Glaesserella parasuis is the causative agent of Glässer’s disease in pigs. Serotyping is the most common method used to type G. parasuis isolates. However, the high number of non-typables (NT) and low discriminatory power make serotyping problematic. In this study, 218 field clinical isolates and 15 G. parasuis reference strains were whole-genome sequenced (WGS). Multilocus sequence types (MLST), serotypes, core-genome phylogeny, antimicrobial resistance (AMR) genes, and putative virulence gene information was extracted.vtaA virulence genes were observed in most isolates (97.2%), except for serotype 8 (ST299 and ST406), 15 (ST408 and ST552) and NT (ST448). A few group 1 vtaA genes were significantly associated with certain serotypes or STs. The putative virulence gene lsgB, was detected in 8.3% of the isolates which were predominantly of serotype 5/12. While most isolates carried the bcr, ksgA, and bacA genes, the following antimicrobial resistant genes were detected in lower frequency;  blaZ (6.9%), tetM (3.7%), spc (3.7%), tetB (2.8%), bla-ROB-1 (1.8%), ermA (1.8%), strA (1.4%), qnrB (0.5%), and aph3''Ia (0.5%).  In silico WGS serotyping identified 11 of 15 serotypes. The most frequently detected serotypes were 7, 13, 4, and 2. MLST identified 72 sequence types (STs), of which 66 were novel. The most predominant ST was ST454. Core-genome phylogeny depicted 3 primary lineages , with LIIIA sublineage isolates lacking all vtaA genes, based on the structure of the phylogenetic tree and the number of virulence genes. At least one group 1 G. parasuis isolates and can be considered an alternative to the more labor-intensive and traditional serotyping and standard MLST. Core-genome phylogeny provided the best strain discrimination. These findings will lead to a better understanding of the molecular epidemiology and virulence in G. parasuis that can be applied to the future development of diagnostic tools, autogenous vaccines, evaluation of antibiotic use, prevention, and disease control.This study showed the use of WGS to type The online version contains supplementary material available at 10.1186/s12917-023-03698-x. Glaesserella parasuis (G. parasuis) is a Gram-negative bacterium that causes Glässer’s disease in pigs. This disease is characterized by arthritis, meningitis, and polyserositis, commonly observed in 4 to 8-week-old pigs, but it can also sporadically occur in older pigs [G. parasuis increased substantially in swine cases received at the Iowa State University Veterinary Diagnostic Laboratory (ISU-VDL) [der pigs . Clinicader pigs , 3. Whilder pigs , it contG. parasuis strains varies quite significantly, from highly pathogenic, such as the well-described Nagasaki strain (serotype 5 ST24), to non-pathogenic strains, such as strains of serotype 3, 6, 9, and 11 [The bacterium is considered endemic in all swine populations, colonizing the upper respiratory tract of pigs. Since the pathogenicity of serotype  ST24, toserotype  ST24, to, and 11 . The maj, and 11 , 9 is an, and 11 –15. Some, and 11 . AlthougG. parasuis. The most important virulence factors described for G. parasuis are the virulence-associated trimeric autotransporter (vtaA) genes [vtaA genes are associated with virulent isolates [Pathotyping is also available for A) genes . There iisolates , 18. Whiisolates –22 have G. parasuis strains circulating in North American pig populations is lacking. The objective of this study was to perform genetic characterization of G. parasuis strains from disease-associated cases using WGS. In-depth characterization using WGS could vastly improve on farm control and prevention programs.The reduction in sequencing costs and advances in bioinformatic pipelines have made whole genome sequencing (WGS) more feasible for diagnostic use. WGS offers higher discrimination between isolates by allowing the extraction of relevant information related to phylogeny, sequence type, putative virulence factors, and antimicrobial resistance (AMR) genes. Current knowledge about the molecular epidemiology of disease-associated n = 108) of the study isolates. LI and LII contained 13.8% (n = 30) and 36.2% (n = 79) of the isolates, respectively. Each lineage contained sub-lineages with only LIIIA sublineage being highlighted due to its unique characteristics  (threshold = 0.1) based on the structure of the phylogenetic tree and the number of virulence genes , in silico whole-genome serotyping pipeline identified 11 of the 15 known G. parasuis serotypes . In addition, LI and LII isolates were significantly associated with respiratory and systemic isolates, respectively (p < 0.05). A total of 80 isolates were isolated from tissues for which histopathological data was available. Of these, 8 did not have detectable lesions and were of serotypes 4, 7 and 13 with majority of pigs having lesions in other sites in which Streptococcus suis isolated. Among the 72 remaining isolates, the following lesions and serotypes were detected: polyserositis (n = 27) bronchopneumonia/pneumonia (n = 26) epicarditis/myocarditis (n = 9); meningitis/encephalitis (n = 3) arthritis/synovitis (n = 4), pleuritis (n = 2) splenitis (n = 1) , CC478 , CC413 , CC417 , CC56 , CC92 , CC452 , CC442 , CC438 , CC245 ,), CC96 , CC433 , and CC246 . The most common CCs were CC157 and CC478 with CC157 comprising of STs, ST416, ST454, ST458, ST462, ST526, and ST157. CC478 was the second most frequently detected clone containing ST459, ST478, and ST624 . However, ST6 strains were also from serotypes 13, ST454 of serotypes 4, 13 and 14, and ST478 of serotypes 7. A high ST diversity within serotypes was observed. For example, within serotype 7 strains, a total of 19 different STs were identified. Similar findings were observed with serotype 13 and 2. The untypable strains were of different sequence types.Through multilocus sequence typing analysis, we uncovered a total of 72 sequence types (STs) within the collection of 218 field isolates. Remarkably, among these, 66 STs were entirely new discoveries, representing 91.7% of the total STs identified, with ST454 emerging as the most commonly encountered one Fig. . The SiminfB locus. Within LI and LII, ST6  and ST454  were the dominant STs, respectively, while LIII showed high diversity with 49 different STs. All six isolates in the LIII A sublineage , and ST454 and ST478 were significantly associated with LII (p < 0.05). From cases with histopathology data, most ST454 and ST478 isolates originated from polyserositis cases.Within the dataset, the three dominant STs were ST454 19.3%, 42/218), ST478 , and ST6 . The remaining STs were each represented by ≤ 7 isolates, and 33/72 STs were represented by a single isolate. The two novel STs (454 and 478) are single locus variants (SLVs) of each other at the /218, ST4vtaA genes. Within group 1 vtaA genes, vtaA5 , vtaA6 , vtaA3 , vtaA8 , and vtaA2  were the most frequently detected , 53.2% (116/218), 43.1% (94/218) and 30.7% (67/218) presence, respectively. On average, LI isolates carried 6.8  group 1 vtaA genes, LII isolates carried on average 6.2 (min: 4 max: 9) group 1 vtaA genes, and LIII isolates carried an average 5.02  group 1 vtaA genes, excluding LIIIA. Sublineage LIIIA isolates lacked all vtaA genes . VtaA1, vtaA4, and vtaA7 were significantly associated with LI, and vtaA6 was associated with LIII.A total of 212 (97.2%) isolates possessed at least one of the group 1 vtaA genes and were of serotypes 7 ST454, ST624, and ST632, serotype 5/12 ST6, ST427, and ST474, serotype 2 ST478 and ST548, serotype 4 ST549 and ST629, serotype 13 ST6, serotype 14 ST442, and NT ST427. From these 23 isolates, six also carried all the group 2 and 3 vtaA genes, and were of serotype 7 ST454 and ST624 and serotype 14 ST442. Some isolates represented by the same ST, carried a varied number of group 1 vtaA genes  and vtaA11 (23.4% 51/218). Finally, within group 3 vtaA genes, vtaA12 was found in 56.4% (123/218) and vtaA13 in 61.5% of isolates, respectively (134/218).Group 2 vtaA1 was associated with serotype 5/12, vtaA2 and vtaA7 were associated with serotype 2, and vtaA5, vtaA6 and vtaA8 were associated with serotype 7 (p < 0.05). Among the dominant STs, vtaA1, vtaA4, vtaA7, and vtaA9 were associated with ST6, and vtaA8 and vtaA9 were associated with ST454 (p < 0.05).Within serotypes, ompP5 gene, and 56.9% (124/218) of the isolates were also positive for the porin protein gene ompP2. Fimbrial genes  and siaB were present in 54.6% (119/218), 69.3% (151/218), 51.8% (113/218), and 84.4% (184/218) isolates, respectively. Among the monomeric autotransporter genes, bmaA5 , bmaA4 , and bmaA6 (51.3% (112/218), were the most prevalent while bmaA1  was the least prevalent. The lsgB gene was the least prevalent gene among the selected putative virulence genes, with a proportion of only 8.3% (18/218) of isolates being positive; all isolates in LIII lacked this gene. Almost all lsgB positive isolates (15/18) were part of L1 -Ib were detected in almost all isolates included in the collection. The remaining genes were present at the following proportions; blaZ , spc , tetB , bla-ROB-1 , ermA , strA ), qnrB , and aph3''Ia .Fourteen AMR genes were detected among the 218 genomes, encoding resistance to antibiotics of nine different classes including 2,5-diketopiperazines, aminoglycosides, beta-lactams, liconsamides, macrolides, lincosamide streptogramins B (MLS), polypeptides, quinolones, sulfonamides, and tetracyclines. Among these, G. parasuis isolates was further analyzed geographically. A total of 76% of isolates (19/25) from Mexico, 48% from the USA (74/154), and 35% from Canada (7/20) were grouped into LIII; 20% (5/25), 37% (57/154), and 30% (6/20) of the isolates from Mexico, USA, and Canada, respectively, clustered in LII. Finally, 4% (1/25), 14.2% (22/154), and 35% (7/20) of Mexico, the USA, and Canada isolates, respectively, were grouped into LI. A few isolates from Chile, Peru, and Denmark clustered in specific lineages  and systemic strains (n = 89) accounted for 80.3% of the isolates. The remaining were from URT (n = 8) or unknown (n = 35). Generally, there was no observed tissue-specific distribution within the lineages. In several isolates (n = 80) from tissues with reported histopathological lesions, serotypes associated with disease were 7, 13, 4, 5/12 and 2.The distribution of G. parasuis continues to challenge the productivity, health, and well-being of post-weaning pig populations, and represents a driver of antimicrobial use on farms [G. parasuis requires a multifaceted approach based on the judicious use of antimicrobials, vaccination, minimizing viral co-infections and environmental triggers, improving husbandry and