To prevent recurrent ankle sprain, it is important to clarify the pathology of chronic ankle instability (CAI). An association has been reported between CAI and abnormalities of foot posture and ankle alignment. There is no consensus on the types of these abnormalities that occur in individuals with CAI. The objective of this systematic review is to clarify the relevance of abnormality of foot posture and ankle alignment for CAI.A systematic computerized literature search was performed of the PubMed, CINAHL, SPORTDiscus, Web of Science, and the Cochrane Register of Clinical Trials databases. The selected studies either compared CAI patients with a control group or CAI ankles with contralateral healthy ankles and specifically reported foot posture and alignment of the ankle in the outcomes. They were written in English and published prior to June 2021. The methodological quality of the included studies was evaluated using a 16-question index. Data were extracted independently by two reviewers, and the certainty of evidence was assessed using GRADE approach.Sixteen studies including 872 patients of high to low methodological quality were included. These showed there was significant anterior displacement and internal rotation of the talus in CAI ankles (low evidence), but there was no consensus on fibular alignment or foot posture.This review showed there was significant anterior displacement and internal rotation of the talus in CAI ankles but found no consensus on the characteristics of fibular and foot alignment. Further investigations are required to clarify the characteristic foot and ankle malalignment in CAI to facilitate the development of efficient interventions.The online version contains supplementary material available at 10.1186/s12891-021-04537-6. Ankle sprain is one of the most common ankle injuries, with a recurrence rate exceeding 50% . Ankle sIndividuals with CAI experience repeated ankle sprains caused by various dysfunctions, such as pathologic laxity, neuromuscular inhibition, balance deficit, and muscle weakness, including arthrokinematic restrictions caused by abnormal foot and ankle joint alignment such as talar anterior displacement or cavus foot deformity . AbnormaRearfoot varus has been reported as a risk factor for CAI , and is This study was conducted and reported according to the PRISMA guidelines for reporting systematic reviews and meta-analysis . A compuThe inclusion criteria were as follows: eligible articles had to be full-text reports of a randomized control trial, cohort study, cross-sectional study, case–control study, or case series published in a peer-reviewed journal; the study compared a group of human participants with CAI with a control group or compared a healthy leg with the contralateral leg with CAI; foot posture and static alignment of the ankle were included in the outcome; and the article was written in English. Because this study included the search period before the publication of recommended criteria by the International Ankle Consortium , we alsoAfter the initial search, articles duplicated between the databases were removed. The titles and abstracts were assessed for relevance to the aim of the review, and the full-text articles were retrieved for those identified as relevant. These were screened against the inclusion and exclusion criteria, and the reference lists of the articles were cross-checked for additional articles. Two reviewers independently screened all the articles identified in the database search, with any disagreements discussed at a meeting between the two reviewers and resolved by consensus. If no consensus was achieved for a specific item, a third reviewer was involved to achieve a consensus between the two raters Fig. .Fig. 1PRAll alignment outcome measures, measurements of foot posture and static alignment of the ankle were extracted from the included articles by two independent reviewers. Information about participants, study objectives, definitions of CAI, independent and dependent variables, measurement methods, statistical analyses, main findings, and conclusions were also collected.Two independent reviewers assessed the risk of bias, with a third reviewer deciding in cases where consensus was lacking. The included studies were evaluated for quality based on an index (Downs and Blacks criteria checklist) developed for nonrandomized studies . BecauseThe results of the literature search and review process are presented in Fig. Six studies including 239 patients (39.8 patients per study) investigated fibular alignment , 28, 35.The two studies involving analysis of the axial plane found the lateral malleolus of the ankle to be significantly posterior in the subjects with CAI , 16. MeaFour studies including 139 patients (34.8 patients per study) investigated alignment of the talus in CAI , 32, 34.The four studies reported consistent results. Regardless of the measurement method and whether or not the ankle was weight bearing, the talus of CAI ankles showed significant anterior displacement 0.9–1.0 mm) and internal rotation (3.85–5.7 degrees) compared with healthy ankles (Table .0 mm andSeven studies including 494 patients (70.6 patients per study) investigated foot posture in CAI –31, 33. The study that used a 2D analysis of sagittal plane radiographs observed mean differences in talocalcaneal, calcaneal, and talometatarsal angles, and tarsal index between the CAI and control groups were 2.0°, 1.5°, 5.4°, and 5.7, respectively . These rThe aim of this systematic review was to clarify the relationships between CAI and abnormalities of foot posture and ankle alignment. The review included 16 studies that examined fibular or talus alignment or foot posture, retrieved through electronic screening. These indicated significant anterior displacement and internal rotation in the talus of CAI ankles compared with healthy ankles. However, there was no consensus regarding fibular alignment and foot posture.The results for fibular anterior/posterior translation in CAI ankles differed across the six included studies. Differences in the reference bone may have been the main cause of this variation. Some studies referenced to the tibia and others to the talus, making it difficult to compare findings across the studies. The 3D analysis found the fibulas of CAI ankles were in a significantly lateral position compared with those of the contralateral side . The autThe studies included in this systematic review consistently showed significant anterior displacement and internal rotation of the talus of CAI ankles. The main cause for this is thought to be damage to a lateral ankle ligament  caused by repeated lateral ankle sprains . These aThere was no consensus among the seven studies in this review regarding the characteristics of foot posture in individuals with CAI. In the study by Larsen et al. , the fooIn CAI, abnormal alignment of the talus may contribute to abnormal arthrokinematics and cartilage loading pattern of the talocrural joint, so assessment of talar alignment using reliable modality is useful in clinical practice. As no consensus has been reached regarding the abnormalities of fibular alignment in CAI, intervention methods must be considered according to the specific alignment abnormality in each case. The tibia may also represent the optimal reference for reliable assessment of fibular alignment. In addition, no clear relationship was apparent between foot posture and CAI from the findings of this systematic review. However, since abnormalities of foot posture are suspected to be associated with various clinical dysfunctions, accumulation of further results using reliable methods is important.This systematic review included 16 retrospective cohort, case–control or cross-sectional studies. Although these provided results regarding the relationship between several aspects of foot or ankle alignment and CAI, the quality of almost included studies was not high, so the evidence underpinning the findings of this systematic review is not strong. In addition, differences in CAI selection criteria among the studies may have influenced the results. For example, some studies included patients with mechanical instability of the ankle joint who resisted conservative treatment, while other studies used a history of ankle sprain and perceived instability as inclusion criteria and did not include the presence or absence of mechanical instability. In addition to mechanical impairments  and perceptual impairments , neurophysiological problems were also associated with the pathogenesis of CAI . Future The aim of this systematic review was to clarify the relevance for CAI of foot and ankle alignment. The review identified that there was significant anterior displacement and internal rotation in the talus of CAI ankles compared with healthy ankles, but there was no consensus on the characteristics of fibular and foot alignment. In addition, the quality of the studies that investigated foot and ankle alignment was not high. Future studies are needed to clarify the foot and ankle malalignments characteristic of CAI, as well as related ligament injuries, using unified selection criteria. This should facilitate the development of efficient interventions.Additional file 1.


Prevention and treatment of the Human T-cell leukemia virus, type 1 (HTLV-1) which was discovered nearly 40 years ago, still remain challenging. The reported high prevalence of HTLV-1 in some countries around the world triggered an open letter to the World Health Organization (WHO), urging action against HTLV-1 infection in 2018. This highlights the importance of virus elimination strategies to eradicate HTLV-1 infection. In Iran, we have documented our experiences with the virus in order to achieve and promote the possible ways to manage, control, and eliminate HTLV-1. Although there has been considerable progress apropos of HTLV-1, a series of additional challenges need to be tackled to control HTLV-1 infection in Iran.  An openom Japan , 4. The In line with HTLV-1 eradication plans, we have documented our experiences with the virus in order to achieve and promote the possible ways to manage, control, and eliminate HTLV-1. HTLV-1 infection is highly concentrated in the northeast of Iran, with a significant prevalence in the cities of Neyshabur 3-5%), Mashhad (2.1%), and Sabzevar 1.66%)  as a national network at Ghaem hospital since 2011 with the aim of increasing public awareness (http://www.htlv-1.ir/). A variety of experts in the fields of neurology, haematology, immunology, and microbiology all make up members of the HTLV-1 Foundation alongside representatives of blood transfusion organizations and the vice chancellors for research and health of MUMS. History has proved that the goals of eradication programs are not achieved, unless they are supported by precise assessment and data analysis. Consequently, we launched a national program to establish the first national HTLV-1 registry in Iran at Ghaem Hospital, MUMS, since 2017. This hospital-based registry, a first in the Middle East region, is considered a major step forward in managing asymptomatic carriers (ACs) and patients with HTLV-1. Ethics approval was obtained from the Biomedical Ethics Committee of MUMS and Ministry of Health and Medical Education, Iran. Registered patients and ACs are mostly referred from the Blood Transfusion Centre, health centres, and outpatient clinics in accordance with a suspicious positive HTLV-1 screening result. These patients are further assessed by enzyme-linked immunosorbent assay (ELISA) and their diagnosis is confirmed via a polymerase chain reaction (PCR) or Western blot tests. Among them, HTLV-1 patients, which show clinical symptoms, are stratified as HAM/TSP or ATL, whilst ACs are considered as HTLV-1 carriers in our registry. Currently patients diagnosed with ATL referred to us directly by hematologists are not included in our registry; however plans are in place to rectify this soon.Our aim of expanding this registry throughout the entire population of Iran began with assembling a data collection team that included trained experts designed to work under supervision of two neurologists and an immunologist, working closely with Mashhad Neurology department. We have carefully and continuously collected demographic, clinical and laboratory (including DNA and RNA) data since 2018 of 100 HAM/TSP patients and 120 ACs in an electronic database. Prior to data collection and blood sampling, our patients signed an informed consent. The confidentiality of these data are maintained, and all the patients sign an informed consent form for having their data gathered and tested.Patients are visited by our neurologist, and history and physical examinations are documented, diagnostic tests are performed free of charge, and the treatment course is followed routinely. The follow-up of the ACs is performed on an annual basis, whereas HAM/TSP patients are followed every six months and the progressive type of HAM/TSP is followed every three months. In our outpatient clinic on every Wednesday, we provide counselling services in addition to visiting the patients and regularly evaluating their treatment program. The HTLV-1 registry program has enabled us to monitor and improve our care for the patients as well as creating a platform for us to conduct better quality research. MUMS annually spends 2 billion Rials  to perform research in the field of HTLV-1-associated diseases. So far, about 7 clinical trials have been designed in our registry program regarding HTLV-1 infection , 12. In Sweeping efforts have been implemented to inform and educate the Iranian population regarding the HTLV-1 virus. In 2009 for the first time, an international congress of HTLV-1 and associated diseases was held in Mashhad. The following year in 2010, a range of informative books and a website were prepared by MUMS to provide general information about HTLV-1 in order to improve social awareness. Furthermore, since 2007, around 10 retraining programs have taken place for health-care workers mainly in the endemic regions of Mashhad and Neyshabur. To contribute on this expanding knowledge on HTLV-1, we plan to collect roughly 10000 samples from a cohort study amongst faculty and staff members of MUMS, allowing us to screen for the HTLV-1 virus and be able to conduct relevant research. Although there has been a considerable progress apropos of HTLV-1, a series of additional challenges need to be tackled to control and eliminate HTLV-1 infection in Iran. Expanding the aforementioned strategies throughout Iran, with its many endemic regions, will help to control and eliminate HTLV-1 in our country. We will continue at every level in the path towards this goal by ensuring high quality clinical data is available for future research.


