In the RB4 dye adsorption design, an adjusted R2 (0.8280) with good correlation was observed, where the optimized conditions were: pH = 2, adsorbent dose 0.6 g, and initial concentration of RB4 dye 5 mg/L. The kinetic behavior and the adsorption isotherm allowed us to conclude that the GA-crosslinked CS beads’ adsorption mechanism was controlled mainly by chemisorption interactions, demonstrating its applicability in systems that require the removal of contaminants with similar structures to the model presented.The use of dyes at an industrial level has become problematic, since the discharge of dye effluents into water disturbs the photosynthetic activity of numerous aquatic organisms by reducing the penetration of light and oxygen, in addition to causing carcinogenic diseases and mutagenic effects in humans, as well as alterations in different ecosystems. Chitosan (CS) is suitable for removing anionic dyes since it has favorable properties, such as acquiring a positive charge and a typical macromolecular structure of polysaccharides. In this study, the optimization of CS beads crosslinked with glutaraldehyde (GA) for the adsorption of reactive blue dye 4 (RB4) in an aqueous solution was carried out. In this sense, the response surface methodology (RSM) was applied to evaluate the concentration of CS, GA, and sodium hydroxide on the swelling degree in the GA-crosslinked CS beads. In the same way, RSM was applied to optimize the adsorption process of the RB4 dye as a function of the initial pH of the solution, initial concentration of the dye, and adsorbent dose. The crosslinking reaction was investigated by scanning electron microscopy (SEM), Fourier transformed infrared spectroscopy (FTIR), and X-ray diffractometry (XRD). The design described for the swelling degree showed an R The dyes used by different industries, such as textiles, leather, paper, and plastics, among others, is of great interest, because the discharge of dye effluents into water generate persistence, bioaccumulation, and toxicity . The dyeWastewater has been shown to contain dye concentrations ranging from 10 mg/L to 200 mg/L . Due to Among many reported techniques, the adsorption process is commonly used due to its remarkable ability to remove dyes from aqueous solutions. However, the use of some sorbents  can be expensive and challenging alternatives to regenerate . In recent years, numerous, inexpensive, and effective alternatives have been developed, including chitosan (CS), which has attracted particular attention because it is renewable, has high availability, and does not generate toxic products ,10,11. Cp-nitrophenol than physical modifications of CS in the form of flakes.However, CS easily dissolves in an acidic medium (pH < 5), which limits its use due to the high protonation of its amino groups, leading to instability and dissolution of the material in the medium . ConsequLikewise, the implementation of low molecular weight (LMW) CS has proven to be better biocompatible and biodegradable than high molecular weight (HMW) CS .Its use at LMW provides a more excellent facility to interact with the dye because its chains are shorter and allow better access to the available adsorption sites . On the Therefore, the purpose of this research was to evaluate the development of low molecular weight CS beads crosslinked with GA to maximize the removal efficiency of reactive blue dye 4 in an aqueous solution.k) with three factors (k = 3), each with two levels ; (b) axial points with coded values α = 2k/4 = ±1.6817; and (c) duplicate of the central point. For the first design, input factors were the concentration of CS, NaOH concentration, and GA concentration. The response factor was the swelling degree. For the second design, input factors were the adsorbent dose, initial concentration of the dye, and the dye solution’s pH. The response factor was dye removal efficiency.A central composite experimental design (CCED) was used to evaluate the swelling degree of CS beads cross-linked with GA and the removal efficiency of the anionic dye RB4. Both designs consisted of conducting 32 randomized experiments, including duplicates. Three series of experiments were developed: (a) a factorial design , response surface methodology, and optimization were performed using Statgraphics Centurion XVI software  . Second-w/v) and 3.3 (% w/v) of CS  solution were prepared in 1 (% v/v) acetic acid solution and homogenized with a digital disperser .Concentrations between 1.7 (% v/v) to 3.3 (% v/v). The crosslinked beads were filtered from the solution, washed vigorously with distilled water to remove any unreacted particles, and dried in an oven at 60 °C for 10 h [The above CS solutions were added dropwise into sodium hydroxide solutions with a concentration range of 0.7 M to 2.3 M, under constant stirring, to allow the formation of CS beads with controlled spherical size. The beads formed were removed from the solution, washed with distilled water until a neutral pH value was obtained. The CS beads were immersed for 12 h in 25 mL of GA solutions with a concentration range of 1.7 (% for 10 h .W0 (g) is the dry mass and W1 (g) is the wet mass after 24 h [One gram of GA-crosslinked CS beads obtained in each experiment was immersed in distilled water at room temperature and neutral pH. After 24 h, the beads were filtered, excess water was removed with filter paper, and weighed to constant weight. The swelling degree was calculated with Equation (2).ter 24 h . FurtherStandard solutions were prepared at different concentrations of the RB4 dye . The above solutions’ absorbances were measured in the UV-VIS spectrophotometer , at a maximum length of 599 nm. The model was estimated using linear regression analysis to obtain the dye concentrations in the adsorption processes.R (%) is the removal efficiency, Co and Ce (mg/L) are the initial concentration and the final or equilibrium concentration of the anionic dye RB4 [A stock solution of 500 mg/L of the RB4 dye was prepared. From the stock solution, solutions were prepared in a range of 5 mg/L to 55 mg/L. Each experiment’s pH was adjusted with minimum and maximum values of 2.0 and 7.0 by adding HCl (0.01 M) and NaOH (0.01 M). Next, 25 mL aliquots were taken, and their initial absorbances were measured at a maximum length of 599 nm. Subsequently, specific amounts from 0.1 g to 0.6 g, corresponding to a dose of adsorbent between 0.1 g/25 mL and 0.6 g/25mL, were added. The samples were placed in the dark with shaking at a speed of 400 rpm to prevent dye photolysis. After 48 h, following beads filtration, the absorbance of the RB4 dye in the supernatant was measured. The dye removal efficiency was determined with Equation (3).c is the area of the peaks representing the crystalline regions and AT is the total area of the crystalline and amorphous region.Scanning electron microscopy (SEM)  and Fourier transformed infrared spectroscopy—attenuated total reflectance (FTIR-ATR)  were used to determine the surface morphology of the adsorbents and the presence of functional groups. The crystalline phase of the cross-linked beads was detected by X-ray diffractometry (XRD)  using radiation of Cu Kα1 (1.540598 Å) and Kα2 (1.544426 Å), in a range of 2θ comprised from 5° to 50°. Equation (4) shows the percentage of crystallinity (Xc %) of each pearl obtained from their diffractograms using the Nara-Komiya methodology .(4)Xc %e (mg/g), was calculated from Equation (5) [Co and Ce (mg/L) are the liquid-phase concentrations of dye initially and at equilibrium, respectively, V (L) is the volume of the dye, and W (g) is the weight of the adsorbent.A 500 mg/L stock solution of the RB4 dye was used to take 25 mL of a 35 mg/L solution, and its initial absorbance was measured. Then 0.4 g/25 mL of GA-crosslinked CS beads were added. This solution was stored in the dark under constant stirring at 400 rpm and room temperature. Twenty absorbance measurements were taken at different time intervals during 48 h, taking small aliquots of the stored solution. The amount of equilibrium adsorption, QRB4 dye solutions of 5 mg/L to 65 mg/L with a pH adjusted to 3.0 were prepared from the 500 mg/L stock solution. The isothermal experiments were carried out under the same conditions as the kinetic experiment. After 48 h of constant stirring, the beads were separated by filtration, and the final absorbances were measured. Subsequently, the adsorption equilibrium models described in The optimization of the GA-crosslinked CS beads was evaluated on the swelling degree. The swelling degree accounts for the expansion of chitosan’s porous structure, which allows the interaction of the dye molecules with the adsorption sites . Table 1p < 0.05) were: CS concentration (A), GA concentration (B), quadratic interaction of CS concentration (AA), quadratic interaction of GA concentration (BB), interaction concentrations of CS, and GA (AB).The contributions of the operational and interaction factors that influenced the swelling degree were evaluated by ANOVA . The fac2, and adjusted R2. R2 (90.74 %) values and adjusted R2 (86.34 %) indicated a good correlation between the input and output variables [The model’s effectiveness was evaluated based on the coefficient of determination, Rariables . AdequatA is the concentration of CS, B is the concentration of GA, and C is NaOH concentration. The Pareto chart plots the standardized effects of each factor and their interactions. The positive (+) or negative (-) effect on the response factor is given by the increase in the factor level . Figure On the other hand, the swelling degree was not affected by the NaOH concentration. This result might be due to the limited range in which this input variable was studied. A reported study indicated that when the NaOH concentration is increased in a range from 1 to 5 M, it is possible to reduce the water content in the porous beads . That isw/v) of CS, 1.7 (% v/v) of GA, and 1.3 M NaOH. Similarly, the mentioned conditions were validated, obtaining a 50% swelling degree.The response surface plot of 2 (0.8634). However, the surface graph shows that the most suitable treatment was found towards a point outside the experimental region, suggesting expanding the study range in the direction where RSM indicated the possible best treatment (towards the region of warm colors). It should be noted that increasing the concentration of CS implies an increase in viscosity and a more challenging bead formation. Therefore, the experimental adsorption design applied in this work was carried out at concentrations of 3.0 (% w/v) of CS, 1.7 (% v/v) of GA, and 1.3 M of NaOH, where the swelling degree obtained was 48%.The GA-crosslinked CS beads response model was recommended for prediction purposes within the selected range since it translated more than 70% of the response in terms of adjusted RpKa of around 6.4 [pKa, the amino groups in the CS molecules ionize into ammonium ions, acting as repulsive forces between polymer chains, allowing a higher swelling degree [On the other hand, the cross-linked beads’ swelling was determined under the influence of acidic pH (3) and basic pH (10), reaching swelling percentages of 90% and 14%, respectively. This behavior was explained because CS is a weak base with a ound 6.4 . Therefog degree .pKa, a more significant number of OH− ions are produced in the medium, and consequently, the protonation of the amino groups is minimal, leading to a low swelling degree [On the other hand, at a pH above g degree .Obtaining the GA-crosslinked CS beads required an acid-base reaction and, subsequently, cross-linking. Initially, the CS amino groups’ protonation was carried out in acetic acid solution a. Subseq2. The value of R2 (99.95%) indicated that the experimental points were correctly adjusted to the