This study highlights the in vitro human monoamine oxidase (hMAO) inhibitory potential and functional effect of those natural flavonoids at dopamine and serotonin receptors for their possible role in neuroprotection. In vitro hMAO inhibition and enzyme kinetics studies were performed using a chemiluminescent assay. The functional effect of three natural flavonoids on dopamine and serotonin receptors was tested via cell-based functional assays followed by a molecular docking simulation to predict interactions between a compound and the binding site of the target protein. A forced swimming test was performed in the male C57BL/6 mouse model. Results of in vitro chemiluminescent assays and enzyme kinetics depicted 1 as a competitive inhibitor of hMAO-A with promising potency  and 3 as a competitive inhibitor of hMAO-B with an IC50 value of 7.19 ± 0.32 μM. Likewise, GPCR functional assays in transfected cells showed 1 as a good hD4R antagonist. In docking analysis, these active flavonoids interacted with a determinant-interacting residue via hydrophilic and hydrophobic interactions, with low docking scores comparable to reference ligands. The post-oral administration of 1 to male C57BL/6 mice did not reduce the immobility time in the forced swimming test. The results of this study suggest that 1 and 3 may serve as effective regulators of the aminergic system via hMAO inhibition and the hD4R antagonist effect, respectively, for neuroprotection. The route of administration should be considered.Natural flavone and isoflavone analogs such as 3′,4′,7-trihydroxyflavone ( According to the United States Centers for Disease Control and Prevention, more than 200 million people, equivalent to roughly 60% of the population, suffer from at least one neurological disorder in the USA. Neurological disorders vary in severity and range from tension-type headaches and anxiety to strokes and dementia. Among these 200 million, more than 3.4 million have epilepsy, 6.2 million have Alzheimer’s or related dementias, and 40 million have anxiety disorders. Furthermore, major depressive disorder is the leading cause of disability for people between 15 and 44, and 19% of adults reported experiencing mental illness in 2019. The causes of these diseases vary, and in some cases, they are not completely known because they are multifactorial.Consequently, treatment is very challenging. The rate at which people are seeking treatment for mental health issues has increased as well. In 2020, 93% more people took a screening test for anxiety disorders than in 2019, and there was a 62% increase in depression screenings for the same period [N-acetyltransferase (AANAT) to N-acetyl serotonin (NAS) and then by N-acetylserotonin O-methyltransferase (ASMT) to form melatonin. 5-HT is also metabolized by monoamine oxidase (MAO) to form 5-hydroxyindoleacetic acid (5-HIAA), which is the main metabolite of 5-HT [+ by quinolinate phosphoribosyl transferase (QPRT) [Tryptophan (TRP) is the most prevalent amino acid, which plays a significant role in protein biosynthesis, and its metabolites are implicated in central nervous system (CNS) disorders ,3. This  of 5-HT . Similare (QPRT) . These te (QPRT) ,9.TRP and its metabolites play an important role in alleviating various diseases ranging from psychiatric/neurological disorders to cancer . This diDifferent classes of antidepressant drugs are available on the market for the treatment of depression with different mechanisms of action, including selective serotonin and norepinephrine reuptake inhibitors , tricyclic antidepressants , serotonin reuptake inhibitors , and monoamine oxidase inhibitors (MAOIs)  . HoweverBrain neurotransmitter levels are regulated by MAO-A (serotonin and norepinephrine) and MAO-B (phenethylamine) or both (dopamine) as preferred substrates . TranylcPueraria lobata [Natural flavonoids have a long history of medical use for the treatment of various medical ailments, including inflammation , cancer a lobata ,27. Thesa lobata ,29, antia lobata , anti-dia lobata ,32, antia lobata ,34, antia lobata , anti-ina lobata , neuropra lobata ,37 and aa lobata . Modern a lobata ,40. What4R antagonists for the management of neurodegenerative diseases, particularly depression. The overall objectives, which are the next step toward the attainment of our long-term goal, are to (i) elucidate the mechanism(s) of hMAO inhibition via in vitro enzyme assays and in silico molecular docking; (ii) evaluate modulating effects on dopamine (DA) and serotonin (5-HT) receptors via GPCR-functional assays and in silico molecular docking; and (iii) determine their in vivo anti-depressant efficacy using depression-like animal models. Our central hypothesis is that natural flavonoids with in vitro hMAO inhibition and hD4R antagonism have in vivo anti-depressant effects. Therefore, to attain the overall objectives and test the hypothesis, we emphasized the “One-compound multiple-targets paradigm” and evaluated the role of three naturally occurring flavonoids, namely, 3′,4′,7-trihydroxyflavone (1), 3′,4′,7-trihydroxyisoflavone (2), and calycosin (3), in hMAO inhibition and GPCR modulation, followed by an in vivo anti-depressant effect in depression-like animal models.Our long-term goal is to discover natural flavonoids as hMAO inhibitors and hD1–3  showed the most promising inhibition of hMAO-A with an IC50 value of 7.57 ± 0.14 µM.The human monoamine oxidase inhibitory activity of the naturally occurring flavonoids 1–3  was test3) and 3′,4′,7-trihydroxyisoflavone (2) showed moderate inhibition of hMAO-A with IC50 values of 113.78 ± 3.39 and 176.79 ± 7.80 µM, respectively. The human MAO-A-specific reference inhibitor clorgyline-HCl had an IC50 value of 0.02 ± 0.00 µM.Calycosin (3) was the most potent among the tested flavonoids, with an IC50 value of 7.19 ± 0.32 µM. Flavonoid 2 showed moderate hMAO-B inhibition with an IC50 value of approx. 70 µM. Interestingly, flavonoid 1 was much less active at hMAO-B inhibition, depicting its selectivity towards hMAO-A. The hMAO-B selective reference inhibitor safinamide mesylate showed an IC50 value of 0.23 ± 0.01 µM.Likewise, for hMAO-B inhibition, calycosin , a kinetic study was conducted in a similar way to enzyme inhibition but at different substrate concentrations  and hMAO-B (flavonoid 3) with Ki values of 2.03 and 2.56 µM, respectively.To better understand the enzyme inhibition mode of the most potent inhibitors . Flavonoids 1 and 2 formed H-bond interactions with Asn181 and Phe208. Additionally, H-bond interactions with Gln215 and Gly443 were also observed for flavonoid 2−hMAO-A binding. Likewise, interactions with FAD and Phe352 (Pi–Pi T-shaped), Tyr407 and Tyr444 (Pi–Pi Stacked), Ile335 and Leu337 (Pi–Alkyl) were commonly observed for 1 and 2 binding with the enzyme , followed by 2 . Those test ligands formed H-bond interactions with Cys172 and Ile199. In addition to this, hydrophobic interactions with FAD and tyrosine residues  were observed for 2, 3, and safinamide binding.As tabulated in 1–3 were first screened for functional effect over human dopamine receptors  and serotonin 5HT1AR at a 100 μM concentration. The screening result is tabulated in 1 showed significant functional effects at the test concentration on hD4.4R. Flavonoid 1 inhibited the response of 100 µM of dopamine by 134.47 ± 25.54%. Based on the screening result, the concentration-dependent response was tested for flavonoid 1. As shown in 1 inhibited the response of the control agonist by 40.9, 57.65, 68.95, and 134.47% at 12.5, 25, 50, and 100 µM, respectively, yielding an IC50 value of 22.47 ± 2.18 µM.Test compounds 1−hD4.4R binding was predicted via molecular docking simulation using AutoDock 4.2  with a low binding score  that was comparable to the reference ligand clorgyline , involving H-bond interactions with Val193 (helix V), Leu187 (ECL2), Ser197, and Ser196 (helix V) and hydrophobic interactions with His414 (helix VI), Leu187 (ECL2), Val193 (helix V), Arg186, and Val116 (helix III).The underlying mechanism of flavonoid Dock 4.2  and compDock 4.2 . Overall1 was tested at a dose of 3, 10, and 30 mg/kg p.o., and none of those doses alleviated the immobility time. Only desipramine at a 15 mg/kg dose p.o. showed a significant reduction in immobility time. Despite being a potent hMAO inhibitor  are the mammalian flavoproteins that play important roles in the deactivation of biological amines such as dopamine, serotonin, norepinephrine, epinephrine, phenylethylamine, benzylamine, melatonin, tyramine, and tryptamine . Catalyt4 and vasopressin V1A receptor antagonists. Additionally, they found that it is an MAO-A inhibitor with an IC50 value of 8.57 ± 0.47 µM [1AR antagonist and a D2L and D4 agonist [1) decreases NO production and exerts an anti-neuroinflammatory effect through the inhibition of the JNK-STAT1 pathway in microglia [1 protects the neuronal cells from H2O2-induced oxidative stress and cytotoxicity [3), a 3′,7-dihydroxy-4′methoxyisoflavone, has exhibited neuroprotective effects mediated through its anti-oxidant and anti-inflammatory actions [3 attenuates the MPTP-induced inflammatory response and alleviates dyskinesia and sensory disturbances in MPTP-induced PD mice, thus suggesting its potential use against PD [1 and 3, along with 2, an isoflavone analog of 1, against hMAO, since the hMAO inhibition potential of 1 and 2 is yet to be explored. Given the evidence that the flavonoids can modulate dopamine and serotonin receptors [Natural products based on flavonoids–scaffolds possess different biological and pharmacological properties such as antioxidant, anti-inflammatory, neuroprotective, acetylcholinesterase (AChE), and butyrylcholinesterase (BChE) inhibition, anti-Aβ fibril formation, β-secretase, and MAO inhibition . Also, r 0.47 µM . Likewisicroglia . Furthertoxicity . From di actions ,54,55. Aainst PD . Coumestainst PD , and amyainst PD . Our preainst PD ,61. Theseceptors , we used1 showed significant inhibition of hMAO-A with an IC50 value of 7.57 ± 0.14 µM. While the isoflavone analogs 2 and 3 showed only a modest inhibition against hMAO-A. However, in the case of the hMAO-B inhibition assay, 3 displayed a notable inhibition with an IC50 value of 7.19 ± 