TSLP. Chrysin attenuated TNFα-induced TSLP expression by downregulating EGR1 expression in HaCaT keratinocytes. We also showed that the oral administration of chrysin improved AD-like skin lesions in the ear and neck of BALB/c mice challenged with 2,4-dinitrochlorobenzene. We also showed that chrysin suppressed the expression of EGR1 and TSLP by inhibiting the extracellular signal-regulated kinase (ERK) 1/2 and c-Jun N-terminal kinase (JNK) 1/2 mitogen-activated protein kinase pathways. Collectively, the findings of this study suggest that chrysin improves AD-like skin lesions, at least in part, through the downregulation of the ERK1/2 or JNK1/2-EGR1-TSLP signaling axis in keratinocytes.Thymic stromal lymphopoietin (TSLP) is an epithelial cell-derived cytokine that acts as a critical mediator in the pathogenesis of atopic dermatitis (AD). Various therapeutic agents that prevent TSLP function can efficiently relieve the clinical symptoms of AD. However, the downregulation of TSLP expression by therapeutic agents remains poorly understood. In this study, we investigated the mode of action of chrysin in TSLP suppression in an AD-like inflammatory environment. We observed that the transcription factor early growth response (EGR1) contributed to the tumor necrosis factor alpha (TNFα)-induced transcription of  Atopic dermatitis (AD), also known as atopic eczema, is a chronic inflammatory skin disease characterized by the development of recurrent eczematous lesions and intense pruritus . The pre+ T helper (Th) lymphocytes, including Th2, Th22, and Th17 cells, as well as pro-inflammatory immune cells, including mast cells, neutrophils, and macrophages/monocytes [The cause of AD has not been identified in sufficient detail; however, the onset of AD is known to be influenced by genetic and environmental factors, epidermal barrier abnormalities, and impaired cutaneous immune functions ,12. In monocytes ,14,15. Tonocytes ,17,18.Thymic stromal lymphopoietin (TSLP) is an IL-17-like cytokine that has been identified and characterized in murine thymic stromal Z210R.1 cells ,20. It iChrysin -mediated EGR1 expression in HaCaT keratinocytes. In addition, we demonstrated that the oral administration of chrysin suppressed EGR1 and TSLP expression in AD-like skin lesions in BALB/c mice. TSLP mRNA levels were enhanced upon TNFα stimulation, as shown using reverse transcription (RT)-PCR  with TSLP-specific SYBR Green-based fluorescent probes. TNFα increased the TSLP mRNA level by 17.9 ± 2.52-fold compared to that in the control; however, upon treatment with 20 and 40 μM chrysin, the TSLP mRNA levels decreased by 7.40 ± 1.45- and 2.97 ± 0.397-fold, respectively, compared to the levels in the control -PCR B. Howeve control C. Chrysi control D. These TSLP expression, we established a series of TSLP promoter deletion constructs: −1384/+18, −1338/+18, −1214/+18, −1017/+18, and −369/+18. These constructs harbored the luciferase reporter gene. Each of these promoter-reporters was transiently transfected into HaCaT cells, and the luciferase activity was measured following TNFα stimulation. As shown in TSLP promoter–reporter activity was persistently repressed in cells transfected with the shortest construct (−369/+18), suggesting that the chrysin response element is located between the −369 and +18 positions.To elucidate the effect of chrysin on the inhibition of TNFα-induced To identify the chrysin response elements, we analyzed the transcription factor-binding sites between the −369 and +18 positions using MatInspector program . The nuclear factor kappa B (NF-κB)-binding site was found to overlap with a putative early growth response 1 (EGR1)-binding sequence (EBS) located in the region between positions −206 and −187 B.TSLP promoter remains elusive. We focused on the role of EGR1 in chrysin-mediated TSLP suppression. To determine whether EGR1 transactivates the EBS in the −369/+18 construct, we co-transfected the −369/+18 construct and an expression plasmid for EGR1 (pcDNA3.1/Egr1) and measured the luciferase reporter activity. Exogenous EGR1 expression increased the promoter–reporter activity of the −369/+18 construct in a plasmid concentration-dependent manner  reduced upon chrysin pretreatment, suggesting that EGR1 interacts with the putative EBS in the −369/+18 region of the TSLP promoter. To determine whether chrysin affects the binding of EGR1 to the putative EBS in the −369/+18 region, we performed the electrophoretic mobility shift assay (EMSA). Nuclear extracts from HaCaT cells treated with TNFα in the presence or absence of chrysin were incubated with a biotinylated EBS oligonucleotide probe, and the DNA-binding proteins were analyzed using streptavidin-conjugated horseradish peroxidase. Unlabeled EBS competitors were administered at a fifty-fold excess (2.5 pmol) concentration to indicate the specific reaction of the DNA–protein complex formation. TSLP expression, we silenced EGR1 expression by expressing the control scrambled shRNA (shCT) or EGR1 shRNA (shEgr1) in HaCaT cells. The knockdown of EGR1 expression was confirmed using RT-PCR  inhibited in HaCaT/shEgr1 cells compared to HaCaT/shCT cells, as revealed by RT-PCR  abrogated after chrysin pretreatment  inhibited the TNFα-induced phosphorylation of ERK1/2 and JNK1/2, but not of p38 kinase  decreased TNFα-induced EGR1 accumulation, as revealed in the Western blot analysis . These data suggest that all three MAPKs mediate TNFα-induced TSLP expression via EGR1, but chrysin selectively inhibits only the TNFα-induced ERK1/2 and JNK1/2 pathways to downregulate TSLP expression.To determine the potential relationship between MAPK activation and TNFα-induced EGR1 expression, we used pharmacological inhibitors of the MAPK pathway. Pretreatment with the MAPK kinase inhibitor U0126, p38 kinase inhibitor SB203580, or JNK inhibitor SP600125 significantly  continued to exhibit chrysin activity, suggesting that the chrysin response element is between the positions −369 and +18. We found a putative EGR1-binding motif, EBS, between the positions −206/−187 in the −369/+18 construct. EGR1, also named zinc finger protein 225 (Zif268), Drosophila Kr finger probe 24 (Krox24), tetradecanoyl phorbol acetate-induced sequence 8 (TIS8), and nerve growth factor-induced clone A (NGFI-A), is an immediate-early response gene that encodes a transcription factor containing three Cys2-His2-type zinc finger DNA-binding domains [Various transcription factors, such as vitamin D3 receptor, NF-κB, and AP1 ,44,52, a domains . It regu domains ,57,58,59Egr1-knockout mice, and the TNFα-induced expression of cytokines, including TSLP, IL-1β, IL-6, CXCL1, CCL2, and CCL5, was inhibited in response to EGR1 knockdown [TSLP promoter was analyzed via transient transfection experiments. We observed that the transient transfection of EGR1 enhanced the promoter-reporter activity of the −369/+18 construct. We further investigated whether chrysin inhibits EGR1 to downregulate TSLP expression.EGR1 is expressed at high levels in damaged skin tissues . EGR1 renockdown . Furthernockdown . These fWe found that chrysin prevented TNFα-induced EGR1 DNA-binding activity, as observed using EMSA. In addition, chrysin inhibited TNFα-induced EGR1 expression in HaCaT keratinocytes. We also confirmed that the oral administration of chrysin attenuated both EGR1 and TSLP expression in vivo in the AD-like skin lesions of DNCB-challenged mice. These findings suggest that chrysin downregulates EGR1 expression to inhibit TSLP expression in the inflammatory microenvironment. One of the best-characterized transcription factors that regulate EGR1 expression is the Ets-like protein-1 (ELK-1), which is phosphorylated and activated by ERK1/2, p38 kinase, and JNK1/2 in response to mitogens and TNFα . Our datNF-κB is a transcription factor expressed ubiquitously in almost all tissues, including skin keratinocytes. TNFα strongly activates the NF-κB signaling pathway in HaCaT keratinocytes . NF-κB mChrysin, DNCB, TB, and H&E staining kits were purchased from Sigma-Aldrich . TNFα was purchased from ProSpec-Tany TechnoGene, Ltd. . A firefly luciferase assay system was obtained from Promega . Anti-TSLP antibody was obtained from Novus Biologicals , and phospho-ERK1/2 (Thr202/Tyr204), phospho-p38 (Thr180/Tyr182), and phospho-JNK1/2 (Thr183/Tyr185) antibodies were obtained from Cell Signaling Technology . Anti-GAPDH and anti-EGR1 antibodies were purchased from Santa Cruz Biotechnology . A secondary antibody conjugated to rhodamine red-X was obtained from Jackson ImmunoResearch Laboratories . Human keratinocyte HaCaT cells were obtained from the Cell Line Service . The cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum  and penicillin-streptomycin (Sigma-Aldrich).EGR1 forward, 5′-CAG CAG TCC CAT TTA CTC AG-3′;EGR1 reverse, 5′-GAC TGG TAG CTG GTA TTG-3;TSLP forward, 5′-TAG CAA TCG GCC ACA TTG CCT-3′;TSLP reverse, 5′-GAA GCG ACG CCA CAA TCC TTG-3;GAPDH forward, 5′-CCA AGG AGT AAG AAA CCC TGG AC-3′;GAPDH reverse, 5′-GGG CCG AGT TGG GAT AGG G-3′.Total RNA was isolated from the HaCaT cells using a TRIzol RNA Extraction Kit , and cDNA was synthesized using an iScript cDNA Synthesis Kit . RT-PCR was performed using reverse transcriptase (Promega) and gene-specific PCR primers. The PCR primers used in this study were as follows:The thermal cycling conditions were as follows: denaturation at 94 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, and elongation at 72 °C for 1 min. The amplified PCR products were separated by electrophoresis in a 2% agarose gel containing ethidium bromide and visualized under UV transillumination.TSLP (id: qHsaCIP0030468), EGR1 (qHsaCEP0039196), and GAPDH (id: qHsaCEP0041396) were obtained from Bio-Rad. The thermal cycling conditions used for PCR were as follows: denaturation at 95 °C for 2 min, followed by 40 cycles of denaturation at 95 °C for 10 s and 60 °C for 45 s. The relative mRNA levels of TSLP or EGR1 were normalized to those of GAPDH using the software provided by the manufacturer.The mRNA levels of the genes were quantified using an iCycler iQ system with an iQ SYBR Green Supermix kit (Bio-Rad). Validated Q-PCR primers and SYBR Green-based fluorescent probes specific for 3VO4, 1 mM NaF, 10 μg/mL leupeptin, and 1 mM PMSF. The proteins were separated by electrophoresis in a 10% SDS-polyacrylamide gel and transferred to nitrocellulose membranes. After treatment with the appropriate primary and secondary