Nicotiana benthamiana as hosts. Recombinant Agrobacterium tumefaciens containing heavy chain (HC) and light chain (LC) domains of anti-human IgE were co-transformed in N. benthamiana. The assembling of the antibody and its expression was detected by SDS-PAGE and Western blot analysis. The functional ability of the anti-IgE antibody was determined via its binding capacity with target IgE by ELISA and the inhibition of basophil activation. The anti-human IgE mAb generated in plants was shown to be effective in binding to its target IgE and inhibit the IgE-crosslink in RS-ATL8 reporter cells. Although, antibody yield and purification process have to be further optimized, this study demonstrates the use of plant expression system as a promising platform for the production of Omalizumab which showed a comparable in vitro function to that of commercial Omalizumab (Xolair) in the inhibition of basophil activation.Omalizumab, the anti-immunoglobulin IgE antibody is the only approved and available monoclonal antibody as an auxiliary medicament for the severe respiratory allergic reactions. It forms small size immune complexes by binding to free IgE, thereby inhibiting the interaction of IgE with its receptors. Additionally, the anti-IgE can also differently shape the airflow by impeding the stimulation of IgE receptors present on structural cells in the respiratory tract. The present study aimed to use plants as an expression system for anti-human IgE antibody production, using   This interaction plays the central role in inducing the B cell class switching to immunoglobulin E (IgE) , affecting both children and adults with a approximate total of 300 million individuals globally . In brie E (IgE) . IgE is eceptors . The fireceptors  , following the binding of IgE to high-affinity IgE receptors on mast cells and basophils. Biologic-targeted treatment has been successfully introduced as an add-on therapy or controller medications to improve asthma condition in patients with moderate-to-severe persistent allergic asthma and chronic spontaneous urticaria . Few monOmalizumab (Xolair), is an anti-human IgE monoclonal antibody (mAb) commercialized in the market worldwide as an add-on therapy for patients with severe allergic asthma and medicaments with inhaled corticosteroids (ICS) and long-acting β2-agonists (LABA) . It is aNicotiana benthamiana plants as an expression platform for producing the anti-human IgE mAb. The main objective was to express and characterize the plant-based anti-human IgE that was compared with commercial Omalizumab (Xolair) using SDS-PAGE, binding by ELISA and basophil activation assay.Omalizumab was recombinantly expressed in the suspension cell culture of Chinese Hamster Ovary (CHO) cell line. Mammalian cells have traditionally been used as the preferred host for mAb expression because they can produce antibodies that are almost identical to those found in the human body while also posing the least risk of non-human glycosylation patterns . HoweverBsaI, NheI, AflII, XbaI, and SacI) and T4 DNA ligase were purchased from New England Biolabs . Standard native human IgG1 was purchased from Abcam . Amintra® Protein A resin for antibody purification was a product of Expedeon . All blue pre-stained protein standard and nitrocellulose membrane (0.45 micron) were bought from BioRad (US). Coomassie brilliant blue was purchased from Applichem . Anti-human kappa antibody was purchased from Southern Biotech  and anti-human gamma antibody was purchased from The Binding Site . Amersham ECL prime western blotting detection reagent was a product of GE Healthcare . Humanized Rat Basophilic Leukaemia cell line (RS-ATL8) used for IgE cross-linking-induced luciferase expression was kindly provided by Prof. Tanapat Palaga, Department of Microbiology, Faculty of Science, Chulalongkorn University. Other chemical reagents were of analytical grade.The pBY3R geminiviral expression vector was provided by Professor Hugh Mason . Taq DNA polymerase used for polymerase chain reaction (PCR) was a product of Vivantis Technologies . Restriction endonucleases . The codon-optimized sequences were synthesized at Biomatik, Canada. The variable regions in the heavy and light chain genes of anti-IgE antibody synthesized were digested using BsaI/NheI and BsaI/AflII restriction enzymes, respectively and the digested DNA products on agarose gel electrophoresis were further purified by using Accuprep PCR/gel purification kit . For the heavy chain, the variable (VH) domain was ligated with constant heavy chain domain (CH1-CH2-CH3), in the pBY3R geminiviral expression vector using T4 DNA ligase . For the light chain, the variable (VL) domain was ligated with constant (CL) regions in pBY3R expression vector. The HC and LC encoding DNA sequences were cloned with the endoplasmic reticulum (ER) retention signal peptide SEKDEL (Ser-Glu-Lys-Asp-Glu-Leu) at the carboxyl terminus (C-terminus) to generate full length HC and LC gene sequences.The gene fragments encoding the variable coding regions of the heavy chain (HC) and light chain (LC) of the Omalizumab (Drugbank accession number: DB00043) and human IgGt region  sequenceXbaI and SacI. Then the A. tumefaciens strain GV3101 was separately transformed with the Omalizumab-heavy chain expression vector pBY3R (pBY3R-Oma-HC) and Omalizumab-light chain (pBY3R-Oma-LC) genes by electroporation. The transformed A. tumefaciens cells were cultured in lysogeny broth media  and confirmed using PCR with gene-specific forward and reverse primers . The positive clones of transformed A. tumefaciens were chosen for further experiments.In this study, the geminiviral replicon system derived from the bean yellow dwarf virus  was usedN. benthamiana using the geminiviral vector. Recombinant A. tumefaciens harboring the plant expression vector containing either HC or LC were inoculated in lysogeny broth supplemented with rifampicin, gentamicin, and kanamycin at a final concentration of 50 mg/L. The cultures were grown in an incubator shaker at 28°C with 200 rpm overnight. The overnight bacterial culture medium was collected and harvested by centrifuging at 1000 g for 10 min. The cell pellet was gently washed and resuspended in infiltration buffer  to obtain a final optical density (OD600) of 0.4 at 600 nm. Agrobacterial suspension containing HC and LC were mixed together in the ratio of 1:1 and co-infiltrated into 30-35 day-old wild-type N. benthamiana leaves by syringe infiltration whereas the large-scale infiltration was performed by vacuum infiltration. After infiltration, the plants were returned to the greenhouse with the controlled condition (temperature at 25 ± 2°C with 16-h light/8-h dark cycle) before being harvested.Plant-derived anti-human IgE mAb was transiently expressed in 2HPO4, and 1.76 mM KH2PO4, pH 7.4) and vortexed for 5 mins The clarified crude extracts were spun down by centrifugation at 26000 g for 30 min at 4°C. The supernatants were used to measure the total soluble protein (TSP) using Bradford assay  following manufacturer’s instructions. The clarified crude extract was further analyzed by SDS-PAGE and Western blot analysis.For time-course experiment of plant-produced anti-human IgE mAb expression levels in plants, infiltration buffer containing the recombinant pBY3R-Oma-HC and pBY3R-Oma-LC (ratio 1:1) were infiltrated into plant leaves. The infiltrated leaves were collected on different days post-infiltration (dpi) that include 2, 4, 6, 8, and 10 dpi to estimate the optimal incubation time that can express high yields of plant-produced anti-human IgE. The infiltrated leaves were ground in liquid nitrogen using a mortar and pestle. The powdered leaf was added with 1:2 w/v cold extraction buffer ; 137 mM NaCl, 2.68 mM KCl, 10.1 mM NaThe harvested leaves after agroinfiltration were collected and extracted in extraction buffer  using an electric drill. Supernatants were collected by centrifugation at 26000 g for 30 min and 0.45µm filtration  was performed to clarify plant crude extract containing anti-IgE mAb. For purification, the anti-human IgE mAb in the clarified crude extract was specifically captured by Protein A affinity chromatography. Briefly, 1.0-1.5 mL of Amintra Protein A resin was packed into a 10 mL polypropylene column to a bed height of 1 cm and equilibrated with 5 column volumes (CV) of the extraction buffer  at a flow rate of 2 mL/min. After column equilibration, the clarified crude extracts were applied at a flow rate of 1 mL/min and the wash step was performed with 10 CV of wash buffer . Elution was done at a flow rate of 1 mL/min using 5 mL of elution buffer  with an incubation time of 5 mins prior to the collection of each 1 mL. The elutes were neutralized with 1.5 M Tris-HCl (pH 8.8). The neutralized elutes were desalted and concentrated with Amicon ultra-centrifugal filter  in 1X PBS buffer (pH 7.4). The protein’s structure integrity in the purified mAb was confirmed by Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting analysis under reducing and non-reducing conditions.The protein structure integrity and product-related variants were characterized using SDS-PAGE and Western blot analysis. Commercial anti-human IgE mAb  was used as positive control. The samples were analyzed under reducing and non-reducing conditions. For non-reducing conditions, the protein samples were mixed with SDS-loading buffer . The protein sample denatured at 95°C for 5 min was separated on 12% freshly prepared polyacrylamide gels. For reducing conditions, the sample was added to SDS-loading buffer containing 22% β-mercaptoethanol, denatured at 95°C for 5 min, and then separated on 12% polyacrylamide gels. The gels were stained with Coomassie brilliant blue , and the bands were visualized.For Western blot analysis, the separated protein samples were transferred on to nitrocellulose membranes . The nitrocellulose membranes were blocked with 5% skim milk in 1X PBS (pH 7.4) for 1 h at room temperature of 25 ± 2°C. The membranes were washed thrice with PBS before incubation with either a 1:5,000 dilution (0.2 mg/L) of HRP-conjugated anti-human gamma antibody  or a 1:5,000 dilution (0.1 mg/L) of HRP-conjugated anti-human kappa antibody  in 3% skim milk prepared in 1X PBS (pH 7.4). After incubation, the membrane was washed thrice with 1X PBST, and the bound antibody was detected using Amersham ECL prime western blotting detection reagent  and the signal was captured on medical X-ray green (MXG) film .2SO4. The absorbance was measured using a microplate reader at an optical density of 450 nm (OD450).The binding efficiency of plant-produced anti-human IgE mAb with IgE was determined using enzyme-linked immunosorbent assay (ELISA). 