Pronematus ubiquitus were assessed. The alternative food sources were selected among those the mite may encounter or be applied to the tomato crop.Biocontrol practitioners have increasingly released generalist predators to control a variety of greenhouse pests. In this study, the effects of alternative food sources on the oviposition rate and the population dynamics of the mite Pronematus ubiquitus (McGregor) is a small iolinid mite that is capable of establishing on tomato plants. Once established, this mite has been shown to control both tomato russet mite, Aculops lycopersici (Tryon) (Acari: Eriophyidae), and tomato powdery mildew (Oidium neolycopersici L. Kiss). In the present study, we explored the effects of a number of alternative food sources on the oviposition rate in the laboratory. First, we assessed the reproduction on food sources that P. ubiquitus can encounter on a tomato crop: tomato pollen and powdery mildew, along with tomato leaf and Typha angustifolia L. In a second laboratory experiment, we evaluated the oviposition rate on two prey mites: the astigmatid Carpoglyphus lactis L. (Acari: Carpoglyphidae) and the tarsonemid Tarsonemus fusarii Cooreman (Acari: Tarsonemidae). Powdery mildew and C. lactis did not support reproduction, whereas tomato pollen and T. fusarii did promote egg laying. However, T. angustifolia pollen resulted in a higher oviposition in both experiments. In a greenhouse trial on individual caged tomato plants, we evaluated the impact of pollen supplementation frequency on the establishment of P. ubiquitus. Here, a pollen addition frequency of every other week was required to allow populations of P. ubiquitus to establish. Amblyseius swirskii (Athias-Henriot), Neoseiulus californicus (McGregor), and Neoseiulus cucumeris (Oudemans), are now among the most commercialized arthropod biocontrol agents worldwide [Aculops lycopersici (Tryon) (Acari: Eriophyidae), and several species of whiteflies [Pronematus ubiquitus (McGregor) is a small (<300 µm) omnivorous mite that feeds on a variety of plant-derived food sources, such as pollen and plant sap [A. lycopersici, and powdery mildew (Oidium neolycopersici L. Kiss) [Typha sp. pollen [In contrast to the long-held conviction that specialist natural enemies are required to guarantee effective pest control , biocontorldwide ,17, and orldwide ,21,22,23iteflies ,25,26. Titeflies ,28. Unliiteflies ,30. For lant sap . This prlant sap  and Tetrlant sap ,33. In alant sap ,35. Rece. pollen .P. ubiquitus can encounter food sources such as tomato pollen and conidia of powdery mildew [P. ubiquitus, collected in Belgium and The Netherlands.The nutritional value of food sources can influence the biological parameters of an organism. When on the tomato crop, y mildew . Other Iy mildew ,37. HoweEphestia kuehniella Zelle [Artemia sp. cysts [P. ubiquitus on a tomato crop, namely the dried-fruit mite Carpoglyphus lactis (L.) (Acari: Carpoglyphidae) and Tarsonemus fusarii Cooreman (Acari: Tarsonemidae). The former is a conventionally used astigmatid prey mite that can support populations of phytoseiid predatory mites on crops [Typha pollen for the establishment of P. ubiquitus on tomato plants. Several food sources have been tested to support the establishment of generalist predators in greenhouse crops ,38,39, ila Zelle , and Artp. cysts ,43. Hereon crops ,45,46,47on crops ,49,50. FP. ubiquitus were collected in Belgium (Merelbeke) and The Netherlands (Venlo), from blackberry (Rubus sp.) and grape (Vitis sp.) plants, respectively. These lines were mass-reared in the production facilities of Biobest N.V. on a diet consisting of T. angustifolia pollen (NutrimiteTM). Tarsonemus fusarii was produced on Aspergillus oryzae fungus grown on rice flakes  as described by Vangansbeke et al. [C. lactis. Both T. fusarii and C. lactis were produced in climate chambers at 22 ± 1 °C, 80 ± 5% relative humidity, and a L16:D8 photoperiod. Two lines of e et al. . A mixtuTomato plants (cv. ‘Marinice’)  were sown in trays. One week after germination, tomato seedlings were transplanted to 1 L pots with potting soil . Plants were subsequently grown in the Greenlab research facilities of Biobest (16.5–22 °C and 70 ± 10% relative humidity).P. ubiquitus populations, collected in Belgium and The Netherlands. Each involved a single adult female P. ubiquitus, randomly selected from the mass-rearing units, which was then placed on a 4 cm2 tomato leaf section on top of moist cotton within a 5 cm Petri dish arena. These leaf discs had Typha angustifolia ad libitum as a food supplement once at the start of the experiment. Each female was allowed to lay eggs for four days. At this time, the total number of eggs laid by each female was counted. These arenas were kept at 23 ± 1 °C, 70 ± 10% relative humidity, and a L16:D8 photoperiod. Each treatment contained 12 replicates. The total oviposition was analyzed using generalized linear models (GLMs) with a Poisson error distribution [In the first experiment, we assessed oviposition of two ribution . In addiP. ubiquitus line collected in Belgium was studied for its oviposition ability by applying the same method as that of Experiment 1. For Experiment 2, the food sources offered for the mites were: clean tomato leaf, tomato leaf infected with powdery mildew, tomato leaf with tomato pollen, and tomato leaf with T. angustifolia pollen. Tomato pollen was obtained from flowers collected from tomato plants (cv. ‘Marinice’)  in the Greenlab facilities. Flowers were dried for 72 h at room temperature (20–23 °C) and relative humidity (30–50% RH). Thereafter, pollen could be removed easily from the flowers before being frozen at −18 °C. Experiment 3 had clean tomato leaf, tomato leaf with T. angustifolia pollen as per the previous experiment, and tomato leaf with the prey mites T. fusarii or C. lactis. For both prey mites, a mix of all life stages was offered. All food sources were offered ad libitum once at the start of the experiment. Each treatment in Experiment 2 had 12 replicates and Experiment 3 had ten replicates per treatment. The total oviposition of Experiment 2 was compared among treatments with a generalized linear model (GLM) with a Poisson error distribution [In these two experiments, the ribution . For Expribution ). T. angustifolia pollen to promote P. ubiquitus (Belgium line) population growth. Cylindrical cages were used for this experiment having a diameter of 40 cm and height of 110 cm. At the start of the experiment the tomato plants had five to six leaves and height of 32 cm. Four frequencies of pollen application were evaluated in comparison with control without pollen supplement. Five treatments were arranged in a randomized complete block design with five replicates: P. ubiquitus without pollen supplement; P. ubiquitus + pollen supplement weekly; P. ubiquitus + pollen every two weeks; P. ubiquitus + pollen every three weeks; P. ubiquitus + pollen every four weeks. Fifty mixed stages of P. ubiquitus were introduced per plant at the first week of the experiment and an additional 100 one week later. A dose of 0.1 g of T. angustifolia pollen was evenly distributed on each plant with a soft hair brush over the entire plant at the frequency defined for each treatment. This amount of pollen per application corresponds to the recommended amount of 500 g/ha for supplementary feeding of phytoseiid predatory mites [P. ubiquitus. Assessments involved counting the number of P. ubiquitus mobiles found on fifteen individual 7 cm2 leaflet sections per plant under a stereoscopic microscope. These leaflets were collected at random through the entire plant. These counts were repeated for five consecutive weeks following the second release. The number of mites was compared among treatments with a linear mixed-effects model (LME) with treatment and time as fixed factors and plant identity as a random factor to correct for repeated measures. Non-significant interactions and factors were removed from the model. In addition, contrasts among predators were determined with general linear hypothesis testing  to determine the ideal frequency of application of ry mites . The firans in R ). All anans in R . P. ubiquitus collected in Belgium and The Netherland . No difference was observed in the reproduction capacity of P. ubiquitus . The highest oviposition was recorded when T. angustifolia was offered as a food source, followed by tomato pollen . Similar to the previous experiment, T. angustifolia induced the highest oviposition rate, followed by the tarsonemid prey mite T. fusarii . Weekly application of pollen resulted in the highest number of P. ubiquitus, followed by applications every other week . We opted to undertake the subsequent trials using only the Belgium strain because we had worked with this strain longer and did not have any reason to believe that the strains would behave differently on the other food sources. In this study, natural and factitious food sources were evaluated for the predatory mite  species ,55. HereTypha angustifolia pollen was found to be the most suitable food source for reproduction compared to a diet consisting of fungus  or prey mites. These results confirm the findings of previous studies showing pollen to be an excellent food for Iolinidae predatory mites, such as P. ubiquitus and the closely related Homeopronematus anconai (Baker) (Acari: Iolinidae) [T. angustifolia pollen. Only a few predatory mites were observed on tomato plants when tomato pollen was the only food source . This confirms that pollens derived from different plant species can differ greatly in their nutritional quality for predatory mites [Overall, linidae) ,37. Howery mites ,56. Furtry mites . Howeverry mites ,58. TherP. ubiquitus on the tomato crop. Finally, pollen quality and quantity depend on the tomato cultivar [P. ubiquitus may be affected by the tomato cultivar itself via plant feeding.Although tomato is a self-pollinating plant, the agitation of the anthers  facilitates shedding of the pollen grains. As a result, tomato pollen tends to be confined in flowers and only a limited amount of pollen will drop down to the leaves below to serve as food for the mites. Tomato growers introduce bumblebee hives in the greenhouse to facilitate crop pollination. By engaging in so-called “buzz-pollination”, bumblebees promote the release of tomato pollen from the anthers ,60, resucultivar ,62, whiccultivar . SimilarOrthotydeus lambi (Baker) was found to reduce the incidence and severity of grape powdery mildew, Uncinula necator (Schwein.) Burrill on both potted and field-grown vine plants [P. ubiquitus to control powdery mildew on tomato plants. The reproductive output of P. ubiquitus on powdery mildew in our laboratory trial was lower than that on a pollen diet and similar to that of the tomato leaf-only treatment. Similarly, Hessein and Perring [Cladosporium cladosporioides (Fres.) de Vries on grape leaf arenas yielded the same low number of H. anconai as compared to a grape leaf-only treatment. In contrast, high numbers of mites were retrieved when grape leaf arenas were supplemented with Typha sp. pollen. Although feeding on powdery mildew conidia and mycelia was observed, the exact mechanisms through which P. ubiquitus controls powdery mildew on tomato, as reported in the study of Pijnakker et al. [P. ubiquitus on powdery mildew is due to feeding on the fungus, plant-mediated effects, or a combination thereof.Tydeid mites are well known to engage in fungivory. For example, e plants ,65. Recee plants  reported Perring  reportedr et al. , remainsr et al. . FurtherP. ubiquitus on C. lactis in our study was negligible. Hence, C. lactis will most likely not be suitable for supporting Pronematus populations in a tomato crop. By comparison, the tarsonemid prey mite, T. fusarii, allowed reproduction in P. ubiquitus, albeit to a lesser extent than T. angustifolia pollen. Body size is an important factor determining the outcome of prey–predator interactions [C. lactis. Typha angustifolia pollen was clearly superior to the other foods tested, and although pollen would be the preferred food supplement based on our laboratory trials and previous results, it may nevertheless be interesting to test combinations of different food sources for rearing or maintaining on the crop. Mixing two diets was reported to yield higher reproduction than offering single diets in predatory mites [Food supplementation has become a standard practice in biocontrol programs in several crops to support the pre-establishment of generalist predators . Astigmatid prey mites are a relatively cheap alternative food source explored as a food supplement on crops, mainly to feed phytoseiid predatory mites [ractions ,68 and mry mites ,70,71.T. angustifolia pollen application frequency on P. ubiquitus establishment on tomato plants. Our results clearly show that pollen needs to be supplemented at least every other week to allow a good population build-up of P. ubiquitus. Populations can be maintained using less frequent applications (every three or four weeks). For the phytoseiid predatory mite Iphiseius degenerans Berlese on cucumber, van Rijn et al. [Typha latifolia recollected from the crop still allowed immature development and reproduction, although at a lower rate compared to freshly applied pollen.Based on the results of the laboratory trials, we tested the impact of n et al.  found thT. angustifolia pollen is a good food source to sustain the reproduction of P. ubiquitus. Application of T. angustifolia pollen allows the build-up of P. ubiquitus populations on tomato plants when applied at a frequency of at least once every two weeks at a dose rate of 500 g of pollen per hectare. Tomato pollen was found to support the reproduction of P. ubiquitus, but to a far lesser extent than Typha pollen. Finally, our laboratory trials indicated tarsonemid prey mites, such as T. fusarii, show potential to support populations of P. ubiquitus, whereas the larger astigmatid prey mite C. lactis was not suitable.In summary, we showed that


