Missense mutations in the lysosomal hydrolase β‐galactocerebrosidase (GALC) account for at least 40% of known cases of Krabbe disease (KD). Most of these missense mutations are predicted to disrupt the fold of the enzyme, preventing GALC in sufficient amounts from reaching its site of action in the lysosome. The predominant central nervous system (CNS) pathology and the absence of accumulated primary substrate within the lysosome mean that strategies used to treat other lysosomal storage disorders (LSDs) are insufficient in KD, highlighting the still unmet clinical requirement for successful KD therapeutics. Pharmacological chaperone therapy (PCT) is one strategy being explored to overcome defects in GALC caused by missense mutations. In recent studies, several small‐molecule inhibitors have been identified as promising chaperone candidates for GALC. This Review discusses new insights gained from these studies and highlights the importance of characterizing both the chaperone interaction and the underlying mutation to define properly a responsive population and to improve the translation of existing lead molecules into successful KD therapeutics. We also highlight the importance of using multiple complementary methods to monitor PCT effectiveness. Finally, we explore the exciting potential of using combination therapy to ameliorate disease through the use of PCT with existing therapies or with more generalized therapeutics, such as proteasomal inhibition, that have been shown to have synergistic effects in other LSDs. This, alongside advances in CNS delivery of recombinant enzyme and targeted rational drug design, provides a promising outlook for the development of KD therapeutics. © 2016 The Authors. Journal of Neuroscience Research Published by Wiley Periodicals, Inc. GALC is required for the hydrolysis of galactosphingolipids, including the major lipid component of myelin β‐galactocerebroside  required for lipid turnover and maintenance of the myelin sheath that surrounds and protects neurons. Although KD is classified as a lysosomal storage disorder (LSD), unlike other LSDs, there is little to no storage of primary substrate  and enters the Golgi apparatus, where it is posttranslationally modified by N‐linked glycosylation at four sites  is an approach commonly used to treat LSDs and is the current standard of care for Gaucher's, Fabry's, and Pompe's diseases as well as for mucopolysaccharidoses (MPS) I, II, and VI Sands, . HoweverSubstrate reduction therapy (SRT) is another approach that has shown promise in treating LSDs such as in type I Gaucher's disease  is typically monitored by an increase in specific hydrolase activity. Partial restoration of enzyme activity to only 10% of wild‐type (WT) levels has been reported to provide sufficient enzyme activity to prevent disease in LSDs  are structural analogs of the sugar moieties on sphingolipid substrates; as such, they make up a family of potent glycosidase inhibitors and have been widely explored as candidate chaperones for LSDs  was shown to be an essential constituent for both α‐gal A and β‐gal inhibition, supporting previous observations  was identified as a GALC inhibitor and conferred the greatest inhibition on the β‐gals, its inhibitory effect on β‐gal identifies a lack of specificity that may compromise its potential as a KD therapeutic. However, Biela‐Banas et al propose that modifications of IGF may result in similar inhibition with increased GALC specificity. Although these findings highlight the subtleties in the azasugar ring architecture that are required for specificity and inhibition, an understanding of each compound's stabilizing effect on GALC at both lysosomal and ER pH  would be beneficial, given that it is widely known that binding can vary greatly between these two cellular environments  possessing two nitrogen atoms in the sugar ring equivalent to those in both IGF and DGJ; a five‐membered pyrrolidine derivative, dideoxy‐imino‐lyxitol  and conferred the highest degree of global stabilization, providing the basis for using these as optimal scaffolds. GALC stabilization was measured by thermal denaturation at two different pH values representing the cellular environments of the lysosome (pH 4.6) and ER (pH 7.2). All but one test compound (DGN) increased the stability of WT enzyme under both conditions in a dose‐dependent manner. However, stabilization of GALC was greater at low pH than at neutral pH, a suboptimal feature for a PC. To confirm specificity, glucose‐configured 1‐deoxynojirimycin (DNJ) was tested and did not stabilize or change the melting temperature of GALC. Crystallographic studies showed that each of the candidate compounds directly bound in the active site of GALC. Analysis of the binding interactions revealed the critical role of the interaction between the ring nitrogen and the side chain of the active site nucleophile (E258) as well as the importance of the ring pucker. This study reinforces the greater potency of IGF over other azasugar derivatives and highlights the specific characteristics of azasugars required for optimal binding specificity and affinity. However, this study also reveals that, because of the electrostatic nature of the interaction, azasugar molecules bind more tightly at lysosomal pH than they do at neutral pH. Thus, PCT molecules that contain nitrogen in the ring have the advantage of high‐affinity binding but have to overcome the tighter binding at low pH to function as efficient chaperones. In these cases, rather than exploiting the acidic pH of the lysosome, the success of these molecules may depend on the ability of excess substrate to compete for binding to the active site.By targeting the active site of the enzyme, chaperones can be rationally designed to enhance specificity. Hill et al.  used ins1‐gangliosidosis , improved lysosomal localization, or reduced accumulation of psychosine  of the identified KD missense mutations are buried deep within the structure and are predicted to cause GALC misfolding  provide an affordable alternative platform in which to evaluate therapeutic approaches effectively  is NB‐DNJ, an alkylated glucose analog of DGJ, and is used for the treatment of adult patients with mild to moderate type I (nonneuropathic) Gaucher's disease. Miglustat is thought to function primarily as an SRT by inhibiting the glucosylceramide synthase (Cox et al., In the case of PCT molecules that mimic the substrate, such as azasugar derivatives, it is conceivable that their effects are bifunctional. The similarity of these PCT molecules to the glycan on the substrate means that, in addition to their ability to bind their respective hydrolases, they also have the potential to bind the glycan‐synthesizing enzyme. For example, miglustat (also known as The combination of PCs with other molecules that promote general protein folding has shown synergistic effects in Gaucher's‐ and Tay Sachs‐derived patient fibroblasts (Mu et al., Despite the recent progress in identifying and characterizing small molecules that elicit chaperoning effects on GALC in vitro, significant challenges still exist that must be overcome to translate PCs into viable treatment options for those who suffer from KD. The significant CNS involvement poses one of the greatest challenges in the development of KD therapeutics. PCT is one strategy that has the potential to overcome this; however, the limitations in our current understanding of both the mechanisms of chaperone–enzyme interaction and the underlying causative mechanisms associated with specific mutations in KD sufferers highlight the requirement for additional investigation. Specific concerns relating to those chaperones investigated to date for KD are listed below.Although specificity for beta‐configured galactosidases has been shown for active‐site‐directed chaperones, obtaining specificity for GALC vs. β‐gal has proved to be extremely difficult. Evaluating this specificity relies on the use of both appropriate assays and substrates when measuring the chaperone response. Also, because of the structural similarity of the active sites of these two enzymes, to confer this specificity it may be essential to focus on modifications of the aglycone region of PC candidates rather than the azasugar portion.To overcome problems experienced with inhibition of the target enzyme, nonactive site inhibitors are used; however, these have the potential to elicit significant off‐target effects. Because these are directed away from the active site, off‐target effects are difficult to predict, monitor, and overcome. During chaperone development, the dosing strategy may become particularly important when using this class of chaperone.It is likely that only a subset of missense mutations will respond to specific chaperones. There are two major implications of this. First, candidate chaperones may be missed if the number of mutations being screened is not sufficiently large or if the selection of potentially responsive mutations is not appropriately made. Second, development and administration of multiple therapeutic interventions may be required for successful treatment. As an alternative strategy, proteostasis regulators, used carefully, may represent a broad‐spectrum therapy that could overcome mutation‐dependent effects.Currently, increased activity is the main readout of chaperone efficacy, which may be misleading. A clear readout of improved lysosomal delivery of functional material is required to assess comprehensively the effectiveness of PCT. Measuring the effectiveness of any of the chaperones discussed here requires the development of alternative assays that are more amenable to high‐throughput approaches than are currently available.The past decade has seen PCT emerge as a viable and promising therapeutic option for LSDs. The dominating presence of CNS pathology in KD has undoubtedly hindered the acceleration of research and clinical translation in this area with respect to other non‐CNS LSDs. Despite this, the use of PCT for KD does show promise, and several GALC candidate chaperones have been identified. Because of the difficulty of establishing a clear genotype–phenotype correlation, it is clear that a detailed mechanistic understanding of both the candidate chaperone and the underlying target mutation will allow enhanced translation of successful therapy. However, it is evident that the measurement of chaperone efficacy should be based on several complementary biological assays that monitor the ability of a chaperone not only to increase enzyme activity but also to promote correct trafficking to the lysosomal compartments. Therefore, although increased GALC activity is an important readout, we believe it should not be the sole determinant of a successful chaperone. The parallel development of multiple therapeutic approaches is likely to be the most successful way to target the complex phenotypes observed in KD, and the identification of new PCT candidates is a critical contribution to future therapeutic interventions for this debilitating disorder.The authors declare that they have no conflicts of interest associated with the content of this Review.Both authors had full access to the data presented in this Review and take responsibility for the integrity of the data and the accuracy of the data interpretation. Both authors contributed to the study concept and design and to the drafting and critical review of the Review.


