Pseudomonas aeruginosa isolates in cystic fibrosis patients are scarce. Furthermore, there is limited knowledge on the effect of MBL production on patient outcomes. Here we describe a fatal respiratory infection due to P. aeruginosa producing VIM-type MBLs in a lung transplant recipient and the results of the subsequent epidemiological investigation.Data regarding the prevalence of metallo-β-lactamases (MBLs) among P. aeruginosa isolates collected in the index patient and among patients temporally or spatially linked with the index patient were analyzed in terms of antibiotic susceptibility profile and MBL production. Whole-genome sequencing and phylogenetic reconstruction were also performed for all P. aeruginosa isolates producing VIM-type MBLs. A VIM-producing P. aeruginosa strain was identified in a lung biopsy of a lung transplant recipient with cystic fibrosis. The strain was VIM-1-producer and belonged to the ST308. Despite aggressive treatment, the transplant patient succumbed to the pulmonary infection due to the ST308 strain. A VIM-producing P. aeruginosa strain was also collected from the respiratory samples of a different cystic fibrosis patient attending the same cystic fibrosis center. This isolate harbored the blaVIM-2 gene and belonged to the clone ST175. This patient did not experience an adverse outcome.P. aeruginosa producing VIM-type MBLs in a lung transplant recipient. The circulation of P. aeruginosa isolates harboring MBLs pose a substantial risk to the cystic fibrosis population due to the limited therapeutic options available and their spreading potential.This is the first description of a fatal infection due to  Pseudomonas aeruginosa is a non-glucose fermenter Gram-negative rod, frequently encountered in the respiratory tract of cystic fibrosis patients. P. aeruginosa is characterized by a low natural antimicrobial susceptibility and by an outstanding ability for selecting and spreading antimicrobial resistance in vivo [ in vivo , 2.P. aeruginosa invasive isolates tested were resistant to two or more antimicrobial groups [P. aeruginosa isolates collected from cystic fibrosis patients in Northern Europe over the period 2006–2012 were analysed, multi-drug resistance was detected in 61.4% of the isolates [In 2018 the European Centre for Disease Prevention and Control (ECDC) reported that 19.2% of the l groups . Higher isolates .P. aeruginosa is mediated by several mechanisms including: i) intrinsic P. aeruginosa resistance, such as the expression of inducible AmpC cephalosporinase, which is associated with a reduced susceptibility to imipenem [OprD porin, which drives imipenem resistance and decreases meropenem susceptibility. In addition, the overexpression of efflux pumps of P. aeruginosa plays a major role in mutation-driven resistance [P. aeruginosa. While at least nine different types of acquired MBLs have been described, the VIM-type is among the most important for geographic dissemination [Carbapenem resistance in imipenem ; ii) acqsistance ; iii) homination . Furthermination .P. aeruginosa isolates collected from cystic fibrosis patients in Northern Europe [P. aeruginosa antimicrobial resistance on the outcome of lung transplant in cystic fibrosis patients is still controversial [P. aeruginosa in a cystic fibrosis patient following lung transplantation. We also detail the epidemiological investigation performed in our medical center after the isolation of P. aeruginosa producing VIM-type MBLs.Carbapenemase production, and specifically MBL production, is rarely documented in patients with cystic fibrosis. Mustafa and collaborators did not detect any carbapenemase production when analyzing n Europe . Furtheroversial . To the oversial –13. HereP. aeruginosa isolates collected in the index patient and among patients linked with the index patient were analyzed. The analysis involved the following steps: i) identification of bacterial isolates and susceptibility profile; ii) detection of carbapenemase activity; iii) whole genome sequencing (WGS) and phylogenetic analysis. Details can be found in the P. aeruginosa with reduced susceptibility to carbapenems. She was exposed to carbapenems approximately 12 months prior to transplant. On day 7 after transplant the patient developed a right pleural empyema, requiring a thoracic drainage and broad-spectrum antimicrobial therapy (amikacin and ceftazidime). Respiratory cultures collected on the same day grew extensively-drug resistant mucoid P. aeruginosa (isolate SK76). SK76 showed a reduced susceptibility to meropenem but results were negative for carbapenemase production . Resistance to carbapenems was mediated by the production of blaVIM-1 carbapenemase  underwent bilateral orthotopic lung transplant in 2018. Pre-transplant respiratory cultures were positive for extensively-drug resistant mucoid and non-mucoid aadA7, aac(6′)-Ib7, blaPDC-9, blaOXA-488, blaOXA-20, sul1, and gyrA. On the contrary, the following resistance genes were detected in SK77: blaVIM-1, aadA7, blaPDC-7, blaPER-1, blaOXA-488, and sul1  analysis showed that isolates SK76 and SK77 belonged to two different and unrelated Sequence Types (STs): ST253 and ST308, respectively . The past medical history of this patient was characterized by: i) chronic colonization with methicillin-susceptible Staphylococcus aureus, mucoid and non-mucoid P. aeruginosa; ii) three respiratory exacerbations requiring treatment with carbapenems in the 12 months prior to the isolation of SK78; iii) end-stage lung disease (FEV1 27%). After the isolation of SK78, the patient was treated with high-dose extended-infusion meropenem and amikacin, without any reported adverse effects. Antimicrobial treatment led to fever remission and global clinical improvement. Twelve months after the isolation of SK78, the patient was still alive with a stable, although severe, pulmonary function (FEV1 27%). Isolate SK78 was characterized by a reduced susceptibility to meropenem and by the following additional resistance genes: aac(6′)-Ib7, blaOXA-50, and gyrA (Table . SK78 belonged to the epidemic high-risk clone ST175 (Fig. The detection of VIM-type MBLs among a cystic fibrosis patient prompted an epidemiological investigation in our medical center. All the respiratory cultures of patients attending the adult and pediatric cystic fibrosis center in 2018 were screened for the presence of MBLs. Similarly, all the patients admitted to the intensive care unit at the same time the index patient was admitted were screened for the presence of MBLs-producing isolates. A P. aeruginosa producing VIM-type MBLs in a cystic fibrosis lung transplant recipient. Among the four cases of MBLs-producing P. aeruginosa infections in cystic fibrosis patients already available in the literature, only two reported outcome data and only one involved a transplant recipient [To the best of our knowledge, this is the first description of a fatal infection due to ecipient –13.P. aeruginosa isolates producing MBLs and the limited therapeutic options available for their treatment. Second, the virulence of P. aeruginosa isolates producing MBLs and their impact on the patient outcomes. Third, the spreading potential of P. aeruginosa isolates producing MBLs. Fourth, the need for stringent and efficient antimicrobial stewardship and infection control policies in cystic fibrosis centers.Our description highlights several substantial issues. First, the broad spectrum of resistance of P. aeruginosa isolates producing MBLs restricts therapeutic options to few molecules, such as colistin, fosfomycin, aztreonam, and cefiderocol [The broad spectrum of resistance of iderocol . Of thesiderocol .P. aeruginosa isolates producing MBLs in comparison to the virulence of sensitive P. aeruginosa strains is a hot topic [P. aeruginosa strains [P. aeruginosa producing blaVIM enzymes. P. aeruginosa producing blaVIM enzymes isolates were strongly associated with the death of 32 patients, leading to a 16.2% attributable mortality [The evaluation of the virulence of ot topic , 16. Acq strains –20. Howe strains , 21, 22.ortality . While tMBLs are generally located on transposable genetic elements, which largely increases the spreading potential of these strains. Furthermore, ST308 is a recently reported emerging high-risk clone, while ST175 is among the three major international high-risk extensively-drug resistant clones and is widely distributed in several European countries . AlthougP. aeruginosa isolates [P. aeruginosa isolates, especially clonal isolates, is known to be associated with increased pulmonary exacerbation rate, exaggerated lung function decline, and progression to end-stage lung disease [P. aeruginosa isolates in cystic fibrosis centers [The selective antimicrobial pressure promoted by broad-spectrum agents favors the emergence of resistant strains. Specifically, previous studies have shown an association between the use of piperacillin/tazobactam, quinolones, and cephalosporins and the emergence of VIM-positive isolates . Further disease . For the centers .blaVIM genes in class 1 integrons in the bacterial chromosome of the P. aeruginosa isolates evaluated. Genes encoding carbapenemases are generally found in class 1 integrons along with determinants of aminoglycoside resistance. These integrons are often inserted into transposable elements, which contributes to the spreading potential of these strains. Furthermore, we did not perform virulence studies. Our epidemiological investigation was restricted to the year 2018: the circulation of P. aeruginosa strains harboring MBLs in our medical center before 2018 was not assessed. Finally, our epidemiological investigation was limited to patients: potential environmental sources of P. aeruginosa isolates producing MBLs were not evaluated and the sources of the isolates SK77 and SK78 were not identified.Despite the relevance of the data presented, this study has several limitations. We did not confirm the location of the P. aeruginosa producing VIM-type MBLs in a cystic fibrosis lung transplant recipient. P. aeruginosa producing VIM-type MBLs represents a matter of concern because of the limited therapeutic options available and its dissemination potential, especially in the setting of fragile hosts, such as cystic fibrosis patients.In summary, we described the first fatal infection due to Additional file 1.