characterization of the disease-causing strains. Whole-genome sequencing analysis provides multiple layers of information that aid in determining the clinical relevance of the isolates and providing epidemiological insight.on farms . EffectiG. parasuis isolates predominantly from North America. The size and GC content were within range with other published G. Parasuis genomes [G. parasuis isolates clustering into two main primary lineages comprised of STs not found in the present study, highlighting the high genetic variation found within the species.In this study, WGS was performed on 218  genomes . Phyloge genomes  that shoWhole-genome in silico serotyping revealed 11 of the 15 known serotypes, with serotypes 7, 13, 4, 2, and 5/12, as the most common serotypes detected. The WGS in silico serotyping scheme was based on the DNA sequence fragment for each serotype published in Howell et al.. The schThe most frequently detected serotypes observed in this study have been previously associated with disease and are commonly detected from clinical cases , 27, 28.eBurst analysis showed that ST454 belonged to the CC157 with its predicted founder ST157, a sequence type first identified in Brazil from a pig with bronchopneumonia (https://pubmlst.org/organisms/glaesserella-parasuis). Surprisingly, ST157 was among the least prevalent STs in this study. In contrast, a clonal complex’s ancestral ST (founder) is typically the most prevalent ST in a population due to the fitness advantage or random genetic drift [Multilocus sequence typing identified 72 sequence types within 218 isolates, of which 91.7% (66/72) were novel. Specifically, this study highlighted the emergence of ST454, a strain detected in outbreaks of high mortality, characterized by per acute disease and polyserositis. The ic drift . It is leBurst analysis revealed that almost half of the isolates had singleton STs, suggesting high heterogeneity and instability in the population structure, as previously described [. identified 122 STs within 150 strains (17), Turni et al., identified 54 STs (41 novel) within 75 isolates from Australia [G. parasuis disease burden for a significant number of production systems. While the intensity of sampling varied across production systems, multiple flows had at least 2 STs  carried between 4 and 9 group 1 vtaA genes. This shows how variable vtaA carriage can be even between strains showing high similarity at the core -genome level. The lack of detection of group 3 vtaA genes 12 and 13 in 44% and 33% of isolates, respectively, was an unexpected finding since previous studies have identified these genes among all G. parasuis strains previously examined but this was based on the translocator domain PCR (17). Some VtaA12 and vtaA13 might be divergent and therefore not identified as vtaA12 or vtaA13 homologs. In this study, genes were only reported if their sequence identity was above 70% and the gene coverage was above 50%. Previous studies have shown divergence within these genes which could have contributed to a lack of detection given our stringent definition for gene reporting .Among the virulence-associated genes, group 1  disease , 35, 36.research , 36, thiage Fig.  lacked afindings . VariabiG. parasuis pathogenesis [bmaA4, bmaA5, bmaA6, ompP2, ompP5, pilA, pilB, pilC and siaB in the current study. In fact, even isolates that were negative for all vtaA genes were positive for some of these, such as ompP5 and siaB. This may suggest that some of these putative virulence genes are conserved in both virulent and nonvirulent strains and may not be good predictors of virulence compared to group 1 vtaA genes. However, this notion should be interpreted with caution since the study isolates were predominantly from clinical cases. lsgB, which has been associated with virulent strains [Monomeric autotransporters, porin proteins, and fimbria are involved in ogenesis , 37, 38. strains , was detbcr (bicyclomycin resistance), ksgA (kasugamycin resistance), bacA (bacitracin resistance), sul2 -Ib (streptomycin resistance) were detected in almost 100% of the isolates. Consistent with this study, bcr, ksgA, and bacA genes were highly prevalent in G. parasuis isolates in a recent study [G. parasuis strains (n = 908) as mostly susceptible to ampicillin, ceftiofur, enrofloxacin, florfenicol, tiamulin and tilmicosin (https://vetmed.iastate.edu/sites/default/files/VDL/pdf/Susceptibility-Summary-Porcine-2021.pdf). The remaining genes; aph3’’Ia, strA, blaROB-1, blaZ, spc, ermA, tetB, tetM and qnrB were detected in lower frequencies. Some studies showed varying frequencies of these genes in G. parasuis [blaZ and tetM, respectively, were in LIII, and were of serotypes 1 ST417 and ST420, 2 ST476, 4 ST418, 7 ST471 and ST473, 13 ST415 and ST421, and NT ST415. Further studies are needed to correlate if the genotypic antimicrobial resistance profile can predict phenotypic resistance in this species.Control and prevention of Glässer’s disease often rely on the strategic use of antimicrobials. Thus, detection of antimicrobial resistance genes could provide value in antimicrobial use decision-making. Among the genes, nt study . Some ofnt study . Furtherparasuis , 41. DueG. parasuis isolates included in the current study were from disease diagnosis cases submitted to the ISU VDL, and thus only captured production systems that submit samples to this laboratory. However, the contributing production systems represented the top pork producers in North America.Although the findings of this study were consistent with the published literature to date, caution is needed when interpreting the results. This is because the G. parasuis strain variation. Results herein will lead to a better understanding of the molecular epidemiology and virulence potential in G. parasuis that can be applied to the development of improved diagnostic tools, evaluation of antibiotic use, tracking of outbreak strains and identification of vaccine candidates for improved management of Glässer’s disease in swine production systems.While WGS is computational demanding, requires high technical skill and is currently more expensive than other typing tools, the value obtained with the information far exceeds these limitations. Still, it is expected that serotyping and virulence marker PCRs will continue to be used to monitor G. parasuis isolates were obtained from porcine cases submitted to the Iowa State University Veterinary Diagnostic Laboratory (ISU VDL) from 2015 through 2022 , they were also obtained from Canada (n = 20), Chile (n = 9), Denmark (n = 4), Peru (n = 6), and Mexico (n = 25).  for definitive species identification prior to sequencing.In addition, a total of 15 reference strains were provided by Dr. Marcelo Gottschalk, each representing one of the 15 G. parasuis from samples was achieved by plating clinical samples onto 5% sheep blood  with Staphylococcus hyicus nurse streak and incubating overnight at 370C. Presumptive G. parasuis isolates were subcultured onto chocolate agar (Thermo scientific™), confirmed using MALDI-TOF MS, and frozen at -800C in brain heart infusion broth with 30% glycerol. Two days before submission for sequencing, bacterial strains were retrieved from the freezer and streaked onto chocolate agar. An overnight pure growth culture lawn was inoculated into 2 mL phosphate-buffered saline (PBS). Genomic DNA was extracted using ChargeSwitch gDNA mini bacterial kit  according to the manufacturer’s instructions [Initial isolation of ructions .The DNA quality (A280/A260) was assessed using a Nanodrop (Thermo scientific™) and quantified using a Qubit fluorometer dsDNA HS kit (Life Technologies). Multiplex genome libraries were prepared using the Nextera XT DNA library preparation kit . The genomic library was quantified using a Qubit fluorometer dsDNA HS kit  and normalized to the recommended amplification concentrations. The pooled libraries were sequenced on an Illumina Miseq sequencer using Miseq Reagent V3 for 600 cycles . Raw reads were demultiplexed automatically on the Miseq.http://www.bioinformatics.babraham.ac.uk/projects/fastqc). Pre-processed reads were assembled using SPAdes Genome Assembler version 3.11.1-Linus [Raw sequence reads were pre-processed using Trimmomatic VO.36  to remov.1-Linus  using asG. parasuis isolates were identified by running kSNP3 with standard mode. The optimal k-mers size was calculated by the Kchooser program, and the whole-genome phylogeny was analyzed based on identified core genome SNPs [Virulence-associated genes in each genome were detected using SRST2  against ome SNPs .G. parasuis pubMLST typing platform [G. parasuis typing database. Novel ST were added to the pubMLST G. parasuis database with their corresponding metadata. Determination of clonal complexes was done using the goeburst algorithm [http://www.phyloviz.net), using the stringent six of 7 alleles identical with the founder ST [G. parasuis dataset and the study isolates were subjected to the goeBURST analysis to determine the study isolates' evolutionary descent and clonal complexes. A clonal complex (CC) was defined if it contained at least three STs, including the founder ST. Sequence types that did not meet this criterion were considered singleton STs.Multilocus sequence typing was performed using SRST2 with an integrated MLST database and definitions. Sequence types (STs) were further confirmed by querying the assembled fasta files using the platform , 47. Asslgorithm , a refinlgorithm , implemeunder ST . The gloA core-genome single nucleotide polymorphism (SNP) phylogenetic tree was generated by Parsnp V1.2  and visuG. parasuis serotypes, STs, AMR, and virulence genes were performed in Microsoft Excel® and R .Data management, descriptive, and inferential analyses of Simpson's index of diversity  was usedvtaA genes (1 – 9) versus tissue of isolation, and ST/serotypes versus lineages and vtaA genes (1 – 9) were evaluated using logistic regression. To account for the statistical model variation of logistic regression, only serotypes and STs containing at least ten isolates, representing at least 10% of the isolates, were selected for association analyses, which included serotypes 7, 13, 4, 5/12, 2, and NT, and STs 6, 454 and 478. Using Poisson Regression, the total number of Group 1 VtaA genes (1—9) was compared across serotypes (or ST).Tissues of isolation, when reported, were classified as lung, upper respiratory tract (URT), systemic tissues or unknown. Likewise, reported lesions were categorized by affected body system, including nervous, respiratory, systemic, and unknown.Associations between serotype/ST, lineages, Additional file 1.Additional file 2.Additional file 3.