antibodies, the blots were developed and observed using an enhanced chemiluminescence detection system .HaCaT cells were lysed in ice-cold cell lysis buffer supplemented with 50 mM Tris-HCl (pH 7.4), 1% NP-40, 0.25% Na-deoxycholate, 500 mM NaCl, 1 mM EDTA, 1 mM NaTSLP promoter fragment spanning nucleotides −1384 to +18 upstream of the transcription start site was synthesized from human genomic DNA (Promega) via PCR using the primers 5′-CGT CCA ACC TCC TTT CTC CG -3′ (forward −1384F) and 5′-TTG GAG TCT CCC TGA TGC TCC AG-3′ . The amplified PCR products were ligated to a T&A vector  and digested using KpnI and HindIII. The products were ligated at the KpnI and HindIII sites of the pGL4-basic vector (Promega), yielding pTSLP-Luc(−1384/+18). Several deletion constructs of the human TSLP promoter fragments were synthesized using PCR, for which the pTSLP-Luc(−1384/+18) construct was used as the template. The forward primer sequences were as follows:−1338F: 5′-GGA CCA GAG CGA TGC AGG-3′−1214F: 5′-CAT GAG CCA AGC CAG GGA G-3′−1017F: 5′-AAA TCT GAG CCC GCC ATC TC-3′−369F: 5′-GGG ACA TAT GCA AGG ACT CC-3′A One reverse primer, +18R, was used to generate the deletion constructs. The amplified PCR products were ligated to the T&A vector and then to the pGL4-basic vector. The insert sequence of each construct was confirmed using DNA sequencing .TSLP promoter–reporter construct using Lipofectamine™ 2000 (Invitrogen) according to the manufacturer’s instruction. After 48 h of transfection, the cells were treated with TNFα in the presence or absence of chrysin (20 or 40 μM). After 8–12 h, the cells were harvested, and the firefly luciferase activity was measured using the Dual-Glo™ Luciferase assay system (Promega) following the manufacturer’s instruction. The relative luciferase activity of the untreated cells was assigned the value 1. Luminescence was measured using a dual luminometer .HaCaT keratinocytes cultured in 12-well plates were transfected with 0.2 µg of each EMSA was performed using a LightShift Chemiluminescence EMSA kit , according to the manufacturer’s instruction. A biotin-labeled deoxyoligonucleotide probe corresponding to the EBS (5’-CAA AAA GGA GGA AGG TGA GGG AA-biotin-3’) was synthesized by Macrogen. Nuclear extracts (3 μg samples) prepared from the HaCaT keratinocytes were mixed with 50 fmole biotin-labeled EGR1-binding oligonucleotide probes with 1 µg poly(dI-dC) . For the competition assay, 2.5 pmol of the unlabeled EGR1-binding oligonucleotide was added. DNA–protein complexes were separated in non-denaturing 6% polyacrylamide gels, and the antibody-reactive bands were visualized using chemiluminescence, according to the manufacturer’s instructions.n = 5 in each group). In addiion to those in the naive group, all mice were sensitized with 4% SDS on both the neck and ear skin to disrupt the skin barrier; after 4 h, the SDS-sensitized areas were challenged with 1% DNCB dissolved in an acetone:olive oil mixture . The DNCB challenge was repeated once daily for 3 days. After a 4-day break, sensitization with 4% SDS followed by the application of 0.5% DNCB was repeated five times per week for 2 weeks (days 8–21). Chrysin powder was dissolved in dimethyl sulfoxide (250 mg/mL) to prepare a stock solution and then diluted using PBS to a final concentration of 25 mg/mL. The mice in Group III were administered chrysin (25 mg/kg) orally from day 7 . On day 22, all mice were euthanized, and tissue sections were prepared. The animal experiments were conducted in accordance with the guidelines for animal experiments and procedures approved by the Konkuk University Institutional Animal Care and Use Committee (IACUC). All experimental methods were confirmed to be in accordance with the relevant guidelines and regulations .BALB/c mice  were obtained from Orient Bio, Inc. . The mice were housed in a specific pathogen-free environment at 20 ± 2 °C and a relative humidity of 50% ± 10%. The mice were randomly divided into three groups (based on the treatment administered): Group I, naive; Group II, DNCB + vehicle; and Group III, DNCB + chrysin (Skin sections of the neck and ear with AD-like lesions were fixed in 100% acetone and embedded in paraffin. Each section was cut (5 μm) using a microtome . The paraffin-embedded ear and neck skin sections were deparaffinized by treating with xylene (three times for 10 min) and hydrated using a graded ethanol series. After deparaffinization and rehydration, the tissue sections were stained with H&E. The infiltrated mast cells were stained with 0.1% TB. Images of each section were captured using a light microscope . Immunostaining of the skin sections from the ear and neck was performed as previously described . BrieflyFor TSLP immunofluorescence staining, the tissue sections were treated overnight with an anti-TSLP antibody (1:100 dilution) at 4 °C. After washing, the cells were treated with a rhodamine red-X-conjugated secondary antibody (1:300 dilution) at 25 °C for 1 h. The nuclei were counterstained with Hoechst 33258 solution for 10 min. After extensive washing with PBS, the slides were mounted using the ProLong Gold Antifade reagent (Invitrogen). Fluorescent images were captured using an EVOS FL fluorescence microscope . p < 0.05.Data are expressed as mean ± standard deviation. Statistical analysis was performed using one-way analysis of variance, followed by Dunnett’s or Sidak’s multiple comparisons test using GraphPad Prism . Statistical significance was set at TSLP expression by downregulating ERK1/2- and JNK1/2-dependent EGR1 expression in the skin inflammatory microenvironment. We believe that the results of this study will improve our understanding of the mode of action of chrysin and its therapeutic efficacy in AD.To the best of our knowledge, this is the first study to demonstrate that chrysin suppresses


Giardia duodenalis is one of the most widely occurring zoonotic protozoan parasites causing diarrheal disease in calves. This study aimed to investigate the prevalence of G. duodenalis in Korean native calves and elucidate the causal factors associated with giardiasis in these animals. We investigated the sequences of three genes  of G. duodenalis in fecal samples collected from 792 Korean native calves during 2019–2020. Data were analyzed with regard to age, sex, sampling season, and the fecal sample type . The samples were screened for the three genes mentioned above, and 44 samples (5.6%) were G. duodenalis-positive. Polymerase chain reaction results showed a significantly higher prevalence of the infection in calves aged ≥1 month and in those with watery diarrhea in spring season. Screening for the gene sequences ssu (87.5%), bg (96.2%), and gdh (96.7%) revealed that most of the G. duodenalis-positive samples belonged to assemblage E. Four of the G. duodenalis-positive samples belonged to the zoonotic assemblage A. This study highlights the importance of continuous surveillance of genetic mutations in G. duodenalis for the detection of emerging variants of zoonotic G. duodenalis in calves. Giardia duodenalis  is a zoonotic protozoan parasite that causes intestinal diseases in various host species. Infection occurs following ingestion of infective stages of this pathogen (cysts) via the fecal–oral route or drinking contaminated water [G. duodenalis and is one of the most important parasitic diseases associated with diarrhea in calves [G. duodenalis show clinical signs such as malabsorptive diarrhea and reduced weight gain [G. duodenalis has a broad host range, including humans, cattle, sheep, goats, pigs, dogs, cats, and rats, and is thus a potential threat to public health [G. duodenalis from cattle is required to elucidate the role of cattle as a potential zoonotic reservoir [ed water ,2,3. Gian calves . Althougn calves . Consequght gain ,6. G. duc health ,8. Previeservoir .G. duodenalis consists of eight distinct assemblages (A to H), which appear to have different host ranges [Recent studies have reported that t ranges ,10. Amont ranges ,7, wheret ranges . Interest ranges ,11.G. duodenalis was 13.1% (2013–2015) and 10.0% (2017) in Korean native calves with diarrhea and normal feces, respectively [G. duodenalis is a major concern of infection in Korean native cattle farms, and zoonotic G. duodenalis (assemblage A) could have emerged in Korea in 2017. Therefore, it is essential to continuously monitor by testing fecal samples from calves and adult cattle to determine the prevalence and identify the genotypes of G. duodenalis responsible for causing zoonotic infections.According to previous studies, the prevalence of ectively ,13. Thesectively , and 2.2G. duodenalis in calves by a single gene locus [G. duodenalis, with multilocus genotyping based on sequences of the small subunit (SSU) rRNA, β-giardin, and glutamate dehydrogenase (GDH). We also analyzed the factors associated with the occurrence of G. duodenalis, including age and sex of calves, sampling season, and the fecal sample type .Most of the previous studies conducted in Korea have identified ne locus ,13. TherA total of 792 fecal samples were obtained from 291 Korean native cattle farms between May 2019 and September 2020. These cattle farms were located in 12 different regions  in the central region of the Republic of Korea . The detssu) and β-giardin gene (bg) fragments from G. duodenalis, nested PCR was conducted for each gene using primer sets described in a previous study [gdh) fragments, semi-nested PCR was performed as reported previously [Genomic DNA was extracted from fecal samples using the QIAamp fast DNA Stool Mini Kit  according to the manufacturer’s instructions. To amplify the SSU rRNA gene  on the 3730XL Capillary DNA sequencer machine (Applied Biosystems) by SolGent Co., Ltd. . If the sequencing result was unclear, the experiment was repeated until a clear result was obtained. The obtained sequences of ssu, bg, and gdh were identified from the public data libraries of the National Center for Biotechnology Information  using the basic local alignment search tool (BLAST) and then aligned using Clustal Omega Alignment Tools . The nucleotide sequences of the G. duodenalis-positive samples were deposited in the NCBI GenBank database: MW876381–MW876412 for the SSU rRNA locus, MW876413–MW876438 for the β-giardin locus, and MW876439–MW876467 for the GDH locus. For phylogenetic analysis, we collected the nucleotide sequences of G. duodenalis variants from various host species