Sporobolomyces pararoseus is regarded as an oleaginous red yeast, which synthesizes numerous valuable compounds with wide industrial usages. This species hold biotechnological interests in biodiesel, food and cosmetics industries. Moreover, the ballistospores-shooting promotes the colonizing of S. pararoseus in most terrestrial and marine ecosystems. However, very little is known about the basic genomic features of S. pararoseus. To assess the biotechnological potential and ballistospores-shooting mechanism of S. pararoseus on genome-scale, the whole genome sequencing was performed by next-generation sequencing technology.S. pararoseus genome into 20.9 Mb containing 54 scaffolds and 5963 predicted genes with a N50 length of 2,038,020 bp and GC content of 47.59%. Genome completeness  and RNA-seq analysis (expressed genes: 98.68%) indicated the high-quality features of the current genome. Through the annotation information of the genome, we screened many key genes involved in carotenoids, lipids, carbohydrate metabolism and signal transduction pathways. A phylogenetic assessment suggested that the evolutionary trajectory of the order Sporidiobolales species was evolved from genus Sporobolomyces to Rhodotorula through the mediator Rhodosporidiobolus. Compared to the lacking ballistospores Rhodotorula toruloides and Saccharomyces cerevisiae, we found genes enriched for spore germination and sugar metabolism. These genes might be responsible for the ballistospores-shooting in S. pararoseus NGR.Here, we used Illumina Hiseq platform to firstly assemble S. pararoseus NGR in biotechnological potential and ballistospores-shooting, which help further research of genetic manipulation, metabolic engineering as well as its evolutionary direction.These results greatly advance our understanding of  Sporobolomyces pararoseus (previously known as Sporidiobolus pararoseus) belongs to the order Sporidiobolales [S. pararoseus includes shades of pink and red due to the presence of lipid droplets full of carotenoid pigments, containing β-carotene, torulene and torularhodin [Genomic studies of the oleaginous red yeasts have gained increased attention due to their great biotechnological potential for biomass-based biofuel production –4. The robolales , which iobolales . Biomassobolales , 8. Cololarhodin –11.Sporobolomyces, Rhodosporidiobolus, and Rhodotorula, are known as competent producers of torulene and torularhodin [S. pararoseus for large-scale torulene and torularhodin production will be one of the major aims of future research efforts.However, there is little information on bioactivity and nutritional value of torulene and torularhodin, perhaps because they are rare in food, but its structure and sparse evidence provide some hints. For example, tests performed on human and mice showed that torulene and torularhodin have anti-prostate tumor activity . Furtherlarhodin . ConsequS. pararoseus is regarded as one of the most efficient microorganisms for bioconversion of crude glycerol into lipids [S. pararoseus also has been considered as potential feed stock for biodiesel industry [Additionally, o lipids . Lipids o lipids . These lo lipids . Microbio lipids , 18. Suco lipids , 20. ThuS. pararoseus. Advances in sequencing technology have drastically changed the strategies for studying genetic systems of microorganisms. Here, we present the first de novo genome assembly of S. pararoseus, as well as genes prediction and annotation. Subsequently, we performed a comparative analysis to investigate candidate orthologous and specific genes between S. pararoseus, R. toruloides and S. cerevisiae. The gene inventories provide vital insights into the genetic basis of S. pararoseus and facilitate the discovery of new genes applicable to the metabolic engineering of natural chemicals.Despite its long history of use for carotenoids fermentation, biodiesel production and ballistospores-shooting, very little is known about the basic genomic features of S. pararoseus NGR was sequenced using the Illumina Hiseq 2500 platform. A total of 8347 Mb raw data was generated from two DNA libraries: a pair-end library with an insert size of 500 bp (2631 Mb) and a mate-pair library with an insert size of 5 kb (5716 Mb). After, removing adapters, low-quality reads and ambiguous reads, we obtained 6073 Mb clean data  for genome assembly. For the genome size estimation of S. pararoseus NGR, we calculated the total 15 k-mer number is 705,505,006 and the k-mer depth is 28.41. According to the 15-mer depth frequency distribution formula, the estimated genome size of S. pararoseus NGR was calculated to be 24.44 Mb. Our final assembly consists of 54 scaffolds, a N50 length of 2,038,020 bp, the longest length scaffold of 4,025,647 bp, the shortest length scaffold of 513 bp, a GC content of 47.59% and a size of 20.9 Mb (85.52% of the estimated genome size). We identified 5963 genes in the genome with an average length of 1620 bp and a mean GC-content of 47.26% that occupied 55.07% of the genome. The results of BUSCO alignment showed that our final assembly contains 1273 complete BUSCOs (95.4%), of which 1268 were single-copy, while 5 were duplicated  using NCBI Nr databases based on sequence homology. In addition, 1940 (32.53%), 3002 (50.34%), 4237 (71.05%), 1806 (30.3%) and 4659 (78.13%) genes could be annotated according to KEGG, KOG, NOG, SwissProt, and TrEMBL databases, respectively. It should be noted that among these genes assigned to Nr database, the top 3 species of matched genes number are R. toruloides , Rhodotorula glutinis  and Microbotryum violaceum . Furthermore, 4057 genes could be classified into three Gene Ontology (GO) categories  genes could be annotated by BLASTN  [crtYB  [crtE , and other genes encoding hydroxylase, monooxygenase, or ketolase/carboxylase which might be responsible for the transformation from torulene to torularhodin; 2) lipid metabolism, including genes encoding acetyl-CoA carboxylase, acyl-CoA oxidase, phospholipid: diacylglycerol acyltransferase, glycerol 3-phosphate dehydrogenase; 3) carbohydrate metabolism, including genes encoding pyruvate dehydrogenase, pyruvate carboxylase and acyl-CoA: diacylglycerol acyltransferase; 4) stress responses, including genes involved in MAPK signaling pathway and calcium signal transduction.Based on KEGG pathways mapping, we annotated the coding genes of candidate for biotechnological potential in the NGR genome. A summary of the candidates  , crtYB (R108013) , crtE (GSporobolomyces (17 species) Rhodosporidiobolus (9 species) and Rhodotorula (16 species) [Sporobolomyces, the NGR showed a closer evolutionary relationship with S. ruberrimus and S. koalae than the other species, particularly for S. johnsonii and S. salmonicolor. The genus Rhodosporidiobolus situates a closer evolutionary relationship with Rhodotorula than with Sporobolomyces. The ballistospores are not uniform in the species of order Sporidiobolales, however, being a specialized