Gammaproteobacteria (likely sulfur oxidizers) that appear to thrive in a variety of chemically distinct fluids. Only the warmest, most hydrothermally-influenced flows were dominated by active populations of canonically vent-endemic Epsilonproteobacteria. These data suggest that the Gammaproteobacteria collected during this study may be generalists, capable of thriving over a broader range of geochemical conditions than the Epsilonproteobacteria. Notably, the apparent metabolic activity of the Gammaproteobacteria—particularly carbon fixation—in the seawater found between discrete fluid flows (the intra-field water) suggests that this area within the Axial caldera is a highly productive, and previously overlooked, habitat. By extension, our findings suggest that analogous, diffuse flow fields may be similarly productive and thus constitute a very important and underappreciated aspect of deep-sea biogeochemical cycling that is occurring at the global scale.Despite years of research into microbial activity at diffuse flow hydrothermal vents, the extent of microbial niche diversity in these settings is not known. To better understand the relationship between microbial activity and the associated physical and geochemical conditions, we obtained co-registered metatranscriptomic and geochemical data from a variety of different fluid regimes within the ASHES vent field on the Juan de Fuca Ridge. Microbial activity in the majority of the cool and warm fluids sampled was dominated by a population of  Free-living microorganisms at hydrothermal vents play a fundamental role in supporting highly productive vent ecosystems and influence local, if not global, geochemistry  where there was visible shimmering and microbial mats , intra-field fluids found among the diffuse vents (~2.5°C), but without any visual biotic or abiotic indications of fluid flow , and background seawater collected hundreds of kilometers outside of Axial Caldera  using a Niskin water sampler. In all cases, we made co-registered geochemical measurements via in situ mass spectrometry or shipboard geochemical assays to provide environmental context.This work attempts to address two questions: First, to what extent are hydrothermally-derived compounds found in the waters surrounding diffuse flows? And second, what is the activity and distribution of vent-endemic and non-endemic microbes throughout these waters, as well as within the diffuse flow fluids? To this end, we collected samples for geochemical analyses and metatranscriptomic library construction from an extensive survey of fluids within and around diffuse hydrothermal flows at the ASHES vent field . However, our findings also demonstrate that low temperature diffuse flow vents in a single vent field can have dramatically different profiles of microbial activity dominated by vent-associated Epsilonproteobacteria. Importantly, these data reveal that the intra-field habitat represents a zone of microbial activity—and likely significant source of primary productivity—that is geochemically distinct from that of either seawater or hydrothermal flows. This intra-field habitat is analogous to plumes from high temperature chimneys, in which chemoautotrophic Gammaproteobacteria are abundant, active, and potentially contribute substantially to net hydrothermal vent productivity.With these samples, we considered the resulting metatranscriptomic and geochemical data from three perspectives: (1) a dataset-wide investigation of the most abundant transcripts that provide a broad view of the active community and their gene expression profiles in different habitat types; (2) a targeted analysis of gene expression relevant to chemosynthesis and elemental cycling; and (3) differential expression analysis of samples collected using the D-ESP to identify the most differentially-expressed genes between our most hydrothermally-influenced diffuse flow site and the intra-field fluids. The data presented here confirm previous findings  vent field . Samples were collected using the Deep-sea Environmental Sample Processor (D-ESP), as well as with ROV-mounted Niskin bottles , intra-field waters (Site 1), or a location where both environments were sampled (Site 2). Sites 1 and 4 were located near the base of Mushroom chimney  or stacked 5 μm (lg) and 0.2 μm (sm) filters (D-ESP). Between one and four samples were collected at each site. Individual metatranscriptomes described herein come from a single filter and therefore represent a single >0.2 μm Niskin sample or either a 0.2 or 5 μm size fraction D-ESP sample. Eleven Niskin and nine D-ESP filters were analyzed for this study. Because the D-ESP samples were passed through two stacked filters, six of the D-ESP filters were paired 0.2 and 5 μm filters (representing three discrete collections), while the additional three D-ESP filters were 0.2–5 μm size fractions (the corresponding 5 μm filter fraction was not sequenced). The Niskin filters, in contrast, represent an entire sample filtered (>0.2 μm). See Table 2S analyses in 1 M zinc acetate (Zn(C2H3O2)2) stored at 4°C, and quantified via a colorimetric assay on a Spectramax Plus 384 absorbance microplate reader  sensor and an Aanderaa oxygen optode (model 3830) placed in line with the D-ESP measured oxygen, salinity, and temperature throughout the entire deployment. Methane concentrations were determined by collecting 250 mL subsamples from Niskin bottles in gastight serum vials and analyzing these shipboard with a gas chromatograph , and the cells were lysed by incubating filters with Trizol™  for 20 min at 25°C then 10 min at 85°C. Nucleic acids were separated using 24:1 chloroform:isoamyl alcohol. RNA was precipitated in 100% isopropanol with 100 mg of Glycoblue™  to improve recovery. RNA was resuspended in RNA Storage buffer . Residual DNA was removed with TURBO DNase™  per the manufacturer's instructions. RNA was quantified with a Qubit 2.0 fluorometer  using the RNA high specificity assay .RNA sample quality control, Illumina TruSeq Stranded mRNA sample preparation , and Illumina HiSeq2000 101 cycle paired-end sequencing were carried out by the University of Utah Microarray Core Facility  using default quality control parameters at submission, with the exception of running Duplicate Read Inferred Sequencing Error Estimation (DRISEE), which is designed to remove artificial duplicate read sequences, but can inadvertently eliminate biologically-relevant sequences from Illumina data  database was used for taxonomic classifications using Best Hit classification , and SEED and Subsystem classification were used for functional gene identification. Read abundances for genes of interest were manually curated by searching for variations of gene names and then compiled. Hit numbers were normalized to total functional or domain-level taxonomic hits. Metatranscriptomes ranged from 7.1 to 22.3 million taxonomic hits, 1–2.5 million SEED hits, and 44–645 thousand L1 Subsystem hits .Sequence data from all 20 metatranscriptomic libraries were analyzed in MG-RAST  and to target the ends of the geochemical spectrum of sites sampled. In total, six metatranscriptomic libraries representing three fluid samples, one from Site 1 (intra-field) and two from Site 4 (diffuse flow) were used in this analysis.A subset of six filters was selected for targeted differential expression analyses between one diffuse and one intra-field site. The selected filters consisted of all D-ESP samples where both the 0.2 and 5 μm filters were extracted and sequenced (one intra-field Site 1 sample and two diffuse Site 4 samples). These samples were selected to minimize variables introduced by different sampling methods  or 200 or more reads from the two Site 4 samples (from four paired filters). The remaining differentially-expressed contigs were identified using blastx . Log odds ratios were calculated to compare relative over- and under-expression of this curated transcript list between the two sites.p = 0.05 for a significance cut-off after correcting for multiple comparisons. Gene categories that were significant after correction with the conservative Bonferroni correction were considered highly significant, while those significant after the less conservative Benjamini-Hochberg test were considered significant. Site 0 (seawater) was not used in statistical analyses because it was represented by a single sample  or they could be significant as a function of 1) site, (2) environment, 3) an outlier single site , (4) sampling method. To test which of these scenarios best explained the variation in our data, we grouped samples accordingly  and performed statistical tests in R . We used  site,  was carried out in R on taxomonic IDs with singleton and doubletons removed. The vegdist function in the vegan package was used to calculate Bray-Curtis dissimilarity matrix, and clustering was carried out using the hclust function and the complete linkage method.Cluster analysis  run concurrently with the D-ESP sampling, oxygen was found to be substantially lower in the Site 4 diffuse flow (0.03 ml L−1) than in the intra-field water at Site 1 (0.96 ml L−1). The temperature was substantially higher at the Site 4 diffuse flow (34.2°C) than the intra-field water at Site 1  revealed an active, autotrophic community dominated by vent-associated s Figure . The moss Figure . These ts Figure .Hierarchical SEED Subsystem functional gene classification showed that all samples had a fairly similar, broadly categorized  profile of transcriptional activity. Most highly expressed categories are shown in Figure p-values).We were interested in examining the expression patterns of key genes related to chemosynthetic metabolisms and related to elemental cycling of carbon, nitrogen, sulfur, and iron, and hydrogen Figure . The relWith respect to nitrogen metabolism, gene expression involved in denitrification was highest in the Site 4 samples Figure . NitrateSulfur metabolism genes were expressed in all samples, but were least expressed in the seawater sample and Site 4 D-ESP samples Figures . The sulFerric iron transporters showed significantly more expression in Sites 1–3 than Site 4 Figure . Genes rin situ by the D-ESP at Site 1 (intra-field) and Site 4 . Figure 10 shows the most abundant differentially-expressed transcripts, plotting relative abundance and log odds ratio of those samples where average Site 1 and Site 4 relative abundance sum to >0.5%. Adenylylsulfate reductase , and particulate methane monooxygenase (pMMO) are among those transcripts most over-expressed in our Site 1 D-ESP samples relative to those from Site 4. In