Dermatophagoides pteronyssinus), American HDM (Dermatophagoides farinae), snow crab (Chionocetes spp.), edible crab (Cancer pagurus), German cockroach (Blatella germanica), and honeybee venom (Apis mellifera). In conclusion, people with a history of allergic diseases, including AD, asthma, and allergic rhinitis, should be cautioned against consuming RJ products because of the potential for cross-reactive responses to ensure the safe and successful use of RJ supplements.Royal jelly (RJ), a creamy substance secreted by honeybees, is the exclusive diet for queen bee differentiation and life maintenance. RJ has been used in cosmetics, beverages, medicines, and supplements worldwide. However, allergy is a concerning issue for RJ, especially in atopic dermatitis (AD) and asthma patients. In some cases, allergic reactions are seen after the first intake of RJ, suggesting the existence of allergens cross-reactive with RJ. Information about the cross-reactive allergens is very important for the safe application of RJ; however, study of this cross-reactivity is quite limited. In this study, we attempted to identify allergens cross-reactive with RJ by using serum samples from 30 AD patients who had never been exposed to RJ. In an enzyme-linked immunosorbent assay (ELISA) experiment, RJ-binding IgE antibodies were detected in the serum of 10 out of 30 patients, and their antibody titers ranged from 4- to 2,048-fold dilution ratios. Additionally, 3 AD patients were determined to be positive in a skin-prick test (SPT) with an RJ solution. Significant correlations were observed between the anti-RJ antibody titer and nonspecific IgE and between the anti-RJ antibody titer and the Eczema Area and Severity Index score. We further examined the cross-reactivity between RJ and 14 typical allergens by using an ELISA-inhibition assay and demonstrated that the following 6 allergens showed cross-reactivity with RJ: the European house dust mite (HDM) ( Royal jelly (RJ) is secreted from the hypopharyngeal and mandibular glands of worker honeybees, and it is the exclusive diet for queen bee differentiation and life maintenance –4. CompaOn the other hand, adverse events induced by RJ have been reported in recent decades, almost all of which are allergy-associated symptoms. These symptoms range from mild to severe and include rhinitis, eczema, contact dermatitis, urticaria, conjunctivitis, hemorrhagic colitis, acute asthma, bronchospasm, and fatal anaphylaxis –21. AlleDermatophagoides pteronyssinus by immunoblot-inhibition assay analysis of the patient’s serum [The existence of allergens cross-reactive with RJ has been suggested for a long time because allergic symptoms have been observed following the first intake of RJ in several cases , 23–30. ’s serum . Li et a’s serum .However, the causality underlying RJ allergic reaction following the first instance of ingestion is not well understood, even though serious allergic reactions have occurred in many cases. Therefore, the discovery of missing information about allergens cross-reactive with RJ is crucial to avoid future unnecessary accidents. In the present study, we aimed to assess whether allergens cross-reactive with RJ exist in AD patients.TM ViewAllergyTM39 , nonspecific IgE titers, Eczema Area and Severity Index (EASI) scores, thymus and activation-regulated chemokine (TARC) levels, eosinophil counts, lactate dehydrogenase (LD) levels, levels of ImmunoCAP® Specific IgE against the American HDM (Dermatophagoides farinae) and honeybee venom (Apis mellifera), and levels of IgE antibodies specific for HDM allergen components were measured. Clinical information and experimental data from the AD patients are shown in The study was approved by the Gifu University Hospital Independent Ethics Committee , and written, informed patient consent was obtained. Serum samples were obtained from 30 outpatients with AD who visited the Department of Dermatology, Gifu University Hospital. Eligibility criteria were age 20 or older and a diagnosis of AD. We confirmed that all patients had no previous contact with RJ in their interviews. All 30 patients met the inclusion criteria and were recruited for the study. Thermo ScientificDermatophagoides pteronyssinus) and the American HDM (Dermatophagoides farinae) were purchased from Biostir Inc. . Custom-made allergen extracts of cat dander (Felis domesticus), timothy grass pollen (Phleum pretense), Japanese cedar pollen (Cryptomeria japonica), and Japanese mugwort pollen (Artemisia indica) were purchased from the Institute of Tokyo Environmental Allergy Inc. . They were prepared at a protein concentration of 3.0 mg/mL with 0.01 M Phosphate-buffered saline (PBS) by Lowry’s method. We adjusted the protein concentration to 160 μg/mL with 0.01 M PBS before use. Lyophilized raw materials from buckwheat (Fagopyrum esculentum) and peanut (Arachis hypogaea) were purchased from Stallergenes Greer plc . The Alaskan pink shrimp , German cockroach (Blatella germanica), silk moth (Bombyx mori), and edible crab (Cancer pagurus) were purchased from Biostar Inc., and we obtained honeybee venom (Apis mellifera) from LATOXAN . Freshly frozen snow crab (Chionocetes spp.) was purchased from Kamasho , and both outer shells and leg muscles were lyophilized and powdered. The lyophilized powders were dissolved in 0.01 M PBS (pH 7.4) at a 1:30 weight-to-volume ratio and stirred overnight at 4ºC. After extraction and centrifugation , the supernatants were filtered with the 0.45-μm membrane DISMIC®-13HP , and their protein concentrations were measured by using the Pierce™ BCA Protein Assay Kit  and adjusted to 160 μg/mL with 0.01 M PBS. Lyophilized RJ was obtained from API Co., Ltd. .Extracts of crude bodies of the European HDM  for 1 h at 37ºC, followed by incubation with a TMB substrate  for 1 h at 37ºC. The reaction was stopped by adding 1 M phosphate, and the absorbance at 450 nm was measured. The plates were rinsed after each incubation step with TBS-T. All samples were assayed in triplicate. Commercial pooled human serum  was used as a negative control, and the TBS-T solution was used as the blank. The titers of antibodies specific for RJ are expressed as the highest dilution ratio in which a positive reaction was observed. A positive reaction, i.e., a positive IgE level, was defined as an absorbance greater than the mean value for the blanks plus 0.01.IgE recognition of RJ proteins by AD patient serum samples and their antibody titers were evaluated by ELISA. ELISA plates  were coated with 7.5 μg/mL lyophilized RJ in 0.05 M carbonate/bicarbonate buffer (pH 9.6) and incubated for 1 h at 37ºC. Blocking was performed with EzBlockChemi  for 1 h at 37ºC. Serially diluted serum samples were added to the ELISA plates and incubated overnight at 4ºC. RJ-binding IgE antibodies were detected by incubation with a horseradish peroxidase (HRP)-conjugated mouse anti-human IgE Fc antibody  diluted 16,000-fold in Tris-buffered saline with TweenTo study the cross-reactivity between RJ and other common allergens, an ELISA-inhibition assay was performed. The experimental procedure was the same as that in the ELISA experiment described above, except for preincubation with allergens. Diluted serum samples were preincubated overnight at 4ºC with an equal volume of an allergen extract solution or a TBS-T solution, which was used as a control. The final protein concentration of the allergen extracts was 80 μg/mL, and the dilution ratios of the serum samples were defined so that the absorbance of the control became approximately 0.10; thus, the dilution ratio of subject no. 39 was 256-fold, that of subject no. 44 was 64-fold, that of subject no. 36 and subject no. 37 was 32-fold, and that of subject no. 48 was 4-fold.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out using a precast 10–20% gradient gel and the Real gel plate SDG-592 . Lyophilized RJ was prepared at a concentration of 2 mg/mL in a sample buffer  and heated at 100ºC for 5 minutes, and then 143 μL of the solution was loaded into one large well (286 μg of RJ/143 μL). Precision Plus Protein™ Prestained Standards Dual Color  was used as a molecular weight marker. The gel-separated proteins were detected by Coomassie blue staining with EzStain Aqua . Following SDS-PAGE, the gel-separated proteins were electrotransferred to polyvinylidene difluoride membranes , which were incubated with EzBlock Chemi at room temperature for 1 h, cut into strips (75 x 8 mm) and incubated overnight in 1 mL of AD patient serum at 4°C. After washing with TBS-T, the membranes were incubated at room temperature for 1 h with an HRP-conjugated mouse anti-human IgE Fc antibody (Abcam) diluted 10,000-fold in a TBS-T solution. The secondary antibody was detected with ECL Prime Western Blotting Detection Reagent , and chemiluminescence was observed with Ez-Capture MG (ATTO).An SPT was performed by using undiluted, 1/10, and 1/100 raw RJ solutions for subject no. 38, subject no. 44, and subject no. 48, who showed a positive response to RJ by ELISA. Histamine dihydrochloride at a concentration of 10 mg/ml and a 0.9% saline solution were used as positive and negative controls, respectively. Skin reactions were estimated at 15 minutes after application. The longest wheal diameter (D) and the diameter perpendicular