5 µg/mL human IgE was coated onto a 96-well ELISA plate  and incubated at 37°C for 4 h. Then, the plate was blocked with 200 µL of 5% skim milk  in 1X PBS (pH 7.4) at 37°C for 2 h and washed three times with 1X PBST. Various concentrations of plant-produced anti-human IgE, commercial Omalizumab (positive control) were diluted in PBS and added to triplicate wells followed by incubation at 37°C for 2 h. After washing three times with 1X PBST (1X PBS containing 0.05% Tween-20), the plate was incubated at 37°C for 1 h with HRP-conjugated anti-human gamma IgG1 antibody  at a dilution of 1:1,000 in 1X PBS. The plate was developed by adding SureBlue TMB 1-Component Microwell Peroxidase Substrate , and the reaction was stopped by 1 M H4 cells per well in 96 well-plate (50 μL per well) and incubated for 3 h. Cells were then sensitized overnight with the pre-mixture of 5 μg/mL of human-IgE  and various concentrations (0.5-512 μg/mL) of plant-produced anti-human IgE (stock 3.4 mg/mL) or Omalizumab (stock 75 mg/mL) diluted in PBS, for 1 h at 37°C. After sensitization, cells were washed once gently with sterile PBS to remove the unbound antibodies. The pre-mixtures that contain only human IgE and only 512 μg/mL of plant-produced anti-human IgE or Omalizumab were used as positive and negative control, respectively. The sensitized cells were then crosslinked with 5 μg/mL of goat anti-human IgE . After 3 h of cross-linking, luciferase activity was measured by the ONE-Glo Luciferase Assay System  using a Varioskan microplate reader  with luminometric mode. The relative luciferase unit (RLU) was calculated by subtracting the signal from the negative control wells. The study was performed in duplicate wells for three independent runs and the average was used for analysis and the values were expressed as mean ± SD.The RS-ATL8 cells, humanized rat basophilic leukemic cells, were cultured in minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS), 1% Penicillin/Streptomycin, 0.5 µg/mL geneticin, 0.2 µg/mL hygromycin B, and GlutaMAX-I. All the reagents for RS-ATL8 cell culture were procured from Gibco; ThermoFisher Scientific, USA. Measurement of luciferase activity was performed following the procedures described previously  with somN. benthamiana, the variable regions of heavy and light chain genes of Omalizumab were fused with human IgG1 along with signal peptide and SEKDEL sequences at N-C terminals respectively and were cloned into pBY3R geminiviral expression vector  stained with Coomassie brilliant blue stain to visualize the antibody fragments and assembling of the purified plant-produced anti-IgE mAb. Western blot analysis was performed to confirm the purified plant-produced mAb by probing with anti-human gamma-HRP and anti-human kappa-HRP. As shown in For the anti-IgE antibody production in plant host n vector Figure 12 fragments exhibit multiple unfolded states in non-reducing SDS (The protein bands were further confirmed by western blot using specific goat anti-human kappa and goat anti-human gamma IgG antibodies antibody. As presented in cing SDS . This coN. benthamiana. The anti-IgE construct with SEKDEL demonstrated higher anti-IgE accumulation (Preliminary studies with anti-IgE gene constructs having with and without SEKDEL at C-terminus were performed to determine the expression levels of anti-IgE mAb in d) was found to be 14.8 µg/mL and 0.9043 µg/mL for plant produced anti-IgE and commercial Omalizumab respectively, using GraphPad Prism 8.1.2 . Human IgG1 that was used as the negative control could not bind to the human IgE receptor protein.The binding efficiency of plant produced anti-IgE was determined by ELISA as depicted in Determination of plant produced anti-human IgE to inhibit IgE cross-linking-induced luciferase expression using RS-ATL8 cell line was performed. The results revealed that plant-produced anti-human-IgE could inhibit IgE crosslink in a dose-dependent manner Figure 5Anti-IgE therapy acts as an essence in the care and treatment of allergic respiratory symptoms and still needs further investigation for unravelling its salient features for application in several other IgE mediated complex disorders . OmalizuAgrobacterium mediated transformation in N. benthamiana host system. The retrieved heavy and light chain sequences from drug bank were codon optimized, synthesized, and constructed into the geminiviral plant expression vector . The difference in the binding properties may be attributed due to purity of the target protein and interference of process related impurities. Although N-glycosylation was not evaluated, glycosylation of antibodies is carried out in a different manner in plant cells than in mammalian cells  modifyinA gradual increase in the prevalence of IgE-mediated diseases including the asthma has put an insight to scrutinize, develop and produce anti-IgE antibody. Although, the anti-IgE mAb namely Omalizumab is commercially available in the market, it involves high costs that cannot be affordable by the middle- and low-income countries. The present study aimed at producing anti-IgE antibody using plant as an expression host. The plant produced anti-IgE antibody expression was optimized and further purified by protein A chromatography, analyzed by SDS-PAGE and western blot. The binding and functionality of the plant produced anti-IgE antibody was compared with commercially available Xolair. However, we acknowledge that there is a need to refine the purification approach for enhancing the protein yield and improve the product quality attributes to meet the industry standards. Further studies shall be performed in animal models to test the safety, efficacy and evaluate whether the plant produced anti-IgE antibody may benefit in clinical application and inexpensive therapeutic treatment of IgE mediated diseased patients.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.in vitro functional study. CK conceptualized the in vitro study. EP designed, conceptualized the in vitro study, and drafted the functional study part in the manuscript. All authors revised the manuscript and approved the final manuscript for submission.WP conceived the study. OH and KR performed the cloning, purification, quantification studies, analyzed the results and drafted the manuscript. AM participated in literature search, edited, and reviewed the manuscript. KT performed the This Research is funded by Thailand Science Research and Innovation Fund Chulalongkorn University (CU_FRB65_hea (55) 064_33_08). The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication.WP is a co-founder/shareholder of Baiya Phytopharm Co., Ltd. Authors KR and AM have potential financial competing interest due to paid employment provided by Baiya Phytopharm Co., Ltd.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Pilot hole preparation has been shown to have an impact on the short and long‐term stability of the screw fixation constructs.Investigation and comparison of two nondestructive modal analysis methods with conventional insertion torque (IT) and pullout tests in optimum pilot hole diameter detection.ult), displacement at Fult (dult) and energy dissipation (ED) were obtained from the destructive pullout test.Twenty conical core titanium screws were embedded in high‐density polyethylene blocks with different pilot hole diameters. The maximum IT was recorded for each screw during implantation. Then, two modal analysis methods including accelerometer  and acoustic modal analysis (AMA) were carried out to measure the natural frequency (NF) of the bone‐screw structure. Finally, stiffness (S), pullout force  and did not deviate from y = x hypothesis in linear regression analysis. The Fult, dult, and ED were measured 4800 ± 172 N, 3.10 ± 0.08 mm and 14.23 ± 1.10 N.mm, respectively.The IT increased, as the pilot hole diameter decreased. The maximum value of IT was observed in the smallest pilot hole diameter. The same trend was found for the Fult and first NF were obtained for the 5.5 mm pilot hole diameter. Both CMA and AMA were found to be reliable methods and can promote the undesirable contradiction between Fult and IT.No significant change was observed in “S” between the groups. The highest F We evaluated the efficacy of a novel method in predicting the pullout strength of bone screws. The analysis showed that the pullout strength is highly affected by the size of the pilot hole. The nondestructive method can be used to predict the pullout strength of the bone screws. The pilot holes are often created using static or punch awls or drills.Researchers have reported that the holding power of the screw depends on the changes induced in bone by the insertion trauma as well as bone reaction around the implant. It is also known that the fixation of the screw in the bone depends on material characteristics of the surrounding bone,The effects of multiple and repetitive reinsertions on pullout strength have been evaluated in several studies, however, the results were contradictory in some cases.New techniques such as modal analysis method have been recently introduced for the fixation strength assessment of dental implants,To further investigate the applicability of the newly introduced modal analysis method, the present study was aimed to evaluate the effect of different pilot hole diameters on the primary stability of pedicle screws in the high‐density polyethylene (HDPE). The primary stability was measured with four different methods in order to verify the accuracy and reliability of the methods. The results of both modal analysis methods that is, classical modal analysis and acoustic one were compared with the conventional pullout strength and IT tests to investigate the optimum pilot hole diameter.22.1Twenty titanium, self‐taped, cylindrical thread and conical core pedicle screws  used in our previous work, were utilized in this study.2.23  were used for screw insertion. The HDPE is uniform, nonporous, homogenous, bone‐like properties and machinable. All these together make HDPE providing creation of accurate pilot hole diameter that is, 0.25 mm accuracy between groups.The HDPE block was cut into the samples of 40 × 40 × 60 mm3 Figure . The You2.3N = 5/group) were chosen to investigate the effect of pilot hole diameter. These dimensions are the final pilot hole diameters. The pilot hole was drilled into the depth of 20 mm, as suggested by ASTM F543.The pilot hole diameter was adjusted to be slightly larger than the core diameter base on the manufacturer's instructions and under provision of ASTM F543.2.42.4.1d) was measured using the recorded acceleration‐time response curve. Decaying oscillation amplitude (δ) was then calculated using Equation t) and a (t + T). Next, the system's damp ratio (ζ) and the NF (ωn) were determined from Equations Classical modal analysis (CMA) was performed for each sample very similar to the previous studies.2.4.2During the tests, the tapping sound was simultaneously recorded using a microphone attached to the accelerometer. The recorded sound