in various countries from the NCBI GenBank database. Phylogenetic trees were constructed using the neighbor-joining method. The sequence data were sampled using 1000 replicates for bootstrap analysis using the Mega-X software (version 10.0.4) with the Kimura two-parameter option.All the fecal samples identified as G. duodenalis by calf age and sex, sampling season, and fecal sample type. The variables were grouped as follows: six age groups , three groups for sex , four groups for sampling season , and four fecal types . The level of significance was set at p < 0.05. The odds ratio (OR) with 95% confidence intervals (CI) was estimated to show the strength of associations. Statistical analysis was performed using SPSS version 26.0 . In addition, the Kappa (κ) values, which range from 1 to 0, were interpreted according to a previous study [A binominal logistic regression analysis was used to compare the prevalence of us study , where <G. duodenalis was 5.6% (n = 44) among 792 calves from 37 farms (12.7% of 271 tested farms), as determined by PCR  and ≥12 weeks , followed by those aged 9–12 weeks  and 5–6 weeks . The calves aged less than 1 month showed a lower prevalence of G. duodenalis compared with those aged more than 1 month. There was no significant difference between the occurrence of G. duodenalis in male (5.7%) and female (5.6%) calves. With respect to seasons, the prevalence of G. duodenalis was 6.7% in spring (March–May), 6.0% in summer (June–August), 7.4% in autumn (September–November), and 1.5% in winter (December–February). Furthermore, G. duodenalis was frequently detected in hemorrhagic (12.5%) and watery (9.8%) diarrheal feces, but rarely detected in pasty (2.9%) and normal (2.3%) feces. Based on the statistical analysis, there was significantly lower prevalence in calves under 2 weeks of age  than in those over 2 weeks old. In addition, G. duodenalis was significantly more prevalent in watery feces  and in fecal samples from the spring .The prevalence of d by PCR . G. duodG.duodenalis-positive fecal samples, 32 fecal samples (72.7%) were ssu-positive, 26 samples (59.1%) were bg-positive, and 30 samples (68.2%) were gdh-positive  were bg-positive and 65.6% (n = 21) were gdh-positive. Only 31.8% (n = 14) of samples tested positive for all three genes. The κ-values of 0.68 and 0.66 suggest a good agreement between ssu- and bg-positive samples and ssu- and gdh-positive samples, respectively. A moderate agreement was observed between bg- and gdh-positive samples (κ = 0.59).Among the 44 positive . Of the ssu-positive sequences, three were different from the 28 sequences but identical to each other, and one sequence showed a marginal difference from the other sequences. Eight and six sequences were identified in bg and gdh sequences, respectively . Four of the ssu-positive samples (12.5%) belonged to assemblage A (bg-positive samples (96.2%) belonged to assemblage E, and only one sample (3.8%) belonged to assemblage A  belonged to assemblage E except one (3.3%), which belonged to assemblage A  and 2017  [G. duodenalis infection in Korean native calves.The present study 2019–2020) indicated that 5.6% of the tested Korean native calves were infected with  n = 90) ,13. The  n = 90) ,19,20,21–2020 ind n = 90) ,22. Therp = 0.006) with prevalence of G. duodenalis in feces. G. duodenalis was more prevalent in calves aged ≥1 month (14.6%) than in those aged <1 month (2.7%). This result was in line with the findings of previous studies in China in 2016 and 2020, which reported the highest prevalence of G. duodenalis infection in calves aged 1–3 months  [G. duodenalis may not be the main pathogen responsible for this disease in these animals [p = 0.998) and female  calves, which was consistent with the findings of a previous study in Korea [G. duodenalis infection was the highest in autumn , followed by spring , summer , and winter . Among the four seasons, the fecal samples of calves from the spring were significantly  associated with the presence of G. duodenalis. The current findings are consistent with the findings of previous studies in Korea [G. duodenalis infection was most frequently detected in the warm season. With regard to fecal sample type, the occurrence rate of G. duodenalis was the highest in calves with hemorrhagic feces (12.5%), followed by watery diarrheal (9.8%), pasty (2.9%), and normal (2.3%) feces. According to the binominal logistic regression model analysis, the watery fecal samples were significantly  associated with the occurrence of G. duodenalis. This result is in accordance with the findings of a previous study in Korea, which reported a statistical association between G. duodenalis detection and the type of fecal sample [G. duodenalis infection [G. duodenalis infection in calves (≥1 month of age) with watery diarrhea in the spring. Moreover, hemorrhagic diarrhea could also be a result of co-infection with G. duodenalis, and thus, the calves must be screened for other pathogenic infections that could cause hemorrhagic diarrhea.As shown in ctively) . Thus, g animals . Howeverin Korea . Furtherin Korea  and Chinin Korea , in whicic feces .5%, follnfection . These oG. duodenalis-positive samples at once. Among the three PCR primer sets used in this study, the SSU rRNA gene primer set was more effective in detecting G. duodenalis than the protein-coding gene (β-giardin and GDH) primer sets. This finding is in agreement with previous studies [ssu, bg, and gdh) showed a markedly good agreement with each other according to their κ-values, laboratory PCR diagnosis of G. duodenalis should be conducted using different primers for detecting all infected calves. Further studies are needed to establish a PCR diagnostic standard that can determine G. duodenalis infection with high sensitivity and specificity.The results of this study highlight that there is no perfect PCR primer set to detect all  studies ,28. AlthG. duodenalis exhibits host-specific characteristics that vary depending on their assemblages [G. duodenalis assemblages. The ssu sequence analysis revealed that four sequences  belong to zoonotic assemblage A, whereas bg and gdh had only one sequence each belonging to assemblage A . The bg (MW876418) and gdh sequences (MW876467) belonging to assemblage A were derived from two samples that also contained two ssu sequences belonging to assemblage A (MW876386 and MW876412). These different phylogenetic results according to the sequences of each locus suggest that assigning one locus is insufficient for laboratory diagnosis and identification of the assemblage of G. duodenalis.emblages ,10. The ssu locus but seven at the bg locus and five at the gdh locus. The gene bg showed the greatest variability, followed by gdh, then ssu. The results implied that bg is the most feasible target for determination of the genetic diversity of G. duodenalis. A total of seven different sequences were identified from the bg gene sequence of assemblage E, three of which were novel types compared with a previous study on calves in Korea [G. duodenalis infecting calves in Korea. Therefore, continuous monitoring is needed to detect emerging novel types of G. duodenalis and obtain updated genetic information to prevent the outbreak of infections caused by a novel G. duodenalis strain. Moreover, many recent studies commonly performed multilocus genotyping with three genes, namely bg, gdh, and triose phosphate isomerase (tpi), which could identify multilocus genotypes (MLGs) of G. duodenalis [tpi sequencing are needed to reveal the MLGs of G. duodenalis from Korean native calves. The prevalence of G. duodenalis belonging to assemblage A (9.1%) was lower in this study than that observed in previous studies in China, the USA, Belgium, and Portugal [G. duodenalis (belonging to assemblage A) were all aged 1–2 months and had watery diarrhea. These results are in contrast to the findings of a previous study, which showed that G. duodenalis assemblage A was identified in calves aged 1 month with normal fecal discharge [G. duodenalis-positive samples (75%) belonging to assemblage A were detected in this season. These results are similar to the findings of a previous study, which reported that all G. duodenalis-positive samples belonging to assemblage A were isolated in autumn [G. duodenalis belonging to assemblage A is not host-specific, some vectors with increased activity in autumn , may play an important role in pathogen transmission. Therefore, further studies are needed to identify the potential vectors for zoonotic transmission of G. duodenalis from cattle farms. The present findings provide valuable information for similar monitoring studies in the future.In assemblage E, we found only one sequence at the in Korea . This reodenalis ,23. ThusPortugal ,17,22,29ischarge . Furthern autumn . As G. dG. duodenalis infection in Korean native calves. The prevalence of G. duodenalis infection was 5.7% (12.7% at the farm level) in this study. Calves aged ≥1 month or producing watery feces in the spring should be carefully monitored by veterinarians to prevent G. duodenalis transmission to healthy calves. In addition, this is the first multilocus genotyping study of G. duodenalis from the feces of native calves in Korea. The results highlight that analysis of the ssu gene sequence is an effective tool to detect the presence of G. duodenalis in fecal samples and determine their assemblages. The bg gene is useful to investigate the genetic diversity in G. duodenalis. A total of four out of 792 samples (0.5%) belonged to assemblage A as identified by ssu sequencing and were potential zoonotic pathogens. The present findings could help to understand the genetic relationships between G. duodenalis variants distributed across Korea.This study provides updated data regarding the prevalence of and the factors associated with