APOE and sex interact to modulate Alzheimer’s disease (AD) risk, however the underlying pathways are unclear. One way that AD risk factors may modulate cognition is by impacting amyloid beta (Aβ) accumulation as plaques, and/or neuroinflammation Therefore, the goal of the present study was to evaluate the extent to which age, APOE and sex modulate Aβ pathology, neuroinflammation and behavior in vivo. To achieve this goal, we utilized the EFAD mice, which express human APOE3 or APOE4 and have five familial AD mutations (FAD) that result in Aβ42 overproduction. We assessed Aβ levels, reactive glia and Morris water maze performance in 6-, 10-, 14-, and 18-month-old EFAD mice. Female APOE4 mice had the highest Aβ deposition, fibrillar amyloid deposits and neuroinflammation as well as earlier behavior deficits. Interestingly, we found that female APOE3 mice and male APOE4 mice had similar levels of pathology. Collectively our data support that the combination of APOE4 and female sex is the most detrimental combination for AD, and that at older ages, female sex may be equivalent to APOE4 genotype.Increasing evidence supports that age,  Importato APOE3 , and APOAD onset . FemalesAD onset . Importacarriers , which mcarriers . Furtherto males . TherefoAPOE4 is associated with greater levels of Aβ  accumulation as plaques, and/or neuroinflammation. In fact, ls of Aβ  and highls of Aβ  comparedls of Aβ  and dysrls of Aβ . These hOE3 mice , 2022. TAPOE and sex interact with age to impact Aβ levels, neuroinflammation, neuronal density and behavior in vivo. To this end, we used EFAD mice as they express human APOE3 (E3FAD) or APOE4 (E4FAD) and overproduce Aβ42. Pathology and behavior were assessed in male and female EFAD mice at 6-, 10-, 14-, and 18-months-of-age using biochemistry, immunohistochemistry, and the Morris water maze test.The goal of our study was to understand the extent that 2.2.1.APOE. Four groups of EFAD (5xFAD+/−/human APOE+/+) mice were used: male E3FAD, female E3FAD, male E4FAD, and female E4FAD mice as described previously , one was used for biochemical and immunohistochemical analysis and the second for behavior analysis using Morris water maze.All experiments follow the University of Illinois at Chicago Animal Care Committee protocols. EFAD mice express five familial AD (FAD) mutations and human eviously . Mice fr2.2.Mice were anesthetized via intraperitoneal injection with ketamine (100 mg/kg) and xylazine (5 mg/kg) and perfused transcardially with ~40 mL phosphate buffered saline with protease inhibitor cocktail. Then, the brains were removed and dissected at the midline to produce two hemi-brains, one each for immunohistochemical and biochemical analysis . The hem2.3.APOE genotype, and sex. Serial sagittal sections  from EFAD mice were stained for NeuN and neuron numbers quantification in layer 5 of the somatosensory cortex, was performed by a design-based stereology system  using an optical fractionator applying the Nv × Vref method  from EFAD mice were immunostained for Aβ deposition, astrogliosis or microgliosis  and staif method .2.4.g, 1 h at 4°C), and the formic acid-soluble fraction was neutralized (with 20 volumes of 1 M Tris base), aliquoted, and frozen at −80°C . In the week prior to sacrifice, mouse behavior was tested using a modified Morris water maze protocol with acquisition trials consisting of 4 × 1 min trials/day for 5 consecutive days with latency to the platform recorded for each trial. A single probe trial was run on day 6 with the platform removed, and the readouts included latency to platform area, platform area crosses and latency to target quadrant .2.6.APOE and sex in SPSS . All other statistical analyses were conducted using univariate general linear models for between subjects’ effects with the independent variables  and their interactions, followed by Bonferroni’s post hoc tests in SPSS. For all statistical tests, p < 0.05 was considered significant. All data are presented as scatter plots with the mean and standard error of mean (SEM). The n size range is indicated in Figure legends. Outliers were excluded using the ROUT method (Q = 5%). All data, detailed n sizes and statistical analysis are provided in 3.APOE and sex modulates Aβ pathology, neuroinflammation and behavior in vivo. To this end, we used EFAD mice that overproduce Aβ42 (via 5xFAD mutations) and express human APOE3 (E3FAD) or APOE4 (E4FAD). EFAD mice were developed to identify how APOE modulates progression of AD-relevant pathology and behavior , middle-aged (14 months) and old (18 months) EFAD mice.The goal of our study was to determine the extent to which age, behavior . Previouto APOE3 , 2022, aOE4 mice . Therefo3.1.APOE4 is associated with higher amyloid deposits in human and in vivo, the extent that APOE and sex modulate extracellular Aβ with age is unclear. Therefore, we initially evaluated cortical extracellular Aβ levels .Extracellular Aβ in fibrillar amyloid deposits are used to diagnose AD and are a current therapeutic target . Althougposition ,C. This position ,C. ThereAD males , howeverAD males . We alsoAD males . In geneAPOE have a strong effect on extracellular Aβ deposition. That interaction results in highest fibrillar amyloid deposits in female APOE4 carriers, equivalent levels in female APOE3 and male APOE4 carriers, and lowest levels in male APOE3 carriers at older ages.Collectively these data support that female sex and 3.2.APOE4 is associated with higher inflammatory responses in younger EFAD mice . However, at 10-and 14-months of age, female E4FAD mice did not improve in performance from day 1 to day 5 . Collectively, our data support that E4FAD females demonstrated earlier behavioral deficits and loss of neurons at 6–10 months of age, compared to the other groups.We next assessed neuronal density (NeuN) in layer 5 of somatosensory cortex in male and female, E3FAD and E4FAD mice . Similar4.4.1.APOE has a pronounced impact on Aβ deposition. Aβ levels are higher with APOE4 in human  in the brain . In geneAPOE4 . HoweverFAD mice . This seung mice , 2022. W4.3.APOE4, compared to APOE3 carriers (APOE4, compared to APOE3 in young (APOE and sex in Morris water maze performance and found that female E4FAD mice had earlier behavioral deficits during acquisition phase of Morris water maze compared to other groups (APOE3 female and APOE4 male had similar behavioral deficits with age.AD is typically characterized by memory loss, and a decline in overall cognitive function . Severalcarriers . This efin young  and old in young  mice. Inin young  and is min young , also rer groups . Consist4.4.APOE, sex and age impacted all aspects of AD pathology and behavior. Future studies could incorporate more detailed evaluation of Aβ production, clearance, and degradation pathways, the full neuroinflammatory phenotype, other AD-prevalent pathologies such as vascular function and metabolism, neuronal function, and a full battery of behavioral tests in the cognitive domain. Furthermore, it is important to validate our findings in other models that incorporate human APOE and human APP. For example, human APP knock in mice have been crossed with APOE targeted replacement mice, and data supports greater behavioral deficits and Aβ pathology with APOE4 (APOE2 genotype which is protective for AD risk is important, with the caveat that these mice have Type III hyperlipoproteinemia (APOE4 and female sex impact AD pathology and cognition. For example, structural differences between apoE isoforms may modulate Aβ levels, glial reactivity, and neuron function [reviewed in APOE3/4 genotype on AD pathology and the interaction with sex? Does the impact of sex and APOE on pathology modify responses to therapeutic treatments?There are some limitations due to the nature of our design. One issue is that we are limited in the extent that we can conclude th APOE4 . In additeinemia . Our stu4.5.APOE4 result in the greatest levels of Aβ pathology, neuroinflammation and behavioral impairments. In addition, we propose that the effect of female sex is analogous to the presence of APOE4 for AD pathology. Through reviewing previous published in vivo and human data, we believe that this important concept may have been overlooked (APOE4 equivalency could therefore impact the optimal treatment window for therapeutics aimed at preventing AD through targeting Aβ and/or neuroinflammation.Our data support that the combination of female sex and erlooked . The femThe original contributions presented in the study are included in the article/The animal study was approved by University of Illinois Animal Care Committee. The study was conducted in accordance with the local legislation and institutional requirements.DB: Writing – original draft, Writing – review & editing, Data curation, Formal analysis, Investigation, Project administration, Supervision. AV-O: Data curation, Investigation, Supervision, Writing – review & editing. ZI: Writing – review & editing, Methodology. CM: Writing – review & editing, Methodology. AH: Writing – review & editing, Methodology. TP: Writing – review & editing, Methodology. JY: Methodology, Writing – review & editing. ML: Conceptualization, Funding acquisition, Project administration, Supervision. LT: Writing – review & editing, Writing – original draft.


