Arthroscopic ankle arthrodesis (AAA) is recognized as the standard treatment for the end-stage ankle arthritis. Two-screw configuration fixation is a typical technique for AAA; however, no consensus has been reached on how to select most suitable inserted position and direction. For better joint reduction, we developed a new configuration  and investigated whether it turned out to be better than the other commonly used 2-screw configurations.In this study, we investigated three kinds of 2-screw configurations: 2 “home run”-screw configuration (group A), crossed transverse configuration , and 2 parallel screw configuration . The effects of the above three insertions on the loading stress of the tibio-talar joint were comparatively analyzed with a three-dimensional finite element model.Group A was better than groups B and C in respect of stress distribution uniformity and superior to both groups B and C in anti-flexion strength and anti-internal rotation strength. Group A was slightly worse than group C but better than group B in anti-dorsiflexion and anti-valgus and varus strength.Two “home run”-screw configuration facilitates the reduction of anterior talus dislocation of end-stage ankle arthritis. Our finite element analysis demonstrates the configuration is superior to crossed transverse and parallel configuration for arthroscopic ankle arthrodesis in terms of stress distribution and initial stability. Ankle arthrodesis is still the most important treatment for the end-stage ankle arthritis . RecentlA healthy male volunteer was selected who was 37 years old and had a height of 175 cm and a body weight of 70 kg; foot tumor, deformation, and other lesions were excluded. The ankle scan was obtained from weight-bearing computed tomography (WBCT) using the PedCAT unit . Triangular mesh models of the tibia, and talus were reconstructed using a visualization software package . The triangular meshed model was imported into a reverse engineering software  and translated triangle mesh surface into NURBS surface and exported as *.iges file. The file was imported into a three-dimensional CAD software (ProE Wildfire), where the talus and tibia components were assembled according to the original anatomical location. There were no contacts between the initial position of the talus and the tibia because of the lack of cartilage in the model. So, the talus was moved up by 1.5 mm as compensation. The cancellous screws were simplified as 6.5-mm-diameter cylinders in ProE Wildfire; and the thread was not modeled, and instead, a contact interface was used to take its effect into consideration.Screw model and the fused tibia-talar joint model were assembled according to three kinds of 2-screw configurations for AAA. Three different forms of assembly were used to represent the 2 “home run”-screw configuration , and tangential direction with a friction coefficient. Furthermore, “finite sliding” was considered to model potential nonlinear effects. The details of all the contact pairs are shown in Table Due to the irregularity of the bone surface, tetrahedral mesh was used for the talus and tibia (C3D4: 4-node linear tetrahedron element), and hexagonal mesh for the screws . In order to improve the efficiency and accuracy of the finite element analysis, the mesh of contact surface of tibia and talus were refined. Group A was meshed a larger and a smaller number of nodes additionally for mesh sensitivity analyses. Both the maximum von Mises stress of the two models were differed from the standard model by < 3%.Considering that the models contained many nodes and elements, the maximum stress of each part, the maximum, and the average von Mises stress of the contact surface were calculated and are shown in Table It is generally assumed that a more stable fixation leads to a higher chance of fusion, so the micromotions at the articular surface, as well as the structural strength of the implant (screw and bone), were focused in this analysis. Because the numbers of the nodes on the fracture contact surfaces of the four models were different, the average absolute displacement U and von Mises stress of the tibia contact surface and displacement of the tibia upper surface (UR) were taken as the evaluation parameters of the initial stability of ankle arthrodesis Tables  and 3.AAA offers fusion rates of greater than 90%, with low complication rates and a shorter time to union. Two cannulated percutaneous screw fixation has commonly been used for stabilization of AAA, and there are many configurations available for delivery of the screws across the tibio-talar interface. The orientation of the screws was still important in determining the stability of a two-screw configuration . CrossedThe talus usually moves forward or dislocation at the end-stage ankle arthritis , 10. TheIn the recent years, the finite element analysis method has been widely applied in the research of orthopedic biomechanics. In analyzing the internal stress distribution and strain state of bones and the stress distribution of internal fixation and quantitatively analyzing the stress concentration and stress shielding, it has achieved the study results difficultly acquired by object reality experiments, as compared with the ordinary mechanical experiment methods; thus, it is of important clinical significance . Our stuAlthough many different biomechanical studies for AAA techniques have been performed, we are not aware of any study comparing new double-screw system with the classic two-screw fixation technique. There are several limitations for our study. First, our sample sizes with only one volunteer’s hindfoot CT. However, the data show that despite the differences in the cause of different volunteer or patient, the affected only is a single amount of data, and the overall trend does not change. Second, the experimental setup does not represent physiologic gait. Nevertheless, dorsiflexion is the major stress for a fused ankle during normal gait . Our douTwo “home run”-screw configuration facilitates the reduction of anterior talus dislocation of end-stage ankle arthritis. The present FEA demonstrates the configuration has a greater primary stability and stress distribution when compared with crossed transverse and parallel configuration. Whether this will improve postoperative rehabilitation after ankle fusion or not is still subject for future clinical and radiographic research.


In addition, measurement of bioaerosols was conducted at an urban area as reference data. Significantly higher concentrations of bioaerosols were found at the mountain site. However, several fold smaller levels of bioaerosols were detected at the seashore site . Concentrations of atmospheric culturable bioaerosols at mountain and seashore sites were measured in field investigations by using a bio-culture sampler. The eastern Korean peninsula was selected for the measurements because of the short distance between the mountain site and the seashore site. Detectable concentrations of culturable fungal and bacterial bioaerosols (maximum 1065 CFU/m The air in the mountains and at the seashore is generally believed to be clean because of the lack of hazardous pollutants. Residents of urban areas frequently visit the mountains and the seashore to breathe clean air in natural environments. However, we hypothesized that significant amounts of airborne microorganisms may be present in these areas. In the mountains many living organisms may release airborne microorganisms into the surrounding environments and the air at the seashore may contain many airborne microorganisms because of humid air conditions. We tried to check our hypothesis by field experiments, providing practical bioaerosol information of mountains and seashore. Atmospheric airborne microorganisms, known as bioaerosols, have been found to be associated with many diseases, such as pneumonia, tuberculosis, allergic rhinitis, asthma, and brucellosis ,4,5,6,7.Measurement of bioaerosols was conducted at a mountain site, a seashore site, and an urban area . All of 3 with positive-hole corrections [We measured outdoor fungal and bacterial bioaerosols from February to April, during mid spring on the Korean peninsula. The sampler , which has been used in previous bioaerosol monitoring studies ,11,12, wrections ,11,13,14In addition to measurement of bioaerosols, we measured concentrations of aerosol particles by using an optical particle counter  to investigate any relevance of aerosol particles to culturable bioaerosol concentrations . At leas3. However, the concentration was 10.7-fold lower at the seashore site (75 ± 13 CFU/m3) on the same day . For bacterial bioaerosols, the concentrations were 775 ± 361 and 150 ± 40 CFU/m3 at the mountain and seashore sites , respectively. The concentrations at the bus terminal (urban site) were between those of the mountain and seashore sites. Differences in temperature and relative humidity among the sites was not significant enough to produce large differences in these concentrations [We conducted measurements of fungal and bacterial bioaerosols for three months. trations . t-test p-value > 0.05) and 7-fold  smaller, respectively, than those at the mountain site. 3 to 1065 CFU/m3, while those at the seashore site ranged from 0 CFU/m3 to 270 CFU/m3. The concentrations of bacterial bioaerosols at the mountain site ranged from 4 CFU/m3 to 1064 CFU/m3, while those at the seashore site ranged from 0 CFU/m3 to 195 CFU/m3. t-test p-value < 0.05). However, for culturable fungal bioaerosols, the concentration at the mountain site was 411 ± 36 CFU/m3 and the concentration was 9-fold lower (46 ± 10 CFU/m3) at the seashore site on the same day , as shown in 3 at the mountain and seashore sites , respectively. Therefore, the overall aerosol particle concentration was not main reason for the difference of bioaerosol concentrations between the mountain site and the seashore site. There could be several explanations for these findings of bioaerosol concentrations at the mountain and seashore sites. Trees and other living organisms in the mountains could produce fungal and bacterial bioaerosols ; therefoCulturable bioaerosol concentrations were measured in mountain, seashore, and urban areas on the Korean peninsula. Significantly higher levels of bioaerosols were found in the air at the mountain site, while several fold smaller amounts of bioaerosols were detected at the seashore site in the field experiments. Further mechanism studies are necessary to investigate the reasons for this phenomenon.