0.32 µM. Compound 2 showed moderate inhibition of hMAO-A with IC50 values of 71.23 ± 0.06 µM. From a structural point of view, calycosin (3) is an O-methylated isoflavone, and 2 is its structural analog. The structural difference between 2 and 3 is that the 4′-methoxy group in 3 is replaced with a hydroxy group in 2. Both showed mild inhibition of hMAO-A, but the effect is good at hMAO-B. Interestingly, 3 showed a ten-times more potent effect (IC50: 7.19 ± 0.32 µM) than 2 (IC50: 71.23 ± 0.06 µM). Comparing the results, we can assume that the 4′-methyl substitution in the isoflavone ring increases the hMAO-B inhibition effect. MAO inhibition is strongly dependent on the presence of a (p-OH-substituted) phenyl at C2, unsaturation at the C2–C3 positions of the structure, the possibility of establishing hydrophobic interactions, and ring planarity [3 showed selective hMAO-B inhibition [50 values vary, which may be due to different experimental conditions. The enzyme kinetic study of 1 and 3 on hMAO-A and hMAO-B revealed the competitive inhibition mode. Interactions with this aromatic cage-forming covalent FAD coenzyme and tyrosine residues of the active site of hMAO-A are considered important for catalytic activity [1 and 2 have many common hydrophobic interactions with hMAO-A active site residues such as FAD, Tyr407, Ile335, and Ile180; however, additional interaction with Phe208 via Pi–Pi T-shaped bond might be responsible for its specificity towards hMAO-A and higher potency. Phe208 and Ile335 are critical residues for inhibitor selectivity [4.4R antagonism by 1, we conducted the forced swimming test to evaluate its anti-depressive activities. Unfortunately, we did not observe any positive effects  plays important roles in cognition, attention, and decision making, and pharmacological activation of D4R could be useful in treating cognitive deficits associated with schizophrenia [4R antagonism may be useful in treating substance use disorder [In the GPCR functional assay, only ophrenia ,70,71,72ophrenia . While Ddisorder  and L-DOdisorder .4Rs are found in the basal ganglia region, which is a key region for dyskinesia [4R antagonists could fill the gap in LID attenuation in PD patients [4R antagonists selectively inhibit the growth of glioblastoma neural stem cells [4R-selective ligands have promise for developing medications for neuropsychiatric conditions. This study highlights the antagonistic effect of 1 on D4R. To our knowledge, this is the first study to report this specific effect of this compound.The etiology of LID is unknown. Previous studies have suggested that LID may be associated with L-DOPA-induced increases in synaptic DA levels in the caudate putamen ,77. In askinesia . So D4R patients . Likewisem cells . TherefoOur study highlights the in vitro human monoamine oxidase inhibition and modulating effect on dopamine (DA) and serotonin (5-HT) receptors via GPCR-functional assays and predicts the binding mode. In silico molecular dynamics studies predicting the stability of ligand–receptor complexes are lacking. Penetration into the central nervous system and the stability of the ligand–receptor complex remain to be studied in vivo. The small number of test compounds limited the structure–activity relationship. The effects of the tested flavonoids at the cellular or organismal level remain unknown because in silico modeling cannot account for interactions between compounds and other unrelated targets. Also, other routes of administration should be considered, as the oral route might not be effective for anti-depressant effects.4.4R signaling and regulation, especially using in vivo models, will be critical to ensuring the activity of these natural flavonoids, particularly 1 in neurogenerative diseases. The anti-depressant effect of 1 should be evaluated from different routes of administration to conclude our hypothesis—natural flavonoids with in vitro hMAO inhibition and hD4.4R antagonism have in vivo anti-depressant effects.In the future, a more detailed understanding of hD1 and 2 were obtained from Indofine Chemical Company, Inc. . Flavonoid 3 was isolated from the n-hexane fraction of Pueraria lobata root. The purity of the isolated compounds was considered to be >98%, as evidenced by spectral data [Flavonoids ral data . An MAO-50 value. The percent of inhibition (%) was obtained with the following equation: % Inhibition = c(A − s)/AcA × 100, where cA is the absorbance of the control and sA is the absorbance of the sample. For hMAO-A, clorgyline was used as a positive control. Whereas for hMAO-B, safinamide mesylate was a reference compound. l-Deprenyl HCl was also used as a positive control for both hMAO-A and hMAO-B inhibition assays.A chemiluminescent assay was performed in a white, opaque 96-well plate using the MAO-Glo kit  to evaluate human monoamine oxidase (hMAO) inhibitory potential. Detailed experimental conditions and procedures were reported previously ,82. The The kinetic analysis of hMAO inhibition was analyzed at different concentrations of hMAO substrate depending on the isozyme  following the same method of enzyme inhibition. The concentrations of the test compounds for the kinetic study are presented in 2+ ion mobilization, depending on the receptor type. All assays were performed at Eurofins Cerep  following their in-house protocol, as stated in our previous reports [Cell-based functional GPCR assays were conducted in CHO cells and Ba/F3 cells transfected with a plasmid containing the GPCR gene of interest. The functional activity of the test compounds (agonist or antagonist) was evaluated by measuring their effects on cAMP modulation or Ca reports ,84,85.1R, D3R, and D4R was assessed by evaluating the effect on cAMP modulation. For this, stable transfectants  were suspended in HBSS  containing 20 mM of HEPES buffer and 500 μM of 3-isobutyl-1-methylxanthine, distributed into microplates (5 × 103 cells/well), and incubated for 30 min at room temperature (RT) in the absence (control) or presence of the test compounds  or reference agonist (DA). In the D3R and D4R assays, the adenylyl cyclase activator NKH 477 was added at a final concentration of 1.5 and 0.7 μM and incubated for 30 and 10 min, respectively, at 37 °C. Then, the cells were lysed, and a fluorescence acceptor (D2-labeled cAMP) and fluorescence donor (an anti-cAMP antibody with europium cryptate) were added. The fluorescence transfer was measured at λex = 337 nm and λem = 620 and 665 nm using a microplate reader  after 60 min of incubation at RT. Agonist effects are expressed as the % of the control response to 10 μM of DA for D1R and 300 nM of DA for D3R/D4R. Similarly, antagonist effects are expressed as the % inhibition of the control response to DA at 300 nM for D1R, 10 nM for D3R, and 100 nM for D4R. The reference agonist DA and antagonists SCH 23390, (+)-butaclamol, and clozapine were used to validate the study.The functional activity of the test compounds on D2R and 5HT1AR was tested by fluorimetrically evaluating their effect on cytosolic Ca2+ ion mobilization. In brief, CHO-D2R and Ba/F3-5HT1AR cells were separately suspended in HBSS  complemented with 20 mM HEPES buffer and distributed into microplates (1 × 105 cells/well). Then, a fluorescent probe  mixed with probenecid in HBSS  supplemented with 20 M HEPES (Invitrogen) (pH 7.4) was added to each well, and the cells were allowed to equilibrate for 60 min at 37 °C. Thereafter, the plates were positioned in a microplate reader , and compounds 1–3 , reference agonist, or HBSS  were added. We then measured the fluorescent intensity, which varied in proportion to the free cytosolic Ca2+ ion concentration. Agonist effects are expressed as the % of the control response to 10 μM DA for D2R and 2.5 μM serotonin for 5HT1AR. Similarly, antagonist effects are expressed as the % inhibition of the control response to 700 nM DA for D2R and 30 nM serotonin for 5HT1AR. Reference agonists (DA and serotonin) and antagonists (butaclamol and (S)-WAY-100635) were used to validate the study.The functional activity of the test compounds on D1–3, respectively). The crystal structures of the reference compounds safinamide, harmine, nemonapride, and clorgyline were also obtained from NCBI under CIDs 131,682, 5,280,953, 156,333, and 4380, respectively. Water and ligand molecules were removed using Discovery Studio . In the case of the human MAO isozymes, the co-factor flavin adenine dinucleotide (FAD) was retained. X-ray crystallographic structures with PBD IDs 5WIU (the best resolution structure of hD4R) were used for the hD4 receptor [Automated single docking simulations were carried out with AutoDock 4.2 . X-ray creceptor . The LamMale C57BL/6 mice  were purchased from Orient Bio Inc. , a branch of Charles River Laboratories , and kept in the University Animal Care Unit for 1 week before the experiments. Five animals were housed per cage and allowed access to water and food ad libitum; the environment was maintained at a constant temperature (23 ± 1 °C) and humidity (60 ± 10%) under a 12 h light/dark cycle (the lights were on from 07:30 to 19:30 h). The treatment and maintenance of the animals were carried out according to the Animal Care and Use Guidelines of Dong-A University, Republic of Korea. All in vivo experiments were performed according to the protocols approved by the Institutional Animal Care and Use Committee of Dong-A University (approved protocol numbers: DIACUC-approved-17-20) and were per the National Institutes of Health guidelines.A forced swim test (FST) was performed in a transparent cylindrical glass cylinder with a diameter of 14 cm and a height of 25 cm filled with 20 cm of water (24 ± 2 °C). Mice were habituated to the experimental environment 1 h before the test. The 3′,4′,7-trihydroxyflavone  or the same volume of vehicle was administered 1 h before FST. As a positive control, desipramine  was administered 30 min before FST. Mice were immersed in water and recorded with a video camera for a total of 6 min. Immobility time was analyzed for the last 4 min of the 6 min using a video-based Ethovision System .t-test using Microsoft Excel 2016 . All in vitro experiments were carried out in triplicate on three individual days and are expressed as the mean ± standard deviation (SD). Differences were considered statistically significant at a p-value < 0.01 compared to the vehicle-treated control group.Statistical analysis was performed with One-way ANOVA and Student’s 1–3. Little is known about the pharmacological importance of these flavonoids in regulating GCPR. GPCR functional screening revealed that 1 possesses hD4.4R antagonist properties. Likewise, 1 showed promising inhibition of hMAO-A, and 3 was the most potent inhibitor of hMAO-B. Due to the multifactorial complexities associated with NDDs such as AD and PD, molecules with multitargeting properties and minimal toxicity hold promise as potential therapeutic approaches. Therefore, the versatility of 1 may help target different root causes of NDD and improve related symptoms. A recent study confirmed that intravenous injections of cannabidiol can induce long-term antidepressant-like effects [1 in the male C57BL/6 mice model at a dose of up to 30 mg/kg did not show an antidepressant effect. So, the effect should be evaluated at different routes of administration or at higher doses following p.o. administration. Further, in vivo studies are needed to support the regulatory role of DAR in vitro and to observe the efficacy of 1 in alleviating DAergic neurodegeneration and disease conditions in animal models.Our study is the first to assess the dopamine (DA) and serotonin (5-HT) receptors modulating activity and hMAO inhibitory potency of natural flavonoids  effects . The fin