contrast, thiosulfate reductase, flagellin and flagellar proteins, nitrate and nitrite reductases, and hydrolase are among the most over-expressed in D-ESP Site 4 samples relative to those from Site 1.Differential expression analysis on selected samples  highlighted transcripts that were differentially expressed between samples collected The overarching goal of this effort was to determine the phylogenetic and functional changes of active microbial communities associated with diffuse-flow vent fluids as they disperse into the environment. Our hypothesis was that microbial activity (for which we used mRNA expression as a proxy) would track fluid geochemistry in a predictable manner. The results of this study, however, do not support the idea that microbial activity can be inferred from the fluid geochemistry. Instead, this work highlights the substantial heterogeneity of these systems and suggests that intra-field areas are a previously overlooked, high-productivity habitat.Epsilonproteobacteria in , which may explain some of the differences observed in gene expression. Surprisingly though, methane concentration and pH from Site 2 and 3 diffuse samples were more similar to intra-field samples than they were to Site 4 diffuse flows. Gene transcription did not appear to be predicted by geochemistry. The active communities from diffuse flow Site 4 were distinct from those of the diffuse flows at Sites 1–3 Figures , 5 despiIt is often assumed that microbial activity is largely controlled by local geochemistry, and well-documented correlations between chemistry and microbiology support that generalization . This is similar to the microbial community identified in a post-eruption “Snowblower” vent sampled shortly after our samples were collected  had a Gammaproteobacteria-dominated activity profile with pH 4.8 and average temperature of 49.7°C . It has previously been demonstrated that vents in close proximity can host significantly different communities despite geochemical similarities  within Axial caldera, and interestingly showed a strikingly different active community far less dominated by either Gamma- or Epsilonproteobacteria than our diffuse flow metatranscriptomes. While oxygen was not quantified in their work, oxygen concentration, which is higher in cold background seawater and lower to absent in hydrothermal fluids, could explain this difference if we assume temperature is a proxy for oxygen concentration. Our Site 4 was 34.2°C, whereas the Marker 113 metatranscriptome was 24.1°C. However, it is impossible to rule out the effect of temperature itself on some other aspect of geochemistry . Additionally, non-geochemical factors such as spatial variation between vent fields . In contrast, these genes were not detected in seawater and showed much lower expression in the Site 4 diffuse flow samples. This implies the community at Sites 1–3 is actively oxidizing methane for energy generation.Thiobacillus denitrificans, and Thioalkalivibrio sp., most of which are known to perform sulfur oxidation. Because sulfide was not detected at Sites 1 and 2 and was relatively low at Site 3 (detection limit: ~1 uM), we suggest this habitat may support oxidation of partially reduced sulfur species such as thiosulfate, or elemental sulfur. Alternatively, this absence of sulfide may be due to active sulfide oxidation keeping concentrations below detection levels.Sulfite reductase alpha subunit  and adenylyl-sulfate reductase  were also highly expressed in Sites 1–3 , not expressed in the seawater sample, and had a much lower expression in Site 4 . Both of these genes catalyze reversible reactions that can be part of either sulfate reduction or sulfide oxidation. Sulfite reductase works between sulfate to sulfide, and APS reductase between sulfite and adenylyl sulfate. From its expression alone, one cannot definitively determine whether it is playing a role in sulfate reduction or sulfide oxidation. However, given that Sites 1–3 are oxic and therefore not conducive to sulfate reduction, it seems likely that expression of these genes is indicative of sulfur oxidation. Additionally, the vast majority of sequences identified as APS reductase and sulfite reductase were most similar to sequences from Gammaproteobacterial symbionts, dsr) genes are used both by anaerobic sulfate reducers and microaerophilic or aerobic sulfur oxidizers for sulfate reduction and sulfur oxidation, respectively. dsr genes were more highly expressed in the more oxic sites (1–3), and we infer that they reflect the prevalence of reduced sulfur compound oxidation. Other sulfur oxidation genes (sox and apr) showed similar expression patterns, further supporting the supposition that this process may be more pronounced in Sites 1–3  was more highly expressed , and dsr genes were less highly expressed. This is consistent with sulfide oxidation to elemental sulfur, and similar to patterns of gene expression found in other low temperature vent environments where transcription from Gammaproteobacterial and Epsilonproteobacterial symbionts were shown to express different profiles of genes for sulfur metabolism  dominated the overall active community at Site 4 and are known to be engaged in sulfide oxidation, hydrogen oxidation, and denitrification  are more similar to the patterns observed in the other intra-field samples , including Gene expression patterns in the D-ESP wand samples (Diffuse Site 4) are markedly different and seem to reflect a higher degree of heterogeneity in sulfur-based energy metabolism. Increased thiosulfate reductase expression potentially indicates thiosulfate oxidation, possibly coupled to nitrate reduction. Increased expression of nitrate and nitrite reductases Figure  is also At the outset of this work, we asked whether microbial activity varies systematically among geochemically distinct environments. Based on the data presented herein, we posit that intra-field water (the fluids within the vent field without any visible signs of vent influence) is not comparable to background seawater, but rather appears to be a diffuse flow-derived non-buoyant plume, the physical extent of which remains unknown. Geochemical and gene expression data presented here are consistent with what has been observed in studies of buoyant plumes emitted from high temperature hydrothermal flows (Dick and Tebo, Deep sea hydrothermal vents and their plumes are known to have a substantial impact on global ocean chemistry (e.g., Resing et al., HO analyzed the data and wrote the manuscript with input from co-authors. PG and HO planned the work and collected and processed the Niskin samples. DR carried out the RNA extractions. CP designed the molecular assays for the D-ESP. WU carried out methane measurements. CS, JB, CP, WU, DP, SJ, and BR built, maintained and operated the D-ESP. MH carried out RuBisCO bioinformatic analyses.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Mineral dust is the most important natural source of atmospheric ice nuclei (IN) which may significantly mediate the properties of ice cloud through heterogeneous nucleation and lead to crucial impacts on hydrological and energy cycle. The potential dust IN effect on cloud top temperature (CTT) in a well-developed mesoscale convective system (MCS) was studied using both satellite observations and cloud resolving model (CRM) simulations. We combined satellite observations from passive spectrometer, active cloud radar, lidar, and wind field simulations from CRM to identify the place where ice cloud mixed with dust particles. For given ice water path, the CTT of dust-mixed cloud is warmer than that in relatively pristine cloud. The probability distribution function (PDF) of CTT for dust-mixed clouds shifted to the warmer end and showed two peaks at about −45 °C and −25 °C. The PDF for relatively pristine cloud only show one peak at −55 °C. Cloud simulations with different microphysical schemes agreed well with each other and showed better agreement with satellite observations in pristine clouds, but they showed large discrepancies in dust-mixed clouds. Some microphysical schemes failed to predict the warm peak of CTT related to heterogeneous ice formation. The insoluble surface of mineral dust particle provides the solid-liquid interface needed for stabilizing newly formed ice embryos and thus enhances the heterogeneous freezing process in super-cooled water. In real atmosphere, at temperatures significantly warmer than ~−37 °C the threshold of homogeneous freezing, multiple types of ice phase clouds  were observed with co-existence of mineral dust particles8. Such ice nuclei (IN) effect in deep convective clouds may further lead to significant enhancement of latent heat releasing, which in turn causes atmospheric dynamic response to it. The enhanced deep convective clouds by dust may have higher rain top height and cloud top height11. However, such microphysical-thermodynamic-combined effects highly depend on cloud evolution stages12 and cloud types15. For clouds with relatively weaker dynamics (e.g. those formed in the outflow of convection core) and small to medium cloud water path (less than 300 g/m2), the cloud droplets without enough uplifting force to reach homogeneous freezing temperatures may glaciate under dust-laden condition rather than in liquid phase at the same height in pristine condition. In addition, the depletion of water vapor and super cool water at warmer temperatures reduces the occurrence of homogeneous nucleation16 at colder temperatures. The net effects of the above processes were a warming of cloud top temperature (CTT) of such clouds, and the associated positive longwave radiation forcing can be as high as 16 w/m2 15.Since 1940s17, the vertical transportation of dust and liquid cloud droplets from lower layer to upper layer is critical for the proposed IN effect. Thirdly, the CTT of ice clouds is controlled by both cloud dynamic effects and potential aerosol IN effects. It is almost impossible to untangle these two effects merely by using satellite observations19.However, large uncertainties existed in satellite observational studies of such IN effect. First, the contacting of aerosol and cloud is questionable if only passive satellite observations without vertical resolution were used . Second, even if clouds and the aerosols did contact with each other, without the information of vertical updraft and horizontal wind, it is unknown whether or not aerosol particles really mixed with the cloud particles and were vertically transported to layers cold enough for ice formation. Since liquid water was necessary for ice