A good compliance in allergen-specific sublingual immunotherapy (SLIT) often comes from good short-term efficacy. We aimed to evaluate the pretreatment parameters which can predict the short-term clinical efficacy in children that underwent SLIT.  517 children with allergic rhinitis (AR) that underwent SLIT were recruited in this study. Baseline clinical characteristics and laboratory parameters were collected, and the clinical efficacy was evaluated using symptom and medication scores. A multivariate logistic regression model and receiver operating characteristic (ROC) curves were established.  A total of 303 (65%) in 466 children that underwent SLIT achieved short-term clinical efficacy. The time of using the air conditioner was negatively correlated with clinical efficacy, whereas the serum-specific IgE (s-IgE) levels, the serum IL-10 and IL-35 levels, and the s-IgE/total-IgE ratio were positively correlated with clinical efficacy.  The time of using the air conditioner, serum-specific IgE (s-IgE) levels, serum IL-10 and IL-35 levels, and s-IgE/total-IgE ratio may be helpful for child selection before SLIT. Allergic rhinitis (AR) is a common pediatric chronic upper respiratory disease worldwide, which affects 10% to 20% of the children, and its prevalence has significantly increased over the last two decades [Allergy immunotherapy (AIT) is effective for both allergic asthma and rhinitis by modifying the natural course of allergic disease and inducing allergen-specific immune tolerance . The safDespite good efficacy, some patients discontinue or reject SLIT because the treatment is slow onset, is long-lasting, and has no surveillance like SCIT. A poor compliance is a general challenge for prolonged medical treatments. On the other side, a good compliance often comes from rapid onset and good short-term efficacy of SLIT. However, few studies have been conducted in the short-term efficacy of SLIT treatment .Therefore, there is an urgent need for identifying predictors for short-term efficacy of SLIT-responsive and SLIT-nonresponsive endotypes. With these predictors, doctors can select patients who are most likely to be responsive to SLIT; thus, the SLIT efficacy and compliance could be significantly enhanced.In this study, we report the short-term efficacy of 517 children who underwent SLIT. The clinical characteristics and laboratory parameters were analyzed for the prediction of short-term efficacy of SLIT, and receiver operating characteristic (ROC) curves were used to determine the sensitivity, specificity, and predicted values for target predictors.A total of 517 children with AR that underwent SLIT between June 2017 and June 2019 were enrolled. The diagnosis of AR was defined according to ARIA criteria, and the typical symptom was recorded [Baseline information such as sex, age, body weight, height, parental allergic history, passive smoke history, daily diet, living environment, physical activity, disease duration, and severity were collected at the beginning of the study. The air conditioner is often used for 10 to 15 hours depending on the weather condition, and the temperature is often set between 24 and 26°C.SLIT were conducted as we previously described . The DerThe symptom and medication scores were assessed at baseline and 12 weeks and 6 months after SLIT. The nasal symptoms in the last two weeks of evaluation  were recorded as follows: 0 = no symptoms, 1 = slight symptoms, 2 = moderate symptoms, and 3 = severe symptoms.The children were allowed to use oral or nasal antihistamines and/or intranasal steroids in case of aggravated symptoms. The medication score was defined as the sum of medication use in the last two weeks  according to the statement of the World Allergy Organization Task Force. The symptom and medication scores were recorded, and a combined Symptom Medication Score (SMS) was obtained by adding together the two scores.The children that obtained a 30% SMS compared to their baseline score were defined as the response group.Peripheral blood cell counts were measured by a Technicon-H1 blood cell counter . The serum total-IgE, eosinophil cationic protein (ECP), and s-IgE in serum were determined using a Unicap ECP kit  at baseline level and 12 weeks and 6 months after SLIT.β, 125 pg/mL.The serum levels of cytokines were determined by ELISA kits  as the instructions provided by manufacturer. The sensitivity for cytokines was as follows: IL-4, 0.22 pg/mL; IL-5, 3.9 pg/mL; IL-12, 5 pg/mL; IL-13, 125 pg/mL; IL-17, 62.5 pg/mL; IL-10, 62.5 pg/mL; IL-35, 62.5 pg/mL; IFN-gamma, 8 pg/mL; and TGF-t-test was used for group comparison. The χ2 test was used to compare categorical variables.Statistical analyses were performed with SPSS 17.0. Data were expressed as the mean and 95% confidence interval (CI) for the mean when the data distribution was normal. The two-sided P < 0.05 was considered statistically significant.According to the results of the univariate analysis, the multivariate unconditional logistic regression model was used to determine the independent predicting factors for the AIT clinical response. The results were expressed as odds ratios (ORs) and their 95% CIs. For all tests, This study recruited 517 children, and the baseline information is reported in As shown in Between children with and without clinical efficacy to 6 months of SLIT, significant differences were found with regard to the following characteristics: parents' educational background, time of using the air conditioner, materials for walls, serum t-IgE levels, serum s-IgE levels, serum s-IgE/t-IgE ratios, and baseline levels of IL-10, IL-35, and TGF-beta .Multivariate logistic regression analysis showed that the time of using the air conditioner was negatively correlated with clinical efficacy, whereas the serum-specific IgE (s-IgE) levels, the serum IL-10 and IL-35 levels, and the s-IgE/total-IgE ratio were positively correlated with clinical efficacy Tables  and 7.P < 0.01), IL-10 (P < 0.01), and IL-35 (P < 0.01). No significant difference was found between other markers. Our ROC analysis of the serum s-IgE/t-IgE ratio showed that a ratio of greater than 12.6% had the best sensitivity (97.2%) and specificity (74.1%) to predict short-term efficacy.Next, we determined the clinical significance of the serum s-IgE levels, s-IgE/t-IgE ratio, and levels of IL-10 and IL-35 for the prediction of short-term efficacy of SLIT. The AUC was 0.916 for the serum s-IgE/t-IgE ratio , 0.770 for serum s-IgE , 0.758 for serum IL-10 levels , and 0.776 for serum IL-35 levels  . When coThe efficacy of SLIT has been proved in previous studies . In genePrevious studies reported inconsistent onset of action for HDM SLIT, ranging from 8 weeks to 6 months –9. One rIn the present study, we found that most children  received good clinical efficacy after 3 months of treatment and the response rate increased to 303 (65%) at 6 months after treatment, proving good short-term efficacy of SLIT. Besides, the treatment was safe and no severe reaction was reported.To confirm predictors for SLIT-responsive and SLIT-nonresponsive endotypes, we compared and analyzed the clinical characteristics and laboratory parameters related to clinical efficacy. We found that parents' educational background, time of using the air conditioner, materials for walls, serum t-IgE levels, serum s-IgE levels, serum s-IgE/t-IgE ratios, and the levels of IL-10, IL-35, and TGF-beta were correlated with clinical responses. However, multivariate logistic regression analysis showed that the clinical efficacy was significantly correlated with the time of using the air conditioner, serum s-IgE levels, and levels of IL-10 and IL-35.Previous studies showed that Der f was found and had high concentration in the air conditioner filters. Therefore, the longer the air conditioner was used, the more severe the symptoms the patients underwent, which also led to poor clinical efficacy .Di Lorenzo et al.'s study reported that a significant positive correlation was found between the serum s-IgE/t-IgE ratio (>16.2) and the clinical response . A pediaThe immunotherapy has been reported to induce downregulation of the Th2 response with a shift toward a Th1 cytokine profile . MoreoveIn summary, our results proved that the serum level of specific IgE (s-IgE), s-IgE/t-IgE ratio, IL-10, and IL-35 and the air conditioner use were significantly correlated with short-term efficacy of SLIT in children, which may be helpful for patient selection before SLIT.