Introduction: Fanconi anemia (FA) is a genome instability condition that drives somatic mosaicism in up to 25% of all patients, a phenomenon now acknowledged as a good prognostic factor. Herein, we describe the case of P1, a FA proband carrying a splicing variant, molecularly compensated by a de novo insertion.Methods and Results: Targeted next-generation sequencing on P1’s peripheral blood DNA detected the known FANCA c.2778 + 83C > G intronic mutation and suggested the presence of a large deletion on the other allele, which was then assessed by MLPA and RT-PCR. To determine the c.2778 + 83C > G splicing effect, we performed a RT-PCR on P1’s lymphoblastoid cell line (LCL) and on the LCL of another patient (P2) carrying the same variant. Although we confirmed the expected alternative spliced form with a partial intronic retention in P2, we detected no aberrant products in P1’s sample. Sequencing of P1’s LCL DNA allowed identification of the de novo c.2778 + 86insT variant, predicted to compensate 2778 + 83C > G impact. Albeit not found in P1’s bone marrow (BM) DNA, c.2778 + 86insT was detected in a second P1’s LCL established afterward, suggesting its occurrence at a low level in vivo. Minigene assay recapitulated the c.2778 + 83C > G effect on splicing and the compensatory role of c.2778 + 86insT in re-establishing the physiological mechanism. Accordingly, P1’s LCL under mitomycin C selection preserved the FA pathway activity in terms of FANCD2 monoubiquitination and cell survival.Discussion: Our findings prove the role of c.2778 + 86insT as a second-site variant capable of rescuing c.2778 + 83C > G pathogenicity in vitro, which might contribute to a slow hematopoietic deterioration and a mild hematologic evolution. Fanconi anemia (FA) is a rare genetic syndrome due to mutations in one of at least 22 genes, whose encoded proteins exert a consolidate role in genome integrity maintenance. Upon interstrand crosslinks (ICLs) impairing replication-fork progression, a nuclear complex of seven FA proteins (FANCore) activates FANCD2 through monoubiquitination, allowing its binding to the lesion and recruiting further players for DNA repair . ConsistGenetic instability is responsible for the main clinical features of FA, such as bone marrow (BM) failure, within the first decade of life in 80% of cases, and heightened predisposition to hematological and solid malignancies with a 30% and 40% incidence in the adulthood, respectively . NeverthSomatic mosaicism, indeed, occurs in a considerable subset of FA subjects, estimated at around 15%–25% of all cases . It may In the standard clinical routine, FA mosaicism is first recognized via the DEB test on cultured T lymphocytes, unveiling a population resistant to clastogenic concentrations typically toxic to FA cells . OptimizFANCA allele characterized by a splicing variant, whose deleterious effect is corrected by a de novo mutation event at least in vitro.Within this framework, we present a young FA patient carrying a 9/l, neutrophils 2.5 × 109/l, hemoglobin (Hb) 13.3 g/dL, mean corpuscular volume (MCV) 95 fL, and platelets 113 × 109/l  smear revealed white blood cells (WBCs) 6.7 × 10 × 109/l . Hematop9/l, neutrophils 1.2 × 109/l, Hb 12.3 g/dL, MCV 103 fL, and platelets 50 × 109/l . Over follow-up, he showed a moderate decline of the overall blood count, especially affecting the platelet population. Currently, 5 years after diagnosis and at the age of 11, WBCs are 4.6 × 10 × 109/l . Hematop2. Following DNA, RNA, or protein extraction, cell cultures were transferred to RPMI or DMEM with 10% DMSO and then frozen in liquid nitrogen for storage.P1’s lymphoblastoid cell lines (LCLs) were generated from primary lymphocytes isolated from peripheral blood . LCLs anFANCA deletion was identified analyzing Ion PGM row data, as described in FANCA cDNA with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence (RefSeq NM_000135). Variants identified were searched in the following annotation databases: Single-Nucleotide Polymorphism Database , Genome Aggregation Database , and Human Gene Mutation Database . Thein silico analyses of splicing mutations were carried out using Splice Site Prediction by Neural Network .Genomic DNA was extracted from patient’s peripheral blood and LCLs. FA genes were analyzed using the Ion PGM system for next-generation sequencing , as described in Total RNA was extracted from the patient’s LCLs and peripheral blood using the High Pure RNA Isolation Kit  and PAXgene Blood RNA Kit IVD , respectively, according to the manufacturer’s instructions. cDNA was synthetized using the Transcriptor First Strand cDNA Synthesis Kit , and RT-PCRs were prepared with KAPA2G Fast HotStart . To verify the deletion from exons 11 to 14, RT-PCR primers 9F 5′-TTGATGTACTGCAGAGAATGC-3′ and 16R 5′-GTGTCTTGGCCAATGAGATG-3’ were used. To assess the c.2778 + 83C>G effect, RT-PCR primers 27F 5′-AGCTGCTTATCTCCAGGCC-3, 28R_+83 5′-CAAACCCTAGACTCAGGACG-3,′ and 30R 5′-GGAAATCCATCAGTGCGTTG-3′3′ were designed within the predicted intronic retention and used in FANCA exon 28 with portions of introns 27 and 28 was created by the amplification of cDNA obtained by patients’ PB or LCLs in order to obtain all the investigated haplotypes. Amplification primers (clonF 5′-ATCATCCATATGGAATGTGGGGTTGTG-3′ and clonR 5′-TGTTGTCATATGGTCAAGATTCCAATC-3′) were designed with an NdeI restriction site (underlined) at the end in order to allow the cloning into the α-globin minigene under the control of the α-globin promoter and the SV40 enhancer .For the analysis of the splicing isoforms, the primers α2F (5′-CTTCAAGCTCCTAAGCCACTG-3′) and β2R (5′-CACCAGGAAGTTGGTTAAATC-3′) were used.Protein whole-cell extracts were prepared using RIPA buffer . In brief, the cells were washed with phosphate-buffered saline and resuspended in RIPA buffer containing protease and phosphatase inhibitors  and benzonase  at 25 U/mL. The samples were lysed and quantified with the Pierce BCA Protein Assay Kit , according to the manufacturer’s instructions. Protein extracts were then loaded on 6% SDS-PAGE gel and blotted with the following antibodies: anti-FANCA , anti-FANCD2 , anti-vinculin  as the loading control, and anti-rabbit . Immunodetection was operated with Immobilon Crescendo Western HRP Substrate  and Pierce ECL Western blotting Substrate  to reveal FANCA, and FANCD2 and vinculin proteins, respectively. The densitometric analysis of the anti-FANCA antibody signal was performed via ImageJ software, based on the use of vinculin for data normalization and the comparison with both positive and negative controls’ results. To analyze FANCD2 monoubiquitination, the cells were treated with 2 mM hydroxyurea (HU)  for 18 h prior to sample collection.LCL survival under MMC treatment  was tested after a few cell passages (Time 0) and a month after the establishment of the cultures (Time 1), as previously described in FANCA gene, both confirmed in the proband  and his mother (I-2) by Sanger sequencing, indicating they are in cis   of patient’s DNA from peripheral blood (PB) revealed two single-nucleotide heterozygous substitutions (c.2778 + 83C > G and c.2778 + 112T > C) in intron 28 of the e in cis . The c.2ite (SS) . The 277ite (SS) . On the ns 11–14 , and subns 11–14 .FANCA patient (P2), compound heterozygote for a large intragenic deletion  and the same c.2778 + 83C > G mutation  . As a comutation , leadingmutation ; Table 2mutation , in P2, mutation .FANCA with 156 additional bp  between exons 28 and 29. Of note, the analysis did not reveal any product with the partial intronic retention. To further confirm these data, we carried out a RT-PCR using a reverse primer specifically designed to anneal over the 83-bp insertion of intron 28  and a novel faint product (559 bp) not detectable in P2 and in the control , later intron 28 . As in tntron 28 , suggestde novo thymine insertion  carrying c.2778 + 83C>G and c.2778 + 112T>C in the homozygous status (data not published). Minigenes expressing the four different haplotypes of intron 28  . We, thude novo compensatory variant in rescuing a FANCA mutant allele, otherwise known for its deleterious effect in FA, at the molecular level. P1 is compound heterozygous for a large intragenic deletion and the c.2778 + 83C > G transversion of FANCA. The latter variant has been identified in linkage disequilibrium with c.2778 + 112T > C in another two apparently unrelated patients , which was regarded as a de novo c.2778 + 86insT event is likely to have arisen in vivo as it was detected in two LCLs established two years apart. Nevertheless, the analysis of the genomic DNA from either proband’s PB or BM cells did not allow detecting the de novo T insertion, suggesting that the number of cells with this mutation is very limited. Moreover, the lack of suitable BM material prevented us from searching the variant in different cell populations and defines the stage of the hematopoietic hierarchy in which the mutational event occurred.The de novo c.2778 + 86insT variant. Indeed, even if over time, the hematopoietic function showed some decline (mainly expressed by the drop of the platelet count from 113 × 10/9 to 50 × 10/9 after a 5-year follow-up), this decrement was less rapid than that observed in FA patients (median age at HSC transplantation is about 8 years)   and, conde novo variant (c.2778 + 86insT) that restores the FA cellular phenotype at least in vitro. Further follow-up will monitor the evolution of the disease in terms of the marrow function, from slow deterioration, stabilization, or even improvement.Considering that the FA phenotype is characterized by extensive variable clinical expression, even in patients carrying the same mutations, we cannot propose any correlation between the relative mild phenotype of the proband with the FANCA alterations identified. However, we could speculate that they contribute to the relatively stable blood counts, diminished or delayed need for HSC transplant, and prolonged survival through the combined effect of a potential hypomorphic germline mutation (c.2778 + 83C > G) and a Nowadays, HSCT is the only curative approach for FA hematological defects, and according to the current guidelines, it should be performed when the marrow function declines below the level of transfusion dependence or in case of evolution to myelodysplastic/acute myeloid leukemia (MSD/AML) . Despite


