may provK(1-127)  . StructuK(1-127) . Such efK N-terminal region and SpoIVFB. Therefore, a cyclic peptide inhibitor may prove to be more effective than a linear one. The desirable characteristics of cyclic peptides as therapeutics, and new methods of producing and screening cyclic peptide libraries, make this an attractive possibility  were collected  and extracts were prepared (Strain BL21(DE3) (Novagen) was used to produce proteins in nsformed  or a sinprepared , then su6 , FLAG2 , GFP  diluted in TBST with 2% milk, 1 hr at 25°C with shaking. Signals were generated using the Western Lightning Plus ECL reagent (PerkinElmer) and detected using a ChemiDoc MP imaging system (Bio-Rad). Unsaturated signals were quantified using the Image Lab 5.2.1 software (Bio-Rad) lane and bands tool in order to determine the Pro-σK(1–127) cleavage ratio or the ratio of disulfide cross-linked complex to the total intensity of the cytTM-SpoIVFB variant monomer, dimer, and complex.Samples were subjected to immunoblot analysis as described . Briefly00), GFP  , GFP  (1:3000), GFP (K  (1:3000), GFP (K  (1:5000)B. subtilis bofA, which are present in the genomes of most endospore-forming bacteria  after chromosomal integration at the amyE locus. Plasmids bearing PbofA-gfpΔ27bofA or its variant, bordered by regions of homology to B. subtilis amyE, were transformed into strain ZR264. Transformants with a gene replacement at amyE were selected on LB agar with spectinomycin sulfate (100 μg/ml) and identified by loss of amylase activity (g for 1 min), supernatants were removed, and cell pellets were stored at –80°C. Whole-cell extracts were prepared as described for E. coli  was excited using a 458 nM argon laser and fluorescence was captured using a BA465–495 nm band pass filter. The lipophilic dye FM 4–64 (1 μg/ml) (AAT Bioquest) was used to stain membranes. FM 4–64 (ex/em ~515/640 nm) was excited using a 515 nm argon laser and fluorescence was captured using a BA560IF band pass filter .K(1–127) cleavage, E. coli BL21(DE3) was transformed with a plasmid, grown in LB (10 ml), induced with IPTG, and equivalent amounts of cells were collected. Cells were mixed with chloramphenicol (200 μg/ml) and 2-phenanthroline (3 mM), collected by centrifugation , washed with 10 mM Tris-HCl pH 8.1 containing 3 mM 2-phenanthroline, and suspended in 10 mM Tris-HCl pH 8.1. Samples were treated with 1 mM Cu2+(phenanthroline)3 or 3 mM 2-phenanthroline (as a negative control) for 15, 30, 45, or 60 min at 37°C, followed by incubation with neocuproine (12.5 mM) for 5 min at 37°C. Cells were lysed and proteins were precipitated by the addition of trichloroacetic acid (5%) and inversion every 5 min for 30 min on ice. Proteins were sedimented by centrifugation  for 15 min at 4°C, the supernatant was removed, and the pellet was washed with cold acetone. The pellets were sedimented by centrifugation  for 5 min at 4°C and the supernatants were discarded. The pellets were dried for 5 min at 25°C and resuspended in buffer  for 30 min at 25°C. Portions were mixed with an equal volume of sample buffer  with or without 100 mM DTT, and were typically incubated at 37°C for 10 min, prior to immunoblot analysis. In experiments with single-Cys V70C cytTM-SpoIVFB E44Q, single-Cys Pro-σK(1–127) variants, and Cys-less variants of BofA and SpoIVFA, samples were boiled 3 min prior to immunoblot analysis, which helped to resolve species (A method described previously  was used species .E. coli BL21(DE3) was transformed with a plasmid, grown in LB (1 L), and induced with IPTG as described above. The culture was split, cells were harvested, and cell pellets were stored at –80°C. Cell lysates were prepared as described (g for 15 min at 4°C) to sediment cell debris and protein inclusion bodies. The supernatant was treated with 1% n-dodecyl-β-D-maltoside (DDM) (Anatrace) for 1 hr at 4°C to solubilize membrane proteins, then centrifuged at 150,000×g for 1 hr at 4°C. The supernatant was designated the input sample and 1 ml was mixed with 50 µl anti-DYKDDDDK magnetic agarose (Pierce) that had been equilibrated with buffer  and the mixture was rotated for 1 hr at 25°C. The magnetic agarose was removed with a DynaMag-2 magnet (Invitrogen) and the supernatant was saved (unbound sample). The magnetic agarose was washed three times by gently vortexing with 500 μl wash buffer , then washed once with 500 μl water. The magnetic agarose was mixed with 50 μl of 2× sample buffer  and boiled for 3 min (bound sample). A portion of the bound sample was diluted tenfold (1/10 bound sample) with 1× sample buffer to match the concentration of the input sample. Samples were subjected to immunoblot analysis.escribed , except g for 2 min at 4°C and the supernatant was saved (unbound sample). The resin was washed three times with 5 ml wash buffer (as above plus 5 mM imidazole), each time rotating the mixture for 10 min at 4°C and sedimenting resin as above. The resin was mixed with 0.5 ml 2× sample buffer and boiled for 3 min (bound sample). A portion of the bound sample was diluted 15-fold (1/15 bound sample) with 1× sample buffer to match the concentration of the input sample. Samples were subjected to immunoblot analysis.Input sample (15 ml) prepared as described above was mixed with imidazole (5 mM) and 0.5 ml of Talon superflow metal affinity resin (Clontech) that had been equilibrated with buffer (as above for magnetic agarose). The mixture was rotated for 1 hr at 4°C. The cobalt resin was sedimented by centrifugation at 708×K (residues 1–114) was subsequently added, which resulted in ‘fb.sigk.’ The ‘fb.sigk.bofa.fa’ model was developed by starting from ‘fb.sigk,’ truncating the Pro-σK N-terminus (residues 1–37), and subsequently adding first BofA and finally SpoIVFA (residues 65–111).The modeling proceeded through stages where initial monomeric models were assembled step-by-step into multimeric complexes guided primarily by the restraints from cross-linking experiments and predicted contacts from co-evolutionary coupling analysis. More specifically, a SpoIVFB monomer was first assembled from the membrane and CBS domains. Two monomers were combined into a dimer and the dimer was assembled into a plausible tetramer. Part of one molecule of Pro-σMethanocaldococcus jannaschii (PDB code: 3B4R) (Thermotoga martima (PDB code: 1O50) (K (residues 40–114) was built based on the structure of RNA polymerase sigma subunit domain 2 (PDB code: 3UGO)  . The stre: 1O50) . The CBSe: 1O50)  and agaie: 3UGO) . SuitablK close to their initial models while allowing subunits to move relative to each other as guided by the restraints. For BofA and SpoIVFA, individual helices were restrained internally but allowed to move relative to each other to find the optimal arrangement in complex with SpoIVFB, again guided by the predicted contact restraints. Contact restraints with respect to SpoIVFB were implemented as minimum distance restraints to the closest residue in any of the four subunits since contact predictions cannot distinguish between chemically equivalent oligomer units. All available experimental cross-links were applied but the list of predicted contacts was edited to exclude certain contacts incompatible with cross-linking data and previous biochemical data on the overall topology of the SpoIVFB-BofA-SpoIVFA complex when embedded into the membrane. Excluded contacts may reflect uncertainties in the prediction method as inter-chain contacts are more difficult to predict reliably. They may also indicate alternate biologically relevant interactions between SpoIVFB, BofA, and SpoIVFA that were not probed in the experiments via cross-linking. Detailed lists of all applied restraints are given in The modeling under restraints was initially carried out using Cα-based coarse-grained models that were allowed to relax under restraints via cycles of energy minimization and short molecular dynamics simulations at elevated temperature as described in more detail previously . RestraiB. subtilis proteins. The contact predictions for the inserted residues were ignored for further modeling.Inter-residue contacts were predicted by trRosetta . A MSA, 2. In addition, Rosetta centroid energy terms were also used: Ramachandran energy (1.0), omega angle potential (0.5), backbone hydrogen bond energy (5.0), and van der Waals energy (1.0) with weights in the parentheses. The minimization was performed for 100 steps. Then, the minimized structure was converted to an all-atom model, and the FastRelax protocol was applied to the model with the Rosetta scoring function (ref2015)   and the  Bacillus subtilis. SpoIVFB activity is tightly controlled, however, its status as a multipass membrane protein has