Fragilariopsis or Nitzschia. Our findings identify a potential future scenario where chytrid representation within these communities increases as a consequence of ice retreat, further altering community structure through perturbation of parasitic or saprotrophic interaction networks.Global warming is rapidly altering physicochemical attributes of Arctic waters. These changes are predicted to alter microbial networks, potentially perturbing wider community functions including parasite infections and saprotrophic recycling of biogeochemical compounds. Specifically, the interaction between autotrophic phytoplankton and heterotrophic fungi e.g. chytrids (fungi with swimming tails) requires further analysis. Here, we investigate the diversity and distribution patterns of fungi in relation to abiotic variables during one record sea ice minimum in 2012 and explore co-occurrence of chytrids with diatoms, key primary producers in these changing environments. We show that chytrid fungi are primarily encountered at sites influenced by sea ice melt. Furthermore, chytrid representation positively correlates with sea ice-associated diatoms such as  Estelle. S. Kilias et al. show that chytrid fungi exhibiting swimming tales are primarily encountered at sites influenced by sea ice melt and that its representation positively correlates with sea ice-associated diatoms. This study predicts that chytrid representation within its Arctic communities may increase as ice retreats further. Climate-driven changes to local physicochemical properties are profound in the Arctic Ocean, for example, leading to increases in temperature, light availability and decreases in sea ice concentration and surface water salinity. These changes have been shown to alter microbial community structure6.The marine environment is a vast reservoir of microbial diversity7. The discovery of this diversity has led to the hypothesis that bona-fide marine fungi  operate as a trophic ‘bridge’ for carbon and nitrogen substrate transfer between recalcitrant phytoplankton such as diatoms and wider nodes on the marine food-web, e.g., zooplankton9. One paraphyletic group of fungi called informally the chytrids, which can form flagellated zoospores capable of chemo- and photo-tactic swimming behaviour11, operates as prominent parasites, saprotrophs, or both (through necrotrophic infection of host) in aquatic systems13. In the following text, the term chytrids will be used as a shorthand exclusively for members of the Chytridiomycota group. Other zoosporic fungi include members of the group Blastocladiomycota or Cryptomycota/Rozellomycota but these groups are not a major focus of this study.Among marine eukaryotic microbes, ‘marine-derived fungi’ (a term referring to the sampling provenance of these microbes) are under-studied and insights into their ecological role are lacking. Recently, marine-derived fungi have received an increase in attention after high-throughput diversity tag-sequencing has revealed an uncharacterised diversity of fungi and fungal-like ribosomal RNA (rDNA) phylotypes in marine environments17. Chytrids have been reported to have an effect on phytoplankton population dynamics through infection, causing changes in the timing and duration of blooms18. Diatoms are known to form vast blooms, influencing environmental nutrient flux by exporting carbon to the deep-sea19. Changes in diatom bloom progression are therefore expected to have cascading implications for the marine ecosystem and associated biogeochemical cycles in many regions of the marine environment. Specifically, parasitism of diatom species by chytrids has been shown to alter diatom bloom dynamics in marine- and fresh- waters13. Documented diatom host genera for chytrids include, for example, Chaetoceros sp., Cylindrotheca sp., Navicula sp., Nitzschia sp. and Pseudo-nitzschia sp.23. In situ experiments, addressing the effect of abiotic and biotic factors on susceptibility of diatom to chytrid infections have shown an influence of light-stress and host densities23, factors which can potentially be driven by ice melt but this relationship has not been demonstrated from wider environmental sampling in ice melt conditions. In the Arctic Ocean, many of the diatom genera  shown to be susceptible to chytrid infections include major contributors to phytoplankton biomass24.Putative chytrid groups have been detected using both microscopy and environmental DNA sequencing in deep-sea sediment, water column and sea ice samplesIn this study, we investigated chytrid-rDNA distribution patterns in the Arctic Ocean in relation to abiotic  and biotic parameters . The strategy includes both, Deep Chlorophyll Max (DCM) and Under-Ice Water (UIW) sampling in order to study the co-occurrence of fungi and phytoplankton in waters influenced by ice melt and surface water freshening. We combine sampling of both the eukaryotic general V4 SSU rDNA and fungal-specific ITS2 region with high-throughput Illumina sequencing, in order to assess the diversity and distribution of fungal sequences and identify environmental parameters that putatively drive this component of the marine microbial community. As part of this work, we test the hypothesis that patterns of sequence variant representation in these groups relate to sea ice melt conditions, identifying a correlation between chytrid fungi and diatom community structure with sea ice melt conditions.5. It has previously been shown that profound sea ice melt in summer 2012 led to increases in the light conditions and nutrient supply, affecting the microbial community and increasing net primary productivity of these waters25. Representative samples from across different Arctic oceanographic regions and encompassing multiple depths were sampled over a time span of three months, allowing us to compare a range of: communities, sea ice melt conditions, and temporal samples. For description of the oceanographic context of the samples please see Fig. In summer 2012, the Arctic Ocean experienced an unprecedented decline in sea ice, resulting in a record sea ice minimum26 targeted PCR followed by high-throughput Illumina sequencing. The non-dimensional metric scaling (NMDS) ordination analysis comparing the ‘V4-identified’ community structure displayed differences in community diversity in the DCM samples . Single oceanographic regions showed some structuring effect, with samples originating from the same region . MANTEL tests of distance matrices demonstrated a correlation between environmental parameters and ‘V4-identified’ community structure. A subsequent principal component analysis for the purpose of identifying environmental factors which showed influence on community structure identified that the linked abiotic characteristics of temperature, salinity and sea ice concentration are significant factors (p ≤ 0.005) for driving the V4 eukaryotic community structure, consistent with other studies31.To understand the wider community dynamic of the environments sampled, the microbial community structure was assessed using eukaryotic V4 SSU rDNAPhaeocystis pouchetii) dominated Atlantic/Atlantic-influenced waters  and picoplankton  dominated Arctic waters (EGC and from S234–S384). The distribution pattern is consistent with previous work showing a correlation with nutrient and light-limited conditions, which have been shown to drive similar patterns of protist biogeography linked with selection for ‘frugal’ picoplankton species, specifically M. pusilla30.The major eukaryotic phytoplankton groups were distributed as show in Fig. Thalassiosira sequence variants (centric diatom) dominate the diatom communities in the Atlantic waters  & Chaetoceros (centric) sequence variants dominate diatom communities in Arctic waters  distribution, where diatom diversity was demarcated between these regions where specific Nitzschia sp. and Navicula sp. Sea ice algae are important primary producers in the Arctic Ocean, capable of growth in low-light conditions and living in brine channels and ice pores24. One characteristic attribute of sea ice algae is the production of extracellular polymeric substances, heterogeneous mixtures of biopolymers , which are exuded in higher concentrations during periods of low-nutrient availability and which allow for cryoprotection and potential floatation under sea ice based on O2 inclusion35. At UIW sampling sites, two pennate sea ice genera dominated the V4 rDNA sequence tag profiles: Nitzschia  and Fragilariopsis  along with Chaetoceros, Navicula, and the sub-ice genus Melosira to a lesser extent .Our fungal-specific ITS2 analyses recovered a large diversity of sequences identified as ‘unclassified fungi’, suggesting the marine environments sampled harbour a diversity of previously unsampled evolutionary distinct fungal taxa or eukaryotic microbes that share similar ITS2 sequence characteristics to fungi Fig. . The higGlomeromycota and Zygomycota were not-detected at most stations and if present represented a low-average relative sequence representation within our samples of 1.2 and 3.8%, respectively. Basidiomycota showed a general low presentation in DCM samples, not exceeding 7% Fig. .38. All three groups were recovered within the ITS2 data set. One plausible explanation for the high distribution in the WSC is the geographic location and proximity to Svalbard, characterised by coastal influence such as river runoffs and debris transport of plant matter.Ascomycota displayed a stable distribution of ~29% in UIW samples Fig. . RepreseBatrachochytrium). Given the high variability of the ITS2 marker and short sequence alignment, we suggest these phylogenies should be interpreted as tentative. Average representation of Chytridiomycota differed strongly in the Fram Strait  were sampled among the wider V4 defined eukaryotic community and were assigned to the class Chytridiomycetes. These sequence variants were placed in a Maximum Likelihood phylogenetic tree (colour coded in green) with published chytrid sequences 14, and Arctic meltpond aggregates (purple)40. The majority of the chytrid sequence variants (17), including the five most highly represented sequences, formed one major branch together with OTU sequences from the western Arctic (100% BS support)14. Furthermore, additional chytrid-like sequence variants recovered in this study branched with Rhizophydium/Kappamyces sequences , which includes a number of sequences previously obtained from meltpond aggregates, which were sampled in the previous year on a similar sampling expedition (ARK26/3)40. Only a few chytrid sequences from European sampling campaigns  branched in proximity to sequences from this or the Arctic Comeau study, with all of them originating from the Norway BioMarKs sampling site. No distinct diversity patterns were observed between DCM or UIW samples.The advantages of V4 tags over ITS sampling is that it allows for improved phylogenetic analysis for the purpose of taxonomic comparisons. This is because SSU rDNA sequences have improved database sampling of taxonomically identified fungi and this gene region allows for improved phylogenetic resolution in comparison to trees generated from ITS sequenceshttps://www.ebi.ac.uk/ena/about/tara-oceans-assemblies). Default settings were used, with tabulated output and the e-value gathering threshold was set at 1e-10. The BLASTn results were then filtered using the tool ‘awk’ to show any results with ≥2000bp hit length and 90% ID in an attempt to find DNA contigs that would allow us to infer a join between the chytrid V4 and ITS2 sequences. This approach returned no hits. To further explore putative ITS2-V4 associations we conducted a separate phylogenetic analysis for both the ITS2 and the V4 markers including ribosomal gene clusters sampled from a range of fungal genome projects of taxa related to the chytrids . These findings suggest that sea ice-associated or sea ice melt influenced waters display an altered ITS2 defined fungal community.The ITS2 fungal community composition varied across sampling sites, with salinity and sea ice concentration identified as significant environmental drivers of community difference . In this study, neither light availability (identified using sea ice concentration as a proxy) nor