Asian Chinese are more susceptible to deposition of visceral adipose tissue (VAT) and type 2 diabetes (T2D) development than European Caucasians when matched for gender, age and body mass index (BMI). Our aims were: (i) characterise the ethnicity-specific metabolomic signature of visceral adiposity measured by dual energy X-ray absorptiometry (DXA) and fasting plasma glucose (FPG), and (ii) identify individuals susceptible to worse metabolic health outcomes.TBF) in each ethnic group. Metabolic risk groups in each ethnicity were stratified based on the joint metabolomic signature for FPG and %VATTBF and clinically characterised using partial least squares-discriminant analysis (PLS-DA) and t-tests.Fasting plasma samples from normoglycaemic (n = 274) and prediabetic (n = 83) participants were analysed with liquid chromatography–mass spectrometry using untargeted metabolomics. Multiple linear regression adjusting for age, gender and BMI was performed to identify metabolites associated with FPG and VAT calculated as percentage of total body fat (%VATp = 3.00E−05) in Caucasians and triglyceride TG(54:4)  in Chinese being the most significantly correlated metabolite based on the p-value. %VATTBF was correlated with 85 and 119 metabolites in Caucasians and Chinese respectively, with TG(56:2)  in Caucasians and TG(58:3)  in Chinese being the most significantly correlated. 24 metabolites associated with FPG were common to both ethnicities including glycerolipid species. 67 metabolites associated with %VATTBF were common to both ethnicities including positive correlations with dihydroceramide, sphingomyelin, glycerolipid, phosphatidylcholine, phosphatidylethnolamine, and inverse correlations with ether-linked phosphatidylcholine. Participant re-stratification found greater total and central adiposity, worse clinical lipid profiles, higher serum glucoregulatory peptides and liver enzymes in normal fasting glucose (NFG) individuals with a prediabetic metabolomic profile than NFG individuals with a normoglycaemic metabolomic profile in both ethnicities.FPG was correlated with 40 and 110 metabolites in Caucasians and Chinese respectively, with diglyceride DG(38:5) (adjusted β = 0.29, TBF, with an ethnic-dimorphic signature for these metabolic traits. These signatures could improve risk stratification and identify NFG individuals with an adverse cardiometabolic and T2D risk profile.Untargeted metabolomics identified common and disparate metabolites associated with FPG and %VAT The prevalence of type 2 diabetes (T2D) has witnessed a drastic surge in China over recent decades. There are now 110 million people with T2D in China , 4, stri1c both provide simple and cost-effective markers of prediabetes, predicting individuals who remain in the prediabetic state for many years vs those progressing rapidly to T2D is difficult from these single markers. Metabolomics provides a useful means to advance the understanding of disease pathophysiology, and may facilitate the identification of at-risk individuals before dysglycaemia occurs and/or predict those who will rapidly worsen to T2D  for polar metabolite and lipid analysis respectively. The reconstitution volume was determined using a previously described workflow [Samples were randomised into 4 batches and extracted on 4 consecutive days. The protocol for metabolite extraction was adapted from a previously reported method . Brieflyworkflow .2O (60:40) with 10 mM ammonium formate and 0.1% formic acid (A), and isopropanol/acetonitrile (90:10) with 10 mM ammonium formate and 0.1% formic acid (B). Analytes were eluted from the column with the following gradient program: 15–30% B (0.0–2.0 min), 30–48% B (2.0–2.5 min), 48–82% B (2.5–11.0 min), 82–99% B (11.0–11.5 min), 99% B was maintained for 3.5 min followed by re-equilibration with 15% B for 3 min. Two microliter reconstituted samples were injected. External mass calibration of the Orbitrap prior to sample analysis was performed by flow injection of the calibration mix solution according to the manufacturer’s instructions. High resolution data  was acquired by full scan from m/z 200–2000 with source voltage of 3500 V electrospray ionisation positive mode (ESI +) or − 3600 V ESI negative mode (ESI −), capillary temperature of 275 °C, and sheath, auxiliary and sweep gas flow rates of 40, 10 and 5 arbitrary units, respectively. Data-dependent MS2 data were collected with a mass resolution set to 35,000 recording a mass range of m/z 200–2000 and maximum trap fill time of 250 ms (full scan mode) or 120 ms (MS2 scan mode). The isolation window of selected MS1 scans was ± 1.5 m/z with a normalised collision energy of 30 units.Lipid analyses were performed using an Accela 1250 quaternary UHPLC system coupled to a Q Exactive hybrid quadrupole-Orbitrap mass spectrometer  with a heated electrospray ionisation source set to 370 °C. An Acquity CSH™ C18 column 1.7 µm, 2.1 mm × 100 mm  was used for lipid separation with a column temperature of 65 °C and mobile phase flow rate at 600 µL/min. The mobile phases consisted of acetonitrile/Hm/z 55 to 1100 with source voltage of 4000 V for ESI + and − 4000 V for ESI − , capillary temperature of 325 °C, and sheath, auxiliary, and sweep gas flow rates of 40, 10, and five arbitrary units, respectively.Polar metabolites were analysed with an Accela 1250 quaternary UHPLC pump coupled to an Exactive Orbitrap mass spectrometry . Chromatographic separation was carried out at 25 °C on a SeQuant® ZIC®-pHILIC 5 µm, 2.1 mm × 100 mm column  with the following solvent system: A = 10 mM ammonium formate in water, B = 0.1% formic acid in acetonitrile. A gradient program was used at a flow rate of 250 µL/min: 3–3% A (0.0–1.0 min), 3–30% A (1.0–12.0 min), 30–90% A (12.0–14.5 min), 90% A was maintained for 3.5 min followed by re-equilibration with 3% A for 7 min. An injection volume of 2 µL was used. The electrospray probe was operated unheated at room temperature (20 °C) to avoid degradation of thermally labile compounds. External mass calibration of the Orbitrap prior to sample analysis was performed by flow injection of the calibration mix solution according to the manufacturer’s instruction. High resolution data  were acquired by full scan from p value ≥ 0.05) generated by the diffreport function from the XCMS package. Manual examination of EIC was conducted to filter out poorly integrated peaks, using build-in function in mzMINE for the polar metabolite data and the EICs generated by the diffreport function in the XCMS package for lipid data. After data cleaning, signal drift and batch effects were corrected by LOESS in the W4M Galaxy environment [2 datafiles  as previously described to generate a MS2-annotated lipid ID library and matched against the processed lipid data matrices based on parent mass and retention times [2-annotated lipid ID were searched against online database including HMDB (https://www.hmdb.ca/), Metlin (https://metlin.scripps.edu/) and Lipid Maps (https://www.lipidmaps.org/) based on m/z with less than 10 ppm mass error. Polar metabolites were annotated using an in-house library based on authentic standards (AgResearch) analysed through HILIC LCMS analysis under conditions identical to the current study. Metabolic features without a hit in the library were searched against online database including HMDB and Metlin based on m/z with less than 15 ppm error.Raw data files were converted to mzXML format with MSconvert (v 3.0.1818) and pre-processed. Three lipid datafiles were corrupted and excluded from analysis. Lipid data were preprocessed with the XCMS package v3.0.2) in the R programming environment (v3.2.2) , whereas in the Rironment , and feaon times . Lipid f1c, BMI and %VATTBF in identifying discriminatory lipids and metabolites between the two ethnicities (with Caucasian as reference level and Asian Chinese as level 1). To characterise the metabolomic and lipidomic signatures for FPG or %VATTBF (as the outcome variable), multiple linear regression was performed on every feature adjusting for age, gender and BMI in an ethnicity-specific manner. All univariate statistical analyses were carried out in R (v3.5.1) and subjected to FDR correction (Benjamini Hochberg procedure) [Multivariate analysis using partial least squares-discriminant analysis (PLS-DA) was initially applied to investigate differences in baseline profiles of the two ethic groups . Logistic regression was subsequently performed to allow for adjustment for age, gender, HDL-C, insulin, FPG, HbAocedure) . Up tillTBF in each ethnicity was built using random forest (RF) [TBF in each ethnicity identified by the aforementioned multiple linear regression, were extracted and imported to MetaboAnalyst for biomarker analysis. Three samples were excluded from the re-stratification due to the absence of their lipidomic profiles. RF analysis was performed on the 100 most important variables ranked by decreases in accuracy through 30 repeats of threefold random sub-sampling cross-validation. Individuals who were predicted as NFG by the metabolomic-based RF model were designated as “mNFG”, whereas those predicted as IFG were designated as “mIFG”. The predicted FPG state was further combined with their actual FPG state as determined by the ADA FPG criteria to create a new stratification system consisted of 4 groups: NFG-mNFG , NFG-mIFG , IFG-mNFG  and IFG-mIFG . The clinical profile  of the metabolic FPG state and the 4 sub-phenotype were then characterised using multivariate PLS-DA  and univariate t-test (R (v3.5.1)).The prediction model of FPG state by a panel of metabolites associated with FPG and %VATest (RF)  in Metabest (RF) . In detaTBF, %VATAAT, HbA1c, FPG, fasting insulin, total TG, ALT, GGT, amylin, GLP-1 and glucagon, and lower HDL-cholesterol than Caucasians.Characteristics of the study population are summarised in Table The score plots from the PLS-DA models for both lipid and polar metabolite profiles showed clear and robust separation between Asian Chinese and Caucasian cohorts , indicating ethnicity influences the fasting plasma metabolome Fig. .Fig. 1PLp < 0.05) . The strongest marker for FPG other than the MS-measured glucose  in Caucasians was DG(38:5) . FPG in Asian Chinese was associated with 110 non-redundant features yielding 101 identified metabolites, including positive correlations with 6 ceramides, 10 DGs, 59 TGs, 4 PCs, 3 PEs, 2 hexoses (MS-measured glucose) and an unknown hexose, and negative correlations with 2 CE, 2 FFAs, 8 SMs, 2 ether-linked PCs, HexCer(d42:2) and L-acetylcarnitine. The strongest marker was TG(54:4) . Only MS-measured glucose, 4 DG species, 16 TG species plus 2 unknowns were common makers for FPG in both ethnic groups (19% overlapped), and many of them contained an oleate moiety . The %VATTBF-associated lipid features, either unique to each, or common to both ethnic groups, have been provided in Fig. FTB in Caucasians was associated with 85 non-redundant features (96% identified), including positive correlations with Cer(d40:0), SM(d36:0), 8 DGs, 4 PCs, 4 PEs and 60 TGs, and negative correlations with 2 ether-linked PCs and 2 SMs. The most strongly associated lipid species based on p-value was TG(56:2) . %VATFTB in Asian Chinese was associated with 119 non-redundant features (92% identified), including positive correlations with PC(38:3), SM(d36:0), SM(d40:0), 4 ceramides, 10 DGs, 8 PEs and 59 TGs, and negative correlations with PC(38:7), PE(P-36:1), 2 CEs, 13 ether-linked PCs and 9 SMs, with TG(58:3) being the most significantly correlated marker . 