made illuminating the molecular basis of SpoIVFB inhibition challenging. Here, Olenic and colleagues combine genetics, cross-linking, and co-evolutionary analysis to develop a structural model of interaction between SpoIVFB and its inhibitors SpoIVFA and BofA. Given the conservation and importance of this family of metalloproteases, this work should have a broad impact influencing our understanding of the regulation of this class of proteins across the tree of life.A member of a family of metalloproteases conserved in all three domains of life, SpoIVFB is required for development in the spore-forming firmicute,  public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:Bacillus subtilis intramembrane protease SpoIVFB" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, including Petra Levin as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Volker Dötsch as the Senior Editor. The following individual involved in review of your submission have agreed to reveal their identity: Volker Dötsch (Reviewer #3).Thank you for submitting your article "Inhibitory proteins block substrate access by occupying the active site cleft of The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:All three reviewers appreciated the rigorous and thorough nature of the experiments. The only additional experiment requested is to assess protein levels for the mutant variants to determine if they accumulate to wild type concentrations. Additional suggestions to improve clarity and presentation are detailed in the reviews below.Reviewer #1 (Recommendations for the authors):As written the manuscript is dense and challenging to absorb and will need to be revised for a broad audience prior to publication. Below are some comments directed at condensing and clarifying different aspects of the manuscript.Specific comments aimed at making the manuscript more accessible:1. Generally, please change all passive voice to active voice in main text . Stating things in first person will eliminate a significant amount of the extra verbiage and streamline the manuscript.2. Similarly, make sure topic sentences summarize the focus of each paragraph. The rationale for all experiments should be clearly stated.3. Line 17-N-terminal pro region needs to be defined in abstract.4. Line 76--This paragraph is a very long and detailed list of all the results. Please condense to important/key take home messages. There is no reason to include these details here as they are in the following Results section.5. Line 100-Please write out acronyms whenever possible. It took a while to figure out what TMS was. Transmembrane sequence doesn't take that much additional space and does not require reader to remember.6. Line 111. There is a lot of detail provided about a system that didn't end being used. Fine to say that authors developed the pET quartet plasmids to overcome technical issues associated with plasmid copy number. (put the details of construction in methods)7. Line 129-what is meant by partial relief? Please provide fraction/percent relief in text.8. Line 141-please be specific about why the lack of conserved residues allows GFP∆27 to be functional. Unclear from text.E. coli." seems to belong in previous paragraph. It is not a topic sentence.9. Line 147 "The Ala substitutions were made in GFPΔ27BofA and co-produced with Pro-σK(1-127), cytTM-SpoIVFB, and SpoIVFA from pET Quartet plasmids in 10. Line 166-please clarify what is meant by "ectopically"? From a non-native locus? At the wrong stage of development?11. Line 228 I believe is the topic sentence for the next paragraph? "Based on these data we hypothesized that BofA48 is a zinc ligand and…"12. Line 240 Please provide rationale for this experiment. Consider using previous sentence (at end of previous paragraph) as start of this paragraph to clarify thinking here.Reviewer #2 (Recommendations for the authors):Much of the work has been performed carefully and conclusions are warranted. However, the instability of the proteins in the absence of the complex makes some of these studies difficult to interpret. The authors should consider comparing expression levels of BofA mutants alone in the absence of other complex proteins. This would help clarify if changes in BofA levels are due to effects of mutations on protein stability or complex stability.It is unclear to me in the crosslinking studies why the P135C-SpoIVFG-BofA complex is not DTT sensitive.Reviewer #3 (Recommendations for the authors):1) The authors use alanine scanning to identify N48 as a critical amino acid for the inhibitory process. They first speculate that N48 complexes the essential zinc ion and replaces the necessary water molecule. This would indeed be a very interesting mechanism. They later however show based on their modelling approach that N48 more likely makes intra-protein contacts to stabilize the conformation of BofA. This is quite confusing and I recommend to remove the speculation about the zinc binding mechanism from the results part and only describe the result that N48 is found to be important. Speculation about the mechanism could then be placed in the discussion.2) Related: it would be nice to have a figure that shows how the side chains of the three important residues interact. At the moment only backbone conformations are shown.3) In some of the figures showing the results of the cross linking experiments the largest density is seen as diffuse bands of molecular weights higher than the complex. . Any explanation? Could this be non specific cross linking effects? Essential revisions:All three reviewers appreciated the rigorous and thorough nature of the experiments. The only additional experiment requested is to assess protein levels for the mutant variants to determine if they accumulate to wild type concentrations. Additional suggestions to improve clarity and presentation are detailed in the reviews below.We thank the reviewers for their appreciation of the experiments.E. coli in the absence of other B. subtilis proteins. Accumulation of the variants was indistinguishable from wild-type GFPΔ27BofA . Therefore, the altered protein concentrations observed for the N61A and T64A variants and for SpoIVFA and cytTM-SpoIVFB in combination with all three variants depended on co-production with the other B. subtilis proteins , perhaps indicative of altered complex formation leading to protein instability (l. 155-159). The new result suggests that the GFPΔ27BofA variants fold almost normally, but cause subtle changes in a BofA structural domain including TMS2 and a membrane-embedded C-terminal region, altering formation of the SpoIVFB inhibition complex (l. 473-487).To address the request for an additional experiment, we created plasmids to express the N48A, N61A, and T64A variants of GFPΔ27BofA in We thank the reviewers for their suggestions to improve clarity and presentation. We describe our responses below.Reviewer #1 (Recommendations for the authors):As written the manuscript is dense and challenging to absorb and will need to be revised for a broad audience prior to publication. Below are some comments directed at condensing and clarifying different aspects of the manuscript.Specific comments aimed at making the manuscript more accessible:1. Generally, please change all passive voice to active voice in main text . Stating things in first person will eliminate a significant amount of the extra verbiage and streamline the manuscript.We changed nearly all passive voice to active voice in the main text.2. Similarly, make sure topic sentences summarize the focus of each paragraph. The rationale for all experiments should be clearly stated.We made sure topic sentences summarize the focus of each paragraph .3. Line 17-N-terminal pro region needs to be defined in abstract.Rather than defining Proregion in the Abstract, we deleted it there and defined it as the “pro-sequence region (Proregion)” in the Introduction (l. 75).4. Line 76--This paragraph is a very long and detailed list of all the results. Please condense to important/key take home messages. There is no reason to include these details here as they are in the following Results section.We deleted the first 10 lines of the paragraph, added a short topic sentence, and left the important/key take home messages.5. Line 100-Please write out acronyms whenever possible. It took a while to figure out what TMS was. Transmembrane sequence doesn't take that much additional space and does not require reader to remember.TMS is defined in the second sentence of the Introduction (l. 29) and used four more times in the Introduction, and 84 times in total, so we did not write it out each time. However, since TMS1 and TMS2 are used frequently, we