temperature showed a strong trend for chytrid distribution.Other abiotic parameters reported to influence chytrid distribution are light, temperature and/or nutrients14 and from those originating from an Arctic meltpond aggregate (purple)40. These results demonstrate that the putative chytrids detected in this study grouped closer to SSU rDNA sequences sampled from cold waters, providing tentative evidence of a set of ‘cold-water chytrids’. Furthermore, marine-derived fungi retrieved from the Western Arctic, displayed little overlap with representatives from Atlantic sites, suggesting that further biogeographic or environment-specific distribution patterns are evident14.Consistent with the ITS data, the V4 SSU rDNA phylogenies identified a proportion of unclassified chytrid-like sequences Fig. . The maj46 and therefore are thought to be associated with physical substrates. One hypothesis for the higher relative representation of basidiomycetes at UIW samples could be that sea ice serves as physical substrate, releasing basidiomycetes, or substrates that basidiomycetes can colonise, during sea ice melt. This explanation is consistent with a study conducted at the North Pole, where a high diversity of basidiomycetes were found in sea ice47. Floating algal aggregates under the sea ice can further form such substrates. As mentioned previously, high production of exopolymers act to increase the ‘stickiness’ of algae, which makes them inherently prone to aggregation and sedimentation after floating under the ice49, suggesting that sea ice algal aggregation may provide a substrate for basidiomycetes and other fungi in UIW environments—allowing for an osmotrophic/lysotrophic trophic interaction50, which otherwise would not be viable on smaller and/or dissolved particles. Consistent with this observation, basidiomycetes in this study demonstrate low-relative representation at the single multi-year ice (MYI) station S360  and/or region. Highest diatom representation in the V4 diversity profile (>40%) were observed at ice-free stations , WSC (S12–S37) and ice-free Barents Sea (S201 and S209), while the sea ice-associated diatom Fragilariopsis was most highly represented (>30%) in the EGC  environment, influenced by Arctic waters, higher sea ice concentrations and lower salinities, possibly as a product of sea ice melt. In comparison with the DCM samples, UIW samples showed a reduced relative representation of diatoms in the V4 diversity profiles, largely composed of ‘sea ice diatoms’ such as Navicula sp., Nitzschia sp., Chaetoceros sp., and Fragilariopsis sp. Out of the five UIW stations, S384 was sampled late in the season, and as such not sampled during sea ice melt but during sea ice formation. As a consequence, the diatom assemblage showed very few characteristic sea ice diatoms (<1%).Previous work has demonstrated a co-association between chytrid taxa and diatoms in the Humboldt CurrentFragilariopsis  showed the strongest potential positive correlations with chytrid sequence variants showing the highest relative representation of sequence reads . Further positive correlations were recovered with Chaetoceros, Nitzschia and Eucampia; diatoms often associated with sea ice, while Thalassiosira  gave rise to predominantly negative correlations. Positive correlation of three chytrid sequence variants were also found with one dinoflagellate and two chrysophytes, however their relative representation did not exceed 0.1%.Correlation analysis to investigate chytrid distribution in association with potential hosts such as diatoms, chrysophytes or dinoflagellates was computed. This analysis included all sampling sites and putative host sequence variants showing a representation of at least 1% at one or more sampling site Fig. . ConsideTo further support our hypothesis that chytrid fungi are forming interactions with pennate diatoms under melting conditions, we searched associated samples that were collected in parallel to the DNA samples using the same filter-based approach used for DNA sampling for the purposes of ‘crude’ SEM analysis. Our analysis of these samples identified one candidate image suggestive of an interaction between a chytrid [zoo-]sporangium and a pennate diatom Fig. . To furtp = 0.05) with different protist community members. A phylogenetic placement of these three sequence variants revealed a close phylogenetic relationship of one sequence variant  with Arctic chytrids from the Comeau study11 , showing the highest correlation. This global wide sample correlation analyses further confirm the chytrid community correlation in terms of co-occurrence with diatoms.In order to further evaluate the role of diatoms as hosts for chytrids we made use of the ‘Ocean Sampling Day 2014’ data set, including 145 surface water sampling sites covering different locations and environmental scenarios. Computation of potential correlations between chytrids and the wider protist community showed a number of associations Fig. . Out of 8. Considering, the importance of diatoms for the food-web, primary productivity and carbon export it is important to investigate the environmental factors affecting chytrid distribution and representation. In Hawaiian waters, eutrophic and mesotrophic conditions were found to correlate with chytrid infection of Cyclotella and Chaetoceros, while higher rates of attached putative chytrid zoosporangia were observed under Thalassiosira/Skeletonema predominance during austral spring in the Southern hemisphere (Humboldt Current)52. In this study, chytrids were primarily found at UIW sampling sites or EGC stations characterised by low salinity and a high prevalence of Chaetoceros, Fragilariopsis and Nitzschia spp., while low-relative representation of chytrid sequence variants were observed in Thalassiosira-dominated, ice-free water sites. This suggests that either abiotic conditions are directly driving the chytrid community composition or indirectly through biotic interactions, for example the composition of the diatom community, which is potentially shaping chytrid community dynamics through heterotrophic interactions.Chytrids have been shown to exert a top-down control on diatom community dynamicsChaetoceros at low temperature (5 °C)23. In marine high-latitude environments, phytoplankton from north-west Iceland were prone to increased viral infection, predation and fungal parasitism under higher light and higher water temperatures22, this is consistent with sea ice melt conditions. In the Western Arctic, a link between chytrid occurrence and algal stress in relation to increased light availability was found, particularly for pennate diatoms like Pleurosigma and Navicula41. These findings imply a potential increase in parasite infection under prolonged light exposure during Arctic summer months. The detection of chytrids in Arctic sea ice16, therefore, suggests that occurrence of chytrids and specific diatom species is linked, either directly due a trophic relationship or indirectly derived by similar patterns of environmental selection. If it is the former and these patterns are driven by a trophic relationship, these findings collectively suggest that stress of host species induced by changes in environmental factors is a modifier of these interactions.In summer, the melting of Arctic sea ice leads to surface water stratification, salinity decrease and the formation of meltponds. In the Arctic Ocean, the distribution and population density of diatom species depends largely on the extent of light transmission through sea ice, the temperature, salinity, nutrient status, as well as the presence of heterotrophic protists and grazers. Changes in these environmental factors are hypothesised to trigger stress responses in diatoms and other primary producers, which in turn can putatively affect the susceptibility of these phytoplankton organisms to parasites. In situ infection experiments of chytrid and diatom species under various stress conditions demonstrate a higher infection density under light-stress conditions and a positive infection prevalence for Nitzschia, Navicula or Fragilariopsis were frequently observed in aggregates from first year sea ice melts from similar samples53. We therefore hypothesise that ice algae aggregates present an attractive micro-habitat for chytrids as shown in Fig. 50 including parasitism. Supportive evidence for our hypothesis is the detection of chytrids in meltpond aggregates from 201140  or indirectly (biotic) influenced chytrid distribution and relative abundance. As such we hypothesise, free-floating algal aggregates under the sea ice, consisting of pennate diatoms forming putative micro-habitats for chytrid trophic associations. Furthermore, we observed a close phylogenetic relationship between chytrid sequence variants from the Arctic Ocean, identifying potential cold-water chytrid phylogroups. In a wider context, these findings suggest that chytrid prevalence in Arctic waters are likely to be altered under continuous sea ice melt conditions and freshening associated with climate change, leading to increased chytrid representation. In order to elucidate the role of chytrids in the Arctic Ocean under continuous sea ice melt, additional environmental studies are needed, including wider seasonal sampling  and different sea ice melt gradients and the sampling of algal aggregates associated with sea ice melt. Future work must broaden our understanding of potential host organisms, including other algae, e.g., chrysophytes or mixo-/heterotrophs like dinoflagellates and key groups of grazers.RV Polarstern on two cruise legs (ARK-XXVII/2 and 3). The first cruise leg, ARK-XXVII/2 includes one station in the Norwegian Sea and a transect through Fram Strait from 11°58.362′ to 11°5.09′ E longitude at ~78°50′ N latitude. The second cruise leg, ARK-XXVII/3 covered the Eastern Arctic Ocean, including sampling in the Barents Sea, and at the edge of the Kara Sea and Laptev Sea and at one high-Arctic sampling station  and −1.8 °C (S360 and S384). Overall, the sampling depth resulted in a lower salinity compared to the DCM samples, ranging between 30.54 (S323) and 33.07 PSU (S360). Sea ice concentration was lowest at S323 with 60% and highest at 360 and 384 with 100%, respectively (Table DNA was isolated with the Power Soil DNA extraction kit (MOBIO) following the manufacturer’s instructions, including a 2 × 20 s bead beating step to force the breakup of fungal cell walls and a second elution step to increase total DNA yield.55. To investigate the branching position of phylogenetically disparate putative chytrid groups and to provide additional data on potential hosts organisms the wider eukaryotic community was sampled using PCR amplification of the SSU rDNA V4 region. The V4 region was amplified with the primer combination D512F (5′-ATTCCAGCTCCAATAGCG-3′) and D978R (5′-GACTACGATGGTATCTAATC-3′)56. PCR reactions were carried out in a 25 µl reaction volume in triplicate, including 12.5 µl NEBNext PCR mix (New England BioLabs), 5 µl of 1 µM primer each (forward and reverse) and 4 ng DNA template. The PCR amplification protocol was the same for both gene targets and consisted of an initial denaturation step at 95 °C for 3 min, followed by 32 cycles of denaturation at 95 °C for 30 s, annealing at 53 °C for 2 min and elongation at 72 °C for 30 s and a final elongation at 72 °C for 5 min. A total of 22 samples were subjected to Illumina sequencing (2 × 300 bp) using the Nano Flow Cell approach. All generated sequence data have been deposited in NCBI Sequence Read Archive (SRA) under the accession number PRJNA561496.To study fungal diversity the ITS2 marker region was amplified with the primer combination ITS3Kyo2 (5′-GATGAAGAACGYAGYRAA-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′)57. In brief, sequence processing was carried out as follows: firstly, demultiplexed raw reads were subjected to DADA2 and quality profiles analysed, and secondly, reads were pre-processed by trimming, filtering and de-replication in order to identify unique sequences. Next, after this de-noising process, sequence variants in each sample were inferred and the paired-reads were merged. Finally, chimeras were identified and removed from the read data before taxonomic