Echinococcus granulosus sensu lato, which employs various strategies to evade the host immune system for survival. Recent advances have revealed the role of annexins as excretory/secretory products, providing new insights into the immune regulation by these proteins in the pathogenesis of CE.Cystic echinococcosis (CE) is a life-threatening zoonotic disease caused by the larval stage of Echinococcus granulosus annexin B proteins EgANXB2, EgANXB18, EgANXB20, and EgANXB23 were cloned, expressed, and analyzed using bioinformatic tools. Membrane binding analysis was used to assess their bioactivity, while their immunoreactivity and tissue distribution characteristics were determined experimentally using western blotting and immunofluorescence staining, respectively. Furthermore, quantitative real-time reverse transcription PCR (qRT-PCR) was used to analyze the mRNA expression profiles of EgANXBs in different developmental stages of E. granulosus. Finally, immunofluorescence staining, cell counting kit 8 assays, flow cytometry, transwell migration assays, and qRT-PCR were used to evaluate the functional effects of rEgANXB18 and rEgANXB20 on mouse peripheral blood mononuclear cells (PBMCs).EgANXBs with conserved protein structures and calcium-dependent phospholipid binding activities. rEgANXBs were recognized by serum from sheep infected with E. granulosus and distributed in the germinal layer of fertile cysts. Interestingly, transcription levels of the four EgANXBs were significantly higher in protoscoleces than in 28-day strobilated worms. Moreover, we demonstrated that rEgANXB18 and rEgANXB20 were secretory proteins that could bind to PBMCs and regulate their function. Specifically, rEgANXB18 inhibited cell proliferation and migration while promoting cell apoptosis, NO production, and cytokine profile shifting. In contrast, rEgANXB20 showed limited effects on apoptosis but inhibited NO production.In this study, we identified four EgANXBs, EgANXB2 and EgANXB23 might play a pivotal role for the development of protoscoleces, while EgANXB18 and EgANXB20, as secretory proteins, appeared to participate in the host-parasite interaction by regulating the function of immune cells.Our findings suggested that among the four identified The online version contains supplementary material available at 10.1186/s13071-023-05967-y. Echinococcus granulosus sensu lato [E. granulosus [Cystic echinococcosis (CE) is a global zoonosis caused by the larval stage of nsu lato . Currentnsu lato . Intermeanulosus . The egganulosus . The proanulosus .2O2 production of human neutrophils [Some proteins within the ESPs have been identified, and their functions have been further investigated, particularly in relation to their interactions with the host immune cells. For instance, AgB has been found to reduce the Htrophils  and promtrophils . Similartrophils . In summEgANXB2, EgANXB18, EgANXB20, and EgANXB23), hydatid fluid  [EgANXB20) or the pulmonary hydatid fluid of patients (EgANXB2 and EgANXB20) [E. granulosus annexins are still limited [Annexins constitute an evolutionarily conserved protein superfamily with calcium-dependent phospholipid-binding properties . Widely gANXB38) , as wellgANXB20) . However limited , and theEgANXB2, EgANXB18, EgANXB20, and EgANXB23 of E. granulosus. Then, the calcium-dependent phospholipid binding activities, secretory properties, distribution, and transcription levels of the four EgANXBs were explored. Furthermore, we investigated the regulatory effects of rEgANXB18 and rEgANXB20 on mouse peripheral blood mononuclear cells (PBMCs) to gain a better understanding of the host-parasite interaction.In this study, we cloned and expressed E. granulosus s.l. from a slaughterhouse in Sichuan province, China. The PSCs and cyst walls were separated and treated as described in previous reports [PSCs were collected from hydatid cysts isolated from the liver of sheep infected with  reports . The via reports . PSCs wiEgANXBs. After PCR, the products were extracted from the gel and cloned into vector pMD19-T  for sequencing. Then, the pMD19-T-EgANXBs plasmids were subcloned into vector pET-32a ( +)  and transformed into Escherichia coli BL21 or Rosetta (DE3) cells (Tiangen), followed by induction with 0.24 mg/ml isopropyl β-d-1-thiogalactopyranoside (IPTG) at 16 ℃ for 12 h. The recombinant (r)EgANXBs were purified using a Ni2+ affinity chromatography column . The imidazole was removed, and the concentration of each protein was determined using a bicinchoninic acid (BCA) protein quantification kit (Bestbio). The purified proteins (0.5 mg/well) were assessed using 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and the gels were stained with coomassie blue.An RNAprep pure Tissue Kit  was used to extract total RNA from PSCs, and cDNA was subsequently synthesized using a RevertAid First Strand cDNA Synthesis Kit . Specific primers . A comparison was made between these sequences and those we obtained here, followed by bioinformatic analysis. Expasy (http://web.expasy.org/protparam/) was used to predict the basic physicochemical properties. Protein signal peptides, transmembrane regions, and subcellular localizations were predicted using SignalP-5.0 Server , TMHMM Server (http://www.cbs.dtu.dk/services/TMHMM-2.0) and WOLFPSORT (https://wolfpsort.hgc.jp/), respectively. The secondary and three-dimensional structures were predicted using SOPMA (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html) and SWISS-MODEL (http://swissmodel.expasy.org/). The multiple sequence alignment of the four EgANXBs was analyzed with Jalview (version 2.11.1) [Echinococcus granulosus [The full-length coding sequences of the four  2.11.1) , and the 2.11.1) . The seqanulosus .2+-dependent phospholipid-binding bioactivity of the four rEgANXBs were conducted according to previously published protocols with slight modifications [EgANXBs, pET32a vector protein (negative control), and bovine serum albumin (BSA) (V) (unrelated control). Both group a and b contained 30 μl of protein (0.5 mg/ml), 20 μl of liposomes (1 mg/ml) , 30 μl of CaCl2 (50 mM), and 20 μl of Tris–HCl (50 mM) in a total volume of 100 μl, while group c excluded CaCl2. After fully mixing, all groups were incubated at 37 °C for 1 h and then centrifuged at 8000 × g for 10 min to separate the supernatant and precipitate. Then, 30 μl of EDTA (50 mM) and 70 μl of Tris–HCl were added to group b precipitate, incubated, centrifuged, and separated as mentioned above. All the supernatants and precipitates were subjected to 12% SDS-PAGE and Coomassie blue staining for analysis.The preparation of liposomes and assessment of Caications . BrieflyEgANXBs were produced by immunizing eight rats, with two rats for each protein, as previously described [EgANXBs in incomplete Freund's adjuvant were performed in the following 3 weeks, with 1-week intervals. One week after the last immunization, anti-sera were collected and purified using a Protein G Resin FF Prepacked Column  to obtain anti-rEgANX rat IgGs.Sheep sera, either positive or negative, were obtained from autopsy-confirmed infected or healthy sheep, respectively. Polyclonal antibodies against the rescribed . The preEgANXBs and cyst fluid, were separated using 12% SDS-PAGE and transferred to Nitrocellulose Membranes . Then, the membrane was blocked using 5% skim milk for 2 h. Sheep negative or positive serum diluted 1:100 in phosphate-buffered saline (PBS), rat negative serum, or anti-rEgANXBs rat IgG (dilution 1:400 in PBS) was added and incubated overnight at 4 °C. After washing, horseradish peroxidase (HRP)-conjugated rabbit anti-sheep IgG (dilution 1:1000 in PBS) or HRP-conjugated goat anti-rat IgG (H + L)  (dilution 1:2000 in PBS) was added for 2 h incubation, and the immunoreactive signals were detected using a Metal Enhanced DAB Substrate Kit .The total proteins of PSCs were extracted using a BBprExtra total protein extraction kit . The extracted proteins, along with rEgANXBs rat IgG (dilution 1:400 in PBS) was added and incubated overnight at 4 °C. Fluorescein isothiocyanate (FITC)-conjugated goat anti-rat IgG (H + L) (dilution1:2000 in PBS) (Invitrogen) was added and incubated for 1 h, followed by staining with 4′,6-diamidino-2-phenylindole for 10 min at 37 ℃ (DAPI) . Images were acquired under a fluorescence microscope .Samples including PSCs, fertile cysts, non-fertile cysts, and 28-day strobilated worms were fixed in 4% paraformaldehyde for at least 24 h. Paraffin embedding and cutting into around 5-μm sections were carried out after dehydration and transparency procedures. Dewaxing, rehydration, and antigen repair (heating in 0.01 M sodium citrate solution at 95 °C for 15 min) were subsequently conducted, followed by quenching the tissue autofluorescence using an autofluorescence quenching kit . The sections were blocked with 5% goat serum (Solarbio) at 37 ℃ for 1 h. Rat negative serum or anti-rEgANXBs in PSCs and 28-day strobilated worms, quantitative real-time reverse transcription PCR (qRT-PCR) was performed. Total RNA was extracted using a cell total RNA isolation kit , and cDNA was synthesized. The specific primers are shown in Additional file GAPDH gene from E. granulosus  was selected as the housekeeping gene. The results were analyzed using the 2−ΔΔCt method and shown as the relative expression to the gene of strobilated worm [To assess the mRNA expression levels of the four 2 incubator. To avoid the effects of endotoxin on cells, endotoxins in the rEgANXB18 and rEgANXB20 preparations were removed using Endotoxin Removal Beads  and tested according to the instructions of the ToxinSensor™ Chromogenic LAL Endotoxin Assay Kit (GenScript). Moreover, rEgANXBs were filtered by 0.45-μm sterile filter membranes and quantified using BCA protein quantification kit (Bestbio).Blood (0.7-1 ml/per mouse) of 60 female BALB/c mice was collected from the tail vein, and the mouse PBMCs were isolated following the manufacturer's instructions of the peripheral blood mononuclear cell separation kit . Cell viability was detected using trypan blue staining. The cells were then resuspended in Roswell Park Memorial Institute (RPMI)1640 medium  supplemented with 10% heat-inactivated fetal bovine serum  and 1% penicillin-streptomycin  before being cultured at 37 ℃ in a 5% COEgANXB18 and rEgANXB20 to PBMCs, immunofluorescence staining was conducted. First, cocultivation of PBMCs (1 × 106 cells/ml/well) with rEgANXB18 (10 μg), rEgANXB20 (10 μg), pET-32a vector protein (10 μg), or 10 μl PBS was conducted in 24-well plates. After 2 h incubation at 37 °C, 5% CO2, the cells were washed and then fixed using 4% paraformaldehyde, permeabilized with 0.25% TritonX-100 and blocked by 5% BSA for 2 h. Then, anti-rEgANXB18/rEgANXB20 rat IgG (1:200 in PBS) or rat negative serum was added and incubated at 4 °C overnight, followed by incubation with cyanine 3 (Cy3)-labeled goat anti-rat IgG (1:500 in PBS)  at 37 ℃ for 2 h and stained with DAPI. The samples were observed under a fluorescence microscope.To analyze the binding of r6 cells/100 μl/well) were seeded into 96-well plates and incubated with rEgANXB18, rEgANXB20 , pET-32a vector protein (1 μg), concanavalin A (ConA) (1 μg), or 1 μl of PBS, respectively. After incubation at 37 °C for 24 h, the