mode of genus Sporobolomyces but absent in Rhodotorula and two characterized species of Rhodosporidiobolus (R. lusitaniae and R. colostri) [Sporobolomyces and Rhodosporidiobolus species shoot ballistospores. However, the ballistospores-shooting ability was gradually lost in R. lusitaniae/R. colostri or other undescribed Rhodosporidiobolus species. Subsequently, some Rhodosporidiobolus species of lacking ballistospores-shooting ability has undergone a series of evolutionary processes to form Rhodotorula species. While these basic hypotheses are non-controversial, further verification basing on discovering more new Sporidiobolales species and obtaining their genome data is required.Among phylum Basidiomycetes yeasts, there are a number of species that grow as pigmented colonies, and are for this reason known as red yeast . Among tspecies) , 25. In olomyces 7 speciesolostri) –28. It sR. toruloides NP11. This motivates us to perform a comparative genomic analysis between S. pararoseus NGR and R. toruloides NP11. In order to exclude the inherent quality of yeast, we added the model yeast S. cerevisiae S288C as a control. As shown in Fig. . 1975 (2077 genes), 4102 (4159 genes), and 4485 (4736 genes) protein families were species-specific in S. pararoseus NGR, R. toruloides NP11 and S. cerevisiae S288C, respectively. As shown in Fig. S. pararoseus NGR, 106 (16.4%), 280 (43.3%) and 261 (40.3%) terms were enriched in the CC, MF and BP, respectively. We found that the significantly enriched GO terms of the S. pararoseus NGR species-specific genes containing, CC: nucleus, membrane, and integral to membrane; MF: protein binding, DNA binding, and zinc ion binding; BP: regulation of transcription-DNA-dependent, transport, transmembrane transport, intracellular protein transport, carbohydrate metabolic process and oxidation-reduction process. Subsequently, we carried out the KEGG pathway mapping of S. pararoseus NGR species-specific genes. As shown in Fig. S. pararoseus NGR species-specific genes including MAPK signaling pathway-yeast, spliceosome, RNA transport, and mRNA surveillance pathways  analysis. Therefore, the Phs1 should be an indirect determinant of the ballistospores-shooting in genus Sporobolomyces.Among the species-specific genes, NGR-1A3721 that assigned to the GO term of spore germination (GO: 0009847) was considered to be one of the candidates for the formation of ballistospores. Moreover, the species-specific genes of the NGR involved in the KEGG pathways of sugar metabolism, including amino sugar and nucleotide sugar metabolism (ko00520), pentose and glucuronate interconversions (ko00040), starch and sucrose metabolism (ko00500), galactose metabolism (ko00052), fructose and mannose metabolism (ko00051), and butanoate metabolism (ko00650) might be related to the ballistospores dissemination as reported in previous studies , 30. RecAM 13481 . HoweverS. pararoseus is recognized as a kind of biotechnologically important oleaginous red yeast, which potentially can be used for biodiesel production as well as other important bio-products, such as carotenoids, enzymes and exopolysaccharide [S. pararoseus NGR will enable direct access to the genes responsible for its biology and biotechnological potential. To date, the only yeast belonging to the Sporobolomyces genus for which genome sequence is available is S. salmonicolor CBS 6832 [S. pararoseus NGR and S. salmonicolor CBS 6832. The genome assembly quality of NGR is better than CBS 6832. The genome GC-content of CBS 6832 (61.3%) is higher than NGR (47.59%), but the predicted genes amount of CBS 6832 (5147) is less than NGR (5963). The S. pararoseus NGR genome will also serve as a useful basis of comparative genomics studies to investigate functional peculiarities specific to this yeast and its relative lineage within the Sporobolomyces clade.ccharide . HoweverCBS 6832 . As showS. pararoseus is the process of ballistospores discharge. Ballistospores discharge is a unique type of spore produced by phylum Basidiomycetes fungi, however, does not occur in other fungal phyla [S. pararoseus NGR was patched on agar medium to form colonies, and the ballistospores are vertically shot into the lid of the plate to form a “mirror” with their colonies. Ballistospores-booting is the main reason for this eukaryotic lineage colonizing in the most ecosystems. The Sporobolomyces species are endowed with many similar phenotypes with Rhodotorula species, such as carotenoids and lipid production, and morphological characteristics. However, an obvious difference between them is that Rhodotorula species are usually considered as marine microorganisms, and does not produce ballistospores. The ancestor of the order Sporidiobolales might be certain Sporobolomyces species and lived on land without the convenience of an aqueous environment. Dissemination of ballistospores is for finding new nutrient sources. As they entered and adapted to the marine environment, they gradually reduce the efficacy of ballistospores-shooting to form the Rhodosporidiobolus species and further lost ballistospores to evolve into the Rhodotorula species. Because of the ballistospores-shooting is widely considered as a biological process of energy consumption. When they exposed to excessive sea water, its energy should be preserved as much as possible to resist cold and high salt stresses, instead of discharging ballistospores. Both cold and salt stresses might play critical roles in positive selection and rapid evolution of genera Sporobolomyces to Rhodotorula species.Moreover, one of the most notable characteristics of al phyla . As showSporobolomyces and Rhodotorula, the free ratio model was used to calculate the substitution rate for each orthologous gene [Rhodotorula species might have evolved a better energy metabolism and osmoregulation system to adapt to the marine environments and delay or prevent potential injury. But, the ballistospores-shooting is not necessary for its spreading in the marine environments. Given that the genes responsible for ballistospores-shooting still remain unknown. Our results provided valuable genetic data for the further characterization of the molecular mechanisms for ballistospores-shooting.The Ka/ Ks ratio is widely considered to be an indicator of selective pressure during evolution . To asseous gene . Among tS. pararoseus genome was reported. It established a genomic basis for further studying on its carotenoids, lipid, carbohydrate metabolism and stress responses. Furthermore, we proposed the evolutionary trajectories that Rhodotorula species were evolved from Sporobolomyces through the mediator Rhodosporidiobolus. Comparative genomic analysis revealed that the species-specific genes of S. pararoseus NGR related to spore germination and sugar metabolism, which might be involved in ballistospores-shooting. In conclusion, our work provides an important foundation for genes with potential biotechnological applications