Giardia duodenalis is a flagellated zoonotic parasite that can infect various animals and humans, causing economic losses in husbandry and detriments to public health. Although it has been reported in pigs worldwide, there are few reports on the prevalence and assemblages of G. duodenalis infection in pigs in China. In this study, the 396 pig fecal samples were randomly collected from seven farms in Zhejiang, Guangdong and Yunnan provinces in southern China, and were examined by means of the nested PCR amplification of β-giardin (bg), glutamate dehydrogenase (gdh), and triose phosphate isomerase (tpi) for the detection of G. duodenalis. Overall, 21 fecal samples were positive for G. duodenalis, with a prevalence of 5.3%. Three risk factors are associated with G. duodenalis infection, namely, region, age and gender. Moreover, 13, six and two samples were successfully amplified at the bg, gdh and tpi gene loci, respectively. Three assemblages of G. duodenalis were identified, including assemblage E (n = 17), assemblage A (n = 3) and assemblage B (n = 1). Assemblage E was the dominating genotype and was distributed in three provinces. These assemblages of G. duodenalis have also been found in human beings, non-human primates, sheep, goats and cattle, which further reveals that farmed pigs pose a potential threat to public health. Giardia duodenalis is a ubiquitous zoonotic intestinal parasite of animals and humans [G. duodenalis is known to cause infection in both humans and animals. While most of the infections are asymptomatic, infection in immunocompromised hosts causes diarrhea, vomiting and weight loss [G. duodenalis is transmitted through the ingestion of food and water contaminated with cysts [Giardia; however, PCR analysis has a higher sensitivity [d humans ,3,4,5,6.d humans . G. duodght loss . G. duodth cysts , or diresitivity ,11.G. duodenalis is classified into eight assemblages (A–H) based on molecular characterization [rization . Among trization ,13,14, brization . Other arization . Howeverrization ,16,17. TEscherichia coli, Rotavirus, Sapovirus, coccidia, Cryptosporidium parvum and G. duodenalis [G. duodenalis infection in pigs in the Xinjiang Uygur Autonomous Region, Henan Province, Shaanxi Province and Shanghai city, with prevalences ranging from 1.7% to 26.9% [G. duodenalis infection in pigs in southern China. Thus, the purpose of this study was to investigate the prevalence and assemblage distribution in pigs in Zhejiang, Guangdong and Yunnan provinces in southern China.Organisms from infected farms such as odenalis ,19 may cto 26.9% ,21,22, tG. duodenalis infection may result in self-limited illness with weight loss and malabsorption, causing a decline in pig production. However, G. duodenalis is a relatively common parasite in pigs all over the world [As the largest pork producer and consumer in the world, China has promoted the rapid development of a large-scale pig industry. Generally, the pigs infected with he world ,25,26,27he world ,29. TherG. duodenalis was 5.3% (95%CI: 3.09–7.51), which was lower than that of previous studies from Shaanxi Province, China  [G. duodenalis in pigs in these reports could be caused by regional differences, but differences in management methods, the number of samples and sampling seasons could also affect these results. Further epidemiological studies should be conducted to collect more data on the infection of pigs with G. duodenalis to analyze the causes of these differing prevalences. These findings provided fundamental information as to the epidemiological situation of G. duodenalis infection in pigs in southern China.In this study, the total prevalence of  45/560) , Shangha 45/560)  and othe 45/560) ,26,27,30 45/560) , Henan P 45/560) , Prince  45/560) . The var2 = 6.158, df = 1, p = 0.02). The significantly different prevalence found in gender groups of pigs  prevalences were observed among different provinces, with the highest prevalence  observed in Zhejiang Province, and the lowest prevalence  observed in Yunnan Province, which was similar to previous reports [G. duodenalis infection in pigs among different areas [G. duodenalis infection in pigs aged 4–6 months  was significantly higher than that in pigs aged 1–3 months    and female pigs  were significantly different  . In fact = 0.01) . This diG. duodenalis-positive by means of the PCR amplification of the SSU rRNA gene at the bg, gdh and tpi gene loci, and three assemblages  were identified , D and E have been reported in pigs worldwide . In thisentified . Among tentified . Two assentified . Furtherin China ,21,23,33In 2016, 396 fecal samples were randomly collected from pigs in seven farms in three provinces, including 124 in Zhejiang province, 72 in Guangdong province and 200 in Yunnan province. All the samples of these pigs were randomly collected from the specific farms in each province. Each fresh fecal sample was collected with a separate sterile glove, marked with the relevant numbers, gender, age and geographical origin, and then placed into the boxes filled with ice. The feces were immediately transported to the laboratory and stored at −20 °C for further analysis.G. duodenalis using nested PCR targeting the bg, gdh and tpi genes. The primers for amplification at the bg locus were G7F (5′-AAGCCCGACGACCTCACCCGCAGTGC-3′), G759-R (5′-GAGGCCGCCCTGGATCTTCGAGACGAC-3′), G99-F (5′-GAACGAACGAGATCGAGGTCCG-3′) and G609-R (5′-CTCGACGAGCTTCGTGTT-3′) [TM  and were observed under UV light.Each stool sample was washed with distilled water and filtered the residue was filtered with a sieve. Then, it was centrifuged at 3000 rpm for 3 min to discard the distilled water. The sediment (200 mg) was used for extracting DNA using an EZNAR Stool DNA kit  following the manufacturer’s instructions. Then, the DNA was stored at −20 °C before PCR amplification. All DNA samples were screened for TGTT-3′) . For othTGTT-3′)  and AL35TGTT-3′) , and poshttps://blast.ncbi.nlm.nih.gov/Blast.cgi accessed on 27 August 2019) and Clustal X 1.83 to identify the assemblages of G. duodenalis.All positive PCR products were sequenced directly by the Genscript Company  and the sequence platform was used for Sanger sequencing. The obtained sequences were proofread by sight using a chromatogram and were aligned with previously reported reference sequences available in GenBank, using the Basic Local Alignment Search Tool (BLASTn)  value < 0.05 was considered statistically significant. Furthermore, 95% confidence intervals (95% CIs) were calculated to explore the correlation strength between G. duodenalis infections and test conditions.SPSS 19.0  was used to analyze all the data relating to G. duodenalis subtypes obtained in this study were deposited under the following accession numbers: MN434084–MN434088 for assemblage E  at the bg gene locus; MN434090 for assemblage E  at the gdh gene locus; MN434089 for assemblage E  and MN434091 for assemblage B  at the tpi gene locus. Other known G. duodenalis assemblages were 100% similar to previous sequences: KT922255 for assemblage A (n = 3); KY633473 (n = 3), and KJ668145 (n = 2) for assemblage E.The nucleotide sequences of G. duodenalis in farmed pigs in the Zhejiang, Guangdong, and Yunnan provinces in southern China, with a total prevalence of 5.3%. Moreover, region, gender and age are risk factors for G. duodenalis infection, which prompted us to pay more attention to these factors. Furthermore, three genotypes  were identified in this study, and all of them were reported as potential zoonotic genotypes. Further studies should be undertaken to collect more samples to assess the risk of animal giardiasis to human beings.The present study revealed the prevalence and assemblages of


Salmonella spp., known to be widespread among wild birds in Poland. The aim of the study was to investigate (1) whether feeders can be a source of Salmonella spp., (2) whether the risk is the same for feeders located in cities and rural areas and (3) whether there is a different level of contamination with Salmonella spp. between old and new feeders. Data were collected in winter 2018 in Poland, and in total, 204 feeders were sampled. Material for analysis consisted of the remains of food and feces. We did not find the presence of Salmonella spp. in any of the tested samples collected from bird feeders. Reasons for the isolation of Salmonella spp. from feeders not being successful lie in the low intensity of bacterial shedding by infected wild birds and low survival of bacteria in the environment.Bird feeders are known to be a transfer site for many important bird pathogens, such as zoonotic Salmonella spp., known to be widespread among wild birds in Poland. The aim of the study was to investigate (1) whether feeders can be a source of Salmonella spp., (2) whether the risk is the same for feeders located in cities and rural areas and (3) whether there is a different level of contamination with Salmonella spp. between old and new feeders. Data were collected in the period 12 January–28 February 2018 in four cities in Poland and nearby rural areas. In total, 204 feeders were sampled. The samples from feeders were taken after a 2-week period of feeding birds. Material for analysis consisted of the remains of food and feces. We did not find the presence of Salmonella spp. in any of the tested samples collected from bird feeders. Therefore, the estimated value of the 95% confidence interval for the binary data was 0.000–0.018. Reasons for the isolation of Salmonella spp. from feeders not being successful lie in the low intensity of bacterial shedding by infected wild birds and low survival of bacteria in the environment in bird feces—which are still not well studied.Bird feeders are known to be a transfer site for many important bird pathogens, such as zoonotic  Along with the progressive urbanization, the observation of plants and animals in the city surroundings is the only opportunity for many people to interact daily with nature . One of Feeding birds entails some threats , the mosSalmonella spp., Campylobacter spp., Mycoplasma gallisepticum, Macrorhabdus ornithogaster, Trichomonas and Poxvirus avian virus [Salmonella. They are commonly found in wild birds, very often with no signs of infection and can be shed in feces [Salmonella spp. in birds [Salmonella Typhimurium (S. ser. Typhimurium), in wild birds with outbreaks of salmonellosis in human populations [S. ser. Typhimurium infection in Great Britain were found in the population of garden birds and humans. These data support the hypothesis that garden birds are the main reservoir of Salmonella infection. Most of the identified outbreaks of passerine salmonellosis occurred in and around the feeding sites of the birds. It has been suggested that bird feeding sites, especially dead birds and their droppings, may play a significant role in the transmission of salmonellosis to humans and increase the risk of infection [Salmonella spp., partly because pollution, microclimate and specific food in urban ecosystems affect bird immunity [Feeders have been found to be an important transfer site for many avian pathogens, such as an virus ,9,10,11.an virus . Among tin feces . Many ouin feces . It has in birds . Some stulations ,16. In 1nfection ,15. It iimmunity . Birds limmunity ,19. Diveimmunity ,20. MoreSalmonella spp.; whether there is a difference between Salmonella spp. presence in feeders located in cities and rural areas; and whether there is a different level of contamination with Salmonella spp. between already used, old feeders (used for approximately 3–10 years) and new ones, provided specifically for the needs of the study. Salmonellosis has been reported as an important cause of mortality of garden birds, as Salmonella ssp. is transmitted to birds from direct contact with other infected individuals or through exposure to contaminated surfaces. During wintertime, birds of various species, especially granivorous and gregarious passerine species concentrate in feeding places [In this regard, the aims of our study were to investigate whether bird feeders can act as a source of infection with bacteria g places ,10, suchData were collected twice during winter, in the period 12 January–28 February 2018, in 4 cities in Poland . Urban sites were matched (up to a distance of 20 km) with a nearby rural area , half located within urban areas and half located within rural areas. Some of the sampled feeders were old , which were provided 3–10 years ago and regularly maintained by people. Other feeders (n = 148) were new and experimental, contained at the bottom four trays with sunflower seeds, very popular during winter bird feeding in Poland . Each exThe samples from feeders consisted of the remains of food and feces. Fecal samples and food remnants were collected from trays on which food was placed, and feces were also collected from the roofs of the feeders. Loose material was poured into sterile containers. Additional material was scraped from the roof of the feeders with sterile feces swabs into sterile containers. Containers were marked with an individual code (unknown for the laboratory) and sent for testing.Isolation of Salmonella was carried out according to the PN-EN ISO 6579: 2003 + AC: 2014-11. Media used for incubations included: Marcy l’Etoile, France);1. Nonselective pre-enrichment medium—Buffered Peptone Water ;3. Solid selective media—Salmonella Chrom Agar SAL-P (Graso), Briliant Green Agar—BGA .Portion of collected material was added to plastic bags and then diluted in a ratio of 1:10 with Buffered Peptone Water. The inoculated bags were homogenized and incubated for 18+/−2 h at 36 °C+/−2 °C. After incubation, samples were cultured on a plate with MSRV medium by adding 100 µL bacterial culture in three drops. The plates were incubated at 41.5+/−1 °C for 48+/−2 h. Typical growth on the plates (grey-white turbid zone extending out from the inoculated drop) were cultured onto agar medium BGA and Chrom agar. Then, plates were incubated at 37+/−1 °C for 24+/−2 h.Salmonella spp.) were calculated using the binomial (Clopper-Pearson) “exact” method [After obtaining all laboratory results, trays for binary data . Therefore, the probability of infection by Salmonella is very low.A wide variety of birds regularly visited the bird feeders, comprising the following 16 species: Chloris chloris, Coccothraustes coccothraustes. Columba livia f. urbana, Corvus monedula, Cyanistes caeruleus, Dendrocopus major, Garrulus glandarius, Parus major, Passer domesticus, Passer montanus, Periparus ater, Pica pica, Poecile palustris, Sitta europea, Streptopelia decaocto and Turdus merula.Salmonella are among the most important zoonotic pathogens. They are widespread in the environment and characterized by high genetic variability. Many species and serovars of Salmonella are found in the digestive system of wild animals, including birds [Salmonella spp. can developed signs of disease, die or remain asymptomatic and shed bacteria, especially under stress conditions, such as cold weather or competition with other birds in feeding sites [Salmonella spp. [Bacteria of the genus ng birds . Salmoneng birds . Therefong sites ,24. Numella spp. ,26.S. ser. Typhimurium in wild birds were observed in Switzerland in the 1950s [S. ser. Typhimurium was also noted in the Norway in the years 1999–2000, when infection with this pathogen was detected in 64.8% of wild Passeriformes found dead in bird feeding stations located across the country [Salmonella spp. An outbreak of S. ser. Typhimurium in humans and cats, associated with infections and mortality in wild birds, was reported in 1999 in the center of Sweden [S. ser. Typhimurium were isolated from wild bird populations and people in Germany [S. ser. Typhimurium found in humans and in wild garden birds were very closely related [S. ser. Typhimurium in the human population with the outbreak of salmonellosis in wild birds [Salmonellosis is often a cause of mortality in wild bird populations . Epizoothe 1950s  and in ehe 1950s . An outb country . It is af Sweden . Similar Germany . Large-s related . Moreoveld birds .Salmonella spp. and in which ways avian bacteria can come into contact with people. One of the potentially perfect and often suggested places to investigate this is bird feeders [Salmonella spp. are known to be widespread in wild bird populations in Poland [Salmonella spp. detection were taken from 124 bird feeders. Surprisingly, Salmonella spp. was not found in any of them. However, in a different study conducted in Scotland [Salmonella spp. bacteria were successfully isolated from bird feeders, also in winter months. This study covered two areas. In the first area, with a history of deaths in wild birds from salmonellosis in previous years, S. ser. Typhimurium was isolated from 33%, 42% and 48% of the pooled fecal samples collected from different parts of bird feeders. In the second area, without a history of salmonellosis in previous years, S. ser. Typhimurium was found in only 2% of feces from one specific part of feeders. Therefore, Salmonella spp. shedding can differ greatly in bird populations living in different areas. It is also interesting that feces from different parts of bird feeders had different bacterial burdens. This could indicate that even small differences in conditions in areas of the feeder can influence the survival of bacteria.For these reasons, it is important to better understand in which places wild birds can become infected with  feeders ,19. It i feeders . It is tn Poland ,26, no en Poland . The epiScotland , SalmoneSalmonella spp. in the environment in bird feces [Salmonella spp. may be hard to find in some conditions at feeding sites. There is very little information about the intensity and time of shedding of naturally occurring bacteria in wild birds. In one study, wild bird feces (of herring gull) have been found to have concentrations of Salmonella spp. in a range from 22 MPN/g to 2.4 × 109 CFU/g [Salmonella spp. shedding intensity and time among different avian taxa. Due to these, we still do not know the exact mechanisms of shedding bacteria by wild birds of species usually visiting feeders. There are also very limited data about the survival of Salmonella spp. in wild bird feces. Some information has been obtained from studies on Canada geese. A 28-day trial conducted in parks in London, England, showed that bacteria from inoculated Canada goose droppings with 104–105 CFU/g of Salmonella spp. survived the 28-day trial, in conditions of heavy rain [Salmonella spp. in the feces of Canada geese is greater than the survival in feces of smaller bird species, observed in bird feeders. Their feces are smaller, with a greater relative surface area, and as a consequence, bacteria shed in them are more prone to desiccation. This hypothesis, however, has not yet been tested experimentally [There is a lack of detailed information about the duration and intensity of bacterial shedding by infected wild birds and the survival of rd feces . This ma09 CFU/g . Another09 CFU/g . It wortavy rain . It is, mentally .Salmonella spp. from bird feeders was not successful in our study could be the temperature in the study period. As commonly reported, the lowest temperature allowing Salmonella spp. survival is 4 °C [Salmonella spp. can survive at an even lower temperature [Salmonella spp. was probably not caused by the temperature in the study period we chose. Bird feeders are not commonly used in Poland during warmer times of the year (spring and summer). However, we do not exclude the possibility that conducting a Salmonella spp. survey in these seasons could produce different results.Another reason that the isolation of  is 4 °C . Howeverperature . Temperaperature . The isoA recent review and metanalysis highlighted that the current available literature is not sufficient to estimate the likelihood of enteric pathogen spillover from wild birds to humans, and the risk of such an event may be overestimated . Our stu