67 lipid species were common markers for %VATFTB in both ethnic groups, encompassing lipid species of ceramide, SM, PC, PEs, ether-linked PCs, DGs and TGs (48.9% overlapped), and many of them notably contained a linoleate moiety  after adjusting for age, gender and BMI. These included DG(36:2), DG(38:2) and 17 TG species plus one unknown lipid species (m/z = 933.8665)  independent of age, gender and BMI, including CE(22:4), Cer(d40:1), Cer(d42:1), PE(36:1), PE(36:2), SM(d42:3), SM(d43:2), SM(t34:1) in addition to 10 DG and 50 TG species plus one unknown lipid species (m/z = 924.8006) , and built RF based on 100 top-ranked metabolites for each ethnicity , participants were stratified into 4 groups for each ethnicity: 2 had predicted FPG states concordant to the actual state , and 2 had a discordant metabolic FPG state from the actual state . 74% of NFG and 77% of IFG individuals had a concordant predicted FPG state  in Caucasian; 26% of NFG individuals were predicted to be mIFG (NFG-mIFG) and 23% of IFG individuals were predicted as mNFG (IFG-mNFG). In Asian Chinese, 70% of NFG and 74% of IFG individuals were predicted to have an mNFG and mIFG state, respectively; 30% of NFG individuals had an mIFG state and 26% of IFG individuals had an mNFG state.The clinical and anthropometric profiles between these newly designated groups were then compared using a multivariate PLS-DA or univariate t-test approach. For the PLS-DA model, we first constructed and compared models using 21 clinical and anthropometric measurements as explanatory variables and mNFG vs mIFG as predicted by the metabolomic signature or NFG vs IFG as stratified by ADA FPG criteria Fig. . Both mop < 0.05)  , liver enzyme , total cholesterol, total TG, LDL-C, glucoregulatory hormones , and lower HDL-C in Caucasians (05) Fig. a. In Asi05) Fig. b. ConverFTB in Asian Chinese (43.9% overlap) than in Caucasians (19% overlap), suggesting visceral adiposity was more closely related to the development of dysglycaemia in Asian Chinese independent of total adiposity.T2D is a progressive disease that may be prevented if at-risk individuals can be identified before disease onset and managed through lifestyle and dietary intervention . As suchFTB markers to both ethnic groups, and %VATFTB was specifically associated with 2 additional lipid classes in Asian Chinese. Both ethnic groups had a TG species (TG(58:2) for Caucasians and TG(58:3) for Asian Chinese) as the most significant marker  correlated with %VATFTB. The similar pattern of lipid profiles associated with %VATFTB across the 2 ethnic groups suggested a homogenous metabolic adaptation to visceral adiposity irrespective of ethnic background.With limited studies investigating metabolic alterations associate with VAT deposition, we here provided evidence demonstrating visceral adiposity profoundly affected lipid metabolism whilst changes of polar metabolites were only weakly associated (i.e. no metabolites passed the significance level after multiple testing correction). 67 lipid species belonging to 7 classes were common %VATFTB also correlated with several ceramide species and dihydroceramide Cer(d40:0), and negatively correlated with SM species in Asian Chinese, highlighting a link between visceral adiposity and altered sphingolipid metabolism. Remarkably, all ceramide species correlated with %VATFTB observed in our study contained a very long acyl chain , all of which are likely to be products of CerS2, a ceramide synthase showing substrate specificity for longer acyl chain species which is predominantly expressed in liver [Dysregulation of lipid metabolism is a characteristic of T2D and suggested to be a metabolic event prior to the onset of dysglycaemia . Meikle in liver , 59. ColFTB in both ethnic groups. The association between PEs and prediabetes/T2D was highlighted in Meikle’s study as well as in another study conducted in a Chinese cohort [Our results confirmed a metabolic shift in the phospholipid profile implicated in T2D development . Howevere cohort , 61. Bote cohort , 57. A ce cohort .We have intriguingly observed an inverse correlation of FA(16:0) and FA(18:2) with FPG in Asian Chinese, as opposed to several studies reporting associations of elevated FFA with IR and development of T2D , 63, 64.FTB was observed in both ethnic groups, in good agreement with a previous finding of an ether lipid signature characteristic for VAT deposition [We observed a negative correlation between FPG and an ether-linked PC in Asian Chinese, consistent with other reports of an inverse association of this lipid class with prevalent and incident T2D, obesity and IR , 57, 69.position . Little position , 71. In position , 72. OurContrary to several studies demonstrating a positive association between CE and prediabetes/T2D, we have unexpectedly found an inverse association. However, the PREDIMED trial has reported an inverse association between CEs and risks of T2D and CVD , 73, andIn addition to the MS-measured glucose itself, FPG was correlated with erythronic acid in Caucasians and an unknown hexose in Asian Chinese. Erythronic acid is a novel hitherto-unreported marker that will require further validation, whereas increased hexose (other than glucose) associated with prediabetes and T2D was in line with the others , 78.FTB—and FPG-associated metabolomic profiles, we were able to create a novel stratification with a subset of normoglycaemic individuals whose metabolomic signature resembled that of prediabetes (termed NFG-mIFG). In both ethnicities they were characterised by higher BMI and W-to-H ratio, and worse lipid , liver enzyme and hormone  profiles compared to NFG-mNFG counterparts. Of particular interest were the higher liver enzymes, which are indicators of inflamed or damaged liver cells. The liver is a key organ in the regulation of energy homeostasis and metabolism, and is proposed as the primary site affected by excess VAT deposition [Taking the metabolomic signature jointly determined by %VATposition . Hepaticposition . ImportaOne strength of our study is the simultaneous measurement of metabolomic profiles from two co-located ethnic groups with the same extraction protocol and analytical platform, allowing direct comparison whilst minimising environmental confounding factors to inform ethnicity-specific changes in metabolism associated with T2D development. The use of unbiased, highly sensitive and complementary methods enabled a more holistic view of metabolic perturbation associated with increased visceral adiposity and FPG. In addition, we have utilised the metabolomic signature characteristics to develop a possible risk prediction of T2D development, identifying individuals with a worse cardiometabolic profile despite having normoglycaemia. Limitations include the nature of cross-sectional studies which precludes conclusion on causality and requires follow-up to confirm our findings. In spite of a wide range of metabolites and lipids measured by untargeted metabolomics, this approach may not be optimal if a particular class of metabolites/lipids is of interest, hence it is inevitable that some of the findings by others using metabolite class-optimised targeted approaches such as the analysis for acylcarnitines were not observed in our study.FTB in both Caucasian and Asian Chinese independent of age, gender and BMI. We have shown plasma metabolomic profile to be profoundly influenced by ethnicity, and are the first to compare an ethnicity-specific signature for T2D risk factors including FPG and visceral adiposity. A similar signature for %VATFTB across both ethnicities but a very different signature for FPG observed in the present study highlighted homogeneous metabolic adaptations and alterations in response to VAT deposition, yet a distinct underlying pathogenesis of dysglycaemia. Predictive modelling using the joint metabolomic signature of FPG and %VATFTB has identified a subset of individuals with worse cardiometabolic risk despite current healthy normoglycaemia. This novel approach of re-stratification using the metabolomic signature aids early identification of those at-risk of T2D. This modelling approach could be applied to a wide range of diseases.Our study has revealed a broad spectrum of lipid species associated with FPG and %VATAdditional file 1. Figure S1: Annotation of glucose peak measured by ESI+ (a) and ESI- (b). Figure S2: Receiver operating characteristic (ROC) curve of RF performed 100 most important variables among the pooled list of those associated with FPG or %VATTBF in (a) Caucasian and (b) Asian Chinese.Additional file 2. Table S1: Internal standard added to extraction solvent for monitoring signal variation during instrumental analysis. Table S2: Data pre-processing parameters for lipidomic profile (XCMS) and HILIC profile (ADAP). Table S3: Differentially expressed lipid species between Caucasian and Asian Chinese. Table S4: Differentially expressed metabolites between Caucasian and Asian Chinese. Table S5: Metabolites associated with FPG in Caucasian and/or Asian Chinese analysed by multiple linear regression adjusted for gender, age and BMI (BH-corrected p<0.05). Table S6: Metabolites associated with %VATTBF in Caucasian and/or Asian Chinese analysed by multiple linear regression adjusted for gender, age and BMI (BH-corrected p<0.05). Table S7: Metabolites associated with FPG and/or %VATTBF in Caucasian analysed by multiple linear regression adjusted for gender, age and BMI (BH-corrected p<0.05). The list of metabolites is ordered by the variable importance in ramdon forest, and the top 100 variables (from unknown X160_HP_241.0719_12.18 to unknown LP_802.5932_348.16) were selected for sample restratification. Table S8: Metabolites associated with FPG and/or %VATTBF in Asian Chinese analysed by multiple linear regression adjusted for gender, age and BMI (BH-corrected p<0.05). The list of metabolites is ordered by the variable importance in random forest, and the top 100 variables (from unknown X160_HP_241.0719_12.18 to PE(P-36:1)) were selected for sample restratification.