Totally 67 mtPPs with 124 phosphorylation sites were identified, which of them included 48 differential mtPPs (mtDPPs). Eighteen mtPPs were reported previously in OCs, and they were consistent in this study compared to previous literature. GO analysis revealed those mtPPs were involved in multiple cellular processes. PPI network indicated that those mtPPs were correlated mutually, and some mtPPs acted as hub molecules, such as EIF2S2, RPLP0, RPLP2, CFL1, MYH10, HSP90, HSPD1, PSMA3, TMX1, VDAC2, VDAC3, TOMM22, and TOMM20. Totally 32 mtPP-pathway systems (p<0.05) were enriched and clustered into 15 groups, including mitophagy, apoptosis, deubiquitination, signaling by VEGF, RHO-GTPase effectors, mitochondrial protein import, translation initiation, RNA transport, cellular responses to stress, and c-MYC transcriptional activation. Totally 29 mtPPs contained a certain protein domains. Upstream regulation analysis showed that TP53, TGFB1, dexamethasone, and thapsigargin might act as inhibitors, and L-dopa and forskolin might act as activators. This study provided novel insights into mitochondrial protein phosphorylations and their potential roles in OC pathogenesis and offered new biomarker resource for OCs.To investigate the existence and their potential biological roles of mitochondrial phosphoproteins (mtPPs) in human ovarian carcinoma (OC), mitochondria purified from OC and control tissues were analyzed with TiO The completion of human genome sequencing has identified about 20,300 human genes . ResearcMitochondria are crucial and multifunctional subcellular organelle, and are associated with multiple cellular activities and diseases, such as metabolic reprogramming of OC cells . Mitocho4) group is added to amino acid residues serine , threonine , and tyrosine , which promotes a conformational alteration via interacting with other hydrophilic and hydrophobic residues [Phosphorylation is a common PTM that phosphate , signaling pathways, protein domains, and upstream regulation, which was used to more accurately determine the roles of phosphorylation in OC mitochondria. The current study deepened understanding of the reported biomarkers in OCs, and indicated the relationship of phosphorylation with those molecules. Simultaneously, those non-reported mitochondrial phosphoproteins (mtPPs) might add novel scientific merits for OCs. One should pay more attention to the hub molecules within PPI network, key molecules in enriched pathways, and important phosphorylations within or adjacent to protein domains, which had important scientific value in cell functions, or drug targets in OCs. The experimental flow-chart was used to identify mtPPs and reported biomarkers .2 enrichment-LC-MS/MS between OC mitochondria relative to controls  derived from CAV1 (Swiss-Prot No.: C9JKI3) . The phosphorylation site was localized to amino acid residue S*6, and the phosphorylation level was significantly decreased in OCs (T) compared to controls (N)   derived from CFL1 (Swiss-Prot No.: E9PS23) . The phosphorylation site was localized to amino acid residue S*3, and the phosphorylation level was significantly decreased in OCs compared to controls   derived from XRN2 (Swiss-Prot No.: B4E0B9) . The phosphorylation site was localized to amino acid residues S*423 and S*425, and the phosphorylation level was not significantly changed in OCs compared to controls  , and PUS1 . Some mtPPs were phosphorylated at residue Thr, such as KIN27 . Some mtPPs were phosphorylated at both residues Ser and Tyr, such as NBAS .A total of 67 mtPPs based on 85 phosphopeptides with 124 phosphorylation sites were identified in the control and OC samples with iTRAQ-TiOcontrols ; and amo C9JKI3) , with a .07E-07) . Another E9PS23) , with a .25E-05) . The thi B4E0B9) , with a .16E-02) . With th.16E-02) . Among 6.16E-02) . In termThose 67 mtPPs between OCs and controls were analyzed with GO enrichment methods, in biological process (BP) , cellulaThe PPI network of 67 mtPPs was constructed with String analysis database . First, CooLGeN (Version GenCLiP 3) was used to map 67 mtPPs to the corresponding molecules  for pathway network analysis. A total of 32 statistically significantly signaling pathway systems (p < 0.05) were identified with 67 mtPPs, and were grouped into 15 clusters . Cluster135 and S138 , TOMM20 at residue S135 , TOMM22 at residue S15 , and VDAC1 at residue S104 , which might be novel therapeutic targets and fundamental improvement of the damaged mitochondria in OC patients. These data showed that mitophagy might play important role in mediating tumorigenesis and tumor progression.When mitochondria were in the status of imbalance, such as stress or damage, defective mitochondria might enter selective degradation process by autophagy, named mitophagy. Mitophagy is a crucial mechanism of quality control in mitochondria and can be regarded as a double-edged sword in cancer cells. The defective mitochondria that responded to oncogenic stresses could either stimulate or block tumorigenesis through mitophagy . Here, tin vivo. At the same time, VEGF can increase vascular permeability and mobility of cancer cells, induce tumor angiogenesis, and maintain the growth of tumors [6 , CAV1 at residue S26 , CTNNA1 at residue S518 , HSP90 at residue S217 , CFL1 at residue S3 , and MYH9 at residue S1943 , which might be novel molecules and regulation mechanism for tumor angiogenesis.VEGF signaling is related to many physiological and pathological processes in human. VEGF is a specific heparin-binding growth factor of vascular endothelial cells, which can induce angiogenesis f tumors . Here, t3 , MYH10 at residue S1975 , MYH10 at residue S1956 , MYH10 at residues S1975 and S1956 , MYH9 at residue S1943 , and CTNNA1 at residue S518 , which might be novel molecules and regulation mechanism for tumor invasion and metastasis.Rho GTPases are the members of small GTPases, and Rho GTPase pathway is closely related to cytoskeleton reorganization, cell morphology, apoptosis, cell migration, and cell cycle. Recent studies showed that Rho GTPases mediated tumor invasion and metastasis by regulating extracellular matrix remodeling, loss of epithelial polarity, lymphatic vasculature, cytoskeleton reorganization, and between-cell junction and adhesion . Here, t2+), so intracellular calcium ions are the necessary condition for occurrence, development, and proliferation of tumors through various pathways [2+ concentrations in cells. This study found that mitochondrial calcium ion transport pathway and calcium signaling pathway were enriched to involve mtPPs, including phosphorylations of VDAC1 at residue S104 , VDAC2 at residue S140 , VDAC3 at residue T6, and ATP2A2 at residue S554 . Imbalance of Ca2+ might be the cause of tumorigenesis, so it is definitely worth further exploring.Multiple signal transduction pathways are initiated by calcium ions , and at residues S88, S92, and S93, HSP90 at residue S217 , and HSPD1 at residue S70 . Enhanced c-Myc-mediated transcriptional activity might induce cancer.C-Myc-mediated transcriptional activity promotes tumorigenesis and development. The major oncogenic mechanisms of c-MYC are related to promotion of cell cycle, drug resistance, reprogramming of cell metabolism, and rapid proliferation . Here, t89. ANKIB1 also contained ankyrin-repeated region circular (sites: 45-197), with phosphorylation at residue S89. EIF2B5 contained W2 domain (sites: 543-720), with phosphorylation at residues S717 and S718. HMGA1 contained HMGI_Y domain (sites: 21-89), with phosphorylation at residue S88. PSMA3 contained proteasome alpha domain (sites: 8-30 and 23-237), with phosphorylation at residue S78. VANGL2 contained TPR regions (sites: 114-578), with phosphorylation at S517. Second, CFL1 and HMGA1 were phosphorylated near protein domains. CFL1 contained actin-depolymerising factor homology domain (ADF-H domain) (sites: 4-153), with phosphorylation at residue S3. HMGA1 contained HMG-Y DNA-binding domain (AT-hooks) (sites: 21-89), with phosphorylation at residues S92 and S93. Third, the phosphorylation sites of the other 20 mtPPs with structural and functional domains were neither presented within domains nor near domains. However, it doesn't mean that those mtPPs were not important. The between-molecule abscopal effects could not been ignored. Additionally, phosphorylation at residue Y was a low abundance event compared to that at residues S and T. Specific tyrosine kinase inhibitors had been discovered to have some pretty amazing therapeutic effects in different types of cancers. NBAS contained ribosomal protein S14 signature (sites: 2207-2229), with phosphorylations at residues S473, S475 and Y477. This protein was associated with transporting proteins from Golgi complex to endoplasmic reticulum.A total of 29 identified mtPPs contained a certain structural and functional domains . Conjoin2+ ATPase. Upstream regulation analysis showed that dexamethasone might be an inhibitor for ATP2A2, and CAV1. L-dopa was also known as L-3,4-dihydroxyphenylalanine. Upstream regulation analysis showed that L-dopa might be an activator for VDAC1, PKP2, CLN6, VANGL2, ATP2A2, and PA2G4. Forskolin was a commonly used chemical toxicant in laboratory to increase cyclic AMP level. Upstream regulation analysis showed that forskolin might be an activator for PGRMC1, OSTF1, BNIP3L, and PRKAR2A.Upstream regulation analysis of 67 mtPPs indicated potential regulatory mechanisms and provided potential anti-cancer drugs . Upstrea397) and PTK2B (pY402) was altered in tumor tissues [Protein phosphorylation regulates multiple cellular processes through complex and highly dynamic signaling pathways. Multiple PTMs are one of the reasons to form proteoforms that are ultimate actors for cellular activity. Distinguishing of different proteoforms benefits for real precision medicine practice . In canc tissues . Taken t3 had a significant effect on glutamine uptake by cells, which facilitated nuclear translocation, and changed actin organization [14 was related to the insulin-resistant state in endometrial tissue of polycystic ovary syndrome patients. Plasma membrane was required for phosphorylation of membrane-associated MARCKS to reduce the amount of F-actin [http://ci.smu.edu.cn/CooLGeN/Home.php). These new identified mtPPs and their phosphorylation sites might be malignancy-associated intracellular events in OCs.Every subcellular component is highly dynamic because of their PTMs, abundance, and the protein expressions depending on cell physiological state. Therefore, it is very important to identify subcellular phosphoproteins for understanding biological functions of proteins in specific compartment. Mitochondria are classically viewed as isolated, spherical, and energy metabolism-related organelles, which is going through a revolutionary change. Literature data showed that mitochondria were associated with energy metabolism, oxidative stress, cell apoptosis, cell cycle, autophagy, and immunity process. In fact, mitochondrial pathways were involved in many diseases, including malignant tumors. For example, high levels of AKAP1 were found in variety of high-grade cancer tissues. AKAP1 scaffolding protein integrated Src and cAMP signaling on mitochondria to regulated oxidative metabolism, organelle biogenesis, and cell survival . These fnization . Blockagnization . Compare F-actin . A totalin vitro, which provided a clue to high amounts of diverse PTMs of HMGA1 protein [A total of 32 statistically significant pathways (p<0.05) was identified to involve mtPPs, which associated closely with the occurrence of cancer to indicate potential molecular mechanisms that mtPPs played in OCs. Additionally, a total of 29 identified mtPPs contained a certain structural and functional domains. This study provided novel insights into phosphorylations of mitochondrial proteins and their potential roles in contribution to molecular mechanisms of an OC. ANK1, ANKIB1, EIF2B5, HMGA1, CS, PSMA3, and VANGL2 were phosphorylated within protein domains. A certain structural and functional domains played important roles in tumor biological behaviors. For example, ANK1 contained ankyrin-repeated region cicular. Ankyrins, which link the membrane proteins to the spectrin-actin cytoskeleton, were encoded by ANK1 gene, and played crucial roles in multiple cellular activities such as proliferation, motility, and membrane domain activation. The 24 tandem ankyrin repeats could recognize a wide range of membrane proteins through non-specific interactions, such as