Dermatophilus congolensis is a bacterial pathogen mostly of ruminant livestock in the tropics/subtropics and certain temperate climate areas. It causes dermatophilosis, a skin disease that threatens food security by lowering animal productivity and compromising animal health and welfare. Since it is a prevalent infection in ruminants, dermatophilosis warrants more research. There is limited understanding of its pathogenicity, and as such, there is no registered vaccine against D. congolensis. To better understanding the genomics of D. congolensis, the primary aim of this work was to investigate this bacterium using whole-genome sequencing and bioinformatic analysis. D. congolensis is a high GC member of the Actinobacteria and encodes approximately 2527 genes. It has an open pan-genome, contains many potential virulence factors, secondary metabolites and encodes at least 23 housekeeping genes associated with antimicrobial susceptibility mechanisms and some isolates have an acquired antimicrobial resistance gene. Our isolates contain a single CRISPR array Cas type IE with classical 8 Cas genes. Although the isolates originate from the same geographical location there is some genomic diversity among them. In conclusion, we present the first detailed genomic study on D. congolensis, including the first observation of tet(Z), a tetracycline resistance-conferring gene. Dermatophilus congolensis (D. congolensis) causes dermatophilosis, a disease mostly found in cattle, goats, sheep, and occasionally in horses .,11.10,11https://cge.cbs.dtu.dk/services/SpeciesFinder/ (accessed on 5 February 2021)) in which all isolates were classified as belonging to D. congolensis. 16s rRNA sequences were also subjected to evolutionary analysis by the Maximum Likelihood method, which separated them into the same two groups as the ANI analysis  and ANI results. The only deflection to the gene order was represented by NCTC13039 and DSM 44,180 = NRBC 105,199 sequences, which are the only other D. congolensis sequences available in GenBank. Of the 40 isolates, only strain BTSK30 showed a discontinuity indicating a genomic variation at region 650–850 K. This suggests that the 40 D. congolensis from the West Indies are slightly different from those initially isolated from different geographical locations, i.e., DSM 44,180 = NRBC from Zambia .Synteny analysis can inform gene conservation among genomes. Although the 40 tet(Z) gene. Cluster 3 comprised isolates from two other geographical locations and included a single isolate with tet(Z). Surprisingly, an additional feature inferred from the cluster separation was the number of CRISPR spacers, where cluster 1 had genomes with the highest number of spacers (27–33), cluster 2 had genomes with 6 spacers and cluster 3 genomes had 5–4 spacers. The map in We further compared the 40 isolates and constructed an SNP-based phylogenetic tree  SNP Mat. The perD. congolensis genomes. Twelve isolates encoded the tet(Z) associated with resistance to tetracyclines  synthase III, OleB—Haloalkane dehydrogenase-like protein, OleC—AMP-dependent synthetase/ligase, OleD—NAD(P) H steroid dehydrogenase-like protein, (2) Auxin biosynthesis cluster contained APRT—anthranilate phosphoribosyltransferase, PRAI—Phosphoribosylanthranilate isomerase, Tsa—Tryptophan synthase alpha chain, and Tsb—Tryptophan synthase beta chain.We employed BlastKOALA to predict functional gene clusters of the 40 isolates compared to the reference genome. Overall, 51% of the genes in the draft genomes fall into 21 functional clusters as analyzed by the Kyoto Encyclopaedia of Genes and Genomes orthology, compared to 54% of the reference genome . The litclusters , with onclusters A, the si cluster B encodedr 2020))  revealedD. congolensis isolated in a single geographical region. The data reported provides a valuable insight into the genome organization as well as the local variability and evolution. The de novo assembled genomes were compared to the existing reference and found not to differ significantly, at least as shown by analysis of the ANI. Computing ANI provides a better resolution of differences or similarities between the strains of the same species (80–100% ANI) [D. congolensis is rather slow because little variation is noticed. Thus, we assume that our observation indicates an evolutionary differentiation. However, a much larger difference would be expected if the bacteria were to evolve much faster. Arguably, this would be more accurately stated if strains from different time points were analyzed. In comparison to the reference genome, no differences were found in the GC composition (58.8–59.2%), but a slight difference in the overall number of CDS in the BTSK isolates and reference genome was observed, where BTSK isolates had an average of 2527 CDS and the reference genome NCTC13039 had 2267 CDS.In this work, we provide a descriptive characterization of the main genomic components of 00% ANI) . In our The few strains whose genomes are available in GenBank to appear to be relatively homogenous, as well as most of our strains. However, depending on local geographical distribution, we saw some more differentiation. Geographical location can impose different evolutionary forces that can lead to alterations in genome sequences. We minimized the number of passages after achieving pure cultures, so as not to exert unnecessary evolutionary forces. Therefore, the sequences obtained reflect the original field isolates well.D. congolensis isolates will be necessary to better understand the diversity.Interestingly, the phylogenetic tree inferred from the genomic data also separates the isolates into 3 clusters according to the three different numbers of CRISPR spacer sequences found in the genomes of isolates. The difference in number and length of CRISPR spacers have been used to differentiate Spiraeoideae-infecting strains of Erwinia amylovora from different geographical locations ,25. AlthThe synteny shows good conservation of the genome structure in our collection as well as compared to the reference strains. We obtained a typical X-alignment with 18 genomes having an inverted alignment which indicates the likelihood of genomic inversion occurring symmetrically at the origin of replication . These rtet(Z) gene and to our knowledge, this is the first observation of tet(Z) in D. congolensis. tet(Z) was first described by Tauch et al. [tet(Z) located the gene on a plasmid (pAG1) [tet(Z) efflux pump in D. congolensis is worrisome because it can lead to therapeutic failure during treatment with tetracyclines, which are the recommended antibiotics for dermatophilosis. However, we have no data on the clinical implications of this resistance in the species and cannot assess whether it will negatively impair the treatment with tetracyclines.It is known that mobile genetic elements (MGE) are critical to adaptive bacterial evolution and particularly play a critical role in the transfer of antibiotic resistance genes and virulence factors. We detected the h et al.  in C. glh et al. , and hash et al. . It is td (pAG1) , and in D. congolensis genomes, it remains a grey area as none of them have been functionally studied in this bacterium. However, assessing the protein identity to D. congolensis suggests the structural likelihood, ideally, the closely related species probably would have given a higher percentage of sequence similarity. Adherence is an important initial step in infection. We identified groEL with an amino acid identity of 72% to M. tuberculosis. It mediates the folding of proteins that are responsible for pathogenesis in M. tuberculosis. groEL knockout strains of M. tuberculosis were reported incapable of forming biofilms [M. tuberculosis virulence, enhancing the growth in human macrophages and in early phases of pulmonary infection in mice. It is possible that such a mechanism may be in play during D. congolensis infection since the infiltration of macrophages has been observed in skin lesions of dermatophilosis in cattle [D. congolensis genomes were significantly identical to those of M. tuberculosis, but, as mentioned earlier, unless experimental evidence is derived it is difficult even to speculate on their function in D. congolensis.Although we derived a considerable list of potential virulence factors found in the biofilms  and granbiofilms , suggestbiofilms . Accordibiofilms , sigA mon cattle . OverallD. congolensis is in the smaller range, approximately 2.6 Mb, which may not allow for the encoding of many secondary metabolite gene clusters. The only well-represented gene cluster was the NRPS class as predicted by antiSMASH [The genome of ntiSMASH . Rarely,ntiSMASH .D. congolensis genome, which has a high GC content of 59.2% and encodes about 2527 genes. It has an open pan-genome, contains at least 49 potential virulence genes, and at least 23 antimicrobial resistance mechanisms, of which the tet(Z) is the only acquired resistance gene, and the first reported acquired resistance in this species. The analyzed draft genomes encode a single CRISPR array of Cas type IE with 8 Cas genes.In conclusion, we provide a molecular characterization of the The samples collected from animals were used for research, therefore, Institutional Animal Care and Use Committee approval was required . Animal owner consent for sample collection was also obtained.n = 85) were collected from cattle with dermatophilosis in different locations on the island of St. Kitts (17.36 N 62.78 W). Bacteria were isolated from scabs according to Quinn et al. [2 emitted by a lit candle inside the jar for 15 min. The topmost content of the tube was collected and spread onto blood agar plates, which were then incubated in the presence of 5% CO2 for at least 72 h. Small, yellow, raised, beta-hemolytic colonies embedded in the agar were presumed to be D. congolensis and were stained with Giemsa to confirm the tramtrack morphology. Identification was confirmed by PCR [Scab or swab samples  (accessed on 18 January 2020) [https://github.com/FelixKrueger/TrimGalore) (accessed on 5 February 2020) with parameters set as follows; quality 30: reads with Phred quality above 30; discard reads whose length is less than 50 bp after quality control; maintain paired-end reads order; retain reads that lost mate because of poor quality.Bacterial DNA was isolated with the AllPrep Bacterial DNA/RNA/Protein Kit  as described by the manufacturer. The quality and concentration of DNA were determined by Qubit 2.0. Illumina’s Nextera XT kit was used to prepare DNA libraries for sequencing. Admera Health, LLC, South Plainfield, NJ, USA was sourced for sequencing on Illumina HiSeq 2 × 150 platforms aiming at a sequencing depth of 30×. Quality control of reads was done with the FastQC software v0.11.5 (ry 2020) . Adapterhttps://narrative.kbase.us) (accessed on 20 February 2021), and the assembly quality was checked in QUAST v4.4 [https://narrative.kbase.us) (accessed on 30 June 2021) [http://enve-omics.ce.gatech.edu/) (accessed on 1 March 2021) [https://crisprcas.i2bc.paris-saclay.fr/CrisprCasFinder/Index) (accessed on 2 March 2020) [https://edgar.computational.bio.uni-giessen.de/cgi-bin/edgar.cgi) (accessed on 12 June 2020) [https://cge.cbs.dtu.dk/services/CSIPhylogeny/) (accessed on 12 June 2020) as described by Kaas et al. [The genomes were assembled with SPAdes v3.13.0 (AST v4.4 . Taxonomne 2021) . The genne 2021) . The Avech 2021) . CRISPR ch 2020) ,11. The ne 2020) . The phys et al. .https://card.mcmaster.ca/analyze/rgi) (accessed on 1 July 2020) [https://cge.cbs.dtu.dk/services/ResFinder/) (accessed on 1 July 2020) [https://castillo.dicom.unican.es/upload/) (accessed on 1 July 2020), a web-graph-based tool for reconstruction of plasmids from next generation sequence pair-end datasets was used to search for the presence of plasmids [https://phaster.ca/ (accessed on 15 August 2020) [We used the Comprehensive Antibiotic Resistance Database   and ResFly 2020)  to look plasmids ,46. PHASst 2020)  identifihttp://www.mgc.ac.cn/cgi-bin/VFs/v5/main.cgi?func=VFanalyzer) (accessed on 19 January 2021) [D. congolensis in VFDB, we run our submitted pre-annotated draft genome in FASTA format against a distantly related bacterium Mycobacterium. Finally, we used antiSMASH (https://antismash.secondarymetabolites.org/#!/start) (accessed on 10 February 2021) [D. congolensis genome. BlastKOALA https://www.kegg.jp/blastkoala/ (accessed on 5 May 2021) was used to determine the metabolic profiles of the isolates.Further, we searched for potential virulence genes using the Virulence Factor Database (VFDB) (ry 2021)  to exami