Ursolic and oleanolic acids are secondary plant metabolites that are known to be involved in the plant defence system against water loss and pathogens. Nowadays these triterpenoids are also regarded as potential pharmaceutical compounds and there is mounting experimental data that either purified compounds or triterpenoid-enriched plant extracts exert various beneficial effects, including anti-oxidative, anti-inflammatory and anticancer, on model systems of both human or animal origin. Some of those effects have been linked to the ability of ursolic and oleanolic acids to modulate intracellular antioxidant systems and also inflammation and cell death-related pathways. Therefore, our aim was to review current studies on the distribution of ursolic and oleanolic acids in plants, bioavailability and pharmacokinetic properties of these triterpenoids and their derivatives, and to discuss their neuroprotective effects in vitro and in vivo. The brain is an essential part of the human body, therefore neurological disorders such as neurodegenerative diseases , stroke/ischaemia and brain cancer usually have a significant effect on vital functions. Noteworthy, oxidative stress has been recognized as a hallmark of the pathogenesis of these disorders. Moreover, the elevated levels of reactive oxygen (ROS) and nitrogen (RNS) species are implicated in the progression of pathological neuro-inflammation . TherefoOver the last decades the attention to triterpene-enriched plant extracts and especially to purified triterpenoids as bioactive phytochemicals has been considerably increased ,6,7,8. PWe retrieved the literature from PubMed, Science Direct, MDPI, Web of Science and Google Scholar. Search terms comprised “ursolic/oleanolic acid”, “triterpene”, “triterpenoid”, “pentacyclic”, “extract”, “plant”, “neuroprotection“, “cancer”, “cell”, “glioma”, “glioblastoma”, “brain”, “ischaemia”, “human”, “rat”, “mouse”, “model”, “bioavailability”, “pharmacokinetics”, “metabolite” and “distribution”. The most recent studies in English with full text available and indexed in Clarivate Analytics Web of Science were included. We also used the reference lists of identified articles to find additional papers. The last search was performed on the 22 February 2021.Triterpenes constitute a significant portion of all phytochemicals, and more than 20,000 triterpenoids have been identified so far. Their biosynthesis is based on squalene cyclization, which involves a chair-chair-chair-boat transition state, and results in compounds, comprised of six isoprene units. Such structures are precursors to steroids in both plants and animals ,18.Ursolic acid (UA) (3β-hydroxyurs-12-en-28-oic acid) and oleanolic acid (OA) (3β-hydroxyolean-12-en-28-oic acid) are pentacyclic triterpenoids which have therapeutic potential . They arOlea europaea, and that plant currently is the main source of commercial OA preparations [The name OA is derived from the name of the plant species arations . OA co-oarations ,18,25,26arations ,28. For arations . UA possarations ,31.Olea europaea were found to accumulate different amounts of OA and UA (Olea europaea than in the bark: 31 mg/g dry weight (DW) of OA and 3.8 mg/g DW of UA. The bark of olive trees also contained substantial amounts of OA (up to 9.8 mg/g DW) [Sambucus nigra and in medicinal or aromatic herbs of the Lamiaceae family , and UA was detected only in dry leaves of Coffea arabica and Nerium oleander  [Paeonia lactiflora was 54.26–618.12 μg/g and 36.23–665.14 μg/g, respectively [The synthesis of OA and UA is characteristic of plants of Araliaceae, Asteraceae, Ericaceae, Lamiaceae, Myrtaceae, Oleaceae, Rosaceae, Rubiaceae, Saxifragaceae and Verbenaceae families. Woody or herbaceous plants that belong to these and some other families contain OA and UA in various parts ,18,25,26A and UA . Triterpmg/g DW) . In contmily see . Moreovectively) . It is wectively .Ocimum basilicum accumulated 3.6 times more OA in the flowers compared to the roots, and 1.8 to 3.5 times more in older leaves, than in the youngest leaves. Conversely, the level of UA was higher in the youngest leaves of Ocimum basilicum when compared to the older leaves. It was also determined that the concentration of UA was 2.7 times higher in the flowers than in the leaves of the plant [The quantity of triterpenes in the plant raw material may also depend on the age of the plant. It was shown that he plant .Calendula officinalis, which accumulated up to 20.5 mg/g DW of UA [Eriobotrya japonica were found to contain different amounts of OA and UA. For instance, dry sepals of E. japonica contained the highest amount of OA and UA (0.68 mg/g and 3.65 mg/g), while the petals had the lowest contents of OA and UA (0.12 mg/g and 0.60 mg/g) [As has been already mentioned, plant flowers are also a rich source of triterpenes. High levels of triterpenes, especially UA, were determined in flowers of DW of UA . Differe60 mg/g) .Malus domestica fruit on average had 9.4 mg/g, Eriobotrya japonica had 8.0 mg/g, Pyrus communis had 7.25 mg/g, Chaenomeles japonica had up to 5.7 mg/g and Prunus persica had up to 2.97 mg/g of UA [Diospyros kaki) fruits have higher content of OA than UA, 88.57 μg/g fresh weight (FW) and 27.64 μg/g FW, respectively [Vitis spp. and Vaccinium spp. can also be used as sources of triterpenes. Dried Vitis vinifera berries have been found to contain up to 79.0 mg/100 g of OA, meanwhile, dried cranberries or blueberries contained both OA and UA  [A large number of triterpenes is characteristic of the fruit peel of the plants in the Rosaceae family. UA and OA have been found to predominate other triterpenes in the cuticular wax of quince, loquat, pear, peach and apple fruits, and UA content was higher than that of OA . Sut et /g of UA . In addi/g of UA ,37. Note/g of UA . Unlike ectively . It is wctively) .Methods for the extraction of UA and OA from plants range from the conventional techniques of stirring, maceration and heat reflux extraction to microwave assisted extraction or ultrasound assisted extraction ,42. The Another critical parameter is the duration of the extraction of UA and OA. Classical methods such as extraction by stirring or maceration usually take 24 h, therefore faster extraction methods, such as microwave assisted extraction or ultrasound assisted extraction, have been developed. The application of microwaves enabled extraction of both UA and OA from the flowers of white dead nettle in 10–30 min, and the extraction efficiency reached 99–100%. Moreover, the efficiency of ultrasound assisted extraction reached 83–91% after 45 min  establisPharmacokinetic and cell membrane permeability studies are crucial in clinical development of new biologically active compounds with a view to understanding their behaviour in vivo and to establish an optimal dosage regimen. Pentacyclic triterpenoids generally suffer from low oral bioavailability , in partow 6.43 and 6.48, respectively [The primary factor affecting bioavailability is the physicochemical properties of the molecule . UA and ectively ) as wellectively . Therefoectively ,48,50. Iectively . Furtherectively ,54. Unfaectively  or incluectively  were invThe second key factor is the ability of the drug to overcome biological barriers . The eviDespite limited absorption, the recovery of intact OA and UA has been reported in animal ,64,65,66High lipophilicity predisposes triterpenoids to liver metabolism. A tissue distribution study in mice has demonstrated that while the concentration of UA in plasma was steadily decreasing, the concentration in the liver was increasing (5–240 min) . In addi50 values obtained by DPPH  radical scavenging potential  and ferric reducing antioxidant power (FRAP)  assays. Although the difference may arise from the distinction of hydrophilicity and the selectivity of the assays [Neurological disorders can be regarded as impairments of the brain or nervous system, resulting in either physical, psychological or both symptoms. Cerebral ischaemia or brain trauma are examples of acute conditions, while gradual memory loss, neurodegenerative diseases or dementia are associated with aging . Proper e assays , it stroe assays . Antioxie assays ,81,82, Se assays ,77,79,81e assays ,80,81,82e assays  and to ae assays ,84. The e assays ,84. Ex ve assays . OA has e assays , and it e assays ,86  content in a multiple sclerosis mouse model after administration for six weeks, as UA acted as an agonist of peroxisome proliferator activated receptor γ (PPARγ) ,93. It i2O2-reduced mitochondrial membrane potential was restored and the activity of succinate dehydrogenase (SDH) was improved [It is noteworthy that the underlying target of UA and OA might be mitochondria, organelles that are involved in both oxidative stress and neuro-inflammation . Recentlimproved . It is wimproved   sensitizers and used as chemotherapy agents in glioblastoma patients with low levels of caspase-8 or p53 activity. In another study [50 value was determined as 163.6 µg/mL (approx. 358 µM). In addition, OA-treated glioblastoma cells exhibited the increase in expression levels of proteins involved in MAPK signalling, and cell cycle arrest at G1 phase.Recently, in a number of reviews UA/OA-containing plant extracts, purified compounds and their natural or chemically synthesized derivatives have been reported to possess anti-tumour activity in skin, breast, lung, gastric, liver, intestine, prostate and pancreatic cancer models in vitro and in vivo with non-toxic or minimal inhibitory effect on normal cells ,113,114.er study , syntheter study . It was 50 value as low as 20 µM [Chamaenerion angustifolium at 50 µM concentration induced both apoptosis and necrosis in U-87 MG cells, and this effect was explained by UA interaction with the PI3K/Akt signalling pathway as predicted by molecular docking [Rosmarinus officinalis at 20 µM has reduced IL-1β or TNF-α-induced rat C6 glioma cell invasion in transwell chambers and inhibited the enzymatic activity and expression levels of MMP-9 via the blockage of the NF-kB-dependent pathway [Sarauja roxburghii at 100 µM concentration has also exhibited cytotoxicity against C6 cells in vitro [UA has been also found to exert a cytotoxic effect in human U-251 MG glioblastoma cells with an ICas 20 µM . The treas 20 µM . In otheas 20 µM  or at 40as 20 µM . However docking . UA  suppressed metabolic activity, decreased ATP content and increased the number of necrotic cells in both rat C6 glioma and primary mouse astrocyte cell cultures to the same extent . Thus, iIn conclusion, various parts of medicinal or aromatic plants, and also the pulps and peels of many fresh fruits, are sources of structurally isomeric pentacyclic triterpenoids UA and OA. Moreover, within the last several years UA/OA-enriched extracts, purified compounds and their synthetic derivatives have become subjects of increasing attention due to their potential anti-inflammatory and anticancer effects. Thus, the reviewed data suggest that neuroprotective activity of UA/OA and their derivatives could be associated with their ability to stimulate the cellular antioxidant defence systems and to downregulate the pro-inflammatory pathways in neurodegeneration or ischaemic brain damage models in vitro and in vivo. Moreover, UA/OA have also demonstrated anticancer activity, which might be related to the potency of these pentacyclic triterpenoids to activate or sensitize intracellular pathways leading to cell death, and to suppress tumour cell growth, proliferation and migration. However, further research is certainly needed to clarify the mechanism of action, since there is obvious variation in experimental data depending on the treatment regimen and applied models. In addition, the chemical modifications of UA and OA, as well as novel drug delivery systems, are subjects for future research aimed at improving the bioavailability and effectiveness of original compounds.