Recent neurosurgical applications based on artificial intelligence (AI) have demonstrated its potential in surgical planning and anatomical measurement. We aimed to evaluate the performance of an AI planning software application on screw length/diameter selection and insertion accuracy in comparison with freehand surgery.A total of 45 patients with 208 pedicle screw placements on thoracolumbar segments were included in this analysis. The novel AI planning software was developed based on a deep learning model. AI-based pedicle screw placements were selected on the basis of preoperative computed tomography (CT) data, and freehand surgery screw placements were observed based on postoperative CT data. The performance of AI pedicle screw placements was evaluated on the components of screw length, diameter, and Gertzbein grade in comparison with the results achieved by freehand surgery.Among 208 pedicle screw placements, the average screw length/diameters selected by the AI model and used in freehand surgery were 48.65 ± 5.99 mm/7.39 ± 0.42 mm and 44.78 ± 2.99 mm/6.1 ± 0.27 mm, respectively. Among AI screw placements, 85.1% were classified as Gertzbein Grade A ; among free-hand surgery placements, 64.9% were classified as Gertzbein Grade A.The novel AI planning software application could provide an accessible and safe pedicle screw placement strategy in comparison with traditional freehand pedicle screw placement strategies. The choices of pedicle screw dimensional parameters made by the model, including length and diameter, may provide potential inspiration for real clinical discretion. Strategies for pedicle screw placement mainly focus on two components: screw dimensions and screw trajectory. For screw trajectory, there are many guidelines, including empirical guidance and radiological instruction. Specific considerations include entry point, parallelization between the screw and superior endplate, mediolateral inclination, and suitable insertion depth –3. RegarRegarding screw dimensions, published papers and reviews have mainly focused on the anatomical dimension of the pedicle; suggestions are variable, depending on specific surgical considerations . For thoRecent neurosurgical applications based on artificial intelligence (AI) have demonstrated their potential in surgical planning and anatomical measurement. Siemionow et al. trained an automatic pedicle screw placement model based on vertebral anatomical features using a neural network . VijayanIn this investigation, we present and evaluate the performance of an AI planning software application in screw length/diameter selection and insertion accuracy in comparison with freehand surgery. Our research hypothesis was that the preoperative AI-based surgical planning software could provide better screw dimensions (with longer lengths and wider diameters being selected), with greater insertion accuracy than that achieved in freehand surgery.Patients who underwent freehand thoracolumbar internal fixation surgeries were included in this investigation. All enrolled patients had lumbar degenerative diseases or thoracic kyphosis. All surgeries were completed by the same surgical team between June and December 2022 in the neurosurgery department at Xuanwu Hospital. The exclusion criteria were as follows: (1) patients with bone tumor, ankylosing spondylitis, diffuse idiopathic skeletal hyperostosis, rheumatoid arthritis, tuberculosis, or secondary osteoporosis; (2) patients undergoing reoperation after initial internal fixation due to postoperative complications; and (3) patients with severe scoliosis before surgery (Cobb angle >45°).Preoperative computed tomography (CT) images of each patient were generally obtained during the 2 days before surgery, while postoperative CT images were normally collected within 3 days after surgery. All CT images were generated in Digital Imaging and Communications in Medicine (DICOM) format using Revolution CT from GE MEDICAL SYSTEMS. The slice thickness of the CT images was 0.625 mm.A novel deep learning model, based on a self-developed 3D-Unet algorithm, was utilized for preoperative pedicle screw planning. The primary version of this model has been published in a previous paper, which mainly focused on comparing the pullout force of the AO insertion method and that of the AI model. In the present study, the aforementioned self-developed screw planning AI model  was incoRegarding screw trajectories, the default screw trajectories were those with the highest BMD and POF. After the default planning calculation was performed, the outcome was displayed in an interactive user interface for further analysis, such as evaluation of insertion accuracy. The screw dimensions used in real freehand surgeries were determined by the surgeons based on experience. Surgeons selected the optimal screw based on simple observation and measurements provided on preoperative CT images; the reference guidelines were the AO spine surgery reference.The insertion accuracy of the results of the AI-based planning and freehand surgeries was evaluated using the Gertzbein–Robbins (GR) classification, consisting of grades A–E: grade A means there is no pedicle breach; grade B means the pedicle breach depth is <2 mm; and grades C, D, and E mean the pedicle breach depth is <4, <6 mm, and >6 mm, respectively. The insertion accuracy of freehand surgical results was evaluated using the Radiant DICOM viewer software package, specifically its three-dimensional multiplanar reconstruction mode. By adjusting the viewing direction of CT planes, screw-axis cross-sectional planes could be displayed for GR classification. The insertion accuracy of the AI-based planning results was directly evaluated using the Surgiplan AI software application, as the screw-axis cross-sectional mode could easily be switched on or off. t-tests were conducted to compare screw length and diameter between the AI model group and the freehand group for both the left and right sides, with p < 0.05 taken to represent a statistically significant difference. Fisher's exact test was conducted to compare GR grades between the AI model group and the freehand group, with p < 0.0001 was taken to represent a significant difference.Statistical analysis was carried out using the GraphPad PRISM software package. Paired t-tests showed that there was a significant difference between the freehand group and the AI group on both length and diameter .A total of 208 screw placements across 45 patients  were examined in this study. Most segments for internal fixation were L4 and L5, covering a total of 79 of the 104 segments . On bothp < 0.0001).Regarding insertion accuracy, among the 208 screw insertions proposed by the AI model, 177 screw placements (85.10%) were classified as GR grade A ; and of all placements made in freehand surgeries, 135 (64.9%) were classified as GR grade A. Only four placements made by the AI model were classified as GR grade C, and the corresponding number for the freehand surgeries was 15. There were no screw placements with GR grades D or E among the AI model placements, but two screw placements with GR grade D were identified in the freehand surgery placements . Table 4Longer and thicker screws have been proven to strengthen the pullout force ; therefoIn the present study, the AI model demonstrated better performance on screw length and diameter selection in comparison with selection based on freehand experience. The average planning time requirement for a single CT DICOM image was just 1–3 min; considering a surgeon's daily workload, this time cost was regarded as acceptable. The benefit becomes more meaningful in consideration of the fact that preoperative screw selection time is approximately 5–10 min when selection is performed manually. Additionally, the AI-based screw placements were validated as having better insertion accuracy was better than that achieved in freehand postoperative results, which means that the screw dimensional parameters determined using the AI model have a guarantee of safety. Furthermore, real-time monitoring of screw insertion accuracy during preoperative planning played a significant role, as doctors could confirm whether the screw selected by the AI model caused a vertebral breach or not, especially using the screw-axis reconstruction plane and three-dimensional view. The AI results should be viewed as a providing a rapid reference for determination of screw length and diameter determination. The model is not perfect; in our investigation, approximately 15% of the results still involved pedicle breach, and surgeons would still need to further confirm the validity of the determination and perform manual modifications