mathematical model of the equation.A calibration curve  was prepw/v), GA at 1.7 (% v/v), and NaOH at 1.3 M. In the adsorption processes, the GA-crosslinked CS beads were prepared from a concentration of CS at 3.0  and the quadratic effects (B2 and C2) are significantly different from zero at a 95% confidence level (p < 0.05). Adequate precision (14.03) indicated an adequate signal, and this model can be used to navigate the design space.According to A (g) is the adsorbent dose, B is the pH, and C (mg/L) is the concentration of the RB4 dye.Based on the experimental data, a second-order polynomial equation (Equation (7)) was used to predict the removal efficiency of the RB4 dye. The mathematical model obtained showed high predictive quality since the predicted values and the experimental values presented a good correlation.pKa values equal to 0.80, 1.44, 7.83, and 12.05. These data show that at pH 4, the two sulfonate groups are dissociated. At a pH 10 and 14, the amino groups and dissociated sulfonate groups coexist. Therefore, the acidity of the sulfonate groups in the structure of the RB4 dye maintains the anionic charges throughout the pH range, and consequently, the dye solution’s pH will affect the adsorption rate. In this sense, in the adsorption with GA-crosslinked CS beads, at a pH of 3, it could be assumed that the sulfonate groups are the species that interact with the adsorbent through an electrostatic interaction [The solution’s pH is a variable of great importance in the dye’s removal efficiency since it can change the adsorbent’s surface charge to influence the adsorption process . When theraction .On the other hand, the adsorbent and adsorbate concentrations influenced the efficiency of the removal process. An increase in the amount of GA-crosslinked CS beads meant more significant adsorption sites on the adsorbent’s surface ,42,43. OFor the modified CS beads’ characterization, morphological analysis was carried out by SEM, FT-IR, and XRD.The SEM analysis showed that the treatment of the GA-crosslinked CS beads modifies their surface texture. −1 due to the superposition of the OH’s stretching vibrations and NH2 functional groups in CS. Bands at 2875, 1022, and 1370 cm−1 are attributed to stretching vibration C–H, C–O, C–N, respectively, indicating the polysaccharide structure of CS [−1 is due to the bending of the primary amine’s N–H bond. A band centered at 1646 cm−1 is assigned to the C=O stretching vibration in the amide group’s CS due to partial deacetylation of chitin to produce CS [−1 that belongs to the imine bond (C=N) typical of crosslinking [−1 overlapping with the vibration band at 1646 cm−1 obtained in the spectrum without cross-linking, and the cross-linking reaction is evidenced by the small increased intensity of the stretch band. A small increase in the 2875 cm−1 band corresponding to the C-H crosslinked bond is overlapped with the -CH2- groups in the GA structure. In general, the spectrum showed an increase in intensity in the bands attributed to the bands generated by the various CS bonds  overlapped with the GA structure’s bonding groups [On the other hand, the FT-IR spectrum of the CS beads without crosslinking A shows cre of CS ,34,47. Tg groups .Finally, the X-ray diffraction pattern of the non-reticulated CS beads  presenteThe high crystallinity is due to the arranged saccharide structure since the hydroxyl and amino groups could form strong intermolecular and intramolecular hydrogen bonds .Furthermore, the structure of CS molecules has a certain regularity. As a result, CS molecules form crystalline regions ,53. In cTo determine the kinetic model that best fits the experimental data and describes the adsorption mechanism, the non-linear models of pseudo-first order (PFO), pseudo-second order (PSO), Elovich, and intraparticle diffusion were analyzed. −1 min−1) corresponds to the initial adsorption rate, and β is related to the degree of coverage and activation energy of the surface [The experimental results showed that the adsorption equilibrium time is reached at 465 min, which corresponded to an 87 % removal efficiency and an adsorption capacity of 1.16 mg/g. Of the models applied in the kinetics, the Elovich model better fit because of the highest determination coefficient and represents the adsorption kinetics of the RB4 dye on the GA-crosslinked CS beads. The constant α  of the RB4 dye with the amino group (NH3+) and the positively charged hydroxyl group (HO+–H) available on the surface GA-crosslinked CS beads are favored when the total "charge" of CS adsorbent is highly cationic, due to the stronger protonation of functional groups at acid pH [The adsorption mechanism of the RB4 dye on the surface of the GA-crosslinked CS beads is shown in  acid pH . Other i acid pH ,60,61,62FK of the model indicated an adsorption capacity of 0.933 when equilibrium was reached. In this sense, the most attractive junction sites occupied the first spaces, and the energy of adsorption decreased with the increasing degree of occupancy of the active adsorption sites [The Freundlich isotherm model provided a better fit with the experimental equilibrium data. This indicated that the system’s adsorption process is not limited by generating the monolayer of molecules of the RB4 dye. Instead, there is a heterogeneous distribution of the active sites on the GA-crosslinked CS beads’ surface. The constant on sites ,63,64. Ton sites ,66. TablThe sorption capacities obtained by various adsorbents in the removal of RB4 are compared in The present study experimentally demonstrated that RSM designs allow locating the most suitable conditions where the response is maximized. The mathematical models obtained in the RSM designs provided good fits with the experimental data at a 95% confidence level. A maximum predicted value of 60.65% was obtained for the experimental design, where the swelling degree was applied as a response variable. Likewise, it was found that the GA cross-linked CS beads increased their degree of swelling at acidic pH due to the protonation of the amino groups on the beads, which acted as repulsive forces between the polymer chains. The complete elimination of RB4 was achieved in the experimental design’s elimination efficiency.−1 that was found to overlap with the vibration band of 1646 cm−1 the functional group C=O. The adsorption kinetics suggested that the interaction mechanism was controlled by chemisorption. The equilibrium data indicated that the RB4 molecules are heterogeneously distributed in the active sites on the CS/GA beads’ surface. Finally, the GA crosslinked beads presented in this work can act as an ecological adsorbent, capable of efficiently removing anionic dyes in water bodies.The crosslinking reaction produced between CS and GA was demonstrated through SEM images, which showed the crosslinking’s rough surface characteristic. Crystallinity and chemical structure changes were confirmed by XRD and FTIR spectra. The existence of amorphous and high crystallinity regions of the CS beads was identified at values of 2θ = 10°, 20°, and 41°. When crosslinking with GA occurred, a loss of crystallinity occurred, eliminating the peak at 10° and decreasing the peak at 20°. The crystallinity index also confirmed the decrease in the non-crosslinking CS’s crystallinity from 25% to 16% with the crosslinking agent’s addition. In the FTIR, beads’ crosslinking was supported by an imine bond stretching band (C=N) at 1648 cm


TAPIR-1 and -2). To test their therapeutic potential, knockdown experiments with siRNA were carried out. The knockdown caused an increase in the p53/TP53 tumor suppressor protein level followed by downregulation of a large number of cell cycle- and DNA-damage repair key regulators. Furthermore, in radiation therapy resistant tumor cells, the knockdown leads to a renewed sensitization of these cells to radiation treatment. Accordingly, in a preclinical PCa xenograft model in mice, the systemic application of nanoparticles loaded with siRNA targeting TAPIR-1 significantly reduced tumor growth. These findings point to a crucial role of TAPIR-1 and -2 in PCa.In search of new biomarkers suitable for the diagnosis and treatment of prostate cancer, genome-wide transcriptome sequencing was carried out with tissue specimens from 40 prostate cancer (PCa) and 8 benign prostate hyperplasia patients. We identified two intergenic long non-coding transcripts, located in close genomic proximity, which are highly expressed in PCa. Microarray studies on a larger cohort comprising 155 patients showed a profound diagnostic potential of these transcripts (AUC~0.94), which we designated as tumor associated prostate cancer increased lncRNA ( Prostate cancer (PCa) is the most commonly diagnosed cancer in men world-wide. The present standard protein-based prostate specific antigen (PSA) screening test is being disputed due to a high rate of false positive findings ,2,3. BetAdvances in sequencing and functional genomics have shown that large, previously unrecognized segments of the human genome are being transcribed. ENCODE predictions suggest that ~80% of the genome’s DNA has been transcribed into RNA ,10,11. ATAPIR-1 and -2, respectively), which are strongly upregulated in prostate tissue specimens of patients suffering from PCa. The finding was verified using microarray studies on a larger validation cohort comprising 155 patients thus indicating the diagnostic potential of these transcripts. Furthermore, the value of TAPIRs to serve as promising therapeutic targets was demonstrated by the following findings: the knockdown of TAPIRs: (a) leads to a prominent cell cycle arrest, (b) reduces tumor growth in a xenograft mouse model and (c) results in a renewed sensitization to X-ray treatment.In this study, we discovered, via transcriptome-wide next generation sequencing (NGS), a genomic locus encoding two yet uncharacterized lncRNA transcripts ENST00000438247.1 and ENST00000366424.2 (designated here as TAPIR-1 and -2). They are strongly upregulated in PCa compared to tumor-free and BPH tissue  and encoded on the minus DNA strand. The locus is flanked by thyroid peroxidase (TPO) and peroxidasin (PXDN) genes. To determine whether TAPIR-1 and -2 represent non-coding transcripts, we used CPAT, a tool that predicts the coding potential of the transcript   and patients who survived or died of other causes  . Analyzing the differential mRNA expression obtained from tissue specimens of the diagnostic mRNA markers DLX1 and HOXC6  an AUC of 0.94 [0.91–0.97] and 0.97 [0.94–0.99] was revealed, respectively  patients were screened using transcriptome-wide deep sequencing (NGS). In total, 2040 differentially expressed genes were found /p.adj < 0.01), 1270 of them being upregulated and 770 being downregulated. Among them we identified an intergenic lncRNA locus on chromosome 2  encoding two transcripts (ENST00000438247.1 and ENST00000366424.2) A. Here, H tissue . Both transcript . Based os ncRNAs . Microars ncRNAs B,C Patiecore >7) D. The ovectively A. A more86–0.95] . Althougive/DoC) B. Notablectively C. The AUectively D. These TAPIR in PCa tissue (median log2 intensity ± sd: 7.68 ± 0.77 (TAPIR-1), 7.76 ± 0.93 (TAPIR-2), 7.70 ± 0.85 (TAPIR-1 und -2); n = 164) and low expression in BPH tissue specimens (median log2 intensity ± sd: 6.28 ± 0.25 (TAPIR-1), 5.90 ± 0.37 (TAPIR-2), 6.09 ± 0.36 (TAPIR-1 and -2); n = 39) and tumor-adjacent tissue (median intensity + sd: 6.26 ± 0.49 (TAPIR-1), 5.90 ± 0.58 (TAPIR-2), 6.12 ± 0.56 (TAPIR-1 and -2); n = 52) . To visualize TAPIR-transcripts ViewRNA® in situ hybridization (ISH) was carried out using formalin-fixed paraffin-embedded prostate tissue sections  and PCa tissue at the same section). Specific staining was observed in PINs and PCa areas and no or weak staining in extended