assignment. Settings for both data sets were the same , except for the length parameter (minLen = 250 bp for the V4 and minLen =  50 bp for the ITS2 data set). Taxonomic classification of the V4 sequences was based on the SILVA reference database version 12858. Since reference sequences of the ITS2 region are not as comprehensive as for the V4 region, taxonomic classification was carried out by combining multiple public databases such as: UNITE, NCBI GenBank nt and ITS2 DB V.Raw sequence data were processed using the DADA2 package in R for sequence variant identification removing the requirement for one universal standard threshold for all cluster groups, i.e., ‘taxa’59. Differences in V4 community composition in comparison to environmental factors were computed using the Bray Curtis and Euclidean distance, respectively and displayed in the form of NMDS plots. To test the significance of environmental factors MANTEL tests were calculated with 10,000 permutations and fitted onto the NMDS plot in the ‘ade4’ R package60. The heatmap was created with the package ‘heatmap3’61. To test for associations between diatom, chrysophyte, dinoflagellate and chytrid distributions within this study, an isometric log transformation was applied to the sequence counts using the ‘robcompositions’ package, with the ‘corrplot’ package being applied afterwards, including a significance test based on the Spearman’s correlation coefficient and 1000 Monte Carlo permutations62. To test for correlations between the distribution of chytrids and protists within the Ocean Sampling Day initiative, sequences from surface water samples were downloaded from the Micro B3 project repository  and processed with DADA2 using the PR2 v4.10 reference database for taxonomic classification. Sequence variant correlations were then computed with SparCC, using the ‘sparse correlations for compositional data’ algorithm, 20 iterations and 1000 bootstrap replication for computing pseudo p-values63.All statistical analyses were performed in R  and the data are shown in Supplementary Data 64 followed by tree calculation of the chytrid V4 sequences (using sequences with ≥0.01% relative representation) and chytrid ITS2 sequences. Topology calculation was combined with 1000 bootstrap (BS) replicates and a node-by-node SH-like approximate likelihood ratio tests. The sequence alignment was further supplemented with chytrid sequences from Richards et al.17, Comeau et al.14 and Kilias et al.40 and with additional reference sequences identified using BLASTn from the GenBank nr database. Alignments are available with and without mask at ‘figshare’ (10.6084/m9.figshare.11335658).Phylogenetic tree were calculated using a maximum likelihood (ML) approach. Evolutionary model evaluation was conducted using the automatic function in IQ-TREEl-lysine-coated glass slide and post-fixed with a 100 µl volume of Osmium tetroxide  in 0.3 M bicarbonate buffer placed on the glass slide for 30 min. After fixation, the glass slide was placed on a critical-point holder and washed twice in the 0.3 M bicarbonate buffer for 5 min. The subsequent dehydration through the ethanol series started with two 5 min washes in 50% ethanol and proceeded with single 5 min washes in 70, 80, 90 and 96% ethanol. The final steps were four 15 min washes in 100% ethanol. After dehydration, the sample was critical-point dried , mounted on an SEM stub, sputter coated with ca. 6 nm of palladium  and observed under scanning electron microscope  at the Electron Microscopy Unit, Department of Biosciences, University of Oslo.To test the presence of putative diatom-infecting fungal-like pathogens in meltpond aggregates, we conducted a scanning electron microscopy (SEM) analysis of the material collected during the Nansen Legacy cruise (AeN2018707) in the summer of 2018. The sampled meltpond was located in the Nansen Basin at the station Sice2b at 83° 19′ 22″ N/29° 28′ 7″ E. The aggregates were collected from the bottom of the meltpond using a sampling tube, fixed immediately in a fixative mixture composed of glutaraldehyde  and paraformaldehyde  in HEPES buffer  and stored at 4 °C. For the SEM preparation, aggregates were collected on a poly-Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Reporting SummaryPeer Review File


The CRF1+ population was found to be composed predominantly of glutamatergic projection neurons with a minority subpopulation of interneurons. CRF1+ neurons exhibited a tonic conductance that was insensitive to acute ethanol. CRF1− neurons did not display a basal tonic conductance, but the application of acute ethanol induced a δ GABAA receptor subunit-dependent tonic conductance and enhanced phasic GABA release onto these cells. Chronic ethanol increased CRF1+ neuronal excitability but did not significantly alter phasic or tonic GABA signaling in either CRF1+ or CRF1− cells. Chronic ethanol and withdrawal also did not alter basal extracellular GABA or glutamate transmitter levels in the LA/BLA and did not alter the sensitivity of GABA or glutamate to acute ethanol-induced increases in transmitter release. Together, these results provide the first characterization of the CRF1+ population of LA neurons and suggest mechanisms for differential acute ethanol sensitivity within this region.The lateral amygdala (LA) serves as the point of entry for sensory information within the amygdala complex, a structure that plays a critical role in emotional processes and has been implicated in alcohol use disorders. Within the amygdala, the corticotropin-releasing factor (CRF) system has been shown to mediate some of the effects of both stress and ethanol, but the effects of ethanol on specific CRF1 receptor circuits in the amygdala have not been fully established. We used male CRF1:GFP reporter mice to characterize CRF1-expressing (CRF1 An improved understanding of CRF1 amygdala circuitry and the selective sensitivity of that circuitry to ethanol represents an important step in identifying brain region-specific neuroadaptations that occur with ethanol exposure. The present findings also have broad implications, including potential relevance to the role of CRF1 circuitry in other contexts that may provide insight into other disorders involving amygdala dysfunction, including anxiety and depression.The corticotropin-releasing factor (CRF) system is a critical component of the stress network and has been implicated in psychiatric disorders including addiction, anxiety, and depression. The present study examines CRF receptor-1 (CRF1) lateral amygdala (LA) neurons and reports differential inhibitory control and acute ethanol effects of CRF1 LA neurons compared with the unlabeled  and the CRF receptor-1 (CRF1) are expressed throughout the amygdala  and haveCrhr1 promoter  investigate phasic and tonic inhibitory transmission in LA CRF1+ and CRF1− cells, and (3) determine the effects of acute and chronic ethanol exposure on inhibitory control within the LA.Previous work using a transgenic mouse line expressing green fluorescent protein (GFP) under the promoter  found th ethanol , 2016. TCrhr1 promoter, as previously described  male transgenic CRF1:GFP mice that express GFP under the escribed . Mice wem): sucrose 206.0; KCl 2.5; CaCl2 0.5; MgCl2 7.0; NaH2PO4 1.2; NaHCO3 26; glucose 5.0; and HEPES 5. After sectioning, slices were incubated in an oxygenated (95% O2/5% CO2) artificial CSF (aCSF) solution containing (in mm): NaCl 130, KCl 3.5, NaH2PO4 1.25, MgSO4 1.5, CaCl2 2, NaHCO3 24, and glucose 10 for 30 min at 37°C, followed by 30 min equilibration at room temperature . Recordings were made with patch pipettes  filled with an intracellular solution containing (in mm): KCl 145; EGTA 5; MgCl2 5; HEPES 10; Na-ATP 2; and Na-GTP 0.2, coupled to a Multiclamp 700B amplifier (Molecular Devices), acquired at 10 kHz, low-pass filtered at 2–5 kHz, digitized at 20 kHz , and stored on a computer using pClamp 10 software (Axon Instruments). Series resistance was typically <15 MΩ and was continuously monitored with a hyperpolarizing 10 mV pulse; neurons with series resistance >15 MΩ or >20% change in resistance during recording were excluded from final analysis. LA neurons containing the CRF1 receptor were identified by GFP expression and differentiated from unlabeled (GFP−) neurons using fluorescent optics and brief (<2 s) episcopic illumination in slices from CRF1:GFP reporter mice. Electrophysiological properties of cells were determined by pClamp 10 Clampex software online during voltage-clamp recording using a 10 mV pulse delivered after breaking into the cell. Drugs were applied either by bath or Y-tube application for local perfusion. Recordings (Vhold = −60 mV) were performed in the presence of the glutamate receptor blockers 6,7-dinitroquinoxaline-2,3-dione  and aminophosphonopentanoic acid  and the GABAB receptor antagonist CGP55845A (1 μm). All recordings were conducted at room temperature, and all solutions (bath and Y-tube) were prepared and maintained at room temperature.Coronal sections (300 μm) were prepared with a Leica VT1000S (Leica Microsystems) from brains that were rapidly extracted from mice after brief anesthesia (5% isoflurane) and placed in ice-cold sucrose solution containing (in mm), AP-5 (50 μm), and CGP55845A (1 μm) were purchased from Tocris Bioscience. SR-95 531 , picrotoxin (100 μm), and 4,5,6,7-tetrahydroisoxazolopyridin-3-ol  were purchased from Sigma-Aldrich.DNQX (10 μn = 4) were anesthetized with isoflurane and perfused with cold PBS followed by 4% paraformaldehyde (PFA). Brains were dissected and immersion fixed in PFA for 24 h at 4°C, cryoprotected in sterile 30% sucrose in PBS for 24–48 h at 4°C or until brains sank, flash frozen in prechilled isopentane on dry ice, and stored at −80°C. Free-floating 35 μm brain sections were obtained using a cryostat and kept at 4°C in PBS containing 0.01% sodium azide.Mice (AB_300798); rabbit anti-α1 and rabbit anti-δ GABAA receptor subunit ; mouse anti-parvalbumin ; mouse anti-calretinin ; and mouse anti-calbindin . Antibodies against native mouse protein were validated by the manufacturer with tissue from knock-out mice, with the exception of anti-δ GABAA. Next, sections were triple washed in PBS for 10 min at RT with gentle agitation followed by a 1 h secondary antibody incubation in PBS (in the dark). The following secondary antibodies were used: Alexa Fluor 488 goat anti-chicken ; Cy-3 donkey anti-rabbit ; and Alexa Fluor 568 goat anti-mouse . Sections were then washed  and mounted in Vectashield .Sections were washed in PBS for 10 min at RT with gentle agitation and then blocked for 1 h at RT in blocking solution ]. Primary antibody was incubated at 4°C overnight with gentle agitation in 0.5% Tween-20 and 5% NGS. The following primary antibodies were used: chicken anti-GFP . All microscope settings were kept the same within experiments during image acquisition. The analyst was blind to the identity of the red fluorescent signal when performing cell counts, and analysis was performed manually in an unbiased manner at four anterior–posterior levels (equidistant sections located −1.00 through −1.70 mm from bregma). Data are presented as the mean ± SE.n = 3) were perfused with ice cold PBS/Z-fix  after anesthesia with isoflurane. Following perfusion, brains were dissected and immersion fixed for 24 h in Z-fix at 4°C, cryoprotected in 30% sucrose in PBS for 24 h at 4°C, and flash frozen in isopentane on dry ice. Brains were preliminarily stored at −80°C until they were sliced on a cryostat in 20-μm-thick sections, mounted on SuperFrost Plus slides , and stored at −80°C.Mice  was performed by first briefly washing prepared slides in PBS. Next slides were submerged in prewarmed target retrieval buffer  and kept at a constant temperature between 95°C and 98°C for 10 min. Slides were then removed and immediately rinsed in distilled water twice, and then dehydrated with 100% ethanol. After dehydrations, slices were demarcated with a hydrophobic barrier pen  and digested with Protease IV for 20 min at 40°C in a hybridization oven. Next, the RNAscope Fluorescent Multiplex Reagent Kit User Manual  was followed entirely. Last, slides were mounted with Vectashield with DAPI . The probes used were Crhr1 , Slc17a7 , eGFP , and a