There was an association between NEK2 mRNA and protein expression (p = 0.023) and elevated NEK2 mRNA levels were related to reduced survival in BP-NEN patients (p = 0.015). Patients with higher PIM1 protein expression had also diminished survival comparing with those with weak or no PIM1 expression (p = 0.037). Elevated NEK2 and PIM1 expression were related to aggressive tumor phenotype and indirectly affected the overall survival of BP-NEN patients. Our pilot study supports the need for future investigation of the biological function of NEK2 and PIM1 in BP-NEN transformation to verify the clinical value of our findings.Dysregulations of the NEK2 and PIM1-3 kinase signaling axes have been implicated in the pathogenesis of several cancers, including those with a neuroendocrine phenotype. However, their impact on bronchopulmonary neuroendocrine neoplasms (BP-NENs) has not been investigated. The aim of this pilot study was to determine mRNA and protein levels of NEK2, PIM1, and PIM3 in a group of 49 patients with BP-NENs: 11 typical carcinoids, 5 atypical carcinoids, 11 large cell neuroendocrine carcinomas, 22 small cell lung carcinomas (SCLC). The expression was measured using TaqMan-based RT-PCR and immunohistochemistry. NEK2 and PIM1 mRNA levels were higher in the SCLC patients than in the other BP-NEN groups ( Neuroendocrine neoplasms (NENs) are rare malignancies arising from cells throughout the diffuse endocrine system, and their incidence has significantly risen in the last years. NENs comprise a heterogeneous group of neoplasms with a wide spectrum of clinical behavior depending on tumor localization and the presence of hormonal hypersecretion. According to the 2015 World Health Organization (WHO) classification, bronchopulmonary neuroendocrine neoplasms (BP-NEN) comprise four tumor entities which differ with respect to their histology, mitotic count, and extent of necrosis: typical carcinoids (TC), atypical carcinoids (AC), small cell lung cancers (SCLC), and large cell neuroendocrine carcinomas (LCNEC) , 4 and NProtein kinases regulate many cellular signaling pathways, and their disruption is often associated with carcinogenesis . AlthougNEK2 is a serine/threonine kinase belonging to the NIMA (never in mitosis gene A)-related family, involved in cell division and cell cycle regulation by centrosome splitting. With the alternate splicing, NEK2 is expressed as three splice variants, namely NEK2A, NEK2B, and NEK2C. NEK2A is the full-length protein, with 445 amino acids (48 kDa), and is the most studied variant . AberranThe human PIM kinase is a highly conserved serine-threonine protein kinase named after the proviral integration site for MuLV, the genomic site where it was discovered. The PIM family, composed of three isoforms, viz. PIM1-3, plays a key role in the control of cell proliferation, survival, and migration .PIM1 kinase is a constitutively active downstream effector molecule of many cytokine signaling pathways controlled by JAK/STAT  transcription factors. PIM1 activity promotes tumor cell growth and survival through the modification of several cell cycle regulators and apoptosis mediators. PIM1 kinase can contribute to tumorigenesis also by enhancing MYC-regulated oncogenic signaling pathways . MoreovePIM3 suppresses apoptosis and promotes cell growth and survival, thereby enhancing cell proliferation of normal and malignant cells. PIM3 is expressed in various tissues, including the heart, brain, lung, kidney, spleen, placenta, skeletal muscle, and peripheral blood leukocytes. PIM3 protein is barely detected in normal adult endoderm-derived organs such as the liver, pancreas, colon, and stomach , 28; howTherefore, the aim of this pilot, clinical-based study was to verify a possible link between NEK2, PIM1, PIM3, and BP-NENs in the context of known prognostic features.n = 16) and NECs (n = 33).A total of 60 formalin-fixed paraffin-embedded tumor blocks (FFPEs) from 49 patients  with a median age of 65 years (60.00–70.00) were provided by Department of Pathology, Chair of Oncology, Medical University of Lodz, Poland. All patients recruited to the study had been newly diagnosed with BP-NENs from 2008 to 2019: 11 patients were diagnosed with TC, 5 with AC, 22 with SCLC, and 11 with LCNEC. All tumors were histopathologically examined and classified according to the WHO 2004 or 2015 classification of lung neoplasms , 36. ForThe sample material comprised either resected specimens, for NETs, or biopsy specimens, for NECs. The primary tumor samples were divided into two or three parts, depending on tumor size, and embedded in separate paraffin blocks. The number of samples used in the study varied from one to two FFPEs per patient.Ethics committee approval was obtained from the Institutional Board of the Medical University of Lodz (Number RNN/145/18/KE).Total RNA was extracted from FFPE tissue using the miRNeasy FFPE Kit (Qiagen). In brief, FFPE slices were processed in 1.5-mL Eppendorf tubes, deparaffinized with 160 μl deparaffinization solution, and then digested with proteinase K and DNase I. Purification of extracted total RNA was performed with RNeasy MinnElute columns according to the manufacturer’s instructions. The yield and quality (the ratio of absorptions at 260/280 nm) of RNA product were measured using PicoDrop spectrophotometer . The purified total RNA was immediately used for cDNA synthesis or stored at − 80 °C until use.Forty-five patients with good quality total RNA were taken for further analysis.cDNA was generated with the Maxima First Strand cDNA Synthesis kit for RT-qPCR (ThermoFisher) according to the manufacturer’s protocol. Briefly, 500 ng of total RNA was used as starting material, to which the reaction components and reverse transcription master mix were added. The reaction proceeded for 10 min at 25 °C followed by 15 min at 50 °C. After inactivation of Maxima Reverse Transcriptase (5 min at 85 °C), the cDNA samples were kept frozen at − 20 C.-μL reactions included 50 ng cDNA, 5-μL TaqMan™ Fast Advanced Master Mix (ThermoFisher), and 0.5-μL appropriate TaqMan Gene Expression Assay. All reactions were run in duplicate on a 7900 HT Fast Real-Time PCR System (Applied Biosystems). The following thermal cycling specifications were performed: 10 min at 95 °C and 40 cycles each for 10 s at 95 °C and 60 s at 60 °C. All reactions were run in duplicate.Measurement of mRNA expression was done using standard TaqMan® Gene Expression Assays (Applied Biosystems): Pim-3 Proto-Oncogene, Serine/Threonine Kinase , Pim-1 Proto-Oncogene, Serine/Threonine Kinase , NIMA Related Kinase 2 , and actin beta  as the endogenous control. TaqMan PCR assays were performed in 10Ct^−ΔΔ method).Kinase expression levels were calculated using the RQ , PIM1 Rabbit Polyclonal Antibody (C-term) (Abgent), and PIM3 Polyclonal Antibody (ThermoFisher Scientific), processed with the EnVision (DAKO) system. Tumor sections were examined for kinase immunoreactivity under a microscope at × 20 and × 40 magnifications. The IHC results were validated using positive and negative tissue controls in all series of immunostained slides. The following positive controls were set up: NEK2 on ovarian carcinoma, PIM1 on breast cancer, and PIM3 on gastric carcinoma. To examine negative control staining, neoplastic tissue slides were evaluated using mouse isotype antibody Ready-to-Use FLEX Negative Control Mouse . The tests were carried out using Autostainer Link 48 .For the semiquantitative immunohistochemical scoring (IRS), we applied a cutoff of 10% immunolabeled cells for BP-NENs. Cytoplasmic staining was considered for the evaluation of NEK2 and PIM3 expression and cytoplasmic and nuclear for PIM1 expression.Negative NEK2, PIM1, and PIM3 staining in tumor sections were defined as IRS 0. All positive sections were further scored according to three grades of staining intensity .Moreover, for further statistical analysis, all sections were additionally divided into a two-point classification: IRS 0 and 1 were placed into one group, and IRS 2 and 3 into a second.The immunohistochemical stainings were examined in standard light microscopy . The selected sections were scanned and representative images were taken using UltraFast Scanner  with DigiPath™ Professional Production Software .t test or one-way analysis of variance (ANOVA) in the case of normal distribution and the Mann Whitney U test  in the case of the non-normal distribution. Differences between categorical variables were evaluated using the χ2, two-tailed Fisher’s, or Yates exact test. Bonferroni’s correction was used for multiple comparisons. Spearman’s rank test was used for correlation assessment. p values < 0.05 were considered statistically significant. The Statistica 13.1 PL package  was used for the analysis.Continuous variables are presented as medians followed by IQR and nominal variables are presented as numbers followed by percentages in brackets. The Shapiro-Wilk test was used to determine the distribution. Continuous variables were compared using Student’s For the outcome analyses, overall survival was defined as the time period from diagnosis to last follow-up (15th October 2019), with censoring of live patients at the last follow-up. Overall survival data are presented as the Kaplan-Meier survival curves and compared within subgroups using the log-rank test. Cox hazards regression analyses of overall survival adjusted for age were performed for each variable.p < 0.001)  in SCLC samples (RQ 2.16 (1.81–3.23)) compared with other BP-NEN groups (RQ 0.85 (0.41–1.26), 0.65 (0.52–0.77), and 1.1 (0.79–1.45), for TC, AC, and LCNEC, respectively) ), which was higher than the PIM1 mRNA level (RQ 1.22 (0.66–2.05)). NEK2 mRNA expression in BP-NENs was low (RQ 0.29 (0.07–1.83)). However, NEK2 mRNA levels were significantly increased in SCLC patients (RQ 4.22 (3.37–5.99)) comparing with other entities: TC (RQ 0.06 (0.03–0.09)), AC (RQ 0.05 (0.03–0.44), and LCNEC (RQ 0.58 (0.29–1.00)) (01) Fig. . Similarly) Fig. .Fig. 1mRp = 0.048) in TC and AC (RQ 3.58 (1.74–4.32) and 3.93 (2.70–4.68), respectively) than in the aggressive BP-NEN tumors (RQ 1.81 (1.10–2.87) for LCNEC and RQ 2.32 (1.88–2.52) for SCLC)  and the age at diagnosis  in BP-NEN patients. PIM1 mRNA levels were also negatively associated with PIM3 mRNA levels .In contrast, PIM3 mRNA levels were significantly higher (LC) Fig. . In addiIn BP-NEN patients, the most frequent immunoreactivity was observed for PIM3 kinase, being present in 57 (97.0%) samples. Moreover, 25 (42.4%) FFPEs demonstrated strong (IRS 3) PIM3 protein expression. Positive immunoreactivity for NEK2 was detected in 54 (91.5%) BP-NEN sections, and they showed mostly (23 (39.0%) sections) moderate intensity of staining (IRS 2). The least frequent immunoreactivity among all BP-NEN specimens was observed for PIM1 kinase (46 (78.0%) samples) and its expression was mainly weak, with 28 (47.5%) FFPEs characterized by IRS 1.PIM1 protein expression was slightly, but not significantly, higher in SCLC, as 45.8% of them (11 sections) showed quite intense immunostaining (IRS 2 in 9 (37.5%) and IRS 3 in 2 (8.3%) samples). IRS 3 for PIM1 was not observed in any other BP-NEN groups, and IRS 2 was detected only in 4 (25.0%) TC, 1 (16.7%) AC, and 2 (15.4%) LCNEC.p = 0.004).Similarly, NEK2 protein expression tends to be slightly elevated in SCLC, being present at IRS 2 and 3 in 70.8% of them (17 samples), but only in 50.0% of TC (8 sections), 50% of AC (3 sections), and 61.5% of LCNEC (8 sections). PIM3 protein expression was rather intensive in BP-NENs, as mentioned above, but did not differ significantly between histopathological groups. In addition, a positive correlation was found between NEK2 and PIM1 protein expression in BP-NEN patients . No significant associations were found between mRNA and protein levels for kinases PIM1 and PIM3.An association was found between mRNA and protein levels for NEK2 in BP-NEN patients . Elevated NEK2 mRNA levels were related to a lower probability of OS in BP-NEN patients ). In addition, patients with higher PIM1 protein expression also demonstrated lower OS than those with weak or no PIM1 expression ). These observations could be mostly attributed to the relationship between high NEK2/PIM1 levels and an aggressive NEC phenotype. However, higher PIM1 protein expression was shown to be associated with worsened OS also in the subgroup of NEC patients ).Within the whole BP-NEN group, 23 deaths were observed and the median (IQR) overall survival was 1.4 years (0.1–11.00). Median overall survival ) was 2.4 years (1.6–2.7) for TC, 4.2 years (3.0–9.7) for AC, 2.1 years (0.9–2.6) for LCNEC, and 0.7 years (0.3–1.2) for SCLC (Table As no deaths were observed among NET patients, this subgroup was further studied to obtain recurrence data. Three cases of tumor relapses were observed: one in TC and two in AC. Unfortunately, no statistical analysis was possible due to the small number of events.The present study is the first to show that NEK2 and PIM1 expression is associated with BP-NEN aggressiveness.Although previous studies have indicated that the NEK2 gene is involved in lung adenocarcinoma , 38 and Recent studies based on immunohistochemical staining found PIM1 to be highly expressed in NSCLC , 42. MorTo our knowledge, the role of PIM3 in lung tumors has not been studied so far. Our results reveal abundant PIM3 expression in all BP-NENs, but surprisingly, no higher expression was found in highly aggressive neuroendocrine cancers compared with less aggressive carcinoids. PIM3 mRNA levels were even significantly higher in lung NETs than in lung NECs, and PIM3 protein expression estimated by IHC was quite intense in all BP-NENs, with no significant differences being observed between histopathological groups. No significant relationships were observed between PIM3 mRNA and protein levels among BP-NEN patients. Similar results were obtained for PIM1, although in SCLC, a correlation was noticeable between increased PIM1 mRNA and protein expression. Disjunctions between the levels of mRNA and the protein product are not uncommon: these have been demonstrated in large-scale proteome- and transcriptome-profiling experiments, and this phenomenon is believed to be connected with post-transcriptional, translational, and protein degradation regulation , 44.Our OS analysis results are in line with global statistical data for NET and NEC patients, suggesting that despite its limited size, our study cohort is representative and our results are reliable. Our survival findings are also supported by the presence of a positive correlation between PIM1 and NEK2 expression in BP-NENs. Hence, our results may suggest a coexistence of PIM1 and NEK2 overexpression in SCLC and their contribution to SCLC tumorigenesis. Unfortunately, it was not possible to perform a statistical analysis evaluating the prognostic value of PIM and NEK2 expression in lung NET patients due to no deaths and a small number of relapses in this group. It should be also noted that due to the very low incidence of BP-NENs, especially lung NETs, it is difficult to conduct a large-scale study on BP-NEN patients drawn from only a single center. In addition, as surgery is not a standard therapeutic option in SCLC, adequate primary SCLC specimens for expression studies are difficult to obtain.To conclude, our study revealed a high expression of NEK2 and PIM kinases in SCLC. Nevertheless, the role of NEK2 and PIM1 overexpression in SCLC tumorigenesis and the clinical relevance of our findings remains unclear. However, data from other cancers indicate that NEK2 and PIM1 kinases possess oncogenic potential and that they interact with diverse downstream signaling pathways. In vitro studies have shown that NEK2 promotes cell proliferation by AKT and Wnt activation in NSCLC  and hepaSuch confirmation is important in the therapeutic context, as NEK2 and PIM kinases have been shown to be potential targets in therapies in different cancers , 55. IndIn conclusion, our data suggests that the aggressive phenotype of BP-NEN is associated with elevated NEK2 and PIM1 expression. Despite being of a preliminary nature, our intriguing findings justify further investigation of the biological function NEK2 and PIM1 in BP-NEN and the translational potential of this knowledge.