and foster comparative genomics studies to elucidate fundamental biological processes and evolutionary consequences of the order Sporidiobolales.Here, the high-quality S. pararoseus NGR was isolated from strawberry fruit in the greenhouse of Shenyang Agricultural University  in Shenyang City, Liaoning Province, China. Species identification was performed through morphological and molecular methods. The available GenBank accession number of S. pararoseus NGR 26S rDNA is HM749332. The strain number is recorded in the China General Microbiological Culture Collection Center as CGMCC 2.5280. S. pararoseus NGR cultures were grown for 72 h in 250 mL Erlenmeyer baffle flasks containing 50 mL of the YPD medium  at 28 °C on a rotary shaker at 180 rpm. Genomic DNA of S. pararoseus NGR was extracted using the DNAiso Reagent kit (Code No.: 9770A)  according to the manufacturer’s protocols. The extracted genomic DNA was subjected to quality control by agarose gel electrophoresis and quantified by Qubit 2.0 fluorometer . The obtained genomic DNA (≥500 ng/μL) was used for whole genome sequencing and PCR verification.http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) was used to plot quality score and sequence length distribution. Finally, the software ABySS (version 1.3.5) [k-mer profile of the reads. With these data, the genome size of the NGR was estimated by k-mer distribution (15 depth frequency) through the program KmerGenie (version 1.5621) with default parameters (inspired by FastQC) [Genome sequencing of the strain NGR was performed utilizing the Illumina HiSeq 2500 platform . In order to obtain a high-quality de novo assembly, the strategy used was to combine data generated from standard short insert paired-end libraries with those from mate-pair libraries. Two DNA libraries were constructed: a paired-end library with an insert size of approximately 500 bp using TruSeq Nano DNA Kit  and a mate-pair library with an insert size of approximately 5 kb using Nextera DNA Library Preparation Kit . The 500 bp library and the 5 kb library were sequenced using the PE125 strategy at the Novogene Bioinformatics Technology Co., Ltd. . After sequencing, quality control of the raw reads was performed, which involved trimming the reads using Trimmomatic (version 0.20)  by removn 1.3.5)  was used FastQC) .http://soap.genomics.org.cn/soapdenovo.html, version 2.0) under k-mer size of 15 [The filtered reads were assembled by SOAPdenovo2  with default parameters  [http://www.repeatmasker.org/, version v4.0.7) with default parameters based on libraries generated by different strategies: de novo-based, signature-based, and homology-based methods [http://tandem.bu.edu/trf/trf.html, version 4.09) with default parameters [http://www.cbs.dtu.dk/services/RNAmmer/, version 1.2) [http://lowelab.ucsc.edu/tRNAscan-SE/, version 2.0.4) with default parameters [http://rfam.xfam.org/, version 13.0) with default parameters [− 5, minimal alignment length percentage larger than 40% . The Gene Ontology (GO) annotations was performed using the program Blast2GO with default parameters [After obtaining the whole-genome sequence of the NGR, genes were predicted using the “Training module” and “Prediction module” of WebAUGUSTUS Service ( = true) . Repetit methods . Tandem rameters . Ribosomion 1.2)  with deframeters . Non-codrameters , 51. Funrameters . BLASTN rameters  and GO-tRhodotorula toruloides NP11 (https://www.ncbi.nlm.nih.gov/assembly/GCF_000320785.1/) and Saccharomyces cerevisiae S288C (https://www.ncbi.nlm.nih.gov/assembly/GCF_000146045.2/) were downloaded from NCBI Assembly database. The NGR, NP11 and S288C genome sequence alignments were performed in an all-against-all comparison using the MUMmer 3 package  with default parameters [https://orthomcl.org/orthomcl/, version 2.0) was used to generate core-orthologs for the NGR, NP11 and S288C whole proteomes datasets with default parameters [http://www.ncbi.nih.gov/BLAST/) and a score for each pair of proteins with significant matches was assigned with a cut-off value of 1 × 10− 7 [https://david.ncifcrf.gov/tools.jsp, version 6.8) with default parameters [Annotations of coding sequences and proteins of rameters . Compararameters . Subsequ × 10− 7 . These srameters , 58. Subhttps://github.com/OpenGene/fastp) with default parameters [RNA was extracted from the NGR cells using Trizol Reagent Kit  and then checked by 1% agarose gel electrophoresis and a NanoPhotometer spectrophotometer . The complementary DNA (cDNA) libraries were constructed using NEB Next Ultra RNA Library Prep Kit for Illumina  following manufacturer’s recommendations. The constructed products were purified and then amplified with PCR to obtain the high-quality cDNA library and sequenced at an Illumina Hiseq 2000 platform using the SE100 strategy at the Novogene Bioinformatics Technology Co., Ltd. . Raw reads of fastq format were firstly processed through the fastp program (version 0.18.0)  with “-rna-strandness RF” and other parameters set as a default , 61. TheBoth orthologous gene alignment and RNA-seq data mapping were employed to evaluate our genome completeness. The program BUSCO (version 3.0.1) was applied to align the orthologs of the NGR to a reference gene set of basidiomycota_odb9 with default parameters .p-value< 0.05 and a higher Ka/Ks value (Ka/Ks > 1) were considered to be the positively selected genes, as described in a previous study [The substitution rates  for each orthologous gene were used to evaluate the overall differences in the selective pressure at the gene level within the three yeast lineages, using the free ratio model in KaKs_Calculator Toolbox (version 2.0) software with default parameters , 67. Theus study .https://www.ncbi.nlm.nih.gov/nuccore). All sequences were processed with the software MEGA (version 7.0) using Muscle alignment with UPGMB clustering method [All 26S rDNA nucleotide sequences for the phylogenetic analyses were from NCBI Nucleotide database  to align the orthologs of the NGR to a reference gene set of basidiomycota_odb9. (XLS 71 kb)Additional file 2. All genes expression profile and functional annotations resulted from RNA-seq analysis. (XLS 1904 kb)Additional file 3. SNP/InDel annotations resulted from RNA-seq data. (XLS 48 kb)Additional file 4. Gene structure optimization resulted from RNA-seq data.Additional file 5. GO categories of 4057 genes resulted from genome analysis.Additional file 6. Mini-satellite DNA annotation file of NGR.Additional file 7. Micro-satellites DNA annotation file of NGR.Additional file 8. All gene annotation information resulted from genome analysis.Additional file 9. Genome annotations file of NGR.Additional file 10. CDS/cDNA sequences resulted from genome analysis.Additional file 11. Protein sequences resulted from genome analysis.Additional file 12. GO/KEGG enrichment results of species-specific genes of the NGR.Additional file 13. Ka/Ks results of single-copy homologous gene (NGR vs NP11).