to D (d) were measured. The results are expressed as the mean calculated as (D + d)/2, with a result of 3 mm or greater being considered positive .TM39, TARC level, and LD level. Welch’s t-test was performed to compare ELISA-inhibition assays with and without preincubation with the test allergens. P values < 0.05, 0.01, and 0.001 were considered statistically significant. All analyses were performed using Ekuseru-Toukei 2012 ver.1.16 for Windows .Spearman’s rank correlation test was used to investigate the relationships between the anti-RJ antibody titer and the following assessment results for AD: the nonspecific IgE titer, EASI score, classification score based on the IgE of ViewAllergyTM39, are shown in Serum samples from AD patients were assessed by ELISA to detect RJ-specific IgE antibodies. In the ELISA experiment, 10 out of the 30 AD patients showed a positive result, and their antibody titers ranged from 4- to 2,048-fold . WesternTM39 test scores. High correlation coefficients were observed for HDM (0.589), Malassezia (0.677), sesame seeds (0.669), shrimp (0.594), crab (0.670), buckwheat (0.597), banana (0.670), and cockroach (0.625).To investigate allergens cross-reactive with the RJ protein, 14 allergens were selected based on the results of the correlation test and their allergenic importance. We performed SPTs with various concentrations of an RJ solution in 3 AD patients . Positive responses were observed in all the subjects tested : subjectRJ allergy is an exclusive adverse reaction caused by RJ. In the case of RJ, allergic reactions sometimes occur after the first ingestion of an RJ product, and therefore, allergens cross-reactive with RJ have been predicted to exist. In the present study, we found that some AD patients who had never consumed RJ had RJ-binding IgE antibodies. We further demonstrated that RJ cross-reacted with allergen extracts from European HDM, American HDM, snow crab, edible crab, German cockroach, and honeybee venom. To our knowledge, this is the first study to investigate the cross-reactivity between RJ components and a wide range of typical allergens in allergic patients.in vitro data suggest that at least some AD patients have cross-reactive antibodies against RJ.Allergy sufferers probably have a higher risk of experiencing allergic reactions to RJ, which can be explained by cross-reactivity. In this study, 33% of AD patients (10 of 30) showed a positive response to RJ in an ELISA experiment. Moreover, significant correlations were observed between the anti-RJ antibody titer and total serum IgE levels and between the anti-RJ antibody titer and EASI scores. The total IgE titer and EASI score are indicators of allergic diathesis and AD severity, respectively. Furthermore, SPTs showed positive results in representative patients. These findings suggest that allergy sufferers should be cautioned about the potential of developing allergic reactions by consuming RJ. These results were consistent with those of previous studies in Australia and Hong Kong, which showed high frequencies of positive results for RJ-binding IgE antibodies in allergic patients , 22. In Dermatophagoides pteronyssinus, to avoid false positives produced by cross-reactivity, the antigen used in the multiple-allergen simultaneous test had to be more highly purified and processed than the commercially available antigen used in the ELISA-inhibition assay. Thus, the result of the serological test used to detect antibodies against an HDM may not reflect all the specific cross-reactivity to RJ.The causal cross-reactive allergen for RJ sensitization was not a single allergen. We confirmed that six out of fourteen typical allergens were promising allergen candidates for cross-reactivity with RJ. The serum from one patient (subject no. 39) exhibited antigenic cross-reactivity between RJ and several allergens, including two mite species, two crab species, a cockroach species, and honeybee venom. Therefore, the RJ-binding IgE antibodies in AD patient serum are thought to be a complex mixture of IgE antibodies with various original specificities. We speculate that HDM allergens largely contribute to the cross-reactivity against RJ because preincubation with HDM allergens drastically decreased the reaction to RJ in an ELISA-inhibition assay. HDM allergens are one of the major inhaled allergens, and serum levels of HDM-specific IgE antibodies are increased in the majority of AD patients , 34. Thihttp://www.allergen.org/) [Honeybee venom also showed positive cross-reactivity, which can be explained by high homology with the major royal jelly protein family (MRJPs). Approximately 90% of RJ proteins are members of the MRJPs, and MRJP1, MRJP2, and MRJP3 were identified as allergen components of RJ in previous studies , 35, 36.en.org/) . Therefoen.org/) , 28–30. The common epitope components shared between RJ and the other allergens are still unclear. One of the possible components could be tropomyosin, a major panallergen in arthropods –43. Howehttps://blast.ncbi.nlm.nih.gov/) using the MRJP1 amino acid sequence to identify epitope components. However, the BLAST search analysis showed no significant similarity among the following organisms: Dermatophagoides pteronyssinus, Dermatophagoides farinae, Pyroglyphidae, Blattella germanica, Chionoecetes, and Cancer pagurus Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S1 Raw Images(PDF)Click here for additional data file.


The serine biosynthetic pathway is a key element contributing to tumor proliferation. In recent years, targeting of phosphoglycerate dehydrogenase (PHGDH), the first enzyme of this pathway, intensified and revealed to be a promising strategy to develop new anticancer drugs. Among attractive PHGDH inhibitors are the α-ketothioamides. In previous work, we have demonstrated their efficacy in the inhibition of PHGDH in vitro and in cellulo. However, the precise site of action of this series, which would help the rational design of new inhibitors, remained undefined. In the present study, the detailed mechanism-of-action of a representative α-ketothioamide inhibitor is reported using several complementary experimental techniques. Strikingly, our work led to the identification of an allosteric site on PHGDH that can be targeted for drug development. Using mass spectrometry experiments and an original α-ketothioamide diazirine-based photoaffinity probe, we identified the 523Q-533F sequence on the ACT regulatory domain of PHGDH as the binding site of α-ketothioamides. Mutagenesis experiments further documented the specificity of our compound at this allosteric site. Our results thus pave the way for the development of new anticancer drugs using a completely novel mechanism-of-action. Many recent findings highlighted the importance of serine metabolism in cancer ,2,3,4. GIn 2011, two independent publications highlighted the oncogenic role of PHGDH ,6. SinceUntil recently, only two different allosteric sites were identified for PHGDH, the ASB  and the ACT (aspartate-kinase chorismate-mutase-tyrA) domains. These two domains, located at the C-terminal part of the protein, have, up to date, never been intentionally targeted to develop PHGDH inhibitors, and their role in the control of PHGDH activity remains poorly understood. In 2016, Wang and coworkers suggested two other allosteric sites of PHGDH that were confirmed by the use of probes targeting these sites. The first, sharing at least five amino acids with the enzyme active site , is located at the interface of the enzyme active site and NAD binding domain, whereas the second, smaller, was identified in the substrate-binding cavity . More re1  revealed the importance of this para-substitution pattern  [para-substitution [2 to PHGDH remained mostly unclear. We previously reported that this series act with a non-competitive mode of inhibition with respect to both substrates and an apparently irreversible profile but without any evidence as to the location of this potentially new allosteric site [Previous studies in our laboratory highlighted compound nstance) . Moreovetitution . Howeverric site .2 using biophysical experiments. Microscale thermophoresis (MST) was thus used to appraise the binding of 2 to PHGDH. The experiments confirmed that 2 binds to PHGDH with a Kd of 35.02 µM, in the same range as the experimentally determined IC50 value. In contrast, the meta-substituted analogue 3, used as a negative control in this experiment, showed no appreciable binding  experiments.The binding of the photoactivatable diazirine probe 11 and the para-chloro substituted compound 2 underwent saturation transfer from the protein via spin diffusion through the nuclear Overhauser effect, corroborating their binding to PHGDH. Furthermore, epitope mapping of both molecules demonstrated a similar pattern of interaction. Epitope mapping corresponds to the relative saturation transfer intensity between the different protons of the molecule. Briefly, the ligands hydrogens in close contact with the protein will undergo a higher saturation transfer than the protons less involved in the interaction and hence display a more intense STD spectrum. Both molecules thus displayed similar binding epitopes, with a high relative saturation transfer for the aromatic hydrogens  was added for 30 