hydrogen bonding, electrostatic interactions, and hydrophobic interactions . EIF2B5  protein .2+-ATPases, which influenced intracellular calcium concentration by blocking cell capacity to pump calcium into the sarco/endoplasmic reticular [in vivo [Furthermore, upstream regulation analysis showed that TP53, TGFB1, dexamethasone, and thapsigargin might act as inhibitors, and that L-dopa, and forskolin might act as activators. TP53 gene was one of the most frequently mutated genes (>50%) in human cancers, it might play a crucial role in cancer formation. TP53 gene also encoded a certain proteins that bond to DNA to prevent mutations of genome. In human, the secreted protein TGF-β1 was encoded by TGFB1 gene. TGF-β1 performed various cellular functions, including cell proliferation, cell growth, apoptosis, and cell differentiation. Upstream regulation analysis showed that dexamethasone might be an inhibitor for ATP2A2 and VCL. Dexamethasone, a kind of corticosteroid medication, was one of essential medicines in the World Health Organization's List, which was usually given to counteract a certain side-effects for cancer patients. Actually, dexamethasone was also used as a chemotherapeutic agent in some malignancies, in which it was given alone or combined with other chemotherapeutic drugs such as lenalidomide, thalidomide, vincristine, bortezomib or doxorubicin . Thapsigeticular . L-dopa,[in vivo . Forskol[in vivo . Therefo2 enrichment-LC-MS/MS method. Many identified mtPPs and their phosphorylation sites have not been reported previously, which benefit for the discovery of novel biomarkers to clarify basic molecular mechanisms of human OC formation, and truly predict OC progression.This study is the first report to investigate globally mitochondrial protein phosphorylations and their potential biological roles in the pathological processes of human OCs with iTRAQ-labeled TiOFrozen OC tissues (n = 8) and control ovaries with benign gynecologic disease (n = 11) were obtained from Department of Gynecology, Xiangya Hospital, Central South University, China. This study was approved by the Medical Ethics Committee of Xiangya Hospital, and the written informed consent was obtained from each patient. Both OC and control tissues were verified with histological analysis. Each tissue sample was immediately placed in liquid nitrogen and then stored at −80°C.Ovarian tissue samples were fully minced in pieces and homogenized in mitochondrial isolation buffer that contained Nagarse, followed by differential-speed centrifugation to obtain crude mitochondria. The crude mitochondria were further purified with Nycodenz-gradient centrifugation. The purified mitochondria samples were verified by western blot and electron microscopy. The detailed experimental procedure was described previously , 5. The An amount (200 μg) of extracted mitochondrial proteins were mixed with 30 μl solution that contained 4% SDS, 100 mM DTT, and 150 mM Tris-HCl pH 8.0. The detergent, DTT, and other low-molecular-weight components were removed by repeated ultrafiltration with the solution of 8 M urea and 150 mM Tris-HCl pH 8.0. The reduced cysteine residues were blocked with adding 100 μl solution that contained 100 mM iodoacetamide, 8 M urea, and 150 mM Tris-HCl pH 8.0, for incubation in darkness for 30 min, followed by washing with 100 μl solution (8 M urea and 150 mM Tris-HCl pH 8.0) for 3 times and then 100 μl dissolution buffer for 2 times. The protein suspensions were digested with 4 μg trypsin (Promega) . The tryptic peptides were desalted on C18 Cartridges, concentrated by vacuum centrifugation. The peptide content was estimated by UV light spectral density at 280 nm. A amount (100 μg) of tryptic peptide mixture of each sample was labeled using iTRAQ reagent. Each sample was labeled by three different iTRAQ reagents. The detailed experimental procedure was described previously [2 beads were added and agitated for 2 h, followed by centrifugation  to keep the beads, washing with 50 μL of washing buffer I (30% acetonitrile and 3% trifluoroacetic acid) (3 x) and then 50 μL of washing buffer II (80% acetonitrile and 0.3% trifuoroacetic acid) (3 x) to remove the remaining non-adsorbed material. Finally, phosphopeptides were eluted with 50 μL of elution buffer (40% acetonitrile and 15% NH3·H2O) (3 x), and then lyophilized.Six iTRAQ-labeled peptides were equally mixed, concentrated by a vacuum concentrator, and resuspended in 500 μl DHB buffer. TiO2-enriched phosphopeptides were analyzed with LC-MS/MS. Briefly, the enriched phosphopeptides were loaded onto a reverse phase trap column  connected to the C18-reversed phase analytical column  in buffer A (0.1% formic acid) and separated with a linear gradient of buffer B (84% acetonitrile and 0.1% formic acid) at a flow rate of 300 nl/min controlled by IntelliFlow technology. The linear gradient was 0–55% buffer B for 220 min, 55-100% buffer B for 8 min, and then 100% buffer B for 12 min. MS/MS analysis was performed on a Q Exactive mass spectrometer (Thermo Scientific), with the following parameters, including positive ion mode, data-dependent top10 method to choose the most abundant precursor ions from the survey scan (300–1800 m/z) for HCD fragmentation, automatic gain control (AGC) target 3e6, and maximum inject time to 10 ms, dynamic exclusion duration 40.0 s, resolution for MS set to 70,000 at m/z 200, resolution for MS/MS set to 17,500 at m/z 200, isolation width set to 2 m/z, normalized collision energy set to 30 eV, the underfill ratio set to 0.1%. MS/MS spectra were used to search protein database with MASCOT engine  embedded into Proteome Discoverer 1.4. The intensities of iTRAQ reporter ions were used to determine differentially phosphorylated proteins between OC and control mitochondrial samples.The TiOhttps://www.ncbi.nlm.nih.gov/pubmed/). Gene ontology, including BP and CC, was analyzed with Cytoscape ClueGO to obtain more insights into the biological information of mtPPs. PPI network was analyzed with String database (http://string-db.org/cgi/input.pl). Pathway enrichment was analyzed by CooLGeN (http://ci.smu.edu.cn/CooLGeN/Home.php) based on Reactome, Wikipathways, NetPath, and KEGG database. Each MS/MS-derived phosphoprotein amino acid sequence was input into the ScanProsite program (http://prosite.expasy.org/scanprosite) to determine its protein domains and relationship of phosphorylation sites and protein domains. The Swiss-Prot accession numbers and corresponding fold-changes between OCs and controls were input to the Ingenuity Pathway Analysis (IPA) data-upload workflow. The upstream regulation analysis was generated to involve those phosphoproteins.The reported biomarkers for OC based on the identified mtPPs were obtained from NCBI (t-test was used to assess mtDPPs between OC and control groups. P-value for GO enrichment analysis was obtained by two-sided hypergeometric test and corrected by Benjamini-Hochberg. P-value for pathway enrichment analysis was obtained by two-sided hypergeometric test and corrected by Q-value. The level of statistical significance was set as p < 0.05.The Student’s All the patients were informed about the purposes of the study and consequently have signed their “consent of the patient”. All investigations conformed to the principles outlined in the Declaration of Helsinki and were performed with permission by the responsible Medical Ethics Committee of Xiangya Hospital, Central South University, China.Supplementary Table 1Supplementary Table 2Supplementary Table 3


This case report shows that keratomas can occur in both hind feet of equine patients and should be considered as a diagnosis for long-standing, intermittent lameness localised to the hooves. A Thoroughbred racehorse presented with long-standing abscessation of the right hind hoof. Owing to the long-standing nature, the abscess draining tract was surgically explored. A focal mass was found within the solar horn. Histopathology revealed the mass to be a keratoma. A similar mass was removed from the left hind hoof a few months later after similar presenting signs. This case shows that keratomas can occur in more than one hoof within a short period and should be considered a differential diagnosis for long-standing lameness localised to the hoof. This case report describes that keratomas can occur in both hind feet of equine patients and should be considered as a diagnosis for long-standing, intermittent lameness localised to the hooves.et al.et al.et al.et al.Most authors consider keratomas to be slow-growing, benign, hyperplastic masses that occupy space in the affected anatomic area , which distinguishes it from an infectious process  in the When examined, the horse was shod with steel shoes with 1° heel wedges. The hoof was considered within normal limits with regard to size, shape and angle to the ground and pastern. The structure of the hoof appeared normal, with no deviations of the white line. The persistent cycle of assumed abscessation and resolution in the lateral quarter, and the positive reaction to hoof testers in the same location, suggested a sequestrum of unknown origin or possible foreign body. At the time of examination, there was a draining tract in the lateral quarter. Surgical exploration of the tract was performed. Radiographs were not taken initially.The procedure was performed with the horse in a standing position. Sedation was achieved using a combination of detomidine  and butorphanol  administered intravenously. The hoof was desensitised with an abaxial plantar nerve block at the level of the sesamoid bones. Standard and looped hoof knives were used to pare away solar horn along the draining tract in the lateral quarter of the right hind hoof. As the tract enlarged, an irregular spherical mass was observed within the cavity. The mass was loosely attached to the surrounding tissue and was easily removed. The wound cavity was curetted to remove all necrotic tissue, packed with gauze swabs soaked in an astringent (copper sulphate) and then bandaged with a water-resistant bandage. The mass was preserved in 10% buffered formalin and submitted for histopathological examination. The horse received a course of phenylbutazone  for 5 days post procedure (2.2 mg/kg body weight per os once a day). The bandage was replaced every 3 days for 2 weeks and then weekly for a further 2 weeks, repeating the initial packing with copper-sulphate-soaked gauze each time. Granulation tissue was evident after the second bandage change and the tissue keratinised, becoming horn like within a few weeks. At 4 weeks post-removal, a bar shoe was fitted and a light dressing was maintained on the cavity and changed every second day. Systemic antimicrobials were not administered. Owing to the histopathological findings, lateral, dorsopalmar, dorsoproximal–palmarodistal oblique and palmaroproximal–palmarodistal oblique radiographic views were taken of the hoof to determine whether P3 was affected and confirm resolution. No radiographic abnormalities were detected on P3.The irregular spherical mass of 3 cm × 2 cm × 1 cm  was submThe horse returned to ridden exercise 1 month after the procedure was performed. It then developed a similar subsolar abscess in the medial quarter of the left hind hoof. The abscess recurred repeatedly over a period of 2 months, with associated intermittent lameness . Based The horse returned to ridden exercise 1 month after removal of the mass. Five months after returning to ridden exercise, the horse placed second in its second racing start and subsequently won its third start.et al.This case is unusual because a keratoma was diagnosed in one hind foot and highly suspected in the other hind foot in a fairly short time period. Keratomas are not commonly reported in more than one hoof (Mair & Linnenkohl et al.et al.There was no evidence of previous trauma to either hoof. As shown in this case, radiographs were not useful as a diagnostic tool, possibly owing to the specific location of the mass. As the solar horn is not as rigid as the dorsal wall, the growing keratoma likely resulted in less pressure necrosis on P3 (Gasiorowski & Richardson Solar keratomas are difficult to diagnose and should be considered when long-standing abscessation or chronic low-grade lameness attributed to the hoof are the presenting complaints. As shown in this case, it is possible that keratomas can occur in both hind hooves of a horse. This suggests that keratomas can occur on any hoof and can potentially occur in multiple hooves of a single horse.