where necessary.The benefit of AI preoperative planning of screw length and diameter was that it supplied doctors with an upper limit for anti-loosening performance with regard to screw dimensional strategy. The AI plan may not be the same as what is finally implanted during the internal fixation, but it provides reasonable support for the selection of longer or thicker screws in comparison with those selected based on experience or using simple measurements. The insertion accuracy achieved by the AI model, on the other hand, solidified its validity, as pedicle breach is a common and conventionally used clinical standard. The AI software application was found to represent a practical and transferable advancement, as it improved the prognosis of internal fixation through a highly efficient and user-friendly method. With regard to intraoperative implementation, several possible methodologies, including surgical guides and surgical robots, have been published. For surgical guides, one previous study has proved that the average horizontal deviation between the preoperative plan and the postoperative result could be smaller than 1 mm . For surIn comparison with similar AI models presented in existing papers, the results of our model, in terms of the selection of longer and thicker screws, were unique and represent a significant advancement. Most existing models have only focused on automatic screw placement based on a doctor's experience with screw trajectory and vertebral anatomy , 19. Then = 30/45); some previous papers have indicated that there are differences between male and female spinal pedicle widths and other spinal topological features (There were still some limitations to this investigation. First, most of the patients included were female (features . In addiThe novel AI planning software described here could provide an accessible and safe pedicle screw placement strategy in comparison with traditional freehand pedicle screw placement strategies. The choices of pedicle screw dimensional parameters made by the model, including length and diameter, may provide potential inspiration for real clinical discretion.


Raphidonema (Trebouxiophyceae) was distributed over both poles and mid-latitude regions and was detected in different ice core layers, corresponding to distinct time periods. Our results indicate that the modern cosmopolitan phylotypes belonging to Raphidonema were persistently present long before the last glacial period. Furthermore, endemic phylotypes originated from ancestral cosmopolitan phylotypes, suggesting that modern regional diversity of snow algae in the cryosphere is a product of microevolution. These findings suggest that the cosmopolitans dispersed across the world and then derived new localized endemics, which thus improves our understanding of microbial community formation by microevolution in natural environments.Recent studies of microbial biogeography have revealed the global distribution of cosmopolitans and dispersal of regional endemics, but little is known about how these processes are affected by microbial evolution. Here, we compared DNA sequences from snow/glacier algae found in an 8000-year-old ice from a glacier in central Asia with those from modern snow samples collected at 34 snow samples from globally distributed sites at the poles and mid-latitudes, to determine the evolutionary relationship between cosmopolitan and endemic phylotypes of snow algae. We further applied a coalescent theory–based demographic model to the DNA sequences. We found that the genus  Global microbial colonization, which gives rise to cosmopolitan species, has been a topic of great interest since 1934, when Baas Becking hypothesized that “Everything is everywhere, but the environment selects” –5. MoreoOur knowledge on the time scale of development of microbial population structures, and how they have been preserved is very limited. Because ice-core samples from glaciers provide a time series of past genome information , analysiTo overcome this shortcoming, we here analyze ancient DNA information derived from an ice-core that dates continuously back to 8000 years before the present, and analyze a comprehensive global modern dataset of snow algae collected from both poles and mid-latitudes Tables –S2. The The ice core was drilled at the top of the Grigoriev Ice Cap in the Tien Shan Mountains in Kyrgyz Republic in August, 2007 Fig. . The iceThe 19 ice-core samples were melted using a device developed by our group that enabled us to obtain water from only the inner portion of the core; the 6-mm-thick outer cylindrical layer remained intact . CompletTo determine whether any contamination had occurred during handling or due to ice-core cracks, a solution of ~2 ng/µl bacterial plasmid vector  was applied to the ice-core surface for 20 h before sampling of the inner portion. The vector contaminant was not amplified from the inner part of ice-core samples, but amplified only for the outer layer of ice-core samples as assessed by 45 cycles of PCR with vector-specific primers (M13-Forward and M13-Reverse primers).Snow samples were collected into sterile 50 ml plastic conical tubes during the melt season from 10 sites on 5 glaciers or snowpacks in No. 31 Glacier in Suntar-Khayata Mountains in Russia; Glacier No. 1 in the Eastern Tien Shan in China; the Grigoriev Ice cap at the Inner Tien Shan in Kyrgyz Republic; Fedchenko Glacier in Central Pamir in Republic of Tajikistan; and Tateyama Mountains in Japan Table . Samplesg for 2 min, and the aqueous phase was collected and transferred into a separate microtube for incubation at 2 °C for at least 1 h. Further purification steps were the same as described by Orlando et al. . If the standard deviation was estimated to be a tiny value, it was omitted. The maximum likelihood estimate provides estimates of t0, N0, and N1. To derive estimates of N0, N1, and t0 in years, the mutation rate and the generation time are needed. Ness et al. [Chlamydomonas reinhardtii to be 2.08 × 10−10 (95% CI: 1.09 × 10–10 to 3.74 × 10–10) per site per generation based on a mutation accumulation experiment followed by whole-genome resequencing of two replicate lines. Because the ITS sequence has 381 base pairs, we assumed the mutation rate to be u = 2.08 × 10−10 × 381 = 7.9 × 10−8 per generation. The generation time of an alga corresponds to the doubling time of algal population size. On the Greenland Ice Sheet, doubling times have been estimated as 3.75–5.5 days [The maximum likelihood estimates of τ, s et al.  estimate5.5 days , while O5.5 days  estimateWe used high-throughput sequencing to obtain DNA sequences of algae from 19 layers of an ice core drilled on a glacier in central Asia, dated from present time to 8000 years ago Fig. . In totaChloromonadinia clade, 1 subgroup of the Monadinia clade  , 3 subgmes, see ). Althouhe world , 57, werRaphidonema group and ‘Chlamydomonas’-snow group B (Sanguina) are the cosmopolitans at both poles [Raphidonema group, we further analyzed the ITS2 sequences obtained from the ice core sample as well as the glacier-surface samples from both poles [Raphidonema group were detected in the older (deep core) layers of the ice core and at the glacier surface of central Asia , accounting for 40% of the total sequencing reads   Fig. –S10. ThiRaphidonema group, because a previous study analyzed their distribution only at the poles [Raphidonema group obtained from each region and the ice core were similar between the USEARCH and DADA2 analyses  and the older (6000–8000 years before present) layers  based on phylogenetic analysis  was assumed to be 7.9 × 10–8/ sequence/generation, and the generation interval was assumed to be 24 days . In Group A, for the cosmopolitans, the estimates of t, N0, and N1 were t, N0, and N1 of other phylotypes and other groups  generally produce zygotes or cysts to become dormant with resistance to desiccation and possibly to UV radiation. Such zygotes/cysts are suitable for long-distance dispersal; however, zygote formation of Raphidonema was reported only once in 1953 [Our results reveal the existence of cosmopolitan phylotypes, which have dispersed globally and regionally to form endemic phylotypes. Although the  species , details species . Based o species , 67, veg in 1953 , but has in 1953 , 70). ThRaphidonema  are actually soil algae that are only occasionally transported to snow [Chlorella and Stichococcus (Trebouxiophyceae), which have broad temperature ranges for growth and are also found on snow/glacier surfaces. We note, however, that the optimum growth temperatures for both species are below 6 °C, and R. sempervirens cannot grow above 13 °C [R. nivale and R. tatrae are 0–15 °C and 0–10 °C, and the optimum temperatures are 5 and 4 °C, respectively [Raphidonema spp. were rarely detected from soils at high elevations, although the samples contained enough Sanguina spp., a well-known genus of snow algae that are associated with red snow, to be detected [Raphidonema sequences in the surface samples indicated that the snow species of the genus probably grow on snow and ice rather than simply being transported from the periglacial soils.A previous study has suggested that snow species of  to snow . This apve 13 °C . In addiectively , 73. Thedetected . TherefoRaphidonema is reported to be a minor genus as compared with other snow/glacier algae  on modern snow surfaces [Raphidonema is categorized as an opportunist, appearing on the surface under certain conditions. The ice core samples analyzed in this study were from central Asia, where the pH of glacial meltwater is higher than in other areas due to abundant carbonates derived from nearby deserts [Raphidonema in this area, although future studies are needed to confirm this point.Although surfaces , the latsurfaces , Raphido deserts . Under t deserts ). Accord deserts , snow grRaphidonema (trebouxiophycean group) was distributed over both poles and mid-latitude regions and was detected in different ice core layers, corresponding to distinct time periods. A phylogenetic network analysis consistently showed that the cosmopolitan phylotypes were located near the root positions, whereas the endemic phylotypes were generally located at the tips, suggesting that the endemic phylotypes originated from the cosmopolitan phylotypes. To examine this hypothesis, we applied a coalescent theory–based demographic model to our data, which confirmed the better fit of an evolutionary scenario with the assumption that the cosmopolitan and endemic phylotypes experienced different population histories, as compared with a scenario with the assumption that these phylotypes shared common population histories. The evolutionary parameters estimated using this model indicated that the modern cosmopolitan phylotypes were present long before the last glacial period (Supplementary Results, Table In conclusion, we examined the genetic structures of snow algae from both poles and mid-latitude high-mountain regions to determine the history of biogeographical distributions and evolutionary relationships between cosmopolitan and endemic phylotypes of snow algae. We found that the genus Although the local adaptations of endemics were inferred from their demographic histories, a precise picture of how newly derived endemics have dispersed and adapted to local environments and of the process by which they have migrated across the global cryosphere is still unclear. Future work will benefit from single-cell genomic technologies to shed light on their morphological and metabolic aspects and will provide new insights into their dispersal and adaptive evolution.Supplemental Information