Firmicutes and Lactobacillus were most abundant on phylum and genus levels, respectively. The above results suggested that GA additive applied in WPS silage was an effective strategy to protect nutrition and improve fermentation quality, and the 1% GA addition showed a better effect.Whole plant soybean (WPS) is a kind of legume resource with characteristics of high nutrition, large biomass, and wide distribution. In the present study, we have investigated the feasibility and effects of gallic acid (GA) on WPS silage quality, nitrogen distribution, tannin content, and bacterial community. The 0.5 and 1% (fresh matter basis) GA were added into WPS for dynamic ensiling . The results showed that the WPS silage with GA addition significantly decreased pH value (6.16–5.38 at ensiling day 30), coliform bacteria count and butyric acid (65.3–62.0 g/kg dry matter at ensiling day 30), and amino nitrogen contents  and promoted lactic acid (9.62–31.5 g/kg dry matter at ensiling day 30), acetic acid (24.1–85.6 g/kg dry matter at ensiling day 30), and tannin  contents. Additionally, the GA addition also contributed to the change of bacterial community, where  Glycine max L. Merrill), one of the most valuable oilseed crops all over the world (10 CFU/g fresh matter (FM) for obtaining well-preserved silage , with the chemical structure of three phenolic hydroxyl groups and one carboxyl group, is also called 3,4,5-trihydroxybenzoic acid or gallo-tannin . Generalin vitro , and thuin vitro . Meanwhiin vitro . As Anurbroilers . To sum Therefore, WPS was ensiled with and without 0.5 and 1% GA for a fermentation period of 3, 7, 14, and 30 days, and then fermentation quality, nitrogen fraction, and bacterial communities of the WPS silage were evaluated.Whole plant soybean was planted without herbicides or fertilizers and harvested with an approximately homogeneous size. During the fruiting period, WPS was hand-collected from QILIN experimental field at the South China Agricultural University  in Guangzhou City, Guangdong Province, China. The samples were then immediately chopped into 2–3-cm lengths by the manual cutter . After homogenization, the triplicate samples of raw material were used for determining the chemical composition and microbial populations. And the ensiling treatments were conducted with or without 1 and 2% GA  on an FM basis. Following mixing WPS and GA thoroughly, about 110 g of silage materials were then packed into polyethylene bags and sealed immediately using a vacuum sealer . A total of 36 silage bags (4 ensiling stage × 3 treatments × 3 replicates) were kept at room temperature (25–30°C) and randomly sampled on days 3, 7, 14, and 30 of ensiling for analysis of fermentation parameters, protein fractions, and bacterial communities.The methods used in this part were similar to our previous study .–1 to 10–6. Yeast and mold counts were incubated and counted using Rose Bengal agar at 28°C for 72–120 h. Lactic acid bacteria (LAB) and coliform bacteria were cultured respectively on de Man, Rogosa, Sharpe (MRS) agar and Violet Red Bile agar at 30°C for 48 h. Another 20 g silage sample were mixed with 180ml distilled water and incubated overnight at 4°C, then one aliquot of the filtrate was used to measure pH value with pH meter .Hence, 20 g of individual silage sample were homogenized with distilled water (180 ml) in an orbital shaker at room temperature, and the supernatants were then serially diluted from 103-N) content was detected by the phenol-hypochlorite colorimetric method  (version 1.9.1) bioinformatic pipeline (version 1.8.2)2. The β-diversity was analyzed with principal component analysis (PCA). And the relative abundances of different bacterial communities at the phylum and genus levels were also analyzed.The WPS silages were sampled and extracted the total bacterial DNA with a DNA Kit  following the attached instructions. And specific conducted steps were similar to P < 0.05 level. All the figures in this paper were downloaded from Omicsmart online platform and further embellished by the software Adobe Illustrator CS 6.0.The effects of GA and ensiling days on the fermentation quality and chemical characteristics of WPS silage were analyzed with IBM SPSS 20.0 for Windows statistical software package. The results were evaluated using two-way analysis of variance (ANOVA), with Duncan’s multiple range tests. Statistical significance was determined at the 10 CFU/g FM, and the counts for yeasts, molds, and coliform bacteria were 5.02, 4.49, and 5.54 log10 CFU/g FM, respectively. The tannin content including total phenol, SP, and HT were 3.55, 1.44, and 1.61 g/kg DM, respectively.The chemical compositions and microbial population of fresh WPS prior to ensiling were listed in P < 0.05) with GA addition on ensiling days 3 and 30, also increased (P > 0.05) on days 7 and 14. The pH value of GA-treated silages was significantly (P < 0.05) decreased in whole ensiling stage. And ensiling days had a highly significant effect (P < 0.01) on pH decline. However, all treatments of WPS silage showed relatively high pH value (5.38–6.56). Meanwhile, GA addition significantly improved (P < 0.05) the LA and AA contents, while these contents significantly decreased (P < 0.05) with prolonged ensiling days (except for 1% GA treatment on AA content). The LAB count decreased significantly (P < 0.05) with GA addition, and the addition of GA also significantly decreased (P < 0.05) other microbes’ number . BA content was significantly decreased (P < 0.05) with GA addition but significantly increased (P < 0.05) with prolonged ensiling days. P < 0.05), while NPN and NH3-N contents significantly increased (P < 0.05). And the addition of GA decreased the NH3-N (P < 0.05) content of WPS silage. Furthermore, the tannin content including total phenol, SP, and HT all significantly (P < 0.05) increased with addition of GA. But with the prolonged ensiling days, their content all significantly (P < 0.05) decreased.The DM content, pH value, organic acids content, and microbial population of WPS dynamically ensiled with or without GA are presented in P < 0.05) with prolonged ensiling days. The result of unweighted principal coordinate analysis (PCoA) was shown in In this study, α-diversity of bacterial community of raw material and dynamically ensiled WPS was shown in Cyanobacteria (58.50%), Proteobacteria (31.44%), and Firmicutes (9.54%) were the top three phyla in the fresh WPS material, while Firmicutes (52.25%), Proteobacteria (31.92%), and Cyanobacteria (14.95%) were also top three dominant phyla in the whole WPS silages. And Pantoea (10.57%), Lactobacillus (6.46%), and Lactococcus (0.95%) were the most dominant genera in the fresh WPS material, but Lactobacillus (28.65%), Lactococcus (7.92%), and Weissella (2.88%) were the most dominant genera in all WPS silages. Cyanobacteria was the most abundant phylum in fresh material on phylum level. The relative abundance of Cyanobacteria decreased, while Firmicutes and Proteobacteria increased rapidly and became the dominant phyla with prolonged ensiling days. And the relative abundance of Cyanobacteria and Proteobacteria decreased while Firmicutes increased with GA addition. Moreover, Pantoea and Lactobacillus were the most dominant genera in fresh WPS. However, the relative abundance of Pantoea decreased  during the ensiling process. The most dominant genus in WPS silage was Lactobacillus, followed by Lactococcus, Weissella, Leuconostoc, Pediococcus, and Enterococcus. And the GA additive increased the abundance of Lactobacillus, while it decreased the abundance of Lactococcus and Weissella.The relative abundances of bacterial communities of fresh material and WPS silages at the phylum and genus levels were presented in Clostridium, which would result in the nutrition loss of effluent and spoilage during the ensiling process. In general, the WPS is nutrition abundant with high protein content and appropriate fiber content. Especially for high proportion of TP, it means that more valuable protein fraction will be higher efficient in utilization for livestock compared to NPN  for satisfactory silage . As McDod to NPN . Howeverd to NPN . The sufd to NPN . Moreovey silage . Howevery silage  and extey silage . To sum Clostridia or yeasts at the early stage of ensiling. P < 0.05) with GA addition. This might be caused by decreasing of cocci , which was less tolerant to low pH , which cannot be restricted in such a high pH condition. According to the above analysis, GA addition is helpful to improve fermentation quality of WPS silage.The DM content was improved with GA addition. It indicates GA had a positive effect on DM preservation. As o low pH . And theactivity . In this3-N  in all silages decreased with prolonged ensiling days from 3 to 60. This might ascribe to the relatively low pH value of Neolamarckia cadamba leaves silage , and it further decreased from ensiling days 3 to 60. Therefore, in this study, the GA and ensiling days had no inhibition effect on α-diversity of bacterial community mainly owing to the high pH value of the WPS silage.In order to detect the microbial community’s compositions and abundance  or monitstridium . And theThe result of unweighted PCoA could reflect the distinction of the bacterial community of each treatment. Especially, a clear separation between GA-treated group and CK group, it suggested that GA had a significant influence on bacterial community. Moreover, a higher extent of separation was formed between fresh material and WPS silages. In the present study, the variational composition of dominant phylum and genus between fresh WPS material and WPS silage in the whole ensiling period indicated that the composition and abundance of bacterial community were much altered before and after ensiling. Same results were also found by Cyanobacteria was the most abundant phylum in fresh material. Cyanobacteria decreased, while Firmicutes and Proteobacteria increased rapidly and became the dominant phyla; these even constituted more than 90% of relative abundance on ensiling day 30. Firmicutes and Proteobacteria were the most dominant phyla and occupied up to 99% of the total relative abundance at the later period of barley silages. Moreover, the relative abundance of phyla between GA-treated group and CK group was much different and which was dramatically altered by the ensiling days. The relative abundance of Cyanobacteria and Proteobacteria decreased, while Firmicutes increased with GA addition. It was indicated that the bacterial community of WPS silage was remarkably altered by the addition of GA. The study of Cyanobacteria, a photosynthesizing phylum of bacteria could exist in diverse environments and mainly be researched on the biotechnical and pharmaceutical industries, such as biofuels or fertilizer generation. In this study, with the effect of GA addition and ensiling days, the dominance of Firmicutes and Proteobacteria on the phylum level might be because of the relative low pH of WPS silages and anaerobic conditions, which were beneficial to the growth of Firmicutes and Proteobacteria. The genus-level bacterial communities of WPS silages are also shown in Pantoea and Lactobacillus were the most dominant genera in fresh WPS. However, the relative abundance of Pantoea decreased during the ensiling process. Pantoea had the ability to reduce NH3-N content and pH value in alfalfa silage. But the specific function of Pantoea needs to be extensively investigated in various types of forage silage. Furthermore, the most dominant genus in WPS silage was Lactobacillus, followed by Lactococcus, Weissella, Leuconostoc, Pediococcus, and Enterococcus. And the GA additive increased the abundance of Lactobacillus, while it decreased the abundance of Lactococcus and Weissella. Generally, Lactobacillus, Weissella, Enterococcus, and Lactococcus are the most commonly used as lactate-producing bacteria in silage and mainly used to occupy the dominant microbe position at the early stage of ensiling and ensure well fermentation quality  due to organic acid accumulation  in all WPS silages might be due to the abundant Weissella. Enterococcus is also applied in grass silage as LAB inoculation, it could reduce ammonia N content of silage and directly affect ruminal fermentation by improving ruminal microbial biomass production . Pediococcus is often found living in association with plant material, dairy products, and foods produced by LAB  contents were increased with addition of GA. Meanwhile, with prolonged ensiling days, the pH value, microbe number, and LA, AA, TP, and tannin contents all decreased, whereas BA, NPN, and NH3-N contents increased. The GA also contributes to the change of bacterial diversity, where Firmicutes and Lactobacillus were most abundant on phylum and genus levels, respectively. The above results suggested that GA additive applied in WPS silage was an effective strategy to protect forage nutrition and improve silage quality, and the 1% GA addition showed a better effect.This present study showed that pH value, coliform bacteria count, and BA and NHThe original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.CW contributed to the investigation, software, data curation, formal analysis, and writing the original draft. MZ and XZ contributed to the investigation, methodology, visualization, and validation. SW contributed to the investigation and methodology. XZ contributed to the investigation, methodology, visualization, and validation. XC contributed to the conceptualization, funding acquisition, project administration, resources and validation. LG contributed to the materials, investigation, funding acquisition and methodology. QZ contributed to the conceptualization, data curation, project administration, supervision, and validation. All authors contributed to the article and approved the submitted version.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.