proliferation of PBMCs was evaluated using the Cell Counting Kit 8 (CCK-8) reagent (Beyotime) according to the manufacturer's instructions. Cell proliferation index was calculated by the formula: OD450 of the treatment/OD450 of the control.PBMCs (1 × 106 cells/ml/well) with rEgANXB18, rEgANXB20 , pET-32a vector protein (10 μg), or 10 μl PBS, respectively. After incubation at 37 °C for 24 h, the cells were collected, washed, and subjected to AnnexinV-FITC  and propidium iodide (PI)  staining to detect cell apoptosis by flow cytometry  [For apoptosis analysis, we cultivated PBMCs  . Cell apEgANXB18/rEgANXB20 on PBMC migration, a Transwell migration assay was conducted. Specifically, cell chambers with an 8.0-µm pore size (Thermo Fisher) were gently placed in a 24-well plate; RPMI1640 medium (1300 μl) containing 10% inactivated FBS was added to the lower chamber, while 200 μl PBMCs (2 × 106 cells) resuspended in RPMI1640 medium without FBS was added to the upper chamber along with rEgANXB18, rEgANXB20 , pET-32a vector protein (10 μg/ml), or 10 μl PBS. PBMCs were incubated for 4 h at 37 °C, and cells that migrated to the lower chamber were collected and counted using a cell counting chamber . Cell migration rate (%) was calculated by the formula: migration rate (%) = cells that migrated to the lower chamber/ 2 × 106 cells × 100%. The experiments for the migration, proliferation and the apoptosis assays were performed in triplicate.To assess the effect of r6 cells/100 μl/well) were seeded into 96-well plates and incubated with rEgANXB18, rEgANXB20 , pET-32a vector protein (1 μg), concanavalin A (ConA) (1 μg), or 1 μl PBS, respectively, at 37 °C for 48 h. NO production was detected using a Total Nitric Oxide Assay kit (Beyotime).PBMCs (1 × 106 cells/ml/well) were seeded into 24-well plates and incubated with rEgANXB18, rEgANXB20 , pET-32a vector protein (10 μg), or 10 μl of PBS at 37 °C for 24 h. The specific primers are shown in Additional file GAPDH (GenBank BC096440.1) gene from mouse was selected as the housekeeping gene, and the relative expression levels of cytokines were analyzed using the 2−ΔΔCt method.For cytokine production analysis, PBMCs , and data are presented as the mean ± standard deviation (SD). An unpaired Student’s t-test was used for comparisons between two groups, and one-way analysis of variance (ANOVA) was applied to compare differences among three or more groups. EgANXBs were successfully cloned and sequenced. EgANXB18, EgANXB20, and EgANXB23 shared the same sequences with their reference sequences, while EgANXB2 (1065 bp) was 21 bp longer than its reference sequence. The molecular weight of four EgANXBs ranged from 35.72 kDa to 39.95 kDa. All four EgANXBs were predicted to be stable, without signal peptides or transmembrane regions . As a positive control, ConA could induce the proliferation of PBMCs (P < 0.001). Moreover, we investigated the effect of rEgANXB18 and rEgANXB20 on PBMC apoptosis. Our data demonstrated that rEgANXB18, at high concentrations (20 μg/ml and 40 μg/ml), significantly promoted PBMC apoptosis (P < 0.001). However, rEgANXB20 showed no effects on PBMC apoptosis  and rEgANXB20  inhibited PBMC migration , while rEgANXB20 inhibited it (P < 0.05)  could markedly promote the relative mRNA expression levels of cytokines interleukin (IL)-10, IL-17A, transforming growth factor beta 1 (TGF-β1), and interferon gamma (IFN-γ) (P < 0.05). However, with increasing recombinant protein concentration, the expression levels of the cytokines showed no significance compared to those in the PBS group  while 20 μg/ml and 40 μg/ml rEgANXB20 inhibited its expression  Fig. A. Meanwhoup Fig. B. By conion Fig. C.Fig. 11E. granulosus survives within its hosts, producing ESPs that are generally considered to play essential roles in parasite invasion, immune escape, and metabolic adaptation [Taeniasolium, as secretory proteins discovered in ESPs, were believed to be involved in modulating host immune responses and facilitating parasite immune evasion [E. granulosus annexins have yet to be clearly determined. In this study, we aimed to expand the current understanding of annexins by characterizing four novel EgANXBs and exploring their potential roles in parasite immune evasion. Annexins belong to an evolutionarily conserved protein superfamily characterized by their ability to reversibly interact with membranes in a calcium-dependent manner [The metacestode of aptation . Some an evasion . Howevert manner .EgANXBs contained the sequence GXGT- {38–40 amino acid residues}-D/E and shared similar conventional three-dimensional structures. Notably, although the GXGT- {38–40 amino acid residues}-D/E sequence was present in EgANXB18, no calcium-binding site was predicted in the protein's three-dimensional structure. Since EgANXB18 was experimentally shown to bind phospholipids in a Ca2+-dependent manner, this unexpected outcome could be attributed to the template selected to construct the model. EgANXB23 was predicted to form a dimer, which is consistent with previous reports indicating that certain annexins, such as ANXA2 and ANXA5, could form dimers and trimers, and even heterotetramers, with other molecules, such as the S100 protein [Annexins are composed of a C- and an N-terminal domain. The C-terminal domain contains four or eight repeats arranged to form five α helices that finally form the overall shape of a slightly curved disc with a central hydrophilic hole , 33. Her protein –36.EgANXBs were confirmed to bind to phospholipids in a Ca2+-dependent manner; intriguingly, although rEgANXB2 and rEgANXB23 could bind to phospholipids, they were not completely released when EDTA was added, likely because of the formation of transmembrane ion channels. Previous studies have shown that several annexins, including ANXA1, ANXA2, ANXA5, ANXA6, and ANXA7 of humans and other species, exhibit transmembrane ion channel activity [2+. However, transmembrane ion channels would keep annexins bound to the membrane, and it was observed that ANXA1 and ANXA2 were resistant to EDTA extraction and required detergent treatment for the release from the membrane [EgANXB2 and rEgANXB23 from E. granulosus possibly attach to the membranes and then insert into them to form transmembrane ion channels, such that EDTA failed to extract them from the liposomes.The four ractivity , 38. Normembrane , 40. AltEgANXBs in different development stages of E. granulosus differed from each other, which was also observed in trematodes [EgANXBs were detected in PSCs compared with those in 28-day strobilated worms, which contrasted with EgANXB3 and EgANXB38 [EgANXBs in this study and PSCs. Furthermore, EgANXB18 and EgANXB20 were identified as secretory proteins despite lacking signal peptides, suggesting that they were probably secreted through unconventional secretion pathways, such as direct translocation across the plasma membrane or secreted via extracellular vesicles [EgANXB2 and EgANXB23 were not detected in cyst fluid, indicating that they were not secretory proteins, or that their concentrations were too low.The distribution of four ematodes –44. SurpEgANXB38 . This suvesicles . Under tClonorchis sinensis annexin B30 [T. solium annexin B1 [E. granulosus annexins B3, B33, and B38 [EgANXB18 and rEgANXB20 were observed to bind to PBMCs and inhibit their proliferation, and high concentrations of rEgANXB18 also promoted PBMC apoptosis, while rEgANXB20 showed no effect. This suggested that rEgANXB18 and rEgANXB20 were more likely to suppress the immune response by affecting proliferation rather than apoptosis.Annexins are generally considered to function intracellularly . Howeverexin B30 , T. solinexin B1 , and E.  and B38 , 21. Fur and B38 –49. SimiEgANXB18 and rEgANXB20 inhibited the migration of PBMCs, implying that they potentially played a role in the modulation of the immune response in hosts.Immune cells can be recruited and migrate to specific tissues when an inflammatory response occurs, which is essential for infection control, tissue repair, and immune surveillance . SeveralEgANXB18 and rEgANXB20 exhibited opposite effects on NO production. Specifically, rEgANXB18 promoted NO production, while rEgANXB20 inhibited it, suggesting that both proteins affected NO production, probably by regulating the expression or activity of inducible nitric oxide synthases (iNOS). Previous studies confirmed that iNOS transcription was induced by IFN-γ and lipolysaccharide (LPS) [EgANXB20 affected the expression of IFN-γ only at the highest concentration, it is likely to function through a different pathway.NO, a multifunctional bioactive molecule, is generally considered to play an important role in anti-parasite infection by inhibiting cellular energy synthesis or destroying proteins, lipids, and nucleic acids . Our stude (LPS) . We obseEgANXB18 significantly increased the levels of IL-10, TGF-β1, IL-17A, and IFN-γ mRNA, with the increase of IFN-γ mRNA being markedly higher than those the others. Previous studies have shown that when PSCs were co-cultured PBMCs from CE patients, the addition of IFN-γ promoted the iNOS and NO production, leading to higher PSC mortality [EgANXB18 participates in anti-parasite infection by inducing IFN-γ expression. In addition, rEgANXB20 promoted the expression of IL-10, IL-17A, and IFN-γ. Moreover, it also significantly increased the expression level of TGF-β1 at low concentrations while showing opposite effects at high concentrations. Taken together, rEgANXB20 induced the expression of both Th1/Th2 and Th17/Treg cytokines, consistent with the cytokine profile observed in patients with CE.Th1 and Th2 immune responses coexist in patients with CE, with Th1 cytokines playing a role in parasite removal and Th2 cytokines being linked to parasite immune evasion . IFN-γ, ortality . TherefoEgANXBs as typical annexins with canonical structures and Ca2+-dependent phospholipid binding properties. Notably, EgANXB2 and EgANXB23 were postulated to have ion channel activities when interacting with membranes. Immunolocalization and qRT-PCR analysis revealed that both the distribution and transcription levels of these four EgANXBs in PSCs were higher than in 28-day strobilated worms, indicating their involvement in the growth and development of PSCs. Furthermore, EgANXB18 and EgANXB20 were found to be secretory proteins that could bind to PBMCs to regulate cell proliferation, apoptosis, migration, and NO and cytokine production. These results suggest potential roles of EgANXB18 and EgANXB20 in immune evasion strategies employed by the parasites.The present study identified four Additional file 1: Table S1. Primers for the PCR amplification of four EgANXBs. Table S2. Primers for the qRT-PCR amplification of four EgANXBs and GAPDH. Table S3. Primers for the qRT-PCR of cytokines.