This is a type of food allergy to red meat and other mammalian products . In Poland, this problem seems to be rare or, more likely, very underdiagnosed. The diagnosis of AGS is difficult. It seems that the knowledge about this syndrome is insufficient. There are no effective diagnostic tools able to clearly diagnose this cross-reactive allergy. This paper presents the clinical application of a non-standard method in the diagnosis of a cross-reactive allergy using the example of AGS. (2) Methods: standard tests for in vitro allergy diagnostics and the non-standard ImmunoCAP inhibition test(IT) were carried out for serum collected from a patient with a red meat allergy. (3) Results: the serum concentration of anti-α-Gal IgE was very high (302 kUA/L), and IgE antibodies toanti-mammalian-meat allergens were found. The level of IgE antibodies to mammalian meat allergens decreased after blocking on α-GAL-CAP. The concentration of anti-α-Gal IgE decreased after blocking on CAPs coated with various mammalian meat allergens. Blocking with allergens of poultry meat did not affect the concentration of anti-α-Gal IgE. (4) Conclusions: the ImmunoCAP ITseems to be a useful tool in the diagnosis of cross-reactive allergies. Based on their clinical history and test results, the patient was diagnosed with AGS caused by a primary sensitization to α-Gal after a tick bite. This is the second case of AGS described in Poland and the first in Pomerania. Alpha-gal syndrome (AGS) is a serious, potentially life-threatening allergic reaction. It is a type of food allergy to red meat and other products made from mammals. AGS is also called α-gal allergy, red meat allergy or tick bite meat allergy. It can manifest as an immediate drug allergy to pharmaceuticals containing α-Gal and a delayed hypersensitivity response to the ingestion of mammalian meat products . The firThe spectrum of AGS symptoms varies greatly. Usually, these include various types of allergic reactions, including skin lesions, digestive tract disorders, edema or anaphylaxis with varying degrees of delay, from several minutes to up to 6 h  after eating a meal with meat. These cases often involve the presence of a cofactor in the form of exercise or simultaneous consumption of alcohol. It is also interesting that in some cases, despite the presence of IgE antibodies to α-Gal, patients without any symptoms of hypersensitivity can eat red meat ,10,11,12The mechanism leading to AGS, especially with regard to the variability of the clinical symptoms and the delay in the response, is not fully understood. Various theories are taken into account, including environmental factors, the efficiency of the immune system and presentation via a different mechanism, and the kinetics of allergen digestion and absorption, especially with regard to animal fats. It is suspected that α-gal is absorbed on the surface of chylomicrons (as a glycolipid), and in this form, it is delivered to immunocompetent cells. This could indeed be responsible for the delay in the reaction, as fats are absorbed from the gastrointestinal tract particularly efficiently between 2 and 6 h after consumption, which explains the important role of alcohol as a cofactor in these reactions ,14,15,16It seems that in Poland, AGS is a very rare phenomenon or its occurrence is very underdiagnosed. To date, only one case of AGS has been described in Poland . There iLaboratory tests were conducted for blood serum taken from a 65-year-old man who had several anaphylactic reactions after eating meat.Patient’s clinical history—detailed analysis;Standard laboratory methods;Non-standard laboratory method—ImmunoCAP inhibition test (IT).There were three stages in this study:The patient’s standard clinical history and detailed personal allergic history were examined. His atopic status was assessed. Detailed questions were asked about repeated anaphylaxis occurrences and the circumstances in which they occurred.ALEX2—multiplex allergy test ;Specific IgE—for the allergens: α-Gal, extracts of mammalian meat allergens , beef gelatin and poultry meat allergens —ImmunoCAP ;Total IgE—ImmunoCAP .Extended laboratory diagnostics were performed according to the data from the patient’s clinical history. Laboratory tests routinely used for in vitro allergy diagnosis were carried out:The ALEX2  is a nano-bead technology, ELISA (enzyme-linked immunosorbent assay) protocol-based IgE multiplex assay. The ALEX2 is a quantitative specific IgE and semi-quantitative total IgE test. The ALEX2 test simultaneously measures serum concentrations of IgE specific for 117 allergen extracts and for 178 allergen components and total IgE. The lowest detectable level of IgE in the test was 0.1 kUA/L. Specific IgE results are presented in classes 0–4, where class 0 , class 1 , class 2 , class 3  and class 4 . The manual Image Xplorer system with Raptor software was used in the experiment.ImmunoCAP  is an in vitro test system for the quantitative measurement of specific IgE and total IgE in human serum or plasma via an ELISA-based fluoro-immuno-enzymatic method (FEIA). Specific IgE results are presented in classes 0–6, where class 0 , class 1 , class 2 , class 3 , class 4 , class 5  and class 6 . The Phadia100 system was used in the experiment.Experimental ImmunoCAP inhibition tests (ITs) were performed according to the procedures designed in our laboratory, the schemes for which are shown in In the experiment, we used “CAPs”, which are components of the ImmunoCAP system. CAPs are the carriers of the allergen. They are a plastic container in which a cellulose matrix coated with allergens is placed. This was the solid phase of the test—the allergen carrier.Scheme A: o215-CAP ;Scheme B: f27-CAP (beef), f26-CAP (pork), f88-CAP (mutton), f213-CAP (rabbit), f83-CAP (chicken), f284-CAP (turkey) and c74-CAP (beef gelatin).The following types of CAPs were used for the tests:g; 15 min). The CAPs were transferred to clean, dry Eppendorf (volume 1500 µL) tubes and the filtrate was discarded. In this way, 9 o215-CAPs and 2 CAPs, each from a different type of CAP used later on in the experiment, were prepared. The number of CAPs needed was determined considering the volume of serum needed for further analysis. The prepared CAPs were used in the next stage of the experiment.The first stage of both schemes was identical. At this stage, solid-phase CAPs were prepared for binding specific IgE antibodies from the patient’s serum with the appropriate allergens coated on the CAPs. Each CAP was placed inside dry, clean Eppendorf tubes (volume 1500 µL). Then, 40 µL of washing solution was added to each CAP. The washing solution that is part of the ImmunoCAP system (Art. No. 10-9422-01) was used. The solution was prepared according to the manufacturer’s instructions. All CAPs sealed inside Eppendorf tubes were centrifuged . After centrifugation, the CAPs were discarded, and the filtrates were collected in a single tube. The IgE concentration anti-α-Gal, anti-f27 (beef), anti-f26 (pork), anti-f88 (mutton), anti-f213 (rabbit), anti-f83 (chicken), anti-f284 (turkey) and anti-c74 (beef gelatin) were measured in the filtrate, using the ImmunoCAP system with Phadia100.Prepared o215-CAPs were used in this branch of the experiment. A total of 40 µL of patient serum was applied to each CAP. CAPs tightly sealed in Eppendorf tubes were incubated in two stages: first for 30 min at 37 °C and then for 24 h at 2–8 °C. After the incubation, the CAPs were centrifuged . After centrifugation, the CAPs were discarded, and the filtrates were collected. The filtrates from the same types of CAPs were combined in one tube. Altogether, 7 types of filtrates were obtained. The concentration of anti-α-Gal IgE  was measured in each of them.In this branch of the experiment, the other prepared CAPs  were used. A total of 40 µL of patient serum was applied to each CAP. CAPs tightly sealed in Eppendorf tubes were incubated in two stages: first for 30 min at 37 °C and then for 24 h at 2–8 °C. After the incubation, the CAPs were centrifuged , the patient developed edema and hives of the scalp, hips and anus, combined with severe pruritus. There was also a rapid fall in blood pressure and loss of consciousness. Specialized treatment in the emergency department was necessary. Subsequent episodes of allergic reactions with a similar course occurred in the patient in September 2010, autumn 2014, December 2017 and several times from July 2019 to January 2020.During this period, there were also several reactions of a lesser severity, which were not accompanied by loss of consciousness, and the spectrum of symptoms was limited to swelling, hives and itching of the previously mentioned body areas.In turn, during the last three episodes, the previously observed symptoms of a