normal glands, whereas connective tissues were unstained , whereas TAPIR-2 showed broad but low expression in brain cerebellum (n = 121), testis (n = 165), brain cerebellar hemisphere (n = 98), normal breast (n = 181) and prostate tissue (n = 100). Moreover, the analysis of expression profiles in tumor tissues  from the Cancer Genome Atlas (TCGA) consortium showed similar expression patterns for both TAPIR transcripts (TAPIR transcripts in PCa (n = 556) and also revealed elevated expression levels in invasive breast cancer (n = 1253).Custom expression microarray analysis revealed a strong expression of  n = 52) B–D. Signnstained . The GTEnscripts . TAPIR-1nscripts . In lineTAPIR-1 and -2, four different specific siRNAs derived from TAPIR-1 and -2  assay  after transfection. Cell cycle analysis revealed an enrichment of cells in G0/G1 accompanied by an equivalent decrease in S and G2/M phases by 50% upon si-A1-mediated TAPIR-1 knockdown. TAPIR-2 knockdown revealed similar but less profound effects on cell cycle distribution. Similar results were observed for MDA-PCa2b cells. Additional experimental evidence of the specificity of the siRNAs could be obtained by DU145 cells that do not express significant levels of TAPIR-1 or -2 lncRNA transcripts. Transfection of siRNAs (siA1 and siA2) in these cells does not alter their proliferation and does not lead to increased levels of p21CIP1/CDKN1A repression or repression of cell cycle key regulators like CCNB1 and CDC25C (TAPIR-1 and -2 (see As seen in A) assay C. RTCA aA) assay D. While d CDC25C . We alsod -2 see . As a reTAPIR-1 and -2 knockdown showed a downregulation of a broad spectrum of cell cycle genes, comprising all central regulators  and Fanconi anemia DNA-repair pathway (pGFdr = 1.1−6)  . Importasfection A, and thn levels A,D in LNnd MKI67 A. Severand CCNB2 ,27,28,291/CDKNA1 .BRCA1 , CHEK1 (Checkpoint kinase 1), and CLSPN (Claspin). Furthermore, numerous key regulators of the DNA-damage response and of the Fanconi anemia pathway were found to be downregulated, suggesting that the DNA repair system is inhibited in response to TAPIR knockdown. All these genes are described as validated p53-DREAM repressed target genes .All gene expression studies are accessible from the Gene Expression Omnibus (GEO) database. NGS of the exploration cohort of PCa and BPH tissue specimens: GSE134073 [reviewer access token: crgtacqetjudhyn], Agilent custom expression microarray of PCa and BPH tissue specimens : GSE134051 [reviewer access token: gtcvummelhmnryv], and Agilent expression microarray analysis following TAPIR-1 and -2, which are strongly upregulated in prostate tissue specimens of patients suffering from PCa. The TAPIR expression allows a remarkably precise diagnostic discrimination between PCa tumor and benign control samples (AUC = 0.94), as shown for a cohort comprising samples from more than 200 patients. Therefore, the development of a diagnostic PCa test is in progress.In this study, we discovered two lncRNA transcripts, designated here as TAPIR lncRNAs may also serve as promising therapeutic targets. The siRNA-based knockdown of TAPIR-1 and -2 results in an upregulation of the tumor suppressor p53 protein, which activates the p53/TP53-DREAM pathway. This mechanism leads to the downregulation/inhibition of CDKs and many other cell cycle regulating proteins, which finally results in cell cycle arrest. Accordingly, in a preclinical PCa xenograft model in mice, the systemic application of nanoparticles loaded with siRNA targeting TAPIR-1 significantly reduced tumor growth in vivo. Furthermore, the activation of the p53-DREAM pathway leads to suppression of essential key regulators of the DNA-damage response, as there are BRCA1, CHEK1, CLSPN, and genes encoding proteins of the Fanconi anemia pathway. Therefore, we studied the effect of TAPIR siRNA when treating radiation therapy-resistant tumor cells. In addition to a strong proliferation inhibition, the knockdown leads to an impressive renewed sensitization of these cells to X-ray treatment. The influence of castration resistance and chemoresistance must be investigated in follow up studies.Furthermore, we show that The following patents have resulted from the work reported in this manuscript: RNA-biomarkers for diagnosis of prostate cancer EP3077537A1 (EU) and EP3077537B1 (US).


Ciona robusta and Ciona intestinalis, can produce hybrids. However, wild sympatric populations display limited gene flow, suggesting the existence of obstacles to interspecific reproduction that remain unknown. Here, we took advantage of a closed culture system to cross C. robusta with C. intestinalis and established F1 and F2 hybrids. We monitored post-embryonic development, survival, and sexual maturation to characterize the genetic basis of simple traits, and further probe the physiological mechanisms underlying reproductive isolation. Partial viability of first and second generation hybrids suggested that both pre- and postzygotic mechanisms contributed to genomic incompatibilities in hybrids. We observed asymmetric fitness, whereby the C. intestinalis maternal lines fared more poorly in our system, pointing to maternal origins of species-specific sensitivity. We discuss the possibility that asymmetrical second generation inviability and infertility emerge from interspecific incompatibilities between the nuclear and mitochondrial genomes, or other maternal effect genes. This work paves the way to quantitative genetic approaches to study the mechanisms underlying genomic incompatibilities and other complex traits in the genome-enabled Ciona model.Reproductive isolation is central to speciation, but interspecific crosses between two closely related species can produce viable and fertile hybrids. Two different species of tunicates in the same ascidian genus,  Reproductive isolation is central to speciation , and resCiona, distinct type A and type B were identified within the species Ciona intestinalis. These types were first thought to represent cryptic sub-species  and Ciona intestinalis  adults were collected in San Diego (CA) and Woods Hole (MA), respectively, and are within the range of known distribution for these species  as food for Ciona juveniles and adults, 107 to 108 cells for each Petri dish, and 109 to 1010 cells for tanks. Stock, starter, and scale-up cultures of algae were kept in 250mL, 500mL and 2L flasks, respectively. Terminal food cultures were kept in 10L carboys. The flasks and carboys were maintained under constant light (Marineland), and were shaken once a day to prevent sedimentation. The cultures were inoculated every 10 to 14 days. Half of the cultures were diluted with autoclaved artificial sea water  for the next round of cultures. We added 1mL Conway medium  per 1L artificial sea water for scale-up and ongoing food cultures of Chaetoceros. Conway medium contains: 45g/L EDTA  100g/L Sodium nitrate , 33.3g/L Boric acid , 20g/L Sodium dihydrogen phosphate , 1.5g/L Manganese (II) chloride , 1.3g/L iron (III) chloride , 21mg/L Zinc chloride , 20mg/L Cobalt (II) chloride , 10mg/L Ammonium heptamolybdate ((NH4)6Mo7O24•4H2O, Acros Organics), 21mg/L Copper (II) sulfate , 10mg/L Thiamine (Acros Organics), 10mg/L Cyanocobalamin (Acros Organics), 200μg/L Biotin (Fisher Scientific).We essentially followed an established protocol (The culture system held 20L glass aquarium tanks (Carolina), 5L polypropylene beakers (Midland Scientific) and 2L polycarbonate aquarium tanks (Eisco), which each could hold 16, 4 and 2 Petri dishes, respectively . The 20L tanks, 5L beakers and 2L small tanks were set in an 18° Chamber. Sea water  was controlled by bio-balls  seeded with bacteria , and salinity was set at 34 ppt. The 20L tanks were cleaned twice a week, and the 5L beakers and the 2L small tanks were cleaned three times a week. The 20L tanks, 5L beakers and 2L small tanks each efficiently supported animal growth and sexual maturation .F1 juveniles were taken from Petri dishes and digested by proteinase (Thermo Fisher Science) to obtain genomic DNA as described previously  and Woods Hole (MA), respectively  at the tip of the sperm duct in Ciona species is the color of siphons , although this feature was deemed quite variable and taxonomically unreliable  locus, which could distinguish C. robusta and C. intestinalis alleles by the size difference of PCR products . We isolated genomic DNA from three F1 juvenile individuals from each type. Six juveniles of either C. robusta or C. intestinalis yielded single bands, albeit of higher molecular weight for the latter . By contrast, six juveniles of either RxI or IxR crosses yielded double bands, showing that these animals had both C. robusta and C. intestinalis Myl2/5/10 alleles, and were indeed hybrids. Consistent with electrophoresis patterns, sequence analysis revealed single alleles for either C. robusta or C. intestinalis, whereas F1 RxI and IxR hybrids produced a mixture of C. robusta- and C. intestinalis-specific sequences . Of note, genomic DNA from both somatic tissue and gametes yielded similar results, whereby homotypic C. robusta and C. intestinalis produced single PCR bands in different sizes, while those of both hybrids produced double PCR bands  animals and eggs from C. robusta collected from the wild, and kept F2 C. robusta animals as controls. We counted the proportion of fertilized eggs out of total eggs to score fertilization rates 1, (R2I1)3, (I1R2)4, (I1I2)1 and (I1I2)2 might have been sterile xR and (IxR)xR hybrids had lower survival rates than F2 C. robusta animals, while an ANOVA did not show significant differences in survival rate on 28 and 50 dpf between (RxI)xR and (IxR)xR hybrids. These observations suggest that BC1 hybrid juveniles experience higher mortality rates, consistent with proposed genomic incompatibilities in second generation hybrids , while the size of BC1 (RxI)xR and (IxR)xR hybrids varied between 0.5 and 8 mm ((RxI)xR; average = 3.82, SD = 1.78, n = 47, (IxR)xR; average = 3.70, SD = 2.22, n = 25). This suggested that growth rates are more variable in the BC1 hybrid population, as expected following the segregation of alleles for a likely multigenic trait such as individual growth rate.As a complement to survival, we measured the body size of BC1 animals at 28 dpf . The sizC. robusta trait. Half of the individuals in both BC1 (RxI)xR and (IxR)xR hybrids had red color in the rim of siphons, as did F1 hybrids, while the other half had transparent siphons, the same as normal C. robusta xR and (IxR)xR hybrids grew to mature adults, respectively, thus allowing us to observe the presence of OPO and the color of their siphons . Except  robusta . This coC. robusta eggs to produce BC2 hybrids, which survived at least 28 dpf, after which we stopped observations. This indicates that the BC1 hybrids that survive, grow and mature are potentially fertile. This possibility is not incompatible with the existence of DMI. Instead, it is consistent with the existence of defined hotspots of unidirectional introgression observed in wild populations xR and (IxR)xR BC1 hybrids grew and matured to produce sperm and eggs . Notably, an ANOVA showed significant differences in the metamorphosis rate between F2 RxI and IxR hybrids. Because of low fertilization and metamorphosis rates, we obtained only 7 F2 RxI hybrid juveniles by 5 dpf, compared to 137 F2 IxR hybrid juveniles .Next, we leveraged the fertility of izations  and 4. Fizations . Specifiizations . The datizations . Thus, wizations . The metuveniles . In tota robusta . There w robusta . Only 1 i.e., influenced by the environment, especially the availability of food, we surmise that most of the observed variation in controlled laboratory conditions is due to polygenic effects.We could measure body sizes for only 3 and 2 F2 RxI