Introduction: Intradiscal mesenchymal stromal cell (MSC) therapies for intervertebral disc degeneration (IDD) have been gaining increasing interest due to their capacity to ameliorate intervertebral disc metabolism and relieve low back pain (LBP). Recently, novel investigations have demonstrated that most of MSC anabolic effects are exerted by secreted growth factors, cytokines, and extracellular vesicles, collectively defined as their secretome. In this study, we aimed to evaluate the effect of bone-marrow-MSCs (BM-MSCs) and adipose-derived stromal cells (ADSCs) secretomes on human nucleus pulposus cells (hNPCs) in vitro.Methods: BM-MSCs and ADSCs were characterized according to surface marker expression by flow cytometry and multilineage differentiation by Alizarin red, Red Oil O and Alcian blue staining. After isolation, hNPCs were treated with either BM-MSC secretome, ADSC secretome, interleukin (IL)-1β followed by BM-MSC secretome or IL-1β followed by ADSC secretome. Cell metabolic activity (MTT assay), cell viability (LIVE/DEAD assay), cell content, glycosaminoglycan production , extracellular matrix and catabolic marker gene expression (qPCR) were assessed.Results: 20% BM-MSC and ADSC secretomes  showed to exert the highest effect towards cell metabolism and were then used in further experiments. Both BM-MSC and ADSC secretomes improved hNPC viability, increased cell content and enhanced glycosaminoglycan production in basal conditions as well as after IL-1β pretreatment. BM-MSC secretome significantly increased ACAN and SOX9 gene expression, while reducing the levels of IL6, MMP13 and ADAMTS5 both in basal conditions and after in vitro inflammation with IL-1β. Interestingly, under IL-1β stimulation, ADSC secretome showed a catabolic effect with decreased extracellular matrix markers and increased levels of pro-inflammatory mediators.Discussion: Collectively, our results provide new insights on the biological effect of MSC-derived secretomes on hNPCs, with intriguing implications on the development of cell-free approaches to treat IDD. Low back pain (LBP) is one of the most common musculoskeletal symptoms, affecting two-thirds of adults in the world population at least once in their lives. According to the Global Burden of Disease Study, LBP is the first cause of disability worldwide, with significant socioeconomic repercussions . Indeed,The main cause of chronic LBP is intervertebral disc degeneration (IDD): a chronic, progressive process hallmarked by catabolic events such as gradual resident cell loss due to accelerated apoptosis and senescence, extracellular matrix (ECM) breakdown, and tissue inflammation . These eIn the last decade, the possibility to restore the NP cell population through cell therapy has gathered significant attention. Indeed, intradiscal cell transplantation, especially involving mesenchymal stromal cells (MSCs), has been shown promising in both preclinical and clinical studies . Indeed,in vitro preconditioning of MSCs with different factors such as 3D culture, drugs, inflammatory cytokines, and hypoxia has also been shown to enhance secretome therapeutic potential  . The comotential .in vitro 3D model.In this study, we hypothesized that MSC-derived secretome may attenuate the degenerative conditions of the harsh IDD microenvironment by acting on several biological processes, such as cell viability, metabolic activity, ECM synthesis, and inflammation. Therefore, the purpose of this study was to compare the potential therapeutic effects of the secretome derived from bone marrow (BM)-MSCs and adipose-derived stem cells (ADSCs) on human primary NP cells (hNPCs) in an The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Campus Bio-Medico University of Rome (n. 09/15 PAR ComEt CBM). Human IVD and lipoaspirate samples were obtained from surgical waste specimens while BM-MSCs were harvested from healthy donors. Written informed consents were collected from all participants, as described below. A summary of all experimental procedures is depicted in 2. ADSCs were isolated from the stromal vascular fraction (SVF) obtained from lipoaspirate (LA) of 3 patients  undergoing intraarticular injection of adipose tissue derivatives for treating knee osteoarthritis. Waste aliquots obtained by LA micro-fragmentation or nanofat processing were used for this study as previously described .BM-MSCs were obtained from 3 healthy donors  through bone marrow aspiration from the iliac crest using 20 mL-syringes preloaded with 500 IU of preservative-free heparin , as previously described . Cells wescribed . Brieflyvia flow cytometry as previously described (5 cells/mL incubated for 2 h at room temperature in the dark with the following fluorescein isothiocyanate (FITC) or phycoerythrin (PE) conjugated antibodies: AntiCD105-FITC , antiCD90-PE , antiCD45-PE , and antiCD73-FITC . A minimum of 25,000 cell events per assay were acquired using CytoFLEX .BM-MSCs and ADSCs were characterized as previously described according to minimal criteria provided by the International Society for Cell and Gene Therapy (ISCT) . The expescribed . Cells wAdherent cells at P4 were induced toward adipogenic, osteogenic and chondrogenic differentiation, as previously described . Adipogeg at 4°C for 10 min to remove gross debris, and then at 2000 g at 4°C for 20 min to remove cellular debris, and further filtered through a 0.22 µm filter. Secretome was stored at −80°C until further use.Secretome collection was performed as previously described . BM-MSCsn = 5, mean age = 57 ± 17 years old, 2. The culture media were changed twice weekly, and cultures were allowed to grow until reaching 80%–90% confluency. Adherent hNPCs at P2 were treated with trypsin-EDTA (Corning), washed, and centrifuged at 1,500 rpm for 5 min. Then, cell mixtures were resuspended in 1.2% low-viscosity sterile pharmaceutical grade alginate  solution at 4 × 106 cells/mL using a syringe with a 21-gauge needle into a 102 mmol/L calcium chloride solution to form semisolid beads (2 and 95% air incubator. The media were changed twice a week. hNPCs were then divided in six groups: 1) DMEM (control group); 2) BM-MSC secretome; 3) ADSC secretome; 4) 10 ng/mL IL-1β ; 5) pretreatment with 10 ng/mL IL-1β for 24 h and then co-treatment with BM-MSCs secretome or 6) ADSCs secretome. Each treatment was performed in triplicate.hNPCs were isolated from IVD tissues of patients undergoing discectomy due to lumbar disc herniation  by evaluating hNPC metabolic activity through MTT assay (Sigma), according to the manufacturer’s instructions. At P2, hNPCs were seeded in wells of 96-well plates (5 × 103 cells/well). After 48 h, cells were treated with BM-MSC and ADSC secretomes at increasing concentrations and then supplemented with 100 μL of MTT -2,5-diphenyltetrazolium bromide) reagent (c = 5 mg/mL). After 4 h at 37°C, MTT solution was removed and 100 μL DMSO were added for 15 min at 37°C to dissolve the formazan crystals. Absorbance readings of DMSO extracts were performed at 570 nm with reference at 690 nm using a TecanInfinite M200 PRO.Cell viability was determined by analyzing membrane integrity through the LIVE/DEAD™ cell viability assay , following manufacturer’s instructions. After 7 days, hNPCs in alginate beads were incubated for 45 min with ethidium homodimer-2 and calcein acetoxymethylester (AM) at room temperature and washed 3 times with PBS and Hoechst 33258  to stain the nuclei. Green, red, and blue fluorescence were detected using confocal immunofluorescence microscopy and analyzed with ImageJ. Beads were dissolved by incubation for 10 min at 4°C in 10 volumes of 55 mmol/L sodium citrate, 30 mmol/L EDTA, 0.15 M NaCl, pH 6.8 and cell proliferation was evaluated at 4, 7 and 14 days using CytoFLEX (Beckman Coulter).At the seventh day of the experiment, after dissolving beads, hNPCs were digested with 100 μL of papain (Sigma) solution  overnight with gentle shaking at 65°C, to assess GAG content. GAGs were measured by reaction with 1,9-dimethylmethylene blue  using chondroitin sulfate (Sigma) as a standard. Measurements of absorption were performed at a wavelength of 530 nm (Tecan Infinite M200 PRO). Data were expressed as quantity of GAG normalized to DNA content of hNPCs cultured for 7 days, comparing the percent variation between the control group and the experimental group.ACAN (Hs0153936), MMP-13 (Hs00233992), ADAMTS5 (Hs00199841), SOX9 (Hs01001343), and IL6 (Hs00174131) gene expression were analyzed, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Hs03929097) was used as a housekeeping gene. The expression level of each gene has been normalized to the expression of GAPDH and calculated as 2−ΔDCt. Values in the experimental group were normalized to expression levels encountered in the control group, which was considered as a baseline.After alginate bead digestion, total RNA was extracted from pellets after 7 days of culture using the TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. cDNA was synthetised using the HighCapacity cDNA Reverse Transcription kit  according to the manufacturer’s instructions. mRNA levels were measured through qRT-PCR using TaqMan Gene Expression Assays and TaqMan Universal Master Mix II (Thermo Fisher Scientific) with UNG-Real Time PCR System Instrument 7900HT FAST according to manufacturer’s instructions. t-test and one-way analysis of variance (ANOVA) with Dunnett’s or Tukey’s post-tests for multiple comparisons whenever applicable. Statistical significance was set as p < 0.05. Formal analysis was performed using Prism 9 . Each experiment was repeated at least three times and representative experiments are shown.Quantitative data are expressed as means ± standard deviation (SD). The normality of data distribution was determined with the Wilk-Shapiro test. Statistical analysis of the results was performed using unpaired Flow cytometry analysis showed that both BM-MSCs and ADSCs were positive for the expression markers CD73, CD90 and CD105 and negative for CD45 . Evidencp < 0.001) as well as to other secretome concentrations  was calculated using control cells as a reference. After 48 h, 20%, BM-MSC secretome significantly increased hNPCs mitochondrial activity (126.3% ± 6.9%) compared to controls (n = 4). Results were normalized to the control group and expressed as percent variation. In basal conditions, both BM-MSCsec and ADSCsec were able to significantly increase cell viability  and ADSCsec (104.3% ± 26.2%), even if the latter did not reach statistical significance (p = 0.021), while the effect of ADSCsec did not reach statistical significance  treated with BM-MSCsec and ADSCsec showed an increase of cell content at 4, 7 and 14 days after treatment compared to the control group  and ADSCsec  compared to the control group (143.3 ± 30.4 × 103 cells/mL). Contrarily, when hNPCs were treated with 10 ng/mL IL-1β, cell numerosity was significantly increased at 7  and 14 days  compared to the controls  and ADSCsec  incremented cell content after IL-1β pretreatment compared to the control group (73.0 ± 18.7 × 103 cells/mL). The same trend was reported at day 7  and 14 .hNPCs in alginate beads were cultured for 7 days and stained for live and dead cells as described above (ificance . In contificance . Neverthol group . Such anp = 0.009) compared to controls (p = 0.005) compared to the control group, but was promptly restored after treatment with both BM-MSCsec  and ADSCsec  treated with BM-MSCsec and ADSCsec showed a significant increase in GAG synthesis normalized to DNA. Considering the GAG/DNA ratio in the control group as a baseline of 100%, hNPCs exposed to BM-MSCsec produced a higher amount of GAG . More specifically, BM-MSCsec determined an increase of ACAN . Furthermore, BM-MSCsec significantly reduced the expression of IL6 , while increasing IL6  and SOX9 levels , whereas significantly reducing IL6 , and MMP13 gene expression . Interestingly, ADSCsec led to a significant decrease of ACAN  and SOX9  gene expression, as well as increased levels of MMP13  and ADAMTS5  compared to the controls.After 7 days, MSC secretomes exerted a beneficial effect on inflammatory and catabolic marker gene expression in hNPCs , catabolic enzymes involved in ECM degradation (ADAMTS5 and MMP13) and the proinflammatory cytokine IL6 were assessed to further investigate the biological effect of MSC-derived secretomes. In a recent study by Zeng et al., umbilical cord-derived MSC secretome was shown to significantly increase the expression of TEK, a marker of NP progenitor cells, as well as pluripotency markers OCT4 and NANOG. They concluded that MSC-derived secretome may play a key role in the promotion of de-differentiation, stemness and multipotency of degenerated NPCs by highlighting its potential “rejuvenating” effects  (2), ADSCsec exerted a catabolic effect on gene expression. In this regard, since paracrine stimulation has a significant impact on MSC activity, the importance of cell preconditioning to increase the therapeutical potential of their secretome is being increasingly evident  on hNPCs in an ammation . Indeed,ammation . Therefoammation . It is wchanisms . In thised cells . Hingerted cells . Furthered cells . Accordistimulus . MSC-derstimulus . Howeverin vitro , ex vivo(ex vivo  and in v(in vivo  followin effects . Interesediators . Compareediators . Therefotors (2) . It is n evident . For exa evident . Similarcretomes . Furtherand IL-6 . Despiteand IL-6 . This vaThe study has some limitations. First, we did not select the patients from whom we collected MSCs neither by age nor presence or absence of comorbidities. Second, hNPCs were derived from patients with varying degrees and levels of IDD. This, together with the presence of additional covariates , may have contributed to generate donor variability, although our results did not seem to be significantly affected by substantial heterogeneity. Finally, since MSCs were not preconditioned as mentioned above, the use of different variables may have greatly improved secretome regenerative capacity.This study compared the potential beneficial effect of MSC secretome isolated from two different sources, namely, BM-MSCs and ADSCs, on hNPCs cultured in a 3D environment. Both secretomes improved hNPC proliferative capacity, viability, and GAG synthesis both in basal conditions and following pro-inflammatory preconditioning with IL-1β. In terms of gene expression of ECM and inflammatory markers, BM-MSCsec appeared to exert a greater regenerative effect compared to ADSCsec, which in turned showed a catabolic expression profile, further suggesting the importance of selecting the right cell source. Despite the lack of consensus regarding culture conditions as well as standardized protocols for preparation, storage, product stability and quality control parameters, the use of MSC-derived secretome may be a promising cell-free approach for the treatment of IDD.