formation even with enough IN20, Cooper21, and Meyers22 relate IN number concentration to temperature and ice super-saturation, respectively. For any given temperature, those parameterized IN from different methods can encompass variations up to order of three in magnitude because of ignoring spatial and temporal variations in IN source particle type23. More observational evidences and model sensitivity studies are needed to quantify heterogeneous ice formation process with acceptable uncertainties.Current parameterizations of heterogeneous ice formation in cloud resolving model (CRM) such as Weather Research and Forecast (WRF) model are generally simple and do not link IN to aerosol properties. For example, the widely-used methods of FletcherIn this study, we developed a satellite-CRM combined methodology to study the mineral dust IN effects on ice cloud top temperature and to assess the model sensitivity to different parameterizations. For possible case of cloud-dust interaction, we combined observations from satellite passive  and active sensors  to obtain horizontal and vertical distribution of cloud and dust aerosol to ensure the contacting between them at first. Then we conducted a simulation of the case using cloud resolving model (WRF) and compared the simulations to satellite observations. After confirming the simulations can capture the main properties of the system, we utilized the simulated three-dimensional (3D) wind field combined with the satellite observed 3D distribution of cloud and aerosol to ensure the mixing of cloud and aerosol. Via doing this, we minimized the error in classifying cloud samples truly mixed with dust. In addition, we studied the sensitivity of WRF simulations of CTT to different microphysical parameterizations, initial conditions and cumulus parameterizations.26. High and thick deep convective clouds with widely spread ice clouds formed in this mid-latitude mesoscale convective system (MCS) with a clear comma structure indicating strong updraft and vorticity. The MODIS observed CTT  and horizontally conveyed to the cloud bottom layer (4.0–5.0 km) with wind speed around 7 m/s Fig. . Since t15. On the other hand, the water vapor at low and mid layers is also important to determine the cloud top height28. The invasion of dry air mass  in Sector 2 with AOD 0.34. This remarkable difference was mainly contributed by the ice clouds with CTT warmer than −38 °C in Sector 4. It indicated that heterogeneous ice nucleation process made strong contribution to ice formation in Sector 4 and caused warmer CTT for given CIWP.Although there were common negative correlations between CIWP and CTT based on satellite observations Fig. , the lind 4 Fig.  than thad 4 Fig. . In Sectlog10CIWP) was much smaller than that observed from the satellite (20.90 °C per unit log10 CIWP). This implies that WRF-MOR simulation can reasonably capture the CTT-CIWP relationship in light dust-laden conditions, but fail in heavy dust-laden conditions.In the WRF-MOR simulation Fig. , the sloFrom the point of view of CTT statistics in each sector, the satellite observed Probability Distribution Functions (PDF) of CTT in this case were divided into two groups. The PDFs in Sectors 2 and 3  of the same case shown in Fig. The largest discrepancies among multiple WRF simulations appeared in Sector 4. Compared to the satellite observations, at temperatures −20 to −40 °C, the WRF-MOR and WRF-Lin significantly underestimated the ice formation (very low PDF of CTT), while the WRF-WSM5, WRF-WSM6 and WRF-Goddard successfully simulated the peak of heterogeneous ice formation, but with some overestimations. This result implies that the ice formation parametrizations in WRF can lead to significant variations in the prediction of ice cloud properties at warm temperatures.This MCS also was observed by another MODIS sensor on the Terra satellite about two hours in advance to the overpass of Aqua. However, we do not have associated observations from CloudSat and CALIPSO to identify the vertical distribution of cloud and aerosol at the overpass time of Terra. Following the same definition of Sectors 1 to 4, results Fig.  from Ter2 slicing algorithm. At satellite pixel level, the cloud top height (CTH) derived form MODIS for cirrus clouds can be 1–2 km lower (i.e 6–12 °C of CTT) than that from CALIPSO/CALIOP. For thick water clouds, the retrieved CTH from MODIS is within 0.25 km (~1.5 °C of CTT) of the Cloudsat/CPR backscatter determinations29. For our case, the ice clouds were related to a deep convective system, and therefore the instantaneous error of CTT form MODIS retrieval should be less than that for cirrus cloud but larger than that for water clouds.Both the satellite observations as well as the model results used in this study were obtained with high uncertainty. The cloud top temperature from MODIS was generated using the COThe uncertainties in WRF modeling can come from different sources. The uncertainties related to microphysical parameterizations have been discussed already. To make assessment of WRF uncertainties from other sources, we further conducted 4 simulations under different initial conditions  and cumulus parameterizations . At the resolution of 2 km, the standard deviation (σ) of WRF modeled CTT due to different initial conditions (e.g. NCEP FNL against ERA) is overall 3.58–9.26 °C Fig. . The σ dIf look at the statistics of CTT at each sector, the sensitivity of modeled CTT to different initial conditions led to some small variations in the CTT PDFs Fig. . None oflog10CIWP was about 5 times steeper than the value in relatively pristine ice clouds. In another word, for given ice water path, the CTT is warmer in dust-mixed ice clouds. And there were two peaks in the PDF of CTT in dust-mixed ice cloud, one is at about −45 °C, and the other is at about −25 °C, which definitely was contributed by heterogeneous nucleation. For pristine ice cloud, only one peak at −55 °C was observed.The effects of dust aerosol acting as ice nuclei on cloud properties is still an open question. Although both ground-based and satellite-based observations showed some hints, large uncertainties existed in the data processing and methodology applied. In this study, we first designed a strict satellite data collocation methodology which combined active cloud radar, LIDAR and passive spectrometer observations to obtain the 3D distribution of cloud and dust. More important, we adopted 3D wind filed derived from cloud resolving modeling to clarify the transportation of aerosol, and to identify the place where the mixing between aerosol and cloud really occurred. We found in a mid-latitude mesoscale convective system, when ice cloud particles were mixed with dust, the linear regression slope of CTT against The mechanism of heterogeneous ice formation remains one of the biggest challenges in cloud modeling. Through this study, the WRF simulation of ice cloud CTT is most sensitive to microphysical parametrizations, followed by the initial conditions. And cumulus parameterizations applied in the outer domain makes no effects on the simulation.log10CIWP, and it completely missed the warm peak of CTT PDF at −25 °C. Meanwhile, the MSM5/6 and Goddard microphysical schemes successfully predicted this warm peak even with some overestimations. It demonstrated those microphysical parameterizations without taking into account the concentration of IN source aerosol (e.g. dust) may yield to large uncertainty in simulations and cannot explain the satellite observations.Overall, WRF simulations with different microphysical schemes agreed well with each other and showed better agreement with satellite observation in pristine ice clouds. However, for the ice cloud mixed with dust, the Morrison schemes failed to capture the slope of CTT-30 for aerosol optical depth and MYD/MOD0631 for cloud properties at resolution of 5 km were used in this study. The standard products 2B-CWC-RO of CPR cloud water content retrieval we used is based on Austin and Stephens32. The vertical mask features of dust and cloud retrieved from CALIOP/CALIPSO are based on the standard product of 33.MODIS standard products of MYD/MOD0434, the RRTM longwave radiation transfer scheme35, the Dudhia shortwave radiation transfer scheme36, and the YSU planetary boundary layer scheme37 were used in the simulation.In this study, the Advance Research WRF model Version 3.4 was used to conduct cloud simulations without considering any dust aerosol effect. There were 55 vertical levels  and three nested horizontal domains with horizontal resolutions of 18, 6, and 2 km respectively in each simulation. The model was forced by the NCEP FNL  Operational Global Analyses data with 1° by 1° spatial resolution and 6-hour temporal resolution. The outputs were saved at 30 minute intervals and the results from the first 24 hours were excluded to avoid spin-up errors. The Kain-Fritsch cumulus scheme38, WSM639, WSM540, and Goddard41 schemes to represent the modeling uncertainty with the standard deviation among them.To investigate the sensitivity of WRF simulations to different microphysical parameterizations, we first used Morrison (WRF-MOR) and then used LIN42 and MERRA43 reanalysis datasets. The associated simulations are marked as FNL-Morrison, ERA-Morrison and MERRA-Morrison, respectively.To investigate the sensitivity of WRF simulations to different initial conditions, we changed the data source of initial condition in the WRF-MOR simulation from NCEP FNL to ERA-Interim34 to Betts-Miller-Janjic scheme44 and Grell-Devenyi scheme45. The associated simulations are marked as Morrison-KF, Morrison-BMJ and Morrison-GD, respectively.To investigate the sensitivity of WRF simulations to different cumulus parameterizations, we changed the cumulus scheme in the WRF-MOR simulation from Kain-Fritsch schemeSupplementary Information