severe systemic reaction were additionally joined by swelling of the lips and edema and urticarial changes in the hands.According to the patient, both the frequency and severity of the symptoms had increased recently. Severe systemic reactions always required treatment in a hospital emergency department. For less severe episodes limited to urticaria and pruritus, short-term treatment with low-dose antihistamines or oral steroids was sufficient.The man could not clearly identify the common cause of the allergic reactions. He associated them with the consumption of small volumes of various types of alcohol or meals with meat. He also drew attention to the possible contribution of physical effort related to practicing sports (such as cycling or skiing).The clinical history of the patient, the spectrum of observed symptoms after the meals with meat, the presence of allergic reaction cofactors  and the absence of features of atopic disease before the first episode of severe allergic reaction raised the suspicion of α-Gal syndrome (AGS).To verify our hypothesis, we extended the clinical interview by asking the patient whether it was likely that he had been bitten by a tick in the time preceding the first episode of acute allergic reaction. The patient confirmed being bitten by a tick in July 2009. It happened near the Tri-City , when he was cycling to work in the morning. The tick was removed and tested for Lyme disease (the tick was not a carrier). Unfortunately, the patient did not have the result of this test and was unable to determine the species of tick that bit him.After obtaining this information, we proposed the patient perform additional laboratory tests, verifying our suspicions as to whether he was allergic to the polysaccharide, α-Gal, found in tick saliva, and the related possibility of severe allergic reactions after eating mammalian meat, especially in the presence of the cofactor.In the first stage of standard laboratory diagnostics for allergic disease, the ALEX2 multiparameter test was performed. The ALEX2 test showed a very high concentration of total IgE. A low concentration of IgE antibodies specific for airborne and food allergens of plant origin as well as hymenoptera venom allergens were detected. A moderate level of IgE antibodies specific for all mammalian meat allergens represented in the ALEX2 test were detected. No IgE antibodies to poultry meat allergens were found. No specific IgE antibodies for the allergen components important in the diagnosis of primary allergy to meat, Bos d 6 and Sus d 1, were detected. IgE results specific for other allergens represented in the ALEX 2 test were negative. The ALEX2 results are presented in The ImmunoCAP method confirmed a very high total IgE concentration. The IgE antibody anti-α-Gal was detected at a very high concentration. The concentration of IgE antibodies for beef allergen extract was very high, as well as that for pork allergen extract. A moderate level of IgE antibodies to mutton and rabbit meat allergens were found. IgE antibodies to poultry meat allergens  and beef gelatin allergens were not detected. The anti-α-Gal IgE to total IgE ratio was 11.61%. All results of these analyses are presented in Branch A In branch A of the ImmunoCAP inhibition test A, we assThe results of the inhibition test on o215-CAP  are shown in Branch B In branch B of the ImmunoCAP inhibition test B, we assThe results of the inhibition test on membranes coated with various meat or gelatin allergens are shown in The patient’s clinical history, sequence of symptoms and subsequent steps of laboratory diagnosis are summarized in AGS is a group of symptoms of a severe allergic reaction to mammalian meat and meat-derived products  which consists of an allergy to the determinant of the carbohydrate α-Gal derived from tick saliva. Primary sensitization develops after a tick bite, and the reaction to meat is associated with the cross-reactivity of anti-α-Gal IgE antibodies with the α-Gal expressed on mammalian meat proteins ,5.AGS was originally thought to be endemic and found mainly in the Southeastern United States. However, there are currently case reports of AGS from all continents . It seemA characteristic clinical feature of AGS is the occurrence of symptoms of severe allergy after eating red meat. It is characteristic that symptoms do not appear immediately after a meat meal, but are delayed for several hours. The presence of a cofactor is also often necessary. The reaction can also develop in people who were not previously allergic ,10,11,12We described a patient who developed a severe allergic reaction a few hours after eating meals with meat, a case which fits perfectly into the area of symptoms of AGS. This 65-year-old man has never suffered from any symptoms of an allergic disease. Only after being bitten by a tick 10 years ago did he develop his first episode of anaphylaxis. It seems, therefore, that the tick bite itself was the factor that initiated these events.It is also significant that the presence of anaphylaxis cofactors often distracts the patient from the true cause of these reactions. In the described case, the man assumed that the cause of his anaphylaxis was the consumption of various types of alcohol. Only the demonstration of the presence of antibodies to mammalian meat allergens and the connection of this result with the clinical history of the patient, supplemented with information about the tick bite, raised the suspicion of AGS in this patient.The quantitative determination of IgE α-Gal antibodies has been successfully used in the diagnosis of AGS. According to Fischer et al.  an anti-Mammalian meat is the most common trigger for allergic reactions in patients with AGS ,5. In ouIt is assumed that α-Gal IgE may also be a target for antibodies in gelatin allergy ,25. HoweDiagnostics of AGS is a difficult and multi-stage process. It often requires combining seemingly unrelated facts and laboratory results. The first signal that led to the suspicion of alpha-gal syndrome in the described patient was the detection, using the ALEX2 test, of the presence of IgE antibodies to mammalian meat allergens in the serum. The ALEX2 test is a multiparameter screening test used in the diagnosis of allergies. The results of these types of tests often need to be confirmed by reference methods according to the clinical symptoms observed in the patient. Based on the patient’s clinical history, we confirmed the ALEX2 results by measured the concentration of specific IgE antibodies to meat allergens using a reference test (ImmunoCAP), and we determined the concentration of anti-α-Gal IgE antibodies because we suspected that AGS was the cause of our patient’s recurrent anaphylaxis. After detecting antibodies to α-Gal in the patient serum, we decided to check whether the presence of antibodies to meat was caused only by cross-reactivity to α-Gal expressed on meat allergens. Since there are no standard tests to study this type of relationship, we performed an experimental inhibition test on factory-allergen-coated CAPs, which is an element of the ImmunoCAP system. We chose these substrates because of their properties. They are usually used to determine the concentration of specific IgE antibodies in the routine diagnosis of allergic diseases. The allergens that are used to coat the ImmunoCAP CAPs are obtained by the manufacturer of the ImmunoCAP reagents under standardized conditions. The manufacturer guarantees the stability and purity of these extracts; thus, the results are repeatable. We assumed that they would be a very good source of well-characterized allergens. The experiments showed that α-Gal antigens bind anti-α-Gal IgE antibodies and IgE antibodies specific for mammalian meat allergens, reducing their serum concentration. Additionally, allergens of various mammalian meats bind anti-α-Gal IgE antibodies, reducing their serum concentration. These results seem to confirm that the cause of the symptoms observed in the described patient is not a primary allergy to meat, but a cross-allergy caused by sensitization to α-Gal that developed following a tick bite.Due to the variability of clinical manifestations, it seems that many difficult or unclear cases of meat allergy remain undiagnosed or misdiagnosed. This may have a significant impact on the therapeutic and dietary management, the quality of the patient’s life and his safety . The diaThe presented clinical case study is an example of the clinical application of the experimental ImmunoCAP inhibition test, the procedure for which was designed by us. Inhibition tests are non-standard analytical methods used in scientific research to study the cross-reactivity of allergens ,29,30,31The results of laboratory analyses, both standard and experimental tests, combined with the patient’s clinical symptoms, allowed us to establish a logical sequence that ultimately led to the development of AGS as a result of sensitization to tick saliva polysaccharides in a primarily non-atopic adult male. It seems that the experimental inhibition ImmunoCAP test used in this study may be a valuable diagnostic tool for testing cross-reactivity of allergens in clinical practice.We advised our patient to exclude mammal meat from his diet and replace it with poultry meat, which is safe in the case of alpha-gal syndrome. Due to his history of anaphylactic reactions, the patient was informed that he should always carry a syringe pre-filled with adrenaline and was trained in its use. The patient is compliant with the dietary recommendations and has had no further episodes of anaphylaxis.The main limitation of the presented study is that only the results obtained for one patient were used to assess the clinical utility of the proposed ImmunoCAP inhibition test (IT). It seems, however, that this test may be a valuable tool in the study of the clinical significance of allergens cross-reactivity, especially if we take into account that we currently do not have any other standardized diagnostic tools in this area. By presenting this description, we want to encourage other researchers to undertake similar activities and search for new, unconventional applications of commonly available laboratory tools.