was transferred to MATLAB R2017  software and data converted from time domain into the frequency domain using fast Fourier transform. The location of the peaks illustrated the natural frequencies at different modes.2.5N = 5/pilot hole diameter) according to the ASTM‐F543 very similar to the previous study,ult), displacement at Fult (dult) and dissipated energy (DE) were also determined for each pullout test.The pullout test was performed (2.6p < 0.05) was used to analyze the differences between the groups in terms of the IT, pullout characteristics and NF using Microsoft Excel . Furthermore, a Tukey–Kramer honesty significant difference (HSD) post hoc test was used to determine significant differences among the results in each test group.Linear regression analysis was used to assess the efficiency of the CMA and AMA methods in the prediction of the NF. Statistical differences between the resulting regression line and the slope intercept of the y = x line were also undertaken. One‐way ANOVA with a confidence level of 95% (3AMA)were obtained in 5.5 and 5.75 mm pilot hole diameters, respectively  placed in 5.5 and 5.75 mm pilot hole diameters and the values for fCMA were quantified 1813 ± 8 and 1471 ± 15 Hz, respectively  and the slope of the regression curve did not deviate from the y = x line  (p = 0.83) . The significant subgroups were examined by the HSD test . According to the Tukey–Kramer test, the Fult, dult, and ED were significantly different between all the subgroups, except between 5 and 5.5 mm pilot hole diameter, while “S” showed a different pattern and was significantly different between the three first pilot holes and 5.75 mm pilot hole diameter  surrounding the screw in the block‐screw interface. The Fly Table , but it From a purely geometrical perspective, the allowable load placed on a screw is dependent on the amount of surrounding material that contacts the thread. Therefore, it could be hypothesized that by increasing the contact area between a pedicle screw thread and the surrounding bone, there is a greater distribution of forces and thus larger pullout strength is obtained.Like any other experimental study, the present study suffered from some limitations as follows. First, this study was carried out using HDPE to provide a uniform model and limit the variation encountered in cadaveric bone densities and qualities. The properties of the HDPE may not replicate the exact material characteristics of the anatomical bones. This can affect the accuracy of the measured load and torque data. However, knowing about this limitation, we intended to look at the data by comparing different cases from the qualitative perspective and not quantitative. Second, the optimum parameters related to hole size are limited to the screw designs used in the experiment. The recommended numbers might vary slightly for other screw designs. Third, the ratio of pilot hole diameter over outer diameter (thread diameter) varied only by 11% and thus wider ranges are necessary for more conclusions. Finally, the damage induced in the block‐screw interface has not been quantified directly according to different pilot hole diameters and the damage was speculated based on dependent variables.5First, modal analysis methods that is, classical or acoustic ones can replace the destructive pullout test and have the potential of using in clinics rather than one‐time IT test. Secondly, the size of the pilot hole created prior to screw insertion plays an important role in the stability of bone‐screw fixation. The critical pilot hole size was defined as the size needed to maintain an optimal balance between low IT and high pullout strength. The desired pilot hole diameter has to meet two terms: First, the more contact area between the screw threads and substrate material and second, minimum damage induced in the screw‐block interface. The 5.5 mm pilot hole diameter in this study met these conditions. By performing nondestructive modal analysis and pullout tests on pilot holes with diameters 77%, 81.5%, 85%, and 88% of the bone screw outer diameter being used, the optimal pilot hole diameter was 85% that is, 5.5 mm hole size.All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Mohammadjavad Einafshar.Mohammadjavad Einafshar led the study. Ata Hashemi contributed in developing the draft and co‐supervised the study. Ali Kiapour supervised the study, reviewed, and commented on the draft of the manuscript. Ata Hashemi contributed as co‐supervisor and Ali Kiapour contributed in conceptualization of the study. All authors read and approved the final manuscript.The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.


The activating molecule in Beclin1-regulated autophagy protein 1 (AMBRA1) is an intrinsically disordered protein that regulates the survival and death of cancer cells by modulating autophagy. Although the roles of autophagy in cancer are controversial and context-dependent, inhibition of autophagy under some circumstances can be a useful strategy for cancer therapy. As AMBRA1 is a pivotal autophagy-associated protein, targeting AMBRA1 similarly may be an underlying strategy for cancer therapy. Emerging evidence indicates that AMBRA1 can also inhibit cancer formation, maintenance, and progression by regulating c-MYC and cyclins, which are frequently deregulated in human cancer cells. Therefore, AMBRA1 is at the crossroad of autophagy, tumorigenesis, proliferation, and cell cycle. In this review, we focus on discussing the mechanisms of AMBRA1 in autophagy, mitophagy, and apoptosis, and particularly the roles of AMBRA1 in tumorigenesis and targeted therapy. The global incidence and mortality of cancers have been dramatically increasing annually . In recevia lysosomes. There are three major autophagy pathways, including macro-autophagy, micro-autophagy, and chaperone-mediated autophagy (CMA), which mainly differ in delivery methods and wrapped cargoes. Macro-autophagy wraps and degrades intracellular cargoes through autophagosomes with a bilayer membrane structure by fusing with lysosomes eventually , identified as an autophagy-associated protein initially, is a fundamental factor in the process of autophagosome formation . FurtherAMBRA1 plays multi-functional roles in intracellular physiological and pathological processes. The aberrant expression and dysregulation of AMBRA1 positively and negatively control tumor formation and progression through diverse signal pathways, such as c-MYC, cyclin D, mTOR, PI3K, STAT3, and TGFβ , 34, 38.AMBRA1 gene is located in chromosome 11p11.2 with 24 exons, and it encodes a protein with a linear sequence of 1,298 amino acids. The subcellular location of AMBRA1 is mainly in cytoplasmic structures, such as autophagosome, cytoskeleton, endoplasmic reticulum, and mitochondria, and it is also found to be localized in the nucleus   , 48, 49.In 1957, autophagy was first noted by Clark in the kidneys of neonatal mice by using an electron microscope  and firsThe paralleled levels of AMBRA1 and autophagy suggest that AMBRA1 is one of the pivotal proteins regulating autophagy. Under normal conditions, AMBRA1 remains in a low or dormant state: 1) AMBRA1 preferentially binds to BCL2 at the outer mitochondrial membrane ; 2) AMBRvia autophagy, namely, mitophagy, is important for the efficient turnover of mitochondria. Experts found that the processes of mitophagy are mainly divided into receptor-mediated mitophagy and ubiquitin-mediated mitophagy in mammals. Emerging evidence indicates that AMBRA1 plays an important role in both processes  also regulates the cross-talk between autophagy and apoptosis . In geneAMBRA1 locus, siRNA interference, Ambra1 heterozygous (+/gtAmbra) mice, and zebrafish embryo transplantation. The study finally identified that AMBRA1 interacts with the phosphatase PP2A and enhances its phosphatase activity on the proto-oncogene c-MYC, which further prevents tumorigenesis and tumor hyperproliferation (Group3), c-MYC correlating with MIZ-1 promotes the transcription of AMBRA1. Consequently, AMBRA1 promotes the activity of c-MYC through SOCS3/STAT3 pathway, which contributes to MBGroup3 stem potential, growth, and migration  by targeting AMBRA1, and similar results were obtained in U937 cells. Moreover, clinical samples analysis revealed that miR-23a-5p is correlated with the NF-κB pathway in relapsed acute promyelocytic leukemia patients , ~22-nt non-coding single-stranded RNAs, are directly associated with some important physiology and disease progression of plants and animals in a post-transcription modification manner. Insights into the roles of miRNAs in cancer have made miRNAs attractive targets for novel therapeutic approaches . By analpatients . MIR7–3H49 cells . A recen in vivo . Since mThe incidence and mortality of cancer are increasing yearly, and cancer is the major source of the global disease burden. A systematic analysis estimates that the burden of cancer will continue to rise for at least the next 20 years . As the in vitro; further work will be focused on validating these findings in vivo. In addition, it would be interesting to understand how AMBRA1 itself is regulated, both with relation to the cell cycle and in light of the multiple well-established functions. Moreover, several AMBRA1 isoforms are annotated in the human genome. It also remains to be determined whether these possible protein isoforms exist in cells and, if so, how their functions differ.AMBRA1 is an intrinsically disordered protein that was associated with various tumor progressions, including autophagy, tumorigenesis, proliferation, EMT, and apoptosis , 45, 48.Currently, the post-translational modifications (PTMs) of AMBRA1 are only focused on phosphorylation and ubiquitylation , 22, 110In sum, targeting AMBRA1 has the potential to inhibit tumorigenesis and tumor progression in some types of malignancies. Furthermore, AMBRA1 tightly correlates with chemoresistance. During chemotherapy, cancer cells could attenuate the cytotoxicity of chemotherapeutic agents through autophagy, thereby promoting cancer survival. Therefore, autophagy inhibition by targeting AMBRA1 might enhance the effect of agents to achieve the therapeutic goal.XL wrote the manuscript. XW read and approved the final manuscript. YL and JL revised the manuscript, and provided suggestions for the final manuscript. All authors read and approved the manuscript and agreed to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work were appropriately investigated and resolved.This work is supported by the National Natural Science Foundation of China (81972361) and Natural Science Foundation of Henan Province (182300410379).