negative control , all from ACD.Using an RNAscope fluorescent multiplex kit , 24; of LA at approximately −1.46 mm from bregma; 5 μm z-stacks). All microscope settings were kept the same within experiments during image acquisition. Background was subtracted from images based on the negative control for each probe, and signal intensity present in DAPI-labeled nuclei after background subtracted denoted positive cells. To perform quantification, ImageJ was used to manually count DAPI-labeled nuclei expressing fluorescently labeled probes in the region of interest (ROI). Next, the percentage of nuclei positive for one or both probes and the percentage of signal colocalization were calculated. The percentage of Crhr1+ nuclei expressing a marker of interest was determined by dividing the number of colabeled nuclei by the total number of Crhr1+ nuclei. Quantification was performed on three to four images (approximately −1.46 mm from bregma) from each mouse in an unbiased manner as probe fluorescence was quantified blindly. Brightness/contrast and pixel dilation are the same for all representative images.Slides were imaged on a Zeiss LSM 780 laser-scanning confocal microscope  and exposed to chronic intermittent ethanol (CIE) vapor (16 h) followed by air (8 h) daily for 4 consecutive days/week for a period of 4–5 weeks . Before n = 11) were unilaterally implanted with custom fabricated microdialysis probes (0.5 mm regenerated cellulose) aimed at the LA . However, as some penetrance into BLA is possible, microdialysis results are described throughout as LA/BLA. Mice were perfused with aCSF at 0.2 μl/min and allowed to recover overnight, as previously described (m) was added to the aCSF perfusate solution for reverse dialysis through the probe, and samples were collected for an additional 1.5 h during the local ethanol exposure period. This dose of ethanol was chosen for consistency with prior experiments using reverse dialysis in rodents, where 1 m was found to induce maximal changes in extracellular GABA and glutamate levels , 2% benzoyl chloride , and 1% formic acid (2 μl), and were subsequently spiked with benzoylated 13C6-labeled internal standards . Samples  were separated by high-performance liquid chromatography and analyzed by positive-ion mode tandem quadrupole mass spectrometry  using multiple-reaction monitoring. The following neurotransmitters were quantified using the standard isotope dilution method (precursor → product): the amino acids aspartate (238 → 105), GABA (208 → 105), glutamate (252 → 105), glutamine (251 → 105), glycine (180 → 105), serine (210 → 105), and taurine (230 → 105). Baseline concentrations were expressed as an absolute value (nanomolar), while changes produced by ethanol reverse dialysis were expressed as relative values (percentage of baseline) over time.Quantification of neurotransmitters was performed using triple liquid chromatography quadrupole mass spectrometry methods as previously described . Brieflyt test or a one-way ANOVA, where appropriate. Frequency, amplitude, and decay of spontaneous IPSCs (sIPSCs) were analyzed and visually confirmed using a semiautomated threshold-based mini detection software . sIPSC characteristics were determined from baseline and experimental drug conditions containing a minimum of 60 events . All detected events were used for analysis, and superimposed events were eliminated. Tonic conductance was determined using Clampfit 10.2 (Molecular Devices) and a previously described method  was used to compare air to CIE-WD mice before and after reverse dialysis of ethanol. All statistical analyses were performed using Prism version 5.02 (GraphPad Software). Data are presented as mean ± SE. In all cases, p < 0.05 was the criterion for statistical significance.Membrane characteristics and excitability were compared between groups using a two-tailed d method  in whichn = 11 images from three mice) to examine colocalization of Crhr1, the transcript for CRF1, and Gfp, the transcript for green fluorescent protein  to examine colocalization of Crhr1 and Slc17a7, the transcript for VGluT1. Consistent with GFP expression and the established glutamatergic makeup of the BLA, Crhr1 and Slc17a7 were similarly expressed in the LA  associated with GABAergic interneurons. Work by + neurons of the LA is unknown. We examined PV and GFP colocalization in the LA of CRF1:GFP mice (n = 16 sections from four mice) as well as calbindin (CB) and calretinin (CR). For the purpose of clarity, we refer to GFP+ and GFP− neurons throughout as CRF1+ and CRF1−, respectively. We observed expression of CB, CR, and PV interspersed with GFP in the LA  glutamatergic population with a smaller (∼20%) population of neurons that express CBPs .The LA is composed of glutamatergic projection neurons as well as local GABAergic interneurons . The resn the LA A–C, but ng cells D. Consisssed GFP E, suggeser <20%; F, sugges+ neurons (n = 28 cells from 14 mice) possessed a significantly smaller membrane capacitance (t(54) = 2.96; p = 0.0046 by unpaired t test, 21.84 ± 7.39 pF effect size; 95% confidence interval, −36.65 to −7.02), increased membrane resistance (t(30) = 2.34; p = 0.0260 by unpaired t test; 99.46 ± 42.48 mV effect size; 95% confidence interval, −186.2 to −12.71), lower time constant (t(54) = 3.08; p = 0.0033 by unpaired t test; 226 ± 73.56 ms effect size; 95% confidence interval, −373.6 to −78.69), and higher resting membrane potential (t(54) = 3.95; p = 0.0002 by unpaired t test; 9.114 ± 2.31 mV effect size; 95% confidence interval, 4.49–13.74) compared with CRF1− neurons  of CRF1+ neurons exhibited spike accommodation  and CRF1− neurons  versus CRF1+ neurons  = 2.73; p = 0.0093; effect size, 4.03 ± 1.48 pA; 95% confidence interval, −7.01 to −1.048; + and CRF1− neurons  = 3.30; p = 0.0053 by unpaired t test; 5.71 ± 1.73 Hz effect size with 95% confidence interval of 2.00–9.42; n = 9 cells from five mice; p = 0.64; p = 0.17; p = 0.88) between CRF1+ and CRF1− LA neurons, respectively.Whole-cell voltage-clamp recordings of sIPSCs were performed to assess baseline phasic inhibitory transmission. CRF1+ (n = 7 cells from five mice) and CRF1− (n = 9 cells from five mice) LA neurons using whole-cell voltage-clamp recordings. The basal holding current was −28.32 ± 20.67 pA in CRF1+ neurons and −17.19 ± 14.65 pA in CRF1− neurons. A GABAA receptor-mediated tonic current was defined as the difference in holding current  before and after application of a GABAA receptor antagonist. Focal application of the GABAA receptor antagonist GBZ (100 μm) produced a significant reduction in holding current in CRF1+ neurons  = 5.56, p = 0.002 by one-sample t test; 9.45 ± 1.70 pA effect size; 95% confidence interval, 5.81–13.09) and a reduction in the amplitude of the holding current or root mean square (rms) noise  = 2.93, p = 0.0264 by paired t test; 1.06 ± 0.364 pA effect size; 95% confidence interval, 0.17–1.94). In CRF1− neurons, focal application of GBZ (100 μm) produced no change in holding current  and a reduction in rms noise of a much smaller magnitude  = 5.24, p = 0.0008 by paired t test; 0.51 ± 0.10 pA effect size; 95% confidence interval, 0.29–0.74). The reduction in holding current was significantly greater in CRF1+ neurons compared with CRF1− neurons (t(14) = 5.56, p = 0.0001 by unpaired t test; 9.45 ± 1.70 pA effect size; 95% confidence interval, 5.81–13.09).We assessed tonic conductance in CRF1A receptors is dependent on specific subunit configurations and/or expression. We performed double-label immunohistochemical studies examining α1 and δ GABAA receptor subunit expression in CRF1+ and CRF1− neurons in the LA (n = 12 sections from four mice). The LA contains a significant number of CRF1+ cells, in contrast with sparse GFP expression in the BLA . Focal application of THIP produced a modest increase in holding current in CRF1+ neurons  = 3.12, p =0.0262 by one-sample t test; − neurons  = 6.82, p < 0.001 by one-sample t test; − neurons compared with CRF1+ neurons (t(18) = 3.03, *p = 0.0072 by unpaired t test; 18.44 ± 6.09 pA effect size; 95% confidence interval, −31.24 to −5.65). Consistent with the observed effects on holding current, focal application of THIP onto CRF1+ neurons resulted in no change in the amplitude of the holding current or rms noise  but significantly increased rms noise in CRF1− neurons  = 4.03, p =0.0014 by paired t test; 1.16 ± 0.29 pA effect size; 95% confidence interval, −1.78 to −0.54). Together, these findings indicate that the δ subunit is expressed predominantly in CRF1− neurons, whereas the α1 subunit is expressed predominantly in CRF1+ neurons, and that δ-containing GABAA receptors contribute to tonic conductance in CRF1− but not CRF1+ neurons.The δ subunit is associated with tonic conductance in a number of brain areas, including the hippocampus, cerebellum, cortex, and amygdala . Thus, wA receptors are sensitive to ethanol, and tonic conductance has been shown to be selectively augmented by acute ethanol (m) did not significantly alter sIPSC interevent interval or sIPSC frequency  = 15.82, *p = 0.0005 by one-sample t test; t(7) = 3.03, #p = 0.01,915 by unpaired t test; 14.08 ± 4.65% control effect size; 95% confidence interval, −25.08 to −3.09). Ethanol did not change sIPSC amplitude , or decay  in CRF1+ or CRF1− neurons, respectively. Additionally, focal application of ethanol did not significantly change the holding current of CRF1+ neurons , but did significantly increase holding current in CRF1− neurons  = 14.11, *p = 0.0001 by one-sample t test, n = 5 cells from five mice; t(8) = 9.09, #p = 0.0001 by unpaired t test; 11.37 ± 1.25 pA effect size; 95% confidence interval, −14.26 to −8.487; + neurons  or CRF1− neurons .GABA ethanol . Focal a+ neurons following ethanol vapor exposure or withdrawal  compared with neurons from AIR mice  = 2.49, p = 0.0187 by unpaired t test; effect size, −28.58 ± 11.49 pA; 95% confidence interval, −52.05 to −5.113; + neurons from CIE-WD mice  and neurons from AIR mice  = 3.34, p = 0.0022 via unpaired t test; effect size, 8.87 ± 2.65 mV; 95% confidence intervals, −14.29 to −3.46; p = 0.3353). In addition, we found no differences in action potentials elicited by ascending current injection among the three exposure conditions  and CIE followed by 3–7 d of withdrawal (CIE-WD). There were no significant changes in membrane properties in CRF1thdrawal A, and, cmodation B. Rheobanditions D. Togeth− neurons following ethanol vapor exposure or withdrawal , CIE mice , or CIE-WD mice  versus neurons from CIE mice (−48.00 ± 1.62 mV) and CIE-WD mice , and sIPSC decay  produced a significant reduction in holding current that was not significantly different among CRF1+ neurons from AIR, CIE, and CIE-WD mice  also produced a reduction in the amplitude of the holding current or rms noise that was not significantly different between CRF1+ neurons from AIR, CIE, and CIE-WD mice  produced no reduction in holding current in CRF1− neurons from AIR, CIE, or CIE-WD mice  and no significant difference among the experimental groups. These data suggest that tonic inhibitory signaling in the LA is insensitive to chronic ethanol exposure and chronic ethanol exposure followed by withdrawal.We also examined the tonic inhibitory conductance in CRF1in vivo microdialysis in CRF1:GFP mice exposed to AIR (n = 4) or CIE-WD (n = 7). Mice were implanted with 0.5 mm microdialysis probes  in the perfusate solution produced significant increases in LA/BLA GABA levels in both AIR and CIE-WD mice as assessed by two-way ANOVA of pre-ethanol and postethanol reverse dialysis (exposure condition × time) with a significant main effect of time  = 5.585, p = 0.0001), but no significant effect of exposure condition or interaction of time and exposure condition  produced significant increases in LA/BLA glutamate levels in both AIR and CIE-WD mice as assessed by two-way ANOVA of pre-ethanol and postethanol reverse dialysis (exposure condition × time) with a significant main effect of time  = 4.747, p = 0.0001), but no significant effect of exposure condition or interaction of time and exposure condition , making the CRF1+ population an important target for the actions of ethanol broadly within the amygdala. The results of the voltage-clamp experiments suggest that this enhanced excitability in the CRF1+ population is not driven by alterations in GABAergic signaling, which may indicate that these changes are instead regulated by ethanol-induced alterations in intrinsic excitability within the LA. Plasticity in glutamatergic signaling within the LA/BLA has been reported following chronic ethanol exposure  remains unchanged, allowing these cells to communicate with downstream amygdalar regions unimpeded. This suggests that neuroadaptations developing in the CeA , GABAergic signaling within the LA is intact despite chronic ethanol exposure and/or withdrawal. This resistance to ethanol-induced plasticity in inhibitory control within the LA may play a significant role in the development of alcohol dependence and alcohol use disorders. Sensory information, including external drug cues and internal states such as craving and withdrawal, is relayed first to the LA from the cortex and thalamus; glutamatergic projections from the LA then synapse with CeA, BLA and lateral paracapsular neurons. Our findings suggest that, despite chronic ethanol exposure, inhibitory control of LA CRF1 the CeA  and BLA  the CeA  on chron