Escherichia coli (E. coli) ribosome can incorporate a variety of non-l-α-aminoacid monomers into polypeptide chains in vitro butwith poor efficiency. Although these monomers span a diverse set ofcompounds, there exists no high-resolution structural informationregarding their positioning within the catalytic center of the ribosome,the peptidyl transferase center (PTC). Thus, details regarding themechanism of amide bond formation and the structural basis for differencesand defects in incorporation efficiency remain unknown. Within a setof three aminobenzoic acid derivatives—3-aminopyridine-4-carboxylicacid (Apy), ortho-aminobenzoic acid (oABZ), and meta-aminobenzoic acid (mABZ)—the ribosome incorporates Apy into polypeptide chainswith the highest efficiency, followed by oABZ andthen mABZ, a trend that does not track with the nucleophilicityof the reactive amines. Here, we report high-resolution cryo-EM structuresof the ribosome with each of these three aminobenzoic acid derivativescharged on tRNA bound in the aminoacyl-tRNA site (A-site). The structuresreveal how the aromatic ring of each monomer sterically blocks thepositioning of nucleotide U2506, thereby preventing rearrangementof nucleotide U2585 and the resulting induced fit in the PTC requiredfor efficient amide bond formation. They also reveal disruptions tothe bound water network that is believed to facilitate formation andbreakdown of the tetrahedral intermediate. Together, the cryo-EM structuresreported here provide a mechanistic rationale for differences in reactivityof aminobenzoic acid derivatives relative to l-α-aminoacids and each other and identify stereochemical constraints on thesize and geometry of non-monomers that can be accepted efficientlyby wild-type ribosomes.The  Ribosome incorporationof aminobenzoic acids is limitedby their suboptimal positioning, failure to trigger the induced stateof the ribosome, and disruption of a water network required for catalysis. The reaction follows an acyladdition–elimination pathway; one tRNA (the A-site tRNA) carriesthe α-amine nucleophile, whereas the other (the P-site tRNA)carries the carbonyl ester electrophile .2 Studies of the ribosomein multiple states of catalysis indicate that bond formation is alsofacilitated by an induced fit, in which substrate-induced conformationalchanges within the PTC fine-tune the relative orientation of the nucleophileand electrophile. The presence of a correctly positioned l-α-peptidyl oligomer in the P-site and an α-amino acidester in the A-site within the inner shell reorients U2506 and U2585,which shield the reaction center from premature hydrolysis by solventnucleophiles and positions the α-amine and carbonyl moietiesin line for nucleophilic attack.4 Bondformation is also facilitated by ordered water molecules within thePTC that function as a “proton wire” or a “protonshuttle” to deprotonate the α-amine nucleophile and activatethe carbonyl electrophile prior to nucleophilic attack, stabilizethe tetrahedral intermediate after nucleophilic attack, and resetthe PTC for another round of amide bond formation.5Ribosomes synthesize natural proteinsby catalyzing amide bondformation between an trophile 1A. The rl-α-amino acids within the A-site,the P-site, and the exit tunnel.6 Evengreater monomer diversity is tolerated in highly optimized in vitro translation reactions that employ chemically acylatedtRNAs. Monomers that can be introduced into polypeptides invitro include d-α-amino acids, α-hydroxyand α-thio acids, β2- and β3-amino acids, α-aminoxy and α-hydrazino acids, and asmall number of aminobenzoic acid derivatives.19 Although these molecules are accepted as ribosome substrates in vitro, the yields of polypeptide products vary greatlyas a function of location, context, monomer identity, and the presenceor absence of supplementary translation factors such as EF-P, andthe factors limiting their incorporation efficiency are unknown.23 Although a single β3-amino acid and an oxazoledipeptide mimic can be incorporated into protein using engineeredribosomes in vivo, the only non-l-α-aminoacid monomers incorporated into protein by wild type ribosomes in vivo thus far are α-hydroxy acids.30A major advance in the cellular biosynthesis ofnovel protein materialswas the discovery that this induced fit and proximity-guided mechanismfor amide bond formation by the ribosome tolerates a diverse rangeof noncanonical l-α-amino acid monomers evaluatedas substrates for wild-type ribosomes in vitro includesa variety of differentially substituted aminobenzoic acid derivatives.13 These monomers are of great interest as bothmaterials and pharmaceuticals. Aromatic polyamides (aramids) representa fundamental building block of both heat- and impact-resistant fabrics,as well as many microbial natural products. They can also fold intounique secondary structures, including β-turns and helices,and can improve the drug-like properties of cyclic peptides.34 Yet aminobenzoic acids differ from l-α-amino acidsin terms of the conformational landscape of the backbone (sp2 versus sp3-hybridized) as well as the nucleophilicityof the attacking amine, and the effects of these differences on intra-PTCreactivity are unknown. Indeed, although certain aminobenzoic acidmonomers can be introduced into short peptide oligomers by the ribosome,the incorporation efficiencies vary widely. Specifically, 3-aminopyridine-4-carboxylicacid (Apy) acid was incorporated into short peptide oligomers almost5-fold more efficiently than ortho-aminobenzoic acid(oABZ); the concentrations of peptide products obtainedfrom otherwise analogous in vitro translation assayswere 0.42 μM for Apy and 0.09 μM for oABZ, compared to 1 μM for Ala.13 Meanwhile, oligomers containing meta-aminobenzoicacid (mABZ) were barely detected by mass spectrometry.20One class of non-l-α-aminoacids and the differences in yields between the aminobenzoic acidderivatives could be related to their positioning within the PTC ofthe ribosome. Here using cryogenic electron microscopy (cryo-EM) wedetermined high-resolution structures of the wild-type E.coli ribosome in complex with full-length tRNAs acylatedwith each of the three aminobenzoic acid monomers—Apy, oABZ, and mABZ—and accommodatedwithin the A-site of the PTC. Our structures reveal that, althoughthe ribosome is capable of accommodating aminobenzoic acid derivativeswithin the A-site cleft, the distinct sp2-hybridized backboneof these monomers and their positioning within the PTC fail to triggerthe conformational changes associated with the induced-fit mechanism.It also depletes the PTC of two of three ordered water molecules usedas proton shuttles during amide bond formation. Based on these observations,we propose a structural and mechanistic basis for the lower incorporationefficiency of aminobenzoic acid monomers relative to l-α-aminoacids and each other and provide guidance on the size and geometryof backbone-modified monomers likely to be accepted efficiently bythe wild-type PTC.We hypothesized that the reduction in ribosomal incorporationefficiencyof aminobenzoic acid derivatives compared to oABZ, and mABZwithin the ribosomal PTC, we obtained cryo-EM structures of the wild-type E. coli ribosome in complex with full-length acylated tRNAsin both the P- and A-sites. To avoid acyl-tRNA hydrolysis during samplepreparation, A- and P-site monomers were appended through amide linkagesusing tRNA molecules carrying a 3′-amino group .Purified E. coli ribosomes were incubated with theP-site substrate fMet-NH-tRNAfMet and an A-site substratein which 3′-NH2-tRNAPhe was acylatedwith either Apy, oABZ, or mABZ . Initial rounds of 2D and 3D classification were performed to selectintact 70S ribosomal particles . The resulting cryo-EM maps containedcomplexes in the classical (nonrotated) state of the ribosome35 with clear density for both P- and A-site tRNAs.To improve the resolution of the Apy, oABZ, and mABZ monomers, we further classified the ribosomal particlesbased on A-site tRNA occupancy. The final maps of 70S ribosomal particlescontaining A-site oABZ-NH-tRNAPhe or Apy-NH-tRNAPhe were refined to a global resolution of 1.9 and 2.1 Å,respectively . This resolution allowed forthe unambiguous modeling of A-site monomers oABZand Apy in a single conformation . This resolution permitted modeling ofthe mABZ monomer in one of two conformations relatedto a 180° rotation about the aryl–carbonyl bond .The A-site tRNA positions the incomingα-amino acid ester within the PTC with the aid of a base-pairinginteraction between C75 of the A-site tRNA and G2553 within the Aloop of the 23S rRNA.or mABZ 2. All th2-hybridized carbonyl carbon of the P-site peptidyl-tRNA. Nucleophilicattack generates a tetrahedral intermediate that, after proton transfer,breaks down to form the product, a peptidyl-tRNA carrying an additionalC-terminal α-amino acid . In the case of the mABZ monomer,the aromatic ring could be modeled in two distinct conformations thatdiffer in a 180° rotation about the aryl-carbonyl bond , the P-site carbonyl oxygen is rotated toward theA-site monomer and lies almost in the same plane as the A-site nucleophile. It is possible that this increased flexibilityis one contribution to the increased reactivity of Apy compared tothat of oABZ (vide infra).The high resolution of the ribosome cryo-EM maps allowed for unambiguousmodeling of  visible 1D. In this 4.9 Å 3. Notablleophile3.5,20,3940 Once an