Physalis peruviana L.) and related taxa germplasm collection. A total of 176 accessions representing, wild, weedy, and commercial cultivars as well as related taxa from the Colombian germplasm bank and other world repositories were screened using GBS. The pipeline parameters mnLCov of 0.5 and a mnScov of 0.7, tomato and potato genomes, and cape gooseberry transcriptome for read alignments, were selected to better assess diversity and population structure in cape gooseberry and related taxa. A total of 7,425 SNPs, derived from P. peruviana common tags (unique 64 bp sequences shared between selected species), were used. Within P. peruviana, five subpopulations with a high genetic diversity and allele fixation  were detected. Conversely, low genetic differentiation (FST: 0.01 to 0.05) was also observed, indicating a high gene flow among subpopulations. These results contribute to the establishment of adequate conservation and breeding strategies for Cape gooseberry and closely related Physalis species.A robust Genotyping-By-Sequencing (GBS) pipeline platform was examined to provide accurate discovery of Single Nucleotide Polymorphisms (SNPs) in a cape gooseberry ( Physalis peruviana L.) is an herbaceous Solanaceae species, native to the Andean region, with enormous potential for biomedical research and commercial purposes [Cape gooseberry , Oomycete (Phytium sp.), and fungal pathogens  [F. oxysporum sp. physali (Foph) [Cape gooseberry production in the Andean region has been diminished due to important phytosanitary problems caused mainly by bacterial ( others) , 15. Amoi (Foph) , represeThe available genetic diversity of cultivated and wild related taxa has helped to establish appropriate conservation, management, and sustainable utilization strategies of different crops . In partP. peruviana populations [Molecular markers have become valuable tools to assess genetic variation of worldwide plant repositories. Population studies, marker assisted selection, mapping and association studies, among other applications, have been used for this purpose . To dateulations –26. HoweFoph resistance [Recently, next generation sequencing (NGS) technologies have accelerated the screening of germplasm collections, identifying thousands of SNP markers in a cost effective and timely way , 28. Notsistance , 32.P. peruviana and 18 wild related species from the Physalis genus, including 95 technical replicates, to leverage the genetic diversity and population structure of this Andean crop towards conservation and sustainable utilization strategies.Although these previous advances in genetic diversity and association mapping have contributed to the exploration of cape gooseberry genetic resources, the species still lacks a more comprehensive GBS-SNP pipeline platform for SNP discovery. This study mainly seeks to provide a robust GBS-SNP calling pipeline for this species and related taxa by obtaining common genomic regions between tomato, potato, and cape gooseberry using the previously developed TasselPipelineGBS . MoreovePhysalis peruviana L.) and 18 related taxa accessions, containing one to seven individuals each, for a total of 644 individuals, were used in this study  following the manufacturer's procedure. DNA quantity was determined using NanoDrop (Thermo Scientific® ND 2000) and λ DNA/HindIII Ladder . DNA quality was assessed using EcoRI restriction enzyme digestions  and visualized on 1% (w/v) agarose gels stained with ethidium bromide (0.5 μg/mL).Solanum lycopersicum) and potato (Solanum tuberosum) [GBS library generation and Illumina sequencing were conducted at the Institute for Genomic Diversity (IGD) from Cornell University . An additional 95 individuals previously sequenced  were incberosum) , 36 as wberosum)  for readDifferent GBS parameters in the DiscoverySNPCaller and GBSHapMapFilters plugins were used for SNP calling and hapmap filtering, respectively , using aThe clustering patterns of the 739 individuals were compared through genetic distances generated by Tassel V4.3.5 in order to verify which parameters were correctly assessed. The parameter selection was based on genetic trees in which most of the technical replicates clustered together with their corresponding counterpart and those which better grouped the accessions according to their passport data . A totalP. peruviana accessions using the best GBS parameter combinations selected from the NJ analysis. The software ADMIXTURE V.1.23 [The population structure and genetic diversity analyses were carried out only for E V.1.23 , was useE), was calculated as ip is the frequency of the thi allele for k alleles. Observed heterozygosity (HO) and HE for each locus were estimated using Genepop version 4.7.5 [ST) and fixation (FIS) indices were calculated using the software mentioned above.Genetic diversity, referred to as expected heterozygosity , after the Illumina reads alignment . These cP. peruviana transcriptome) were selected to evaluate the clustering pattern in cape gooseberry and related taxa.A total of 54 genetic trees were generated, corresponding to each parameter and whether or not common tags were evaluated , using tP. philadelphica accessions and one accession from P. ixocarpa, and A2 harbors two accessions from both P. angulata, and P. floridana, and one accession from both P. pruinosa and P. viscosa. We also observed three P. peruviana accessions  in group A2. Furthermore, most cape gooseberry accessions , clustered in five different groups. Clusters B, C, D and E concentrated most accessions from the cape gooseberry producing regions, while cluster F harbors a high number of wild accessions from the Antioquia and Nariño departments of Colombia.The best NJ tree includes six clusters in which clear patterns emerged for the population under study . ClusterMoreover, the PCA revealed that the first three principal components explained 29% of the total variance, in which the first component contributed for almost all the variance observed, with 21.5%, followed by the second and third component with 4.7% and 2.8%, respectively .P. philadelphica accessions and one accession from P. ixocarpa, while Group A2 includes P. angulata, P. floridana, P. pruinosa and P. viscosa as well as three P. peruviana accessions  and P. peruviana to reduce the bias caused by the large amount of repetitive sequences, structural variations, and complex polyploid genomes present in plants [In NGS and GBS studies, one of the most important applications is the ability to accurately and comprehensively identify genetic variation. Obtaining unbiased results usually requires a complex multi-step processing pipeline that includes pre-processing, read alignment, and variant calling. Each of these steps uses its own set of modifiable parameters, creating a significant amount of possible distinct pipelines, which vary significantly in the resulting called variants . Therefoassembly , for rean plants . In partn plants , 51. In n plants , 53. As These parameters were selected based upon their ability to call SNPs in terms of read and taxon coverage, using additional values below and above to those reported in previous studies for cape gooseberry , 32. SimMoreover, the use of reference genomes to identify additional sources of diversity that went undetected when using a single reference, as has been previously used in similar species like tomato , was an P. peruviana and related taxa, as reported in previous studies [P. philadelphica and P. ixocarpa accessions from the others  and Tolima (09U291) departments of Colombia, and Poland (09U289). In another study based on orthologous genes Wei et al. [P. peruviana accessions cluster together with related taxa (such as P. philadelphica and P. angulata). One of these accessions corresponds to the 09U289 (PI28570597GI) from Poland, supporting the grouping pattern obtained in this study. Additionally, one possible explanation of finding P. peruviana accessions grouping together with related taxa could be associated to misclassifications within the cape gooseberry germplasm collection as previously reported [P. peruviana population structure and diversity analyses.The PCA revealed a grouping pattern in which most cape gooseberry cultivated accessions clustered together. However, the NJ tree uncovered additional subgroups (subpopulations B-F) within i et al. , found treported , 31. ForP. peruviana accessions neither by geographical origin nor by state of cultivation, as observed in other studies [E values indicates a high diversity within this species, possibly as the sum of multiple factors including mixed ploidy, heterozygosity, mating system, and marker informativeness. The HE values in this study agree with those found for the same species using SNP, SSR and COSII markers [P. peruviana germplasm collections. For instance, low HE values (0.22–0.25) have been uncovered in a small cape gooseberry population using random amplified polymorphic DNA and SSRs markers [ST values Click here for additional data file.S2 Table(XLS)Click here for additional data file.S3 Table(XLS)Click here for additional data file.