min. The labeling with the photoactivatable diazirine was performed by irradiation at 350 nm for 10 min. Finally, the enzyme-inhibitor complex underwent trypsin digestion, and the resulting peptides were analyzed by nanoUPLC/MS. As a result, several peptides were identified, some of them being covalently modified by the diazirine 11  domain [2. Indeed, only the percentage of labeling of this particular peptide was reduced (from 43% to 6%) upon addition of 2, thus suggesting that this particular peptide sequence is most probably part of an allosteric binding site for the α-ketothioamide 2  domain  of PHGDHoamide 2 .E. Coli, this domain is a regulatory domain where L-serine binds to induce a negative feedback inhibition of the enzyme  was produced. Interestingly, this mutant retained its enzyme activity in the same range to that of the native PHGDH. However, the inhibitory potency of 2 on the mutant was found to be completely abolished , a well-known PHGDH inhibitor acting at an allosteric site distant from the site identified herein [Finally, to further document our hypothesis that bolished , hence dd herein , displayGeneral Chemistry—All reagents were purchased from chemical suppliers and used without purification. The characterizations of the products were performed as reported previously .PHGDH Assay—Enzymatic assay was carried out following a previously described procedure .PHGDH-tr Purification—pET28a human PHGDH-tr (Genecust) were transformed into BL21 Escherichia coli, the protein was produced and tested following a reported procedure . ProteinMST assay—MST measurements were adapted from a previously reported procedure . Measure11 and compound 2 at various concentrations in classical assay buffer . The mixture was then irradiated on ice using Stratalinker UV Crosslinker 1800 at 350 nm for 10 min.Diazirine irradiation—Human PHGDH (5 µg) was incubated 30 min at room temperature with compound Mass spectrometry experiments, Orbitrap Lumos—Mass spectrometry experiments were carried out on an Orbitrap Lumos, following a previously reported procedure, with some modifications . OxidatiEpitope mapping by Nuclear Magnetic Resonance—All experiments were adapted from a previously published procedure . For 1D 2 on PHGDH. Interestingly, this led to the identification of a previously unknown allosteric site on PHGDH delineated by the 523QHVTEAFQFHF533 C-terminal peptide sequence, that is part of the PHGDH ACT domain, as the target binding site. We further confirmed our hypothesis using a complementary protein truncation experiment.In this study, we aimed to detail the site of action of a series of α-ketothioamides on PHGDH to fuel the development of new and potent anticancer drugs acting along a potentially completely novel mechanism-of-action. First, we characterized the binding of our lead compound 2 using microscale thermophoresis experiments. Then, a crosslinking strategy using an original photoactivatable diazirine probe was used to decipher the binding of Although to our knowledge, the structure of the allosteric site identified here is not known, our data pave the way to the development of novel anticancer agents acting along a particularly original mechanism-of-action.


The delineation procedure relies on a convolutional neural network paired with a set of common image processing operations and permits explicit measurement of relative elevation at the center of each ice wedge polygon. The resulting map visualizes meter- to kilometer-scale spatial gradients in polygonal geomorphology across an extensive landscape with unprecedented detail. This high-resolution inventory of polygonal geomorphology provides rich spatial context for extrapolating observations of environmental processes across the landscape. The map also represents an extensive baseline dataset for quantifying contemporary land surface deformation  at the survey area, through future topographic surveys.It is well known that microtopography associated with ice wedge polygons drives pronounced, meter-scale spatial gradients in hydrologic and ecological processes on the tundra. However, high-resolution maps of polygonal geomorphology are rarely available, due to the complexity and subtlety of ice wedge polygon relief at landscape scales. Here we present a sub-meter resolution map of >10 Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.11871369 Nonetheless, variability in this microtopography often drives sharp spatial gradients in fluxes of energy5, water7, and carbon11 between the atmosphere and the subsurface. The spatial distribution of polygons of different morphologies within a single landscape can be highly complex, as thousands of polygons may occupy a single square kilometer of terrain, and gradation between endmembers (such as LCPs and HCPs) may occur over distances that vary from meters to kilometers13. The difficulty of accounting for the influence of such complexity on environmental processes is compounded by the transient nature of the microtopography itself, which can deform over timescales of years to decades in response to increased ground temperatures19. High-resolution inventories of spatial and temporal heterogeneity in ice wedge polygon form are therefore important for quantifying and predicting future trends in landscape-scale processes, including mobilization of soil organic carbon, which may exert important feedbacks on global climate change over the coming century20.One of the challenges to upscaling hydrologic and ecological measurements in tundra settings is accounting for spatial heterogeneity associated with ice wedge polygons. Segmenting the ground into discrete units some tens of meters across, ice wedge polygons are some of the most conspicuous features of the tundra surface. However, mapping ice wedge polygons across broad spatial scales is challenging, due to the subtlety and complexity of polygonal microtopography. Commonly, features demarcating the edges of polygons—such as rims of soil in low-centered polygons (LCPs), or sunken troughs in high-centered polygons (HCPs)—represent less than 50 cm of relief from the surrounding terraine.g.21). The spatial extent of previous maps has varied from ~1 km2 or less, to the entirety of the Alaska North Slope. These landcover maps play an essential role in dividing the Arctic into functional terrain units, for extrapolating point- to plot-scale measurements of hydrologic and ecological processes across landscapes. However, to our knowledge, no dataset yet published quantifies gradational  change in polygonal morphology over spatial scales greater than several square kilometers. This gap prevents quantitative assessment of the degree of heterogeneity within each polygonal class, and also limits the precision with which contemporary rates of land surface evolution in polygonal terrain can be quantified.To date, most efforts to map ice wedge polygon morphology have relied on optical or multispectral imagery, and have been designed to classify terrain into distinct bins, such as LCPs, HCPs, or intermediate polygons , derived from a set of two airborne lidar surveys, one conducted during August 2012 and the other during August 2014. The primary laser pulsing system on the lidar instrument  operated within the near-infrared spectrum (1064 nm) at a frequency of 400 kHz (a second pulsing system fired at 38 kHz operated at 515 nm to measure bathymetric profiles of surface water bodies). Deployed in an aircraft at a nominal altitude of 400 m, it generated a point cloud representation of the land surface with an average return density greater than 20 points m26. Landcover by gravels and sands was mapped by hand atop Google Earth imagery by drawing polygons surrounding exposed bars adjacent to the two major streams of the survey area, which were distinctly brighter than the dark green to brown tones of the nearby tundra soils. These polygons were subsequently converted into rasters, snapped to the original DEM, for use during mapping.In addition to the DEM, our procedure employed two secondary geospatial datasets, demarcating zones of the survey area in which either open water or exposed sands and gravels associated with the Sagavanirktok and Kuparuk Rivers were present at the surface. These data were used to indicate zones of the DEM in which our algorithm was instructed not to delineate polygonal ground, as ice wedges would not be expected to form in such terrain. Landcover by both water and coarse sands and gravels were mapped as 50 cm horizontal resolution rasters, snapped to the original DEM. The landcover raster representing water was generated prior to this study by extracting zones of divergence between elevation as estimated by the red and green lasers on the lidar instrument13, which is briefly summarized here. At its core, the method relied on a convolutional neural network (CNN) to infer whether or not each pixel of the DEM represents a polygon boundary. The binary map of polygon boundaries produced by the CNN was then processed to segment the DEM into discrete polygons. .Ice wedge polygons were delineated within the DEM using a novel, deep learning-based approachi.e., a distance comparable to the width of a typical ice wedge polygon). This mesoscale topography was subtracted from the DEM to isolate microtopography