Chlamydia pneumoniae is an obligate intracellular bacterium that causes respiratory infection. There may exist an association between C. pneumoniae, asthma, and production of immunoglobulin (Ig) E responses in vitro. Interleukin (IL-4) is required for IgE production.C. pneumoniae-induced production of IgE and IL-4 responses in peripheral blood mononuclear cells (PBMC) from asthmatic subjects. Whereas macrolides have anti-chlamydial activity, their effect on in vitro anti-inflammatory (IgE) and IL-4 responses to C. pneumoniae have not been studied.We previously demonstrated that doxycycline suppresses C. pneumoniae BAL69, +/- azithromycin  for 10 days. IL-4 and IgE levels were determined in supernatants by ELISA. IL-4 and IgE were detected in supernatants of PBMC (day 10).PBMC from IgE- adult atopic subjects (N = 5) were infected +/- C. pneumoniae, both IL-4 and IgE levels decreased. Addition of azithromycin  decreased IL-4 levels and had no effect on IgE levels.When azithromycin  was added, IL-4 levels decreased. At low dose, IgE levels increased and at high dose, IgE levels decreased. When PBMC were infected with in vitro.These findings indicate that azithromycin decreases IL-4 responses but has a bimodal effect on IgE responses in PBMC from atopic patients  Chlamydia pneumoniae is an intracellular bacterium that infects humans and causes respiratory infections [C. pneumoniae infection triggers the production of pathogen specific IgE, which may lead to inflammatory responses [fections ,2 in astfections . This baesponses .C. pneumoniae, can be treated with antibiotics  that may have beneficial effects in patients with asthma [h asthma ,7. Tetrah asthma .et al. reported that azithromycin inhibits production of IL-6, in hepatocytes infected with C. pneumoniae and cytomegalovirus [Macrolides can affect the innate immune system ; alteratalovirus .C. pneumoniae infection and may be due to past exposure [C. pneumoniae [Previous studies in our laboratory reported that treatment of asthmatic adults with minocycline had improvement in their asthma symptoms, independent of the presence of exposure . This obeumoniae ,12.C. pneumoniae-induced IgE and IL-4 responses in PBMC obtained from patients with asthma [et al recently reported that doxycycline suppressed C. pneumoniae induced interferon-gamma responses in PBMC in asthmatic children [C. pneumoniae infection and/or antibiotic treatment had no effect on IgE production in vitro [Other studies in our laboratory demonstrated that doxycycline suppressed h asthma . Smith-Nchildren . Howeverin vitro .in vitro effect of azithromycin on C. pneumoniae mediated IL-4 (Th2-type) cytokine responses and IgE responses in non-asthmatic atopic adults.The present study describes the C. pneumoniae and/or M. pneumoniae (determined by PCR), and peripheral blood (10mL) was collected. All clinical data was reviewed at the time of enrollment. The study was approved by the SUNY Downstate Medical Center Institutional Review Board . Written informed consent was obtained from all participants.Adult subjects  were recruited from the outpatient department at SUNY Downstate Medical Center . Inclusion criteria included: non-asthmatic adult with atopy  without clinically defined persistent asthma symptoms , with loTotal serum IgE levels were determined in serum using the UniCap Total IgE fluoroenzyme immunoassay  as previously described . Tests wC. pneumoniae-specific IgG antibodies were measured using the microimmunofluorescence (MIF) test , as previously described [escribed .C. pneumoniae AR-39  was propagated in HEp-2 cells as previously described [escribed .2 atmosphere for up to 10 days. Cell viability was determined at 0, 48 and 240 hrs , in the absence of any infection with C. pneumoniae.PBMC were separated from blood on a Ficoll-Paque  gradient (density 1.077) and put into cell culture a previously described , at 37°CC. pneumoniae (by adding purified EB for 1hr), or mock-infected (MI) and/or stimulated in the presence or absence of azithromycin (0.1 or 1.0 ug/mL) (Sigma) for either 48 hrs (IL-4) or up to 10 days (IgE) at 37°C in cRPMI in a humidified 5% CO2 atmosphere, as previously described [10 and 10d p.i. for IgE 10) used for analysis were selected by kinetic and dose response studies (using MOI of 0.01–10) for optimization of the assay. Two types of controls were used in infection experiments: identical volumes of heat-inactivated purified C. pneumoniae [C. pneumoniae [Following a 2 hr incubation to allow adherence, PBMC cultures were infected with escribed . All antescribed  to deterescribed , for supeumoniae  and ideneumoniae  based onin vitro quantitative determination of human cytokine or IgE content in cell culture supernatants, solid-phase sandwich ELISA assays were performed using either cytokine  or IgE ELISA test kits , according to the manufacturer’s recommended procedure, as previously described [For the escribed . Cell cuescribed  or 10 daC. pneumoniae and M. pneumoniae DNA according to Apfalter et al. [C. pneumoniae or M. pneumoniae to represent a positive result.Extractions of bacterial DNA from NP swab specimens  and PBMCr et al.  and Warir et al. , using TU test. A P value of <0.05 was considered significant. All statistical analyses were performed using Windows v.12.0 software .Data are expressed as means ± SD unless elsewise indicated. Data between cases were analyzed using the Mann-Whitney Patient demographic and clinical characteristics is shown in C. pneumoniae-specific IgG MIF titers. All patients tested negative for C. pneumoniae and M. pneumoniae by qPCR (NP swabs).Non-asthmatic patients had negative C. pneumoniae, IL-4 levels decreased (17%). Addition of azithromycin  decreased IL-4 levels   was added to cultures, IL-4 levels decreased  . When PBctively) . Thus, aC. pneumoniae, IgE levels decreased (63%). Addition of azithromycin  had no effect on IgE levels (0–3%) (C. pneumoniae-induced production of IgE in vitro.When azithromycin (0.1 ug/mL) was added to cultures, IgE levels increased (50%), but when 1.0 ug/mL was added IgE levels decreased (38%) . When PBs (0–3%) . Thus, ain vitro C. pneumoniae mediated cytokine and IgE responses and its contribution to inflammation are complex and not fully understood. In our initial report, we demonstrated that doxycycline suppresses C. pneumoniae mediated interferon-gamma responses in PBMC from children with asthma [in vitro effect of azithromycin on IL-4 and IgE response in C. pneumoniae-infected PBMC obtained from non-asthmatic adults. Successful treatment of C. pneumoniae infection with azithromycin may differ depending on asthma status.The effect of azithromycin on h asthma ; azithroh asthma . The curh asthma ; we furtC. pneumoniae infected PBMC from non-asthmatic subjects. Recent studies of Smith-Norowitz et al reported that doxycycline can suppress in vitro C. pneumoniae mediated interferon-gamma responses in asthmatic children [C. pneumoniae-induced cytokine and IgE responses by doxycycline in vitro was independent of anti-chlamydial activity in PBMC from asthmatic patients [in vitro effect of azithromycin in PBMC differs depending on asthma status. Thus, the relevance of these properties may be different based on asthma status as well as atopy status and needs to be considered for treatment guidelines.Our first finding was that addition of azithromycin decreased IL-4 responses in children . Howeverchildren ; in vitrchildren . Earlierpatients . Those fpatients . The stuC. pneumoniae-induced IgE production. The observed IgE production by azithromycin-treated infections might have been induced by other unknown contributors and not solely by C. pneumoniae infection. The IL-4 responses observed in our cultures represent a pathway for IgE production; it is possible that the failure of azithromycin to inhibit IgE responses was due to IL-4. The current findings are in agreement and supported by our previous studies [C. pneumoniae-induced IgE responses in PBMC from patients with asthma [We also found that treatment of cells with azithromycin could not suppress  studies  that reph asthma .C. pneumoniae respiratory infections in humans [C. pneumoniae infection eradication [in vitro findings in asthmatic patients by examining the in vitro effect of azithromycin on C. pneumoniae mediated IL-4 and IgE responses in non-asthmatic atopic adults. Several important conclusions can be drawn from our data: (1) azithromycin decreases IL-4 responses but has a bimodal effect on IgE responses in PBMC from atopic patients in vitro and may have immunomodulatory properties; (2) at low concentrations, azithromycin does modulate C. pneumoniae-induced production of IL-4 but not IgE. Thus, depending on the concentration, azithromycin may inhibit production of proinflammatory mediators [The antibiotics azithromycin (macrolide), clarithromycin, and levofloxacin are used to treat n humans . Howeverdication . The curediators ; this maediators .Some limitations to our study should be mentioned. We are aware of the small study number. Lastly, we did not elucidate the mechanism for the antibiotic effect on IL-4 production.C. pneumoniae in non-asthma and demonstrate the importance of disease-associated immune/inflammatory responses in non-asthmatic controls.These observations may have broad implications for the study of diseases such as