hybrid individuals at 28 and 50 dpf, preventing robust statistical analysis. By contrast, 85 and 57 F2 IxR hybrid individuals measured at 28 and 50 dpf showed a range of body sizes similar to that of BC1 hybrids . This isC. robusta trait. Three-quarters of the F2 IxR hybrids had red color in the rim of siphons, as did F1 hybrids, while the other quarter had transparent siphons, the same as normal C. robusta 10x(I2R1)20 and (I3R4)11x(I4R3)21) 10x(I2R1)20 and (I3R4)11x(I4R3)21, respectively . By contrast, at the marker 2 locus, there were 2 and 1 larvae homozygous for the C. robusta allele, 13 and 18 heterozygous larvae, and 9 and 5 larvae homozygous for the C. intestinalis allele out of 24 and 24 verified samples in (I1R2)10x(I2R1)20 and (I3R4)11x(I4R3)21, respectively . Biased genotype in Myl2/5/10 showing less C. intestinalis type and marker 2 showing less C. robusta type, suggests that these genes of homozygous type are linked to loci depleted in F2 hybrids populations. Because Myl2/5/10 and marker 2 genes are on different chromosomes and neither are located in the inferred hotspots of introgression , their relationships to documented “hotspots” of introgression 21) . All theectively . The proectively . At thisCiona robusta and Ciona intestinalis to establish hybrid lines, further probe the reproductive isolation of these recently distinguished species, and explore opportunities for quantitative genetics using the genome-enabled Ciona model. Taking advantage of a simple inland culture system, we monitored post-embryonic development and survival, and successfully raised F1 and F2 hybrid and backcrossed animals to maturity. The partial viability of first and second generation hybrids provided insights into the genetics of simple traits, such as the presence of OPO, which appears to be a dominant C. robusta-specific trait. On the other hand, siphon pigmentation showed an overdominant phenotype in hybrids, suggesting more complex genetic interactions, although the trait distribution in F2 hybrids could be explained by allele segregation at one locus. Moreover, simple quantitative traits, such as body size, showed an increased variability in F2 hybrids as expected for polygenic traits following allele segregation. These observations suggest that quantitative genetics approaches could be used to study complex traits that differ between C. intestinalis and C. robusta, such as tolerance to high water temperature (In this study, we crossed the ascidian species Ciona species. These observations are consistent with previous reports (C. robusta and C. intestinalis genotypes are viable, which would permit at least low levels of gene flow between populations, and is consistent with the existence of previously reported hotspots of introgression (Despite preliminary evidence of allele segregation in F2 hybrids, the representation of genotype combinations is likely to be biased due to genomic incompatibilities, which might hinder quantitative analysis of polygenic traits. Indeed, our observations suggest that both pre- and postzygotic mechanisms contribute to genomic incompatibilities in hybrids, and thus act as obstacles to interspecific reproduction between these two Ciona robusta and Ciona intestinalis (C. intestinalis maternal lineages. Indeed, this asymmetry suggested that mechanisms of genomic incompatibility involve interactions between the nuclear genomes and maternal determinants inherited in a trans-generational manner, such as mitochondrial DNA (C. intestinalis animals, we cannot formally rule out the possibility that the C. intestinalis maternal lineage itself causes reduced fitness in our culture system, independently of alleged incompatibilities between the C. robusta and the C. intestinalis genomes. Thoroughly addressing this possibility will require the development of culturing conditions more compatible with C. intestinalis.Asymmetric fertilization success in reciprocal interspecific crosses  was prevC. intestinalis fitness sufficed to explain our results, we would expect to also observe a markedly lower fitness of F1 RxI hybrids compared to F1 IxR hybrids, which emerge from the C. robusta maternal lineage. To the contrary, we could obtain F1 RxI hybrids and raise them to maturity to produce backcrossed animals, which suggested a more pronounced reduction of fitness in second generation RxI hybrids from the C. intestinalis maternal lineage. Moreover, we reasoned that the second generation in the RxI lineage, which necessary follows gametogenesis and possible recombinations between C. robusta and C. intestinalis chromosomes in F1 RxI hybrids, is the first generation where the C. intestinalis mitochondrial genome could encounter homozygous C. robusta alleles in the nuclear genome, which encodes the majority of mitochondrial proteins. For instance, incompatibilities between the nuclear and mitochondrial genomes in hybrids were reported in various organisms, including fungi (C. robusta and C. intestinalis genomes limit the fitness of the RxI hybrid lineage, in a way that depends on the environment, on possible maternal effects genes and/or on specific interactions between the nuclear and mitochondrial genomes. Finally, it is tempting to speculate that these asymmetric incompatibilities provide a mechanistic explanation for the unidirectional introgressions observed in wild populations (Nonetheless, if low ng fungi , insect


Hybrid physics-machine learning models are increasingly being used in simulations of transport processes. Many complex multiphysics systems relevant to scientific and engineering applications include multiple spatiotemporal scales and comprise a multifidelity problem sharing an interface between various formulations or heterogeneous computational entities. To this end, we present a robust hybrid analysis and modeling approach combining a physics-based full order model (FOM) and a data-driven reduced order model (ROM) to form the building blocks of an integrated approach among mixed fidelity descriptions toward predictive digital twin technologies. At the interface, we introduce a long short-term memory network to bridge these high and low-fidelity models in various forms of interfacial error correction or prolongation. The proposed interface learning approaches are tested as a new way to address ROM-FOM coupling problems solving nonlinear advection-diffusion flow situations with a bifidelity setup that captures the essence of a broad class of transport processes. Numerical simulations are the workhorse for the design, testing, and implementation of scientific infrastructure and engineering applications. While immense advances in computational mathematics and scientific computing have come to fruition, such simulations usually suffer a curse of dimensionality limiting turnaround. The field of multifidelity computing, therefore, aims to address this computational challenge by exploiting the relationship between high-fidelity and low-fidelity models. One such multifidelity approach becomes crucial, especially for multi-query applications, such as optimization, inference, and uncertainty quantification, that require multiple model evaluations in an outer-workflow loop. To this end, sampling-based approaches have been often introduced to leverage information from many evaluations of inexpensive low-fidelity models fused by only a few carefully selected high-fidelity computations. An excellent review of the state-of-the-art multifidelity approaches for outer-loop contexts can be found in .zonal multifidelity approach that we tackle in this work. In general, boundary-layer control poses a grand challenge in many aerospace applications including lift enhancement, noise mitigation, transition delay, and drag reduction. Among many other actuator technologies, blooming jets  form orthonormal basis functions for a q-dimensional subspace complementing that spanned by Φ and β defines the corresponding projection coordinates. Similar to Φ, Ψ can be computed through the POD algorithm. Note that Φ and Ψ are orthogonal to each others . Indeed, Ψ represents the next q basis functions generated by POD after the first r being dedicated to Φ. Those are also constructed a priori during an offline stage using the collected set of snapshot data. We highlight that the Galerkin ROM equations only solve for α to keep the computational cost as low as possible.proposed , where bβ has to be constructed so that the uplifting approach can be employed. To accomplish this, a mapping from the first r modal coefficients to the next q modes is assumed to exist. Nonlinear Galerkin projection has been pursued to express this mapping as r modal coefficients as input, and return the true coefficients of the first r + q modes. Thus, it provides a closure effect for the first r modes and a superresolution effect for the next q modes, simultaneously in a single network as follows,r + q spatial modes have to be built and stored beforehand, which introduces slightly more storage costs. For the present study, we explore the specific case where q = r, but a generalization is straightforward.Therefore, a complementary model for  systems . InsteadIn the following, we formulate the four methodologies outlined in ζ as below α, β, and γ are the corresponding projection coordinates. We also suppose that Galerkin ROM resolves the Φ set of modes . We reiterate here that the Galerkin ROM yields inaccurate solution (sketched by the noisy (rough) curve of predicted α). Consequently, the quality of the direct prolongation mapping is compromised. The PCI aims at correcting the reconstructed solution at the interface. Even though the PCI technique acts only on a small portion of the domain , it might amount to excessively large input and output sizes.The ROM-FOM coupling philosophy as well as the introduced interface learning approaches are summarized in the cartoon shown in α. This closure simply compensates the effects of truncated modes  onto the dynamics of Φ. This yields a better estimate of α, as illustrated by the smooth curve in ζ are only considered to improve the prediction of α. However, their contributions to the solution manifold reconstruction are not included, resulting in a substantial reconstruction error (projection error). To deal with this caveat, UROM seeks to add a superresolution enhancement by incorporating the Ψ set of basis into the reconstruction step by learning the dynamics of the corresponding β coordinates. This is represented by a higher resolution (denser) reconstruction in UPI case, compared to CPI, and DPI. Note that the PCI is still showing the highest resolution (the densest reconstruction) as it nudges the prediction at the interface to its FOM counterpart .On the other hand, CPI treats the Galerkin ROM deficiencies at the ROM level. In particular, it introduces a closure effect to better predict d = 1). Second, we consider the Marsigli flow problem represented by the two-dimensional Boussinesq equations to demonstrate the ROM-FOM coupling for multiphysics systems. In particular, a ROM solver is devoted for the mass and momentum transport equations while a FOM is reserved for the energy transport.We demonstrate the ROM-FOM coupling methodologies using two examples of varying complexities. In the first one, we describe a fluid flow scenario over a bizonal domain with heterogeneous physical properties using the one-dimensional Burgers problem. For this case, we shall see that the interface between different sub-domains is defined by a single point  solver over the whole domain, the quality of the solution will be dominated by stiffness of the left zone. In other words, a smaller time step would be required to satisfy numerical stability when using an explicit temporal integration scheme. Specifically, assuming that we utilize the