associated with individual polygons. The raster of microtopographic data was then converted into a grayscale image, in which local peaks >0.7 m in relief were assigned a maximum brightness, and pits >0.7 m in depth were assigned minimum brightness. These bounds were chosen to enforce a consistent relationship between image intensity and microtopographic relief, while capturing the vast majority of microtopography encountered on the tundra.In the first stage of the workflow, mesoscale topographic trends were removed from the DEM, which was then converted into 8-bit grayscale imagery, in preparation for processing by the CNN. Mesoscale topography was first estimated by applying a two-dimensional averaging filter across the DEM with a radius of 20 m . The CNN was purposefully designed using a simple architecture , generating an intermediate raster in which the area of an individual polygon comprised a valley of negative signal intensity. At this stage, to minimize the likelihood of over-segmentation, any local valley with a minimum depth <1.5 m was fused with the nearest larger valley using morphological reconstruction27. This operation was based on the rationale that most real instances of ice wedge polygons contain at least one point greater than 1.5 m in distance from the closest boundary. Subsequently, a watershed transform was used to delineate the divides between valleys, thereby segmenting the intermediate raster into discrete regions, each representing a potential ice wedge polygon.Following application of the CNN, the binary map of polygon boundaries was processed in several stages to extract discrete polygons. In the first stage, all continuous regions identified as “boundary” but with an area less than 20 mi.e., each string of pixels on the divide between two adjacent regions) was analyzed individually. Any edge along which less than half of the pixels had been recognized as an ice wedge polygon boundary by the CNN was eliminated, and the adjacent regions were merged. Effectively, this procedure tended to preserve the boundaries of real ice wedge polygons, while lumping non-polygonal terrain into regions much larger than a typical polygon. Non-polygonal terrain was then removed from the final raster by eliminating all regions greater than 10,000 m2, a threshold which was chosen to be greater than the area of a real ice wedge polygon. Finally, any remaining polygons delineated by the algorithm overlapping surface water or the coarse sediments of the Sagavanirktok and Kuparuk rivers were treated as false positives and removed. Typically, all post-processing subsequent to the application of the CNN required 15 seconds or fewer per square kilometer.In the final stages of processing, each edge in the segmented image , UTM coordinates of the polygon centroid (m), and relative elevation at the polygon center (m). A single tab-delineated file summarizing these attributes across the entire survey area is also included.The final map and ancillary datasets have been uploaded to a PANGAEA repository2 subsets of the survey area. The DEM used as input to the mapping procedure is presented as a single band raster with units of meters above mean sea level, in double-precision floating-point format. The raster of microtopographic imagery is presented as single-band, 8-bit grayscale imagery. Additionally, the full resolution lidar point cloud used to create the DEM is available in LAZ format.All rasters included with the dataset are presented in geotiff format at 50 cm horizontal resolution. For usability, rasters are also broken into multiple files corresponding to 1 kmBesides the DEM and microtopography, two additional rasters are included with the dataset: a labeled raster of the unique IDs for each polygon, and raw output from the convolutional neural network. These rasters are provided for use with the script for manual validation of results. The raster of unique IDs is presented as a set of single-band, 16-bit, labeled images, in which the pixels representing each identified polygon are assigned a unique integer label. Because some polygons straddle the divisions between adjacent square kilometer regions, each labeled raster contains a 100 m (200 pixel) buffer around the edges. The raster of raw CNN output is presented as a binary image in which pixels recognized as polygon boundaries are labeled “true.”Finally, code and instructions for repeating the procedures for CNN training, polygon delineation, measurement of microtopography, technical validation, and vectorization of polygon boundaries are included in a repository accompanying the main dataset.6 polygons identified in the survey area, 1200 (~0.1%) were selected randomly for this procedure. One at a time, the boundaries of a polygon were projected atop the 8-bit microtopographic imagery and visually inspected. Each polygon was classified as whole, fragmentary, conglomerate, or false. For this analysis, a fragmentary polygon was defined as a machine-delineated polygon including less than 90% of the true area of an ice wedge polygon, as determined by the observer. Conglomerates were defined as polygons comprising portions of multiple ice wedge polygons. This definition includes some ambiguity, as field observations indicate that ice wedge polygons are often subdivided into secondary and tertiary polygons, associated with relatively recent ice wedge cracking30. In general, to maintain consistency, an ice wedge polygon that appeared to contain subdivisions was labeled as conglomerate if any of the interior boundaries, discernible to the inspector, were more prominent than the exterior boundaries of the polygon delineated by the algorithm. Finally, a polygon delineated by the algorithm was classified as false if it occupied terrain in which no ice wedge polygon network was discernible to the observer.Accuracy of the polygonal delineation procedure was determined through manual validation of the results. Of the 1.16 × 10Overall, the manual validation revealed that the delineation algorithm was highly skilled. Using the above criteria, 91% of polygons were classified as whole Table . Among ee.g.6). In this context, we anticipate that future topographic measurements at the survey area may reveal greater coverage by discrete ice wedge polygons than is visible in our datasets from 2012–2014.While completing the validation procedure, we observed rare instances of well-defined polygons (discernible to the inspector) which the algorithm failed to identify. These cases tended to be limited to terrain with unusually subtle microtopographic relief. Specifically, most instances were located within the drained basins of former lakes, some of which were characterized by large, flat polygons demarcated by narrow and shallow troughs. We attribute this mistake to the faintness of the microtopography associated with such polygons, rendering the boundaries difficult for the algorithm to distinguish from other local depressions on the tundra surface. We also note that, among terrain mapped as non-polygonal tundra within our datasets, scattered evidence of ice wedge-related trough development was commonly observed. However, trough segments in the affected regions failed to form a coherent network dividing the surface into discrete polygons. These isolated troughs indicate that: (1) the fraction of ground surface explicitly covered by polygonal microtopography is only a subset of the terrain affected by ice wedges; and (2) HCP formation among previously non-polygonal terrain may be incipient in many zones across the survey area, reflecting a trajectory of land surface evolution that recently has been observed in other landscapes underlain by massive ground ice, where no polygonal microtopography was discernible at the surface only years ago (


F1 fraction (<2 kDa) exhibited highest radical scavenging and α‐glucosidase inhibitory activity. While F2 fraction (2–10 kDa) showed the higher Fe2+ chelating and α‐amylase inhibitory activity, F1 fraction of flavourzyme showed the highest α‐glucosidase inhibitory and F2 fraction of alcalase and flavourzyme exhibited highest α‐amylase inhibitory activity. Hydrolysate and F1 fraction of alcalase and F2 fraction of trypsin showed the highest DPP‐IV inhibitory activity. RP‐HPLC results showed that trypsin hydrolysate had higher levels of high‐hydrophobic peptides. The amino acid composition of the F1 fractions showed high levels of hydrophobic amino acids. Thus, CGPHs may be used as a potential source of antioxidant and antidiabetic peptides in food industry and pharmaceutical application.In the present work, defatted corn germ was hydrolyzed by three proteases and further separated by sequential ultrafiltration with different molecular weight cutoff . Corn germ protein hydrolysate (CGPH) and their fractions were investigated for antioxidant activity, α‐glucosidase, α‐amylase, and DPP‐IV inhibitory activity. The degree of hydrolysis (DH) after 2 hr was 17.5%, 11.14%, and 2.05% for alcalase, trypsin, and flavourzyme, respectively. Trypsin hydrolysate showed the highest DPPH and ABTS Corn germ protein hydrolysate (CGPH) by three proteases and their fractions were investigated for antioxidant activity, α‐glucosidase, α‐amylase, and DPP‐IV inhibitory activity. F1 fraction (<2 kDa) exhibited