Kluyveromyces marxianus is a promising host for producing bioethanol and heterologous proteins. It displays many superior traits to a conventional industrial yeast species, Saccharomyces cerevisiae, including fast growth, thermotolerance and the capacity to assimilate a wider variety of sugars. However, little is known about the mechanisms underlying the fast-growing feature of K. marxianus. In this study, we performed a comparative genomic analysis between K. marxianus and other Saccharomycetaceae species. Genes involved in flocculation, iron transport, and biotin biosynthesis have particularly high copies in K. marxianus. In addition, 60 K. marxianus specific genes were identified, 45% of which were upregulated during cultivation in rich medium and these genes may participate in glucose transport and mitochondrion related functions. Furthermore, the transcriptomic analysis revealed that under aerobic condition, normalized levels of genes participating in TCA cycles, respiration chain and ATP biosynthesis in the lag phase were higher in K. marxianus than those in S. cerevisiae. Levels of highly copied genes, genes involved in the respiratory chain and mitochondrion assembly, were upregulated in K. marxianus, but not in S. cerevisiae, in later time points during cultivation compared with those in the lag phase. Notably, during the fast-growing phase, genes involved in the respiratory chain, ATP synthesis and glucose transport were co-upregulated in K. marxianus. A few shared motifs in upstream sequences of relevant genes might result in the co-upregulation. Specific features in the co-regulations of gene expressions might contribute to the fast-growing phenotype of K. marxianus. Our study underscores the importance of genome-wide rewiring of the transcriptional network during evolution. Saccharomyces cerevisiae is the most commonly used eukaryotic cell factory, thanks to its safe status, ease of genetic manipulation and a wealth of genomic, genetic, and biochemical knowledge . S. cerevisiae S288c strain was used. FIM1 and S288c were growing in YPD liquid medium  under aerobic condition at 30°C.K. marxianus FIM1 by a Yeast Genomic DNA Extraction Kit . Contigs were assembled using 300-bp pair-end reads by SOAPdenovo , KCTC 17555 (GenBank assembly accession GCA_000299195.2), and CCT 7735 (GenBank accession CP009303–CP009311) were performed as described above. Annotation GFF files of DMB1, KCTC 17555, and CCT 7735 genome were provided as Annotations of DMB1 (GenBank assembly accession Kluyveromyces lactis NRRL Y-1140 (GenBank assembly accession GCA_000002515.1), Kluyveromyces aestuarii ATCC18862 (GCA_000179355.1), Eremothecium cymbalariae DBVPG7215 (GCA_000235365.1), Eremothecium gossypii ATCC10895 (GCA_000091025.4), Lachancea thermotolerans CBS6340 (GCA_000142805.1), Naumovozyma castellii CBS4309 (GCA_000237345.1), Naumovozyma dairenensis CBS421 (GCA_000227115.2), Kazachstania africana CBS2517 (GCA_000304475.1), Saccharomyces cerevisiae S288c (GCA_000146045.2), Tetrapisispora phaffii CBS4417 (GCA_000236905.1), Torulaspora delbrueckii CBS1146 (GCA_000243375.1), Zygosaccharomyces rouxii CBS732 (GCA_000026365.1), Pichia pastoris GS115 (GCA_001708105.1), and Yarrowia lipolytica CLIB122 (GCA_000002525.1) were downloaded from NCBI and protein sequences were extracted. First, an all-against-all BLAST was applied to these protein sequences with E-values < 10–5. Second, global protein similarities were calculated using InParanoid . RNAseq was performed in Genergy Biotechnology company .K. marxianus FIM1 and S. cerevisiae S288c (Saccharomyces Genome Database) using HISAT2.1 . qPCR was performed on a LightCycler 480  with the SYBR Premix Ex Taq II . The mRNA level of the individual gene was normalized against the average level of MPE1, TRK1, and SWC4. Primers used for RT-qPCR were listed in To analyze mRNA levels in lag phase, an overnight culture of Aspergillus niger (r) was applied to infer the function of KMS families. For each gene family, its expression level was the sum of FPKM values of genes contained in the family. Value of r was calculated between a KMS family and a gene with known function at different time points after culturing. If r > 0.8, the function of the known gene can be considered as the candidate function of the KMS family. The known gene with the highest r value was chosen for the predicted function.Inspired by the idea of using gene co-expression network to predict the function of hypothetical genes in us niger , PearsonK. marxianus FIM1 strain was performed in this study. Eight nuclear chromosome sequences were obtained with a total size of 10.9 Mbp. The genome was annotated via “Yeast Genome Annotation Pipeline” for gene identification and via non-redundant (nr) database for functional annotation (CP015054–CP015061 and assembly accession GCA_001854445.2).A genome sequencing of the notation . Five thnotation . Two copK. marxianus-specific genes, a concept of “gene family” was introduced. Each gene family is a set of homologous genes among 15 yeast Saccharomycetales species, including 13 species in Saccharomycetaceae, one in Phaffomycetaceae and one in Dipodascaceae as outgroups. Totally, 636 gene families contain a single copy of a gene in each species. Single-copy genes were concatenated in each species into a supergene, followed by reconstructing a phylogenetic tree using the maximum likelihood method  genes (There are 4076 families in  species . Notablyc) genes . Four KMK. marxianus strains, including DMKU3-1042, NBRC1777, DMB1, CCT 7735 and KCTC17555 was investigated  in the family. By using the expression level of a gene family at 1 h as control, changes of the expression level of the family at later time points were calculated using the GFOLD algorithm. Expression levels of 22 KMS families were upregulated and those of 5 KMS families were downregulated at later time points (r) was calculated between a KMS family and a gene with known function, based on the hypothesis that strongly co-expressed genes are likely to function in the same or closely related biological pathways  were used to infer the possible function of a KMS family. The predicted functions of KMS families and corresponding r values were listed in Out of the 53 KMS families, 49 families have not been functionally annotated. The function of each KMS family was predicted based on the similarity between its expression and the expressions of genes with known functions. e points . Upregulpathways . The knoTo demonstrate the reliability of prediction based on co-expression, changes in mRNA levels of selected KMS genes upon heat stress were investigated. FIM1_4171 was predicted to be required for the growth under stress. The mRNA level of FIM1_4171 increased by fivefold after growing in 45 degrees . On the K. marxianus and S. cerevisiae, S. cerevisiae cells were also collected at 1, 4, 6, 12, 24, 48, and 72 h after culturing in the YPD, and then subjected to the RNA-seq. Gene family contains a set of homologous genes and is suitable for many-to-many comparison between K. marxianus and S. cerevisiae. There were 3676 gene families that contained both K. marxianus and S. cerevisiae genes   in each cluster was listed in To perform a comparative transcriptomic analysis between ae genes . Out of (C1∼C12) , using tK. marxianus and S. cerevisiae families showed dramatic changes since 12 and 24 h, respectively. The time points were in line with the late exponential phase, as showed by the growth curves of K. marxianus and S. cerevisiae  were located in upstream regions in K. marxianus  revisiae , 8B. Tworevisiae . Regarding phase . The co-ransport . TherefoKluyveromyces marxianus tightly co-upregulated genes involved in respiratory chain and FOF1 ATPase-mediated ATP biosynthesis during the fast-growing phase  ng phase . In contnchanged . The resarxianus  was grearevisiae , suggestnto heat . Heat lento heat . Consistarxianus .K. marxianus proposed here was based on the transcriptome of cells cultured in the rich YPD medium under aerobic condition. Rich media supported the fast growth of K. marxianus  ions from wastewater in the presence of molasses  can be found below: WM, YY, JZh, and JQ performed the genome sequencing and assembly. WM, TL, and JQ carried out the genome annotation and upload to NCBI. XY, HR, and TL carried out the strain cultivation and RNA-seq sample preparation. WL and JQ reconstructed the phylogenetic tree. WM and JQ analyzed the gain/lost genes and high-copied