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Neisseria infections coupled with emerging resistance to antimicrobials, alternative approaches to limit the spread are sought. We investigated the inhibitory effect of oropharyngeal microbiota on the growth of N. gonorrhoeae and N. meningitidis and the impact of the essential oil-based mouthwash Listerine Cool Mint® (Listerine). Oropharyngeal swabs from 64 men who have sex with men (n = 118) from a previous study (PReGo study) were analysed . These included 64 baseline and 54 samples following three months of daily use of Listerine. Inhibition was confirmed by agar overlay assay, and inhibitory bacteria isolated using replica plating and identified using MALDI-TOF. The number of inhibitory isolates were compared before and after Listerine use. Thirty-one pharyngeal samples (26%) showed inhibitory activity against N. gonorrhoeae and/or N. meningitidis, and 62 inhibitory isolates were characterised. Fourteen species belonging to the genera Streptococci and Rothia were identified. More inhibitory isolates were observed following Listerine use compared to baseline, although this effect was not statistically significant (p = 0.073). This study isolated and identified inhibitory bacteria against pathogenic Neisseria spp. and established that daily Listerine use did not decrease their prevalence. These findings could provide a new approach for the prevention and treatment of pharyngeal Neisseria infections.With increasing incidence of pathogenic  Neisseria includes two human pathogenic species, N. gonorrhoeae and N. meningitidis [Neisseria meningitidis is a coloniser of the nasopharynx from where it can become invasive, causing sepsis and cerebrospinal meningitis [Neisseria gonorrhoeae, on the other hand, causes infections in the urinogenital tract, rectum, and pharynx [N. gonorrhoeae infections can also result in neonatal conjunctivitis if untreated women transmit the pathogen during delivery [N. gonorrhoeae have been on the rise [The genus ngitidis . Neisserningitis . Neisser pharynx . N. gonodelivery . Over ththe rise . Unfortuthe rise ,7.N. gonorrhoeae, bacteriocins could offer a new avenue to combating antibiotic resistant strains [Neisseria species [Considering the alarming rate at which gonococcal isolates are developing resistance against conventional antibiotics, the search for novel antimicrobials from other sources, including natural sources, is increasingly gaining attention ,9. Scree strains . To the  species ,15.® (herein after referred to as Listerine) , was bactericidal against N. gonorrhoeae and other bacterial species [N. gonorrhoeae [® versus Biotène. We hypothesised that this effect might have been mediated by Listerine reducing the abundance of commensal bacteria responsible for colonization resistance. It is worth noting that, indigenous human microbiota use a number of modalities, such as the secretion of bacteriocins, and competition for space and resources to resist the colonisation and invasion of the host by exogenous bacteria (termed colonisation resistance) [In this study, we screened for the presence of such inhibitory bacteria in samples from a randomized controlled trial we performed to investigate the efficacy of another antibiotic sparing agent , to reduce the incidence of bacterial sexually transmitted infections (STIs) in men who have sex with men (MSM) (the PReG0 study) . In-vitr species . Surprisorrhoeae . Chow etorrhoeae , in anotistance) . The effistance) . The useistance) . Listeriistance) .N. gonorrhoeae and N. meningitidis in the participants of the PreGo study and secondly, to determine if the use of Listerine has an effect on the prevalence and number of these inhibitory bacteria.Despite these findings, Listerine and other antiseptic mouthwashes have been proposed and are being used as an intervention to reduce gonorrhoea transmission in MSM ,23. TherThis sub study analysed samples from the Preventing Resistance to Gonorrhoeae (PReGo) study  . This waThis study being a continuation of the PReGo study was also covered by the ethical approvals from the Institutional Review Board of the Institute of Tropical Medicine, Antwerp (Ref: 1276/18) and the Ethics Committee of the University of Antwerp (Ref: 19/06/058).Neisseria species, N. gonorrhoeae WHO P, a WHO reference strain [N. meningitidis M00003/, a clinical isolate, obtained from an oropharyngeal swab from an asymptomatic man participating in the Resistogenicity Study at ITM [This study tested the inhibition of the growth of two pathogenic e strain  and N. my at ITM . Some of2. Growth inhibition of the N. gonorrhoeae and N. meningitidis strains was assessed by agar overlay assay as previously described [N. gonorrhoeae/N. meningitidis, in phosphate-buffered saline (PBS) approximately 106 CFU/mL, was diluted in 10 mL of melted GCB agar supplemented with 100 µL IsoVitaleX . This was overlayed (in turns) on two sets of the triplicate replicas and further incubated for 24 to 48 h. Following incubation, inhibitory colonies were observed and isolated from the third set of replicas. Each inhibitory colony that was obtained was purified on Columbia blood agar and reconfirmation of the isolates at the species level was performed by MALDI Biotyper IVD  (library updated to v.10.0.0.0_8326-9468 (IVD).The 118 oropharyngeal swabs analysed were previously cultured on Columbia blood agar plates and stored at –80 °C in skimmed milk. A loopful of each sample was diluted  and inoculated on Columbia Nalidixic Acid Agar (CNA) plates selective for gram positive cocci to obtain single colonies. Replica plating of each subculture was made on GC agar plates following the method of Lederberg and Lederberg . Specifiescribed ,32. BrieThe number of inhibitory colonies were compared between baseline and samples following three months of daily Listerine use. Due to the cross-over study design, the ‘month 3′ specimens could be from the month 3 or 6 visits but in all cases, they were specimens that were taken after 3 months exposure to Listerine. Results were presented as counts and percentages of samples positive. The paired samples t-test was used to compare the mean number of inhibitory colonies before and after the receipt of Listerine at a 95 % significant level. The statistical analyses were performed in STATA MP v.16 .Neisseria species, N. gonorrhoeae WHO P and N. meningitidis M00003/1. Our screening was restricted to gram positive commensal bacteria as the initial growth of the samples was on CNA agar, a selective medium for this group. Of the 118 samples, 31 (26%) had inhibitory colonies, while 87 (74%) had none. Eight of the 31 samples had only one inhibitory colony, while 26 had multiple inhibitory colonies  and genus Rothia (2 species) , S. sanguinis  and S. mitis . One isolate each belonging to R. mucilaginosa, S. gordonii, S. infantis and S. peroris were identified. It is worth noting that more than one inhibitory species was identified in the same participant in some cases (All 62 inhibitory isolates were identified by MALDI-TOF MS and they belonged to the genus species) . The mosme cases .p = 0.073, 95% CI −1.2–0.5). Likewise, the number of inhibitory species were greater in samples following Listerine treatment (13 species) as compared to baseline samples (9 species). Notably, R. mucilaginosa was the only isolate that was present in the baseline samples but absent in the treatment samples. The two R. dentocariosa isolates and S. gordonii, S. infantis, S. peroris and S. vestibularis were present only in the post-Listerine samples  were assessed in this study. Surprisingly, more inhibitory isolates were in samples after Listerine treatment  as compared to baseline samples  , althoug samples . It is w samples .We also noted the number of inhibitory colonies in participants who tested positive for pharyngeal gonorrhoea by PCR in the PReGo study . In totaNeisseria species provide strong motivation to search for novel approaches to prevent and treat these infections [Neisseria species. The primary aim was to identify commensal bacteria as putative candidates for possible use, either as probiotics in preventing pathogenic antibiotic resistant Neisseria colonisation of the human host or their secretory products as therapeutics. We identified 14 different species of Streptococci and Rothia from 31 individuals capable of inhibiting the growth of N. gonorrhoeae and/or N. meningitidis in vitro. More interesting is the fact that 13 of the 14 species are non-pathogenic bacteria, with some even designated by the Food and Drug Administration (FDA) with the GRAS  status by their virtuous nature [N. gonorrhoeae [N. gonorrheae [The high levels of antimicrobial resistance in pathogenic fections ,7. This s nature  or listes nature , indicatorrhoeae ,35. Wortnorrheae .N. gonorrhoeae. These include other Neisseria spp. [Lactobacilli, Staphylococci, Streptococci, and Escherichia which are part of the human microbiome [Previous studies have found that a range of bacteria inhibit the growth of ria spp. ,37 as wecrobiome ,31,38,39crobiome .Neisseria spp. Listerine Cool MintÒ is a 22% hydroalcoholic solution containing menthol, thymol, methyl salicylate and eucalyptol. This or other antiseptic solutions are commonly used by high-risk populations such as MSM on HIV PrEP to prevent STIs, including gonorrhoea [Streptococcus anginosus and Fusobacterium nucleatum [® (22%) may therefore explain some of our findings. It is important to note that, maintaining a healthy microbial community in the oropharynx is important not only to prevent colonisation and infection of the host, but also in various metabolic and immunological processes [A secondary aim of this study was to evaluate if Listerine mouthwash reduced the prevalence of bacteria with inhibitory effects against pathogenic norrhoea ,42. Unfonorrhoea ,24,43. Wucleatum . Other sucleatum ,27. The rocesses . We wereN. gonorrhoeae presents the possibility of using these commensal isolates as probiotics [N. gonorrhoeae infections [The identification of human microbiota with inhibitory activity against a multidrug resistant obiotics . If bactfections .N. gonorrhoeae, N. meningitidis and commensal Neisseria spp. strains including clinical strains currently in circulation in order to have a more comprehensive overview of the spectrum of strains inhibited.We recommend further research should be undertaken to investigate the interference mechanism of the 14 species of bacteria to identify and characterise all plausible bacteriocins, determine their amino acid residues, bioactivity in vivo, minimum inhibitory, bactericidal concentrations, tolerance, absorption, distribution, elimination and susceptibility to resistance-induction. A limitation in the study was the use of selective media for gram positive bacteria and serial dilution of the sample which might have resulted in exclusion of other inhibitory bacteria with therapeutic potentials. We recommend plating of multiple dilutions on nonselective media to increase the possibility of isolating additional inhibitory bacteria. We also recommend testing the inhibitory isolates against different Neisseria spp. from the human oropharyngeal microbiome. We also established that long-term use of the essential oil-based mouthwash Listerine does not reduce the prevalence of oropharyngeal commensals with inhibitory activity against N. gonorrhoeae and N. meningitidis.This study successfully isolated and identified inhibitory bacteria against pathogenic