forward in time and central in space finite difference scheme (FTCS) to solve −6 will be required for the left part of domain, while a time step of 2.5 × 10−4 would be sufficient if we were able to only resolve the right part. Therefore, domain decomposition approaches might be adopted to segregate partitions with varying numerical requirements. Despite the effectiveness and efficiency of these approaches, idle delays can arise in order to accommodate information transfer from the left zone to the right zone through their common interface. Instead, low-fidelity proxy models can be utilized to avoid such lags by approximating the effective dynamics of stiff regions and providing sound interface boundary conditions to the rest of the computational domain.In order to represent a zonal multifidelity simulation, we consider the following one dimensional (1D) viscous Burgers problem,x ∈  and a right zone with x ∈ , where n1 + n2 = n + 1. We build a reduced order model for the left sub-domain as follows, a;b〉 = aTb).The discretized domain is divided into a left zone with −6. For initial condition, we consider a square-like wave defined as,xb = 0.75 is considered as the location of the interface and ϵ = 0.005 is used to define the sharpness of the shock at xb. Dirichlet boundary conditions are assumed at both boundaries  = u = 0). We compute the evolution of the velocity field for t ∈ , and collect snapshots every 100 time steps. That is snapshots are collected every 2.5 × 10−4 time units .For data generation, we solve the full order model representing the 1D Burgers equation  over thex ≤ 0.75) for t ∈ . For POD basis generation, we use only 200 snapshots distributed evenly from t = 0 to t = 1, to reduce the computational cost of solving the corresponding eigenvalue problem. Once ROM is constructed, it is integrated in time with a time step of 2.5 × 10−4 to match the time step in the right part of the domain (to be handled via a FOM solver). During the deployment phase of the coupled system, the ROM feeds the FOM solver with the boundary condition at xb. On the other hand, the effects of the interface on the ROM dynamics are considered during the offline stage of data generation and basis construction.For ROM construction, we consider the truncated solution snapshots for the left part of the domain . We highlight that numerical experiments reveal that the performance in our case is not significantly sensitive to the neural network hyperparameters. Thus, we manually tune the hyperparameters during the training and validation stages by repeating the previous procedure multiple times while varying the seeds for the random number generator and using averaged loss values for assessment. We adopt an LSTM architecture of 2 layers with 20 cells in each layer. In the meantime, we note that automated hyperparameter search tools can be utilized for optimized architectures. Testing of the proposed schemes is performed for t ∈ , corresponding to a temporal extrapolation behavior with respect to the training interval.We utilize labelled data at the time interval of xb, we notice that the Θ in the DPI map  represents the projection of the truncated velocity field in the left zone onto the POD subspace. The shaded area in Since the coupling between ROM and FOM is represented by the physical interface at r = 2, are not very accurate due to the mutual effects of modal truncation and model nonlinearity in Galerkin ROM. On the other hand, the PCI solution gives almost perfect match with FOM. As the PCI approach nudges the prolonged ROM solution to its FOM counterpart, it gives even higher accuracy than TP. That is TP is limited by the maximum quality that can be obtained using a rank-r approximation. For CPI, since the LSTM introduces a closure effect, it steers the ROM results to match the TP solution. Finally, the UPI recovers some of the smaller scales (truncated modes) so it yields better reconstruction than TP since it spans a larger subspace. For r = 2, UPI uplifts the solution to a rank-4 approximation, while for r = 4, it is uplifted to a rank-8 approximation.It can be seen that DPI results, especially for ℓ2 norm of the deviation of the predicted velocity at the interface compared to the FOM solution in r ∈ {2, 4, 8}. We see that the error in the CPI case almost matches that of TP, while PCI gives the highest accuracy since it is trained to learn the correction with respect to the FOM solution. Also, CPI results are significantly close to TP, illustrating the closure effect introduced by CPI to account for the effect of modal truncation on ROM dynamics. Another interesting observation is that UPI quality at a given value of r is equivalent to TP with twice that value. This indicates that UPI is able to give a superresolution effect up to 2r (since we select q = r). Also, we notice that at r = 2, DPI yields lower ℓ2 norm than TP. This is because TP solution is obtained by the projection of the FOM solution onto the POD subspace generated using data at t ∈ . So, this subspace is optimal only for t ∈ , while testing is performed up to t = 2. Therefore, TP solution no longer represents the best rank-r approximation beyond t = 1.For quantitative assessment, we document the x ≤ 1) using a high fidelity FOM solver applied only onto this subdomain. This is fed with a boundary condition u from the low-fidelity interface learning approaches described before. r = 2, compared to the FOM predictions. Again, we observe that the solution with PCI boundary is similar to this FOM solution. Also, CPI matches the TP results, but they both smooth-out the surface peak because of the low rank limitations. Although it seems that the accuracy of UPI cannot exceed that of CPI with r + q assuming optimal performance for both CPI and UPI, there is a computational side in this comparison. The CPI with r + q modes implies the solution of a Galerkin ROM with a dimension of r + q, while the UPI requires the solution of a Galerkin ROM with r. We have seen that for fluid flows with quadratic nonlinearity, the computational cost of solving a Galerkin ROM scales cubically with the number of modes. Thus, the implementation of CPI with r + q with q = r is about 8 times more costly than UPI with r + q. At this point, we highlight that the selection of r and q is highly dependent on the problem in hand, the corresponding decay of POD eigenvalues, and the level of accuracy sought. A compromise between computational cost of solving a Galerkin ROM with r and the corresponding stability, the amount of information captured by r and r + q modes, and reliability of UPI with r + q is always in place.Finally, we investigate the coupling efficiency by solving the right part  for both ω and ψ as follows,We utilize the 2D Boussinesq equation to illustrate the ROM-FOM coupling for multiphysics situations. In particular, we suppose that we are more interested in the temperature field predictions. Thus, we dedicate a FOM solver for β represent the projection of the temperature fields onto the reduced subspace defined asθ and Φθ are the corresponding orthonormal POD modes. The predetermined coefficients in Thus, the Galerkin ROM of β values. This constitutes a two-way ROM-FOM coupling problem, in contrast to the one-way coupling in the aforementioned 1D Burgers example. We also highlight that the computational cost of the projection step  ∈  ×  with a a vertical barrier dividing the domain at x = 4, keeping the temperature, θ, of the left half at 1.5 and temperature of the right half at 1. Initially, the flow is at rest  = ψ = 0), with uniform temperatures at the right and left regions  = 1.5 ∀ x ∈  and θ = 1 ∀ x ∈ . Free slip boundary conditions are assumed for flow field, and adiabatic boundary conditions are prescribed for temperature field. Reynolds number of Re = 104, Richardson number of Ri = 4, and Prandtl number of Pr = 1 are set in Eqs  slides below the lower density fluid (on the left) causing an undercurrent flow moving from right to left. Conversely, an upper current flow moves from left to right, causing a strong shear layer between the counter-current flows. As a result, vortex sheets are produced, exhibiting the Kelvin-Helmholtz instability.The problem is defined by two fluids of different temperatures, in a rectangular domain (t in Eqs  and 24)x, y) ∈ [a scheme  is used 6. Building and training of LSTMs with an input and output vector sizes of two millions become prohibitive. Even the training of convolutional neural network with such high resolutions  requires excessive computational resources. Therefore, we present results for ROM-FOM coupling using approaches that operate in ROM space . We note that 800 time snapshots are 454stored for POD basis construction. For the Galerkin ROM solver, r = 8 modes are retained. We also utilize the dataset of the stored 800 snapshots for LSTM training and validation . A two-layer LSTM with 20 cell in each layer constitutes our LSTM architecture. During the testing phase, the trained neural networks are deployed at each and every timestep. This corresponds to the application of the presented approached 16000 times.Considering the dimensionality of this problem, we emphasize that the PCI approach becomes highly unfeasible. For example, with the current mesh resolution , the dimension of the state space of the prolonged interface is ≈ 2 × 10t = 8) computed from DPI, CPI, and UPI approaches compared to the FOM field. We emphasize that the ROM-FOM coupling results correspond to the solution of the vorticity equation with a ROM solver, which feeds the FOM solver with streamfunction to solve the temperature equation only as opposed to the FOM results which comes from the solution of both the 2D Boussinesq equations using a FOM simulation. Although the CPI results are better than those of DPI, we can observe that the fine details of the flow field are not accurately captured. That is 8 modes are not sufficient to sufficiently represent the flow field. This is a common problem for convection-dominated flows which exhibit slow decay in the POD eigenvalues and the generated global basis functions suffer from modal deformation [r = 8 and q = 8 recovers an increased amount of the fine flow structures that are not well-represented by the first r = 8 modes.ormation . On the θ  framework to enhance the ROM approximation of interface conditions. A demonstration with a bizonal 1D Burgers problem is considered to assess the performance of the introduced learning schemes for multi-component systems. For 1D problems, we find that a prolongation followed by a machine learning correction interface (PCI) yields very good predictions. However, this might be unfeasible for 2D and 3D cases, where a correction in ROM subspace is essential. For such, a machine learning correction in ROM space followed by a prolongation interface (CPI) can produce sufficient accuracy. For complex systems where the projection error is significantly large, an uplifted prolongation interface (UPI) methodology can be adopted to recover some of the truncated scales. This is further illustrated using the lock-exchange problem governed by the 2D Boussinesq problem, where the ROM and FOM solvers address the vorticity and temperature equations, respectively. The coupling from ROM to FOM is represented by the 2D streamfunction fields reconstructed from the ROM solver, saving the run time for Possion equation, which is the most demanding part of an incompressible flow solver. Owing to the relative simplicity, robustness and ease of these interface learning methods, we expect a growing number of applications in a large variety of interfacial problems in science and engineering. Of particular interest, this ROM-FOM coupling could be a viable method for developing next generation digital twin technologies.