did write out each at first occurrence (l. 74 and 95).6. Line 111. There is a lot of detail provided about a system that didn't end being used. Fine to say that authors developed the pET quartet plasmids to overcome technical issues associated with plasmid copy number. (put the details of construction in methods)E. coli in Materials and methods (l. 617-625).We deleted the details (l. 99-101). We describe plasmid construction in Supplementary File 1 and induction of protein production in 7. Line 129-what is meant by partial relief? Please provide fraction/percent relief in text.We deleted “partial relief” from the revised paragraph topic sentence (l. 122-123). We define “partial relief” and the percent by stating “partially relieved cytTM-SpoIVFB inhibition, resulting in 14% and 5% more cleavage…” (l. 125-128) and “partially relieved inhibition, resulting in 12% more cleavage…” (l. 130-131).8. Line 141-please be specific about why the lack of conserved residues allows GFP∆27 to be functional. Unclear from text.We revised the sentence and added a sentence to clarify the observations that suggest BofA “TMS1 plays a minor role in SpoIVFB inhibition and that any residues compatible with TMS formation may suffice for that role.” (l. 140-144).E. coli." seems to belong in previous paragraph. It is not a topic sentence.9. Line 147 "The Ala substitutions were made in GFPΔ27BofA and co-produced with Pro-σK(1-127), cytTM-SpoIVFB, and SpoIVFA from pET Quartet plasmids in We agree. We moved the sentence to the end of the previous paragraph (l. 149-151).10. Line 166-please clarify what is meant by "ectopically"? From a non-native locus? At the wrong stage of development?amyE chromosomal locus” (l. 177). We also revised the paragraph to better explain the experiment.We removed “ectopically” and clarified that we mean “non-native chromosomal locus” (l. 172) and “from the non-native 11. Line 228 I believe is the topic sentence for the next paragraph? "Based on these data we hypothesized that BofA48 is a zinc ligand and…"We removed the hypothesis that BofA N48 is a zinc ligand from the Results, at the suggestion of Reviewer #3.12. Line 240 Please provide rationale for this experiment. Consider using previous sentence (at end of previous paragraph) as start of this paragraph to clarify thinking here.As mentioned in our preceding response, we removed the hypothesis that BofA N48 is a zinc ligand. We broadened the hypothesis – “TMS2 of BofA occupies the SpoIVFB active site cleft in the inhibition complex” and explained the rationale for the hypothesis – “the GFPΔ27BofA N48 side chain is important for inhibition  and located near the middle of predicted TMS2 .” (l. 225-228). We then explain in general terms a strategy to begin testing the hypothesis based on a model of the SpoIVFB active site cleft and reasoning that BofA TMS2 occupancy of the cleft may be detectable using a disulfide cross-linking approach (l. 228-236). Because this approach may be unfamiliar to readers and we use it in many subsequent experiments, we added a sentence explaining its implementation in the context of the SpoIVFB inhibition complex (l. 236-239). Finally, we describe the first experiment to begin testing the hypothesis (l. 240-249) and the result (250-261).Reviewer #2 (Recommendations for the authors):Much of the work has been performed carefully and conclusions are warranted. However, the instability of the proteins in the absence of the complex makes some of these studies difficult to interpret. The authors should consider comparing expression levels of BofA mutants alone in the absence of other complex proteins. This would help clarify if changes in BofA levels are due to effects of mutations on protein stability or complex stability.E. coli in the absence of other B. subtilis proteins and found that accumulation of the variants was indistinguishable from wild-type GFPΔ27BofA . Since the Ala substitutions had little or no effect on GFPΔ27BofA stability, altered complex formation leading to protein instability likely explains the altered protein concentrations observed upon co-production with the other B. subtilis proteins  (l. 155-159).We think the reviewer meant “the potential instability of the proteins in the absence of the complex…”. As mentioned in our response to “Essential Revisions”, we expressed the N48A, N61A, and T64A variants of GFPΔ27BofA in B. subtilis, the three variants of GFPΔ27BofA accumulated normally at 4 h poststarvation, yet allowed premature cleavage of Pro-σK, despite reducing the level of SpoIVFB and in some cases SpoIVFA . These observations support altered complex formation leading to instability of SpoIVFB and in some cases SpoIVFA, without additional experiments.During sporulation of E. coli in the absence of other B. subtilis proteins  and at 4 h during B. subtilis sporulation , but subtle changes alter formation of the inhibition complex, relieving SpoIVFB inhibition and reducing its level. In the Discussion (l. 473-516), we integrate these observations with others that can be explained by proposing that “the BofA N48, N61, and T64 side chains are critical for interactions that stabilize a structural domain including TMS2 and a membrane-embedded C-terminal region, which in turn interact with SpoIVFA and SpoIVFB , accounting for most of the steric hindrance mechanism of inhibition.” (l. 473-476).Together, the data suggest that the GFPΔ27BofA variants fold almost normally in It is unclear to me in the crosslinking studies why the P135C-SpoIVFG-BofA complex is not DTT sensitive.We think the reviewer is referring to the bands labeled “complex” in lanes 8 and 9 of Figure 4B. We find that cross-link reversal by DTT treatment is variable. For example, in Figure 6—figure supplement 4D (60 min), we observed slightly more reversal of the same cross-link by DTT treatment. In Figure 6—figure supplement 4A (60 min), we observed less reversal of the E44C cytTM-SpoIVFB cross-link to MBPΔ27BofA C46 by DTT treatment than in lanes 1 and 2 of Figure 4A. We observed many other examples in our cross-linking experiments, but we do not understand the variability. We decided not to comment on the variability in the manuscript, since we think it would distract readers. In general, some cross-links appear to be less sensitive to DTT treatment, perhaps owing to the chemical environment of the cross-link.Reviewer #3 (Recommendations for the authors):1) The authors use alanine scanning to identify N48 as a critical amino acid for the inhibitory process. They first speculate that N48 complexes the essential zinc ion and replaces the necessary water molecule. This would indeed be a very interesting mechanism. They later however show based on their modelling approach that N48 more likely makes intra-protein contacts to stabilize the conformation of BofA. This is quite confusing and I recommend to remove the speculation about the zinc binding mechanism from the results part and only describe the result that N48 is found to be important. Speculation about the mechanism could then be placed in the discussion.As mentioned in our response to comment 12 of Reviewer #1, we removed the hypothesis that BofA N48 is a zinc ligand from the Results. There, we broadened the hypothesis – “TMS2 of BofA occupies the SpoIVFB active site cleft in the inhibition complex” and explained the rationale for the hypothesis – “the GFPΔ27BofA N48 side chain is important for inhibition  and located near the middle of predicted TMS2 .” (l. 225-228).In the Discussion, we note that “In addition to steric hindrance, inhibition of soluble metalloproteases often involves a residue of the inhibitory protein, prodomain, or prosegment with a side chain that coordinates the catalytic metal ion in place of a water molecule necessary for substrate peptide bond hydrolysis 6970.” (l. 459-462), then we explain why neither BofA C46 nor N48 likely acts as a zinc ligand (l. 463-472).2) Related: it would be nice to have a figure that shows how the side chains of the three important residues interact. At the moment only backbone conformations are shown.We added a figure with images of the BofA model showing the N48, N61, and T64 side chains . We note that in the model, N48, N61, and T64 are in proximity and “likely able to interact, but the constraints from experiments and co-evolutionary couplings are not sufficient for predicting the exact side chain orientations with high certainty. So, even though the N61 side chain is shown pointing away from the N48 and T64 side chains in the model, slight structural rearrangements within the modeling constraints could allow the N61 side chain to interact more directly with the N48 and/or T64 side chain(s).” (l. 410-414).As mentioned in our response to the first comment of Reviewer #2, we propose in the Discussion that “the BofA N48, N61, and T64 side chains are critical for interactions that stabilize a structural domain including TMS2 and a membrane-embedded C-terminal region, which in turn interact with SpoIVFA and SpoIVFB , accounting for most of the steric hindrance mechanism of inhibition.” (l. 473-476).3) In some of the figures showing the results of the cross linking experiments the largest density is seen as diffuse bands of molecular weights higher than the complex. . Any explanation? Could this be non specific cross linking effects?E. coli proteins is the most likely explanation, so we annotated the figure and added “The star (*) indicates likely nonspecific cross-linking of the single-Cys cytTM-SpoIVFB variants to E. coli proteins.” in the legend. We note that the four novel species starred in Figure 6—figure supplement 3 E and G likely have a similar explanation (l. 361-363).We think the reviewer is referring to diffuse bands migrating between 45 and 55 kDa primarily in the lanes treated with Cu but not DTT in Figure 4—figure supplement 4. We agree that nonspecific cross-linking of the single-Cys cytTM-SpoIVFB variants to