Enterococcus faecium (E. faecium) in UC remains unclear. Nevertheless, the potential mechanism of the protective effect of E. faecium remains unknown. In this study, a dextran sulphate sodium-induced (DSS-induced) colitis model was used to detect the underlying mechanism of E. faecium in maintaining gut homeostasis. ELISA was performed to detect the levels of cytokines . Furthermore, 454 pyrosequencing was used to investigate the microbiota composition in fecal samples. The results illustrate that E. faecium administration could prevent DSS-induced gut inflammation and intestinal flora imbalance. At the same time, the damage to intestinal mucosal barrier and tight junctions was partially repaired. These results demonstrate the preventive effect of E. faecium in DSS-induced intestinal injury. The present study provides new insights into the medicinal value of E. faecium for UC.The involvement of gut microbiota composition in ulcerative colitis is strongly supported by previous research. Growing evidence suggests that probiotic therapy protects against inflammatory bowel disease in animal models and patients. However, as a probiotic, the role of  Inflammatory bowel disease (IBD) is complex and presents many challenges to gastroenterologists . UlceratAlthough there have been many studies on UC, its pathological mechanism remains poorly understood . PreviouEnterococcus faecium (E. faecium) is a type of probiotic that inhabits the human gastrointestinal tract [Caenorhabditis elegans, Pedicord et al. observed that E. faecium colonization improves host intestinal epithelial defense programs in mice [E. faecium is associated with gut protection. In this study, the relationship between E. faecium administration and gut barrier integrity was analyzed.The intestinal microbiota, which seems to be a hidden organ composed of trillions of microorganisms, makes essential contributions to intestinal homeostasis . Probiotal tract . A previal tract , 12. Con in mice . IncreasE. faecium could have gut-protective effects [E. faecium in UC.The protective mechanism of probiotics against UC is multifaceted. The inhibition of intestinal pathogenic bacteria by probiotics plays a crucial role in the protective effect of probiotics in UC. Although previous studies have illustrated that  effects , 12, the effects . It is oIn this work, we investigated the role of E. faecium on dextran sulphate sodium-induced (DSS-induced) intestinal injury in order to provide new methods for UC treatment.E. faecium  and phosphate-buffered saline (PBS) (n = 16). On the eighth day, the bleeding scores and colon length were assessed. Half of the mice were sacrificed on day 8, and the other mice were sacrificed on day 14 to observe the effect of E. faecium on survival. All animal studies were approved by the Institutional Animal Care and Use Committee of Nanjing Medical University.Male C57BL/6 mice  were purchased from Shanghai Jiesijie Experimental Animal Co., Ltd. . Before the experiment, animals were adaptively fed for a week. During the experiment, the mice were weighed daily to observe the changes in body weight. All mice were maintained under standard conditions in an animal house at Nanjing Medical University. Experimental ulcerative colitis was induced by dextrose sulfate sodium . Mice were randomly chosen to receive different treatments via gavage from day 1 to day 7, including Stool consistency was assigned grades according to the following criteria: normal stool consistency with negative hemoccult: 0, soft stools with positive hemoccult: 1, very soft stools with traces of blood: 2, and watery stools with visible rectal bleeding: 3.Bleeding score was assigned grades according to the following criteria: normal stool consistency with negative hemoccult: 0, soft stools without positive hemoccult: 1, soft stools with positive hemoccult: 2, very soft stools with traces of blood: 3, and watery stools with visible rectal bleeding: 4.Colons from mice were excised, flushed, and collected. Colon tissue was fixed in Bouin's fluid (Sigma-Aldrich) and embedded in paraffin. Five micrometer sections of paraffin-embedded colons were prepared for further experiments. Alcian blue staining and HE staining were used to examine mucosal damage. For immunofluorescence (IF), the sections were incubated with anti-ZO-1 antibody  and anti-claudin antibody  at 4°C. All the results were observed by microscopy. The above histological analysis was performed in a blinded manner to avoid bias.E. faecium  were given a probe after fasting for 12 h by oral gavage, and urine was collected after 12 h. High-performance liquid chromatography was performed to measure the urine concentrations of lactulose and mannitol. The recovery rates of lactulose and mannitol were examined to evaluate intestinal permeability.Disaccharide permeability probes (50 mg/ml mannitol and 100 mg/ml lactulose) were used to detect intestinal permeability. DSS-treated mice administered phosphate-buffered saline or Fecal samples were collected from the metabolism cage on the 8th day after DSS treatment. After mixing with distilled water, the fecal samples were centrifuged . The samples were then extracted with ether and sulfuric acid. The ether phase was collected and used to examine the total SCFA concentrations by an Agilent 6890 N gas chromatography machine (Agilent Technologies CA USA).Blood samples were collected from the left ventricle on the 8th day after DSS treatment. A protein-based enzyme-linked immunosorbent assay (Elisa) was used to detect the levels of cytokines . In brief, after the antibody was coincubated overnight with the plate, the sample to be tested was added, followed by a reaction solution for coloration, and the level of cytokine was detected by colorimetry. Limulus test kit  was used to detect LPS.454 pyrosequencing was used to determine the microbiota composition by targeting the V3-V4 region of the bacterial 16S rRNA gene. Sequences were binned for a minimal sequence length of 300 bp, a minimal base quality threshold of 30 cycles, and a maximum homopolymer length of 6 cycles. Sequences were further clustered into operational taxonomic units (OTUs) or phylotypes. According to the results, Seqmatch and Blastall were used to assign the closest taxonomic neighbors and relative bacterial species.t-test. Differences with P < 0.05 were considered statistically significant.Data are presented as the mean ± SD. Between-group comparisons were performed by one-way ANOVA followed by Dunnett's multiple comparison test or Student's E. faecium in protecting the intestine from injury. As shown in E. faecium reduced the lethality rate remarkably compared with the DSS + PBS group. Consistent with the survival curve, mice treated with E. faecium lost significantly less body weight after DSS treatment (E. faecium treatment. Meanwhile, DSS induced colon necrosis and shorter colons, which were restored by E. faecium treatment (Figures E. faecium (E. faecium administration prevents DDS-induced colon damage in vivo.DSS-mediated colitis was used to detect the mechanisms of reatment . To furt Figures . Evaluat faecium . These rE. faecium may protect against DSS-mediated colitis. Previous studies have shown that intestinal mucosal damage is an important phenotype in DSS-induced intestinal injury. To investigate the intestinal mucosal after E. faecium treatment, we performed HE staining and alcian blue staining. As shown in Figures E. faecium. In addition, the levels of mucins were upregulated in the E. faecium-treated group. Furthermore, intestinal permeability was measured to test the effect of E. faecium administration on DSS-induced intestinal mucosa damage. As shown in E. faecium treatment. These results suggest that treatment with E. faecium may prevent intestinal mucosa damage.As the above research shows, E. faecium on the repair of tight junctions, we performed immunofluorescence to detect the expression and distribution of ZO-1 and claudins, which are tight junction-associated markers. As shown in E. faecium treatment. This result indicated that E. faecium was involved in the repair of the tight junction complex.To further explore the effect of E. faecium treatment on gut inflammation. Compared with the control group, the levels of plasma cytokines, including TNF-α, IL-1β, IL-6, and IL-10, were significantly decreased in the E. faecium-treated group (A previous study demonstrated that the relief of inflammation is related to intestinal permeability. Therefore, we tested serum samples from mice by ELISA to assess the protective effect of ed group .E. faecium in DSS-induced intestinal inflammation, the plasma LPS of mice was analyzed. As shown in E. faecium treatment. Furthermore, compared with the control group, the concentration of total SCFAs was significantly upregulated after E. faecium treatment (To further identify the potential mechanism of reatment .E. faecium treatment. As shown in E. faecium treatment. Specifically, the proportion of Ochrobactrum sp. and Acinetobacter sp. was reduced upon E. faecium treatment. Meanwhile, the proportion of Butyricicoccus sp. Lactobacillus sp., and Bifidobacterium sp. was upregulated. These results revealed the pivotal role of E. faecium in restoring the imbalance of intestinal flora induced by DSS.Trillions of microorganisms colonize the human gut. These microorganisms are related to regulating the immune system and balancing the internal environment. Therefore, in this study, we performed 16S rDNA sequencing analysis to detect changes in the microbiota after E. faecium treatment on a DSS-induced UC model was investigated. The remarkable finding of this study was that E. faecium markedly protected against intestinal damage induced by DSS and significantly decreased mortality. Based on alcian blue staining and gut permeability, E. faecium treatment contributed to intestinal mucosal injury. Meanwhile, the upregulation of ZO-1 and claudin confirmed that E. faecium was able to restore intestinal barrier function by increasing the concentration of tight junction proteins. Consistent with previous studies, E. faecium treatment reduced the intestinal damage induced by DSS, which further supports the hypothesis that E. faecium colonization improves host intestinal epithelial defense programs in rats [α, IL-1β, IL-6, and IL-10 are upregulated in patients with IBD [α, IL-1β, IL-6, and IL-10 was downregulated after E. faecium treatment.In this study, the effect of  in rats . Previewwith IBD –16. In tE. faecium treatment increased the levels of SCFAs. Moreover, our data also showed that E. faecium treatment can restore the inhibition of Butyricicoccus sp., which contributes to butyrate generation and was decreased in IBD [Increasing evidence has confirmed the anti-inflammatory functions of SCFAs. Previous studies have suggested that SCFA-producing bacteria are significantly downregulated in pediatric CD patients . A studyd in IBD , 19.Ochrobactrum sp. and Acinetobacter sp. were increased in UC patients, while Lactobacillus sp., Butyricicoccus sp., and Bifidobacterium sp. were decreased [Lactobacillus sp. and Bifidobacterium sp., can prevent or attenuate UC [Lactobacillus sp., Butyricicoccus sp., and Bifidobacterium sp., were upregulated in the E. faecium-treated groups, demonstrating the potential therapeutic value of E. faecium.Preview studies suggest that ecreased –24. The ecreased . Meijer nuate UC . In thisE. faecium administration prevents the intestinal mucosa and reduces damage to tight junctions, as summarized in E. faecium could reduce intestinal damage and improve the imbalance of the intestinal flora induced by DSS. This study explored the potential mechanisms of E. faecium treatment and provided new insight into the intervention and treatment of UC.In conclusion, in this study, it was confirmed that