The emergence and rapid worldwide spread of a novel pandemic of acute respiratory disease – eventually named coronavirus disease 2019 (COVID-19) by the World Health Organization (WHO) – across the human population has raised great concerns. It prompted a mobilization around the globe to study the underlying pathogen, a close relative of severe acute respiratory syndrome coronavirus (SARS-CoV) called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Numerous genome sequences of SARS-CoV-2 are now available and in-depth analyses are advancing. These will allow detailed characterization of sequence and protein functions, including comparative studies. Care should be taken when inferring function from sequence information alone, and reverse genetics systems can be used to unequivocally identify key features. For example, the molecular markers of virulence, host range and transmissibility of SARS-CoV-2 can be compared to those of related viruses in order to shed light on the biology of this emerging pathogen. Here, we summarize some recent insights from genomic studies and strategies for reverse genetics systems to generate recombinant viruses, which will be useful to investigate viral genome properties and evolution. Coronaviridae, subfamily Coronavirinae, genus Betacoronavirus, in the species severe acute respiratory syndrome-related coronavirus [Betacoronavirus, the subfamily Coronavirinae is composed of the genera Alphacoronavirus, Gammacoronavirus and Deltacoronavirus. Seven types of CoVs are known to cause human disease. Several alphacoronaviruses (HC 229E and HC NL63) and betacoronaviruses (HC OC43 and HC HKU1) are endemic and cause mild respiratory tract infections [Coronavirus disease 2019 (COVID-19)  belonginonavirus . This paonavirus . On 11 Mfections . Previoufections . In Janufections . Therefofections . The genViral replication takes place in the cytoplasm. The genome organization of the virus is summarized in Clearly COVID-19 is different from the disease caused by SARS-CoV-2’s close relative, SARS-CoV. Indeed the case fatality rates for COVID-19 are lower, and the disease can be mild or asymptomatic . Investiin vitro ligation of DNA fragments covering the full-length genome, or vaccinia virus-based expression vectors [Reverse genetics is a powerful technique for the generation of an infectious virus from the cloned full-length cDNA/synthetized DNA of a given virus. Manipulation of the DNA by well-established molecular biology methods allows modification of the sequence before virus production, if this is desired. It allows unequivocal identification of molecular markers for a given virus, including the genome features, virulence, host range, etc. of a given virus, and as such it is a key component in the study of coronaviruses . Ultimat vectors . The sys vectors . A ligat vectors . Similar vectors . Both of


Immunoglobulin E (IgE) is pivotal for manifestation and persistence of most immediate-type allergies and some asthma phenotypes. Consequently, IgE represents a crucial target for both, diagnostic purposes as well as therapeutic approaches. In fact, allergen-specific immunotherapy – aiming to re-route an IgE-based inflammatory response into an innocuous immune reaction against the allergen – is the only curative approach for IgE-mediated allergic diseases known so far. However, this requires the cognate allergen to be known. Unfortunately, even in well-characterized allergics or asthmatics, often just a small fraction of total IgE can be assigned to specific target allergens. To overcome this knowledge gap, we have devised an analytical platform for unbiased IgE target epitope detection. The system relies on chemically produced random peptide libraries immobilized on polystyrene beads (“one-bead-one-compound (OBOC) libraries”) capable to present millions of different peptide motifs simultaneously to immunoglobulins from biological samples. Beads binding IgE are highlighted with a fluorophore-labeled anti-IgE antibody allowing fluorescence-based detection and isolation of positives, which then can be characterized by peptide sequencing. Setting-up this platform required an elaborate optimization process including proper choice of background suppressants, secondary antibody and fluorophore label as well as incubation conditions. For optimal performance our procedure involves a sophisticated pre-adsorption step to eliminate beads that react nonspecifically with anti-IgE secondary antibodies. This step turned out to be important for minimizing detection of “false positive” motifs that otherwise would erroneously be classified as IgE epitopes. In validation studies we were able to retrieve artificial test-peptide beads spiked into our library by using IgE directed against those test-peptides at physiological concentrations (≤20 IU/ml of specific IgE), and disease-relevant bead-bound epitopes of the major peanut allergen Ara h 2 by screening with sera from peanut allergics. Thus, we established a platform with which one can find and validate new immunoglobulin targets using patient material which displays a largely unknown immunoglobulin repertoire. Still, allergy diagnostic testing has been vastly improved in the past decades due to molecular allergology providing single allergen molecules, either naturally purified from the source or obtained by recombinant DNA technology  levels but do not react with the common allergens the patients usually are tested for in commercially available routine allergy diagnostic tests –3. In thchnology . The avachnology , 9. Multchnology –13, therchnology , 15. YetTherefore, smart approaches are needed in order to determine unknown antibody reactivities and to identify the interaction sites – the so-called epitopes – on the target antigens/allergens. In most cases, these epitopes are protein-derived entities, either linear chains or three-dimensional structures composed of amino acids. A promising line of action consists in offering a broad variety of peptidic targets to the antibody (mixture) in question, and to check for reactivity.vice versa by offering a defined IgE reactivity to a pool of peptide-presenting phages created by cloning random oligonucleotides into the permissive site of the scaffold protein gene  libraries constitutes a promising approach. The OBOC technology based on the “split-and-mix” synthesis was invented by Furka et al. , 26 and Neither the phage display nor the OBOC technique have been used to reveal epitopes of unknown serum IgE reactivities. We therefore wanted to address this question via the OBOC strategy. In the study presented here, we have developed the methodology for the identification of hitherto unknown IgE reactivities toward unknown allergens via detection of bead-bound linear peptide epitopes.2 resin was custom-made by Rapp Polymers , with the following specifications: approximately 7.1 million polystyrene beads per gram dry powder, 0.36 mmol amino functions/g, bead diameter 60–70 μm under dry conditions, approximately 50 picomoles of functional amino groups per bead. These beads were used as resin material for the on-bead peptide synthesis applying the fmoc solid phase peptide synthesis technique with an automated multiple peptide synthesizer .TentaGel S NHMonoclonal, humanized IgE antibodies directed against the c-myc epitope as well as anti-human IgE antibodies with different fluorophore labels were obtained from various sources as summarized in http://www.mimotopes.com.Peptide sequences to be synthesized onto the beads were chosen according to known target structures recognized by human IgE antibodies. As negative control, a scrambled version of each specific peptide was produced with an online tool at 2 resin (corresponding to approximately 1.5 × 106 beads) were swollen in 5 ml of a 7:3 mixture of dichloromethane  and dimethylformamide  and transferred in portions of approximately 2.2 × 105 beads (corresponding to 10 μmol amino functions) to 2 ml filter bottom reaction columns (Intavis). In the following, all reagent amounts are given per reaction column. All reaction steps were performed at room temperature. The resin was prepared for synthesis by washing three times with 800 μl of DMF. Fmoc deprotection was achieved by treating the resin two times for 8 min with 400 μl of a mixture of 20% (v/v) piperidine  in DMF and subsequent washing seven times with 750 μl of DMF. Coupling was done by reacting a 10-fold excess of fmoc-protected amino acid building blocks  with the resin. For this, the resin was incubated two times for 25 min each with a mixture of 77 μl of a 0.6 M fmoc amino acid building block solution in DMF, 25 μl of a 4 M solution of 4-methylmorpholine (Sigma-Aldrich) in DMF and 75 μl of a 0.6 M solution of 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (IRIS Biotech) in DMF. After washing three times with 750 μl of DMF, unreacted amino termini were capped for 5 min with 400 μl of a 5% (v/v) mixture of acetic anhydride (Merck) in DMF. Subsequently the resin was washed and extracted additional six times with 750 μl of DMF.200 mg of the TentaGel S NHin vacuo. Side chain protecting groups were cleaved off by treatment with 2 ml of cleavage cocktail  for 3 h at room temperature. After incubation, the cleavage cocktail was discarded and the resin beads were washed five times with 10 ml of DMF, five times with 10 ml of pure ethanol , five times with 10 ml of dichloromethane and again five times with 10 ml of DMF. Beads were treated with 30% (v/v) of H2O/DMF, 60% (v/v) of H2O/DMF and with neat H2O for 5 min each at room temperature. Subsequently, the resin beads were washed ten times with 10 ml of phosphate buffered saline , resuspended in 2 ml of PBS containing 0.05% (w/v) of NaN3  and stored at 4°C. The migration of the beads from organic solvents to an aqueous buffer system results in the swelling of the beads and a final diameter ranging from 90 to 110 μm.After synthesis the resin was treated three times for 8 min with 400 μl of a mixture of 20% (v/v) piperidine in DMF, washed seven times with 750 μl of DMF and five times with dichloromethane and dried 6 beads and 0.5 mmol amino functions) of the TentaGel S NH2 resin were swollen in 20 ml of a 1:1 mixture of dichloromethane and DMF for 1 h. The resin was distributed equally into 19 polypropylene vials , beads were allowed to settle and the supernatants were removed carefully. To each vial, 433 μl of a 0.6 M solution of one of 19 different fmoc amino acid building blocks in DMF, 433 μl of a 0.6 M solution of 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate in DMF and 47 μl of N-ethyldiisopropylamine (Sigma-Aldrich) were added. Vials were incubated on an end-over-end mixer at room temperature for 2 h. This way, every proteinogenic amino acid, except cysteine, was coupled to one portion of resin beads. After that, all 19 resin portions were combined in a 20 ml reaction column equipped with a 35-μm filter bottom (Intavis), mixed well, and the supernatant was removed. The resin was washed five times for 2 min at room temperature with 10 ml of DMF each. 10 ml of a solution of 20% (v/v) piperidine in DMF were added and incubated two times for 15 min at room temperature. After that, the resin beads were washed six times for 2 min with 10 ml of DMF, and the resin was again distributed equally to 19 polypropylene vials. This cycle of splitting and mixing the resin beads was repeated eight times to create different random 8mer peptides on the resin beads  and stored at 4°C after addition of NaN3 to a final concentration of 0.05% (w/v), in order to prevent growth of microorganisms.Amino acid side chain de-protection after the last cycle of peptide synthesis, as well as migration of the beads from organic into an aqueous PBS buffer system, were performed as described above for the synthesis of beads with defined peptide sequence. The total OBOC library was