Immunoglobulin E (IgE) antibodies play a central role in the allergic response: interaction with FcεRI on mast cells and basophils leads to immediate hypersensitivity reactions upon allergen challenge, while interaction with CD23/FcεRII, expressed on a variety of cells, regulates IgE synthesis among other activities. The receptor-binding IgE-Fc region has recently been found to display remarkable flexibility, from acutely bent to extended conformations, with allosteric communication between the distant FcεRI and CD23 binding sites. We report the structure of an anti-IgE antibody Fab (8D6) bound to IgE-Fc through a mixed protein-carbohydrate epitope, revealing further flexibility and a novel extended conformation with potential relevance to that of membrane-bound IgE in the B cell receptor for antigen. Unlike the earlier, clinically approved anti-IgE antibody omalizumab, 8D6 inhibits binding to FcεRI but not CD23; the structure reveals how this discrimination is achieved through both orthosteric and allosteric mechanisms, supporting therapeutic strategies that retain the benefits of CD23 binding. FcεRI is principally expressed on the surface of mast cells and basophils. Allergen mediated cross-linking of FcεRI-bound IgE on the surface of these IgE-sensitized cells triggers degranulation and release of inflammatory mediators2. CD23 is expressed in membrane-bound (mCD23) and soluble forms, the latter existing as monomeric or trimeric fragments5. CD23, expressed on B cells and a range of other cell types, regulates a diverse set of immunological functions, including cellular adhesion, antigen presentation, regulation of growth and differentiation of B and T cells, rescue from apoptosis, release of cytotoxic and inflammatory mediators, transcytosis of IgE-immune complexes, and regulation of IgE synthesis5. CD23-deficient mice or those strains carrying mutated CD23 variants show a hyper-IgE phenotype8 whereas transgenic strains that overexpress CD23 show reduced levels of IgE10. Moreover, B cells, rather than FcεRI-bearing cells, are the major cell type controlling serum IgE levels in a CD23-dependent manner11.The interactions between immunoglobulin E (IgE) and its two receptors, FcεRI and CD23 (FcεRII), play pivotal roles in allergic disease18, and the crystal structure of IgE-Fc revealed an acutely bent conformation, in which the (Cε2)2 domain pair folds back against the Fcε3-4 region, with an angle of 62° between the local two-fold axes of the (Cε2)2 domain pair and Cε4 domains20. Crystal structures of unliganded and receptor-bound forms of IgE-Fc, and the Fcε3-4 region, reveal the Cε3 domains to adopt a range of “open” and “closed” orientations relative to the Cε4 domain pair29. The interaction between IgE-Fc and FcεRIα involves an “opening” of the Cε3 domains, which engage FcεRI at two distinct sub-sites on the Cε2-proximal region of each Cε3 domain25, and IgE-Fc becomes even more acutely bent (54°) in the receptor-bound complex19. CD23 engages IgE-Fc at a region of the Cε3 domain distal to the FcεRIα binding site, and near the interface with the Cε4 domain29. Crystal structures of IgE-Fc and the Fcε3-4 region in complex with CD23 reveal this receptor to engage a range of “closed” Cε3 domain conformations29. The open and closed Cε3 domain conformations involved in FcεRIα and CD23 interactions, respectively, preclude simultaneous engagement of both receptors by IgE-Fc; binding of FcεRIα and CD23 are thus regulated in an allosteric manner30.IgE-Fc, the region of the antibody responsible for effector functions, comprises two identical chains, each composed of three immunoglobulin-like domains: Cε2, Cε3 and Cε4. IgE, and IgE-Fc, adopt a compact, bent structure in solution24. An anti-IgE-Fc Fab (aεFab) captured IgE-Fc in an extended conformation, and the crystal structure of the aεFab/IgE-Fc complex revealed a fully extended, linear IgE-Fc molecule, in which the local two-fold axes of the Cε2, Cε3 and Cε4 domain pairs were coincident, and the (Cε2)2 domain pair no longer contacted the Fcε3-4 region24. This extreme conformational flexibility is suggested to underpin the different biological functions of IgE, with acutely bent FcεRI-bound IgE positioning the Fabs in an appropriate orientation for cross-linking by allergen, and the fully extended molecule, in the membrane-bound form (mIgE) as part of the B-cell receptor for antigen, extending the Fabs away from the membrane, to facilitate antigen capture24.Unexpectedly, IgE-Fc was recently observed to undergo a large-scale conformational change2. The therapeutic monoclonal anti-IgE antibody omalizumab  is approved for the treatment of moderate-to-severe persistent allergic asthma and chronic idiopathic urticaria32. Omalizumab prevents IgE from engaging both FcεRI and CD23, decreases serum IgE levels in vivo by up to 95% and down-regulates surface expression of FcεRI on basophils35. The structural basis for the mechanism of action of omalizumab has recently been elucidated36; omalizumab inhibits the binding of IgE to FcεRI allosterically, as antibody binding causes the Cε3 domains to adopt a conformation that is too open to permit simultaneous engagement of both FcεRI sub-sites on IgE-Fc36, while the binding of IgE to CD23 is inhibited orthosterically37. Another anti-IgE antibody, MEDI4212, also inhibits the interaction between IgE-Fc and its two receptors21; binding to FcεRI is inhibited due to steric overlap, while binding to CD23 is inhibited as the Cε3 domains are “locked” in an open conformation21.The interaction between IgE and FcεRI is a long-standing target in the treatment of allergic disease39, but was not taken forward into late clinical trials. Indeed, there may be advantages to a therapeutic strategy that combines IgE neutralization and modulation of IgE production simultaneously.A large body of literature supports roles for CD23 both as an essential regulatory molecule in normal immune responses, and as a marker of, and mediator in, the pathophysiology of a number of human disorders. A primatized anti-CD23 antibody, lumiliximab/IDEC-152, showed modest clinical efficacy in early clinical trials for allergic asthma, where it reduced circulating IgE levels by about 40% during the period of active treatment33, thus preventing mast cell degranulation. However, 8D6, and 8D6/IgE-immune complexes, are able to engage IgE that is already bound to mCD23 on B cells33. An antibody with the binding characteristics of 8D6 could thus exploit the ability of CD23 to down-regulate IgE production, such as cross-linking of mCD23 by IgE-immune complexes, which inhibits B-cell proliferation and IgE synthesis41.8D6 is a monoclonal anti-IgE antibody that, like omalizumab and MEDI4212, inhibits the interaction between IgE and FcεRI24, with the (Cε2)2 domain pair instead contacting the Cε3 domains. The more compact IgE-Fc molecule demonstrates that not only is IgE dynamic, with the ability to flex from an acutely bent to an extended structure24, but the fully extended molecule itself is also conformationally diverse.Here we report the 3.7 Å resolution crystal structure of IgE-Fc in complex with two 8D6 Fab fragments. IgE-Fc adopts a nearly linear conformation, exposing both faces of the Fcε3-4 region, to which the Fabs bind through a mixed protein-carbohydrate epitope. This extended IgE-Fc conformation is more compact than the conformation captured by aεFabThe structure of the 8D6 Fab/IgE-Fc complex reveals that FcεRI binding is inhibited both allosterically and orthosterically. By contrast, the CD23 binding site is accessible, and solution studies demonstrate that the affinity of IgE-Fc for CD23 is not substantially altered when in complex with 8D6. We thus reveal the structural basis by which IgE-Fc binding to its two principal receptors can be selectively modulated, offering insights into an alternative therapeutic approach to the treatment of allergic disease.D of about 20 pM, with kon = 5 × 106 M−1 s−1 and koff = 8 × 10−5 s−1, where the slow dissociation rate is the main contributor to the high affinity interaction.We performed SPR-based molecular interaction studies to measure binding stoichiometry, kinetics and affinities for the interaction of the 8D6 Fab with IgE-Fc. The 8D6 Fab was covalently coupled to an SPR sensor chip using a standard amine coupling protocol. IgE-Fc, over a range of concentrations, was flowed over the 8D6 surface, and association and dissociation rate constants were extracted from the binding curves . These SPR-based interaction analyses confirmed the expected 2:1 stoichiometry of 8D6 for IgE, and showed that both sites have subnanomolar binding affinities, which are higher than those for the clinically approved omalizumab36.The homodimeric character of IgE commonly results in two separate epitopes for anti-IgE antibodies and the asymmetrical structure of IgE-Fc sometimes results in two different affinities for the two epitopesWe solved the crystal structure of the 8D6 Fab/IgE-Fc complex, in which two 8D6 Fab fragments are bound to IgE-Fc, at 3.7 Å resolution. IgE-Fc adopts a fully extended conformation, exposing both faces of the Fcε3-4 region, to which the 8D6 Fabs bind Fig. . Each 8D24 2 domain pair and Cε2-Cε3 domain linker move as a rigid unit relative to the Cε3 domains  adopts a variety of conformations , His422, His424 and Leu425 (FG loop)  is sandwiched between the Pro365 and Lys391 (Cε3) side chains, and the interface also includes a number of hydrogen bonds  pack against Gln301, Lys302 and Leu305 (Cε2). Although the two IgE-Fc chains are not identical, similar contacts are maintained between the heavy chain and Cε2 domain at each interface; main chain atoms for residues Gly26-Phe29 (CDRH1) shift to preserve this interaction, for example, the Tyr27 Cα atom position differs by 1.5 Å , Tyr36 (CDRL1) and Asn96 (CDRL3) form a cleft which accommodates the side chain of Arg393 from the same Cε3 domain contacted by the heavy chain , Asp32 and Gly33 (CDRL1) contact two mannose residues (Man906 and 907) from the α(1–3) branch, and a hydrogen bond forms between the Asp32 main chain and the α(1–3) linked mannose residue, Man906. For the other