The paleogeographic reconstruction of the successive inland positions of a retreating ice sheet is generally constrained by mapping moraines. However, deltaic complexes constructed by sediment-charged meltwater can also provide a record of the retreating ice-margin positions. Here, we examine a serie of ice-contact, ice-distal glaciofluvial and paraglacial depositional systems that developed along the Québec North Shore (eastern Canada) in the context of falling relative sea level during the northward retreat of the Laurentide Ice Sheet (LIS). Ice-contact depositional systems formed when the LIS was stillstanding along the Québec North Shore. Subsequent inland retreat of the ice margin generated glacial meltwaters feeding sediment to glaciofluvial deltas, leading to their rapid progradation. The retreat of the ice margin from drainage basins was marked by the onset of paraglacial processes such as the shutdown of delta progradation, severe fluvial entrenchment, and deposition of shallow-marine strata. Four end-member scenarios describe the spatial and stratigraphic distribution of these three depositional systems . They reflect both the inherited drainage basin physiography and the retreat pattern of the ice margin. Applied to twenty deltaic complexes, these end-members allowed us to refine the model of LIS-margin retreat over southeastern Québec. Final deglaciation relates, however, to major continental-scale re-organization of ice flows and drainage basins as well as to global climate change. In eastern North America, fragmentation of the Late Wisconsinan Laurentide Ice Sheet (LIS) and drainage of Lake Agassiz are suspected to have triggered the 8.2 ka cooling event4. The subsequent disintegration of the residual Québec-Labrador ice sector probably had a major impact on climate and ocean circulation6. Defining the final LIS retreat pattern and chronology is thus a crucial issue when deciphering Early Holocene climate forcings.The pattern of final inland glacial retreat of ice masses is generally less constrained than those tied to earlier, marine-based deglacial stages that offer better constrained temporal framework9 but with poorly constrained chronologies awaiting systematic direct dating on the basis of cosmogenic exposure ages2 and luminescence10. In contrast, marine and coastal depositional systems tied to earlier deglaciation stages are characterized by detailed temporal framework due to the abundance of datable material13. In particular, delta morphostratigraphies that are in part controlled by change in meltwater and sediment supply, itself intimately tied to the history of ice-margin retreat throughout drainage basins17, constitute integrative systems which the unravelling delivers crucial information about their formative processes and penecontemporaneous deglacial evolution. Indeed, such sedimentary archives have a great potential to provide additional and independent age constraints for ice-sheet retreat history in delta river drainage basins.Morainic complexes, eskers and glacial lineations deposited during the retreat of the Québec-Labrador ice sector are well-expressed landforms2 . Interpreting deltaic complexes along ~1500 km of coastline allows us to map the positions of Late Pleistocene–Early Holocene inland ice margins of the retreating Québec-Labrador ice sector of the LIS during the progressive deglaciation of the adjacent drainage basins of these deltas, together representing an area of more than 250,000 kmca. 24 ka  ago, the LIS began to recede by collapse and iceberg calving13  20. Although the deltaic complexes of the QNS are characterized by a common deglacial framework and RSL fall history —based on radiocarbon dating—, they may vary in terms of depositional architectures. Figure 20. As related depositional systems have specific and unique morphological and surficial expressions, they can confidently be identified on satellite and aerial imagery and/or on digital elevation models.Deltaic complexes of the QNS typically show three superimposed or laterally juxtaposed depositional systems: 1) ice-contact deposits, formed by ice-contact subaqueous fan that sometimes evolved as ice-contact delta, 2) ice-distal glaciofluvial delta, and 3) paraglacial suites21. Note that backstepping grounding zone wedges may in some case have been emplaced at earlier —and deeper— stillstand positions22. Ice-contact subaqueous fan and ice-contact delta architecture includes boulder-bearing pebbly topsets20 deposited from either subaqueous or subaerial meltwater outflows23, and foreset beds alternating siltstones and thick graded sand beds deposited by low- and high-density turbidity currents, respectively24, and commonly supercritical25. The topmost surface of ice-contact deposits is flat to very gently-sloped (<1°) and stands at or slightly below the marine limit , glaciofluvial deltas rapidly expanded on the St. Lawrence coastal shelf owing to high rates of delta progradation20. Even though the ice margin might have been located tens of kilometers further inland, sediments were transferred to the coast throughout the structural valleys via the riverine network and permitted a perennial delta accretion. The stratigraphic architecture of glaciofluvial deltas typically shows flat- to gently-sloped topsets deposited by braided streams, upper foresets consisting of stacked graded sand beds deposited by high-density turbidity current, and gently-sloped lower foresets formed by low-density turbidity currents. In spite of high rates of RSL fall forced by the glacio-isostatic rebound28, no coeval fluvial entrenchment was identified at least in the early part of this stage. The absence of incision in pre-existing sediments is due to the large amount of glacially-derived sediment supply that maintained a fluvial equilibrium profile steeper than the shoreline trajectory31. The extensive  top surfaces of the glaciofluvial deltas, which locally preserve relict braided channels and isolated beach ridges, stand well below marine limit . Coastal deposits consist of stepped beach and shoreface wedges deposited along inherited foreset slopes of the ice-contact deposits and/or glaciofluvial delta20. Occasionally, coastal deposits forming thicker (>15 m) sand wedges correspond to coastal spit platforms characterized by composite offlapping geometries .In the ber Figs , 2 and 3Overfilled dammed valley scenario is initially similar to the Underfilled dammed valley scenario, namely the development of an initial ice-contact depositional systems at the valley outlet and then of a fjord lake. However, a significant and/or protracted glaciofluvial sediment supply relative to the lake volume subsequently results in the complete infill of the lake. Sediment supply allows the fluvial system to be re-established up to the open coast through the entire valley. The afterward retreat of the LIS margin from the drainage basin forces the fluvial entrenchment and the cannibalization of ice-contact and glaciofluvial sediments by coastal processes.The Overfilled dammed valley scenario, fjord-mouth deltaic complexes show an initial outwash fan where the lower reach of its foreset slope is buried beneath glaciofluvial deltaic sediments. The elevation difference between the top surfaces of the outwash fan and the glaciofluvial delta reflects the duration of the lake infill according to the local RSL curve. The archetypal delta of this scenario is the Sault-au-Cochon deltaic complex20  the modern shoreline.In this scenario, no ice stillstand occurs at the valley mouth (absence of sedimentary deposits and/or bedrock sill?). As a consequence, the ice margin retreats inland, turning the valley into a fjord during marine invasionUndammed fjord, early deglaciation scenario, as no ice-contact system has been deposited at the fjord mouth, the deltaic complex is twofold, only comprised of a glaciofluvial delta overlain and reshaped by long-lasting paraglacial processes that resulted in well-developed paraglacial suites that include severely entrenched meander belts and stepped marine terraces. The elevation difference between marine limit and the top surface of the glaciofluvial delta relates to the duration of the in-valley deltaic progradation before expanding onto the open coast domain. Such systems are typified by the Portneuf and St. Jean River deltas . We argue, however, that this error can be considered as negligible as the drainage divides lie in relatively high-relief areas.When present, ice-contact deposits lying on the QNS provide information on the time interval corresponding to LIS-margin stillstand at the valley outlet, either from altitude of the top surface (date inferred from RSL curve) or radiocarbon dates  between 9.7 and 8.2 ka likely reflects the transition from frozen to warm basal thermal conditions of the Québec-Labrador ice sector which could be associated with a phase of development of ice streams1. This cold to warm based ice transition occurring slightly after 10 ka according to these authors. This hypothetical cold- to warm-based transition is in agreement with Tarasov and Peltier46 who suggest an abrupt increase of the proportion of warm-based ice at 8 ka. Between 9.7 and 8.2 ka, a rapid (130 m.yr−1) retreat rate characterizes the Ste. Marguerite inter-lobe area that subsequently decreased down to 80 m.yr−1 between 8.2 and 7.0 ka. Comparatively, retreat rates of 80 m.yr−1 and 160 m.yr−1 took place throughout this entire period for the eastern and western lobes, respectively. Compared to the post-8 ka enhanced retreat (900 m.yr−1) corresponding to periods of enhanced melting19, our reconstruction shows more steady overall rates of retreat during the LIS deglaciation.Our deglaciation model indicates low retreat rates of the older, linear ice margin from 12.5 to 9.7 ka. The older dates correspond to the Younger Dryas (YD) Cold Event dated from 12.8 to 11.7 ka, and bracketed further south by the Mars-Batiscan (12.2 to 11.5 ka) and St. Narcisse (12.8 to 11.5 ka) moraines, the latter being likely equivalent to the Québec North Shore moraine37). This model can also be further expanded to deltaic complexes lying in the eustatically-dominated zone  for which the morphostratigraphic record of ‘lowstand deltas’ is preserved at depth owing to the postglacial eustatic transgression16. Our approach of reconstructing past ice-margin configurations from the delta morphostratigraphic record can provide valuable inputs to data-calibrated models to infer the general retreat pattern of the LIS43.The correspondence between our morphostratigraphy-based reconstruction and other published reconstructions is a validation of the here-proposed method, which thus appear robust as far as a great number of deltaic complexes have been integrated into our model. Our reconstruction complements and strengthens previous ice-margin retreat models based on glacial landforms and surface exposure ages. However, our aim here is to provide a method for constraining retreat patterns solely based on delta morphostratigraphies which, beyond the particular case of the LIS retreat in south-eastern Québec, should be applicable to other studies of formerly glaciated regions. Particularly, the deltaic systems of eastern Hudson Bay were supplied in sediments by an extensive drainage basin formerly occupied by the northern Québec-Labrador residual ice sector almost up to its final disintegration (coastal deglaciation around 8 ka, glacial retreat after 7 ka