suspended in 10 ml of PBS  were mixed with the respective scrambled version as irrelevant bead matrix. Ratios of “relevant” to “irrelevant” beads ranged from 1:10 to 1:100.For screening experiments, mixtures of these beads  were transferred into 2 ml reaction columns  with a filter on the bottom of the vessel and a luer outlet. The reaction columns were closed with a luer sealing plug during the incubation of the beads with different reaction solutions. For washing purposes, the sealing plug was removed and the washing buffer was pressed through the beads with a fitting stamp. Contact between the tip of the stamp and the bead-covered filter was carefully avoided to prevent the loss of beads sticking to the stamp’s tip.The beads were first washed with 2 ml of PBS containing 0.1% (v/v) Tween-20 (Sigma-Aldrich) (PBST) followed by two times washing with the same amount of PBS. After washing, free areas on the polystyrene bead surface were blocked by incubating the beads in 2 ml of blocking solution (0.05% (w/v) fish gelatin  in PBS) for 1 h at room temperature. Constant mixing of beads in blocking solution was achieved by mounting the tube onto an overhead shaker  performing a semicircle movement at 10 rpm. This was followed by thorough washing (six times with 2 ml of PBST and two times with 2 ml of PBS). After that, beads were ready for the incubation with the respective IgE antibody preparation used in the specific screening experiment . IncubatThe beads were examined visually on a standard inverted fluorescence microscope  equipped with a UV light source and filter systems compatible with the fluorophores used. In addition, bead fluorescence intensity and distribution was documented with a MORE life cell imaging microscope  equipped with a Clara CCD camera system , using appropriate filter sets to capture green, red and far red fluorescence signals (FITC-channel: detection filter wavelength: 535 ± 50 nm/rhodamine-channel: detection filter wavelength: 630 ± 75 nm/Cy5-channel: detection filter wavelength: 700 ± 75 nm). Analysis of the respective pictures (including measurement of fluorescence intensities) and image processing was conducted using the software ImageJ v1.52p   according to the manufacturer’s instructions, with minor modifications. 300 μl of serum from an allergic donor was loaded onto a protein G-sepharose column. The flow-through of the column was collected as IgG-negative/IgE-positive fraction. IgG bound to the sepharose was eluted with 400 μl of acidic elution buffer into a tube containing 30 μl of basic neutralizing buffer, resulting in functional IgG in a buffer of neutral pH.Untreated serum from an allergic donor and the corresponding IgE-fraction obtained as flow through of the protein G-sepharose column were adjusted to the dilution of the eluted IgG-fraction by adding PBS. This results in an approximate 1.4-fold dilution and in an antibody concentration of 70% compared to the original serum. 180 μl of each of these diluted immunoglobulin preparations  were mixed with 20 μl 0.5% (w/v) fish gelatin in PBS, resulting in a final antibody concentration of approximately 65% compared to the untreated serum. These dilutions were then used for the incubation with pre-blocked peptide-bearing polystyrene beads as described above, using a 1:1,000 dilution of the secondary, anti-IgE antibody.4 to 2 × 105 beads were transferred into the reaction column used for screening. The screening protocol was identical to the procedure described for the “artificial” peptide libraries, except for the use of 400 μl of diluted serum/primary antibody and secondary antibody solution (due to higher bead numbers). Antibody concentrations and types of secondary antibodies were varied to identify optimal conditions. After the last washing step, beads in the reaction column were resuspended in 2 ml of PBS, transferred in portions into 12-well or 24-well plates and examined as described above.Combinatorial OBOC libraries ) were spiked with beads bearing defined peptide sequences . The ratio “defined peptide bead to OBOC library bead” was between 1:500 and 1:1,000, some preliminary testing was done with ratios of 1:10 to 1:15. The beads were mixed as described above for the “artificial” peptide libraries, and a total of 2 × 106 beads of an OBOC peptide library were incubated in a reaction column of 20 ml capacity (Intavis) with 15 ml of blocking solution over night at 4°C with semicircle mixing at 10 rpm. Washing steps (before and after blocking) with PBST and PBS were performed as described above, with 15 ml of washing solution per washing step. This was followed by 3 h incubation at room temperature with 3 ml of phycoerythrin-labeled anti-IgE antibody at a concentration of 500 ng/ml and another washing two times with 15 ml of PBST and two times with 15 ml of PBS. Beads were resuspended in a total of 5 ml of PBS and transferred in portions into a 15-ml polystyrene tube. A small sample of the bead suspension was analyzed in the fluorescence microscope to verify successful staining. Afterward, the beads were stored in the 15-ml tube at 4°C in the dark, until separation of fluorescence positive and negative beads was performed, which should be done within 2 weeks after the staining procedure . This separation was performed on a BioSorter  equipped with a Fluidics and Optics Core Assembly (FOCA) of 500 μm, using PBS as sheath fluid. Beads were re-suspended in PBS, and concentration was adjusted until a stable event rate of 10–20 events/second was achieved. An unstained bead sample was run as control to set gating conditions. Non-fluorescent, i.e., non-IgE cross-reactive beads (“phycoerythrin-negative”) were sorted with the coincidence mode set to “Pure.” Data graphs were generated in FlowJo software v.10.7.1 . The fluorescent beads were discarded, the non-fluorescent beads were stored at 4°C until use in screening experiments.To minimize false-positive results due to binding of secondary, anti-IgE antibody directly to individual beads in the OBOC population, such anti-IgE-cross-reactive beads were identified in a pre-adsorption step and removed from the OBOC library before screening with the actual IgE samples. For this, 1 × 10As an alternative approach to the pre-cleaning step, i.e., the removal of pre-adsorbed anti-IgE-cross-reactive beads from the OBOC-library via BioSorter separation before the actual screening process, the pre-adsorption step was performed using anti-IgE labeled with a different fluorophore than the one used in screening. Afterward, anti-IgE-cross-reactivity was determined in the eventual fluorescence readout.5 beads of an OBOC peptide library, spiked with beads bearing defined peptide sequences (c-myc or Ara h 2) in a ratio of 1:1,000, were incubated in a 3-ml reaction column with 2 ml of blocking solution for 3 h at room temperature under constant mixing. After removal of the blocking solution, 400 μl of fluorescein isothiocyanate (FITC)-labeled anti-IgE  was added, and the incubation was continued for another 3 h. Beads were washed  and incubated with 400 μl of anti-c-myc IgE (200 ng/ml) or human serum (160 ng IgE/ml) in blocking solution over night at 4°C under constant mixing. Beads were washed again , incubated with 400 μl of phycoerythrin-labeled anti-IgE  for 3 h at room temperature, washed and processed for microscopy as described above.For this, 2 × 10For bead selection and isolation, the standard fluorescence microscope was used. Fractions of the bead mixture which had been examined before in 24- or 12-well plates were transferred into a 6-well plate (Costar Corning) containing 1.8 ml of PBS plus 200 μl of PBST per well. The dimensions of the wells of this plate and the sample dilution allowed access to selected single beads with minimal contact to neighboring beads. This facilitated manual bead manipulation by use of a micropipette. Beads were re-evaluated and adequate beads were chosen for isolation according to their fluorescence properties (for details see section “Results”). Each single chosen bead was removed by using a standard 10 μl pipette with low retention pipette tip. Non-relevant beads were pushed aside and the chosen bead was aspired together with ≤5 μl of the surrounding liquid and transferred into a separate well until further processing for peptide sequencing.After manual isolation of a fluorescence positive bead, the bead was placed onto a trifluoroacetic acid-treated glass fiber disc . Location of the bead on the filter was verified via microscopic observation. The filter was loaded into the reaction chamber of an automated peptide sequencing system . Peptide sequences were determined by direct on-bead Edman degradation followed by HPLC separation of the step-wise cleaved-off phenylthiohydantoin-derivatized amino acids. Identification of the individual amino acids was done by reference to a phenylthiohydantoin (PTH) amino acid standard .One-bead-one-compound libraries have the advantage to test millions of peptides in parallel for binding of a defined target molecule. To adapt and optimize this system for the detection of new IgE epitopes, we had to establish an assay format where IgE binding to a specific peptide immobilized on polystyrene beads can be reliably detected. To achieve this, we started with an unambiguous system of a defined IgE antibody – peptide epitope pair in an “artificial peptide library” based on a matrix of irrelevant peptide-carrying beads. We decided on a commercially available, recombinant human IgE antibody directed against the Myc-protein derived peptide c-myc (peptide sequence E-Q-K-L-I-S-E-E-D-L). Beads carrying the c-myc peptide, as well as “irrelevant” matrix beads carrying a scrambled version of the c-myc epitope (sequence E-I-E-D-K-L-S-L-Q-E), were produced by classical fmoc solid phase-based peptide synthesis. These beads were used to optimize assay conditions, especially in terms of anti-IgE antibody employed , first in order to achieve highest possible sensitivity, and second to take into consideration that the structure of the fluorescent dye coupled to the anti-IgE antibody has an influence on the screening of OBOC libraries .The first experimental set-up encompassed a 1:10 mixture of c-myc beads and scrambled c-myc beads and an incubation with anti-c-myc IgE antibody in a defined blocking buffer. By successively testing a variety of monoclonal and polyclonal anti-human-IgE antibodies with different green fluorophores , and to different reactivities against our chosen epitope peptide. A further reason for low IgE detectability may be the presence of IgG with identical epitope recognition in the sample. Along that line, we had realized already from our epitope mapping experiments that we could improve the IgE signals by depleting the serum samples of IgG. Hence, we wanted to port these findings to our bead-based IgE detection, in order to further improve the system.We therefore compared the bead-based IgE detection of untreated serum with serum which had been depleted of IgG by a protein G-sepharose matrix. We used a serum containing approximately 50 ng/ml of Ara h 2-specific IgE (patient 2), which had shown a specific, albeit not very strong, epitope reactivity in our previous mapping experiments. Yet, the total, untreated serum could not detect any signals above background in our artificial library of Ara h 2/scrambled Ara h 2 peptide beads in combination with a monoclonal, phycoerythrin-labeled anti-IgE antibody . FluoresIn addition to enhancing the specific IgE binding, removal of IgG from the serum enabled us to test the anti-IgE antibodies for potential cross-reactivity with IgG. We used the IgG fraction separated from serum of a donor where IgG reactivity with the Ara h 2 epitope had been demonstrated before in our epitope mapping experiments. We incubated these IgG with the artificial Ara h 2 bead library, followed by the