Alcaligenes faecalis NYSO were studied in the present investigation.Due to the multitude industrial applications of ligninolytic enzymes, their demands are increasing. Partial purification and intensive characterization of contemporary highly acidic laccase enzyme produced by an Egyptian local isolate designated Alcaligenes faecalis NYSO laccase has been partially purified and intensively biochemically characterized. It was noticed that 40–60% ammonium sulfate saturation showed maximum activity. A protein band with an apparent molecular mass of ~ 50 kDa related to NYSO laccase was identified through SDS-PAGE and zymography. The partially purified enzyme exhibited maximum activity at 55 °C and pH suboptimal (2.5–5.0). Remarkable activation for enzyme activity was recognized after 10-min exposure to temperatures (T) 50, 60, and 70 °C; time elongation caused inactivation, where ~ 50% of activity was lost after a 7-h exposure to 60 °C.2+, Zn2+, Co2+, Ni2+, Mn2+, Cd2+, Cr2+, and Mg2+ caused strong stimulation for enzyme activity, but Fe2+ and Hg2+ reduced the activity. One millimolar of chelating agents  caused strong activation for enzyme activity. Sodium dodecyl sulfate (SDS), cysteine-HCl, dithiothreitol (DTT), β-mercaptoethanol, thioglycolic acid, and sodium azide caused strong inhibition for NYSO laccase activity even at low concentration. One millimolar of urea, imidazole, kojic acid, phenylmethylsulfonyl fluoride (PMSF), H2O2, and Triton X-100 caused activation. The partially purified NYSO laccase had decolorization activity towards different dyes such as congo red, crystal violet, methylene blue, fast green, basic fuchsin, bromophenol blue, malachite green, bromocresol purple eriochrome black T, and Coomassie Brilliant Blue R-250 with various degree of degradation. Also, it had a vast range of substrate specificity including lignin, but with high affinity towards p-anisidine.Some metal ions CuAlcaligenes faecalis NYSO strain would support several industries such as textile, food, and paper and open the possibility for commercial use in water treatment. It will also open the door to new applications due to its ligninolytic properties in the near future.The promising properties of the newly studied laccase enzyme from  In the past few years, enzymatic bioremediation has become an attractive alternative to further support the bio-treatment techniques, where the most currently available enzymes provide simpler systems than a whole organism . Most xeMany of the industrial applications of the ligninolytic enzymes  require only crude preparations of the enzyme, and in such cases, the enzyme purification is not obligatory. However, in certain cases, purified enzymes are also used for some applications . The purAlcaligenes faecalis NYSO, and its decolorization activity towards different dyes.Due to the high industrial applicability of laccases, this study addressed the partial purification, stability characteristics of a laccase produced by A. faecalis is a Gram-negative, alkali-tolerant, rod-shaped bacterium with flagella, commonly found in soil water, and other environments, and belong to the Alcaligenaceae family. In this study, A. faecalis strain NYSO (KP859538) isolated from a discharged effluent of tanning and leather industry, Alexandria, Egypt, was used and identified by 16S rRNA as described previously by Abdel Galil et al.  × 100The decolorizing ability of the partially purified laccase was evaluated by using different synthetic dyes (0.2 g%) such as malachite green  and D% = dye decolorization (%) after time (t)  and at relatively high T . Also, it is more stable against thermal denaturation and has high thermal stability and a long half-life when T is below 70 °C . Most of the reported bacterial laccase showed preference to work optimally at nearly similar T and pH –29, but t pH 8.0 . Thermalt pH 8.0  explaineg et al.  noticed Furthermore, the investigated NYSO laccase exhibited a high pH stability, especially in acidic condition (pH 4–5) for 24-h incubation. In contrast, the enzyme was unstable at pH 6.0 where about 49.1% of activity remained after 5.0-h incubation. These results are in agreement with the results obtained by other investigators , 31. The2+, Cr2+, Mn2+, Cu2+, Zn2+, Ni2+, Cd2+, and Co2+), specially Cu2+, caused strong stimulation for the studied enzyme, while Fe2+ and Hg2+ ions caused a reduction in activity. The high extent activation of laccase by Cu2+ might be caused by the filling of type-2 copper-binding sites with Cu2+ [2+, Mn2+, Zn2+, Cu2+, Ni2+, Mg2+, and Co2+ which is matching with our results, while the inhibitory effect of Fe2+ and Hg2+ on laccase activity was reported by many investigators [2+, Co2+, and Ni2+ metal ions caused a reduction in Streptomyces psammoticus laccase activity while Fe2+ caused activation.Some metals ; these findings were in congruence with the result obtained by Niladevi et al. . Howeverproducts . Howeverproducts .A. faecalis NYSO). The theme chosen for this study and noted promising characteristics of the studied enzyme opens the door for many applications. The promising unusual features can be summarized in the following points: it is able to work in a highly acidic condition up to pH 2.5 and has high stability against thermal denaturation up to 70 °C for 1 h and high stability at pH range 4.0–5.0. The enzyme showed the highest storage stability at − 20 °C and 4.0 °C up to 46 days, while at 25 °C the enzyme activity decreased to more than half after 8 days’ storage time. Some metals  caused strong stimulation for the studied NYSO enzyme, while Fe+2 and Hg+2 ions caused a reduction of the activity. Also, chelating agents  caused strong activation for enzyme activity at 1 mM. Cysteine-HCl, DTT, β-mercaptoethanol, sodium azide, thioglycolic acid, and SDS caused strong inhibition for NYSO laccase even at low concentrations. Additionally, the partially purified studied enzyme showed decolorization activity towards wide variants of synthetic dyes. The enzyme exhibited the highest activity with p-anisidine, and its catalytic efficiency of phenolic substrates could be ordered as follows: p-anisidine > vanillic acid > p-phenylenediamine > 4-aminophenol > 4-nitrophenol > tannic acid > hydroquinone > ABTS > catechol > guaiacol > pyrogallol > syringaldazine. The aforementioned promising features of NYSO laccase make it suitable for various industrial processes, decolorization of industrial effluent, and wastewater treatment.Laccases are one of the most widely used enzymes in industry. Thus, this study has focused on addressing the major characteristic properties of newly partially purified YNSO laccase produced by alkali-tolerant Egyptian isolate (


L. elongatus using different protocols of hormonal induction.Thus, fifteen wild mature females maintained at the Experimental Fish Station, Salto Grande,SP, Brazil were submitted to three different hormonal treatments: CPE (fractioned dose:0.5 and 5.0 mg kg-1); mGnRHa (single dose: 3.5 µg kg-1) andmGnRHa (single dose: 5.0 µg kg-1). The spawning rate and absolute fecunditywere similar among the treatments, but fertility rates were higher for CPE treatment (23.60± 9.40) then for mGnRHa treatments (close to or zero zero). Although females ovulatedin all treatments, none of them provided viable embryos, showing hatching rates close to zeroor zero. Both mGnRHa treatments were more potent for inducing the ovulatory process then CPEtreatment, which was evidenced by the fact that the formers showed higher volume density ofpostovulatory follicles (POF). Accordingly, E2 and 17α-OHP plasmalevels were higher for the mGnRHa treated females compared to the CPE one at the time of ovulation.In this study we confirmed previous scientific evidence that, regardless of whether promotingovulation, the use of conventional CPE and GnRH doses are not appropriate for some South Americanmigratory species, due to the non-attainment of viable embryos. Moreover, we have broughtnew information about the relationship between reproductive performance and gonadal steroidsconcentrations using different hormonal therapies, contributing to understand the reasonsfor Leporinus elongatus embryo loss in induced spawning.Several studies have been developed to support the replacement of the crude carp pituitaryextract (CPE) by synthetic products for induced reproduction of South American rheophilicspecies. However, results have been quite heterogeneous and there is no consensus or a routineuse of synthetic products in these species. Thus, the aim of this study was to evaluate the ovulatoryprocess in  Leporinus elongatus is a medium-sized, total spawner rheophilic fish,commercially relevant and known to be a good model for studies concerning the reproductive biologyof rheophilic species . Moreover, this species was one of the ten most produced fish in Brazil in 2014  has grown rapidlydue to its numerous advantages, but mainly because they are not species-specific moleculesand present high structural similarities among fish. Moreover, due to their synthetic nature,they pose no risk of transmitting diseases, such as carp pituitary extracts (CPE) do, and sincethey act at higher levels of the hypothalamic-pituitary gonad axis, they stimulate the releaseof LH and FSH, as well as other pituitary hormones, which may serve important reproductive functions(review in-1) . A highproportion of oocytes retained in the ovaries after stripping (week ovulation) also seems tobe a constant in treatments using CPE in South American rheophilic species  under averagetemperature of 27°C and natural photoperiod. Fish were manually fed a pelleted balancedcommercial diet  corresponding to 3.0% of total bodyweight twice a day.Wild males and females (at a sex ratio of 1:1) were maintained at Duke Energy’s ExperimentalFish Station (22 ° 54'23 .81 “S and 50 ° 00 '05.06”W). The animals were kept in 200 m-1, 12hinterval, diluted in 0.5 mL saline - 0.9%); 2-) mammalian gonadotropin-releasing hormoneanalogue (mGnRHa)  – single dose  and 3-) mammaliangonadotropin-releasing hormone analogue (mGnRHa)  –single dose .Fifteen mature females with a mean biomass of (mean ± SEM) 1.5 + 0.4 kg were randomlyselected for the induced breeding experiments. At the time of spawning, broodstock were transportedto the lab for acclimatization and maintained in 500 L tanks with constant water circulation.Females were randomly submitted to three different hormonal treatments: 1-) crude carp pituitaryextract (CPE) (Fish braz®) – two doses  was estimated according to the methodproposed by-1. To determine the fertilization rate, 8-12hours post-fertilization, 100 eggs from each female were randomly sampled and counted, andthose which were normally dividing were scored (viable embryos). After 17 post-fertilization,overall hatching rate was determined by counting the number of hatched eggs/number of fertilizedeggs X 100.Soon after fertilization, ~50 ml of eggs from each female were distributed into four funnel-shapedplastic incubators with a capacity of 18 L. Then, 18 g of eggs were placed in each incubator witha constant water flow of 5 L/minAll spawned females were euthanized after spawning with an overdose of anesthesia  and had their ovaries collected. The gonadosomatic index (GSI)was determined according to-1.Water parameters were measured throughout the latency period and egg incubation  using a YSI model 55 oximeter and a YSI model 63 multiparametersonde . The experiments were performedunder natural photoperiod and average water mean temperature, pH and dissolved oxygen wererespectively 26.5 ± 1.25°C, 6.9 ± 0.4 and 6.03 ± 0.54 mg LFor the histological evaluation (volume density), cranial, medial, and tail regions of theovary tissues were fixed in Bouin solution for routine