R,2S)-2-but-3-en-yl)(methyl)amino)cyclohexanecarboxylic acid (SBV2-114) was developed. A thorough pharmacological characterization of SBV2-114 using a cell-based [3H]GABA uptake assay at heterologously expressed BGT1, revealed an elusive biphasic inhibition profile with two IC50 values (4.7 and 556 μM). The biphasic profile was common for this structural class of compounds, including EF1502, and was confirmed in the MDCK II cell line endogenously expressing BGT1. The possibility of two binding sites for SBV2-114 at BGT1 was assessed by computational docking studies and examined by mutational studies. These investigations confirmed that the conserved residue Q299 in BGT1 is involved in, but not solely responsible for the biphasic inhibition profile of SBV2-114. Animal studies revealed anti-seizure effects of SBV2-114 in two mouse models, supporting a function of BGT1 in epilepsy. However, as SBV2-114 is apparent to be rather non-selective for BGT1, the translational relevance of this observation is unknown. Nevertheless, SBV2-114 constitutes a valuable tool compound to study the molecular mechanism of an emerging biphasic profile of BGT1-mediated GABA transport and the putative involvement of two binding sites for this class of compounds.Focal epileptic seizures can in some patients be managed by inhibiting γ-aminobutyric acid (GABA) uptake via the GABA transporter 1 (GAT1) using tiagabine (Gabitril®). Synergistic anti-seizure effects achieved by inhibition of both GAT1 and the betaine/GABA transporter (BGT1) by tiagabine and EF1502, compared to tiagabine alone, suggest BGT1 as a target in epilepsy. Yet, selective BGT1 inhibitors are needed for validation of this hypothesis. In that search, a series of BGT1 inhibitors typified by (1The online version of this article (10.1007/s11064-020-03017-y) contains supplementary material, which is available to authorized users. A receptors -4-(2-methoxyphenyl)-4-piperidinol (NNC 05–2090) and A 804598 were purchased from Tocris Bioscience . GABA (35.0 Ci/mmol) and Micro-Scint-20 were purchased from PerkinElmer .Dulbecco’s Modified Eagle Medium (DMEM) with GlutaMAX-I, Hank’s Balanced Salt Solution (HBSS), fetal bovine serum (FBS), and penicillin–streptomycin (P/S) were purchased from Life Technologies . PolyFect Transfection Reagent was purchased from Qiagen . HEPES (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid), Poly-R)-4-butyl)(methyl)amino)-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol ((R)-EF1502), -2-but-3-en-yl)(methyl)amino)cyclohexanecarboxylic acid ((SBV2-114), -5a), -2-but-3-en-1-yl)(methyl)amino)cyclohexane-1-carboxylic acid -4a), -2-but-3-en-1-yl)(methyl)amino)cyclohexane-1-carboxylic acid -5a), -2-propyl)(methyl)amino)cyclohexane-1-carboxylic acid -5c), and N-(1-benzyl-4-piperidinyl)-2,4-dichlorobenzamide (BPDBA) were synthesized in-house as previously described GABA competition uptake assay was performed as previously described [3H]GABA was used for a 15 min incubation period at 37 °C instead of 30 nM [3H]GABA and 3 min incubation. Studies related to the inhibition curves with GABA and BPDBA at hBGT1 were obtained both with and without the presence of 10 μM ATP during the [3H]GABA uptake assay.The GABA uptake to ensure that the compounds reached their site of action. All compounds, except BAPTA/AM, were also present at the same concentration during the course of the actual [3H]GABA uptake assay.To examine the influence of Ca3H]GABA uptake data was normalized to the percentage of total uptake in the individual experiments in the presence of the lowest compound concentration or no compound present. Data presented is the pooled data of at least three independent experiments with three technical replicates. Concentration–response curves (CRCs) were fitted with GraphPad Prism  by non-linear regression to the sigmoidal concentration response model:50 is the concentration giving a response halfway between Top and Bottom,and nH is the Hill slope.The [50_1 and LogIC50_2 are the concentrations that give half-maximal inhibitory effects. nH1 and nH2 are the Hill slopes and Frac is the proportion of maximal response due to the more potent phase.And the biphasic model:3H]GABA uptake assay was identified by the extra-sum-of-squares F test (for convenience referred to as F test).The best fit for CRCs obtained with the GABA uptake assay was performed. This entailed 12 concentrations to generate a detailed CRC. This study revealed that SBV2-114 inhibited mBGT1 (stably expressed in HEK-293 cells) and hBGT1 (transiently expressed in tsA201 cells) in an unusual biphasic manner (confirmed by F tests) with IC50 values in the low (m/hBGT1: 3.8/4.7 μM) and high μM range (m/hBGT1: 402.7/555.9 μM) corresponding to the high and low affinity components, respectively  indicating that the biphasic inhibition profile at BGT1 was not influenced by the expression level of BGT1.Previous studies in our group have revealed that SBV2-114 has a potency in the mid μM range for mBGT1 and displays about 26, 43 and 13 times reduced inhibitory potency at mGAT1, mGAT2 and mGAT3, respectively . In thision CRCs . In compc manner . Furthercanine BGT1 GABA uptake at hBGT1-CHO in a biphasic manner in two out of four experiments, and with a reduced % inhibition at the low IC50 (Frac = 0.10). Additionally, SBV2-114 displayed a biphasic inhibition profile at hGAT3, but also with a reduced Frac value compared to hBGT1-tsA, and in one out of four experiments at GAT2 . These data collectively show that SBV2-114 generally inhibits the [3H]GABA uptake by m/hGATs in an atypical manner.To test whether the biphasic inhibition profile was confined to BGT1, the detailed pharmacological characterization of SBV2-114 was extended to mGAT1-3 stably expressed in HEK-293 cells  and hGAT3H]GABA uptake assay -4a, -5a, and -5c GABA uptake assay, we wanted to address the molecular details. To this end, the possibility of SBV2-114 binding to two non-equal binding sites in BGT1 was investigated. Further, the influence of Ca2+ on the inhibition profile was examined due to the link between intracellular Ca2+ changes and altered [3H]GABA uptake GABA uptake results for the hBGT1 Q299L mutant showed a clear biphasic inhibition profile and no significant altered activity for SBV2-114, which would contradict the second binding mode and the relevance of Q299 for binding (data not shown). Additionally, we also investigated the activity of SBV2-114 in the corresponding hGAT3 mutant L314Q mimicking the orthosteric Q299 site of hBGT1. Through this mutation we were able to strengthen the biphasic inhibition profile of SBV2-114 and introduce activity in hGAT3 L314Q similar to hBGT1 underlining the potential relevance of Q299 for hBGT1 activity. In detail, the profile of SBV2-114 at GAT3 L314Q had a higher F value compared to at hGAT3 , and hence, statistically a more pronounced biphasic CRC, and a higher Frac value compared to both hGAT3 and hBGT1  and the protonated nitrogen undergoes hydrogen bonding with Ser395 .In terms of the allosteric site (third binding mode), the best poses showed that the carboxylic group undergoes hydrogen bonding with the side chains of tyrosine 453 (Tyr453), arginine 61 (Arg61) and glutamine 290 (Gln290), whereas the protonated nitrogen forms a hydrogen bond with aspartate 452 (Asp452) .A link between changes in intracellular Careported , which p2+ response, and where the Ca2+ originates from. To this end, tsA201 cells expressing hBGT1 were pre-incubated with either the cell impermeant Ca2+ chelator BAPTA to abolish any possible Ca2+ influx or the cell permeant BAPTA/AM to chelate all intracellular Ca2+ increases in response to SBV2-114. It is clear from these experiments that the pre-incubation itself (30 min at 37 °C), both in the assay buffer (control) and assay buffer supplemented with BAPTA/AM, affected the inhibition curve of SBV2-114 at hBGT1 in the [3H]GABA uptake assay  and the BAPTA/AM experiment . The same scenario was observed in the BAPTA experiment (data not shown). Consequently, it is impossible to conclude on the effect of chelating Ca2+ in response to SBV2-114 alone. Nevertheless, although the pre-incubation affected the Frac values and Hill coefficients, the preferred fit was still biphasic in both cases (F test), altogether indicating that the Ca2+ response elicited by SBV2-114 is not responsible for the biphasic inhibition profile.Next, we wanted to investigate if the biphasic CRC of SBV2-114 was dependent on the Casay Fig. , however2+ in the biphasic profile in another way, we tested if we could abolish the biphasic profile of SBV2-114 by inhibiting any potential release of Ca2+ from the endoplasmic reticulum (ER), elicited by SBV2-114, by inhibiting phospholipase C (PLC), or by blocking any SBV2-114 mediated Ca2+ influx through the P2X7 receptor [3H]GABA uptake. Inhibition of PLC with 5 μM U-73122 or P2X7 with 1 μM A 804595, however, did not convert the biphasic profile into a monophasic curve. Also, we still observed that the pre-incubation had an impact similar to pre-incubation with BAPTA/AM on the SBV2-114 inhibition curve at hBGT1 compared to Table 2+ increase is involved in the biphasic CRC of SBV2-114 at BGT1.To address the involvement of Careceptor . For thi2+ increases with ATP. Even though ATP modulated the BGT1 mediated [3H]GABA uptake and decreased the total [3H]GABA uptake by 9% , the presence of ATP during the [3H]GABA uptake had no effect on the type of inhibition profile of BPDBA and GABA at hBGT1 .In order to avoid this pre-incubation, we tested if we could induce a biphasic inhibition profile of BPDBA and GABA by triggering Ca50 of 139 mg/kg. However, SBV2-114 produced rotarod impairment with a TD50 of 163 mg/kg, and with 95% confidence intervals overlapping with the ED50. In the s.c.PTZ seizure test we only examined a single higher dose of 150 mg/kg SBV2-114. At this dose, SBV2-114 did not block s.c.PTZ-induced seizures or produce rotarod impairment , the adrenergic receptor alpha2A, and the dopamine transporter (DAT)  consequently hampering the usability of SBV2-114 to study BGT1 function in vivo. Additional studies are needed to investigate if interaction with any of these targets are implicated in the biphasic inhibition profile of SBV2-114 at hBGT1 in which case they would have to be endogenously expressed in our cell lines. Most likely, the involvement of any serotonin receptors and the adrenergic receptor alpha2A can be disregarded, since they are not endogenously expressed in HEK-293 cells GABA uptake assay, it is clear that SBV2-114 inhibits the GATs in an atypical manner. We have demonstrated an apparently novel biphasic inhibition profile of SBV2-114, which is most evident at BGT1. At BGT1, SBV2-114 has an IC50 value in the low μM range (m/hBGT1: 3.9/4.7 μM) inhibiting a fraction of 0.42–0.45 of the BGT1-mediated GABA uptake, while the second IC50 is in the high μM range (m/hBGT1: 402.7/555.9 μM). Additionally, we show that the high affinity component of the biphasic profile of SBV2-114 was most evident when the Frac value (% inhibition) and potency was high, as is the case for BGT1 compared to the other GATs. We further show that (R)-EF1502 and other SBV2-114 analogues, all sharing the β-amino acid moiety attached to di-aromatic lipophilic side-chains, inhibit BGT1 in a biphasic manner. The reason why there are no prior notions of (R)-EF1502 having a biphasic profile is likely due to the fact that the [3H]GABA uptake assay is typically performed with 8-point CRCs and not 12 and is thus problematic to capture. By contrast, BGT1 inhibitors such as BPDBA and NNC 05-2090, which comprise different scaffolds compared to SBV2-114, as well as GABA itself, display regular monophasic inhibition curves, supporting the finding that the biphasic profile at BGT1 is reserved to the class of structurally related compounds to SBV2-114 only. Importantly, we show that this biphasic inhibition profile of SBV2-114 at BGT1 in [3H]GABA uptake is indeed a true phenomenon as it occurs regardless of species, origin of the transporters (recombinant vs endogenous), expression levels, and kinetics.Based on the detailed molecular characterization of SBV2-114 using the [human serotonin transporter comprises an additional allosteric binding site in the extracellular vestibule above the orthosteric site [3H]GABA uptake data clearly shows that SBV2-114 inhibits GATs in an atypical manner, and hence, it is possible that other interacting residues not specific to BGT1 are accountable for the biphasic inhibition profile of SBV2-114.Recently, it was demonstrated that the highly related ric site . This pr2+, altered [3H]GABA uptake GABA uptake, supporting that a change in intracellular Ca2+ levels modulate BGT1 activity as reported for GAT1 and GAT3 GABA uptake data presented here, since nM concentrations of SBV2-114 decreased the total [3H]GABA uptake. Another example comes from a truncated version of the human DAT, which displayed a biphasic inhibition profile of [3H]DA uptake presumably due to two DAT populations with different affinities for dopamine GABA for 3 min in the presence of U-73122 and A 804598. All experiments were performed in triplicate in two-four independent experiments. The curve fittings preferred according to the extra-sum-of-squares F test were in all cases the biphasic fit, P<0.001. (a) Control: F value=8.77, U-73122: F-value=9.35; (b) Control: F value=34.71, A 804598: F-value=10.48 (EPS 239 kb)Supplementary file2 Supplemental Fig. 2 Inhibitory activity of GABA and BPDBA at hBGT1 transiently expressed in tsA201 cells in the presence of 10 μM ATP. GABA (a) and BPDBA (b) were tested for their ability to inhibit uptake of 30 nM [3H]GABA for 3 min. All experiments were performed in triplicate in two-three independent experiments. The curve fittings preferred according to the extra-sum-of-squares F test were in all cases the monophasic fit, P>0.05 (EPS 268 kb)Supplementary file3 (DOCX 17 kb)Below is the link to the electronic supplementary material.