Fab (106 Å2 interface), Asp32, Gly33 and Asp34 (CDRL1) contact equivalent mannose residues, but a hydrogen bond forms between the Asp34 side chain and the α(1-2) glycosidic bond between Man906 and Man907 , but the most dramatic change occurs in the Tyr27 (CDRH1) side chain, which flips from one face of the main chain to the other to contact Gln301 and Lys302 from the Cε2 domain. Conformational flexibility in CDRH1 appears to be facilitated by the presence of two glycine residues, at positions 26 and 31, whereas in the complex, steric constraints are imposed by the Cε3 domain residues Pro365 and Ser366  involves an acutely bent conformation for IgE-Fc, in which the Cε3 domains adopt an open conformation, and engage the receptor through two distinct sub-sites19. The anti-IgE 8D6 antibody inhibits binding of IgE to FcεRI, and is also unable to engage FcεRI-bound IgE33. Comparison of the IgE-Fc/FcεRIα and 8D6 Fab/IgE-Fc complexes reveals that the Cε3 domains adopt a conformation that is too closed to permit simultaneous engagement at both FcεRIα sub-sites 2 domain pair and FcεRIα  on RPMI 8866 cells, obviating the need for secondary IgE detection antibodies. We observed that IgE-Fc alone was able to bind mCD23, but failed to do so in the presence of MHM6 (anti-CD23) and omalizumab (anti-IgE), known inhibitors of the IgE/CD23 interaction47  with the same affinity as IgE-Fc captured via an N-terminal His-tag  was similar to binding of derCD23 to unbound IgE-Fc (KD = 1.7 ± 0.2 μM)  Fig. . The lacμM) Fig. .Figure 629. Omalizumab inhibits the interaction between IgE and CD23 orthosterically37, while MEDI4212 (anti-IgE) “locks” the Cε3 domains in an open conformation, precluding the interaction with CD2321 24, and a partially bent conformation, with markedly open Cε3 domains, was observed in the crystal structure of IgE-Fc in complex with an omalizumab-derived Fab36.IgE-Fc adopts a predominantly bent conformation in solution2 domain pair and Cε2-Cε3 domain linker rotate and move closer to the Cε3 domains, transforms one fully extended form to the other. Furthermore, the unique Cε3 BC loop conformation loop conformation found in the 8D6 Fab/IgE-Fc complex suggests that local conformational changes in IgE-Fc contribute to global structural changes.Here, we report the 3.7 Å resolution crystal structure of IgE-Fc in complex with the Fab fragment of the anti-IgE antibody 8D6, in which two 8D6 Fabs bind to a fully extended IgE-Fc molecule. The 8D6 Fab/IgE-Fc complex reveals a more compact form of fully extended IgE-Fc, in which the Cε3 domain contacts the Cε2 domain from the same chain, and the Cε2-Cε3 domain linker from the other chain. The 8D6 Fab/IgE-Fc complex reveals that not only is IgE-Fc flexible, adopting both acutely bent and extended conformations, but the fully extended conformation itself is also conformationally diverse. A spiral motion, resembling that of a corkscrew, in which the Cε3 domains close, and the (Cε2)24. Given the potential for extreme flexibility, it is therefore possible that IgE-Fc can freely adopt this compact, extended conformation in solution.It remains to be determined whether the compact, extended form observed is induced, or selected, by 8D6, but the extended conformation previously observed in the aεFab/IgE-complex was captured by the Fab24, but the extended conformation captured by aεFab occupied a high-energy basin24. Given the proposed role for the extended form of IgE as part of the B-cell receptor for antigen, the more compact form found in the 8D6 Fab/IgE-Fc complex provides clues about how a fully extended IgE conformation might be stabilized, even transiently, on the B-cell surface, to facilitate antigen capture.In a recent molecular dynamics study exploring the unbending of IgE-Fc to an extended conformation, the acutely bent conformation occupied the lowest energy basin2, and is inhibited by the clinically approved therapeutic antibody omalizumab31, and other anti-IgE antibodies such as MEDI421221, aεFab24 and 8D633; however, the mechanism of inhibition for these antibodies differs. In the complex of an omalizumab-derived Fab with IgE-Fc, the Cε3 domains adopt a markedly open conformation, even more open than that for FcεRI-bound IgE-Fc36, which precludes simultaneous engagement of the two FcεRI-binding sub-sites36, while the complex of MEDI4212 with Fcε3-4 reveals steric overlap with FcεRI21. In the aεFab/IgE-Fc complex, structural rearrangements in the Cε2-Cε3 domain linker, disruption of a key receptor interaction, and steric clashes between the extended IgE-Fc conformation and FcεRI, preclude receptor binding24. In the 8D6 Fab/IgE-Fc complex, the Cε3 domains adopt a conformation which is too closed to permit simultaneous engagement of the FcεRI-binding sub-sites, and the Cε2 domains in the extended IgE-Fc conformation would also clash with the receptor.The interaction between IgE and FcεRI, a prerequisite for mast cell degranulation, is a long-standing target in the treatment of allergic disease33. We have demonstrated here that 8D6-bound IgE-Fc binds to mCD23 on cells, and that the affinity of the 2:1 8D6/IgE-Fc complex for a soluble fragment of CD23 (derCD23) is similar to unbound IgE-Fc. Consistent with these observations, the crystal structure of the 2:1 8D6 Fab/IgE-Fc complex reveals that one of the CD23 binding sites on IgE-Fc is fully accessible, with only minor clashes at the second site; a minor conformational change in IgE-Fc, involving the flexible 8D6 epitope, which includes the oligosaccharide moiety, would render the second CD23 binding site accessible.Unlike the clinically-approved omalizumab, 8D6 does not inhibit the interaction between IgE and CD2341. Inhibiting the interaction between IgE and FcεRI, but not CD23, is thus a potentially promising therapeutic approach in the treatment of allergic disease. Here, we have revealed the structural basis by which binding of IgE to these two key receptors can be selectively modulated.In its role as a regulator of IgE levels, engagement of mCD23 by IgE-immune complexes inhibits B-cell proliferation and IgE synthesis49. Recombinant 8D6 Fab was expressed in Freestyle HEK293F cells using a previously published method50 with the exception that after a four hour transfection at high cell density, the cells were diluted four-fold in the presence of 3.75 mM sodium valproate, and after seven days the culture supernatant was harvested and protein purified by KappaSelect affinity chromatography. His-tagged IgE-Fc used for the SPR analysis contained the same sequence as that used for the crystallographic study , in addition to an N-terminal tag (6xHis and Xpress tags), was expressed in Freestyle™ HEK293F cells, and purified by Ni2+-affinity chromatography.Human IgE-Fc used for the crystallographic study and FACS analysis, and human derCD23 used for the SPR analysis, were expressed as previously describedThe 8D6 Fab/IgE-Fc complex for the crystallographic study was purified as follows: 8D6 Fab and IgE-Fc proteins were mixed at a molar ratio of 2.5:1 at 4 °C for 16 hours and the resulting complex purified by size exclusion chromatography using a Superdex 200 column.For the flow cytometry analysis, IgE-Fc was concentrated to 50 µM, and dialysed overnight at 4 °C against a buffer of 100 mM sodium bicarbonate and 50 µM NaCl, pH 9. A 2.5 molar excess of Alexa Fluor® 647 succinimidyl ester (Life Technologies) was added to IgE-Fc, gently agitated for 3 hours, and then dialysed against PBS overnight. Absorbance of the sample at 280 nm and 647 nm was used to determine the labeling efficiency.Experiments were performed on a Biacore T200 instrument . Specific binding surfaces were prepared by coupling 8D6 Fab to a CM5 sensor chip, using a standard amine coupling protocol . For measuring the affinity of the first binding site of 8D6 Fab to IgE-Fc, His-tagged IgE-Fc, at concentrations ranging from 100 nM to 190 pM, prepared by a 2-fold serial dilution in HBS-P buffer , was injected at 30 μL/min, with a 2 min association phase followed by a 12 min dissociation phase. For the sandwich binding experiments, approximately 100 resonance units of His-tagged IgE-Fc was captured on an 8D6 Fab surface; after a 2 min stabilization period, 8D6 Fab was injected at 30 μL/min with a 2 min association phase followed by a 12 min dissociation phase. The sensor surface was regenerated by two 30 sec injections of 0.2 M glycine-HCl, pH 2.3.2. DerCD23 binding to (1) N-terminally His-tagged IgE-Fc captured using an anti-His-tag antibody , (2) IgE-Fc captured on an 8D6 Fab surface, resulting in derCD23 binding to a 1:1 8D6 Fab/IgE-Fc complex, and (3) IgE-Fc captured on an 8D6 Fab surface, and then bound to a second 8D6 Fab by injecting a saturating concentration (100 nM) of 8D6 Fab, resulting in derCD23 binding to a 2:1 8D6 Fab/IgE-Fc complex. DerCD23 solutions over a range of concentrations  were injected at 20 μL/min with a 60 sec association phase, followed by a 90 sec dissociation phase. All measurements were done in duplicate, using titration series of “low-to-high” analyte concentration followed by a “high-to-low” series51 at 25 °C. In all cases, standard double referencing data subtraction methods were used51 and kinetic fits were performed using Origin software (OriginLab).For measuring the binding of derCD23 to IgE-Fc or 8D6 Fab-bound IgE-Fc, all experiments were performed in HBS-P buffer with 5 mM CaCl6 cells from a CD23 expressing cell line (RPMI 8866), maintained as described previously52, were incubated with 5 μg/mL of IgE-Fc Alexa-647 with or without 10 μg/mL 8D6 (anti-IgE). MHM6 (anti-CD23) and omalizumab (anti-IgE), two known inhibitors of IgE binding to CD2347, were used as controls. After 30 minutes of incubation on ice, cells were washed by centrifugation at 300 × g for 5 mins at 4 °C, collected using a BD FACSCanto and analysed using FlowJo software (Tree Star Inc).To examine the effect of 8D6 on IgE binding to membrane CD23, 0.5 × 10280 of 11. 