The foremost concern of a surgeon during pedicle screw fixation is safety. Assistive modalities, especially intraoperative electromyographic monitoring (EMG) can function as an essential tool to recognize screw malposition that compromise neural integrity, so that the screws can be repositioned immediately rather than later. We intend to study the efficacy of intraoperative EMG monitoring to detect potential pedicle breach and evaluate whether reoperation rates were significantly reduced.Retrospectively, patients who underwent posterior stabilization with pedicle screws for various pathologies were analysed and those with screws among L1-S1 levels were shortlisted. They were divided into two groups. Group 1 included patients in whom trigger EMG (t-EMG) was used to confirm appropriate screw placement and Group 2 included those in whom it was not used. Responses to t-EMG and corresponding stimulation thresholds were recorded for Group 1 patients. The sensitivity and specificity of the test was calculated. Reoperation rates due to postoperative neurologic compromise caused by malpositioned screws were compared between both the groups.n = 296; Screws = 1856], 145 screws (7.8%) showed a positive response for t-EMG at stimulation thresholds ranging between 2.6 to 19.8 mA. The sensitivity and specificity of t-EMG to diagnose potential pedicle breach was found to be 93.33% and 92.88% respectively. Only one patient among Group 1 required reoperation. However, among Group 2 , six patients required reoperation. This indicated a significant decrease in the number of malpositioned screws that caused neurological compromise [p = 0.02], leading to subsequent decrease in reoperation rates [p = 0.04] among Group 1 patients.A total of 518 patients had 3112 pedicle screws between L1-S1 levels. Among Group 1 [Trigger EMG is well efficient in detecting potential pedicle screw breaches that might endanger neural integrity. In combination with palpatory and radiographic assessment, it will certainly aid safe and secure pedicle screw placement. It can also efficiently reduce reoperation rates due to neurologic compromise provoked by a malpositioned screw. Utmost care needs to be taken during pedicle screw placement to prevent potential damage to neural structures that closely approximate the bony pedicle. Pedicle screw technique has evolved over time and experience; however, “free-hand” technique is most widely practiced –5. This Even though, intraoperative confirmation of secure screw placement can be obtained using fluoroscopic images, neural integrity or compromise needs to be checked. Moreover, completely depending on fluoroscopic images may not be appropriate, especially when proper anatomy cannot be visualized. Therefore, we believe that intraoperative electromyographic monitoring (EMG) can be an essential tool to recognize screw malposition that compromise neural integrity , 9. SuchDegenerative spondylosis,Spondylolisthesis (≥ grade 1),Spondylodiscitis andTumours.We retrospectively reviewed data of all patients who underwent posterior instrumentation with pedicle screw constructs for various pathologies at our institution over a period of time. Their preoperative x-rays and MRI were evaluated. The indications of surgery included:Only those patients with pedicle screws in L1-S1 levels were shortlisted and a sample containing 518 patients was obtained. They were divided into two groups based on intraoperative EMG monitoring. EMG monitoring was utilized based on the availability of neuromonitoring system and the operating personnel. Those patients in whom intraoperative EMG was used were Group 1  and the remaining patients were Group 2 (Control group).Those patients with dysmorphic pedicle features, such as in congenital scoliosis,Those in whom minimally invasive percutaneous screw insertion was performed andThose with pedicle screws in vertebrae other than L1-S1.We excluded:Surgeries were performed by five experienced orthopaedic surgeons and their procedure to apply pedicle screw was unanimous. Although, we do both open surgery with free hand technique and minimally invasive surgery (MIS) with percutaneous screw insertion under fluoroscopic guidance, we only included patients in whom open surgery with free hand technique was used to place the screws. All surgeons identified bony landmarks including pars, transverse process and facet joints before making the entry point. Partial infero-lateral corticotomy of the superior facet was performed in line with the mid-point of transverse process to identify the appropriate entry point. Following entry, the pedicle was probed and walls were felt using a pedicle sound to assess structural integrity. If satisfactory tactile perception was obtained, pedicle screws were placed. This technique of pedicle screw placement was customary during the study period.Group 1 patients had recording electrodes placed in muscles of interest; hence, handling of nerve roots may produce spontaneous response in the corresponding muscle. However, this recording cannot be continuous due to the use of muscle relaxants for anaesthesia; therefore, trigger EMG (t-EMG) was used to identify nerve root compromise following screw placement. Neuromuscular blocking agents were not used for at least 30 min prior to testing t-EMG. Disposable pedicle screw stimulating probes  which had a fully long insulated shaft terminating in a three millimetre ball tip was used to pass an electrical stimulus of up to 20 mA to the screw. Electrophysiological monitoring was conducted using Cascade Elite Intra-Operative Neuro-Monitoring (IONM) system . A positive response below 20 mA was considered as pedicle breach and a negative response was considered as accurately positioned. A positive response alerts the surgeon to attempt direct visualization of the medial pedicle wall. If the screw had violated the medial cortex, they were either revised or removed based on surgeon’s decision. Screw placement was also checked intraoperatively using x-ray images after completing the construct.Group 2 patients had pedicle screws applied with similar technique except for the use of EMG. Screw positioning was checked only by means of intraoperative x-ray image after completing the construct. Post-operative X-rays were taken for all patients among both the groups. Further evaluation by CT scanning was arranged if there was clinical evidence of neurological compromise. If malpositioning was determined by post-operative CT evidence, reoperation was performed to revise or remove the screw. Patient demographics, clinical presentation, indications for operation and operative variables were reviewed for each case. The efficacy of intraoperative EMG monitoring to identify screw breach and subsequent decrease in reoperation rates were evaluated.p) value of <0.05 was considered statistically significant.Statistical analysis was performed using SPSS software v.18.0 for Windows . Student’s t-test was used for continuous variables and Fisher’s exact test for categorical variables. A probability  = 63.6 ± 11.5 years; 3112 pedicle screws] based on our inclusion criteria. Patients who received EMG monitoring throughout the procedure and t-EMG to determine screw placement were in Group 1 , leading to subsequent decrease in reoperation rates [p = 0.04] among Group 1 patients . Screws breaching the pedicle pose a potential threat to adjacent neural structures and the most alarming consequence is neurological deficit. However, this can be avoided by meticulous technique. Recent advances in image guided navigation modalities that aid the surgeon to perform safe pedicle screw placements are gaining popularity , 12. Yet. Screws Continuous EMG monitoring acts as a real time monitor of spontaneous activity . Any irrOur analysis showed that a positive response to stimulation thresholds below 10 mA was highly suggestive of pedicle breach; but, a positive response for stimulation thresholds above 10 mA was frequently false positive . BesidesThe one false negative response we encountered was perplexing as intraoperative EMG was negative but the patient had developed symptoms of neurological compromise which was later confirmed with CT, to have resulted from a malpositioned screw that had breached the medial cortex which required repositioning. This could have been avoided if intraoperative direct palpation of the medial cortex was routinely performed; however, we reserved it for those showing a positive response to t-EMG. Besides that, intraoperative perception that a screw had breached the medial cortex is not feasible always, especially if a thin layer of bone is still present over the screw . FurtherOur results of sensitivity and specificity of t-EMG to detect potential pedicle breaches, is only with relation to symptomatic pedicle breaches where patients present with neurological deficits, and does not account for undetected pedicle breaches. Unlike other studies, our results showed high sensitivity for t-EMG monitoring (93.33%). This is because we had only one false negative response among 1711 screws and the false negativity of t-EMG was confirmed only with post-operative clinical examination. Various studies reported high specificity but not high sensitivity percentage; but unlike our study, they used post-operative CT to evaluate the status of the pedicles , 17, 21.Considering all factors, depending solely on EMG monitoring may not be appropriate but when combined with other modalities, surgeons can benefit the most , 19. BotOur analysis may be subject to secular influences regarding certain factors due to the retrospective nature of this study. Regarding selection of samples, we only included patients with pedicle screws among L1-S1 segments, excluding dysmorphic pedicles. However, it should be understood that, not all pedicles are anatomically similar and there can be variants or anomalies , 25. TheWe studied the efficacy of intraoperative EMG monitoring to detect potential pedicle breach during lumbar pedicle screw placement. Our results suggest that, t-EMG can be considered highly sensitive and specific for identifying potential pedicle breach by a malpositioned screw that can cause neurologic compromise; but, undetected breaches may still exist. However, t-EMG monitoring in combination with palpatory and radiographic assessment will certainly aid safe and secure pedicle screw placement. It can also efficiently reduce reoperation rates due to neurologic compromise provoked by a malpositioned screw.