Ambrosia artemisiifolia) is one of the most important allergen sources, worldwide, causing severe respiratory allergic reactions in late summer and fall, in sensitized patients. Amb a 1 has been considered as the most important allergen in ragweed but 12 ragweed pollen allergens are known. The aim of our study was to investigate IgE reactivity profiles of ragweed allergic patients and to associate them with clinical symptoms.Ragweed (n = 150) were analyzed using immunoblotted ragweed pollen extract. Immunoblot inhibition experiments were performed with two Amb a 1 isoforms and CCD markers and basophil activation experiments were performed with IgE serum before and after depletion of Amb a 1‐specific IgE.IgE sensitization profiles from clinically well‐characterized ragweed allergic patients (By IgE‐immunoblotting 19 different IgE reactivity patterns with and without Amb a 1‐sensitization were found. The majority of patients (>95%) suffered from rhino‐conjunctivitis, around 60% reported asthma‐like symptoms and about 25% had skin reactions. Patients with complex IgE sensitization profiles tended to have more clinical symptoms. Serum with and without Amb a 1‐specific IgE induced basophil activation.Ragweed pollen allergic patients exhibit complex IgE reactivity profiles to ragweed allergens including Amb a 1 isoforms and cross‐reactive carbohydrates indicating the importance of Amb a 1 isoforms and additional allergens for diagnosis and allergen‐specific immunotherapy of ragweed allergy. Asthma symptoms were reported in 23.7% of ragweed allergic patientsAmb a 1, formerly antigen E, is a major allergenBut meanwhile, other 11 ragweed pollen allergens have been described in the WHO/IUIS allergen nomenclature database.Accordingly, ragweed pollen is a complex allergen source containing several isoforms of Amb a 1 and other allergens with varying cross‐reactive potential. Currently, commercially available component resolved diagnosis offers only natural or recombinant Amb a 1 and Amb a 4. Whether these allergens alone are sufficient for an accurate diagnosis remains unclear. Compared with birch pollen allergy where 8 allergens were described but only one allergen is dominant (Bet v 1),We aimed to investigate IgE reactivity profiles of ragweed allergic patients, by performing an extensive IgE‐immunoblot study combined with IgE inhibition experiments with purified ragweed allergens and basophil activation experiments using sera from clinically well‐characterized patients. The importance and novelty of this study lie in the better illustration of the heterogeneity and complexity of ragweed pollen allergy. Our results reveal several different IgE sensitization profiles indicating that molecular forms of diagnostics and immunotherapy may need to include Amb a 1 isoforms and additional ragweed pollen allergens.2Only essential information was included in this section, more details can be found in Supporting Information 2.1Patients included in this study were recruited from an allergy center in Timisoara, Romania, where they were clinically evaluated by an allergist, based on ARIAAll recruited patients reported severe symptoms only during the ragweed pollen season although they were not all monosensitized to ragweed.Serum samples were collected after written informed consent was obtained. The study was approved by the Local Ethics Commission of Scientific Research of the Pius Brinzeu Emergency County Hospital Timisoara .2.2Aqueous ragweed pollen extract, natural Amb a 1.01 (nAmb a 1.01), recombinant Amb a 1.03 (rAmb a 1.03), recombinant glycoprotein HHM2 (horse heart myoglobin) and recombinant Der p 2 were produced as described.2.3Carbohydrate‐specific IgE levels were quantified by ImmunoCAP measurements using biotinylated ProGlycAn P  coupled to Streptavidin ImmunoCAPs (o121) .2.4125I‐labeled anti‐human IgE  and visualized by autoradiography.Ragweed pollen extracts, separated under reducing conditions by SDS‐PAGE were blotted onto nitrocellulose membranes and incubated with patients' sera, diluted 1:10. Bound IgE was detected with For the comparison between ragweed pollen extract and the Amb a 1 isoforms, four concentrations of extract  and 1 μg of nAmb a 1.01 and rAmb a 1.03 were separated by 18% SDS‐PAGE followed by Coomassie Brilliant Blue staining.For IgE immunoblot inhibitions, 1:10 diluted sera from ragweed allergic patients and individuals exclusively sensitized to CCDs , co‐sensitized to other ragweed allergens and negative to CCDs, were repeatedly incubated, first on nAmb a 1.01 coated plates, then on rAmb a 1.03 coated plates (5 μg/ml), following the schedule: 2 × 3 h, overnight, 4 × 2.5 h, overnight, 2 × 3 h, overnight, 3 h, at 4°C. IgE antibody removal was confirmed by ELISA.2.6Rat basophil leukemia cells transfected with human FcεRI (RS‐ATL8)2.7p value was ≤0.05.Statistical analysis was performed using SPSS version 25.0 (IBM Corp.). Differences between patients with only Amb a 1 sensitization and patients with Amb a 1 and/or other ragweed pollen allergens sensitization were evaluated by Chi‐square test and were considered statistically significant if the 33.1In this study, 150 ragweed allergic patients were analyzed. Symptoms reported during the ragweed pollen season were recorded and no significant differences between mono‐ and polysensitized patients were observed  and buffer without serum (B) were analyzed for IgE reactivity to nitrocellulose‐blotted ragweed pollen extract, but only patients negative to CCDs by ImmunoCAP measurements  were considered for IgE sensitization profiles analysis, to reduce the role of CCD‐specific IgE. IgE reactivity profile of the CCD positive patients is available in Figure The IgE reactivity profiles of these 130 patients could be summarized into 19 different sensitization patterns P1‐P19 Figure . Seven dInterestingly, 13 patients displayed no IgE reactivity even after long exposure of the autoradiographies.Serum from the non‐allergic individual (NC) and buffer without serum (B) showed no reactivity.The protein composition of ragweed pollen extract was visualized by SDS‐PAGE and subsequent staining of proteins Figure . Bands cIgE reactivity to Amb a 1 was further tested using immunoblot inhibition experiments. Sera from 10 patients corresponding to different patterns: 7 (P6), 14 (P9), 35 (P8), 51 (P2), 54 (P13), 57 (P10), 64 (P9), 74 (P2), 81 (P7), 89 (P14) and displaying more than the two bands around 38–40 kDa were pre‐incubated with two Amb a 1 isoforms. Different levels and patterns of inhibition were observed Figure . nAmb a Based on earlier work,3.3Patients with negative CCD‐specific IgE results were grouped according to IgE sensitization patterns and symptoms. The most frequent IgE‐sensitization patterns were P5 (16.9%) and P1 (11.5%) Table . While pAmong the patients with IgE reactivity on blot , 23.9% belonged to patterns P1 and P2, whereas the majority of patients  displayed reactivity to Amb a 1 and/or other ragweed allergens.Stratification based on the number of reported symptoms showed that most of the patients had three (46.1%) or two (36.9%) symptoms Figure .Each IgE pattern was associated with a certain number of reported symptoms. Most of the patterns were associated with two or three symptoms Figure . For P5,n = 60)  , while 64.5% were sensitized to Amb a 1 and/or to other ragweed allergens (patterns P3‐P19) Figure . This pe) Figure  and was ) Figure . Also, s) Figure , but theAmong patients with different combinations of symptoms, a higher percentage showed reactivity towards Amb a 1 and other ragweed allergens. However, this difference in the proportion of patient reactivity within symptom groups was not statistically significant Figure .3.4Sera from patients 54 (pattern P13), 81 (pattern P7) and 89 (pattern P14) were subjected to depletion of Amb a 1.01‐ and 1.03‐specific IgE. They displayed signals on the blot corresponding to Amb a 1 and to other ragweed allergens (the band from 24 kDa and the two bands between 10 and 15 kDa) and showed total or partial inhibition with nAmb a 1.01 and rAmb a 1.03 Figure . Sera beThe allergenic activity of sera after Amb a 1‐specific IgE depletion was analyzed comparing sera before and after IgE removal in basophil activation experiments Figure . Sera be3.5Sera from seven ragweed allergic patients positive to CCDs and three individuals sensitized to CCDs but not to ragweed (PC1‐3) were pre‐incubated with nAmb a 1.01, rAmb a 1.03 and two CCD markers and tested for IgE reactivity to nitrocellulose‐blotted ragweed pollen extracts. Pre‐incubation with HHM2 inhibited the ragweed extract‐specific IgE reactivity almost completely for patient 98, comparable with PC1 and PC2. Partial inhibition was observed for patients 63, 75, 82, 99 and PC3. ProGlycAn P inhibited IgE reactivity for patient 98, also for PC1 and PC2. Partial inhibition of IgE binding was observed for patients 58, 63, 75, 82, 99 and PC3 Figure .4Ragweed pollen allergy represents a major health issue in the infested areas with increasing relevance due to climate change and urbanization.First, we analyzed symptoms related to ragweed pollen sensitization of 150 ragweed allergic patients ,Interestingly, the two Amb a 1 isoforms showed different abilities to inhibit IgE binding to natural Amb a 1 in the blotted extracts as observed for patients 35, 54, 74 and 81 Figure  indicatiNo inhibition could be observed for signals around 55, 24, 14 and 10 kDa, except a narrow band around 28 kDa (patient 89), representing probably the α‐chain of Amb a 1, indicating that these bands belong to other ragweed pollen allergens. The band around 55 kDa may be attributed to the newly discovered enolase Amb a 12 (48 kDa),The analysis did not reveal a dominant IgE recognition pattern. Pattern P5 including Amb a 1 and other ragweed pollen allergens  Viravaxx , WORG Pharmaceuticals  and serves as a consultant for Viravaxx and WORG Pharmaceuticals. Elijahu Babaev and Frank Stolz were working at the time of the study at Biomay AG, however, there are no direct conflicts with the data presented in this study. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be considered a potential conflict of interest.Supporting Information S1Click here for additional data file.Supporting Information S2Click here for additional data file.Figure S1Click here for additional data file.Figure S2Click here for additional data file.Figure S3Click here for additional data file.