histological procedures, accordingto the metodology applied byThe volume density was determined using light microscopy and a 320-intersection grid. Threefields from each region of the ovary were randomly selected, with a total of 2880 points scored for each animal at magnificationX 4. For this analysis, the methodology applied by2) and 17α-hydroxyprogesterone (17α-OHP) assays. The plasmasteroid level was measured through ELISA (Enzyme Linked Immunosorbent Assay) . Plasma samples were run in duplicate withan acceptable limit of < 20.0 for the intra-assay coefficientsof variation  and needles. Blood was centrifuged at 1300 g for 10 minutes. Theplasma was separated into aliquots and frozen at -80°C for the subsequent 17β-estradiol(E2). The volume density was analyzed by comparing different treatments with a one-wayanalysis of variance (ANOVA). In order to analyze the gonadal steroids, two-way ANOVA forrepeated measures was used. The Tukey’s test was used as a post hoc analysis. A thresholdof P < 0.05 was set to infer statistical significance. All statisticalanalyses were based onData normality was verified using the Cramer-von Mises test. Homoscedasticity was checkedthrough the Fmax test. ANOVA test was used to analyze all parameters of reproductive performance,except for the percentage of spawning, which was analyzed through the Chi - square test (X−1: 15.40 hours and 5 µg/kg−1: 15.20 hours)   than for both mGnRHatreatments (3.5 µg/kg−1: 24% and 5 µg/kg−1: 29%) compared to the CPE treatment (6.5%)    compared to theCPE treatment (1%)  than in the CPE one (74.17 pg mL−1)  and ovulation (74.17 pg mL−1)  and ovulation(3.5 µg/kg−1: 132.06 pg mL−1 and 5 µg/kg−1: 150.60 pg mL−1) in the mGnRHa treatments  induced fish were similar during the first dose  than in the CPE one (229.06 ng mL−1) ; however, none of the treatmentsprovided viable embryos.L. elongatus ovulation and viable embryos  has been very successful in Colossoma macropomum, butprovided low quality embryos when compared to CPE  provoked ovulation, but not viable embryos(Rhamdia quelen (two doses: 2 µgmGnRHa kg-1 + 1 mg metoclopramide kg-1 and 20 µg mGnRHa kg-1 + 10 mg metoclopramide kg-1 metoclopramide) ) in Schizodon fasciatus to hormonal induction, especially with GnRH, which provides inconstant and very heterogeneousresults of difficult reproducibility, especially if we consider that the doses applied in thisstudy (3.5 or 5.0 μg mGnRH kg-1 + 10 mg kg-1 metoclopramide)and for the congener L. macrocephalus (7 μg mGnRH kg -1+ 10 mg kg -1 metoclopramide) which did not provide viable embryos either (-1)  kg to CPE . In a cramide) . When asciatus. Taken 2 at the time of ovulation, which remained similar over time for the mGNRHa treatments.In this concern, future approaches that correlate maternal plasma E2 levels withembryonic survival rates would be necessary to evaluate a possible maternal E2transfer to eggs which may cause toxicity to them. It has already been demonstrated that unbalancedmaternal plasma reproductive hormones are transferred to eggs levels in non-viable eggs in relation to viable eggs 5 hours after fertilization and, coincidently, that treatment was the only one to present a reductionof EL. elongatus ovulatory process when compared to CPE, as it significantlyincreased the FPO volume density and gonadal steroid levels at the time of ovulation. However,the results obtained here reinforced the existing hypothesis of an ovarian hyperstimulationwith consequent egg toxicity due to the hormonal treatment in this rheophilic species and otherSouth American ones, that must be addressed in futures studies. Recent reports on embryonicmortality obtained in spawning induced South American migratory species (in vitro) in order to confirm which substances used in hormone therapies (and their derivatives), aswell as which levels are indeed toxic to the eggs. This aspect needs to be addressed for the developmentof aquaculture in countries which depend on these species, since rheophilic fish are known tobe among the most important fish for commercial purposes in South American countries (In conclusion, the use of mGnRHa associated with metoclopramide was more potent for inducingthe untries  and the


Arabidopsis thaliana leaf, root, and seed tissues by optimized sucrose density gradient centrifugation of protease protected plant extracts. The method co-purified non-translating 30S and 40S ribosome subunits separated non-translating 50S from 60S subunits, and resolved assembled monosomes from low oligomeric polysomes. Combining ribosome fractionation with microfluidic rRNA analysis and proteomics, we characterized the rRNA and ribosomal protein (RP) composition. The identity of cytoplasmic and chloroplast ribosome complexes and the presence of ribosome biogenesis factors in the 60S-80S sedimentation interval were verified. In vivo cross-linking of leaf tissue stabilized ribosome biogenesis complexes, but induced polysome run-off. Omitting cross-linking, the established paired fractionation and proteome analysis monitored relative abundances of plant chloroplast and cytoplasmic ribosome fractions and enabled analysis of RP composition and ribosome associated proteins including transiently associated biogenesis factors.Conventional preparation methods of plant ribosomes fail to resolve non-translating chloroplast or cytoplasmic ribosome subunits from translating fractions. We established preparation of these ribosome complexes from  Ribosomes are highly conserved ribonucleoprotein complexes that translate messenger (m) RNA into proteins by ribozyme catalysis . Owing tThe recently elucidated structure of spinach chloroplast ribosomes confirms 25 prokaryote type SSU RPs that are assembled with 16S rRNA and 33 LSU RPs in a complex with 4.5S rRNA, 5S rRNA, and 23S rRNA. Plant 23S rRNA has two hidden breaks at positions 515 and 1755 , resulti2 pre-rRNAs of the LSU branch. Assembly of RPs in the nucleus leads to pre-ribosomal subunits that are larger than the mature SSUs and LSUs, for example, the 66S-preribosomal subunit of the final 60S LSU branch. The two independent, but coordinated pathways culminate in the export of the yet immature pre-60S and pre-40S subunits from the nucleus to the cytoplasm. The last steps of 60S and 40S maturation occur in the cytoplasm, where protein factors protect and proof read yet immature subunits and facilitate the final transformation of pre-ribosomal complexes into translation-competent subunits.Several authors reviewed the complex pathways of eukaryotic ribosome biogenesis and specialized reviews were dedicated to the biogenesis of yeast, mammalian, and plant eukaryotic ribosomes ,16,17,18Arabidopsis thaliana , 2 M KCl [1 L], 0.5 M ethylene glycol-bis (β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) pH 8.3 [0.5 L], 1 M MgCl2 [0.5 L], 20% (v/v) polyoxyethylene 10 tridecyl ether (PTE) [25 mL], and 10% sodium deoxycholate (DOC) [25 mL]. A 20% detergent mixture [50 mL] consisted of 20% (w/v) Brij-35, 20% (v/v) Triton X-100, 20% (v/v) Igepal CA 630, and 20% (v/v) Tween 20. This mixture was initially heated to not more than 60 °C and dissolved completely. Before dispensing volumes for REB preparation, all stock solutions were gently mixed avoiding bubble formation. The 20% detergent mixture was re-heated to 45 °C to ensure complete and homogenous solution of all components.Pre-ribosome extraction buffer (REB) and stock solutions that are required for the final, ready to use REB  were pre−1 cycloheximide [50 mL in ethanol], 50 mg mL−1 chloramphenicol [50 mL in ethanol], 200 mg mL−1 heparin [1 mL], 0.5 M phenylmethylsulfonyl fluoride (PMSF) [15 mL in isopropanol], and 1 tablet mL−1 cOmpleteTM protease inhibitor mixture . These solutions were prepared without autoclaving and stored at −20 °C. Before pipetting, these solutions were carefully mixed avoiding bubble formation and kept on ice. Cycloheximide, chloramphenicol, heparin, and DTT inhibit eukaryotic and chloroplast translation elongation by arresting ribosomes. PMSF and cOmpleteTM protease inhibitor mixture suppress protease activities.The above-described stock solutions were combined to obtain pre-REB using volumes and temperatures reported in g and 4 °C. The supernatant was loaded onto a lilac QIAshredder mini spin column , centrifuged and the eluate was loaded onto previously prepared sucrose gradients in ultracentrifugation vials (cf. below).The ribosome extraction procedure was according to previously reported protocols ,66 with 2 , was dissolved in ddH2O using a water bath set to less than 50 °C. The dissolved final sucrose solution was filtered using 0.22 µm qpore sterile filtration unit  and stored at room temperature. Prior to gradient assembly, four sucrose gradient solutions were prepared with concentrations of 15% (w/v), 30% (w/v), 45% (w/v), and 60% (w/v) sucrose. The volume parts of the 2 M sucrose stock-solution, the 10x salt and buffer solution, ddH2O, cycloheximide, and chloramphenicol solutions that are required for 1.8 mL of 15% and 60% sucrose step gradient solutions and for 3.6 mL final volumes of the 30% and 45% sucrose solutions are reported in 2M sucrose, that is, 68.5% (w/v) sucrose gradient solutions that are best prepared in large volumes. These prepared volumes of sucrose gradient solutions should ideally be sufficient for at least 50 large or small ultracentrifugation tubes.Sucrose gradients were assembled in large (13.2 mL) or small (5 mL) thin-wall polypropylene ultracentrifugation tubes . Gradient assembly is, in our hands, the critical step for reproducible separation of ribosome complexes. To ensure reproducible gradient performance, we recommend in-parallel preparation of large sets of sucrose gradient ultracentrifugation tubes from the same 15%, 30%, 45%, and 60% (w/v) sucrose gradient solution. The volumes for large and small ultracentrifugation tube are reported within All preparation steps were performed in a cold room, unless indicated otherwise. All sucrose gradient solutions were temperature equilibrated to 4 °C and thoroughly mixed before pipetting. The only exception was the 60% solution, which, owing to the high viscosity and for proper dispensation, was equilibrated and pipetted at room temperature. The four sucrose concentrations steps were pipetted in ascending order, starting with the 60%  or 2 h at 50,000× g and 4 °C using the SW55Ti rotor , as previously described [The extract of ribosome complexes, that is, the eluate of QIAshredder mini spin columns, was loaded without delay onto the sucrose gradients prepared in ultracentrifugation vials. Prior to loading, the prepared sucrose step gradients were taken from −80 °C storage and kept cold at 4 °C overnight, to allow diffusion and formation of a continuous gradient. Any abrupt movement or vibration while handling the thawing and thawed tubes must be avoided to minimize gradient perturbation. The extracts of ribosome complexes or 500 µL REB non-sample controls, that is, blanks, were loaded gently and in steps with a 100 µL or 200 µL pipet onto the top of the thawed sucrose gradients by letting the liquid run slowly down the tube walls. The surface of the sucrose gradients must not be perturbed by rapid, vigorous pipetting. All sample tubes of a run were equally balanced using REB. We centrifuged with either a SW41Ti rotor of 13.2 mL nominal tube capacity or using a SW55Ti rotor of 5.0 mL nominal tube capacity. The rotors were swinging-bucket and operated by an Optima L80-XP ultracentrifuge . The rotors are limited to six buckets per ultracentrifugation run. Each run had a non-sample gradient overlaid with 500 µL REB and a gradient with a reference preparation of ribosome complexes from Arabidopsis rosette leaves at developmental stage 