Anti-IgE therapeutics interfere with the ability of IgE to bind to its receptors on effector cells. Here we report the crystal structure of an anti-IgE single-domain antibody in complex with an IgE Fc fragment, revealing how the antibody inhibits interactions between IgE and the two receptors FcεRI and CD23. The epitope overlaps only slightly with the FcεRI-binding site but significantly with the CD23-binding site. Solution scattering studies of the IgE Fc reveal that antibody binding induces a half-bent conformation in between the well-known bent and extended IgE Fc conformations. The antibody acts as functional homolog of CD23 and induces a closed conformation of IgE Fc incompatible with FcεRI binding. Notably the antibody displaces IgE from both CD23 and FcεRI, and abrogates allergen-mediated basophil activation and facilitated allergen binding. The inhibitory mechanism might facilitate strategies for the future development of anti-IgE therapeutics for treatment of allergic diseases. IgE is linked to allergic diseases and there is a great interest in developing anti-IgE therapeutics. Here the authors characterize the binding of human IgE Fc to a single domain antibody (sdab) and show that the sdab induces a closed conformation, which prevents and disrupts IgE binding to its receptor FcεRI and abrogates allergen mediated activation. Although the least abundant type of antibodies, IgE exhibits a variety of structural peculiarities with major functional consequences. IgE acts as a key molecule in a network of proteins, including the high-affinity IgE receptor FcεRI, the low-affinity receptor CD23, and galectins, e.g., galectin-32. Upon crosslinking by allergens, IgE bound to FcεRI on mast cells and basophils triggers degranulation, release of proinflammatory mediators, and immediate reactions2.Allergic diseases can be linked to IgE antibodies present in the circulation and on the surface of a variety of cell types5. The α-chain of the FcεRI displays an affinity for IgE in the range of 1011 M–1, providing the basis for long-term stability on effector cells and half-life of ~10 days6.IgE is an evolutionarily conserved and heavily glycosylated heterotetramer Fig.  with the7. The only approved anti-IgE antibody, omalizumab, primarily prevents interaction of free IgE with its receptor on effector cells10 and eventually reverses phenotypic and functional effects of IgE such as enhanced FcεRI levels on effector cells12. Not all patients with allergic asthma benefit from treatment13 and failure may also be caused by pharmacologically active IgE:omalizumab complexes14 that hamper correct dosing of anti-IgE15. Second-generation anti-IgE molecules such as ligelizumab and MEDI4212 are currently under investigation, but initial results suggest limited improvement. Basic structural and functional aspects of anti-IgE, e.g., the mechanism of rapid improvement in chronic urticaria, remain unclear17.Strategies to reduce increased levels of IgE and to limit effector cell degranulation included the development of antagonistic anti-IgE antibodies and antibody alternatives including a DARPin and aptamers19. Furthermore, the Cε3–4 sub-fragment adopts different conformational states ranging from closed to open depending on the spacing of the Cε3 domains and their distance to the Cε4 domains20. This conformational flexibility allows the Cε3 domains to rotate (swing) closer to or farther away from each other  are the antigen-binding moiety of heavy chain antibodies occurring in camelid species and cartilaginous fishes26, was produced in bacteria and purified from supernatant in yields of up to 80 mg l−1. The IgE Fc Cε2–4 was purified from the supernatant of mammalian cell culture  in the sdab are responsible for the majority of the IgE contacts. The sdab bridges the Cε3 and Cε4 domains from each ε-chain in the IgE Fc dimer Fig.  with theIn order to validate the crystal structure we mutated residues within sdab CDRs observed to contact IgE 2(mannose)5 (NAG2MAN5) hepta-saccharide, which represents the major form in natural IgE derived from myeloma cells as well as allergic patients . Sera of patients with elevated level of specific IgE (>100 kUA l−1) against the honeybee venom allergen Api m 1, the birch pollen allergen Bet v 1 and the major yellow jacket venom allergen Ves v 5 were incubated with the respective allergen and the IgE:allergen complexes applied to CD23. The sdab efficiently prevented binding of the IgE:allergen complexes to CD23 2 that is larger than the omalizumab epitope23. Both epitope and inhibition mode of sdab 026 are different from those previously observed for anti-IgE molecules. The sdab blocks interaction with FcεRI by trapping IgE Fc in a closed conformation of the Cε3 domains incompatible with FcεRI binding and a slightly more extended conformation of the Cε2 domains.Key to intervention in allergic diseases is the reduction of IgE in the serum and eventually on effector cells. In this study, we show that the anti-IgE sdab 026 targets IgE by binding to an epitope within the Fc domains that does not significantly overlap with the FcεRI-binding site. Notably the epitope is a conformational epitope involving two domains and a buried surface of ~800 Å34 the shared motif of the sdab and CD23 epitopes and the high affinity of the 026 sdab render an effect of the stalk on the inhibitory activity of the sdab unlikely. As observed in the ELIFAB analyses the inhibition of IgE binding to CD23 and displacement of IgE from CD23 strongly supports the epitope inferred from the structure and the mode of action of the anti-IgE sdab. Recently it has been verified that omalizumab also blocks the interaction with CD2334, most likely due to minor overlap of the epitope with the binding site23.The sdab epitope shows significant overlap with the CD23-binding site. Although additional importance of the stalk region of CD23 in binding to IgE has been postulated35. The inhibition of CD23 binding by patients’ serum IgG correlates with the activity of blocking IgG induced by successful allergen immunotherapy36 and leads to reduced T-cell activation37. Combining immuno-therapy with omalizumab results in clear benefits for treatment efficacy38. The main target of anti-IgE approaches however remains interference with FcεRI-mediated immediate effects.Binding of allergen:IgE complexes to CD23, a C-type lectin and low affinity IgE receptor, is crucial for facilitated antigen presentation and transport across the epithelium, and amplifies the generation and epitope spread of specific IgEIgE Fc adopts conformational states within the Cε3–Cε4 domain pairs ranging from an open conformation when in complex with FcεRI to the closed conformation when bound to CD23. Binding of FcεRI and CD23 to IgE is mutually exclusive as each is dependent on the conformation of Fc. FcεRI binding to free IgE moves the two Cε3 domains away from each other, decreases the angle between Cε3 and Cε4 and makes the binding site for CD23 inaccessible. Conversely, binding of CD23 to free IgE increases the angle between Cε3 and Cε4, moves the Cε3 domains to a more closed conformation and prevents IgE Fc interactions with FcεRI. In complex with the sdab, the Cε3 and Cε4 domains adopt the closed conformation, which is incompatible with FcεRI binding. CD23 is contacting residues in Cε3 and Cε4 within the same chain and enforces the closed conformation by pushing the Cε3 domains closer together. With a similar result, the sdab binds Cε3 and Cε4 from different ε chains forcing a closed conformation by pulling the Cε3 from one chain toward the other.Most of the structures available for IgE Fc contain only the Cε3–4 domains, and likewise we did not obtain crystals with the entire IgE Fc probably since the flexibility of the Cε2 domains in the IgE Fc hampers crystallization. The Cε3–4 fragment is often used in a stabilized form having artificial disulfide bridges or reduced glycosylation as a result of mutagenesis or choice of the expression host. Here we use the authentic IgE Fc and thereby minimize the risk of trapping the molecules in a rare or artificial conformation.Being unable to crystallize sdab in complex with the full IgE Fc we used SAXS to analyze the complex and free IgE Fc in solution. This allowed insights into the positioning of the Cε2 domain in solution for both the free Fc as well as in the complex with the sdab. Two different approaches to SAXS based modeling suggested that with respect to the sdab complex, the IgE Fc Cε2 domains appear to adopt a primary conformation in between the two well-established bent and extended IgE Fc conformations. In unbound IgE Fc, the dominating conformation appears to be the bent conformation. In addition, EOM analysis suggested that one minor conformation somewhat different from the major conformation may be present in both cases.31, and a similar, but lower activity has been reported for omalizumab. Our data suggest that the 026 sdab displaces IgE from FcεRI more efficiently than omalizumab. An even four-fold molar excess of omalizumab Fab fragment over the sdab did not result in significant IgE displacement. Apparently, the sdab removes IgE from the surface of effector cells as shown for basophils obtained from patients with different types of allergies. The reduction of IgE correlates with a decreased sensitivity of the effector cells to allergen-dependent activation as shown in a cohort of birch pollen-sensitized patients. It is imperative to consider that sensitivity of effector cells is shaped by the characteristics of surface IgE, e.g., ratio of specific and total IgE, affinity and repertoire complexity39. Although we used the dominant birch pollen allergen, Bet v 1, patient-specific sensitization profiles influence the reduction of cellular sensitivity, an effect likely to be even more pronounced in multisensitized individuals.Recently, the displacement of IgE from FcεRI by a DARPin IgE antagonist was described40. Although the removal of IgE from both circulation and effector cells could represent a benefit, the in vivo effect has not been fully proven yet. It has been shown that reduction of IgE on effector cells is counterbalanced by increased sensitivity41. Additional studies are therefore needed to address the displacement of IgE and its consequences and implications in detail.With respect to the accessibility of one sdab-binding site on the IgE even when bound to the FcεRI, the disruptive activity of the sdab is most likely driven by the conformational rearrangement upon sdab binding to the FcεRI bound IgE. With the capability of the E2_79 DARPin, omalizumab and the 026 sdab for displacing IgE it appears reasonable that the majority of anti-IgE molecules might exhibit similar activity, at least to a certain extent. Increasing this activity could become an important strategy for improving efficacy of anti-IgE therapeutics42. Targeting IgE with sdabs could offer a variety of benefits. Their 10-fold smaller size as compared to IgG allows efficient targeting of less accessible sites and the monovalent format prevents formation of larger complexes. State-of-the-art strategies for half-life extension and multiple targeting can easily be applied to sdabs. Delivery in functional form via mucosal and airway tissues is possible using sdabs43 and could improve the use of difficult routes for anti-IgE application44.Single-domain antibodies exhibit high production yield in simple expression systems and extraordinary stability, properties that render them interesting for numerous biotechnological and biomedical applicationsThe sdab’s mode of action identified in this study could also open up for the development of novel anti-IgE molecules of even lower molecular mass. It seems reasonable that high affinity targeting of an epitope similar to the sdab epitope by smaller molecules might drive similar conformational rearrangements. Thus, our description of the 026 sdab mode of action is likely to accelerate the development of anti-allergy and asthma drugs in the future.