For an investigation into the effects of glycosylation site modification on hemagglutinin (HA) on the biological characteristics of the H5N6 subtype avian influenza virus (AIV), the HA sequences of H5N6 AIVs from Global Initiative on Sharing All Influenza Data (GISAID) and the isolates in China were analyzed for genetic evolution and glycosylation site patterns. Eight recombinant H5N6 AIVs with different glycosylation site patterns were constructed, and their biological characteristics were determined. The results showed that H5N6 AIVs containing a 129-glycosylation site on HA are becoming prevalent strains in China. Acquisition of the 129-glycosylation site on the HA of H5N6 AIVs increased thermostability, decreased pH stability, and attenuated pathogenicity and contact transmission in chickens. Most importantly, H5N6 AIVs escaped the neutralization activity of the Re-8-like serum antibody. Our findings reveal that H5N6 AIVs containing the 129-glycosylation site affect antigenicity and have become prevalent strains in China.IMPORTANCE H5N6 avian influenza viruses (AIVs) were first reported in 2013 and have spread throughout many countries. In China, compulsory vaccine inoculation has been adopted to control H5 subtype avian influenza. However, the effect of vaccination on the antigenic drift of H5N6 AIVs remains unknown. Here, we found that H5N6 AIVs with the 129-glycosylation site on hemagglutinin were the dominant strains in poultry in China. The neutralization assay of the serum antibody against the H5 subtype vaccine Re-8 showed a significantly lower neutralization activity against H5N6 AIVs with the 129-glycosylation site compared to that against H5N6 AIVs without the 129-glycosylation site, indicating that the 129-glycosylation site may be a crucial molecular marker for immune evasion. The H5N6 avian influenza virus (AIV) broke out in China in 2013 and then spread throughout Asia . MoreoveHemagglutinin (HA) is the main antigenic protein on the surface of AIVs and is responsible for binding sialic acid receptors . AIVs ma17–In this study, we analyzed the glycosylation site pattern on HA of H5N6 AIVs and constructed a series of recombinant viruses with glycosylated or deglycosylated sites at residues 63 and/or 129 on the HA protein and evaluated their antigen variation, thermostability, pH stability, pathogenicity, and contact transmission in chickens.n = 2,107), followed by Europe (n = 51), and there were a few isolates in Africa (n = 1) and North America (n = 3). Analysis of the HA sequences of H5N6 viruses from Asian countries revealed that the number of H5N6 AIVs with a 129-glycosylation site on HA isolated in China was the highest (n = 421); however, a small number of H5N6 viruses with the 129-glycosylation site were isolated from Bangladesh (n = 35), Vietnam (n = 45), Japan (n = 2), and other countries (n = 10) , in which most isolates were distributed in Asia ((n = 10) . Further(n = 10) . H5N6 vi(n = 10) . The Re-(n = 10) . Hence, in vitro compared to the wild-type strain rB-63−129− , and the rB-63+129− also showed a significant decrease in the TCID50 at 37°C on day 1 posttreatment compared to the TCID50 of wild-type strains  displayed pathogenicity in chickens slightly lower than that of the wild-type rB-63−129− . The conFor understanding the effects of glycosylation on antigenicity, eight mutated viruses and four isolated viruses with or without the 129-glycosylation site were selected . The croNext, the neutralization titers of the antisera against the wild-type and mutant strains were determined. The neutralization titers of the antisera induced by rB-63−129− against the recombinant viruses without the 129-glycosylation site (rB-63−129− and rB-63+129−) were 2,560 and 2,560, respectively, whereas the neutralization titers against the recombinant viruses with the 129-glycosylation site (rB-63−129+ and rB-63+129+) were 320 and 320, respectively, with an 8-fold decrease. Similarly, the neutralization titers of the antisera induced by rW-63−129− against the recombinant viruses without the 129-glycosylation site (rW-63−129− and rW-63+129−) were >5,120 and >5,120, respectively, whereas the neutralization titers against the recombinant viruses with the 129-glycosylation site (rW-63−129+ and rW-63+129+) were 40 and 40, respectively, with more than a 128-fold decrease. Additionally, the antisera induced by viruses with the 129-glycosylation site  showed good cross-neutralization titers against their derived viruses with or without the 129-glycosylation site. For antisera against the vaccine strains, the cross-neutralization titers of Re-8 antisera showed a high level against rW-63+129− and rW-63−129−  and a low level against rW-63+129+ and rW-63−129+ , a more than 32-fold decrease. However, Re-11 antisera had similar cross-neutralization titers against rW-63+129+, rW-63−129+, rW-63−129−, and rW-63+129− . SimilarFor determining whether the Re-11 antisera contained antibodies against the 129-glycosylation site on HA, the expression of HA in H5N6 AIVs was detected by Western blotting. When incubated with Re-11 antiserum, the expressions of HA proteins of the viruses with the 129-glycosylation site (rW-63+129+ and rW-63−129+) were significantly higher than those of rW-63+129− and rW-63−129− in cell lysate or purified virus  and D. HFinally, for exploring the impact of the 129-glycosylation site on viral antigenicity from a structural perspective, a glycan model of the H5N1 virus was used, and HA trimers were generated using SWISS-MODEL . The res50, both in viral thermostability at 42°C and 56°C and in low-pH stability (pH 4.5 to 5.5).A study showed that more than 50% of H5N6 AIV isolates from Guangdong Province contained the D63N mutation (63-glycosylation site) and D129N/S or D130 deletion/E/T mutation (129-glycosylation) . Our mol50 of H5N6 AIVs acquiring the 129-glycosylation site compared to the TCID50 of their parent strain, H5N6 AIVs with the 129-glycosylation site can survive in the environment as H5N6 AIVs without the 129-glycosylation site.However, the addition of a 129-glycosylation site on HA improved HA thermostability at 56°C, resulting in a delayed HA titer decrease. Therefore, the emergence of the 129-glycosylation site endows the HA protein of H5N6 AIVs with the ability to maintain stability at high temperatures, which might be applied to achieve long-term preservation of the influenza vaccine. Because the temperature had no effect on the TCIDAn acid-stable HA protein is a molecular requirement for the pandemic influenza virus, which is necessary for pH1N1 influenza virus pathogenicity and airborne transmissibility . For H5 The compulsory immunization program has been implemented since 2004 in China, and the vaccination strategy has not been adopted in other Asian countries. Our molecular epidemiological study revealed that the H5N6 virus containing the 129-glycosylation site appeared and became a prevailing strain in China; however, there were few or no H5N6 viruses with the 129-glycosylation site in other Asian countries, based on the GISAID database, indicating that the H5N6 virus containing the 129-glycosylation site may be selected by vaccine pressure. Re-8-like antisera showed a high level of neutralization activity against H5N6 viruses without the 129-glycosylation site on HA and a low level of neutralization activity against H5N6 viruses with the 129-glycosylation site on HA, indicating that H5N6 viruses with the 129-glycosylation site are novel variants of the immune escape strain. Although Re-11 antisera had similar neutralization titers against rB strains with and without the 129-glycosylation site, the neutralization titers of Re-11 antisera against rW strains with the 129-glycosylation site were higher than those against rW strains without the 129-glycosylation site. Western blotting confirmed the presence of massive antibodies against the 129-glycosylation site epitope in the Re-11 antisera. The simulated 3D structure showed that the 129-glycosylation site appeared on the head domain of the HA protein and covered some area of antigenic epitope B, which may induce antibodies against the 129-glycosylation site epitope and prevent the neutralization activity of Re-8 antisera. The data suggest that the H5N6 virus with the 129-glycosylation site may be a strain that adapted under the vaccination pressure induced by the Re-8-like vaccine. Re-11 antisera had good neutralization activity against epidemic strains with the 129-glycosylation site, which probably generates novel immune escape strains under vaccination pressure.In summary, based on GISAID data analysis, H5N6 AIVs with the 129-glycosylation site on HA are the prevailing strains in China. The additional 129-glycosylation site on the HA of H5N6 AIVs increased HA thermostability, decreased pH stability, and attenuated both pathogenicity and contact transmission levels in chickens. Thus, continuously monitoring the immune escape strain under the vaccination pressure is essential.All experiments involving H5N6 AIVs were performed in animal biosafety level 3 (ABSL-3) facilities and were approved by the Biosafety Committee of Yangzhou University. All animal studies followed protocols approved by the Jiangsu Province Administrative Committee for Laboratory Animals (SYXKSU-2016-0020).Commercial antisera Re-8 and Re-11 were purchased from YeBio , and polyclonal antisera against mutated viruses were collected from immunized specific-pathogen-free (SPF) chickens with inactivated viruses. Monoclonal antibodies against H5 HA protein were prepared as described previously .The six H5N6 AIVs used in this study were isolated from clinical samples of diseased poultry collected between 2016 and 2020 . The virTwo H5N6 AIVs B strains with glycosylation sites 63−129− on the HA protein and W strain with glycosylation sites 63+129+ on the HA protein were chosen for recombinant virus construction. The amplified HA genes were inserted into the pHW2000 vector and confirmed using sequence analysis. Glycosylated and deglycosylated HA plasmids were constructed using the Mut Express II fast mutagenesis kit V2 . The primers for mutation construction are listed in Table S1.Recombinant viruses were generated according to reported protocols . Brieflyhttps://www.gisaid.org). Sequences with a similarity greater than 99.5% were removed using BioAider (v.1.334) (https://itol.embl.de/).The HA sequences of H5N6 AIVs from different regions and countries by the end of 2020 were downloaded from GISAID (der v.1.3 (34). Thv.1.334) . DiffereCEF cells were infected with recombinant viruses at a multiplicity of infection (MOI) of 1. At 24 h postinfection, the cells were lysed with 200 μL RIPA lysis buffer  on ice for 15 min, and the purified viruses were lysed using the same method. Cell lysates and purified viruses were divided into two aliquots: one was treated with 2 μL PNGase F at 37°C for 2 h, and the other was the control and treated with phosphate-buffered saline (PBS). Proteins were loaded into lanes on SDS-PAGE gels and transferred to nitrocellulose membranes. The membrane was blocked in 5% skimmed milk and incubated with Re-11 polyclonal antibody or monoclonal antibody against HA, followed by incubation with conjugated goat anti-mouse or chicken IgG antibodies . Protein bands were detected by electrochemiluminescence  .2. The virus titers in the supernatant were monitored periodically by determining the TCID50 in MDCK cells  of test viruses in 0.2 mL PBS. Four hours postinoculation, five uninfected SPF chickens were placed together with the infected chickens. Mortality was monitored for 2 weeks, cloaca and oropharyngeal swabs of the directly exposed group were collected to determine virus shedding, and all swabs were inoculated with chicken embryos . DGalpb1-4DGlcpNAca1-2[DGalpb1-4DGlcpNAcb1-6] DManpb1-3[DGalpb1-4DGlcpNAcb1-2DManpa1-6] DManpb1-4DGlcpNAcb1-4[LFucpa1-6] DGlcpNAcb1-OH was used as a glycan array compound (The antigenic variation among the viruses was analyzed by cross HI titers, and antigenic cartography was performed according to the methods in Smith et al. . HA trimcompound , 43. Thecompound .P < 0.05.Statistical analyses were performed using one-way analysis of variance. GraphPad Prism version 9 software was used to generate the graphs. Statistical significance was set at MZ708707 to MZ708720.The sequence data from this study were deposited in NCBI databases with the accession numbers