1.10  for qualescribed . We sele-1 flow rate with continuous absorbance measurement at λ = 254 nm using a Brandel BR-188 density gradient fractionation system . Considering the solvent delay between the UV-detector and fractionator, ~100 s equivalent to 1.25 mL, up to 40 fractions were collected at room temperature from large ultracentrifugation tubes, and up to 20 fractions from small tubes. After completion of fractionation, the collected ~250 µL fractions were stored at −80 °C until further use.After ultracentrifugation, sucrose gradients with separated ribosome complexes were retrieved from the top of bottom-pierced ultracentrifugation tubes. Approximately 250 µL fractions were collected at 0.75 mL minw/v) sucrose gradients from a rotor set, the fractionation system was equilibrated by first mounting a clean ultracentrifugation tube filled to approximately 80% of the volume with 15% (w/v) sucrose gradient solution. After piercing the clean tube at the bottom, the 15% (w/v) solution was pumped through the system and an absorbance baseline was recorded. Subsequently, a 15–60% (w/v) sucrose gradient tube was mounted and pierced at the bottom and high-density chase solution was used to push and empty the gradient from the bottom to top. The chase solution contained 62% (w/v) sucrose in 1x salt and buffer solution, ddH2O, and 1 µg mL−1 bromophenol blue. This solution was filtered using 0.22 µm pore size sterile filters and stored at 4 °C. Air bubbles must be avoided when mounting the tubes and preparing the fractionation system. Before and after fractionation of each gradient, the system is best cleaned sucrose-free by flushing the system in both forward and backward flow mode with at least 10 mL DEPC–water.In detail, prior to the sampling of each of the six 15–60% . Briefly, 350 µL of TRIzol and 150 μL of chloroform/isoamylalcohol (v/v) were added to the 250 µL of a single collected fraction, then vortexed for 10 s and centrifuged at 11,000× g, 4 °C for 10 min. The aqueous phase was transferred to new tubes, where 350 μL of isopropanol and 100 μL of 0.8 M sodium acetate were added. The samples were thoroughly mixed and incubated at 4 °C for 30 min, with subsequent centrifugation at 11,000× g, 4 °C for 10 min. The supernatant was discarded. The pellet was washed with 1 mL of 70% ethanol, followed by centrifugation at 11,000× g, 4 °C for 10 min. In the end, the pellet was resuspended in 20 µL of DEPC H2O.Total RNA extracts were prepared using guanidinium thiocyanate-phenol-chloroform extraction with TRIzolThe extracted RNA samples were loaded onto an agarose gel and analyzed using an Agilent 2100 Bioanalyzer and RNA 6000 nano kit, according to the instructions of the manufacturer  and described previously ,68. The A 2 mg/mL bovine serum albumin (BSA) stock solution was prepared from chromatographically purified BSA  as an internal reference standard for proteomic analyses. Then, 250 μL from a single fraction and 6 µL of BSA stock solution were mixed with 600 μL of methanol. After thorough mixing, 150 μL chloroform and 450 μL water were added. After subsequent vortex-mixing and immediate centrifugation for 20 min at 14,000 rpm and 4 °C, a white disc of protein formed between the lower organic layer and the upper aqueous layer. We discarded the upper aqueous layer, added 650 μL of methanol to the tube, and inverted the tubes three times. The tubes were centrifuged again for 20 min at identical settings. All liquid was carefully removed, and the pellet was air-dried. The precipitated dried protein was resuspended in SDS-PAGE sample buffer for electrophoretic analysis and can be submitted directly to proteomic analysis by liquid chromatography mass spectrometry (LCMS).We optimized this step for ribosome-associated proteome analysis to reduce the effects of varying amounts of residual sucrose in the final protein preparations. Here, 3 kDa cut-off Amicon Ultra-0.5 Ultracel-3 membrane centrifugal filters were used for protein purification and elution steps . Approximately 250 µL of a single sucrose fraction of interest was mixed with 6 µL of 2 mg/mL BSA internal standard. The mixture was then added to the filters and a volume of 500 µL adjusted by 1x salt and buffer solution for sucrose gradients. Centrifugation was for 20 min at 14,000 rpm at 4 °C. We repeated the buffer-washing step four times and adjusted the sample volume to 500 µL with sucrose free 1x salt and buffer solution. In a final step, 20 µL of the concentrated proteins was retrieved for proteomic analysis.w/v) acrylamide SDS-PAGE. Polyclonal anti-RPL13B antibodies (AS13 2650/anti-L13-1), directed against the 23.8 kDa RPL13B/eL13 LSU protein encoded by At3g49010, and anti-RPS14 antibodies (AS12 2111/anti-RPS14-1) directed against the 16 kDa RPS14-1 (uS11) SSU protein encoded by AT2G36160), were obtained from Agrisera AB, Vännäs, Sweden [Western blot analysis was performed as described previously  using 12, Sweden ,21. The w/w) of the protease. The digested peptides were acidified to pH < 3.0 with 10% trifluoroacetic acid (TFA). Prior to the chromatography, the peptide mixture was purified and desalted on C18 SEP-Pak columns , which were attached to a QIAvac 24 Plus (QIAGEN) vacuum manifold. The columns were equilibrated with 1 mL 100% methanol, once with 1 mL 80% acetonitrile (ACN) and twice with 1 mL of 0.1% TFA. The peptides were applied to the C18 SEP-Pak column and allowed to pass through slowly. The column was washed twice with 1 mL of 0.1% TFA. The peptides were eluted with 800 µL of a mixture of 60% ACN and 0.1% TFA, dried in a speed vacuum concentrator, and stored at −80 °C prior to mass spectrometry analysis. The peptides were resuspended in 30 μL of resuspension buffer, that is, 5% (v/v) acetonitrile and 2% (v/v) trifluoroacetic acid.The following protocol entails protein digestion peptide clean-up and instrumental analysis. The disulfide bridges of proteins were reduced using a 200 mM DTT, 50 mM Tris-HCl pH 8.0 solution by incubation for 2 h at 25 °C. The thiol groups were alkylated using 200 mM iodoacetamide (IAM) by incubation for 1 h at 25 °C in the dark. The enzymatic digest was performed for 16 h at 30 °C using a Trypsin/endoproteinase LysC mix  in a ratio of 25 parts of the protein to 1 part (−1 in a solution consisting of 3% (v/v) acetonitrile and 0.5% (v/v) formic acid. Peptide elution was facilitated by increasing the acetonitrile gradient from 3% to 24% (v/v) over 90 min, from 24% to 36% for the next 30 min, and from 36% to 85% for the last 6 min at a flow rate of 0.3 μL min−1. Peptide ions were detected in full scan mode, with range of mass-to-charge ratios from 300 to 1600 at a resolution of 120,000, automatic gain control (AGC) target of 3 × 106, and maximum injection time IT of 100 ms. Each dd-MS2 scan was recorded in profile mode at a resolution of 15,000 with AGC target of 1 × 105, isolation width mass-to-charge ratio 1.2 m/z and maximum IT of 150 ms.Measurements were performed by a Q Exactive HF Quadrupol-Orbitrap Mass Spectrometer  coupled to an ACQUITY UPLC M-Class System . Here, 8 μL samples were loaded onto an ACQUITY UPLC M-Class HSS T3 column, 75 μm inner diameter, 20 cm length, and 1.8 µm bead size  at a flow rate of 0.4 μL minArabidopsis thaliana TAIR10 protein sequences  were used by the search engine Andromeda [Peptides for which MS/MS spectra had been recorded were excluded from further MS/MS scans for 30 s. Raw files were submitted to MaxQuant software for protein identification and quantification . Arabidondromeda  for idennces 35,36 entriesndromeda ,73,74,75The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE  partner 2-transformed ratios and corresponding gene identifiers were uploaded into the bioinformatics tool agriGo v2.0 to perform a gene ontology enrichment analysis [LFQ intensities of all proteins in a single fraction from the generated data sets were normalized to the abundance of the BSA standard in each fraction. For comparison between fractions, the relative abundance of each protein was calculated as the ratio of the normalized LFQ intensity in a single fraction to its average normalized LFQ intensity across all fractions of a data set. Loganalysis ,78.Our analyses demonstrate that the established methodology allows separation of cytosolic and chloroplast non-translating ribosome complexes from respective monosomes and low oligomeric polysomes. The non-translating chloroplast and cytosolic 50S and 60S LSUs are separated, but most cytosolic and organelle ribo-complexes co-purify. The co-purification restricts relative quantification of ribosome complexes from leaf material using UV absorbance profiles of sucrose density fractionations ,5,6,34. In vivo formaldehyde crosslinking can be used for qualitative analysis of ribosome-associated proteins, as was exemplified by selected Arabidopsis pre-60S ribosome biogenesis factor homologs. However, in vivo cross-linked ribosome complexes clearly do not represent the in vivo composition of ribosome complexes and cannot serve as a proxy for analyzing the abundance and RP composition of non-translating ribosome complexes. In addition, the accumulation of translation initiation factors in the cross-linked 80S fraction raises the concern of crosslinking artifacts that may create non-native ribosome complexes.Omitting crosslinking from the currently established combination of methods allows to assess the relative abundance of non-translating ribosome complexes and of RPs with high genomic coverage of cytosolic and organelle RP families and paralogs. Ribosome associated proteins that may be present and may change abundance in these complexes can be assessed by purification and concentration of sucrose density fractions using ultra-membrane centrifugation. Nevertheless, we cannot exclude that weakly bound RPs and ribosome associated proteins may still be lost or de-enriched from current preparations of ribosome complexes.With this caveat in mind, indications of altered RP or RP paralog composition of non-translating plant ribosome complexes can be attempted. Proteome analyses of RP and RP paralog composition may support and extend our knowledge of altered ribo-proteomes. Such analyses may prove fruitful and important in view of recent studies that revealed unexpected and selective roles of core RPs from non-plant eukaryotic ribosomes in cell homeostasis and organism development . We argu