8D6 Fab crystals were grown by sitting drop vapour diffusion at 18 °C in MRC 96 well plates. The reservoir contained 50 µL of 0.1 M ADA (N-(2-acetamido) iminodiacetic acid) pH 6.5 and 12% (w/v) PEG 4000. The drop contained 250 nL and 400 nL of protein solution and reservoir, respectively. Crystals were cryoprotected in 0.1 M Tris-HCl pH 7.0, 20% (w/v) PEG 3350, 0.2 M (NH4)2SO4 and 18% (v/v) ethylene glycol before flash-cooling in liquid nitrogen. Purified 8D6 Fab/IgE-Fc complex was buffer-exchanged into 100 mM Tris-HCl pH 7.4 and 50 mM NaCl, and concentrated to an OD280 of 16.4. Crystals of the 8D6 Fab/IgE-Fc complex were also grown by sitting-drop vapour diffusion. The reservoir contained 75 µL of 0.1 M Tris-HCl pH 8.0 and 45% (v/v) pentaerythritol propoxylate (5/4PO/OH). The drop contained 100 nL and 50 nL of protein solution and reservoir, respectively. Crystals were flash-cooled in liquid nitrogen using mother liquor as a cryoprotectant.For crystallization trials, the 8D6 Fab was buffer-exchanged into 20 mM Tris-HCl pH 8.6, and concentrated to an OD54, and further processed to 2.4 Å resolution with programs from the CCP4 suite56. The structure was solved in space group P43, with three Fab molecules in the asymmetric unit. The structure was solved by molecular replacement with PHASER57 using protein atoms from PDB entry 1I7Z58 as a search model. Data for the 8D6 Fab/IgE-Fc complex were collected at beamline I04-1 at the Diamond Light Source. Intensities were integrated with XDS53 and further processed to 3.7 Å resolution with programs from the CCP4 suite56. The structure was solved in space group P212121, using a combination of molecular replacement with PHASER57 and manual domain placement, using a partially refined 8D6 Fab structure and protein atoms from PDB entry 2WQR as search models19, and the asymmetric unit contained one 2:1 8D6 Fab/IgE-Fc complex. Both structures were refined with PHENIX59. Manual model building was performed with Coot60. Overall structure quality was assessed with MolProbity61 and POLYGON62 within PHENIX. Data processing statistics are summarized in Table 63, figures were produced with PyMOL64, and movies were generated using Chimera65, PyMOL64 and the eMovie plugin66 for PyMOL.Data for the 8D6 Fab were collected at beamline I04 at the Diamond Light Source . Intensities were integrated with XDS using the xia2 packageCoordinates and structure factors have been deposited at the Protein Data Bank with accession codes 6EYN (8D6 Fab) and 6EYO (8D6 Fab/IgE-Fc complex).Supplementary InformationSupplementary Movie S1Supplementary Movie S2


Platelet-derived growth factor (PDGF) signaling is dysregulated in a wide variety of diseases, making PDGF an attractive therapeutic target. However, PDGF also affects numerous signaling cascades essential for tissue homeostasis, limiting the development of PDGF-based therapies that lack adverse side-effects. Recent studies showed that fibroblast-mediated assembly of extracellular matrix (ECM) fibronectin fibrils attenuates PDGF-induced intracellular calcium release by selectively inhibiting phosphoinositol 3-kinase (PI3K) activation while leaving other PDGF-mediated signaling cascades intact. In the present study, a series of recombinant fibronectin-derived fusion proteins were used to localize the sequences in fibronectin that are responsible for this inhibition. Results demonstrate that attenuation of PDGF-induced intracellular calcium release by the fibronectin matrix mimetic, FNIII1H,8-10 requires α5β1 integrin ligation, but is not dependent upon the matricryptic, heparin-binding site of FNIII1. Intact cell-binding fibronectin fragments were also unable to attenuate PDGF-induced intracellular calcium release. In contrast, a novel integrin-binding fragment that adopts an extended and aligned conformational state, inhibited both PI3K activation and intracellular calcium release in response to PDGF. Taken together, these studies provide evidence that attenuation of PDGF-induced intracellular calcium release by fibronectin is mediated by a novel conformation of the α5β1 integrin-binding, FNIII9-10 modules, that is expressed by fibrillar fibronectin. Platelet-derived growth factor (PDGF) evokes a diverse array of intracellular signaling cascades that promote a variety of cell and tissue behaviors, including cell growth , survivaFibronectin is an extensible, modular glycoprotein that binds to and influences the behavior of cells, other ECM components, and growth factors . FibroneCell adhesion to fibronectin is mediated primarily by binding interactions between α5β1 integrins and the Arg-Gly-Asp (RGD) sequence within the tenth type-III repeat of fibronectin (FNIII10) . The fleIntegrin binding specificity is also affected by the conformation of the central cell-binding region of fibronectin ,22. FibrA recombinant, fibronectin-derived fusion protein, FNIII1H,8-10, was developed previously to mimic the conformation and biological effects of the ECM form of fibronectin . FNIII1HFibronectin was purified from outdated human plasma  using gelatin-Sepharose  affinity chromatography . CollageFN-null MEFs were cultured routinely under serum- and fibronectin-free conditions on collagen I-coated tissue culture flasks in a 1:1 mixture of Aim V  and SF Medium . Fibronectin-null MEFs do not produce endogenous fibronectin but are able to polymerize exogenously-added fibronectin into a fibrillar ECM . Thus, tRGD; FNIII1HRGD; FNIII1H,8RGD); the fibronectin matrix mimetic in which the PHSRN synergy site in FNIII9 was mutated -tagged fusion proteins were produced in  FNIII10 ; FNIII9- FNIII10 ; FNIII1H FNIII10 ; FNIII1H FNIII10 ; FNIII1HNIII1H,8 ; FNIII1HH,8 [RGD . For som1326-Thr1961) was produced using the sense primer: 5′-ACCATGGCTGTTCCTCCTCCCACTGACCTGCG-3′ (NcoI site in bold) and antisense primer: 5′-GGCGGCCGCCAGTGGAGGCGTCGATGACCACAGG-3′ (NotI site in bold). The human fibronectin cDNA pCEF103 construct was used as a template. PCR-amplified DNA was cloned into the pEcoli-Cterm 6xHN plasmid , expressed in E. coli strain BL21(DE3), and isolated on nickel-NTA-agarose . A fibronectin fragment spanning FNIII8 through FNIII13  was produced. PCR was used to amplify human fibronectin cDNA, with pGex2T/FNIII8-10 serving as a template [GGTACCGTCACCTGGGCTCCACC-3′) contains a KpnI site (shown in bold) at the 5′ end. The antisense primer for III8c-10 (5′-CCCCCCGGGCTATGTTCGGTAATTATTGGAAATTGGCTTGCTG-3′) contains an Xma1 site (shown in bold) at the 5′ end. PCR-amplified DNA was cloned into pGex2T  and transfected into DH5α bacteria [A fibronectin fragment comprised of a carboxyl-terminal fragment of FNIII8 with intact III9 and III10 modules  (Gibco #11965-092) onto black, clear-bottom, 96-well plates precoated with collagen. Four hours after seeding, fibronectin or fusion proteins were added to the media. Following an overnight incubation (~23 h post-seeding), cells were well-spread and confluent. Media was removed and cells were loaded with 4.6 μM Fluo-4 AM  in a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-based imaging buffer  [FN-null MEFs were seeded at 1.1 × 10 pH 7.4) . After 40). Data are presented as the relative change in fluorescence (ΔF/F0) [0 from at least 3 experiments performed in at least triplicate. ΔF/F0 values from individual trials were normalized to a control condition , which was set to 1.To quantify changes in intracellular calcium in response to PDGF, raw data were filtered to reduce noise inherent in the fluorescence signal by taking a 30 s running average of the raw data . The PDG (ΔF/F0) . Data ar® West Pico Chemiluminescent Substrate (Thermo Fisher Scientific). The average net band intensity for duplicate samples was determined by densitometry using Carestream Molecular Imaging Software . Relative band densities were averaged across multiple experiments.Cells were solubilized in Laemmli buffer and lysates (~40 μg protein per lane) were analyzed by SDS-PAGE and immunoblotting, as described previously . Immunob1379 in FNIII9 and R1493 in FNIII10. The rotational angle was defined as the dihedral angle formed by the Cα carbons of R1379 in FNIII9 and R1493 in FNIII10 and the centers of mass of FNIII9 (S1396) and FNIII10 (T1486) [All calculations were conducted using version 9.1.107 of Maestro  with the Amber* force field. The crystallographic structure of FNIII8-10 was obtained from FNIII7-10  (Protein4 cells/cm2) in 96-well plates, pre-coated with saturating concentrations (125 nM) of FNIII8-10, FNIII9-10, or FNIII10, and incubated for 15 min at 4 °C. For competition assays, cells were pre-incubated with various concentrations of FNIII8-10 or FNIII8c-10 for 15 min prior to seeding onto FNIII8-10-coated wells. Cell attachment was determined by staining with 0.1% crystal violet, as described previously [FN-null MEFs were preincubated in suspension with anti-β1 or -β3 integrin blocking antibodies (25 μg/mL), immunoglobulin G or M (IgG or IgM) isotype controls (25 μg/mL), or 10 mM ethylenediaminetetraacetic acid (EDTA), for 40 min at 37 °C. Cells were seeded  relative to control treatment. Experiments were performed in quadruplicate  or duplicate (immunoblotting) for a minimum of 3 independent experiments. Statistical analysis was performed using Prism, Version 4 . Unless otherwise indicated, statistical significance was determined using an analysis of variance (ANOVA) with a Bonferroni’s post-test. Results were considered significant if Fibronectin matrix assembly by either FN-null MEFs or human dermal fibroblasts reduces the magnitude of intracellular calcium release in response to PDGF exposure . A simil613-Trp614-Arg615-Pro616-Lys617 (RWRPK) [FNIII1 contains a matricryptic, heparin binding sequence, Arg (RWRPK) ,49. Muta (RWRPK)  and the  (RWRPK) ,49. To dThe anti-fibronectin monoclonal antibody, 9D2, binds to and blocks the biological activity of the RWRPK site of FNIII1 in full-length fibronectin ,50. ThusThus far, the results indicate that neither the matricryptic site of FNIII1 nor integrin-binding fragments inhibit calcium release in response to PDGF. Therefore, to further explore the mechanism of FNIII1H,8-10 inhibition, we analyzed a series of FNIII1H,8-10 variants for changes in activity. Replacing FNIII8-10 with the non-integrin-binding modules, FNIII2-4 , resulted in a loss of activity C, pointiRGD) or FNIII8  creates constructs