Aeromonas veronii is a bacterial pathogen in aquaculture, which produces virulence factors to enable it colonize and evade host immune defense. Given that experimental verification of virulence factors is time-consuming and laborious, few virulence factors have been characterized. Moreover, most studies have only focused on single virulence factors, resulting in biased interpretation of the pathogenesis of A. veronii.A. veronii was first constructed followed by prediction and mapping of virulence factors on the network. When topological characteristics were analyzed, the virulence factors had higher degree and betweenness centrality than other proteins in the network. In particular, the virulence factors tended to interact with each other and were enriched in two network modules. One of the modules mainly consisted of histidine kinases, response regulators, diguanylate cyclases and phosphodiesterases, which play important roles in two-component regulatory systems and the synthesis and degradation of cyclic-diGMP. Construction of the interspecies PPI network between A. veronii and its host Oreochromis niloticus revealed that the virulence factors interacted with homologous proteins in the host. Finally, the structures and interacting sites of the virulence factors during interaction with host proteins were predicted.In this study, a PPI network at genome-wide scale for A. veronii by involving in signal transduction pathway and manipulate host biological processes by mimicking and binding competitively to host proteins. Our results give more insight into the pathogenesis of A. veronii and provides important information for designing targeted antibacterial drugs.The findings here indicate that the virulence factors probably regulate the virulence of The online version contains supplementary material available at 10.1186/s12866-021-02261-8. Aeromonas veronii is one of the main pathogenic bacteria that affect aquatic animals in freshwater and seawater [A. veronii can result in ulcerative syndrome, hemorrhagic septicaemia and mass mortality in aquatic animals such as Oreochromis niloticus [A. veronii, hence, it is classified among quarantine objects of water quality and food safety in some countries [A. veronii remain elusive.seawater . Infectiiloticus , which lountries , 4. Pathountries . AlthougA. veronii.Virulence factors can be classified into three categories based on their subcellular localization, including cytosolic, membrane associated, and secreted virulence factors . CytosolA. veronii PPI network at genome-wide scale is still not available.Protein-protein interaction (PPI) networks are powerful tools in predicting potential virulence factors , 10. ForArabidopsis thaliana [Saccharomyces cerevisiae [Caenorhabditis elegans [Drosophila melanogaster [Escherichia coli [Homo sapiens [Ustilaginoidea virens [Phomopsis longicolla [Several high-throughput experimental methods, such as yeast two-hybrid screening and tandem-affinity purification coupled with mass spectrometry, have been developed to identify large-scale PPIs . Due to thaliana , Saccharrevisiae , Caenorh elegans , Drosophnogaster , Escherihia coli , and Hom sapiens . To comp sapiens . Two pro sapiens . The PPIa virens  and Phomngicolla , have bengicolla , 27.A. veronii were predicted and mapped onto the PPI network. The importance of the virulence factors were first evaluated based on network topology properties. Two modules enriched by the virulence factors that played important roles in A. veronii infection were identified. The molecular mechanisms of pathogenicity was explored by constructing the interspecies PPI network between A. veronii and its host O. niloticus. Three-dimensional structures and interacting sites were added to the interspecies PPI network to provide more interaction details that would enhance understanding of host-pathogen interactions. Finally, key residues of the virulence factors that are involved in the interaction with different host proteins were identified. These data could be leveraged for accelerated development of new antibacterial agents.In this study, potential virulence factors of the aquatic pathogen A. veronii, the two commonly used interolog and domain-based methods were applied. With the interolog method, 13,201 A. veronii PPIs involving 1904 proteins were obtained. Among these, most PPIs (79.74%) were derived from the model organism E. coli, with only 0.47% constituting the A. veronii PPIs derived from A. thaliana. When the domain-based method was used, 8328 A. veronii PPIs among 1479 proteins were obtained after filtering with strict standards. Thus, a total of 21,418 A. veronii PPIs were predicted by the interolog and domain-based methods, involving 2494 proteins  terms of the PPIs were first calculated. The PPIs in the A. veronii PPI network had significantly higher biological process, molecular function or cellular component similarities compared with those in any random network , suggesting that the PPIs in the A. veronii PPI network had the tendency to be co-expressed. Although the percentages of PPIs decreased as absolute PCC values increased in both the A. veronii PPI network and the random networks  values in the A. veronii PPI network. When the degree and betweenness centrality were compared between the virulence factors and other proteins in the A. veronii PPI network, the results showed that the virulence factors had significantly higher degree and betweenness centrality than the other proteins . Notably, 16 and 15 out of the 17 virulence factors were annotated with the terms “phosphorelay signal transduction system” and “regulation of transcription, DNA-templated”, respectively (one virulence factor was not annotated with any term). When the topology characteristics of the module in the A. veronii PPI network was analyzed, an average degree of the proteins in the module was 23.61, which was higher than that in the A. veronii PPI network (17.18). After removing the 17 virulence factors, the average degree of the proteins in the module increased (24.38). These results indicate that the module connect other modules and has a great effect on the A. veronii PPI network. Analysis of the other module revealed that it was enriched by the virulence factors , thioredoxin 1 (Trx1), thioredoxin 2 (Trx2), S-adenosylmethionine synthetase (MetK), catalase, ATP-dependent Clp protease proteolytic subunit (ClpP), and peroxiredoxin 2 (Prx2), had higher degree in the interspecies PPI network  secreted virulence factors were first predicted, out of which, 36 virulence factors were found to interact with 1461 PIs Fig. . In the O. niloticus proteins, such as heat shock protein, elongation factor Tu, DNA-directed RNA polymerase subunit, Trx2, ribosomal protein S3, SdhA, peroxiredoxin 1 (Prx1), transcriptional regulator, MetK and ClpP, could interact with at least 5 proteins in A. veronii. In vertebrates, Trx2, MetK, ClpP, and Prx2 perform their functions by forming homo-dimers or homo-oligomers [A. veronii Trx2, MetK, ClpP, and Prx2 were homologous and could interact with O. niloticus Trx2, MetK, ClpP and Prx2, respectively. These results indicate that the virulence factors mimic and bind competitively to homologous proteins in host to interfere with host biological processes.Many ligomers –31. MoreO. niloticus proteins were predicted and the data stored at https://drive.google.com/drive/folders/18cHNUOSSJ5ugmFUL1_1QLVossf5ybYUY?usp=sharing. Figure A. veronii Trx1 and four O. niloticus proteins, including Trx2 , implying that A. veronii could directly manipulate host metabolic processes, component organization, and homeostasis to achieve successful infection. Group A Streptococcus have been reported to deliver virulence factors into host cells during infection to modulate host metabolism by causing endoplasmic reticulum stress to induce asparagine formation. The formed asparagine can then be sensed by group A Streptococcus to increase its growth rate [Each virulence factor interacted with an average of 11 proteins in wth rate . Thus, twth rate . In thisA. veronii probably hijack host pathways by mimicking host (O. niloticus) proteins, which is a common strategy used in pathogen-host interactions [The findings from this study revealed that virulence factors of ractions . Virulenractions . In thisractions , with thA. veronii proteins. Six organisms with large-scale experimental PPIs were selected as model organisms, including A. thaliana, S. cerevisiae, C. elegans, D. melanogaster, E. coli and H. sapiens. Protein sequences of these six model organisms were downloaded from the UniProt [A. thaliana and H. sapiens were obtained from the TAIR [A. veronii and the six model organisms. A stringent threshold  was set. Furthermore, the orthologs were analogized to predict A. veronii PPIs based on experimentally verified PPIs of the six model organisms.The interolog method was first used to infer the interactions between  UniProt  database UniProt , IntAct  UniProt , DIP [50 UniProt  databasethe TAIR  and HPRDthe TAIR  databasethe TAIR  was usedA. veronii PPIs. Experimentally verified domain-domain interactions as templates were collected from the 3did [A. veronii proteins were identified by PfamScan [e ≤ 1.00 × 10− 3). Three strict standards were adopted to improve the prediction accuracy of A. veronii PPIs [A. veronii PPI network was constructed based on the A. veronii PPIs predicted by the interolog and domain-based methods.The domain-based method was also used to infer the 3did  and iPfathe 3did  databasePfamScan   annotations, similar gene expression patterns, and the same subcellular localization. To assess the reliability of the predicted GOSemSim , includiA. veronii proteins and the known virulence factors by BLASTP. An A. veronii protein was predicted as potential virulence factor if the sequence identity was ≥40% and the coverage was ≥80% when aligned with a known virulence factor.Virulence factors known to affect pathogen-host interactions were collected from the PHI-base database . SequencA. veronii PPI network were calculated by the Cytoscape plugin NetworkAnalyzer [A. veronii PPI network and the number of interactions between the random proteins was also counted. This process was repeated 1000 times. The A. veronii PPI network was divided into modules by the Markov cluster algorithm (http://micans.org/mcl/). Only modules with at least five nodes were further analyzed. Fisher’s exact test was used to identify the modules enriched by the virulence factors and for annotation of the functions of modules.The degree and betweenness centrality of virulence factors and other proteins in the Analyzer , which iAnalyzer , 63. TheO. niloticus proteins only if it is translocated into host cell. Thus, secreted virulence factors were first predicted by EffectiveDB [O. niloticus proteins were downloaded from the UniProt [O. niloticus and the six model organisms , and potential domains of O. niloticus proteins were identified by PfamScan [O. niloticus proteins were predicted based on experimentally verified PPIs of the six model organisms and experimentally verified domain-domain interactions. Fisher’s exact test was used to perform functional enrichment analysis of O. niloticus proteins.A virulence factor has the potential to interact with ectiveDB , which i UniProt  database UniProt  was usedPfamScan . The intO. niloticus protein interactions were first searched in the PDB database [Homologous template complexes of virulence factor-database  by BLASTdatabase –69: (1) database  based onAdditional file 1: Table S1. Gene expression data under different conditions in Aeromonas veroniiAdditional file 2: Table S2.Aeromonas veronii protein-protein interaction network.Additional file 3: Table S3. Interspecies protein-protein interaction network.