We tested IgE functionality and IgG blocking capabilities using appropriate model cell lines. In vitro studies showed IgE engagement with FcεRI and CD23 and Bet v 1-dependent degranulation. Overall, we hereby present fully functional, human IgE, IgG1, and IgG4 sharing the same variable region against Bet v 1 and showcase possible applications in first mechanistic studies. Furthermore, our IgG antibodies might be useful candidates for passive immunotherapy of birch pollen allergy.Birch pollen allergy is among the most prevalent pollen allergies in Northern and Central Europe. This IgE-mediated disease can be treated with allergen immunotherapy (AIT), which typically gives rise to IgG antibodies inducing tolerance. Although the main mechanisms of allergen immunotherapy (AIT) are known, questions regarding possible Fc-mediated effects of IgG antibodies remain unanswered. This can mainly be attributed to the unavailability of appropriate tools, i.e., well-characterised recombinant antibodies (rAbs). We hereby aimed at providing human rAbs of several classes for mechanistic studies and as possible candidates for passive immunotherapy. We engineered IgE, IgG Birch pollen allergy is among the most common allergies in Central and Northern Europe, with a prevalence of up to 16% . TherefoThe molecular key player in any allergy is IgE, which interacts with IgE receptors FcεRI and CD23 on the surface of effector cells . BindingThe low-affinity IgE receptor CD23 is mainly expressed on B cells, but its expression can also be found or stimulated on various other cell types, such as monocytes, macrophages, follicular dendritic cells, and epithelial cells ,14. In c4 antibodies to the allergen [1 and especially IgG4 antibody serum levels are considered to be useful biomarkers for the success of AIT [Currently, the only effective treatment of birch pollen allergy—besides symptomatic relief medication—is active allergen immunotherapy (AIT) . In thisallergen  and raiss of AIT ,19. Bases of AIT .4 and its many unusual properties [1 isotype, despite observed increases in the levels of both subclasses in individuals undergoing AIT. In addition, few mechanistic studies directly compare IgE, IgG1, and IgG4, likely due to limited availability of well-characterised recombinant antibodies produced in sufficient amounts.Although advances have helped understand the mechanisms of AIT , questiooperties  rather t1 and IgG4 [Polymerase Incomplete Primer Extension (PIPE) cloning can be employed for rapid combination of any known variable antibody regions with the constant region sequences of any isotype to generate functional antibodies . This haand IgG4 . Furtherand IgG4 ,26.1 and IgG4 isotypes from a known IgE antibody against the major birch pollen allergen Bet v 1 [We now applied PIPE cloning with the objective of deriving IgG Bet v 1  and eluc1, or γ4 heavy chain sequences by PIPE cloning , and 161 (IgG4) kDa , 168 (IgGgG4) kDa . TherefoWe confirmed antibody specificity to immobilised Bet v 1 by dot blot, ImmunoCAP ISAC112  microarray and in-house ELISA .1 control strips gave a faint background signal that can be attributed to unspecific binding of the secondary antibody. Therefore, another HRP-labelled antibody was used for IgG1 detection in all further experiments .All M0418 isotypes recognised membrane-bound Bet v 1—but not the negative control allergen β-lactoglobulin (BLG)—in a dot blot a. The IgM0418 IgE binding to Bet v 1 was furthermore confirmed in the ImmunoCAP ISAC112 microarray , where binding appeared to be exclusive to Bet v 1, i.e., it did not bind any of the other 111 allergens that were immobilised on the chip b.Specificity was furthermore confirmed in an ELISA c, where Finally, we confirmed concentration-dependence of Bet v 1 binding in an ELISA for all M0418 isotypes d. DifferNext, we were interested in possible inhibitory functions of our IgG antibodies as well as utilising our IgE for Bet v 1—specific IgE quantification from human plasma.Therefore, we created a quantitative blocking ELISA, in which we used our recombinant M0418 IgE to set up a standard curve and determine the IgE plasma levels of four birch-pollen-allergic and non-allergic donors, respectively. The levels of IgE that were measured in the patients’ blood samples were between 0.2 and 2 nM, while those in serum samples from non-allergic donors were below the limit of detection in this assay a. In add1 and IgG4—but not their respective non-specific isotype controls—could decrease the relative amount of plasma IgE binding to immobilised Bet v 1 at 1000 nM  IL-4-primed human monocytic U937 cells that upregulate cell surface CD23 , and (b)Both of the cell lines expressed CD23, but not FcεRI, as confirmed by flow cytometry . In U937RPMI-8866 cells showed a high level of unspecific internalisation and, thus, internalised Bet v 1 independently of IgE c,d.Finally, we aimed to investigate the main effector function of our M0418 IgE, allergen-dependent degranulation. A well-established system for degranulation studies is the rat basophilic leukaemia cell line RBL-SX38, which is transfected with a fully functional human FcεRI . We wereInterestingly, Bet v 1 could crosslink FcεRI-bound allergen-specific IgE-but not non-specific isotype control IgE-and cause degranulation in RBL-SX38 cells a. Degran1 and IgG4 blocked Bet v 1 degranulation in a concentration-dependent manner  were cultivated in RPMI 1640 medium with GlutaMAXTM , supplemented with 10% FBS , and maintained in T75 flasks  at 37 °C, 5% CO2.Expi293F cells  were cultivated in 30 or 125 mL of Expi293 Expression Medium  in 125 mL or 500 mL baffled Erlenmeyer flasks with vented caps , respectively, at 37 °C, 8% COns. LAD2   was produced in escribed ,52. CommBet v 1 was labelled with a pHrodoTM Green STP ester kit  according to the manufacturer’s instruction. Briefly, 200 µg of Bet v 1 was labelled at a concentration of 1 mg/mL in 0.1 M bicarbonate buffer. For the labelling reaction, a molar dye: protein ratio of approximately 10:1 was used. The reaction mixture was incubated at room temperature for 1 h, protected from light. Tube was inverted every 15 min. to ensure mixing. After labelling, pHrodo Green-Bet v 1 was dialysed against phosphate-buffered saline (PBS) in order to remove excess dye using a Slide-A-Lyzer™ MINI Dialysis Device with a cut-off of 3.5 kDa , overnight at 4 °C. Buffer was exchanged and the sample was dialysed for another 4.5 h at 4 °C. Labelled Bet v 1 was stored at 4 °C until used.® buffer at 37 °C, followed by 20 min. enzyme deactivation at 80 °C. Digested PCR products were then mixed in a 1:1:1:1 ratio and incubated at room temperature for at least 2 h up to overnight for ligation. NEB 10-beta Competent E. coli  was transformed with the resulting pVitro1-hygro-mcs constructs according to the manufacturer’s instructions. Selection was carried out on LB-agar plates supplemented with 150 µg/mL hygromycin B . Colonies were screened for constructs via colony PCR. The integrity of construct antibody sequences was finally confirmed by Cycle sequencing . The resulting antibody sequences are summarised in M0418 variable region sequences were obtained from a previously published human IgE sequence . If nece1 and IgG4 purification, 1 mL; GE Life Sciences) and an AEKTA chromatography system . The antibodies were eluted from the column with 0.1 M glycine, pH 2.5, step elution (IgE) or 0.1 M citric acid, pH 3, gradient elution (IgG antibodies), respectively. Eluate was collected in 1 mL fractions and pH was immediately neutralised with 100 µL of Tris-HCl, pH 9.3. Antibody-Containing fractions were pooled and dialysed overnight at 4 °C against PBS using Tube-O-Dialyzer medi dialysis devices with a molecular cut-off of 15 kDa . Concentration was then determined by measuring the UV absorption at 280 nm with a UV/VIS spectrophotometer , using the respective extinction coefficients, as determined from the protein sequence. Antibody solutions were sterilised by passing them through a 0.2 µm PES filter  and stored at 4 °C until use.Expi293F cells were transfected with 30 µg of plasmid DNA according to the manufacturer’s instructions, with some modifications: 50 µg/mL of hygromycin B  were added 72 h after transfection to further enhance production. Supernatant was harvested on day 7 after transfection by two consecutive centrifugation steps at low speed , followed by a final centrifugation step at 3500 x g for 35 min. Supernatant was then filtered through a 0.22 µm PES membrane filter  and diluted 1:2 with PBS prior to purification. The antibodies were purified via affinity chromatography using either a HiTrap KappaSelect column  or a HiTrap Protein A HP antibody purification column (IgGThe circular dichroism (CD) spectra of antibodies were measured in 75 mM phosphate buffer (pH 7.2) with a 0.1 mm quartz cuvette with on a Jasco J-1500 spectropolarimeter  at room temperature. Sample concentrations were between 0.2 and 0.6 mg/mL. Spectra were obtained from 260–180 nm, data pitch 0.2 nm, at a scan speed of 20 nm/min., with a response  of 8s. Data represent the average of 5 accumulated measurements and they are given as mean residue ellipticity.1 and IgG4 or the respective commercially available isotype controls  were mixed with Laemmli loading buffer with and without β-mercaptoethanol (reducing vs. non-reducing conditions), respectively, and denatured at 95 °C for 5 min. Samples were loaded into 50 µL pockets of a 4–15% gradient polyacrylamide gel  and gel was run in SDS-Tris-glycine buffer at 200 V for 30–40 min. For size determination, PageRuler Plus prestained protein ladder  was run on each gel. After electrophoresis, gel was stained with SimplyBlue safe stain  according to the manufacturer’s instructions.2–3µg of M0418 IgE, IgGThe samples in PBS were injected onto a SEC column  at a flow rate of 0.5 mL/min. at 7 °C, detected at 280 nm, and coupled to a multi angle light scattering detector .1 and human myeloma IgG4, cat# 16-16-090705, 16-16-090707-1M, and 16-16-090707-4M, respectively, Athens Research & Technology, Athens, GA, USA) were spotted onto a nitrocellulose membrane  in duplicate and dried overnight. Membrane was cut into strips and blocked with wash buffer (PBS with 0.1% Tween 20) supplemented with 5% skimmed milk powder  for 1 h. The membrane was washed with wash buffer three times and incubated either with antibody diluted with wash buffer to a final concentration of 1 µg/mL  or wash buffer only for 1 h. Strips were briefly rinsed with wash buffer, three times, and then washed for 5 min. The strips were then incubated with the respective HRP-labelled detection antibodies diluted with wash buffer  for another 1 h. Strips were again briefly rinsed, three times and then washed for another 10 min. For signal detection, Clarity Max Western ECL substrate  was prepared and then added to the membrane according to the manufacturer’s instructions. Membrane pictures were acquired using a ChemiDocTM Touch Imaging System . All incubation and wash steps were carried out at room temperature.Bet v 1 , BLG (1 µg), and isotype antibodies , strictly following the manufacturer’s instructions. A total of 1 µg M0418 IgE was used in the test, diluted with non-allergic serum.1, or IgG4 and the respective myeloma isotype controls, see 1, 1:1000, cat# A10648, Invitrogen, Carlsbad, CA, USA; anti-hu-IgG4, 1:24,000, cat# 9200-05, Southern Biotech, Birmingham, AL, USA, respectively) for another 1 h. The plate was again washed three times and 100 µL TMB/well  was added. The reaction was stopped with 100 µL/well 1 M H2SO4 and absorbance at 450 nm as well as 630 nm (reference wavelength) was measured using an Infinite 200M PRO plate reader . All of the incubation and wash steps were carried out at room temperature, unless stated otherwise. Each condition was tested in triplicate and each experiment was repeated at least three times. The values corrected by the reference wavelength were used for analysis. The limit of detection (LOD) was defined as the mean of the blank plus three times the corresponding standard deviation. Raw values below the LOD were excluded from the analysis. Measurements from each well were blank-corrected and the mean values of replicate wells of each experiment were calculated with Excel 365 .Maxisorp 96-well plates  were coated with 0.3 µg of Bet v 1 or BLG  in bicarbonate buffer, pH 9.6, overnight at 4 °C. Liquid was removed and the plate washed with wash buffer . Plate was then blocked with 200 µL/well wash buffer with 1% BSA for 2 h. Plate was then washed twice and sample or control antibodies  or wash buffer, respectively, for 1 h. The plate was washed four times and 100 µL of 1:2 diluted plasma samples from birch pollen allergic individuals was added to sample wells. A 1:2 dilution series of M0418 IgE was prepared in the standard row wells, ranging from 0.5 nM to 0.007 nM and 3 nM to 0.05 nM, respectively; matching the expected IgE levels in the plasma samples). After 1.5 h of incubation, the plate was washed four times and HRP-labelled detection antibody added . After another 1 h of incubation