that selectively ligate αvβ3 integrins [FNIII8-10 contains three FNIII domains that work together to promote α5β1 integrin ligation ,27. FNIIntegrins ,42. NoneBinding of α5β1 integrins to FNIII9-10 requires the RGD loop of FNIII10 and the PHSRN “synergy sequence” of the neighboring FNIII9 module ,25,26,51The cell-binding region of fibronectin is conformationally labile . Moreove199) was significantly reduced in cells cultured in the presence of either fibronectin , PLC-γ1, or extracellular signal-related kinase (ERK)-1/2 . In the In contrast to several previous studies demonstrating a role for the matricryptic site of FNII1 in mediating the effects of ECM fibronectin ,41,49,50Changes to FNIII8-10 conformation have been observed in response to a variety of conditions, including force-induced unfolding of fibronectin , inclusiOur results indicate that both the synergy site and the RGD sequence are necessary for attenuation of PDGF-induced intracellular calcium release. The crystal structure of FNIII7-10 shows that the RGD and PHSRN sites are separated by a distance of ~32 Å , while ocollagen A1 and fibronectin 1 [The increase in cytosolic calcium concentration evoked by PDGF activates downstream signaling by altering the conformation of intracellular signaling molecules. As one example, calcium is bound by calmodulin, which results in calmodulin undergoing a conformational change to adopt its active conformation . In turnnectin 1 . PDGF-innectin 1 .The ECM form of fibronectin selectively attenuates PI3K activity and intracellular calcium release in response to PDGF . In contThe discovery that cell-assembled ECM fibronectin attenuates PDGF-induced PI3K activity and intracellular calcium release may be important in understanding the pathogenesis of conditions that are characterized by both altered cell responsiveness to PDGF and aberrant fibronectin matrix assembly. As one example, fibrosis is characterized by substantial remodeling of the ECM, excess proliferation of myofibroblasts that are abnormally responsive to PDGF, and enhanced deposition of ECM proteins . TogetheA novel, recombinant, cell-binding fragment of fibronectin was shown to inhibit both PI3K activation and intracellular calcium release in response to PDGF. This fragment was produced by removing β-strand A from the amino-terminus of FNIII8. Molecular dynamics simulations of FNIII8c-10 indicated that as a result of the FNIII8 truncation, FNIII9 and FNIII10 adopt an aligned, extended conformation with a unique rotational angle between the RGD and PHSRN sites. PDGF-induced intracellular calcium release was attenuated by soluble fibronectin fragments that bind to α5β1, and not αvβ3, integrins. Previous studies [


This study compared the biomechanics of reinserted pedicle screws using the previous entry point and trajectory with those of correctly inserted pedicle screws.The study used 18 lumbar vertebrae (L1–6) from three fresh calf spines to insert 6.5 × 40-mm pedicle screws. A control screw was inserted correctly along the axis of one pedicle, while an experimental screw was reinserted completely using the previous entry point and trajectory in the other pedicle. The experimental screw was removed after being completely inserted in group A and after 80% of the total trajectory inserted in group B. And the experimental screw was removed after 60% of the total trajectory was reached in group C. The biomechanical values of the pedicle screws were measured.PA = 0.463, PB = 0.753, PC = 0.753). Stiffness measurement increased for the reinserted screw compared with that of the control screw. Fracturing was observed between the vertebral body and pedicle.There were no significant differences in pedicle screw axial pullout strength between reinserted screws and correct screws in the 3 groups (Theoretically, a surgeon can remove the pedicle screw when necessary, inspect the trajectory, and reinsert the screw using the previous entry point and trajectory. Pedicle screw instrumentation is considered standard for treatment of spinal degenerative diseases, fractures, tumors, and deformities –3. AlthoMany studies reported that the biomechanical strength of a redirected pedicle screw is less than that of a correctly placed screw , 8–11. B3), range of motion , neutral zone (14–30%), and stiffness (0.07–0.38) in 12 calf spines and found that they were similar to those of human spines. They suggested that calf spines could be used to assess spinal implants as an alternative to use of human cadavers.Wilke et al.  measuredThe purpose of this research was to compare the biomechanics of reinserted pedicle screw using the previous entry point and trajectory with that of correctly inserted pedicle screws using calf lumbar vertebrae.This study used 18 lumbar vertebrae (L1-6) from 3 fresh calf spines (age about 6–9 months). Due to similar BMD, un-mature calf models were used. Muscles, ligaments, and intervertebral discs were removed, preserving only normal osseous structures. The vertebrae were stored at − 20 °C until the day before testing and thawed at room temperature. The vertebrae were radiographed in the anterior-posterior and lateral planes to exclude fracture, tumor, and congenital disease. And the length and diameter of pedicles were measured by a ruler.Before testing, the specimens were divided randomly into 3 groups according to the lumbar sequence. A control screw was inserted correctly along the axis of one pedicle, while an experimental screw was reinserted in the other pedicle, using the previous entry point and trajectory. Each screw was inserted using the same depth and angle. If a control screw was inserted in the right pedicle, a control screw would be inserted in the left pedicle of the next vertebra, so that the screws intersected Fig. . The conEach pedicle screw was inserted using the freehand technique under direct visualization . The entIn group A, the experimental screw was removed after being completely inserted in one pedicle and then reinserted completely using the previous entry point and trajectory after inspecting the trajectory. In group B, the experimental screw was removed after 80% of the total trajectory was reached and was then reinserted completely. In group C, the experimental screw was removed after 60% of the total trajectory was reached and was then reinserted completely Fig. .Fig. 2RaFor each pedicle screw, we assessed biomechanical properties using a material testing machine , with the screw pulled out along its long axis Fig. .Fig. 3PhP value < 0.05.All analyses were performed using SPSS 19.0 software . Differences in biomechanical properties were analyzed using the paired Wilcoxon rank sum test. Significance was set for a As shown in Table Pedicle screw instrumentation is often used to treat spinal disease. Most surgeons insert pedicle screws using the freehand technique. Malposition of pedicle screws is a major complication during screw insertion. The reported rate of pedicle screw malposition is 5–41% , 6, 7.After a pedicle screw breaches the lateral wall, the nerve can be damaged by bone chips. To avoid this situation, some surgeons remove the pedicle screw after passing through the posterior vertebral body. If the pedicle lateral wall is in good condition, the screw can be reinserted using the previous entry point and trajectory. Moreover, if a shorter pedicle screw is used because of insufficient preoperative preparation, the surgeon may remove the pedicle screw and reinsert a longer pedicle screw. Many studies have examined the biomechanics of pedicle screws , 13, 14.BMD shows a high correlation with pedicle screw axial pullout force , 16. In The results indicated that there was no significant difference in pedicle screw axial pullout strength between reinserted and correctly inserted screws in the 3 groups. Second, stiffness increased in the reinserted screw compared with that in the control screw. Third, fracture occurred between the vertebral body and pedicle.The purpose of pedicle screw instrumentation is to increase the stability of the spine. Pullout strength is one of the main indexes used to evaluate stability. In the present study, the pullout strength of reinserted pedicle screws was less than that of control screws in group A and B. However, reinserted screws were stronger than control screws in group C. No group showed statistically significant differences. The question is whether a pedicle screw should be removed after insertion to inspect the trajectory or to replace it with a longer screw. The pullout strength of reinserted screws was only slightly less than that of correctly inserted screws. Accordingly, the pedicle screw can be removed if necessary. Maeda et al.  found thP = 0.753), the results are worth noting. The reasons for these findings are unknown. It is possible that the specimen was insufficient. Further study is required. Nonetheless, we concluded that the stiffness of inserted screws did not decrease.Pedicle screw loosening is a frequent complaint . StiffneIn every specimen, fractures developed between the vertebral body and pedicle when the terminal end of the pedicle screw was removed through the edge of the vertebral body. The vertebral body consists mainly of cancellous bone, while the major structure of the pedicle is cortical bone. Thus, fracture can easily occur at the interface. Clinically, when the pedicle screw is pulled out accidentally, the possibility of fracture should be considered, especially in the presence of osteoporosis.A limitation of this study is that we were only able to evaluate the biomechanics of the calf spine because human spines were not available. Although Wilke et al.  studied In spite of the limitations of this study, the pullout strength was not found to be significantly greater between correctly inserted screws and reinserted screws using the previous entry point and trajectory. Theoretically, a surgeon can remove the pedicle screw if necessary, inspect the trajectory, and reinsert the screw using the previous entry point and trajectory. Despite this finding, pedicle screws should be inserted carefully and removal should be avoided as much as possible.