aminoacyl-tRNA is accommodated in the A-site, the ribosometransitions to an induced state in which U2506 is repositioned toconfine the side chain of the A-site α-amino acid within theA-site cleft and away from the reaction center .20 In addition, U2585,which normally moves in tandem with U2506 in the induced fit mechanismto expose the P-site carbonyl for attack, remains in the uninducedposition within van der Waals distance of the P-site fMet . Taken together, the structures of ribosomes containingaminobenzoic acid monomers in the A-site all adopt conformations closerto the uninduced state of the PTC, rather than closing around thesubstrates and helping position water molecules for catalysis, asobserved with l-α-amino acids.44Nucleotide U2506 in the PTC has been implicated in the inducedfit mechanism necessary for efficient amide bond formation by theribosome. In the uninduced state, when the A-site is not occupiedby a natural aminoacyl-tRNA, U2506 forms a wobble base pair with G2583.n center 4A. U2506e A-site 4B–D. TheoABZ,where the final cryo-EM map reached a global resolution of 1.9 Å,a slight torsion (∼12°) of the aromatic ring relativeto the carbonyl group was evident. The global resolution for the complexescontaining Apy and mABZ were slightly lower , yet in both cases the aromatic ringand exocyclic amino substituent are clearly visible. The high resolutionalso revealed mABZ to be bound in two different conformationsrotated by 180°, in both cases with the nucleophilic amine pointedaway from the P-site monomer . This packingprevents U2506 from adopting the position seen with natural acyl-tRNAsubstrates and thus fails to position the attacking nucleophile withrespect to the carbonyl carbon of the P-site monomer.The high-resolution structures reported here also identify a secondexplanation for the diminished reactivity of aminobenzoic acid monomersin ite tRNA 5A,B. Thel-α-amino acids. The absence ofthe conformational changes associated with the induced fit mechanismprovides a second explanation for the diminished reactivity of aminobenzoicacid monomers in in vitro translation assays. A finalmitigating factor is intramolecular H-bonding between the amino groupand carbonyl oxygen.The inabilityof U2506 to rotate into the induced conformation likely also preventsmovement of U2585 away from the carbonyl carbon of the P-site esterlinkage, which in turn prevents its rotation into a position suitablefor efficient amide bond formation 5. In theE. coli ribosome, insertion of residue R163 in the stallingsequence into the A-site cleft PTC causes U2506 and U2585 to adoptconformations consistent with the uninduced state, resulting in translationalstalling.45 In the case of mABZ, these factors, combined with the fact that the nucleophilicamine is pointed away from the P-site, render this monomer virtuallyunreactive. The directionality of the nucleophilic amine is reminiscentof the case of d-α-phenylalanine, for which a recentcrystal structure revealed that the A-site amino group is also pointedaway from the P-site carbonyl carbon resulting in the slower peptidebond formation of this enantiomer despite the ribosome PTC adoptingthe induced state.46 On the other hand, although theinability of the ribosome to adopt the induced conformation in thepresence of both Apy and oABZ helps to explain theirlower reactivity compared to l-α-amino acids, the reasonfor the reactivity difference between Apy and oABZremains more subtle. In the cryo-EM maps, the P-site fMet in the Apycomplex appears to be more dynamic than that in the oABZ complex. With the increased dynamics suggested by the structureof the Apy ribosome complex, the P-site monomer may possess enoughconformational flexibility to adopt a position compatible with nucleophilicattack, as opposed to the P-site fMet in the case of oABZ, which appears to be well ordered and trapped in the uninducedconformation.The lack of conformational changes associatedwith the inducedfit mechanism is also seen in structures of the ribosome with stallingpeptides. In a cryo-EM structure of SecM in the wild-type Supplementary Figure S14.5 In the stepwise proton shuttle mechanism, two water molecules and2′-OH groups of A2451 and the P-site A76 facilitate protontransfer via distinct 8- and 6-membered rings.44Amide bond formation within the PTC requires more than the attackof a nucleophilic amine on a proximal ester carbonyl. A key considerationfor efficient catalysis is the shuttling of protons between the attackingand leaving groups involved in the reaction. In the case of naturalα-amino acid esters, the nucleophilic amine in the A-site existsin the ammonium form at physiological pH and must be deprotonatedprior to nucleophilic attack on the P-site carbonyl carbon to forma tetrahedral intermediate. Following this step, this amine must bedeprotonated once again, and a proton must be transferred to the 3′-oxygenleaving group of the P-site A76 to facilitate breakdown of the tetrahedralintermediate. There is significant evidence that these proton transfersare mediated by water molecules located at discrete positions withinthe PTC. In the proton wire mechanism, three water molecules are usedfor proton transfer events  as shown in Supplementary Figure S14).The inability of the PTCto adopt an induced-fit conformation with aminobenzoic acid monomersbound in the A-site disrupts the water positions associated with boththe proton wire and the 8-membered ring proton shuttle models. Forinstance, nucleotide A2602, which coordinates both W1 and W2 of theproton wire, is disordered and could not be modeled with high confidence.Also, no density for the N-terminus of uL27 is seen in these structures.As a result, W1, which is proposed to act twice as a general base, is notobserved. Density corresponding to W2, the proposed general acid thatstabilizes the oxyanion of the tetrahedral intermediate, is also absentin these structures. The only water observed with confidence is W3,which facilitates protonation of the P-site A76-O2′ and thusbreakdown of the tetrahedral intermediate  forms of the oABZ and Apy methyl esters were determined to be 2.8 and−3.0, respectively, using computation . The pKa value of the acidic (ammonium) form of mABZ wasfound to be 3.5. These low pKa valuesimply that none of the aminobenzoic acid monomers require the assistanceof W1 as a general base prior to or subsequent to nucleophilic attack.These monomers are expected to exist predominantly in the neutralform at neutral pH, both before and after the tetrahedral intermediateforms. The proton lost from the attacking amine in the tetrahedralintermediate could protonate the tetrahedral intermediate and therebysubstitute for the absence of W2. This loss would be easier from thetetrahedral intermediate containing Apy due to its significantly lowerpKa value (−3.0 versus 2.8). Thisanalysis provides a testable explanation for the enhanced reactivityof Apy relative to oABZ and provides design rulesfor monomers that diverge significantly from natural α-aminoacids in terms of structure, conformation, and/or electronics.Why then do Apy and 48 and efficiency of delivery of charged tRNAs to the ribosome by EF-Tu,49 would also influence overall yields of the finalpolymer. Nevertheless, the structures of the aminobenzoic acid monomerswithin the A-site of the ribosome presented here provide valuableinsights into the requirements for the efficient incorporation ofthe monomer incorporation by the ribosome. For example, monomers relatedto oABZ and Apy but possessing an expanded or alternativearomatic core may allow the monomer to enter the A-site cleft withoutsteric constraints with U2506. Monomers could be chosen to allow U2506to rotate into its correct position in the induced fit mechanism,thereby triggering the movement of U2585 and the other conformationalrearrangements necessary for amide bond formation to take place efficiently.Alternatively, the conformational rearrangements associated with inducedfit could be promoted using ribosome evolution strategies that exploitrecently mapped intra-ribosome allosteric relationships to avoid universallyconserved nucleotides.50 Regardless, thethree ribosome models reported here provide an unprecedented structuraland mechanistic rationale for differences in reactivity among non-l-α-amino acid monomers and identify clear stereochemicalconstraints on the size and geometry of nonproteinogenic monomersthat are likely to be processed efficiently by wild-type ribosomes.Theincorporation of even a single unnatural monomer into a peptide chainnecessitates the successful execution of several steps, beginningwith the charging of tRNAs with the monomers and their incorporationinto elongated polymer chains. While the structures presented herereveal the details of how the aminobenzoic acid derivatives are positionedwithin the PTC and their inability to trigger the induced state ofthe ribosome necessary for efficient amide bond formation, they onlyprovide insight into the possible defects in that single step. Otherfactors, such as translocation of the growing polymer chain from theA-site to the P-site, positioning of the monomer incorporated in thegrowing polymer within the P-site, and interactions within the exittunnel, remain unknown. Furthermore, factors affecting other aspectsof translation, such as efficiency of charging monomers onto tRNA,editing activity of mischarged tRNAs by aminoacyl-tRNA synthetases,