monoclonal, phycoerythrin-labeled anti-IgE antibody. Although one can presume, due to our previous data, that IgG-binding to the Ara h 2 beads occurred, no corona-positive red fluorescence was detected, and total fluorescence values were not different from background values. This demonstrates that IgG-cross-reactivity of the monoclonal anti-IgE antibody was negligible.In total, these experiments show that serum depleted of IgG may improve the detection of target-specific IgE, especially when IgE with only low/medium abundance are analyzed.via peptide sequencing. We suppose that this is due to the presence of peptide sequences within the library that directly cross-react with the secondary (anti-IgE) antibody, even without an IgE bound to the beads. In fact, when we incubated the OBOC beads with anti-IgE antibodies alone, without prior addition of IgE or serum, corona-positive fluorescent beads were detectable. This effect was seen – albeit to a varying degree – with all the different anti-IgE antibodies we tested, polyclonal as well as monoclonal, and irrespective of the fluorophore used , differing only in their fluorophore label.This strategy proved successful in essence, but the parallel use of two fluorophores, and the inherent bead auto-fluorescence problem associated with the green channel, brought about difficulties in the visual inspection and identification of true positive beads. As exemplified in Although our approach to pre-adsorb anti-IgE cross-reactive sequences in an OBOC library before the screening process worked reasonably well, we decided to evaluate another, different strategy to solve the cross-reactivity problem. In this approach, rather than leaving the pre-adsorbed beads within the library and having the green (auto) fluorescence interfere with the subsequent bead isolation process, we wanted to remove the cross-reactive beads from the library pool before the actual screening procedure. To do so, beads of the peptide library were again pre-incubated with high concentrations of a fluorophore-labeled secondary, anti-IgE antibody. Afterward the fluorescence-positive, anti-IgE cross-reactive beads were separated from the fluorescence negative beads (with no intrinsic affinity to the secondary antibody) by sorting with a BioSorter . This prFor our final screening experiments, we used an OBOC peptide library where the anti-IgE cross-reactive beads had been removed via BioSorter separation after treatment with a phycoerythrin-labeled monoclonal anti-IgE antibody. One portion of this pre-cleaned OBOC library was spiked with c-myc beads (ratio c-myc to OBOC = 1:500) and incubated with anti-c-myc IgE (100 ng/ml). A second portion of the pre-cleaned OBOC library was mixed with Ara h 2 beads  and incubated with serum from a peanut-allergic donor (specific anti-Ara h 2 IgE = 90 ng/ml). IgE-binding to the beads was detected with the same phycoerythrin-labeled monoclonal anti-IgE antibody that had been used for the pre-cleaning process.Under microscopic examination, both screening set-ups showed a small number of red fluorescent beads, in line with the low abundance of beads in the library that were carrying specific epitopes. Due to the brightness of the fluorescence, and the low background in the red channel, the individual positive beads could easily be detected and singularized. For each of both set-ups, 10 red-fluorescent beads were isolated and their peptides were sequenced. The correct c-myc sequence could be confirmed for 9 out of the 10 c-myc candidates, and for the Ara h 2 screening set-up, also 9 out of 10 isolated beads could be confirmed to carry the Ara h 2 peptide. Moreover, after repeating the Ara h 2 screening experiment in an identical set-up, but using a different batch of the same monoclonal anti-IgE antibody for detection, 10 of 10 isolated beads carried the Ara h 2 epitope sequence.In conclusion, we demonstrate here that we have established a method of pre-cleaning and screening an OBOC library that can be used to identify specific IgE-epitope bearing beads within a library of ≥100,000 different peptides.in vitro routine allergy diagnostic tests include only a limited number of clinically relevant allergen sources and often lack relevant single allergenic components. In-vivo allergy diagnostic tests (skin prick tests) represent the main approach to confirm clinical suspicion of allergic sensitization, but they are mainly based on the application of crude allergen extracts, and standardization remains difficult (2LEN) has established a protocol for a standard prick test panel with a total of just 18 inhalant allergens  can be introduced to the respective patients. Thereby a specific anti-inflammatory treatment can be provided and – if necessary – can be combined with treatment of disease-relevant target molecules (target treatments), like the application of biologicals against certain cytokines . In any In principle, the specificity of high affinity IgE can be deduced from their selective recognition of cognate allergen or allergen epitope upon its being offered for binding. Unfortunately, most bioinformatical approaches to predict an epitope on the basis of the paratope/CDR sequence , 47 did 10 different permutations when 19 amino acids are used. This translates into about 2 times 10 billion different phages or 240 kg of OBOC beads. Such huge numbers of phages and amounts of beads are impossible to handle and to screen. Yet, typical epitopes contain a core region of 4–7 amino acids (mean 5.5)  , 50 whicIn order to establish a robust screening procedure that uses OBOC-libraries for epitope discovery, we decided for a stepwise, systematic approach to solve any intrinsic and extrinsic problems. The main problem to be overcome turned out to be the necessity for high sensitivity of the assay system in order to enable us to detect a specific reactivity with IgE at physiological concentrations. In healthy people the total serum IgE level is usually below 100 IU per ml. A total serum IgE value ≥100 IU/ml (corresponding to 240 ng/ml) is considered indicative of allergy/atopy, and in some allergic patients serum IgE against one specific target may reach 1,000 IU/ml  and above. Still, these immunoglobulin concentrations are in a range where detecting their specific reactivities on a one-bead-level may become a problem. Addressing the sensitivity issue, we tested a variety of different reporter systems attached to anti-IgE antibodies to visualize binding of IgE to the beads. Initial attempts with enzymatic reporters that produce insoluble dyes failed to provide the desired sensitivity and/or specificity, even when tyramide signal amplification  or fluorA further problem which we had not initially anticipated was the capacity of certain beads in the OBOC library to directly acquire secondary antibody. Although the extent of direct binding to the beads varied with the secondary antibody(-conjugate) used, it appears likely that the main determining factor for this cross-reactivity resides in the peptide sequence motifs presented on specific beads. This includes peptides which by chance resemble part of the IgE heavy chain or motifs that have affinity to some region of the secondary antibody or of the fluorophore. Analysis of such undesired binders revealed that they mostly contain aromatic amino acids and proline. Similar results were reported in another study, where OBOC beads were screened with a probe equipped with the fluorophores ATTO590 and TexasRed. Here, the amino acids leucine/isoleucine, histidine, phenylalanine and tyrosine were enriched in false positives reacting with the dye . These dIn order to reduce undesired direct secondary antibody labeling of beads without tampering with library composition we followed two avenues of resolution for the problem, both of which proved practicable. In the first approach, the complete OBOC-library was pre-incubated with an excess of secondary antibody equipped with a green fluorescent dye, then incubated with the IgE pool and finally exposed to the same secondary antibody labeled with a red fluorophore. In this case the decision between false and true positives has to be made by comparing green and red fluorescence of individual beads upon microscopic inspection. In practice, this turned out to be a rather time-consuming process with a certain operator variability. Focusing on beads with a strong corona in the red fluorescence channel and negligible green fluorescence yielded an excellent true-positive rate – as verified by sequencing – in our experimental set-up. However, it must be surmised that the same strategy decreases the sensitivity of the procedure and reduces the probability to positively identify epitopes of rare IgE species. It therefore seemed reasonable to remove any cross-reactive beads from the OBOC library before the actual screening process. This was achieved by sorting the fluorescently pre-adsorbed OBOC library with a large particle sorter (BioSorter). While this approach led to satisfactory results in our set-up, it must be conceded that the limited availability of suitable sorting devices renders this procedure not highly practicable for general use. Here certainly remains room to improve the protocol, either by adaption of more accessible sorting devices  to the specific particle size used in the libraries, or by refining the back-up protocol employing two differently labeled anti-IgE antibodies with the use of fluorophores less sensitive to the interfering bead auto-fluorescence.in silico. Even known peptide sequences on defined c-myc or Ara h 2 beads could not be resolved by MALDI-TOF-MS. We therefore switched to Edman degradation-based peptide sequencing on an automated protein sequencer. Single isolated beads were directly subjected to the sequencing process, without the necessity of first removing bound antibodies or cleaving the peptide off the bead. Although the low amount of peptide present for sequencing  is close to the sensitivity limit of the sequencer, the peptide sequence on the isolated beads could be successfully resolved in the great majority of cases. Furthermore, beads with higher peptide capacity  are available. All procedures described here could be adapted without much effort to those beads, in case of ambiguous results in terms of peptide sequencing during OBOC library screening.The final step to validate our OBOC library screening procedure was the verification of the true positives after bead isolation by peptide sequencing. First attempts using mass spectrometric analysis (MALDI-TOF-MS) of fragments after ammonolysis with aqueous or neat ammonia, however, were not successful. Although some studies report mass spectrometrical sequencing of peptides released from beads upon ammonolysis , 55–57, Taken together, we here present a detection system to identify unknown IgE reactivities by using chemically synthesized one-bead-one-compound libraries. We are confident that this technology will aid in the identification of novel allergens for asthmatic individuals with high total serum IgE and with no specific allergic reaction detectable to date.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ethics Committee of the University of Lübeck. The patients/participants provided their written informed consent to participate in this study.TK designed the study, designed and performed the experiments, analyzed and interpreted the data, and wrote the manuscript. NR and BM designed and performed the experiments, analyzed the data, and wrote the manuscript. HS, CS, and SM performed the experiments, analyzed the data. UJ recruited and characterized peanut-allergic patients, collected the samples, and discussed the data. AF conceived and supervised the study, discussed the data, and wrote the manuscript. All authors read, revised and approved the manuscript for submission.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.