Roseibium album  strains that were obtained from the octocoral Eunicella labiata. Genome annotation revealed multiple symbiosis factors common to all genomes, such as eukaryotic-like repeat protein- and multidrug resistance-encoding genes, which likely underpin symbiotic relationships with marine invertebrate hosts.Here, we announce the genomes of eight  Roseibium album  that has frequently been isolated from sessile, filter-feeding invertebrates such as sponges, corals, and oysters  is a Gr oysters , 4–6. To labiata , and we  labiata .R. album strains were isolated from E. labiata specimens that had been collected off the coast of Faro, Portugal, after plating of host-derived homogenates on half-strength marine agar medium followed by incubation at 18°C for 4 weeks (http://bbtools.jgi.doe.gov). Filtered reads were assembled into contigs using SPAdes v3.14.1 R. album  repeats , as reve repeats . MoreoveR. album  strains have been deposited in GenBank by the JGI. Accession numbers are listed in The genome sequences of the eight


The tumor microenvironment acts a pivotal part in the occurrence and development of tumor. However, there are few studies on the microenvironment of papillary renal cell carcinoma (PRCC). Our study aims to explore prognostic genes related to tumor microenvironment in PRCC.PRCC expression profiles and clinical data were extracted from The Cancer Gene Atlas (TCGA) and Gene Expression Omnibus (GEO) database. Immune/stromal scores were performed utilizing the ESTIMATE algorithm. Three hundred fifty-seven samples were split into two groups on the basis of median immune/stromal score, and comparison of gene expression was conducted. Intersect genes were obtained by Venn diagrams. Hub genes were selected through protein-protein interaction (PPI) network construction, and relevant functional analysis was conducted by DAVID. We used Kaplan–Meier analysis to identify the correlations between genes and overall survival (OS) and progression-free survival (PFS). Univariate and multivariate cox regression analysis were employed to construct survival model. Cibersort was used to predict the immune cell composition of high and low risk group. Combined nomograms were built to predict PRCC prognosis. Immune properties of PRCC were validated by The Cancer Immunome Atlas (TCIA).We found immune/stromal score was correlated with T pathological stages and PRCC subtypes. Nine hundred eighty-nine differentially expressed genes (DEGs) and 1169 DEGs were identified respectively on the basis of immune and stromal score. Venn diagrams indicated that 763 co-upregulated genes and 4 co-downregulated genes were identified. Kaplan-Meier analysis revealed that 120 genes were involved in tumor prognosis. Then PPI network analysis identified 22 hub genes, and four of which were significantly related to OS in patients with PRCC confirmed by cox regression analysis. Finally, we constructed a prognostic nomogram which combined with influence factors.Four tumor microenvironment-related genes  were identified as biomarkers for PRCC prognosis.The online version contains supplementary material available at 10.1186/s12885-021-08319-0. The incidence of renal cell carcinoma (RCC) is approximately 431,288 in 2020, accounting for 2.2% of common malignant neoplasm on a global scale . In geneTumor microenvironment (TME) is an intricate system comprised of tumor cells, surrounding immune and inflammatory cells, tumor-related fibroblasts and nearby stromal tissues, and varieties of cytokines and chemokines . RelevanIn this study, we extracted PRCC datasets from TCGA and GEO and obtained relevant immune and stromal score calculated by ESTIMATE. After a series of bioinformatics analyses, several tumor microenvironment-related genes in connection with the prognosis of PRCC patients were selected and validated, and were used to establish nomograms as potential tools to evaluate the outcomes of PRCC.http://xena.ucsc.edu/). Another dataset (GSE2748) was obtained from the GEO database. And corresponding clinical information including overall survival (OS) time and status was extracted from the ONCOMINE database (www.oncomine.org/). Immune and stromal scores were calculated by ESTIMATE packages of R.Level_3 gene expression patterns for PRCC was downloaded from TCGA via UCSC Xena  was used to analyze the molecular interactions of DEGs and construct a PPI network visualized by Cytoscape (v3.7.2). Then we used the MCODE tool to find densely connected hub genes based on topology.The String database (http://david.abcc.ncifcrf.gov), comprising three aspects: biological process, cellular component, and molecular function. And Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was applied for searching functional pathways of hub genes. P < 0.05 was deemed statistically significant.Gene ontology (GO) analysis was performed by DAVID (http://cibersortx.stanford.edu) was used to estimate the composition of 22 immune cell types of PRCC. The percentages of these types were visualized in a bar graph by using ggplot2 package of R. Immunophenogram of TCIA (https://tcia.at/), influenced by four factors: MHC molecules, immunomodulators, effector cells and suppressor cells, was applied to evaluating the immune properties of PRCC. And IPS scores are positively associated with the immunogenicity.CIBERSORT algorithm .P < 0.05). And the average immune scores of type 2 were higher than type 1 (P < 0.05). Similarly, type 2 had higher stromal scores compared to type 1  and stromal score (− 1821.04 to 1469.55) of 291 samples of TCGA and 34 samples of GEO were calculated. We attempted to analyze the relations between immune/stromal score and TNM, stage, subtype and grade . We used IPS to predict the potential response of ICI (immune checkpoint inhibitors) for PRCC patients and investigated the association between IPS and 4-genes model of PRCC. Statistical analysis indicated that the IPS of low risk group was higher than high risk group  adaptive immune responses . BesidesThe interaction of PRCC and its TME affected the whole process from tumor occurrence and progress to metastasis and recurrence, which created plenty of opportunities for diagnosis, treatment and prognosis of PRCC patients. In present study, we attempted to find tumor microenvironment-genes associated with PRCC patients’ survival. Our results may provide some related data to future research on the relations of PRCC and TME. However, the mechanism of PRCC and its microenvironment could be extremely complex, our analysis based on the TCGA and GEO database and bioinformatic tools is only one part of it. Further research and analysis based on large samples will be essential.By analyzing the TCGA and GEO database and applying ESTIMATE algorithm and a series of bioinformatic methods, we obtained tumor microenvironment associated genes , which were related to the clinical outcome of PRCC patients. These genes could be used as biomarkers for predicting PRCC prognosis.Additional file 1: Fig. S1 Comparison of differentially expressed genes. A. Volcano plots of differentially expressed genes based on immune score. B. Volcano plots of differentially expressed genes based on stromal score. C. Venn plots of co-downregulated genes. D. Venn plots of co-upregulated genes. GO analysis of 763 co-upregulated genes. E. Biological process. F. Cellular component. G. Molecular function.Additional file 2: Supplementary Table S1 763 upregulated common genes and 4 downregulated common genes between TCGA and GEO samples.Additional file 3: Supplementary Table S2 120 genes associated with PRCC overall survival.Additional file 4: Fig. S2 PPI networks of significant module. A. module1, B. module2. The node color changes gradually from pink to red indicating the ascending order of the degree of the genes. C KEGG pathway analysis for 17 hub genes.Additional file 5: Supplementary Table S3 Univariate cox proportional hazard regression analysis of 22 genes based on TCGA database.Additional file 6: Supplementary Table S4 Multivariate cox regression results of 4-gene prognostic model.Additional file 7: Fig. S3 ROC curve analysis. The predictive ability of 4-gene model in different ages (A), genders (B), stages (C) and subtypes (D) of PRCC. AUCs of 4-gene model were compared with age, gender, TNM, stage, tumor grade of TCGA (E) and GEO (F).Additional file 8: Fig. S4 The association between 4-gene model and clinicopathologic features. A. age, B. gender, C. T, D. M, F. N, G. stage, H. subtype.