This study evaluated the minimal invasiveness, safety, and accuracy of robot-assisted pedicle screw placement procedure using a modified tracer fixation device.Patients were randomly assigned to conventional fixation group (25 patients) and modified fixation group (27 patients).P > 0.05). The length of unnecessary incision, amount of bleeding, and fixation duration for tracer fixation respectively were 6.08 ± 1.02 mm, 1.46 ± 0.84 ml, and 1.56 ± 0.32 min in the modified fixation group and 40.28 ± 8.52 mm, 12.02 ± 2.24 ml, and 5.08 ± 1.06 min in the conventional group. The difference between both groups was significant (P < 0.05). However, no significant difference between the two groups was observed in terms of the accuracy of pedicle screw placement (P > 0.05).No baseline statistical difference was observed between the groups (The modified minimally invasive procedure for tracer fixation results in minimal trauma and is simple, reliable, and highly safe. Additionally, the procedure does not compromise the accuracy of pedicle screw placement. Thus, it has great clinical applicable value.Chinese Clinical Trial Registry: Registration number, ChiCTR1800016680; Registration Date, 15/06/2018. Pedicle screw fixation plays an important role in reconstruction procedures for spinal stability. Given the complex morphology and variability of the pedicle, screws penetrating it may injure surrounding tissues, thus resulting in severe complications in conventional spine surgery procedures, especially those involving freehand pedicle screw insertion –5. To imBriefly, robot-assisted spinal operation involves the preoperative or intraoperative acquisition of images and image-guided surgery after registration and calibration , 10.A tracer intraoperatively provides the origin of a coordinate system for a navigation system. A conventional tracer system includes a nonmetallic tracer and metallic spinous process clamp, which fixes the tracer on the spinous process of a patient. This tracer system is composed of a relatively stable combination and intraoperatively prevents the movement of the coordinate system origin, thereby preventing pedicle screw misplacement. An additional 3–4 cm incision is necessary for fixing the metallic clamp to adjacent spinous process, so it may lead to greater trauma, increase the risk of infection and affect fluoroscopy.To solve the disadvantage of conventional tracer fixation, we developed a novel device for tracer fixation . This deThis study is a prospective, randomized, controlled trial.All the participants who participated in this study needed to undergo pedicle screw placement and were admitted to the Department of Orthopedics of the Karamay Central Hospital of Xin Jiang between July and December 2017. The patients suffered from thoracolumbar vertebral fracture and lumbar degenerative disease. The study was approved by The Karamay Central Hospital’s institutional review board (2018-05-23) and followed the principles of the Declaration of Helsinki of the World Medical Association. Each participant provided a written informed consent before enrollment.The inclusion criteria were as follows: 1) > 18 years and 2) diagnosed with thoracolumbar vertebral fracture without nerve injuries (AO classification: type A or type B)  and lumbA total of 52 patients with pedicle screw insertion between July and December 2017 were divided into traumatic and degenerative groups. Then, each group was randomly assigned either to the conventional tracer fixation group (including 25 patients and 118 pedicle screws) or minimally invasive tracer fixation group (including 27 patients and 121 pedicle screws) through the random number table method. The patients were concealed before the randomized allocation. All the surgeries were performed by a senior doctor. Additionally, the pedicle screws were provided by Wego Medical Instruments Limited Company, and the third generation of TiRobot system came from TINAVI Medical Technologies Co., Ltd. .The third generation of the TiRobot system is the orthopedic surgery robot independently developed in China. It consists of a robot-arm system with six degrees of freedom, infrared optical tracking system (NDI), surgical planning, and navigation system. The surgical process was divided into five phases: robot setup; collection; registration and calibration of the 3D images, parameters, and trajectory designs of the screws; robot-assisted pedicle screw placement; and verification.The patients were under general anesthesia at the prone position. The bilateral shoulders and iliac crests were cushioned with a soft pillow to prevent abdominal compression. Before each operation, we turned on and set up the robot in a usual left unfold position. The TiRobot system, covered with a sterile plastic sheeting, was placed along the right side of the operating table, whereas the infrared optical tracking system was assembled at the head side of the patient, facing to the operating area. The patient tracer was fixed firmly on the adjacent spinous process. Three-dimensional images were acquired with a 3-D C-arm prior to calibration. The parameters and trajectory for the pedicle screw placement were set up, and the robot arm system was manually moved to the rough position of the screw insertion. Then, the robot system was run. The arm was allowed to move at the planned trajectory automatically. When the positioning accuracy indicated < 1.00 mm on the screen of the workstation, we incised the skin under the sleeve guiding, inserted the sleeve and reached the scheduled entry point, placed the guide wires, and performed screw insertion after the verification of the 3-D C-arm. We confirmed the pedicle screw position. Computed tomography (CT) scan was performed and the screw position was verified after the operation.The conventional tracer fixation device is a metallic spinous process clamp Fig. . For traThe modified minimally invasive tracer fixation device was linked to the bottom of the support frame of the tracer. Then, place the coupling device near the spinous process of the patient and insert a Kirschner wire into the porous channel via the guide channel. Finally, the tracer system was fixed firmly on adjacent spinous process of the vertebral body to insert the screw into.The basic data were recorded and collected, including the age; sex; diagnosis; pedicle diameter; angle (e angle) between the longitudinal axis of the pedicle and sagittal axis of the vertebral body. Endpoints included primary endpoint (incision length) and secondary endpoints . For the assessment of the accuracy of the pedicle screw placement procedure, postoperative CT scans were performed on the third day after surgery, and the screw positions were evaluated separately by three independent observers: two experienced orthopedic surgeons and a radiologist who did not know the purpose of the study and assigned patients’ group. The lowest grade among the three evaluators was taken as the breach grade, so the underestimation of the breach rates of the screws was prevented. The pedicle screw positions were classified according to the Gertzbein and Robbins criteria as follows : grade AP < 0.05 indicates statistically significant differences for all the tests.All statistical analyses were performed using the SPSS 21.0. The continuous variables were aggregated as the mean and standard deviation and analyzed using the independent Student t-test. Categorical variables were aggregated as the relative number and analyzed by Chi-square test. P > 0.05; Table A total of 52 patients met the criteria for this study . The two groups had no significant differences in baseline variables, such as age, sex, diagnosis, pedicle diameter, and e angle  The material is improved. The original metallic material is changed to the current photosensitive resin, which not only can be repeatedly sterilized but also has the characteristics of being safe, nontoxic, and nonallergenic. Meanwhile, the material can perform 3D printing to realize rapid production and personalized customization. (2) Nonmetallic materials are able to effectively avoid image interference from metal artifacts of the fixation device in fluoroscopy. (3) The Kirschner wires are used to fix the base and the tracer, reducing the injury and infection risk. (4) The device without screw fixation effectively avoids the screw failure on spinous process clamp. This phenomenon will cause difficulty in removing the clamp from the spinous process.The main drawback of this fixation method is that the fixators use the Kirschner wire without the limiter. The depth of the inserted Kirschner wire mainly depends on the experience of surgeon, and the Kirschner wire can possibly enter the spinal canal, leading to serious consequences. Therefore, adding the limiter to the fixator to avoid the risk of nerve injury is necessary.This study included 52 patients. No significant difference was observed between the minimally invasive and conventional tracer fixation procedures in the baseline variables, such as age, sex, diagnosis, pedicle diameter, and e angle. Compared with the conventional group, minimally invasive tracer fixation group did not suffer from unnecessary surgical wound, showed less bleeding, presented statistically significant differences. Thus, the minimally invasive tracer fixation method accorded with the philosophy of contemporary minimally invasive surgery particularly by further reducing the risk of injury and infection. Furthermore, this method can relieve post-operative pain and shorten hospital stays. This minimal advancement promotes further expansion of indication of robot-assisted minimally invasive surgery, such as robot-assisted percutaneous vertebroplasty/percutaneous kyphoplasty (PVP/PKP), or robot-assisted operation will be meaningless for PVP/PKP surgery, as shown in Fig. P > 0.05) between both groups. This result revealed that the modified minimally invasive tracer fixation device does not compromise accuracy during pedicle screw placement compared with conventional fixation and inherits the natural advantage of high accuracy in robot-assisted spine surgery. The device is highly clinically feasible with the characteristics of short tracker fixation duration, simple, and reliable fixation method. Furthermore, no image artifact displayed excellent picture quality despite the Kirschner wire development during image collection and automatic registration. In fact, no complications of the spinal cord and nerve root injury were found for both groups in the intraoperative and postoperative periods. This result demonstrates that the device is highly safe and relieves the concerns of patients, surgeons, and robotic engineers on severe complications of spinal surgery. Moreover, this finding promotes the pervasive application of the device in robot-assisted pedicle screw placement surgery.No significant differences were observed in the accuracy of pedicle screw placement (The minimal invasiveness, accuracy, and intelligence of surgery are the trend landmark of the development of surgical robot. As an important link to reflect the minimally invasive and accurate robot, fixation method of the tracer is of great significance during robot-assisted pedicle screw placement. The modified minimally invasive tracer fixation device presented the advantage of minimal invasiveness, firm fixation, simple, and reliable operation. Meanwhile, this fixation had no influence on the accuracy and safety of robot-assisted pedicle screw insertion. Thus, its great value in clinical application is proven. However, this study is a single-center study, and the sample size is relatively low. It is necessary for multi-center, large-sample studies to further explore.