wash step was repeated and 100 µL TMB/well  were added. Reaction was stopped with 100 µL of 1 M H2SO4 and absorbance at 450 nm and 630 nm (reference wavelength) was measured using an Infinite 200M PRO plate reader . IgE in plasma of four non-allergic donors was quantified in the same manner, but without the addition of any blocking antibodies. All of the incubation and wash steps were carried out at room temperature, unless stated otherwise. Each condition was tested in duplicate and each experiment was repeated at least three times. Reference-Wavelength corrected values were used for analysis. Values below the LOD were excluded from the analysis. Measurements from each well were corrected by the blank and duplicate means were calculated with Excel 365 . Further statistical analysis was carried out with GraphPad Prism version 8 .Maxisorp 96-well plates  were coated with 0.17 µg Bet v 1/well in coating buffer  overnight at 4 °C. The plates were washed with wash buffer (TBS with 0.05% Tween 20) once and then blocked with wash buffer supplemented with 1% BSA for 2 h at room temperature. Sample wells  were washed with wash buffer four times and incubated with 10, 100, or 1000 nM of M0418 IgG5 LAD2 cells (500 µL of 3 x 105 cells/mL suspension) was incubated with 0.5, 5, or 50 nM of M0418 IgE, 6 ng/mL of IL-4 (positive control) or medium, respectively, in a 24-well plate. The cells were harvested after four days of incubation at 37 °C, 5% CO2 and washed with PBS twice. Cells were then stained with Zombie Violet  for 15 min., followed by co-incubation with APC-Cy7-labelled anti-FcεRI antibody , Biolegend, San Diego, CA, USA) or the respective isotype control antibody  for another 15 min. The cells were then washed with Hank’s Balanced Salt Solution , centrifuged at 300 x g for 5 min., and then re-suspended in 200 µL of HBSS for flow cytometric analysis. 10,000 events were acquired using a FACSCanto II  and further analysis was carried out in FlowJoTM version 10  and Graphpad Prism version 8 .A total of 1.5 x 10TM, cat# 01-2222-42, Thermo Fisher Scientific, Waltham, MA, USA). LAD2 cells were included as a FcεRI-positive, CD23-negative staining control. For the determination of Bet v 1 internalisation, U937 cells and RPMI-8866 cells were harvested and washed with assay buffer (HBSS supplemented with 1% FBS) once. 100 µL containing 1 x 105 cells were seeded into FACS tubes and 5 µg of M0418 IgE or isotype control IgE  and subjected to flow cytometric analysis. 10,000 events were acquired using a FACSCanto II  and further analysis was carried out in FlowJoTM version 10  and GraphPad Prism version 8 .U937 cells were primed with 10 ng/mL IL-4 for 48 h, twice, to upregulate CD23, as previously described . Express4 RBL-SX38 cells (in 100 µL medium) were seeded into the wells of a flat-bottom 96 well cell culture plate  and incubated overnight to attach to the plate. The cells were briefly washed with assay buffer (HBSS supplemented with 1% of FBS) and sensitised with 5 nM of M0418 IgE or isotype control IgE . The samples were diluted with 25 μL of assay buffer and 50 µL of fluorogenic substrate  were added. The plate was incubated at 37 °C for 1 h in the dark and the reaction quenched with 100 μL of 0.5 M Trizma® base , pH 8.2, per well and fluorescence signal was measured using a FLUOstar Omega Microplate Reader ; excitation at 350 nM, emission at 450 nm. In the case of blocking experiments, stimulation was carried out with 400 nM of Bet v 1 with or without 1, 10, or 100 nM of M0418 IgG1 or IgG4, respectively. All of the measurements were made in quadruplicates and experiments were repeated at least three times. The signal gain was adjusted to 90% for the well giving the highest signal (Triton-X-lysed cells). The limit of detection (LOD) was defined as the mean of the respective blank (regular buffer vs. Triton-X) plus three times the corresponding standard deviation. Raw values that were below the limit of detection were eliminated from the analysis. All other values were then blank-corrected, normalised to the 100% degranulation control (Triton-X lysed cells) and their mean values were used for further statistical analysis. All of the incubation steps were carried out at 37 °C, 5% CO2, if not indicated otherwise. Basic calculations were carried out in Excel 365  and further statistical analysis was done in GraphPad Prism version 8 .To determine cell degranulation, β-hexosaminidase release of RBL-SX38 cells was measured, as described previously . Brieflyescribed ) for 2–3Plasma samples from birch pollen-allergic individuals were obtained with their informed consent. Experiments with blood samples were in accordance with the Helsinki declaration of 1975 and they were approved by the institutional ethics committee of the Medical University of Vienna (EK number 2007/2016).Q = 1%) was carried out. Data were then analysed via repeated measures one-way or two-way ANOVA or a corresponding mixed-effects model, as appropriate, followed by Dunnett’s multiple comparisons test. All of the statistical analyses were carried out using GraphPad Prism version 8 .For outlier identification, ROUT analysis (1, and IgG4 antibodies specific to Bet v 1. Our IgE engaged with high and low affinity FcεRs and stimulated FcεRI upregulation in culture, while our IgG antibodies effectively inhibited IgE-mediated degranulation in vitro. Therefore, these basic tools may help to systematically investigate Fc-mediated effects of IgE versus IgG antibodies in allergy or tolerance at a cellular level. Future studies may prove our IgG1 and IgG4 to be useful candidates for passive immunotherapy of birch pollen allergy.Overall, we present, for the first time, the rapid PIPE cloning of fully human, variable region-matched recombinant IgE, IgG


Lactobacillus plantarum and L. uvarum strains for the valorisation of W to provide safer food and feed stock. The influence of different treatments on biogenic amine formation, mycotoxin content, and free amino acids, as well as acidity, microbiological parameters, and sugar concentration, were analysed. This research showed that a combination of extrusion and fermentation with selected strains can change several aspects of W characteristics. There was a significant effect of applied treatments on acidity and the microbiological parameters of W, as well as biogenic amines content. The lowest total mycotoxin concentration (29.8 µg/kg) was found in extruded  and fermented with L. uvarum sample. Finally, the combination of the abovementioned treatments can be confirmed as a prospective innovative pre-treatment for W, capable of potentially enhancing their safety characteristics and composition.Processed wheat bran (W) is of great importance for food and feed. Consequently, the biosafety of W should be evaluated and improved with valorisation strategies. This study tested a design combining extrusion  and fermentation with  After 15 min, the samples were filtered through a filter paper and a 0.22 μm nylon syringe filter before analysis. A 2 mg/mL standard solution of sugars mixture  was prepared following dilution with distilled/deionized water.For determination of sugars concentration, 2–3 g of sample were diluted with ~70 mL of distilled/deionized water, heated to 60 °C in a water bath for 15 min, clarified with 2.5 mL Carrez I . The UPLC apparatus was a Shimadzu LC-20AD  equipped with Evaporative Light Scattering Detector ELSDLTII  detector. Chromatographic conditions were as follows: the eluent was a mixture of 75 parts by volume of acetonitrile and 25 parts by volume water, flow rate was 1.2 mL/min, 20 μL was injected. The YMC-Pack Polyamine II 250 × 4.6 mm, 5 μm  column was used. Column temperature was set at 28 °C. The limit of detection (LOQ) for sugars—0.01 g/100 g sample.Free amino acids (FAA) were analysed by GC-FID instrument  with flame ionization detection after an ion-exchange solid phase extraction and chloroformate derivatization using EZ:faast technology . Standard solutions of the amino acids aspartic acid (Asp), alanine (Ala), glycine (Gly), valine , leucine (Leu), isoleucine (Ile), threonine (Thr), serine (Ser), proline (Pro), asparagine (Asn), methionine (Met), glutamine (Glu), phenylalanine (Phe), lysine (Lys), histidine (His), arginine (Arg), tyrosine (Tyr), tryptophan (Trp), and cysteine (Cys) were used, in addition to the internal standard DL-Norvaline (NVAL). The analysis is detailed in .® HS C18 column  was used. Ammonium acetate (0.1 mol/L) and acetonitrile were used as the mobile phases by a flow rate of 0.45 mL/min.The extraction and determination of biogenic amines (BA) in wheat samples followed the procedures developed by Ben-Gigirey et al.  with somThe limit of detection (LOQ) for biogenic amines concentration—0.1–0.6 mg/kg, for free amino acid—0.01–0.1 mg/100 g. Size of subsamples—5.The following mycotoxins were analysed in samples: alternariol; alternariol monomethyl ether; 17-dimethylaminoethylamino-17-demethoxygeldanamycin; 15-acetyldeoxynivalenol; deoxynivalenol; deoxynivalenol-3-glucoside; 15-acetoxyscirpenol; enniatin A and A1; fumonisin B1 and B2; meleagrin; sterigmatocystin; ochratoxin A and B; T-2 toxin; HT-2 toxin; fusarenone; neosolaniol; aflatoxin B1.g, 5 min) and the supernatants were transferred to 15 mL centrifuge tubes and stored for 15 min at −80 °C in a Heto PowerDry® freeze dryer . After removal, the extracts were immediately centrifuged  at 10 °C. Then, 2500 µL of extracts were transferred to 15 mL PP tubes and were evaporated to dryness at 50 °C under a gentle nitrogen stream. The dry residues were reconstructed in 100 µL of 50% acetonitrile in water with 0.1% formic acid and shaken 20 min. Then diluted by 400 µL water with 0.1% formic acid  and shaken for 10 min. Extracts were filtered through centrifuge filters  and transferred into the autosampler for analysis.Sample preparation. Samples (5.00 ± 0.01 g) were accurately weighed in 50 mL PP tubes. The quality control (blank) samples were supplemented with mycotoxin standard solutions at the appropriate spiking levels. Then water (10 mL), acetonitrile (10 mL), and formic acid (20 µL) were gradually added to the tubes and extraction was started by mixing for 10 min on mechanical shaker. One portion of the QuEChERS buffer salt kit was added to each of the tubes and the extraction was continued for additional 10 min. The obtained mixtures were centrifuged  HPLC coupled with a Thermo Scientific TSQ Quantiva MS/MS detector. The separation was performed on a Phenomenex Luna C18 reversed-phase analytical column . The autosampler was maintained at 4 °C and the column temperature was 40 °C. The sample injection volume was 40 µL. Ion monitoring was conducted in both positive and negative ion modes and the mass analysis was performed in selected reaction monitoring (SRM) mode. The following instrumental settings were used: spray voltage 3.5 kV (positive ion mode), 2.5 kV (negative ion mode), vaporiser temperature 350 °C, ion transfer temperature 300 °C, sheath gas 55 arbitrary units (arb), auxiliary gas 25 arb, and sweep gas 5 arb. Data processing was performed with Xcalibur™ software . For more detailed information, see Additional Information in n = 5) ± standard deviation (SD). The normal distribution of data was checked using Descriptive Statistics tests in the statistical package SPSS for Windows . In order to evaluate the effects of the different treatment, the data were analysed by multivariate analysis of variance (ANOVA) and Tukey’s honestly significant difference (HSD) procedure, as well as post hoc tests. A linear Pearson’s correlation was used to quantify the strength  of the relationship between the variables [p ≤ 0.05.The results were expressed as the mean (ariables . The cor