Trichoderma biofertilizer (TF) on nitrogen (N) use efficiency and associated mechanisms in wolfberry (Lycium chinense) in coastal saline land. As with plant biomass and fruit yield, apparent N use efficiency and plant N accumulation were also higher with TF plus 75% CF than 100% CF, indicating that TF substitution promoted plant growth and N uptake. As a reason, TF substitution stabilized soil N supply by mitigating steep deceases in soil NH4+-N and NO3–N concentrations in the second half of growing seasons. TF substitution also increased carbon (C) fixation according to higher photosynthetic rate (Pn) and stable 13C abundance with TF plus 75% CF than 100% CF. Importantly, leaf N accumulation significantly and positively related with Pn, biomass, and fruit yield, and structural equation modeling also confirmed the importance of the causal relation of N accumulation coupled with C fixation for biomass and yield formation. Consequently, physiological and agronomical N use efficiencies were significantly higher with TF plus 75% CF than 100% CF. Overall, partial substitution of CF by TF improved N use efficiency in wolfberry in coastal saline land by stabilizing soil N supply and coupling N accumulation with C fixation.A two-year field trial was conducted to investigate the effects of partial substitution of chemical fertilizer (CF) by  Wolfberry may be an ideal economic crop to plant in coastal saline land, because it is a halophyte naturally distributed in coastal zones. However, despite high adaptability to salt, saline stress also inhibits wolfberry photosynthesis and growth, and cultivation methods need to be developed to achieve high fruit yields in saline land  with the TF on N use efficiency in wolfberry was investigated by a two-year field trial in coastal saline land. In particular, mechanisms were examined that affected soil N supply, C and N coupling, and root N absorption. This study may deeply disclose the mechanisms by which TF substitution improve crop growth under salt stress, and also can provide technical guidance for the cultivation of crops in saline land.Wolfberry  in Beijing. Apple residue is a typical organic waste in Yantai city, China and has been utilized as the material to prepare TF by us. A Trichoderma potato glucose agar plate was punched to obtain three 1 cm diameter blocks, which were shaken in potato glucose liquid medium at 150 rpm and 28°C for 5 d to prepare fermentation broth. Similar with the method in previous studies  NF (no N application); (2) 50% CF plus TF ; (3) 75% CF plus TF (combined application of 75% CF and TF); (4) 100% CF. In each treatment, four replicate plots (5.5 m × 4 m) were randomly distributed in the field. Plots were separated by 0.3 m wide and 0.3 m high ridges to avoid border effects, and 2 m remained around the plots as an isolation belt. The NF plots were designed as the control without N application. The N application rate was 400 kg ha-1 in 100% CF plots, whereas the TF application rate was 5,000 kg ha-1 with reduced chemical nitrogen application in 75% CF or 50% CF plus TF treatments. The amount of TF applied was based on a conventional organic fertilizer dose, and the total N application rate in plots with 75% CF plus TF was the same as that in 100% CF plots. Phosphorus application rate was 50 kg ha-1 in all plots. Urea and calcium superphosphate were applied as chemical N and P fertilizers, respectively. In April, 2020, twelve two-year bare-rooted wolfberry plants were planted in each plot, and plant and row spacing were 1.5 m. In April, 2020 and 2021, fertilizer was applied to circular furrows, which were 20 cm around plants and 20 cm deep. During the growing seasons in 2020 and 2021, three plants were randomly selected in each plot for measuring gas exchange and modulated and prompt chlorophyll fluorescence parameters. The topsoil (0–20 cm) was also collected in each plot by a five-point sampling method with a soil auger  in the growing seasons. At the end of October in 2020 and 2021, one plant was selected in each plot and excavated to measure biomass, N and C contents and leaf stable N and C compositions.The field trial was conducted in moderate saline land in the Yellow River Delta, Dongying, China . The site has a warm temperate continental monsoon climate, and annual average temperature and precipitation are approximately 13.5°C and 700 mm, respectively. Soil texture was silty loam, and soil chemical properties were the following: electrical conductivity, 433.7 µs cm2.34+-N and NO3–N concentrations and moisture. Soil NH4+-N and NO3–N were extracted with 2 M KCl for 1 h, and filtrates were analyzed for NH4+-N and NO3–N concentrations by a continuous flow analyzer . Another soil subsample was air-dried, ground, and sieved through a 150 μm screen, and soil total N was determined by an elemental analyzer . Soil available K was extracted by using ammonium acetate to measure its content through flame photometry  was packed in tin capsules, which were sealed and measured using an Elemental Analyzer  coupled with an isotope ratio mass spectrometer . Stable C and N isotope compositions were expressed using standard delta notation:  = Rsample/Rstandard - 1, where Rsample and Rstandard were isotopic ratios in samples and standards, respectively. Total N and C in leaf powders were also measured using an elemental analyzer (VarioMACRO cube).2.62 concentration in the leaf cuvette were set at 25°C and 400 μmol mol-1, respectively. Actinic light intensity was set at 1200 µmol m-2 s-1 in the field trial. Photosynthetic rate (Pn) and stomatal conductance (Gs) were simultaneously recorded, and modulated chlorophyll fluorescence was also recorded to calculate photosynthetic electron transport rate (ETR) according to a previous study  equipped with a fluorescence leaf chamber . Temperature and COus study .abs) was calculated according to A multifunctional plant efficiency analyzer  was used to record prompt chlorophyll fluorescence in the first 1 s of illumination with red light, and the PSII performance index , and differences were considered significant at P-values, χ2 values, a goodness-of-fit index (GFI), and the root mean square error of approximation (RMSEA).Structural equation modeling (SEM) was used to evaluate direct and indirect relations among N accumulation, C fixation, and yield in different fertilization treatments and was performed using AMOS Graphics 23.0 . The fit of the resulting model was evaluated using 33.1Fruit yield, plant biomass, and N content increased significantly with application of N fertilizer, and the increase was significantly higher in plots with TF plus 75% CF than in plots with 100% CF and TF plus 50% CF (3.213C, except for a significant increase in TF plus 75% CF plots in 2020 and 2021 (15N increased significantly with N fertilizer application, but there was no significant difference in leaf δ15N between plots with TF plus 75% CF and those with 100% CF (Applying N fertilizer did not significantly affect leaf δ3.3abs generally increased with N fertilizer application. Changes in those parameters were relatively greater in TF plus 75% CF plots than in 100% CF and TF plus 50% CF plots, although differences were not always significant in the monthly samplings (In 2020 and 2021 growing seasons, Pn, Gs, ETR, and PIamplings Figure 33.4In regression analysis, leaf N accumulation was significantly positively related to Pn, plant biomass, and fruit yield (3.54+-N and NO3–N contents increased significantly in June in plots with N fertilizer application and then gradually declined during the growing seasons (4+-N and NO3–N contents after June were not as great in TF plus 75% CF plots as in 100% CF plots, and soil NH4+-N and NO3–N contents remained generally higher in TF plus 75% CF plots in the second half of the seasons (Compared with NF and 100% CF plots, soil total N content increased significantly in TF plus 75% CF plots at the end of the growth seasons in October 2020 and 2021 (4-1 reduced wheat yield and root weight density in contrast to an optimal application rate at 240 kg ha-1. However, this study focused on the approach of TF substitution to reduce chemical N application. TF substitution could reduce 50% CF investment without influencing wolfberry growth and yield in contrast to 100% CF application, and notably, TF substitution with 25% reduction of chemical fertilizer improved wolfberry growth and yield (15N has a positive relation with crop growth and yield (15N increased in plants with N fertilization. However, the relatively minor difference in leaf δ15N did not match the greater yield and biomass in 75% CF plus TF plots than in 100% CF plots (15N by decreasing discrimination against 15N, whereas high N uptake indirectly increases discrimination against 15N by increasing available N (15N discrimination. 15N in salt-tolerant cultivar RIL47 of durum wheat in contrast to salt-sensitive cultivar RIL 24 under salt stress. Overall, TF substitution increased wolfberry N uptake and use in coastal saline land.High N application is commonly used for planting wolfberry by farmers because N in saline soil is liable to loss, and maybe, a lower chemical N application rate is appropriate considering that excessive N availability can lead to a systemic repression of root growth with yield reduction . For exand yield . In thisCF plots 2B. Nitrilable N . Thus, g3 volatilization from saline soil (Trichoderma spore powder (3 volatilization loss from saline soil by increasing soil nitrification which also increased N supply. Recent studies also show that addition of organic materials such as crop straw can optimize soil N supply by regulating N transformations (Similar to the accumulation of plant N, the stock of soil total N also increased in 75% CF plus TF plots compared with 100% CF plots at the end of growth seasons (ine soil . Notablye powder . In addirmations . Therefo2 diffusion into leaves (2 supply with high Gs tend to decrease δ13C by increasing 13C fractionation, whereas strong CO2 fixation can depress discrimination against 13C and increase δ13C (13C without reduction in Gs indicated greater CO2 fixation in plants with 75% CF plus TF than with 100% CF (Plant N use for growth and yield formation is dependent on photosynthetic C fixation. The greater increase in Pn with 75% CF plus TF than with 100% CF suggested that TF substitution helped increase photosynthetic capacity in wolfberry . In accoase δ13C . Thus, t 100% CF 3B. Over5In summary, partial substitution of CF by TF improved N use efficiency in wolfberry in coastal saline land by stabilizing soil N supply and increasing the synergism between N accumulation and C fixation.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.KY designed the experiment, performed the experiment, and wrote the manuscript. YR and HS participated in data analysis. HM, SY, YZ, SL, and YS participated in the experiment. All authors contributed to the article and approved the submitted version.