Type I hypersensitivity  is an unsuitable or overreactive immune response to an allergen due to cross-link immunoglobulin E (IgE) antibodies bound to its high-affinity IgE receptors (FcεRIs) on effector cells. It is needless to say that at least two epitopes on allergens are required to the successful and effective cross-linking. There are some reports pointing to small proteins with only one IgE epitope could cross-link FcεRI-bound IgE through homo-oligomerization which provides two same IgE epitopes. Therefore, oligomerization of allergens plays an indisputable role in the allergenic feature and stability of allergens. In this regard, we review the signaling capacity of the B cell receptor (BCR) complex and cross-linking of FcεRI which results in the synthesis of allergen-specific IgE. This review also discusses the protein-protein interactions involved in the oligomerization of allergens and provide some explanations about the oligomerization of some well-known allergens, such as calcium-binding allergens, Alt a 1, Bet v 1, Der p 1, Per a3, and Fel d 1, along with the effects of their concentrations on dimerization. Hypersensitivity reactions are generally classified into Type I, Type II, Type III, and Type IV. Type I hypersensitivity is an immediate reaction due to immunoglobulin E (IgE) antibodies against a soluble allergen . It reveFcεRI has a high affinity for the IgE antibody or allergen-IgE complex. It is reported that cross-linking of at least two FcεRI-bound IgEs by an allergen are needed to the degranulation of mast cell and basophils . PreviouIgE synthesis in allergic reactions is induced following B cell activation through allergens binding to BCR . The stiEpitopes of a polyvalent antigen are able to cross-link at least two BCRs which in turn enhance B cell activation, proliferation, and differentiation into IgE-secreting cells . HoweverFor the successful cross-linking of the FcεRI-bound IgE antibodies on the effector cells, allergens must have at least two IgE-binding sites (IgE epitopes) . FurtherMost of multivalent antigens are large molecules, but some allergenic proteins have low molecular weight. Monomeric allergens rarely have two identical epitopes due to their low molecular weight. Since the oligomer structure of an allergen can present a repetitive array of the identical IgE epitope, oligomerization of allergens displaying only one epitope is sufficient to cross-link FcεRI-bound IgE antibodies on effector cells and initiates an allergic reaction via the release of immune agents , 19, 20.Although there are some reports pointing to the importance of the oligomerization phenomenon in allergenic properties of soluble antigens , the postimothy grass (Phl p 7), birch (Bet v 4), and common lambs quarters (Che a 3) have been characterized and their structures in three dimension have been determined [Some members of the calcium-binding protein family show important characteristics which can participate in allergic reactions in polysensitized individuals , 22. Caltermined , 23, 24.It is demonstrated that calcium-binding polcalcin possesses very high allergenic properties, while the calcium-depleted form of polcalcin  fails to bind to IgE . As showSeveral studies have been performed on calcium-binding effects on the oligomerization of polcalcin , 26. It Alternaria spores with unknown functions [Alternaria alternata fungus that induces an allergic reaction in approximately 90% of individuals suffering from Alternaria allergic [Alt a 1 is a protein in the cell wall of unctions , 31. It allergic –34. In aallergic .εRI-bound IgE [The natural form of Alt a 1 is a dimer protein with a molecular weight of 30 kDa, showing two bands of 16.4 and 15.3 kDa, under reducing conditions on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) . Five diound IgE , 36.Alternaria spore. It is constant even in the low acidic environment such as bronchial epithelium with a pH value of 6, whereas this complex is broken down at pH less than 5.5 [Alternaria alternata allergies patients. Although some evidence pointing to the oligomerization of Alt a 1, as a classic and dimeric structure, is an important prerequisite for its allergenicity [In addition to the disulfide bond, hydrophobic and polar interactions, hydrogen bonds , and N- and C-terminal regions contribute to the stabilization of the dimer structure of Alt a 1  proteins, is a major allergen of h pollen , 42. Thih pollen , 44. Varh pollen , 45. Schh pollen . Thus, tThe dimer form of Bet v 1 is generated by the interaction of two monomer forms of Bet v 1 through their N-terminal β-strands. This interaction is then strengthened by disulfide bonds and the salt bridge between Glu 127 and Lys 137 . Per a 3 is identified as a major allergen from the American cockroach, which was recognized by the serum IgE from cockroach-allergic patients . MindykoFel d 1, a 35-kDa tetrameric glycoprotein, has been reported as the main allergen in cat-allergic patients, which reacts with 90% of the serum IgE from these patients. Allergens from domestic cats cause approximately 10% of mild rhinitis but also life-threatening asthmatic reaction in the western world . HeterodThe interface generated between two monomer forms of Fel d 1 is constructed of a central hydrophobic core surrounded by a hydrogen bond made by charged residues. Three calcium-binding sites exist in tetrameric Fel d 1; one site is within the dimerization interface, and other sites are located on the side surface of each dimer in tetrameric Fel d 1. Three linear IgE epitopes were identified on the Fel d 1 allergen. These epitopes are located on the first and the end segments of H1–H2, H3–H4, and H5–H6, respectively .In spite of amino acid-base interactions and peptide bonds, allergen concentration is a key factor in dimerization of monomeric allergens. Previous studies have shown that many allergens are able to make dimers or oligomer structures. The accumulations of monomeric antigens at higher concentrations provide a chance to increase their dimerization and thereby participate in enhancing protein immunogenicity , 62. AltRouvinen et al. demonstrated that approximately 80% of allergens are able to produce symmetric dimers or oligomers in crystals. Allergens frequently create a transient dimer structure at high concentrations; this observation is confirmed by the fact that their hypoallergenic variants have a monomer structure . KuriyanOligomerization of allergens play undeniable roles in the allergenic features and stability of allergens. In the present study, the protein–protein interactions were reviewed, which are responsible for allergen oligomerization (including amino acid-base interactions and peptide bonds). There are numerous studies pointing to at least two epitopes on the surface of allergens are necessary for the successful and effective cross-linking, however, some reports showing small proteins with only one IgE epitope (which form homo-oligomerization) can also cross-link FcεRI-bound IgE by providing two same IgE epitopes.In conclusion, homo-oligomer structure in an allergen, which is mainly mediated by protein–protein interactions, has a key role in allergenicity of an allergen through providing at least two epitopes and enhancing the cross-linking of the BCRs on IgE-B cells and FcεRI-bound IgE on effector cells, but is not necessary. In addition, there are some reports revealing many important allergens are protein monomers.