−1 in 20 mM HEPES, 50 mM NaCl, pH 7.2, and crystallized by vapor diffusion in sitting drops formed by mixing protein and reservoir solution containing 0.1 M imidazole pH 7.0, 11–12% polyethylene glycol (PEG) 20,000 in the ratio 1:1. Crystals were cryocooled in liquid nitrogen after transfer to a cryo-protectant composed of 0.1 M imidazole pH 7.0, 12% w/v PEG 20,000 and 30% glycerol.The 026 sdab:IgE Fc complex was concentrated to 5 mg ml45 and processed with XDS46. The structure was determined by molecular replacement with PHASER47 using the pdb ID: 2WQR as search model21. The model was rebuild in Coot48 and refined with Phenix.refine and iMDFF50. Figures were prepared with the PyMOL Molecular Graphics System (Schrödinger LLC).Data were collected at Diamond I2451. Point mutations were introduced by PCR Human embryonic kidney cells  were cultivated in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% (v/v) heat-inactivated fetal calf serum (FCS), 100 IU ml−1 penicillin and 100 μg ml−1 streptomycin. HEK-293 cells were transfected with the expression vector using Nanofectin  according to the recommendations of the manufacturer. After selection by addition of zeocin stably transfected cells showed an expression yield of 10–15 mg l−1. Cell culture supernatant obtained in roller flasks (Greiner) was collected and subsequently subjected to purification of the IgE Fc via nickel based affinity chromatography. The supernatant was loaded on a 1 ml HisTrap excel column  equilibrated with PBS . After washing with 10 column volumes (CV) of PBS and 20 CV of 5% PBS/imidazole  the IgE Fc was eluted in a 5–100% gradient of PBS/imidazole. Pooled fractions were dialyzed against Buffer A  and subsequently applied to a 1 ml Mono S 5/50 column  equilibrated with Buffer A. After washing with Buffer A, the protein was eluted with 20 CV of Buffer B  with a 0–50% gradient. The IgE Fc was further purified by size exclusion chromatography using a 24 ml Superdex 200 20/300 GL column  equilibrated in 20 mM HEPES, 50 mM NaCl, pH 7.2.The IgE Fc constant region cDNA was obtained from an IgE expression vector initially constructed from a human cDNA library. The cDNA was introduced into an expression vector providing a human immunoglobulin signal sequence via SmiI and MssI restriction enzymesThe DNA of the sdab 026 (patent WO 2012’/175740 A1) was obtained as a synthetic gene and cloned into the bacterial expression vector pET22+ providing a pelB signal sequence and a C-terminal histidine tag . The sample obtained from size exclusion chromatography was dialyzed against Buffer A and applied to the column. After washing with 5 CV of Buffer A, the protein was eluted with a 15 CV gradient from 0–100% Buffer B . IgE Fc Cε2–4 from the Source 15Phe purification was mixed with purified 026 sdab and the complex was purified by size-exclusion chromatography as described above.Prior to SAXS data collection IgE Fc was further purified to remove the Cε3–4 fragment by hydrophobic interaction chromatography using a 1 ml Source 15PHE column  equilibrated in Buffer A (1.6 M NH−1) was coated on microtiter plates (Greiner) at 4 °C and blocked with 40 mg ml−1 milk powder in PBS. Thereafter, 026 sdab at a concentration of 10 µg ml−1 was incubated for 2 h at RT. After washing, binding to IgE Fc was detected using alkaline phosphatase-conjugated anti-human kappa antibody and anti-human IgG antibody diluted 1:30,000 and 30 µl of substrate solution.The binding of 026 sdab to IgE Fc was assessed in ELISA. Purified IgE Fc (50 µg ml−1) at 4 °C and blocked with 40 mg ml−1 milk powder in PBS. After incubation with IgE Fc, the anti-IgE antibodies were incubated in a final volume of 20 µl for 4 h at room temperature. After washing bound antibodies were detected using an alkaline phosphatase-conjugated anti-human IgG antibody diluted 1:20,000, and 30 μl of substrate solution .For comparative assessment of anti-IgE antibodies, ELISA microtiter plates were coated with 026 sdab  onto a CM5 sensor chip  using NHS/EDC coupling procedures. Analyses were performed at 25 °C in a buffer containing 10 mM monosodium phosphate, 40 mM disodium phosphate and 100 mM NaCl, pH 7.4 supplemented with 0.01% Tween-20. For kinetic analyses the 026 sdab was injected at concentrations from 0.003 to 50 µg ml−1 (sdab 026 and mu112) and from 0 to 90 µmol l−1 , respectively, in IL-3-containing stimulation buffer (Bühlmann) for up to 60 min on a shaker. Samples were stained with anti-CCR3-PE in a 37 °C water bath for 15 min and anti-IgE-FITC (both BioLegend) for 20 min at 4 °C. Erythrocytes were lysed for 7 min followed by centrifugation at 500 × g for 5 min. Cell pellets were resuspended in 100 µl washing buffer and the mean fluorescence intensity (MFI) of IgE on CCR3+SSClow basophils was measured by flow cytometry .Peripheral EDTA blood from allergic donors was preincubated with 026 sdab, mu112 or omalizumab (Novartis) at concentrations from 0 to a maximum of 45 µmol l−1 sdab 026 for 60 min on a shaker. Afterwards, Bet v 1 (Biomay) diluted at different concentrations (0.01–1000 ng ml−1) in stimulation buffer was added. In each case one sample was left unstimulated to exclude background activation. Next, all samples were incubated with 15 µl of staining reagent (Bühlmann) consisting of anti-CCR3-PE and anti-CD63-FITC in a 37 °C water bath for 15 min. As above, erythrocytes were lysed for 7 min followed by centrifugation at 500 × g for 5 min. Cell pellets were resuspended in 100 µl washing buffer and basophil activation was measured by flow cytometry . Basophils were identified as CCR3+SSClow cells and CD63+ basophils were considered as activated. All results were analyzed using BD FACSDiva software (BD Biosciences).To analyze the impact of IgE026 on basophil activation, peripheral EDTA blood from six patients with birch pollen allergy was incubated ±45 µmol l52.CDsens analyses were performed as basophil allergen threshold stimulation. Basophil allergen sensitivity was measured as the allergen concentration eliciting 50% (EC50) of maximum CD63 upregulation. The CDsens is defined as inverted value for EC50 multiplied by 100, and was calculated by the following formula: CDsens = 1/EC50 × 10053. After washing with incomplete Tyrode’s buffer, receptor cross-linking was performed by incubation with goat anti-human IgE for 60 min at 37 °C. Release of β-hexosaminidase from viable vs. lysed cells was assessed for 60 min at 37 °C using p-nitrophenyl N-acetyl-glucosaminide (Sigma-Aldrich) as a substrate. After stopping the reaction by addition of carbonate buffer  the absorbance was recorded at 405 nm.In order to analyze in vitro degranulation RBL-SX38 cells (kindly provided by J.P. Kinet) were sensitized with rIgE Fc54. To allow for formation of IgE:allergen complexes 20 μl of indicator serum containing either high Api m 1-, Bet v 1- or Ves v 5-specific IgE concentrations  was preincubated with the respective allergen at 37 °C for 1 h in the presence of different 026 sdab concentrations. Following preincubation, IgE:allergen complexes were transferred to plates coated with CD23  and incubated for 1 h at RT. IgE:allergen complexes bound to immobilized CD23 were detected by adding biotin-conjugated anti-human IgE antibody (BD Biosciences), streptavidin-peroxidase (Sigma-Aldrich), and 3,3′,5,5′-tetramethylbenzidine (TMB) substrate . All samples were analyzed in duplicates.In order to evaluate the capability of 026 sdab to inhibit the binding of IgE:allergen complexes to CD23 the ELISA-based ELIFAB assay, a cell-free variant of the FAB assay, was performed−1 for Api m 1 (Latoxan), 8 μg ml−1 for Bet v 1 (Biomay) and 11 μg ml−1 for Ves v 5, respectively.In a second set of ELIFAB experiments, preformed IgE:allergen complexes bound to CD23 were incubated with 5 µl 026 sdab up to 60 min evaluating the ability of 026 sdab to displace IgE:allergen complexes from CD23. Displacing IgE:allergen complexes from immobilized CD23 was determined at a 026 sdab concentration of 12 μg ml55. The data were collected using a PILATUS 1 M pixel detector and λ = 0.992 Å in a temperature controlled capillary at 4 °C. The sample-to-detector distance was 2.872 m, covering a range of momentum transfer 0.04 < q < 5 nm−1 (θ is the scattering angle). Samples in 20 mM HEPES, 50 mM NaCl, pH 7.2 were investigated in the concentration ranges 0.5–1.7 and 2.2–9.0 mg ml−1 for the IgE Fc:026 sdab complex and IgE Fc, respectively. Data were collected with 10 exposures of 2 s. Radial averaging, buffer subtraction and concentration scaling were performed using the beamline pipeline56. Bovine serum albumin  used for calculation of the molecular weight  and bent (RCSB: 2WQR) conformation of IgE Fc was performed using DynDom60. Ensemble optimization of both unbound IgE Fc and the IgE Fc:026 sdab complex was performed using the EOM 2.0 programs RANCH and GAJOE33. A total of 5000 models were generated utilizing the same rigid bodies as for CORAL rigid body modeling, but with a randomly generated alpha carbon trace connecting the two rigid bodies, instead of a distance restrain. GAJOE was then used to select the ensemble of models best representing the scattering curve.The SAXS measurements of IgE Fc and IgE Fc in complex with sdab 026 were performed in batch mode at the BM29 beamline at the European Synchrotron Radiation Facility (ESRF), Grenoble, FranceThe study was approved by the Ethics Committee of the Medical Faculty of Philipps University, Marburg, Germany; all patients provided written informed consent to participate in the trial.Coordinates and structure factors have been deposited in the Protein Data Bank under accession code 5NQW. Other data are available from the corresponding authors upon reasonable request.Supplementary informationPeer Review File