Mayflies (Ephemeroptera), stoneflies (Plecoptera), and caddisflies (Trichoptera) (EPT) are aquatic insects that are well known to the general public and are commonly used as indicators of environmental quality in water management. Knowledge of how EPT communities react to human-induced gradients in real environments can be important, for example, during the assessment of the implications of newly planned or currently active human disturbances for natural or cultural landscapes. We sampled a stream ecosystem affected by mining and smelting industries and communal wastewaters with pronounced concentrations of cadmium, lead, and zinc, as well as high levels of pesticides, pharmaceuticals, illegal drugs, and sewage-derived organic matter. Changes in other environmental factors such as increases in temperature were also studied at the affected sites. The abundance and species richness of stoneflies fell rapidly at the study sites. The richness of mayfly families also declined, from four to one, even though overall mayfly abundance was not affected. Conversely, the abundances of caddisflies were higher at the affected sites, and their richness did not decrease. This study will provide feedback for ecotoxicologists who perform better controlled and manipulated tests in laboratories, although any such test results are limited by simple artificial environments.–1 for zinc) and sediments  were found at the most contaminated sites. The highest loads of pesticides, pharmaceuticals, and illegal drugs were found under the WW effluent. Other associated factors such as the physicochemical parameters of the water and alterations to microhabitats were also evaluated and taken into account. Although EPT richness was lower at affected sites, abundances did not fall. Stoneflies were dominant at unaffected sites, while caddisflies dominated at affected sites. Only baetid mayflies were detected at the sites contaminated by trace metals and WWs; ephemerellid, heptageniid, and leptophlebiid mayflies were absent from these sites. The site contaminated by trace metals was also inhabited by numerous limnephilid caddisflies, in which limb malformations were detected in up to 11.8% of all specimens of a single taxon. Downstream from the entrance of the WWs, the locality was dominated by hydropsychid caddisflies. The increasing prevalence of predator or passive filter-feeding strategies in these EPT communities was significantly related to increasing water conductivity and acute ecosystemic exposure to ‘poorly treated’ WWs.Abundances of EPT larvae sampled in a Central European locality affected by mining and smelting, as well as by the continual inflow of treated communal wastewaters (WWs), were recorded. High concentrations of trace metals in water (maximum 1200 µg·L The aquatic insect orders of mayflies (Ephemeroptera), stoneflies (Plecoptera), and caddisflies (Trichoptera)  are frequently used indicators of environmental quality, particularly in running waters ,2. TheirGiven their rapid expansion through the natural environment, the ecological impact of human-induced gradients needs to be monitored, and possible threats are objectively predicted. The contamination of stream ecosystems by toxicants is a global issue; nevertheless, engineering work severely modifies the morphology of whole catchment areas and causes substantial changes in hydrological regimes, habitat structure, and water quality ,9,10. HeThe experiment described in this work explored an EPT community in a temperate zone in the European Central Highlands ecoregion , specifiThe contamination of streams by trace metals  is a global phenomenon, and many studies tackling the responses of macroinvertebrate communities have identified mining as the cause of this type of pollution ,17,18,19Effluent from wastewater treatment plants (WWTPs) often represents an ‘entrance gate’ for many pollutants, including sewage-derived particulate organic matter (SDPOM), nutrients, pesticides, active pharmaceutical compounds (PhACs), synthetic organic compounds, phthalates, and trace metals into streams ,25,26,27This article attempts to decipher the relationships between these human-induced gradients and EPT taxa and their feeding strategies which could be beneficial and permeable in such an impaired environment. It provides a list of taxa that can be considered tolerant to particular types of pollution, and the possible susceptibility of missing taxa is compared and discussed with previous reports. Since EPT taxa usually represent the essential part of macroinvertebrates in streams ,4,5 and (i)Fontinalis antipyretica) and species of Marchantiophyta. Despite not monitoring fish populations by electrofishing, brown trout  were observed during the macrozoobenthic sampling (Lampetra planeri), brook trout , and European perch (Perca fluviatilis) at this site four years before our study began. Insects were prevalent in the macrozoobenthic samples, although only a few small oligochaetes, crustaceans, and molluscs were found;Site 1 was a stretch of the Obecnický brook in the Brdy highlands above the Obecnice reservoir F. antipyretica). Fish stocks consist of brown trout, brook lamprey, and stone loach (Barbatula barbatula) , and molluscs (Pisidium sp. and Ancylus fluviatilis) were relatively abundant;Site 2 consists of a stretch of the Obecnický brook below the Obecnice reservoir Phoxinus phoxinus), brown trout, and European perch Rutilus rutilus), chub , gudgeon (Gobio gobio), brown trout, European perch, common bream (Abramis brama), common rudd , stone loach, and rainbow trout (Oncorhynchus mykiss) , and the water louse (Asellus aquaticus) were very abundant. Molluscs were detected in low numbers.Site 4 was a stretch of the Litavka river downstream from its confluence with the Příbramský brook  Obecnický brook and (ii) Litavka river—are situated in central Bohemia  N 49.7186939, E 13.8732253–N 49.7083778, E 13.9831206 and (ii) N 49.6563228, E 13.8557881–N 49.9602636, E 14.0847414; The whole sampling campaign was conducted in 2020, specifically on 11 May, 24 June, 11 August, and 24 September. The macrozoobenthos were sampled using a Surber sampler  at four sampling sites and three points (triplicates) at each site  pesticide and PhAC concentrations in water (measured from samples taken on 11 August 2020), (iii) cadmium, lead, and zinc concentrations measured in sediments and water (measured from samples taken on 24 September 2020), and (iv) daily volumes of sewage  recorded up to 15 days prior to each sampling day at the Příbram WWTP . Detailed monthly data of water and sediment analyses from Sites 1, 2, and 3 (2009–2020) and data of actual water discharge from the same sites were provided by the Czech Hydrometeorological Institute  and Povodí Vltavy, State Enterprise . In addition, the water temperature was continuously monitored during the sampling campaign by four data loggers (TFA) placed approximately in the middle of each site.Environmental data were taken for both the whole and subplot levels. The environmental variables (EVs) differing only at the whole-plot level (hereafter referred to as ‘whole-plot EVs’) consisted of (i) physicochemical parameters—conductivity, pH, oxygen concentrations, and water temperature—measured using a HACH–1] , measured continuously for 0.5 min above the Surber sampling spot with a flowmeter  placed close to the bottom, in the water column and close to the water surface; (iii) widths and depths at each subplot; and (iv) classification as riffles, pools, inflows into pools, or backs of pools.The EVs also differed at the subplot level (hereafter referred to as ‘subplot EVs’) in terms of their (i) choriotopic compositions, which were empirically estimated for each Surber sampling spot; (ii) current velocities [m·sData were analysed using Canoco 5 version 5.12   and R-stp values. Forward selection (FS) was used for selecting significant whole- and subplot EVs. ‘Hybrid analysis’ was chosen when tests on visualised constrained axes were not significant (p > 0.05). The significance of differences in choriotopic compositions between sites was tested in a similar way; individual values were transformed by the angular (inverse sine) function and the transformed values were used as response variables. This transformation was also applied when the choriotopic compositions were used as explanatory variables.The significances of axes constrained by (i) site identity, (ii) the identity of sampling times, (iii) whole-plot EVs, and (iv) subplot EVs  were tested using Monte-Carlo permutation tests (999 permutations used). Permutations were carried out according to the experiment’s hierarchical design: (i) when the whole-plot EVs or the identity of sites were used as explanatory variables, both whole-plots and subplots in the four blocks defined by the four sampling times used as covariates were permuted using cyclic shifts; (ii) when the subplot EVs were used as explanatory variables, only the subplots in the four blocks defined by sampling times were permuted using cyclic shifts; and (iii) when the identity of sampling times was used as an explanatory variable, both whole plots and subplots in the four blocks defined by the four sites were permuted using cyclic shifts. A false discovery rate was used for adjusting p value adjustment was applied.Generalised linear models (GLMs) were employed to model trends in shifts of multiple averaged feeding strategies along environmental gradients or along the resulting ordination axes. The significance of differences between sites and sampling times in EPT abundances and richness was tested using GLMs with negative binomial and Poisson distributions, respectively (the quasi-likelihood estimation method was applied in the case of the Poisson distributions). The significance of improvements using Generalised linear mixed-effects models (GLMMs) rather than GLMs was tested by likelihood ratio tests. A post hoc test with Tukey’s method for 3 in 2020  = 0.025 and explained variation = 6.0%, pseudo-F = 2.9, p (adj.) = 0.049, respectively). Samples from Site 1 compared with those from Site 2 had a much greater proportion of smaller mineral particles  and ‘mosses’ (Marchantiophyta only at the Site 1), and a lower proportion of light deposits (‘coarse particulate organic matter’—CPOM and ‘fine particulate organic matter’—FPOM), debris, and xylal  and the Leuctridae family (stoneflies) were detected at disturbed Sites 3 and 4. At these sites, Habrophlebia lauta (Leptophlebidae) and Seratella ignita (Ephemerellidae) all but completely disappeared, while Paraleptophlebia submarginata (Leptophlebiidae), Ecdyonurus torrentis, and members of the genus Rhithrogena (both Heptageniidae) were totally absent, although these latter taxa were detected upstream at the interconnected Sites 1 and 3 , several species belonging to the genera Protonemura, Amphinemura, and Nemoura (Nemouridae), all very abundant at Sites 1 and 2, along with Diura bicaudata and Perlodes aff. microcephalus (Perlodidae) were completely absent in the samples taken from disturbed Sites 3 and 4  and Sericostoma flavicorne/personatum (Sericostomatidae), very abundant at both Sites 1 and 2, were completely missing from the samples from Sites 3 and 4 , whilst Rhyacophila nubila gr. was commonest in samples from Sites 1 and 2.Of the caddisflies, Site 1 had a greater richness of trichopterans than Site 2; the abundant family Philopotamidae, for example, was no more detected at Sites 2, 3, and 4; finally, members of the Goeridae were detected only sporadically at particular sites H. On the 3 and 4 . One of t Site 4 . The tott Site 4 H. Even tpossible , RhyacopF = 1.9, p = 0.001; test on all axes: pseudo-F = 4.0, p = 0.001; F = 0.7, p = 0.024; test on all axes: pseudo-F = 1.8, p = 0.001; p < 0.05); those selected by FS are shown in The differences in species compositions between sites were significant . Significant relationships with ‘conductivity’ (32.5% of explained variability), ‘total volume of released sewage for three days prior to sampling time’ (5.9% of explained variability), ‘oxygen concentration’ (2.8% of explained variability), and ‘total concentration of PhACs’ (2.3% of explained variability) were detected (p < 0.05). ‘Passive filter feeder’ and ‘predator’ feeding strategies were dominant in the WW-polluted environment; the ‘grazer and scraper’ feeding strategy seemed to be limited by the most polluted WW; and the ‘gatherer/collector’ and ‘shredders’ feeding strategies were generally suppressed in the most polluted environment .The differences in compositions of the averaged feeding strategies between sites were significant in general; however, only the composition at Site 4 was significantly different from all other sites . Peculiarly fused terminal parts of walking legs  or overall shortened walking legs were observed in the two given limnephilid taxa; deformed and shortened anal claws were observed in R. cf. aurata , although at the latter site, the total number of sampled specimens was substantially lower than at Site 3 (7 vs. 76 specimens at Site 3). There were no malformed specimens in the abundant R. cf. aurata in Site 4; however, there was one malformed specimen out of a total of 20 specimens of R. nubila gr. at Site 4.A substantial proportion of final instar caddisfly larvae in . aurata . MalformDrusus annulatus, H. cf. tesselatus, and Limnephilidae gen. sp. inc. in the given samples . Several dead and decaying pupae of Hydropsyche sp. were also found in the samples from Site 2 taken in June.We also observed a drift of dead limnephilid pupae at Site 3 in September during the macrozoobenthic sampling. We found dead and decaying larvae and pupae of The EPT community can significantly reflect the quality of the environment . Even thBaetis fuscatus group . The total abundance of B. fuscatus gr. compensated for the decrease in abundances of other mayfly taxa so that this small-sized baetid could act as a proxy for other mayflies in human-disturbed sites. The other most commonly detected baetids, B. rhodani and B. vernus, did not show any substantial changes in abundance between sites. Nevertheless, trace metal pollution can have an effect later on in the life cycle of baetids and other aquatic insects by rapidly causing increased mortality in emerging imagines (or subimagines) [The changes in EPT abundance and richness related to pollution by trace metals observed in our study generally resemble reports from other sites, the greatest similarity being in the susceptibility of mayflies—and especially heptageniids—to this type of pollution . Sites 3magines) ,48.Amphinemura sulcicollis, Leuctra fusca, and L. inermis, taxa that are similar to those found in our study [–1 of cadmium, copper, and zinc, respectively, for 10 days in microcosm conditions. By comparison, numbers of heptageniids and Baetis tricaudatus fell significantly in only half of these concentrations [Stoneflies are generally the aquatic insects most sensitive to conditions modified by humans ,50. Therur study ,19. Littur study , althougtrations .Therefore, the significant reduction in stonefly abundance and richness at both contaminated sites cannot be conclusively attributed to pollution by trace metals. The absence there of most of the stoneflies detected upstream could also be due to water warming since most stoneflies prefer lower temperatures than those recorded at Sites 3 and 4 ,42. HoweLeuctra fusca gr.  of small sizes were detected at Site 4, contaminated by both trace metals and municipal WWs. It is important to note that the organic pollution produced by the WWs sometimes reaches high levels. According to unpublished data provided by the Czech Hydrometeorological Institute and Povodí Vltavy, State Enterprise, the average estimated five-day biochemical oxygen demand (BOD5) was equal to 2.39 mg·L–1 at Site 4; however, the estimated maximum was 17.00 mg·L–1 just four months before the beginning of our sampling campaign. In the Příbramský brook .According to our data, the less sensitive stoneflies were able to withstand the heavy industrial trace metal pollution but were then eliminated by the municipal WW, despite the advanced technology used in the WW treatment . A syneris study ) and L. t Site 3  but not Chaetopterygopsis maclachlani and Rhyacophila polonica detected during our study [Hydropsyche sp., leptocerids, Rhyacophila sp. [Psychomyia pusilla [Caddisflies were the least harmed insects in the EPT community. Their richness was not significantly different between sites, and their abundance was significantly higher at Site 4 than at Sites 1 and 2 ; Table 5ur study ,56), andhila sp. ,18,46).  pusilla ). Nevert pusilla ,58,59.–1). Slower water speeds caused by water abstraction [The vast majority of EPT taxa situated on the right-hand side of the CCA biplot A were catraction  combinedtraction ,60 favoutraction ,62.O. albicorne and S. flavicorne/personatum, which were dominant in the samples from Sites 1 and 2. The warmer temperatures (and poorer oxygen conditions) are expected to have a negative effect. Haidekker and Hering [O. albicorne tolerates well mean summer temperatures up to 16 °C. However, this figure was exceeded at Site 4 by 1.24 °C  were most abundant downstream from the WW effluent. The taxa are shown in the upper right-hand corner of C. trimaculatus with a more pronounced microhabitat preference for macrophytes [P. flavomaculatus at Site 4 than Site 3. The most abundant hydropsychids can use multiple food resources and, aside from predating on small benthic organisms and consuming detritus, they also graze on algae [H. siltalai in September at Sites 3 and 4 with the presence of aquatic mosses . Hydropsychids, polycentropodids, rhyacophilids, and psychomyiids . Nevertheless, Krno [Lithax niger is a bryophyte dweller and reports that Rhyacophila obliterata can only live on emergent mosses, whilst R. nubila, R. polonica can also live on submerged mosses. Similar findings have been reported by Glime [The proportion of mosses in the choriotope (explained community variation = 5.3%) and mean current velocity (explained variation community variation = 5.2%) had the highest parsimonious and significant power to explain EPT community composition at the subplot level . These tss, Krno  suggestsby Glime . Conversby Glime  considerH. pellucidula/incognita : fitted47 m·s–1 . The may35 m·s–1 . By contAsellus aquaticus and gatherers/collectors by chironomids and oligochaetes , the abundance of winged adults of the aquatic insect directly utilising CPOM may be substantially lowered and could cause an alteration in nutrient and energy flows through the environment.The analysis of shifts in ecological traits and, above all, their relationship to environmental characteristics has recently become a key topic in community ecology . SubstanB. fuscatus gr., which probably colonised this site after drifting from an upstream stretch after a high discharge event (up to 8.5 times the average discharge). This hydrological situation occurred at the same time as the release of a high volume of sewage. The sewage release ended four days before the sampling time. The greatest similarity in the overall EPT community of samples to the samples from Site 3 is shown in the overlap of the ellipses in Despite that, 3-day sewage volumes positively correlated to the responses of passive filter feeders, predators, and shredders and negatively to the responses of gatherers/collectors , the greL. fusca gr. and L. geniculata can be considered tolerant to trace metal pollution but sensitive to WW pollution or its combination with trace metals. The municipal WW combined with trace metal contamination led to the greatest changes in the EPT community, including changes in the composition of feeding strategies. Caddisfly passive filter feeders and predators—mainly hydropsychids, polycentropodids, and rhyacophilids—dominated the most polluted environment. EPT shredders and collectors/gatherers dominated in unaffected sites. Our results demonstrate how the EPT community reacts to human-induced gradients in natural environments, which is important knowledge for assessing the implication of planned or current human disturbances in natural or cultural landscapes.This study highlights the impacts on the EPT community of trace metal pollution originating from mining and smelting and effluent from a municipal WWTP, which releases continuously treated WW and, periodical, ‘poorly’ treated WW. According to our data and previously published studies, trace metal pollution can have quite different consequences for the EPT community than, for example, pesticides and several caddisflies, especially limnephilids, were found to be very abundant at the affected site. Additionally, malformations in caddisfly larvae and mortality of caddisfly pupae were observed. Even though the affected sites were warmer, the absence of ephemerellid, heptageniid, and leptophlebiid mayflies is attributable to trace metal pollution, given the results from previous studies. The factors responsible for the decline in the stonefly community are less clear, and, except for higher water temperature, other kinds of pollutants and habitat degradation can be assumed to have a negative impact. Nevertheless,