In low and middle income countries macroeconomic volatility is common, and severe negative economic shocks can substantially increase poverty and food insecurity. Less well understood are the implications of these contractions for child acute malnutrition (wasting), a major risk factor for under-5 mortality. This study explores the nutritional impacts of economic growth shocks over 1990–2018 by linking wasting outcomes collected for 1.256 million children from 52 countries to lagged annual changes in economic growth. Estimates suggest that a 10% annual decline in national income increases moderate/severe wasting prevalence by 14.4–17.8%. An exploration of possible mechanisms suggests negative economic shocks may increase risks of inadequate dietary diversity among children. Applying these results to the latest economic growth estimates for 2020 suggests that COVID-19 could put an additional 9.4 million preschoolers at risk of wasting, net of the effects of preventative policy actions. Economic shocks may lead to food insecurity and therefore acute child malnutrition (wasting). Here, the authors use data from Demographic Health Surveys to estimate impacts of past economic shocks on wasting and project possible effects of shocks related to the COVID-19 pandemic. Other estimates suggest 140 million people may have fallen into $1.90/day poverty in 20202.Macroeconomic volatility is far more common in low and middle income countries (LMICs) for a variety of economic, political, and environmental reasons. However, the economic impacts of the COVID-19 pandemic have been exceptional, forcing governments throughout the world to shut down large parts of their domestic and international economies in both 2020 and 2021, thereby transforming a health crisis into a complex economic crisis of exceptional scale, scope, and duration. Virtually all LMIC economies contracted sharply in 2020, and most have experienced further shocks due to the Delta variant in 20213. Infants and young children are at the greatest risk of wasting – and of mortality due to wasting–because of their immature immune system and their high nutrient requirements for growth and development. Although less prevalent than stunting–an indicator of chronic undernutrition–wasting is a much stronger predictor of child mortality. A pooled analysis of ten prospective cohort studies estimated that severe wasting had a hazard ratio of 11.6 compared to 5.5 for severe stunting (height-for-age Z-scores < −3), while moderately wasted children (−2 > WHZ > −3) were 3.4 times and mildly wasted children (−1 > WHZ > −3) 1.6 times as likely to die before their fifth birthday compared to non-wasted children4.While there is not yet any evidence to suggest that COVID-19 has directly resulted in significantly higher mortality risks for young children in LMICs, the indirect effects of COVID-related economic shocks pose serious risks for malnutrition, morbidity, and mortality, particularly among children under 5 years of age. In the short term, nutritional insults often manifest in the form of acute weight loss, typically measured as low weight-for-height z scores (WHZ) that are used to classify wasting into different levels of severity. Wasting is usually the result of both severe reductions in food intake and recent or repeated episodes of infectious diseases, and the dynamic interaction between poor diets and infections11, or the numerous studies estimating the impacts of negative economic shocks on child mortality.9 Moreover, while LMICs have made significant progress in the past few decades in reducing stunting, progress in reducing wasting is uneven at best, with some programmatic improvements in treating wasting12, but limited improvement in prevention. Globally, more than 45 million under-5 children were estimated to be moderately (WHZ < −2) or severely (WHZ < −3) wasted in 2020, and as much as 75% of wasted children residing in South Asia and sub-Saharan Africa (particularly the Sahel and Horn of Africa), where prevalence of wasting remains remarkably high13.Despite the serious risks that wasting poses for morbidity and mortality in young children, the underlying economic causes of wasting are under-researched compared to the many studies linking longer-term economic growth to stunting14 that collected information on 1.256 million children in 52 countries over three decades (1990–2018). We link these child and household level data to national level estimates of short- and medium-term changes in Gross Domestic Product per capita (GDP) or Gross National Income per capita (GNI), which is equal to GDP plus net income earned abroad. With some modifications, we follow the empirical strategies of previous papers16 that combined macroeconomic data with multiple rounds of DHS data to examine the mortality effects of economic shocks: fixed effects estimators that control for region-specific time trends to net out the biasing effects of many potential confounding factors.In this study, we explore the impacts of short-term economic growth shocks on the risks of child wasting. While are data are historical and pre-date COVID-19, understanding whether the economic shocks currently being experienced in LMICs are likely to significantly increase risks of wasting is an important motivation of this study. To do so we use an extensive set of 177 Demographic Health Surveys (DHS)1 predicts that an extra 9.4 million children could become wasted in 2021 as a result of COVID-related economic contractions in 2020.We show that departures of annual economic growth from long term growth trends  increase the risk of child wasting in the subsequent calendar year, and by a significant margin: the model predicts that a 10% decline in GNI per capita predicts a 14.4–17.8% increase in moderate/severe wasting in children under-5. Our findings also support the hypothesis that increases in child wasting are associated with increased risks of children having poor dietary diversity and infectious diseases symptoms such as diarrhea or fever. Moreover, applying these growth elasticities to the latest GDP growth estimates for 2020Although this estimate may be an upper bound given the various interventions that governments have implemented to protect their economies during the pandemic, the effectiveness of these interventions in preventing household economic hardship and wasting remains uncertain. Unless protected urgently and holistically, children in LMICs could well be neglected victims of an economic pandemic affecting poor populations throughout LMICs, with no immediate end in sight.Our results are structured in three parts: (1) Descriptive results designed to outline the data structure and patterns in wasting and economic shocks; (2) Main regression results for child wasting; (3) predicted impacts of COVID-19 on wasting; (4) sensitivity tests and extensions; and (5) an exploration of potential mechanisms.17 Wasting is very common in South Asia (notably India) as well as the Sahel and parts of the Horn of Africa, but generally much lower in the rest of sub-Saharan Africa despite many African countries having high rates of stunting, a measure of chronic malnutrition. Supplementary Fig. 18 (27%), but then declines steadily to level off at around 15% from age 3 onwards. In sub-Saharan Africa wasting progression follows a very different pattern: children are commonly born wasted, wasting rates are then typically steady for the first few months of life, but then rise and peak in the 10-12 months of age (at 11–17% in most sub-regions) before declining thereafter. However, in the Sahel these dynamics are noticeably accentuated: 21% of children are born wasted, but by 11 months wasting prevalence reaches 30% and thereafter declines to 10% by 36 months.Our sample of 1.256 million children 0-59 months of age spread across 52 LMICs exhibits large variation in wasting prevalence , country fixed effects, and various region-specific temporal effects to control for wasting-age dynamics . Results from an earlier version of this study were also used to estimate the potential global change in numbers of wasted children using September 2020 GNI growth projections from IFPRI19.In total, the model estimates that an addition 9.37 million pre-school children could have become wasted in 2021 as a result of the growth shocks experienced in 2020. It is also notable that despite the use of a slightly different growth elasticity (GDP instead of GNI), a different measure of growth shocks , and a slightly different sample size, our results are very close to the 9.3 million projection previously reported using GNI shocks in combination with IFPRI MIRAGRODEP GNI projections for 2020One commonality across projections is the huge impact of large negative economic growth in India, which suffered an unusually large GDP shock in 2020 (−14.9%), had exceptionally high under-5 wasting prevalence prior to COVID-19 (20.8%), and an exceptionally large population of children under the age of 5 . Clearly, the massive risk of increased wasting in India is an issue that warrants closer empirical investigation and extraordinary policy responses, especially in light of the extremely severe third wave of COVID-19 cases in the second quarter of 2021.A final point of some note is that these estimates likely represent upper bounds, as they reflect historically derived wasting-growth elasticities. The COVID-19 crisis motivated a number of extraordinary