Schedonorus-Lolium complex of the subtribe Loliinae (Poaceae) includes several economically important forage and turf grasses. This complex encompasses Lolium Linnaeus, 1753, Festuca Linnaeus, 1753 subgenus Schedonorus  Petermann, 1849 and Micropyropsis Romero Zarco et Cabezudo, 1983. New FISH results of 5S and 18S–26S rDNA sequences are presented for three species and the results are interpreted in a review of distribution patterns of 5S and 18S–26S rDNA sequences among other species in the complex. Micropyropsistuberosa Romero Zarco et Cabezudo, 1983 (2n = 2x = 14) displayed a distribution pattern of rDNA sequences identical to that of F.pratensis Hudson, 1762, supporting a close phylogenetic relationship at the bottom of the phylogenetic tree. “Loliummultiflorum” Lamarck, 1779 accessions sourced from Morocco showed a different pattern from European L.multiflorum and could be a unique and previously uncharacterised taxon. North African Festucasimensis Hochstetter ex A. Richard, 1851 had a marker pattern consistent with allotetraploidy and uniparental loss of one 18S–26S rDNA locus. This allotetraploid has previously been suggested to have originated from a hybrid with Festucaglaucescens . However, the distribution patterns of the two rDNA sequences in this allotetraploid do not align with F.glaucescens, suggesting that its origin from this species is unlikely. Furthermore, comparisons with other higher alloploids in the complex indicate that F.simensis was a potential donor of two sub-genomes of allohexaploid Festucagigantea (Linnaeus) Villars, 1787. In the overall complex, the proximal locations of both rDNA markers were conserved among the diploid species. Two types of synteny of the two markers could, to a considerable extent, distinguish allo- and autogamous Lolium species. The ancestral parentage of the three Festuca allotetraploids has not yet been determined, but all three appear to have been sub-genome donors to the higher allopolypoids of sub-genus Schedonorus. Terminal locations of both the markers were absent from the diploids but were very frequently observed in the polyploids.The  Lolium Linnaeus, 1753 with ten diploid species and fescues of the genus Festuca Linnaeus, 1753 subgenus Schedonorus  Petermann, 1849 are closely related and, together with Micropyropsis Romero Zarco et Cabezudo, 1983, form the “Schedonorus-Lolium complex”, belonging to the family Poaceae Barnhart, 1895, subtribe Loliinae Dumortier, 1829  can overcome such problems, and provide enhanced pictures of chromosome architecture, leading to clear karyotype and genome discrimination (rDNA), one coding for 5S and the other for 35S rRNA arrays are universally present in plants. Tandemly repeated blocks of these genes are located independently at particular chromosomal sites and provide species-specific markers  and tandemly repeated blocks of these units form the nucleolar organizer regions (NORs) or secondary constrictions on chromosomes. FISH mapping of 5S and 35S rDNA sequences is widely used to compare the chromosomal structural changes of related species and to infer the karyoevolutionary variations that accompany species diversification , a 1.8 kb fragment from Trifoliumrepens Linnaeus, 1753 containing almost the entire 18S rDNA sequence representing the 35S rDNA and pTr5S (GenBank accession number AF072692), a 596 bp DNA fragment encoding the T.repens 5S rRNA. 35S and 5S rDNA probes were directly labelled with fluorochromes Fluor-X-dCTP and Cy-3-dCTP , respectively by nick translation according to manufacturer’s specifications. Double target FISH using the above DNA probes, post-hybridisation washing and counterstaining of somatic chromosomes with DAPI were carried out as described earlier . At least five good quality early to late metaphase cells from each plant were used for analysing hybridization signals.The DNA probes used for  earlier . ChromosFISH mapping using 35S and 5S rDNA sequences as probes on pro-metaphase or metaphase chromosomes of Loliumperenne Linnaeus, 1753 (2n = 2x = 14) are given in Fig. rDNA signals representing three loci were located proximally on three pairs of chromosomes  or secondary constrictions, were frequently decondensed and sometimes stretched in our flame-dried somatic chromosome preparations. These loci are positioned pericentromerically, and the cloudy decondensed and stretched 35S rDNAFISH signals could be observed joining the two condensed parts of NOR-bearing chromosomes  of north European/Mediterranean origin produced rDNAFISH signals identical to the pattern observed for L.perenne  of Moroccan origin displayed only two pairs of NORs  and AFLP fingerprinting revealed a close phylogenetic relationship with European broad-leaved fescues, especially with hexaploid F.gigantea, . Instead, the distribution patterns are consistent with the possible involvement of F.simensis in the formation of 6x F.gigantea (Linnaeus) Villars, 1787. Festucapratensis Hudson, 1762 is a putative diploid sub-genome donor of allohexaploid F.gigantea (F.simensis (present results) and F.gigantea  consistently occurred in all Lolium species. The number of 35S loci displayed has previously been noted as a distinguishing feature between F.pratensis (one locus) and Lolium species (more than one locus) , L.persicum Boissier et Hohenacker, 1854, L.temulentum Linnaeus, 1753, L.remotum Schrank, 1789, L.rigidumvar.rottbollioides Heldreich ex Boissier, 1884 and L.canariense Steudel, 1855, three in L.perenne and L.multiflorum (European origin) to four or five in L.rigidumvar.rigidum Gaudin, 1811. These results were consistent with those of angiosperms in general, where numbers of 5S sites vary considerably less than 35S sites (A single 5S e locus) . All the5S sites .rDNA loci can be located on the same chromosome (syntenic) or on separate chromosomes (non-syntenic)  have synteny  group, represented by L.persicum, L.temulentum, L.remotum, and subspecies and races of L.rigidum, both types of rDNA sequences were adjacent on the same chromosome arm, with 35S always distal to 5S. L.canariense shows the diploid Micropyropsis-F.pratensis arrangement with proximally located 5S and 35S rDNA loci on separate chromosomes as well as an additional pair of 35S loci  . The Mac35S loci . Howevereny Fig. . The synSchedonorus. All the species are allopolyploid (x = 7) and no apparent changes in the ancestral karyotype.The data presented in Fig. olyploid  and showF.mairei St. Yves, 1922 and F.glaucescens to eight in decaploid F.letourneuxiana (Festucaarundinaceavar.letourneuxiana (St. Yves) Torrecilla et Catalán, 2002) while 35S numbers ranged from one in tetraploid F.simensis to six in F.letourneuxiana . In contrast, terminal 35S loci were more frequent. Five species mapped at least one 35S locus in the terminal region while four displayed exclusively terminal 35S loci  or two 35S loci adjacent to each other on the same arm (F.mairei)  Fig. . Three piosperms . None ofhic form .F.mairei and F.glaucescens have been suggested as the ancestral parents of allo-octoploid F.atlantigena (Festucaarundinaceasubsp.atlantigena (St. Yves) Auquier, 1976) based on the formation of fertile interspecific hybrids between the two suggested ancestral parental species , as the putative sub-genome donors to the allohexaploid and octoploid species, provide a novel example of sequential allopolyploidisation. The putative progenitors of all three allotetraploids remain unknown. However, nuclear and chloroplast DNA sequence analyses (FISH mapping (F.mairei and F.glaucescens. The tetraploid species that became the sub-genome donors for higher ploidy fescues had terminal 5S and 35S loci that were largely conserved in the derivative species (Fig. Schedonorus-Lolium complex diploids studied so far, none have shown terminal localization of either marker, and neither were their DNA sequences consistent with them having been progenitors of these tetraploids (F.scariosa Lagasca y Segura ex Willkomm, 1861, belonging to the sub-genus Scariosae outside the Schedonorus-Lolium complex, was a potential ancestral parent for allotetraploid F.mairei. The likelihood of involvement of diploid sub-genome donor species from outside the Schedonorus-Lolium complex should be further explored using molecular and cytogenetic methods, including genomic in situ hybridization.The three allotetraploids (analyses , support mapping  indicateies Fig. . Among traploids . Harper Lolium and the post-polyploidisation changes in the Festuca species have apparently occurred without any obvious changes in the symmetrical bi-armed karyotype that is a consistent feature of the Schedonorus-Lolium complex. Such lability in the absence of obvious structural changes might be attributable to paracentric chromosome rearrangements and/or the activity of transposable elements (The variations in numbers of 35S sites in elements .rDNA sequences to seven of the ten known Lolium species and has added F.simensis to the list of seven polyploid fescue species already characterised. It has also explored the distribution patterns of rDNA loci within the Schedonorus-Lolium complex and considers some possible evolutionary trends. While these patterns can be used to deduce relationships among the higher polyploid Festuca species, the diploid progenitors of the allotetraploid species remain unidentified and enigmatic.This report has extended the distributional data on the FISH. AVS and NWE provided significant help in improving the manuscript. All authors read and approved the final manuscript.HAA designed the study with AVS and WMW. HAA performed the experiment, analysed the data and wrote the manuscript with co-writing from WMW. NWE isolated the DNA and labelled all the probes for


Cynara cardunculus L. is a cardoon species native to the Mediterranean region, which is composed of three botanical taxa, each having distinct biological characteristics. The aim of this study was to examine wild populations of C. cardunculus established in Portugal, in order to determine their genetic diversity, geographic distribution, and population structure. Based on SSR markers, 121 individuals of C. cardunculus from 17 wild populations of the Portuguese Alentejo region were identified and analysed. Ten SSRs were found to be efficient markers in the genetic diversity analysis. The total number of alleles ranged from 9 to 17 per locus. The expected and observed means in heterozygosity, by population analysed, were 0.591 and 0.577, respectively. The wild population exhibited a high level of genetic diversity at the species level. The highest proportion of genetic variation was identified within a geographic group, while variation was lower among groups. Geographic areas having highest genetic diversity were identified in Alvito, Herdade da Abóboda, Herdade da Revilheira and Herdade de São Romão populations. Moreover, significant genetic differentiation existed between wild populations from North-Alentejo geographic locations  and Centro Hortofrutícola, compared with other populations. This study reports genetic diversity among a representative number of wild populations and genotypes of C. cardunculus from Portugal. These results will provide valuable information towards future management of C. cardunculus germplasm. Cynara cardunculus L. is a perennial species native to the Mediterranean area and is well adapted to hot and dry climates. It belongs to the Asteraceae family, and it comprises three botanical varieties: C. cardunculus var. scolymus (L.) Fiori (globe artichoke), C. cardunculus var. altilis (DC). (cultivated cardoon) and C. cardunculus var. sylvestris (Lamk) Fiori (wild cardoon) , ca weight) , 23. Cyn weight) , 25.C. cardunculus genetic diversity. Genetic markers, such as microsatellites , are informative molecular markers and are useful in breeding programs for marker-assisted selection. The technical ability to characterize germplasm . The modal value of this distribution is the true K, or the uppermost level of structures, here designating three clusters.(TIF)Click here for additional data file.S1 TableThe multiplex loci combination and the respectively dyes used during our work, are also indicated.(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 TableThe asterisk mark shows the largest values in the Delta K column.(XLSX)Click here for additional data file.