The skin harbors diverse communities of microorganisms, and alterations to these communities can impact the effectiveness of the skin as a barrier to infectious organisms or injury. As the global availability and adoption of antibacterial products increases, it is important to understand how these products affect skin microbial communities of people living in rural areas of developing countries, where risks of infection and injury often differ from urban populations in developed countries. We investigated the effect of antibacterial soap on skin microbial communities in a rural Malagasy population that practices subsistence agriculture in the absence of electricity and running water. We quantified the amount of soap used by each participant and obtained skin swab samples at three time points: prior to soap use, immediately after one week of soap use, and two weeks after soap use was discontinued. Soap use did not significantly impact ecological measures of diversity and richness . However, the amount of soap used was a predictor of community-level change (beta diversity), with changes persisting for at least two weeks after subjects stopped using soap. Our results indicate that the overall species richness of skin microbial communities may be resistant to short-term use of antibacterial soap in settings characterized by regular contact with the natural environment, yet these communities may undergo shifts in microbial composition. Lifestyle changes associated with the use of antibacterial soap may therefore cause rapid alterations in skin microbial communities, with the potential for effects on skin health. The microbes  that live in and on the human body are abundant and diverse, with important implications for health –4. TheseLess is known about the microbial communities on human skin, despite it being the largest organ and home to a variety of bacteria and other organisms important for health . The skiEnhydrobacter was found to be common on the skin of the Chinese participants but not on the skin of Westerners [Propionibacterium, one of the groups of Amerindians’ forearms dominate by Staphylococcus, and the other group of Amerindians’ forearms significantly more diverse than the other two groups [In addition to changes that occur throughout development, human skin bacterial communities also undergo alteration due to environmental variables. The body can be viewed as an island that is in constant interaction with different microbes over time, with a wide variety of environmental and biological factors influencing the diversity and abundance of these microorganisms , 15. In sterners . Anothero groups . These dBehavioral practices can also impact skin microbial communities , 23, 24.While people in developed countries often have access to antibacterial products and are encouraged to maintain high levels of personal hygiene through public health programs and cultural norms, these products are much less common in low and middle-income countries (LMICs). As some LMICs transition toward increased development, access to and use of antibacterial soaps may increase. In addition, as LMICs see an increase in antibiotic resistance, the addition of more antibiotic products, including soap, is of concern. The net benefits of antibacterial soap on human health is unclear and potentially even harmful, as evidenced by the US Food and Drug Administration’s (FDA) recent ruling to prohibit the sale of soaps and shampoos containing certain antibacterial ingredients . While sWe aimed to assess the impact of antibacterial soap on skin bacterial communities in a population in rural Madagascar. This population is valuable in this context for several reasons. First, subsistence agricultural is commonly practiced without powered machinery, putting people in close contact with elements of the natural environment, including domesticated and wild animals. Due to this increased diversity and intensity of environmental exposures, the effects of antibacterial soap on skin microbial communities may differ from effects in Western populations. Second, this population lacks access to running water, and instead uses naturally flowing, mountain-derived water for bathing. Use of this common water source, which contrasts with Western plumbing and bathing practices, may homogenize microbial communities across individuals. Third, this rural community lacks access to Western hygiene products, including antibacterial soap. Hence, skin bacterial communities may be less disturbed on average, providing context for understanding how antibacterial soap alters microbial communities in novel ways. Finally, this population is increasingly exposed to aspects of the Western lifestyle through economic development. Moving forward, demand for Western products, including soap, is likely to increase, making it important to investigate how the use of antibacterial soaps affects skin microbial communities, and in turn, health.We investigated the hypothesis that antibacterial soap use impacts skin microbial communities in this rural population. By comparing an experimental group with access to antibacterial soap to a control group without access to antibacterial soap, we tested the following predictions:Individuals using antibacterial soap exhibit a greater change in taxonomic richness  after one week of soap use, as compared to a matched control group that was not given access to antibacterial soap.Change in alpha diversity of microbial taxa covaries with the amount of soap used, consistent with a dose-response relationship.Individuals using antibacterial soap exhibit a greater change in taxonomic richness after having stopped soap use for two weeks, compared to individuals that did not use antibacterial soap over the same time period.Individuals using antibacterial soap exhibit a greater change in the composition of skin microbial communities across sampling periods (beta diversity).Changes in beta diversity covary with amount of antibacterial soap used, consistent with a dose-response relationship.Because the composition of the microbial community may inhibit the invasion of new microorganisms, changes in community composition following antibacterial soap use would persist for at least two weeks after discontinuing soap use.The study was conducted in Mandena, Madagascar (approximately -18°42'00” S 47°50'00” E), located in the SAVA region . The village consists of approximately 3,000 people and is adjacent to Marojejy National Park. We first recruited individuals at a village-wide meeting, where the project was introduced and interested individuals were invited to participate in the study on future dates. Upon their return to our health clinic and after obtaining informed written consent through Malagasy translators, we obtained basic health measurements for each participant, including temperature, blood pressure, heart rate, height, and weight. Participants with clinically elevated health measurements, open wounds, infirmity, or illness were excluded from enrolling in the study. Of the eligible participants, we enrolled 20 adult males that ranged in age from 18 to 75 years. After obtaining a second informed written consent facilitated by Malagasy translators, ten individuals were randomly assigned to the experimental group to use antibacterial soap for one week, while the other ten individuals were assigned to the control group that would not be administered antibacterial soap. We selected individuals at random because most participants reported using only local soap, which we observed was used for bathing, washing clothes, and cleaning dishes. We administered Santex antibacterial soap   to the eWe sampled each individual at four skin sites  using sterile, dual-tipped rayon swabs . All skin swab samples were obtained by rubbing the swab across the site for 30 seconds, with all sampling conducted by authors MBM and JJY. We provided individuals in the experimental group with both written and verbal instructions to maintain their typical bathing schedule, but to use the soap on specific body parts at each bath time  and to refrain from sharing the soap with family members. We did not provide the control group with instructions regarding soap use because avoiding hygienic practices in this rural environment could put these individuals at risk of disease, and we wished to minimize disruptions to the typical behaviors of our participants. All subjects were swabbed at three different time points to investigate how antibacterial soap use impacts skin microbial composition. The first sampling event, defined as Time Period 1, took place before the introduction of soap ; the second, Time Period 2, occurred after one week (7–8 days) of antibacterial soap use (to investigate the immediate effects of soap use); and the third, Time Period 3, occurred two weeks (14–15 days) after discontinuation of antibacterial soap use  . We collThe swab samples were stored in a refrigerator powered by a generator when available, or inside a plastic cooler at room temperature with a daytime mean of 20°C . Because Mandena is not supported by electricity and a laboratory was inaccessible, we stored the samples in a uniformly cool environment in the field and during transit, rather than exposing samples to repeated freeze-thaw cycles. A previous study found that differences in short-term storage conditions did not significantly alter microbial communities, and that samples stored in non-freezing conditions were still useful for analyses . SamplesDNA extraction followed the MO BIO Powersoil DNA Isolation Kit  protocol with two modifications . The ext44,308,999 300 bp MiSeq read pairs were joined in QIIME , with join_paired_ends.py, using the fastq-join method with a maximum percentage difference (-p) of 25%. Assembled amplicons were further quality filtered with split_libraries_fastq.py , resulting in 20,583,303 sequences used for downstream analysis. 16S rRNA OTUs were picked using the closed reference method with SortMeRNA and greengenes database (version 13.8) at 97% similarity. A minimal sampling depth (-e) of 8,000, and otherwise default parameters, were used for subsequent diversity analyses with core_diversity_analyses.py of the QIIME package. All QIIME pipeline scripts were run on the Duke Compute Cluster. All sequence reads have been deposited at GenBank under the accession KBXC00000000.To ensure there were no biases between the experimental group and control group, we were first compared the two groups with two separate t-tests, one comparing Shannon diversity index between the two groups and the other comparing observed OTUs between the two groups. We found no significant differences between the two groups for either measure of alpha diversity. To investigate the impact of antibacterial soap on alpha diversity of skin microbial communities, we compared the change in bacterial taxa for all individuals between Time Period 1 and 2, grouping together individuals that used soap  and individuals that did not use soap (control group). We quantified alpha diversity as taxonomic richness (number of OTUs), and as combined taxonomic richness and abundance (Shannon diversity index). We used paired t-tests with an alpha of 0.05 for observed OTUs and Shannon diversity indices for both the experimental and control groups, comparing alpha diversity in Time Period 1 and 2 matched by subject (Prediction 1). We repeated this to compare between Time Period 2 and 3, following discontinuation of soap use. To investigate if change in alpha diversity covaries with amount of soap used (Prediction 2), we used linear regression models including both the control and experimental group to assess whether the amount of antibacterial soap used had an impact on the number of OTUs or the Shannon diversity index from Time Period 1 to 2. We repeated this process from Time Period 2 to 3 to investigate the relationship between discontinuing soap use and resulting alpha diversity (Prediction 3). We included amount of soap used and subject’s age as predictors of change in alpha diversity.We defined beta diversity as the UniFrac distance between two of the same samples  from different time periods . A greatRamlibacter and Variovorax for Time Period 1 (before soap use) and Acidovorax for Time Period 2 (after one week of soap use) (The amount of soap used significantly predicted beta diversity from Time Period 1 to Time Period 2 . The coeoap use) .We found no significant differences in alpha diversity (number of OTUs or Shannon diversity index) between samples collected at Time Periods 1 and 2 . The conWe found that soap use impacts the community composition of microbes on human skin, but may not affect the taxonomic richness of these communities. Changes in community composition persisted for at least two weeks in the experimental group, which suggests that continued use of antibacterial soap may have long-term effects on skin microbial communities. Similar effects have been documented for the use of other hygienic products, such as antiperspirant and deodorant .Although we expected that use of antibacterial soap would change the taxonomic richness of skin microbes (Prediction 1), we failed to find any evidence of that relationship, as measured by the number of OTUs and Shannon diversity index across the four body sites . MoreoveDespite the lack of change in alpha diversity, we found support for predictions related to changes in beta diversity. There was greater dissimilarity in microbial communities across time in samples from the experimental group than in samples from the control group , indicatRamlibacter, Variovorax, and Acidovorax, all of which are part of the family Comamonadaceae, which consists of environmental bacteria from water and soil, as well as certain pathogens [Our LefSe analysis  indicateathogens . It may athogens , 19. Whiathogens . This coImportantly, time period was not a significant predictor of beta diversity based on our general linear mixed model , indicatIn 2013, the FDA urged continued research on the possible effects of antibacterial soap use, especially soaps containing the active ingredients Triclosan and Triclocarban. Three years later, the FDA prohibited the sale of hygiene products containing these antibacterial agents . As the Other studies have identified a wide variety of associated environmental health concerns from antibacterial product use . These eThere is a growing interest in the microbiomes of non-Western populations –10, 47, In conclusion, we found that antibacterial soap impacts the structure of microbial communities, and that these changes persist for at least two weeks. These findings suggest that antibacterial products have a lasting impact on skin microbes, and are especially important in the context of changing lifestyles throughout the developing world, with increases in wealth likely to be associated with increased demand for hygienic products such as antibacterial soaps. While the efficacy of antibacterial soap and bactericidal products has been under scrutiny, the health-related effects of these products on skin microbial communities has yet to be examined thoroughly. Future research would benefit from considering additional environmental and behavioral factors that have the potential to impact the skin microbial communities in rural settings, such as interactions with non-human animals, occupation, and use of shoes.S1 Text(DOCX)Click here for additional data file.S2 Text(DOCX)Click here for additional data file.S1 Data(CSV)Click here for additional data file.S2 Data(CSV)Click here for additional data file.S3 Data(CSV)Click here for additional data file.S4 Data(CSV)Click here for additional data file.