highest radical scavenging and α‐glucosidase inhibitory activity. Hydrolysate and F1 fraction of alcalase and F2 fraction of trypsin showed the highest DPP‐IV inhibitory activity. Its goes mainly into animal feed, and very low amount has been used as ingredient in food formulations , PAHBAH (4‐hydroxybenzhydrazide), trolox , 1,10‐phenanthroline, acarbose, chromogenic substrate Gly‐Pro‐p‐nitroaniline (Cat no. G0513) were purchased from Sigma‐Aldrich company. l‐Histidine (Cat no. 104351) was purchased from Merck company. Diprotin A (Ile‐Pro‐Ile) was purchased from Cayman chemical company. All other chemicals were of analytical grade.Corn germ was obtained from Glucosan Ind Co. DPPH , ABTS  diammonium salt)), ferrozine (3‐(2‐Pyridyl)‐5,6‐diphenyl‐1,2,4‐triazine‐4′,4′′‐disulfonic acid sodium salt), PNPG (4‐nitrophenyl α‐Bacillus licheniformis), trypsin from porcine pancreas (Cat no. T4799), porcine pancreatic α‐amylase (Cat no. A3176), rat intestinal α‐glucosidase (Cat no. I1630), and dipeptidyl peptidase‐IV (DPP‐IV) (Cat no. 317640‐M) are obtained from Sigma‐Aldrich. Flavourzyme 500 MG from Aspergillus oryzae (Cat no. P6110) was purchased from Novozymes company.Enzymes: Alcalase 2.4 L . The DCG was hydrolyzed according to the method of He, Girgih, Malomo, and Aluko  with alc2.3The pH‐stat method of Adler‐Nissen  was usedtoth of this equation was 7.75 meq/g . CGPHs were dispersed (10 mg/ml) and filtered through 0.2 μm cellulose acetate filters. RP‐HPLC was carried out following the procedure reported by Connolly et al.  with som2.5CGPH was fractionated by the ultrafiltration membranes with the molecular weight cutoff (MW) of 100, 10, and 2 kDa (Sartorius ‐VivaFlow 200) subsequently and namely F1, F2, F3, and F4. F1 corresponds to peptide with molecular weight lower than 2 kDa, F2 to peptide with MW between 2 and 10 kDa, F3 to peptides with MW between 10 and 100 kDa and F4 to undigested proteins and other compounds fragments with MW more than 100 kDa. All fractions were lyophilized and stored at −20°C for using all analysis.2.62.6.1The DPPH radical scavenging effect of CGPH and fractions were measured according to the method of Zheng et al. . The DPP2.6.2+ radical were performed according to the method of Ngoh and Gan (The scavenging effect of CGPH and fractions on ABTS and Gan . The ABT2.6.34 (1.865 mM), and 100 μl of 1,10‐phenanthroline solution (1.865 mM) were mixed thoroughly and were allowed to stand for 10 min and then 100 μl of H2O2 (0.03% v/v) was added to the mixture. The mixture was then incubated at 37°C for 60 min, and after that, the absorbance was measured at 536 nm. Histidine was used as a standard curve . The result was expressed as µmol of histidine equivalents/g dry matter of samples.The hydroxyl radical scavenging effect of CGPH and fractions were measured following the method of Wang et al. . Briefly2.6.42+ ions by hydrolysates and fractions was estimated by the method of Sarteshnizi, Sahari, Gavlighi, Regenstein, and Nikoo (2O and 50 μl FeCl2 (2 mM). After 3 min, 100 μl of 5 mM ferrozine aqueous solution was added and the mixture was allowed to react for 20 min. The absorbance of complex was measured at 562 nm. A standard curve was obtained by using EDTA (20–250 μmol/L). The result was expressed as µmol EDTA equivalents/g dry matter of samples.Chelation of Fend Nikoo . The sam2.6.550 value of acarbose was used as the positive control. The following equation was used to assess percentage inhibition of α‐glucosidase activity:Inhibition of α‐glucosidase  by CGPH hydrolysates and fractions were measured using the method of Connolly et al.  with som2.6.6g to separate the undigested starch. Twenty microliters of supernatant was mixed with 1 ml of PAHBAH and heated to 70°C for 10 min. Finally, solution was cooled at room temperature and absorbance was measured at 410 nm. The IC50 value of acarbose was used as the positive control. The following equation was used to assess percentage inhibition of α‐amylase activity:α‐Amylase inhibition was determined according to the method of Alu'datt et al.  with som2.6.7p‐nitroaniline released was read at 405 nm. The IC50 value of diprotin A was used as positive control. The inhibitory activity of sample on DPP‐IV was calculated by the following equation:DPP‐IV inhibition was determined following the procedure reported by Nongonierma and FitzGerald . Briefly2.7Samples were hydrolyzed with 6 M HCl at 110°C for 24 hr. Subsequently, the digested samples were lyophilized. Amino acid profile of the samples was determined using an OPA method by HPLC (Knauer), RP‐C18 Hypersil ODS column , and fluorometric detector RF‐530 (Shimadzu‐japan) .All analyses were carried out in triplicate and data presented as mean ± 33.1DH of all enzymes treated DCG was increased sharply within the initial 30 min of reaction. Thereafter, the rate of DH was decreased Figure . The red3.2The ratio of hydrophilic to hydrophobic peptide groups is one of most important parameters that can effect on functional properties. Reversed‐phase HPLC was separated peptides based on the hydrophobic or hydrophilic character that resulted in later elution of hydrophobic peptide compared hydrophilic peptides . Hydrolysates prepared using trypsin had the highest ABTS+ RSA with 267 µmol trolox eq/g sample, while flavourzyme hydrolysates showed the lowest values (167 µmol trolox eq/g sample) , while trypsin and alcalase hydrolysates significantly had the lower activity  Figure . The sim) Figure  on highey Figure . Hydrolyy Figure . In geney Figure .+ RSA was also increased after separation with UF membranes. ABTS+ RSA was observed in all fractions. F1 and followed by that F2 fraction showed the higher ABTS+ RSA than hydrolysates. F1 fraction of alcalase exhibited the highest activity against ABTS+ radical (333.84 µmol trolox eq/g) compared with other fractions (p < .05). Similar results were obtained and F1 fraction showed significantly higher (p < .05) OH RSA. However, there was no significant difference between trypsin and flavourzyme  had higher activity than the higher molecular weight. ABTSe Figure . Similare Figure , corn gle Figure , rapeseee Figure , wheat ge Figure , and pere Figure  had showp < .05). Decreasing chelating activity may be attributed to the inability of small peptides to form the complex with metals , and F1 of flavourzyme hydrolysate (the highest OH RSA) is described in Table Type of amino acid present in hydrolysates also could influence antioxidant activity. The amino acid composition of the F1 of trypsin hydrolysate (the highest DPPH and OH RSA), F1 of alcalase hydrolysate (the highest ABTS2+ chelating abilities of peptides from F2 trypsin hydrolysate may return to high content of acidic and basic amino acids of peptides (glutamic acid and glutamine (Glx), aspartic acid, asparagine (Asx), arginine, and lysine) (Table The higher Fee) Table . Ambigaie) Table  reportede) Table .3.450 = 110 μg/ml) was higher than CGPH (20 mg/ml). Since synthetic inhibitors are pure compounds but CGPHs are mixtures of proteins and peptides . However, the least inhibition was related to F2 of trypsin (15.9%). These results indicate that short peptides are more potent inhibiting α‐glucosidase activity. Similar results were obtained by Uraipong and Zhao  on rice Inhibitory activity of peptides may be influenced by the composition of the amino acid residues  could confirm that the interaction of metal ion chelator peptides with calcium ion in the enzyme structure is one of the reasons for its further decrease in enzyme activity by F2 fraction.One of the effective approaches to control of type 2 diabetes is considered via inhibition of α‐amylase. CGPH obtained by all tree enzymes exhibited the strongest α‐amylase inhibitory activity in which higher than 50% Figure . AnotherAmino acids found in peptides such as arginine, lysine, aspartic acid + asparagine, glutamic acid + glutamine, proline, leucine, glycine phenylalanine, serine, tryptophan, and tyrosine likely to bind to active domains and have a potential to inhibit the enzyme (Admassu et al., 3.6Inhibition of DPP‐IV proteinase and enhanced insulin secretion is another approach to lowering postprandial serum glucose. The DPP‐IV inhibitory ability of CGPH and its fractions is shown in Figure 42+ chelating and α‐amylase inhibitory activity, hydrolysate and F1 fraction of alcalase and F2 fraction of trypsin showed the highest DPP‐IV inhibitory activity. The amino acid composition of the F1 fractions showed high levels of hydrophobic amino acids such as valine, isoleucine, leucine, and proline. In general, CGPH could be considered as a natural source for ingredients in the functional food and medicinal industries with antioxidant and antidibetic potential.It was demonstrated that DH and antioxidant and antidiabetic potential of CGPHs can vary depending on the enzymes used. RP‐HPLC chromatograms showed that trypsin hydrolysate had higher levels of high‐hydrophobic peptides, which may be related to its higher antioxidant effect. F1 fraction exhibited highest radical scavenging and α‐glucosidase inhibitory activity. While F2 fraction showed the higher FeThe authors notify that there are no conflicts of interest.Study's protocols and procedures were ethically reviewed and approved by Tarbiat Modares Research Council.