Giardia lamblia. The disease became a global health issue due to development of resistance to commonly used drugs. Since many plant-derived products have been used to treat many parasitic infestations, we aimed to assess the therapeutic utility of Artemisia annua (A. annua) for giardiasis. We showed that NO production was significantly reduced whereas serum levels of IL-6, IFN-γ, and TNF-α were elevated in infected hamsters compared to uninfected ones. Additionally, infection resulted in increased numbers of intraepithelial lymphocytes and reduced villi heights, goblet cell numbers, and muscularis externa thickness. We also showed that inducible NO synthase (iNOS) and caspase-3 were elevated in the intestine of infected animals. However, treatment with A. annua significantly reduced the intestinal trophozoite counts and IEL numbers, serum IL-6, IFN-γ, and TNF-α, while increasing NO and restoring villi heights, GC numbers, and ME thickness. Moreover, A. annua treatment resulted in lower levels of caspase-3, which indicates a protective effect from apoptotic cell death. Interestingly, A. annua therapeutic effects are comparable to metronidazole. In conclusion, our results show that A. annua extract is effective in alleviating infection-induced intestinal inflammation and pathological effects, which implies its potential therapeutic utility in controlling giardiasis.Giardiasis is an intestinal protozoal disease caused by  Giardia lamblia , which is a flagellated protozoan infecting the small intestine of humans and animals [G. lamblia annually [Giardiasis is one of the most common human intestinal protozoal infections . It is c animals . Approxiannually . The preannually . In Egypannually ,6,7,8.Giardiasis is transmitted through the ingestion of cysts in food and water. Infection can remain asymptomatic or can present in the form of acute or chronic diarrhea that is accompanied by abdominal pain and nausea . ChildreG. lamblia-infected intestines have been described and the organism is usually found on the villus surface or between the intestinal villi [G. lamblia is associated with mucosal inflammation, intraepithelial inflammatory cell infiltration, and changes in the villus architecture [Pathological changes in al villi . Infectiitecture . Additioitecture . Moreoveitecture ,18. Furtitecture .G. lamblia with elevated IL-6 mRNA levels in infected mice [G. lamblia infection [G. lamblia infection in mice [G. lamblia trophozoites [G. lamblia, due to its cytotoxicity [G. lamblia trophozoites compete with the host for arginine [Previous studies of giardiasis in rodents have highlighted the essential role of IL-6 in promoting immunity against ted mice . Interesnfection ,21. IgA  in mice ,23. Studhozoites ,25. In ftoxicity ,26. Intearginine , or incrarginine . The prearginine . TherefoG. lamblia to these compounds [Drugs such as furazolidone, quinacrine, paromomycin, benzimidazole compounds, 5-nitroimidazole compounds, and nitazoxanide have long been used in treatment of giardiasis . Howeverompounds ,34. ConsArtemisia have long been used to treat various human diseases [Artemisia annua (A. annua), is highly effective in the treatment of quinine-resistant malaria and is a potent antioxidant due to its high phenolic content [A. annua is effective against trypanosomiasis [A. annua [Medicinal plants such as diseases ,36. Thisdiseases ,38. Arte content ,40. Sevesomiasis , schistosomiasis , toxoplasomiasis , leishmasomiasis ,45, and somiasis . MoreoveA. annua .A. annua in an animal model of giardiasis. We show that A. annua is effective in reducing G. lamblia trophozoite count, inflammation, and parasite-induced intestinal pathological changes. Interestingly, all these effects were comparable to metronidazole, the currently used anti-giardiasis drug, which suggests that A. annua could be a potential and safe therapeutic for giardiasis.In the current study, we evaluated the efficacy of This study was conducted at the animal house and Medical Parasitology Department, Faculty of Medicine, South Valley University, Qena, Egypt. All experiments were performed according to the regulations of Animal Care and Use Committee at the Faculty of Medicine, South Valley University, Qena, Egypt. The study design was approved by the Institutional Research Committee at the Faculty of Medicine, South Valley University, Qena, Egypt.Thirty-two male golden hamsters, aged 3–4 weeks and weighing 150–200 g each, were procured from the animal house at Theodore Bilharz Research Institute (TBRI), Giza, Egypt. Hamsters were bred under specified pathogen-free conditions. To ensure that hamsters were free from intestinal parasites, stool samples were examined for three consecutive days before starting the experimentsThe plant material was collected during the flowering stage from a cultivated field near South Valley University. A voucher specimen of the plant (code: Aa.78) was kept in the herbarium, Department of Pharmacognosy, Faculty of Pharmacy, South Valley University, Egypt. Collected fresh plant was washed with running tap water to remove any associated soil particles. The plant was allowed to dry at room temperature before being ground into a fine powder.A. annua ethanolic extract was prepared according to the method described previously [A. annua ethanolic extract was prepared by maceration of 200 g powdered plant material with 1 L of 95% ethanol. Plant material was allowed to macerate for 16 h at room temperature and then filtered. The process was repeated three times. The combined filtrates were evaporated to dryness in a rotary evaporator under reduced pressure to produce the crude dry ethanolic extract. The obtained dry extract (16 g) was stored at 4 °C for subsequent preparation of the required animal experiment doses.eviously . A. annuG. lamblia cysts were collected from stools of patients of the outpatient clinics in the University hospitals, South Valley University, Qena, Egypt. All stool specimens were processed immediately at the parasitology laboratory. Giardia cyst viability was confirmed using 0.1% eosin vital staining. Cysts were then counted in 0.1 mL of sediment and the concentration process was repeated with more stool samples until the suspension contained about 10,000 viable cysts/mL of phosphate-buffered saline. Animals of different groups were each infected orally with 1 mL of phosphate-buffered saline containing 10,000 cysts [00 cysts .A. annua (AA) group were infected with 1 mL phosphate-buffered saline containing 10,000 giardia cysts orally [A. annua ethanolic extract for 3 consecutive days, respectively [A. annua ethanolic extract were selected according to previously published reports [Animals were equally divided into 4 groups, 8 animals each. UI group included uninfected animals. Animals of IGL, metronidazole (Met) treatment positive control group, and s orally . Three wectively ,51. Staiectively . Doses o reports .G. lamblia trophozoites in five successive fields/animal as previously described [After sacrificing hamsters, the small bowel was removed and the duodenal contents were subjected to parasitological examination in order to count the number of escribed ,54. Blood samples were collected and sera were separated and stored at −20 °C until used. Levels of NO were determined by measuring NO end products (NOx) in serum using Biodiagnostic colorimetric assay kit . The levSpecimens of 2–5 cm from the proximal part of the small intestine (duodenum and jejunum) removed from sacrificed hamsters were fixed in 10% formalin and embedded in paraffin. Sections of 5 µm thickness were stained with hematoxylin and eosin or Periodic Acid Schiff (PAS) and hematoxylin as previously described .Three animals/group were used for the assessment of these parameters. A total of 15 images of random fields/group were used. Images at 100× magnification were used for assessment of the villi length and at 400× magnification for measuring muscularis externa thickness using Fiji ImageJ software, version 1.52p.Three animals/group were used for the assessment of IEL and goblet cell numbers. Images captured at 200× were used for assessing IEL. We counted 2500 enterocytes and IELs and then the ratios of IEL/100 enterocytes were calculated . For gob2O2 in ethanol. Sections were incubated with anti-caspase-3 antibodies  or anti-inducible nitric oxide synthase (iNOS) polyclonal antibodies  for 60 min at room temperature. Next, sections were washed with TBS containing 0.05% Tween-20 (TBS-T). Sections were then incubated with HRP-conjugated goat anti-rabbit secondary antibodies  at a dilution of 1:5000 for 1 h at 4 °C. After washing in TBS-T, the color was developed by incubating sections with 0.05% diaminobenzidine (DAB) and 0.01% H2O2 for 3 min. For negative control, we omitted the primary antibodies during staining of some slides. Images were captured using Leica light microscope equipped with a digital camera in the Histology and Cell Biology Department, Assiut University, Faculty of Medicine, Assiut, Egypt.Paraffin sections of the small intestine of different groups were deparaffinized and rehydrated with descending percentages of ethanol. Sections were boiled in citrate buffer (pH 6.0) in a microwave for epitope retrieval. Endogenous peroxidases were then blocked with 3% Hp < 0.05.The collected data were analyzed by SPSS , version 20 for Windows. All values were expressed as mean ± standard deviation (SD). Differences between groups were determined using one-way ANOVA test to compare the mean values between treated and control groups for different variables by the Bonferroni post hoc test. Differences were considered significant when A. annua (AA) treatment groups for trophozoite count revealed that these animals had significant reductions in trophozoite count compared to the infected untreated group (IGL) (98.3% and 92.5% reductions respectively)  and ctively) . These dG. lamblia infection has been well established [G. lamblia (IGL group) significantly reduced NO compared to uninfected animals (UI). In contrast, treatment of infected hamsters with metronidazole (Met group) significantly increased NO compared to IGL group (p < 0.001) and UI group (p < 0.001). Similarly, treatment with A. annua extract (AA group) significantly induced NO production compared to IGL group (p < 0.001). However, there was no significant difference between metronidazole and A. annua treatment on NO production  compared to uninfected animals (UI group) (p < 0.05). Treatment of infected animals with metronidazole (Met group) or A. annua extract (AA group) resulted in a significant reduction in serum IL-6 compared to IGL group (p < 0.05) (A. annua treatment (p < 0.05)  showed significant increase in the serum IFN-γ compared to the uninfected animals (UI group) (p < 0.05) (A. annua compared to IGL group (p < 0.05) (A. annua extract. As shown in p < 0.05). However, treating infected animals with metronidazole or A. annua extract significantly reduced serum TNF-α levels compared to the infected untreated hamsters by 4-fold and 3-fold, respectively. Next, we measured serum IL-6 levels in different animal groups. Zhou et al. previously reported increased IL-6 levels during the course of giardiasis in mice . Therefo < 0.05) b. Howeve < 0.05) b. A rece < 0.05) c. On the < 0.05) c. Howeve < 0.05) c. We alsA. annua treatments on giardiasis-induced small