social protection measures designed to bolster household incomes, employment, and small businesses, and these may have protected nutritionally vulnerable households and their children to some extent.Additional results testing robustness to variations in specifications and samples are reported in the Supplementary Information. Supplementary Fig. 22, while rural populations  tend to be more protected because of the inelastic demand for food. For GDP shocks, but not GNI shocks, we also find some evidence that the wasting status of girls is somewhat more sensitive to shocks than for boys. This heightened vulnerability of girls to economic shocks is consistent with an earlier literature suggest that girls in India are more adversely affected during times of deprivation23.Supplementary Tables Table The results in Table In addition to that suggestive evidence on dietary impacts, GDP shocks are also significantly and negatively associated with diarrhea and fever prevalence, although only at the 10% level, and the result does not robustly extend to GNI shocks. Elasticities of low maternal BMI with respect to growth shocks were imprecisely estimated and not statistically different from zero. Hence, evidence from this analysis on the hypothesis that economic shocks affect child infections or maternal weight is inconclusive.This study examined the impact of macroeconomic shocks–defined as annual differences from long-run economic growth–on the risk of child wasting in LMICs. We find robust evidence that the elasticity of wasting with respect to lagged growth shocks is negative and relatively large in magnitude. The results are robust to variations in specification, although point estimates for GDP shocks tend to be larger than those of GNI shocks (though less precisely estimated). We find some evidence of significantly greater sensitivity of urban children to macroeconomic shocks, and some evidence that girls are more sensitive to GDP shocks. We also find some indications that poor diets and increased infectious disease risks might be important mechanisms linking growth shocks to wasting, bearing in mind that the results are sensitive to the choice of GNI or GDP shock measures.11. Moreover, since wasting is a major risk factor in early childhood mortality24, our results help explain previous findings demonstrating that adverse economic shocks in LMICs significantly increase child mortality rates9.This study makes a novel contribution to the existing literature on economic growth and nutrition, which has been largely confined to studying the impacts of longer term economic growth on child stunting4. The true extent of these economic shocks has become increasingly apparent, with incorporation of April 2021 IMF GDP growth estimates predicting that approximately 9.37 million children could become wasted in 2021, but also some further deterioration in economic output in LMICs as the Delta variant has spread far and wide. Globally, the impacts of COVID-19 on wasting very much depend on what happens in India (and some other large countries). Disconcertingly, previous research suggests that state-level macroeconomic shocks in India do indeed predict higher levels of mortality among young children, especially girls15.Our findings are also highly relevant in the context of the complex and ongoing COVID-19 crisis because they predict that aggregate income shocks alone–even without COVID-related health disruptions–will have severe impacts on wasting and increase the risks of elevated child mortality rates25 as well as welfare evidence from earlier macroeconomic crises in LMICs populations22. This is likely because the farm economy is relatively resilient to macro shocks, with demand for food being less adversely affected than demand for most non-farm goods and services.Our empirical results also suggest that macroeconomic shocks might raise wasting risks among urban children more than they do among rural children, consistent with recent evidence on the impacts of COVID-19This study has limitations. Although we have a large sample of children, the number of economic shocks we study (country-year dyads) is relatively small and offers limited opportunities for exploring heterogeneity across shocks or across countries. Macroeconomic shocks can be caused by very diverse underlying factors , and the effects of any given shock could affect the welfare of different socioeconomic groups quite heterogeneously, depending on factors such as economic structure and the extent of social protection measures. Of course, if the impacts of economic shocks are indeed highly heterogenous, this might lead to attenuation of the relevant regression coefficient, perhaps towards statistical insignificance. The fact that we do find such statistically significant coefficients in the wasting regressions gives us some confidence that acute malnutrition is indeed generally quite sensitive to recent macroeconomic shocks in a general sense, especially where wasting is prevalent in normal times.26, and indeed some early country case studies of COVID-19’s economic impacts suggest that losses in household income will be much larger than losses in national income averaged over the population27. Contextually, the COVID-19 crisis has also generated unprecedently large government responses to limit the economic damage to households, although the scale of the response in LMICs has thus far been inadequate relative to the economic damage28.Another limitation is that it is also well known that national income measures are imperfect predictors of household welfare30. Reassignment of health staff to COVID-19 tasks, restrictions on mobility, lack of transport, and fear of using health services during lock-downs will also affect access to and utilization of health and nutrition services. A recent COVID-19 study estimated that different scenarios of reduced coverage of health and nutrition services combined with assumptions about increases in wasting could result in 168,000 additional deaths in under-five children in LMICs 19. That study estimated that increased wasting would account for close to 25% of the additional deaths.A final limitation is that this study only assessed the impacts on wasting due to economic shocks. While economic channels are clearly important in 2020 and 2021, the prolonged COVID-19 crisis will also affect child wasting and mortality through health system disruptions, including suspension of a range of essential nutrition and health actions such as antenatal care, vitamin A supplementation, and immunization, and the prevention and treatment of severe acute malnutrition and infectionsOur study complements these findings by identifying an important channel of impact: negative economic shocks substantially increasing the risk of all types of wasting. This implies that efforts to protect health systems also need to be bolstered by nutrition-sensitive social protection programs to protect incomes of poor and vulnerable populations, including newly poor populations who will often reside in urban areas. Another potential problem is that wasting is often seen as a public health issue rather than a broader problem of underdevelopment (unlike stunting), although our results suggest that wasting is very much affected by economic shocks, not just health-related problems. Wasting and other important forms of malnutrition are multidimensional problems requiring concerted multisectoral solutions, especially in the wake of COVID-19. Unfortunately, COVID-19 continued to ravage many LMICs in 2021 and early 2022 (the time of writing). Much stronger international support is needed to solve the immediate health and nutrition crises affecting these countries, as well as the ongoing economic crises affecting LMICs.To explore the impact of macroeconomic shocks on child wasting we combined a large multi-country child-level DHS dataset with national-level macroeconomic data. The DHS are well suited for this kind of analysis because of their high degree of standardization and coverage of a wide range of LMICs, their collection of a rich array of nutrition, health, demographic and socioeconomic data, their representativeness at both national and subnational levels, and their repeated application within countries over different periods of time. Our dataset comprises 177 DHS rounds that collected data on child weight in multiple rounds for children 0–59 months of age in 52 LMICs between 1990 and 2018 (See Supplementary Table 31. We then defined three standard measures of wasting: any wasting (WHZ < −1), moderate/severe wasting (WHZ < −2), and severe wasting (WHZ < −3).DHS data were used to calculate WHZ scores relative to WHO reference weight-for-height measures of healthy breastfed children in multiple countriesr = 0.90), is imperfect. In this study we sourced both of these indicators from the UN National Accounts Database,32 although we also tested robustness to a World Bank source for these same indicators33.Our key explanatory variable is the lagged annual change in economic growth, although economic growth can be measured through both GNI and GDP. GDP measures the value of goods and services produced within a country and includes national output, expenditures, and income, while GNI equals GDP plus wages, salaries, and property income of the country’s residents earned abroad and at home. A priori, it is not obvious which is a better predictor of changes in household incomes, and