Daldinia fissa) extract showed the highest inhibitory activity against MAO-B, and 5-hydroxy-2-methyl-chroman-4-one (HMC) was isolated as a ~ 4-fold selective inhibitor of MAO-B (IC50 = 3.23 µM) compared to MAO-A (IC50 = 13.97 µM). HMC is a reversible competitive inhibitor with a Ki value of 0.896 µM. No cytotoxicity was observed in normal and cancer cells at 50 µM of HMC. HMC showed blood–brain barrier permeability and high gastrointestinal absorption in silico pharmacokinetics. The docking simulation results showed that the binding affinity of HMC for MAO-B  was higher than that of MAO-A  and that HMC formed a hydrogen bond interaction with Cys172 of MAO-B (distance: 3.656 Å), whereas no hydrogen bonding was predicted with MAO-A. These results suggest that HMC can be considered a candidate for the treatment of neurodegenerative diseases, such as Alzheimer’s disease and Parkinson’s disease.Inhibitory activities against monoamine oxidases (MAOs) and cholinesterases (ChEs) and antioxidant activity were evaluated for 195 extracts from Ukraine-derived endogenous lichen fungi (ELF). Among them, an ELF13 (identified as  Thamnolia vermicularis (Sw.) Schaer. exist in a variety of environments, such as gravel, frost boils tundra, and mossy bushes , one methylene proton [H-3 (δH 2.73)], one methine proton [H-2 (δH 4.55)], and one methyl doublet proton [H-2 (δH 1.49)] (13C NMR and HMBC spectroscopic data displayed one carbonyl carbon [C-4 (δC 199.6)], one oxygenated carbon [C-2 (δC 75.3)], one methylene carbon [C-3 (δC 42.8)], three quaternary carbons , three aromatic carbons , and one methyl carbon [C-2 (δC 21.0)] (C2 showed the peak of m/z 179.2 [M+H]+ (C2 was identified as 5-hydroxy-2-methyl-chroman-4-one (HMC)   suggested that C2 exists as an excess of the (S) enantiomer of 5-hydroxy-2-methyl-chroman-4-one [The H 1.49)] . The 13CC 21.0)] . The LR-2 [M+H]+ . Compounn-4-one) , based oH-7 δH 7., H-8  or HL-60  cells with the treatment of 50 µM HMC .S)-HMC located well at the binding site of HRM complexed with MAO-A and at the binding site of P1B complexed with MAO-B. The AutoDock Vina showed that the binding affinity of the compound for MAO-B  was higher than that of MAO-A , and that the compound could interact with MAO-B by a hydrogen-bond with the Cys172 residue at a distance of 3.656 Å, whereas no hydrogen bond interaction was predicted for MAO-A (R)-enantiomer was analyzed, the binding affinities for MAO-B  and MAO-A  were similar or comparable to (S)-enantiomer (i = 5 or 17 nM) and P1B (Ki = 500 nM) were used for MAO-A and MAO-B, respectively, and their binding scores were calculated to be −8.1 kcal/mol and −8.7 kcal/mol, respectively -HMC for MAO-B and MAO-A were estimated to be 0.971 ± 0.121 and 1.061± 0.172 Å, respectively, and those of (R)-HMC for MAO-B and MAO-A were 0.860 ± 0.097 and 1.017 ± 0.181 Å, respectively.In molecular dynamics, for both MAO-A and MAO-B complexes, the RMSD values increased and reached a stable state after 125 ps. The RMSD values in complexes with MAO-B and HMC enantiomers were lower than those in MAO-A . The aveThe pharmacokinetic analysis of HMC using SwissADME’s web tool showed that it has high gastrointestinal absorption and can cross the blood-brain barrier, and does not inhibit cytochrome P450 . For LipDaldinia fissa forming a symbiotic relationship with the lichen Thamnolia vermicularis (Sw.) Schaer.In this study, the inhibitory activities of 195 ELF extracts from Ukraine-derived lichens against MAO-A, MAO-B, AChE, and BChE were evaluated as well as their antioxidant activities. Among them, several effective extracts were selected: two extracts for MAO-A, five for MAO-B, two for AChE, one for BChE inhibitory activities, and three for antioxidant activity. Among them, ELF13 extract showed the highest inhibitory activity against MAO-B. ELF13 was an endogenous fungus 50 14.3 µM [50 value 4.27 µM, among lichen compounds and their synthetic analogues [50 values of the compounds were higher than that of HMC, which showed strong and ~4-fold selective MAO-B inhibitory activity (IC50 = 3.23 µM).Extracts and compounds of lichens were shown to have effective inhibitory activities against various enzymes, including MAO and AChE in a recent review . However 14.3 µM . A synthnalogues . The inhSaccharomyces cerevisiae growth and the formation of soybean callus [50 = 8.93 µg/mL, 50.16 µM)) [HMC is known to have several biological activities, such as antifungal and phytotoxicity , and an n callus . Melleinn callus , HCV pron callus , hepatitn callus , and humn callus . The pot.16 µM)) .50 = 0.063 µM) is a reversible inhibitor of MAO-B and is used in the treatment of PD and AD. The potency of HMC is lower that of lazabemide; however, it can be served as a lead compound or a scaffold for the development of promising derivatives through the large-scale cultivation of fungi as a natural compound.Lazabemide (ICS)- and (R)-enantiomers at the MAO-B active site were different, few differences in binding affinities were predicted. Collectively, binding affinity of HMC to MAO-B is greater than that of MAO-A, and the affinities of (S)-enantiomer to the enzymes are comparable to those of (R)-enantiomer. Molecular dynamics also supported the experimental data and the docking simulations well. In addition, HMC crosses the blood–brain barrier and shows no violations of Lipinski’s rule of five, and no intracellular toxicity, indicating pharmacological potential. This indicates that HMC can be considered a candidate for the treatment of neurodegenerative diseases.The docking simulations revealed that HMC interacted with the Cys172 of MAO-B to form a hydrogen bond, while no hydrogen bond interaction with MAO-A was predicted. Therefore, hydrogen bond interaction might play an important role in the strong binding and inhibitory activity of HMC against MAO-B. Although the positions and orientations of two (50 = 3.23 µM) with a moderate selectivity over MAO-A (IC50 = 13.97 µM), and is a reversible competitive inhibitor of MAO-B. HMC bound to MAO-B with a binding energy of −7.3 kcal/mol, which was greater than its affinity with MAO-A , indicating that HMC is a more potent and selective inhibitor for MAO-B than MAO-A. In addition, HMC showed pharmacological advantages as it has high gastrointestinal absorption, passes through the blood–brain barrier, and is non-toxic. The results in this study suggest that ELF can be an excellent resource in exploring new pharmaceuticals.HMC, isolated from an ELF extract, showed a strong inhibitory activity against MAO-B (IC


Nucleic acid aptamers show clear promise as diagnostic reagents, as highly specific strands were reported against a large variety of biomarkers. They have appealing benefits in terms of reproducible generation by chemical synthesis, controlled modification with labels and functionalities providing versatile means for detection and oriented immobilization, as along with high biochemical and temperature resistance. Aptamers against immunoglobulin targets—IgA, IgM, IgG and IgE—have a clear niche for diagnostic applications, therefore numerous aptamers have been selected and used in combination with a variety of detection techniques. The aim of this review is to overview and evaluate aptamers selected for the recognition of antibodies, in terms of their design, analytical properties and diagnostic applications. Aptamer candidates showed convincing performance among others to identify stress and upper respiratory tract infection through SIgA detection, for cancer cell recognition using membrane bound IgM, to detect and treat hemolytic transfusion reactions, autoimmune diseases with IgG and detection of IgE for allergy diseases. However, in general, their use still lags significantly behind what their claimed benefits and the plethora of application opportunities would forecast. X-. X-17].  aptamer . The X-rng agent . Since tng agent . The aptng agent . The samng agent . Two metApparently, the highest affinity aptamer selected for Fc domain is an RNA aptamer, called R18, which was found to have an equilibrium dissociation constant of 15 pM . This taβ 1-42 (Aβ 1-42) in direct and sandwich assay formats, respectively, through aptamer labeling of rIgG . This enabled the label-free determination of 6X-histidine and amyloid  of rIgG C 71]. T. Tβ 1-42The nonproteinaceous aptamers enable in a particular, but very relevant example the detection of protein targets by using non-selective protein dyes, i.e., R18 aptamer could be used for sensitive detection of rIgG employing Coomassie Brilliant Blue (CBB) dying followed by optical detection . Apparen−3–10−6 ng of IgG could be determined with high sensitivity, and the method is adaptable to detect proteins, peptides, nucleic acids, toxins and ions with the attachment of the DNA template to the aptamer and making quantitative analysis with PCR . . 125]. T2(dppz)]2+ complex as indicator of the aptamer–IgE binding. The ruthenium complex is able to intercalate in the double stranded region of the aptamer in the absence of IgE. However, in the presence of IgE, the structure of the aptamer changes owing to the target–aptamer interaction, that triggers a luminescent signal change . . 