intestinal lesions, we stained sections taken from the proximal parts of the small intestine with hematoxylin and eosin (H&E) stains. Small intestinal sections taken from UI animals showed normal intestinal villi and crypts  (A. annua (AA) restored the normal villi length (p < 0.001). We did not find a significant difference between the villi length in Met and AA group.Next, we measured the villi length and found a significant reduction in the villi length in sections taken from IGL group animals compared to UI animals (< 0.001) . HoweverG. lamblia trophozoites in the intervillous spaces . However, treatment with metronidazole (Met) or A. annua (AA) significantly decreased the number of IELs in epithelial cells (p < 0.001). We did not find a significant difference between the effect of metronidazole and A. annua on the number of IELs.Next, we counted the IELs/100 epithelial cells on images of H&E-stained sections taken at 200× magnification according to previously published methods . As showA. annua treatment can restore goblet cell numbers, we stained our small intestinal sections with Periodic Acid Schiff reagent (PAS) and hematoxylin. In uninfected animals, goblet cells are distributed among the epithelial cells in the epithelium covering the villi  significantly reduced goblet cell number compared to the uninfected (UI) animals (p < 0.001). On the other hand, treating infected animals with metronidazole (Met) or A. annua (AA) significantly increased the goblet cell number compared to the infected untreated animals (p < 0.001). No significant difference was found between the effect of metronidazole and A. annua on goblet cell number.To confirm our observations, we counted the number of goblet cells/100 epithelial cells in images of small intestinal sections stained with PAS and hematoxylin. A total of 2500 epithelial cells were counted according to previously published methods . Infecti animals  . On the other hand, treating infected animals with metronidazole (Met) or A. annua (AA) extract significantly increased the muscularis externa thickness compared to the infected untreated animals (p < 0.001). No significant difference was observed between the effects of the two treatments.To confirm our results, we measured the thickness of the muscularis externa, using Fiji ImageJ software version 1.52p, in 15 pictures of random fields taken at 400 × magnification. As shown in G. lamblia infection and that NO is important for elimination of the parasite [A. annua would modulate the expression of iNOS, we employed immunohistochemistry to examine iNOS expression in tissue from the small intestine. In uninfected animals, we showed that iNOS is expressed in connective tissue cells in the core of the villi  is highly expressed following parasite ,60. To ihe villi a, arrowshe villi b, arrowshe villi c, arrowsing iNOS d, arrowsG. lamblia trophozoites induce apoptosis in cultured enterocytes [A. annua extract would attenuate giardia-induced apoptosis in small intestine. We immunostained our small intestinal sections with anti-caspase-3 antibodies. In uninfected control animals, weak caspase-3 signals were observed in the enterocytes, lamina propria cells . This could be attributed to either the inhibitory effect of A. annua on G. lamblia trophozoite growth or inhibition of arginase release by macrophages. This assumption is partially supported by the work of Yang and colleagues who showed that A. annua derivatives inhibited activation of macrophages in vitro [A. annua treatment, were associated with decreased expression of iNOS. The increased NO production in A. annua-treated animals could be explained by Li etal.’s study. This study demonstrated that neuronal nitric oxide synthase (nNOS) plays a more significant role in giardiasis than iNOS [A. annua could increase NO levels through increased expression of nNOS rather than iNOS. However, this speculation needs further investigation.Several studies have suggested that NO plays an important role in the elimination of hozoites ,65. To sf growth . Additioarginine . Furtherarginine . In lineficiency ,60. Howein vitro . Howeverhan iNOS . TherefoG. lamblia. Our findings are in line with previous reports that demonstrated increased cytokine production in TLR2-deficient mice which led to reduction in parasite burden and alleviated giardiasis [G. lamblia infection [G. lamblia infection [G. lamblia infection was associated with pathological changes in the small intestine [A. annua reduced serum IL-6 levels, TNF-α, and IFN-γ compared to those of infected untreated animals. However, these levels were higher than those of metronidazole-treated animals. Therefore, we believe that treatment with A. annua would be advantageous over metronidazole since IL-6 blocks epithelial cells apoptosis and helps in epithelial repair [Our results also showed significant increase in proinflammatory cytokines, namely; IL-6, TNF-α, and IFN-γ in animals infected with ardiasis ,70,71. Anfection . Moreovenfection . Howeverntestine . In our l repair ,74.G. lamblia led to shortening of the intestinal villi, shedding, and desquamation of enterocytes. These effects could be due to the secreted parasite proteins with proteolytic activities. These proteins could lead to disruption of cell–cell junctions and enterocyte damage in vitro [+ T cells destroyed the apical membrane of enterocytes, with loss of brush border and reduced disaccharidases activities [G. lamblia infection increased caspase-3 levels, which could account for tissue damage seen in our model. Taken together, small intestinal damage observed in our model could be due to the parasite secretory products and/or because of elevated cytokine levels.Our histological analyses show that infection of hamsters with in vitro . Additiotivities . Other etivities . Moreovetivities ,79. Furttivities ,81,82. IA. annua extract markedly decreased desquamated enterocytes with nearly normal villi covering. This effect could be attributable to the direct toxic effect of A. annua on G. lamblia which could protect enterocytes from the proteolytic enzymes secreted by the parasite. This assumption is partially supported by the work of Jiao and colleagues who demonstrated that A. annua leaf extract killed Eimeria tenella, an intracellular parasite that infects chicken enterocytes [A. annua decreased the expression of the inflammatory markers; nuclear factor-kappa B(NF-kB) and IL-17. Moreover, Sun et al. showed that artesunate, an A. annua ingredient, induced apoptosis of lamina propria macrophages and dendritic cells and decreased mucosal expression of TNF-α in a rodent model of colitis [A. annua. Treating infected animals with A. annua extract also decreased serum IFN-γ levels. This effect of A. annua could account for alleviation of morphological changes that occurs during G. lamblia infection. On the other hand, Jiao et al. have demonstrated that A. annua leaves induced apoptosis of Eimeria tenella-infected enterocytes [A. annua inhibited the viability of resistant cancer cells [A. annua extract reduced the amount of caspase-3 in enterocytes. In line with this finding, Yuan et al. demonstrated that Artemisia derivatives protected mice liver against the injurious effect of acetaminophen through decreased cellular levels of caspase-3 and -8 in addition to lower serum TNF-α levels [A. annua components could inhibit growth or induce apoptosis of infected/stressed cells as suggested by Deng et al. and Jiao et al. reports [A. annua derivatives as shown by us and others [A. annua attenuated giardia-induced enterocytes damage through direct toxic effect on G. lamblia, anti-inflammatory and possible anti-apoptotic effect. However, these mechanisms of A. annua therapeutic effect need further investigation.However, treating infected animals with erocytes . In the  colitis . Numerou colitis ,85. Thererocytes . Lang aner cells . In the α levels .Therefor reports ,88, wherd others . AltogetG. lamblia infection [+ and CD8+ cells contribute to increased IELs during giardiasis [+ T cell from Giardia-infected animals to normal mice was associated with epithelial tissue damage [G. lamblia increased proliferation of CD4+ T cells and increased secretion of IFN-γ from both intestinal and blood lymphocytes [A. annua showed lower number of IELs compared to infected untreated animals. This observation could be attributed to the reduction of the parasite load in treated animals.In the current study, intestinal sections of infected untreated animals showed increased IEL numbers, which is in line with a previous report . IELs arnfection . This rinfection . Unlike nfection . Both CDardiasis . Howevere damage . Additiophocytes . On the G. lamblia reduced goblet cell numbers, increased excretion of cysts in stool, and decreased trophozoite count in the intestine [G. lamblia significantly reduced goblet cell number compared to uninfected animals. This reduction in the number of goblet cells could be due to the breakdown of mucin by trophozoites’ proteases [Goblet cells are essential for protection of the intestinal epithelium from luminal pathogens by secreting mucins which form a protective viscous barrier against invading pathogens ,92. In gntestine . Similarroteases , and henA. annua led to restoration of goblet cell number. This could be attributable to the direct cytotoxic effect of A. annua on trophozoites as we mentioned earlier. Therefore, we can also assume that increased goblet cell number and hence mucus production by A. annua impaired colonization of the intestine by giardia trophozoites. This assumption is supported by the in vitro experiments of Roskens and Erlandsen who demonstrated that mucin inhibited the attachment of trophozoite to the substratum [In our study, we showed that treating infected hamsters with bstratum .A. annua ethanolic extract similar to a number of studies [A. annua ethanolic extract ensures sufficient concentration of all active ingredients, particularly polysaccharides, coumarins, saponins, phytosterols, essential oils, polyphenols, and flavonoids [A possible limitation of our study should be noted here.We used a high dose of  studies ,97. Usinavonoids . This isavonoids .G. lamblia lowered NO production, while increasing IL-6, IFN-γ, and TNF-α. Additionally, G. lamblia infection reduced villus height, goblet cell number, and muscularis externa thickness, while increasing IEL numbers. Interestingly, treatment with A. annua reduced inflammation and pathological damage to the intestine and the therapeutic effect was comparable to that of metronidazole. Treatment of infected animals with A. annua extract also reduced small intestinal trophozoite count. Therefore, we believe that A. annua extract could be a potential therapeutic option for giardiasis. In conclusion, infection of hamsters with G. lamblia to commonly used anti-giardia agents necessitates the development of novel therapeutics. In this study, we show that A. annua extract is effective in controlling giardiasis in a hamster disease model. Furthermore, we demonstrate that the A. annua exerts its therapeutic effect by reducing trophozoite count, alleviating inflammation, and reversing pathological damage to the intestinal wall. Therefore, we believe that A. annua could be a potential effective therapeutic for the management of G. lamblia resistant strains.The increase in resistance of