the correlation between the two indicators in our sample, while high ), medical facility births and vaccinations (% children fully immunized for age), improved sanitation and water supplies, household demographics , child sex and rural location. In addition to these DHS controls we also followed a previous cross-country DHS study on growth shocks by testing sensitivity to two additional national-level controls that could influence economic growth but independently affect child health: climate shocks, as captured by the one-year lag of total annual rainfall and average monthly temperatureFinally, we used child morbidity symptoms and dietary indicators, as well as low maternal body mass index (BMI < 18.5) to explore whether disease, dietary or maternal nutrition mechanisms might explain the impacts of growth shocks on child wasting. For morbidity symptoms we use the DHS measure of whether a child was reported to have had diarrhea or fever-only in the past two weeks. For diets we use the standard minimum dietary diversity (MDD) indicator corresponding to a child having consumed at least four of seven food groups in the previous 24 h measured for children 6–35 months. We also use the individual food groups to test sensitivity of their consumption to growth shocks.Our analysis of these data was conducted in three stages. All analysis was conducted in STATA™ Version 16.First, we estimated a new set of household weights to make the regressions more representative of under-5 children in the DHS countries in our analysis. In a typical single-country analysis one need only use the standard DHS survey weights for each household to render statistics nationally representative, but in a multi-country analysis a more complex approach is needed, especially when the main explanatory variable of interest is itself measured at the country-year level. This is because: (a) countries differ in terms of their total population of under-5 children; (b) different countries have different numbers of rounds, and survey sizes that do not reflect population sizes; and (c) different rounds within a country can have very different sample sizes. Were we not to construct explicit weights to reflect these facts then the regression coefficients for growth shocks would remain implicitly weighted by the somewhat arbitrary number of observations in each individual survey and the number of surveys in each country, resulting in some growth episodes being over-weighted or under-weighted relative to their country’s population of under-5 children or relative to the number of survey rounds per country.35 statistics on the population of children <5 years of age to create a country-level population weight. India, for example, accounts for 20% of all child-level observations in our sample, but India contains 34% of all under-5 children in our DHS sample of countries  to plot wasting by child age and region, and examined the distribution of growth shocks across DHS rounds.In the second step of our analysis we used different descriptive analysis techniques to explore patterns and trends in the data by mapping moderate/severe wasting, using non-parametric regressions , but also mathematically appropriate since a WHZ sample that is distributed more closely to the various wasting thresholds should see larger absolute changes in wasting. This specification also has a benefit for interpretation since the coefficient represents the elasticity of wasting prevalence with respect to economic growth shocks. The one-year lag in growth shocks is also justified on several grounds. First, the timing of wasting measurement varies within years but growth shocks can occur at different times within a year; specifying a lagged growth shock always ensures that the macroeconomic shock precedes wasting measurements. Second, caregivers may take actions to prevent or delay the translation of economic shocks into nutritional insults for their children, so the impacts of economic shocks may be quite delayed. Third, although diets and diseases could emerge quickly in the wake of economic shocks, changes in weight can a longer period of time.16, although the application to wasting poses some additional challenges highlighted below.The remaining variables in the model are control variables. The regression model closely approximates a difference-in-difference model in that it uses fixed and temporal effects to net out numerous time-invariant factors that influence wasting as well as common trends over time, such as improvements and expansions in programs aimed at preventing and treated acute malnutrition. This approach is similar to a previous study examining the impacts of growth shocks on retrospective mortality measures from the DHS38, whereas in some parts of the Sahel and Horn of Africa it is reported to increase in drier seasons40. Second, and relatedly, wasting may have quite different etiologies in different regions. For example, low birth weight is more prevalent in South Asia than in sub-Saharan Africa despite similar poverty levels.41 Moreover, DHS evidence suggests that there may be difference in long-term wasting trends across regions; wasting rates have also been stubbornly immune to longer-term economic growth in South Asia, but have declined notably in some West African countries, such as Ghana.The first is that wasting is seasonal and seasonal variations in wasting are region-specific. In South Asia, the available evidence suggests that wasting sharply increases in the monsoonTo control for these complex differences in the trends and timing of wasting, we first created a more refined series of regional dummy variables that account for the diversity of wasting prevalence within sub-Saharan Africa: the Sahel and Horn of Africa , Western and Central Africa , and Eastern and Southern Africa . The remaining regions are more standard: South Asia, South-East Asia, Middle East, North Africa, Eastern Europe, Central Asia, Latin America, and the Caribbean. We then interacted these regional dummy variables with three kinds of temporal effects: 5-year time brackets to flexibly control for secular changes in wasting in different regions; month of child measurement dummies to control for seasonality of wasting; and child age dummies (in months) to control for differences in the region-specific progression of wasting in early life.w) for child i in country c and region r at time t is a function of lagged annual economic growth (gt-1) interacted with average wasting prevalence across all rounds , country fixed effects (C), and three types of region-specific temporal effects: child age effects (A.R), seasonality effects (S.R), and trend effects (T.R). Since the specification includes country fixed effects, the coefficient on economic growth represents the elasticity of wasting risks with respect to deviations of lagged growth from long-term growth rates, which we, therefore, define as growth shocks. Standard errors (ε) are clustered at the country level for the calculation of 95% confidence intervals (CIs) in all tables, although several graphs report 90% CIs to reflect one-sided tests of the null hypothesis that the growth elasticities are significantly below zero.The remaining variables in Eq.  include Although all our regressions follow the basic structure of Eq.  we conduFirst, we explored the influence on key results of excluding various sets of control variables, as well to exclusion of extreme growth outliers. Our assumption is that many DHS controls are pre-determined and not influenced by recent growth shocks, including household assets, parental education, sanitation, and water sources, but access to health services might indeed be seriously affected by shocks.42. However, little variation across age ranges was uncovered, so those results are not reported herein (results are available on request).Second, we tested variation in wasting impacts by child age since previous research has shown that wasting prevalence is higher in young children (e.g. <2 years) and that wasting risks in younger children appear are more sensitive to various nutritional insults43 and that urban populations are typically more vulnerable to economic crises than rural populations22.Third, we introduced interactions with urban and girl dummies to test for differential impacts of growth shocks on boys and girls and rural and urban children since previous research on the health and nutrition impacts of economic shocks suggests impacts may differ by genderFinally, we explored potential mechanisms linking growth shocks to wasting, including symptoms of common childhood infections such as diarrhea and fevers, maternal nutrition (low BMI), as well as minimum dietary diversity for children 6–35 months of age, and its individual food group components. These regressions follow the same structure as Eq. , but witFurther information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