143]. AOne of the main benefits of electrochemical methods is the label-free transduction mechanisms. A relevant example is the use of polyaniline (PANI) nanowires covalently modified with aptamers for label-free, real-time, sensitive IgE detection based on relative conductance changes upon IgE binding. In the presence of IgE the conductance was increased, because the negatively charged IgE was specifically accumulated on the surface of the p-type nanowire blocked previously with BSA, a compulsory step to ensure applicability. However, the variation of the signal response was very considerable , which mElectrochemical impedance spectroscopy (EIS) also ensures label-free detection of IgE. Generally, the charge transfer resistance is measured, which scales with the bound protein, particularly IgE, to the aptamers confined at the electrode surface. The electrodes can be prepared conveniently by photolithography in the form of arrays, as shown by Xu et al. It was found, that in the case of EIS detection, the aptamers have genuine advantage over antibodies in terms of lowered background noise, minimal non-specific adsorption and larger relative signal C 146]. . 146]. SAnother type of electrochemical label-free methodology based on chemically-modified nanopores that could reach single molecule detection capability was introduced by the group of Gu. For this purpose, glass nanopores of ca. 60 nm were modified with aptamer strands that in case of nanoconfinements have clear advantages over antibodies in terms of their small size . Measurip-aminophenyl phosphate (p-APP) conversion to p-aminophenyl (p-AP), an electrochemically active product detected by differential pulse voltammetry (DPV) at the underlying electrode surface (+ on the electrode surface detected by stripping voltammetry (Voltammetric measurements have widely been used for IgE by coupling with (bio)catalytic or redox labels in sandwich assays. Papamichael and coworkers developed an alkaline phosphatase enzyme (ALP)-based sandwich assay, where the antigen of IgE was immobilized onto an electrode surface followed by the binding of IgE, which enabled capping with the biotin-modified aptamer. Extravidin–alkaline phosphatase conjugate was further reacted with biotinylated aptamer, and then the IgE was quantified by enzyme hydrolyzed  surface A 151]. . p-aminotammetry B. The amtammetry C. The naAn interesting example of using a self-reporting format that is apart from the majority of voltammetric methods that use a tracer affinity reagent was introduced by Li et al. An aptamer is used that is linked through a thiol label to a gold electrode while the other terminus is modified with an ALP. In the absence of IgE, the ALP converts 1-naphthyl phosphate (1-NP) to the electroactive naphthol, which is oxidized on the electrode surface. Interestingly, the IgE binding “buries” the enzyme and as such its catalytic activity is suppressed in an IgE concentration-dependent manner. Surprisingly, this relatively simple assay construct led to a remarkably good LOD of 22.7 fM .Such self-reporting approaches, in which the conformational switch of the aptamer triggers a signal, are more common using redox labels (most often methylene blue (MB)) that are approached or distanced from the electrode surface upon the selective target binding and as such turn-on or turn-off the current, respectively . ApparenIt is not required to chemically modify the aptamer with the methylene blue for electrochemical detection, since this cationic dye can bind to the G-rich sequence of the aptamer. This property of MB was used in a sandwich assay-based method, in which the anti-IgE-captured IgEs were capped with aptamer-modified gold nanoparticles. The high probe density on the gold nanoparticles led to an accumulation of the added MB that could be detected by DPV F. ObviouStreptavidin functionalized and AgNP decorated graphene was used as a redox labeling in square wave anodic stripping voltammetry measurements. A sandwich immunoassay was built up for selective and sensitive detection of IgE with 3.6 ng/mL (19 pM) LOD. + 3−/4− and the electrode, thereby the peak current in DPV was significantly dropped and 42 fg/mL (0.22 fM) detection limit was achievable [m-aminobenzoic acid) (PABA) electrochemical deposited on the electrode surface followed by immobilization of the aptamer and the zwitterionic peptide [The development of an antifouling sensing interface to allow for ultrasensitive and specific detection with voltammetry received also attention. Macroporous gold surface was prepared byelectrochemical gold deposition on multilayer polystyrene nanospheres followed by the removal of the nanospheres. Thiol-labeled aptamers and zwitterionic peptides were immobilized to prevent non-specific adsorption owing to its hydrophobicity, neutral charge and a well-defined secondary structure, which ensures closely packed monolayer. IgE prevents the electron transfer between the [Fe(CN)hievable . The mac peptide .Given the large mass change upon binding IgE, capillary electrophoresis-based separation of the aptamer–IgE complex from free aptamers is readily possible. Moreover, the use of fluorescent-labeled aptamers made the detection by laser-induced fluorescence (CE-LIF) especially convenient. Such separations and detections can be carried out in ca. 1 min, with two well separated peaks corresponding to the free and IgE bound forms of fluorescent-labeled aptamer that enabled their quantitfication with five orders of magnitude dynamic range and 46 pM LOD . AspectsMicrofluidic CE devices have also been fabricated since they have several advantages over conventional CE including reduced reagent necessity, minimized cost and analysis time, thereby they show excellent prospects for aptamer-based protein detection coupled with electrophoresis. Wide range of microfluidic devices were used including gel-shift detection platform incorporating competitive inhibition strategy , DNA mobIn this review, the current state of the art in immunoglobulin specific aptamer-based biosensors is compiled. The main uses of the aptamers selected for immunoglobulins are very broad and include: (i) affinity purification; (ii) reporting molecules replacing secondary antibodies; and (iii) selective capturing or detecting reagents. In terms of affinity purification, their high, but easily controllable, affinity makes them suited for the purification of antibodies under mild conditions to preserve the activity of the antibodies.This compilation shows a clear dominance of the aptamer-based detection of IgE as compared with other immunoglobulin targets. This is by far not justified by the diagnostic importance of IgE as compared with other immunoglobulins, but, given the excellent affinity and selectivity of IgE aptamers, by their use as a convenient model to develop new detection concepts and analytical platforms. Many of these detection concepts exploit the intrinsic properties of the nucleic acid aptamers that show appealing advantages and versatility compared to antibody-based reagents and as such they might provide a useful snapshot on the general possibilities in this field. Particularly, the amenability of aptamer reagents for PCR amplification and their negative charge opened new detection routes hardly possible by conventional immunoassays. While aptamers in the present report routinely enabled subnanomolar LODs, the innovative use of their properties led to LODs in the fM range.Despite all their claimed advantages, the use of aptamers as diagnostic reagents for antibody determinations still strikingly lags behind that of the conventionally used reagents. Presently, the standard diagnostic methodologies targeting immunoglobulin biomarkers are largely based on antibodies. Thus, infection-generated IgG and IgM are detected from serum and plasma samples by sandwich ELISA or lateral flow immunoassays; the detection of the extraordinary IgG antibody expression caused by cancer and autoimmune disease is determined by sandwich immunoassays ; and conIndeed, aptamers, beside their many advantages, also have some drawbacks starting from the inherently probabilistic nature of the selection processes, which cannot guarantee that sufficiently high affinity aptamers, fully lacking of cross-reactivity to coexistent species in complex sample matrices, such as whole blood, serum, saliva, etc., can be delivered. The large negative charge density and lack of hydrophobic moieties in the case of natural nucleic acid aptamers are also rather prohibitive in recognizing either negatively charged or hydrophobic immunoglobulin epitopes. In terms of integration in sensor platforms, as their structure is more flexible and as such susceptible to environmental conditions , the immobilization, chemical modification and the medium in which the assay is performed need more consideration than in the case of antibody-based reagents.However, the advances in the field have largely addressed these drawbacks. Most importantly, the implementation of non-natural nucleic acids diversified the range of non-covalent interactions, increased the success rate of the selection  and provided nuclease resistance. The IgA aptamer presented is a relevant example in this respect. The counter-selection emerged also as a powerful tool to custom adjust the selectivity of the aptamers for the application, by removing those sequences that bind to critical interferences and matrix components.Overall, the number of methodologies for selection, control and screening seems up to the challenge of generating aptamer-based diagnostic reagents for antibodies, admittedly at the expense of a more complex and costly selection process. However, this is perhaps a small price to pay given the benefits in terms of their chemical synthesis (solely based on the knowledge of the nt sequence), accordingly batch-to-batch consistency, excellent temperature and biochemical stability ideal for integration in long shelf-live cartridges, and the overall versatility of nucleic acid chemistry in terms of controlled labeling.


Computer-aided navigation and robotic guidance systems have become widespread in their utilization for spine surgery. A recent innovation combines these two advances, which theoretically provides accuracy in spinal screw placement. This study describes the cortical and pedicle screw accuracy for the first 54 cases where navigated robotic assistance was used in a surgical setting. This is a retrospective chart review of the initial 54 patients undergoing spine surgery with pedicle and cortical screws using robotic guidance with navigation. A computed tomography (CT)-based Gertzbein and Robbins System (GRS) was used to classify pedicle screw accuracy. Screw tip, tail, and angulation offsets were measured using image overlay analysis. Screw malposition, reposition, and return to operating room rates were collected. 1 of the first 54 cases was a revision surgery and was excluded from the study. Ten screws were placed without the robot due to surgeon discretion and were excluded for the data analysis of 292 screws. Only 0.68% (2/292) of the robot-assisted screws was repositioned based on surgeon discretion. Based on the GRS CT-based grading, 98.3% (287/292) were graded A or B, 1.0% (3/292) screws were graded C, and only 0.7% (2/292) screws was graded D. The average offset from preoperative plan to actual final placement was 1.9 mm from the tip, 2.3 mm from the tail, and 2.8° of angulation. In the first 53 cases, 292 screws placed with navigated robotic assistance resulted in a high level of accuracy (98.3%), adequate screw offsets from planned trajectory, and zero complications. In spine surgery, cortical and pedicle screws are routinely placed for posterior spinal fixation to stabilize and promote fusion. Originally, screws were inserted via the freehand technique, which presents the risk of misplacement. Misplaced screws are not only biomechanically disadvantageous but also carry an increased risk for neurological deficit, vascular damage, and morbidity –3. AdvanThe demonstration of improved accuracy, operative efficiency, and patient safety is required to establish the validity of this recent robotic technology. The purpose of this study is to describe the cortical and pedicle screw accuracy and screw offset for the first 54 cases in which navigated robotic assistance was used.This retrospective chart review was exempt from the Italian Ethics Committee. Demographic data and preoperative/postoperative computerized tomography (CT) scans of 54 patients who underwent lumbosacral pedicle screw placement with minimally invasive navigated robotic guidance using preoperative CT were analyzed.®; Globus Medical, Inc. Audubon, PA, USA)  Fig.  uses radA CT scan of the operative spinal levels was taken prior to the patient entering the OR, and screw placement planning was completed based on that scan. The CT data set was transferred into the robotic positioning system in preparation for surgery.A dynamic reference marker was inserted into the posterior superior iliac spine, and a surveillance marker was inserted into the iliac crest for registration. The fluoroscopy registration fixture was attached to the C-arm and subsequently activated through a registration marker to ensure the robotic camera could verify its position. Fluoroscopy anteroposterior and lateral images were taken at each operative level to align the preoperative CT with the patient’s actual anatomy. Landmark checks were completed to ensure registration was calculated successfully. A surgeon-controlled foot pedal positioned the robot arm to the most cephalad planned pedicle trajectory. Stab incisions were made on the skin using a scalpel. Pedicle and cortical screws were inserted using navigated instruments guided by the robotic arm. This sequence was repeated until all screws were placed. Based on the surgeons’ discretion, 17 patients underwent a laminectomy and 8 patients underwent a discectomy. Rods were then placed and locking caps were tightened in standard fashion. Intraoperative fluoroscopy images were taken to verify screw and rod position. Screw placement was qualitatively assessed using a postoperative CT scan.A CT-based Gertzbein and Robbins System (GRS)  was usedp < 0.05 for all statistical analysis.Statistical analysis was performed using SPSS Statistics Version 25 software . Data were presented as mean ± standard deviation. Pearson correlation coefficients were calculated for the relationship between body mass index (BMI) and screw offset . Fisher’2 (range 18.8–40.8 kg/m2) , spondylolisthesis (34.6%), or degenerative spondylolisthesis (36.5%). The average age was 49.8 ± 11.3 years (range 23–77 years), and 29% were female. The average body mass index was 25.5 ± 4.0 kg/mA total of 302 screws were placed, including 12 cortical screws and 290 pedicle screws. Ten pedicle screws were placed manually due to surgeon discretion and were excluded from the study for data analysis of 292 screws. Of the 292 screws, 32.9% (96/292) were placed at L5, and 28.1% (82/292) were placed at L4. The mean operative time was 103.3 ± 42.3 min, mean estimated blood loss was 9.72 ± 42.8 cc, and mean radiation time was 17.6 ± 17.4 s (Table n = 12) and pedicle screw (n = 280) GRS grades were similar, resulting in 83.3% A, 16.7% B, 0% C, 0% D, and 0% E for cortical screws, and 85.3% A, 12.9% B, 1.1% C, 0.7% D, and 0% E for pedicle screws. BMI was not correlated with screw tip, tail, or angular offset . Screw offset was divided into two groups with cutoffs of 1.5 mm for tip and tail offset and 2.0° for angular offset. Of the clinically acceptable screws (Grades A and B), 49.8% and 64.8%, respectively, displayed a tip and tail offset greater than 1.5 mm. In comparison, 100% and 100% of inaccurate screws  displayed a tip and tail offset greater than 1.5 mm. For angular offset, 57.1% of clinically acceptable screws (Grades A and B) had an offset of greater than 2° compared to 80% of inaccurate screws  were graded A or B, 1.0% (3/292) screws were graded C, 0.7% (2/292) screws were graded D, and 0% screws were graded E. The GRS grades per level treated are shown in Table Only 0.68% (2/292) of the robot-assisted screws were repositioned intraoperatively based on surgeon discretion. There were no reported adverse events, complications and no returns to the OR for misplaced screws.Pedicle screw pullout strength decreases by up to 71% when the lateral wall is perforated, making accurate screw placement of utmost importance . Robotic®. They found a 2.6 ± 1.5 mm tip offset, 3.3 ± 2.0 mm tail offset, and 5.6 ± 4.3° angular offset that was higher than what was found in the current study. The pedicle screw accuracy rate was 96.6% based on GRS grading, which was consistent with the 98.3% accuracy rate found in this study.Few studies have presented the offset value from planned screw trajectory to final placement. Godzik et al.  reportedIn 2015, Van Dijk et al.  studied Interestingly, results from this study found no correlation between GRS grading and screw offset from planned trajectory, meaning that a screw that deviates off the planned trajectory does not constitute inaccurate screw placement. In other words, despite a deviation from the planned trajectory, pedicle screw accuracy was at 98% based on GRS grading. This emphasizes the importance of surgical proficiency while using the robot. The robot has shown to place screws in the pedicle with high accuracy, but ultimately it is up to the surgeon to plan and execute placement effectively.The current study was not a comparative study, but rather an exploratory study on the effectiveness of navigated robotic assistance in posterior screw placement. Multiple studies have examined the screw accuracy of robot-assisted screws versus freehand screw placement with fluoroscopy guidance and found improved screw accuracy, lower radiation, improved outcomes, and fewer revisions from screw malposition with navigated robotic assistance –22.In a meta-analysis by Fan et al. , robot-aFeng et al.  comparedIn a separate study conducted by Zhang et al. , a robotOther studies report an average fluoroscopic exposure time anywhere from 16.8 s to 3.3 min per pedicle screw fixation case –28. The Although this is a single-surgeon, single-site, retrospective study without comparison to a cohort, the results are consistent with findings from the literature. The method of evaluating screw offset is limited in the subjective nature of manual image overlay; however, the assessor was blinded to treatment. Future studies should report radiation exposure in both dose (mSv) and time to make comparisons with other studies easier. Larger sample sizes are needed to evaluate the effectiveness and cost–benefit analysis of this novel multi-functional robotic navigation system.This navigated robotic guidance system for percutaneous screw placement exhibited adequate screw offsets from planned trajectory, while demonstrating safety and effectiveness through a 0% return to OR rate and a 98% accuracy rate based on GRS grading, respectively. There was no correlation between GRS grading and screw offset in the studied population.