There has been increased development of robotic technologies for the accuracy of percutaneous pedicle screw placement. However, it remains unclear whether the robot really optimize the selection of screw sizes and enhance screw stability. The purpose of this study is to compare the sizes (diameter and length), placement accuracy and the loosening rate of pedicle screws using robotic-assisted versus conventional fluoroscopy approaches for thoracolumbar fractures.A retrospective cohort study was conducted to evaluate 70 consecutive patients [34 cases of robot-assisted percutaneous pedicle screw fixation (RAF) and 36 of conventional fluoroscopy-guided percutaneous pedicle screw fixation (FGF)]. Demographics, clinical characteristics, and radiological features were recorded. Pedicle screw length, diameter, and pedicle screw placement accuracy were assessed. The patients’ sagittal kyphosis Cobb angles (KCA), anterior vertebral height ratios (VHA), and screw loosening rate were evaluated by radiographic data 1 year after surgery.P < 0.001] and a higher “grade A + B” pedicle screw placement rate . The mean screw diameter was 6.04 ± 0.55 mm in the RAF group and 5.78 ± 0.50 mm in the FGF group (P < 0.001). The mean screw length was 50.45 ± 4.37 mm in the RAF group and 48.63 ± 3.86 mm in the FGF group (P < 0.001). The correction loss of the KCA and VHR of the RAF group was less than that of the FGT group at the 1-year follow-up [(3.8 ± 1.8° vs. 4.9 ± 4.2°) and (5.5 ± 4.9% vs. 6.4 ± 5.7%)], and screw loosening occurred in 2 out of 34 patients (5.9%) in the RAF group, and 6 out of 36 patients (16.7%) in the FGF group, but there were no significant differences (P > 0.05).There was no significant difference in the mean computed tomography (CT) Hounsfield unit (HU) values, operation duration, or length of hospital stay between the groups. Compared with the FGF group, the RAF group had a lower fluoroscopy frequency [14 (12–18) vs. 21 (16–25), Compared with the fluoroscopy-guided technique, robotic-assisted spine surgery decreased radiation exposure and optimizes screw trajectories and dimensions intraoperatively. Although not statistically significant, the loosening rate of the RAF group was lower that of than the FGT group. Thoracolumbar fractures account for 30–60% of spinal fractures and are commonly seen in high-energy trauma patients , 2. BursIn general, misplacement and inappropriate choice of pedicle screws negatively affect the internal fixation stability , 8. InseSome studies have reported that robot-assisted techniques provide more precise screw placement –14, and This was a retrospective cohort study. A consecutive sample of 70 patients with A3 and A4 thoracolumbar fractures from May 2018 to October 2020 was enrolled. Of the 70 patients reviewed, 34 and 36 were treated with robotic-assisted percutaneous pedicle screw fixation and fluoroscopy-guided percutaneous pedicle screw fixation, respectively, based on the following inclusion criteria: (1) thoracolumbar fracture classified as A3 or A4; (2) short-segment fixation with two additional screws at the single fracture vertebra; (3) no other fractures or organ injuries; and (4) absence of neurological deficits. The exclusion criteria were as follows: (1) incomplete clinical information; (2) combined anterior-posterior surgeries; (3) revision surgeries; (4) fracture displacement in the vertebral pedicle; and (5) structural spinal deformity, spinal tumour, or infection. Written informed consent was obtained from all patients. The present study was reviewed and approved by the Ethics Committee and Institutional Review Board of the Second Affiliated Hospital of Soochow University.Patients were placed in the prone position after general anaesthesia. The CT scan of the surgical region was uploaded to the workstation  before surgery, and the perfect trajectories of the vertebrae based on the surgeon’s requests were planned Fig. A–D. AftePatients were placed in the prone position after general anaesthesia. The fractured vertebrae were confirmed by fluoroscopy. Six transpedicular puncture needles were inserted through six small incisions using fluoroscopy, after which guide wires were inserted. The appropriate pedicle screws were then placed through the guide wires. The position of each screw was examined by fluoroscopy, followed by the insertion of titanium rods with the assistance of screw extenders.Patient demographics and clinical characteristics were recorded. The mean CT Hounsfield Unit (HU) values were obtained from the measurement of preoperative CT measurements Fig. . The durTwo spine surgeons with more than 10 years of experience completed the measurements independently. The average value of the measured data was the final data. All data were measured using radiology software (Neusoft PACS/RIS) to reduce variability.Based on the Gertzbein and Robbins scale , the accThe pre- and postoperative sagittal kyphosis Cobb angles (defined by the upper endplate of the first vertebra above the fractured vertebra and by the lower endplate of the first vertebra below the fractured vertebra) and anterior vertebral height ratios  of the fractured vertebra were measured using radiographic images Fig. .Pedicle screw loosening was defined as a “double-halo sign” on plain X-ray or a “lucent zone” around on CT images, which were obtained 1 year postoperatively Fig.  17, 18], 1817, 1t tests for normally distributed continuous variables, Wilcoxon rank-sum tests for variables with skewed distributions, and chi-square tests for categorical data. A p value < 0.05 was considered statistically significant. All analyses were performed using SPSS for Mac .Descriptive statistics are presented as the mean ± standard deviation for normally distributed continuous variables; the median and interquartile range was used for nonnormally distributed continuous variables and the count (frequency) was used for categorical variables. Comparisons of baseline characteristics, which were stratified by group, were performed using Student’s Overall, 70 patients who underwent short-segment fixation were included in the study. Patient demographics and clinical characteristics were compared between the RAF and FGF groups. There were no significant differences between the groups in terms of age, sex, BMI, mean HU values, mechanism of injury, fracture level and type, and TLICS score. The baseline data of the two groups were comparable Table .P < 0.001) but higher hospitalization costs (P = 0.001) than the FGF group (Table The RAF group had significantly fewer radiation cycles (up Table . No signup Table .P < 0.001) (Table There were 136 screws placed in the RAF group and 144 placed in the FGF group (screws in the vertebra above and below the fractured vertebra). Screws placed in the RAF group had both a larger screw diameter  and screw length  than screws placed in the FGF group (P < 0.05) ; the remaining screws were graded B (5.4%), C (2.9%), or D (1.0%), but no screw was graded E. In the FGF group, 80.1% of the 216 screws were graded A; the remaining screws were graded B (9.3%), C (6.9%), D (2.8%), or E (0.9%). The RAF group had a significantly higher ratio of clinically acceptable screws  than the FGF group (89.4%) (The preoperative kyphosis Cobb angle (KCA) and anterior height ratio of fractured vertebra (VHR) were similar between the two groups. After surgery, the KCA was significantly corrected, and the VHR was well improved in both groups. The correction loss of the KCA (3.8 ± 1.8°) was less than that of the FGT group( 4.9 ± 4.2°), and the VHR of the RAF group (5.5 ± 4.9% ) was less than that of the FGT group (6.4 ± 5.7%) at the 1-year follow-up, but there were no significant differences (Table Screw loosening occurred in 2 out of 34 patients 5.9%) in the RAF group, and 6 out of 36 patients (16.7%) in the FGF group. There was no statistically significant difference in terms of the screw loosening rate between the two groups , and the correction loss of the KCA (3.8 ± 1.8° vs. 4.9 ± 4.2°) and VHR (5.5 ± 4.9% vs. 6.4 ± 5.7%) of the RAF group was less than that of the FGT group at the 1-year follow-up, although there were no significant differences in the groups. Of note, 5 out of 8 patients with loosened pedicle screws (62.5%) in this study had showed the obvious screw pull-out phenomenon (data not shown). Three possible interpretations were considered to explain why the significant differences were not seen in our study. First, the mechanisms that contribute to pedicle screw loosening are certain to be multifactorial, in addition to the screw placement and sizes mentioned, including high forces of spinal rods, bone mineral density (BMD), postoperative physiological movements, and paraspinal muscle function –30. PrevMoreover, we also found that robot-assisted technology helped to reduce intraoperative radiation exposure, which is consistent with previous studies . AnotherOur study has several limitations. First, the sample size of fracture cases was relatively small, and all patients were recruited from a single centre. Future studies with larger sample sizes are needed to replicate the findings. Second, data on the trajectory angles, the radians and forces of spinal rods, and misaligned spinal rods, all of which can cause screw loosening, were not available. Finally, further experiments are necessary to design an adequate trajectory, which can significantly increase the stability of the pedicle screw.Compared with fluoroscopy-guided techniques, robotic assistance can help a surgeon to optimize screw trajectories and dimensions intraoperatively. Although not statistically significant, these findings are a possible way to increase bone purchase and reduce the screw loosening rate for thoracolumbar fractures. In addition, with increased experience and proficiency in robot-assisted techniques, surgeons should be more confident in their screw selection, especially when using sizes that they may have previously considered too large.