genes. WM, JQ, HL, JZe, and YY analyzed the RNA-seq data. HR performed the RT-qPCR analysis. WM analyzed the motifs of the upstream sequences. YY, WM, and HL prepared the manuscript. HL, JQ, and YY organized this research project. All the authors have read and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Acute cardiac tamponade is a rare but fatal disease that requires urgent intervention. The underlying cause of acute cardiac tamponade varies widely . If the 9/L with 89% abnormal lymphocytes, a hemoglobin level of 102 g/L, and a platelet count of 16.8x109/L. Coagulation tests, including prothrombin time, international normalized ratio (1.09), and partial thromboplastin time (30.4 s), were normal. An electrocardiogram showed AF with low QRS voltage. A chest X-ray showed marked cardiomegaly  visited our hospital complaining of malaise and dyspnea. She had not been prescribed antiplatelet or anticoagulant agents. No bleeding diathesis or cardiac disease had been identified. Her initial treatment for CLL was cyclophosphamide monotherapy, which was discontinued because of progression of the disease. The treatment was changed to ibrutinib monotherapy and she achieved partial remission. On admission, she was afebrile, but her respiratory rate was increased to 24 breaths/min and her systolic blood pressure was 96 mmHg. On physical examination, jugular vein distention was prominent and heart sounds were muffled. Laboratory tests showed a leukocyte count of 43x10iomegaly , which wiomegaly . Emergenytoplasm . Flow cyIn this case, cardiac tamponade and AF were ibrutinib-associated adverse events because they improved immediately after ibrutinib discontinuation. The mechanism of bleeding is due to the off-target effect of ibrutinib hampering platelet aggregation . Ibrutin


Oxidative stress plays a fundamental role in the pathogenesis of Parkinson's disease (PD). Oxidative stress appears to be responsible for the gradual dysfunction that manifests via numerous cellular pathways throughout PD progression. This review will describe the prooxidant effect of excessive consumption of processed food. Processed meat can affect health due to its high sodium content, advanced lipid oxidation end-products, cholesterol, and free fatty acids. During cooking, lipids can react with proteins to form advanced end-products of lipid oxidation. Excessive consumption of different types of carbohydrates is a risk factor for PD. The antioxidant effects of some foods in the regular diet provide an inconclusive interpretation of the environment's mechanisms with the modulation of oxidation stress-induced PD. Some antioxidant molecules are known whose primary mechanism is the neuroprotective effect. The melatonin mechanism consists of neutralizing reactive oxygen species (ROS) and inducing antioxidant enzyme's expression and activity. N-acetylcysteine protects against the development of PD by restoring levels of brain glutathione. The balanced administration of vitamin B3, ascorbic acid, vitamin D and the intake of caffeine every day seem beneficial for brain health in PD. Excessive chocolate intake could have adverse effects in PD patients. The findings reported to date do not provide clear benefits for a possible efficient therapeutic intervention by consuming the nutrients that are consumed regularly. Duplication, triplication, and point mutations in the N-terminal region  are linked to familial PD [α-synuclein monomers and tetramers are the physiological forms, while oligomers and fibrils are the pathogenic forms [α-synuclein monomers may lead to the formation of oligomers and fibrils as a key pathogenic event in the early stages of PD [Parkinson's disease (PD) is the second most common chronic progressive neurodegenerative disorder. PD is characterized by the selective loss of dopaminergic neurons of the substantia nigra (SN) pars compacta, which conditions deficiency of dopamine secretion in the basal ganglia of the midbrain with the ability to produce classic motor symptoms: bradykinesia, tremor, rigidity, posterior postural instability, gait disturbances, smell, memory, and dementia . PD invoposition . α-Synucic forms . Abnormaes of PD . The fires of PD , 10. Bases of PD .α-synuclein, parkin, and ubiquitin hydrolase which are directly related to PD [α-synuclein oligomers produce superoxide (O2‐) radicals that bind to transition metal ions such as copper and iron [α-Synuclein toxicity may contribute to elevated cellular oxidative stress. Oxidative stress may trigger α-synuclein toxicity [α-synuclein oligomers cause the impairment of proteasomes and lysosomes' degradation activity, increasing protein accumulation and aggregation. The accumulation of α-synuclein is associated with a decrease of dopamine release [ROS are necessary components for cellular homeostasis. However, when ROS are produced in excess, they induce transcription errors that cause dysfunction in the expression of different proteins, including C-terminal ed to PD . A recenand iron . α-Synuctoxicity . In PD,  release . The mit release .In this review article, we will briefly discuss the role of lipoperoxidation, oxidative damage, DNA repair, mitochondria, endogenous antioxidants, and the anti- and prooxidant effects of some natural foods for daily consumption and some food alternatives with antioxidant potential in PD. These dietary alternatives at low or increased levels can have beneficial or detrimental effects to increase or decrease the signs and symptoms of PD.α-synuclein by promoting the formation of secondary β-sheets and toxic soluble oligomers in a dose-dependent manner. Therefore, oxidative stress can also influence α-synuclein toxicity and mediate the pathogenesis of PD [Oxidative stress induces toxicity in the cell by the oxidation of lipids. Lipid oxidation leads to the accumulation of intracellular aggregates, mitochondrial dysfunction, excitotoxicity, and apoptosis. Oxidative damage is a common phenomenon in neurodegenerative diseases. However, it is unclear whether oxidative stress is a cause or a consequence. The formation of modified lipids via oxidation can produce postmitotic cellular dysfunction, and the dysfunction is capable of leading to necrosis or apoptosis of neurons. Lipoperoxidation of polyunsaturated fatty acids (PUFAs) in cell membranes initiates the cumulative deterioration of cell membrane functions by causing decreased fluidity, reduced electrochemical potential, and increased permeability of the cell membrane . Postmoris of PD . In the is of PD . Lipoperis of PD .PD is characterized by defects in the ability to repair acute or chronic oxidative damage to neurons . The 8-h2+ homeostasis. Therefore, mitochondria are crucial to cell survival. Intracellular Ca2+ stimulates the electron transport chain in the mitochondria producing ATP and ROS as subproducts. The endoplasmic reticulum is a quality control organelle that organizes protein synthesis, folding, and transport. Crosstalk between the endoplasmic reticulum and mitochondria increases with oxidative stress and mitochondrial stress, which can cause endoplasmic reticulum dysfunction. Research in the postmortem brain tissue has previously reported impaired mitochondrial function and elevated oxidative stress caused by α-synuclein aggregates, autooxidation, and degradation of dopamine in the SN [Mitochondria are organelles that produce ATP  and play a critical role in energy metabolism, the redox state, and Can the SN . Chronicn the SN . Antioxin the SN . When ann the SN . Random n the SN . OS incrn the SN .Prooxidant foods are compounds that promote oxidative stress by increasing ROS generation or by decreasing antioxidant systems . Diet cade novo protein synthesis, resulting in enzyme dysfunction with the ability to cause cellular damage [Meat products are the primary source of protein, amino acids, vitamins , and minerals such as iron and zinc . Howeverr damage . One of r damage . The oxir damage . Oxidizer damage .mortadella, and anchovies [Lipids are a necessary part of nutrition, providing large amounts of energy and essential fatty acids and promoting food acceptance . Lipids nchovies –65. Lipinchovies , 67. Durnchovies . The heanchovies , 70. Baynchovies . The effnchovies , 73. Humnchovies , 75. Hownchovies –78. The nchovies . Also, tnchovies –82.2‐production [Eating a diet rich in carbohydrates can promote cellular signaling of inflammatory effects . High caoduction . High gloduction . The incoduction , 94. Somoduction . It has oduction . Howeveroduction . Diet wioduction . A diet oduction , 98. PD oduction . There ioduction . Howeveroduction , 102.Due to the increase in life expectancy and generational change, PD has become a common health problem and care of a PD patient has become a treatment challenge. PD is a costly disease for health services characterized by the accelerated appearance of clinical manifestations. PD becomes devastating for patients and their families. In the field of neurodegenerative diseases, the existing therapies only limit the activity of the disease. Alternative treatments need to be evaluated for both their beneficial and harmful properties . A combiβ-barrel motif and two functionally important large loops, called zinc and electrostatic loops that coat the metal-binding region. SOD1 catalyzes the reaction of the O2‐ anion in molecular oxygen (O2) and hydrogen peroxide (H2O2) in a bonded copper ion [μM) [2‐ converting it catalytically into oxygen and H2O2. Some studies suggest that abnormalities in SOD1 or SOD2 may contribute to the development of PD [Cells have developed antioxidant defense systems to protect themselves against their destructive products. The antioxidant defense system consists of enzymes that involve BER, SOD, glutathione peroxidase (GPx), peroxiredoxins, and GSH . Due to pper ion . The intion [μM) . SOD1 reion [μM) . Reportsion [μM) , 116. SOnt of PD .In vitro studies showed that melatonin has prooxidant effects mainly at lipids and proteins [μM) are reported to promote ROS production by inducing cytotoxicity and apoptosis in human leukemia cells [Melatonin is a natural hormone mainly secreted by the pineal gland that regulates different physiological functions. Melatonin is also synthesized by other organisms, such as bacteria, invertebrate animals, and plants . The conproteins . Howeveria cells . Similaria cells . The proia cells . The benComplementary to the usual pharmacological therapy for PD, it is suggested to add some other natural compounds as adjuvants. Vitamins are natural bioactive products with antioxidant properties . Vitamin2O2 stimulates the formation of OH radicals [Vitamin C (ascorbic acid) is an essential water-soluble vitamin that is widely distributed in various tissues. Vitamin C is abundant in vegetables, fresh fruits, and animal livers. Vitamin C contains two molecular subforms in the body. The reduced form of vitamin C is ascorbic acid and the oxidized form of dehydroascorbic acid. Vitamin C is essential for the nervous system's physiological function and antioxidant function by inhibiting oxidative stress, reducing lipoperoxidation, and eliminating free radicals . Vitaminradicals . Thereforadicals . At suffradicals . Postmorradicals , 156. Inradicals . 25-Hydrradicals .α, IL-6) and work as a hepatoprotective agent in hepatitis and liver fibrosis in rat models [In addition to the protein obtained from food, there is also whey protein (WP) supplements used to treat some metabolic disorders . WP is at models . Other st models . Rat stut models . Falim et models . Reyes et models , 168. PDt models –171, altt models . Clinicat models .Theobroma cacao tree. Polyphenols, especially flavonoids, are the main components of health interest in cocoa and its derivatives [In vitro studies have shown the beneficial antioxidant effects of cocoa [in vivo studies [β-phenylethylamine, traces of a type of amine with neurotransmitter activity [β-phenylethylamine in the brain reaches its maximum concentration in dopaminergic regions [β-phenylethylamine causes inhibition of mitochondrial complex I favoring the generation of OH and psychomotor dysfunction [β-phenylethylamine in mice causes alterations of akinesia, catalepsy, and other motor disorders found in PD [β-phenylethylamine [Chocolate is produced from the cocoa bean of the ivatives . Currentivatives . The antivatives . In vitrof cocoa , 178. Ho studies . It is d studies . Dark ch studies . Dark ch studies , cancer  studies , cardiov studies , and neu studies . The stu studies . A singl studies . An impoactivity , 189. St regions . Studiesfunction . Furthernd in PD , 193. Duhylamine . There iα-synuclein in PD is intrinsically disordered. This protein erroneously folds and forms distinctive amyloid fibrils in neuropathological inclusions. Initial oligomerization and eventual fibrillation are believed to be critical steps leading to neuronal dysfunction and death [α-synuclein in aggregates is hyperphosphorylated in serine 129, and antibodies to phospho-Ser129-α-synuclein (p-α-syn) are useful in detecting these inclusions [α-synuclein in serine 129 accelerates its oligomerization and fibrillation in vitro. Consequently, this posttranslational modification is of pathogenic and therapeutic interest in α-synucleinopathies [α-synuclein activity in PD [α-synuclein aggregates with improved neuronal integrity and suppression of the neuroinflammatory response [α-synuclein-mediated toxicity by maintaining active PP2A [Drosophila [Normally, soluble nd death . Postmorclusions . The phoopathies . Dephospopathies . Carboxyopathies . In addiopathies , caffeinty in PD . In 2013response . The stuive PP2A . Decaffeosophila . Caffeinosophila . Growingosophila . Acute cosophila . A 150 mosophila . Extensiosophila .Hydrastis canadensis, Coptis chinensis, Berberis aquifolium, and Berberis vulgaris [Interest in scientific research on the properties of natural antioxidants in chronic degenerative diseases has been steadily increasing over the past two decades . Medicinvulgaris . Berberivulgaris , hepatitvulgaris , and hypvulgaris . Studiesvulgaris , 219. Exvulgaris . Howevervulgaris , 222.Curcuma longa plant that has been extensively researched for its antioxidant and anti-inflammatory properties [In vitro studies report that the antioxidant properties of curcumin contribute to its neuroprotective effects [α-synuclein protein [Drosophila model of PD has shown that the administration of curcumin decreases ROS and neurodegenerative severity and improves motor skills [Curcumin is a compound derived from the operties . In vitr effects . Cogniti effects . Curcumi protein . A studyr skills .Another bioactive molecule of natural products is quercetin. Quercetin is one of the main flavonoids widely distributed in apples, berries, onions, tea, tomatoes, and other plant products . The antPD is a common neurodegenerative disorder. PD incidence generally increases with age. Potential risk factors in developing PD include environmental toxins, drugs, pesticides, brain microtrauma, focal cerebrovascular damage, and genomic defects. Previous studies suggest that the intake of certain products may be associated with an increased risk of PD. Many foods for daily consumption have benefits because of their content of amino acids, vitamins, minerals, and micronutrients. However, increased use of some prooxidant foods may increase the risk of developing or increasing PD symptoms. Processed meat is characterized by high sodium content, advanced glycation end-products, cholesterol, and free fatty acids. The overconsumption of meat conditions a prooxidant effect. The alteration or fragmentation of the structure of meat products can cause oxidation of proteins by carbonylation. On the other hand, fruits and vegetables stand out for their antioxidant effect due to their amounts of vitamins and minerals. The evidence about the beneficial effect of coffee intake and the health risk by consuming large amounts of chocolate in PD patients is noteworthy. Short-term, medium-term, and long-term follow-up clinical studies are required to establish the useful quantities of the food substances, alone or in combination, to determine the bioavailability and nutritional content of each type of food in PD.


To report multimodal imaging findings in two cases of AIDS-related cryptococcal chorioretinitis associated with uveitis and vasculitis. Patients. Both patients were diagnosed with Cryptococcus neoformans meningitis in the setting of untreated HIV infection with CD4+ T cell count < 100/mm3. Ocular manifestations occurred during the course of the antifungal therapy for meningitis.  Findings on clinical examination, color fundus photography, fluorescein and indocyanine green angiographies, and optical coherence tomography.  In both cases, fundus showed vitritis. Fluorescein angiography allowed the characterization of vasculitis lesions, and indocyanine green angiography indicated choroidal involvement. In combination with optical coherence tomography, ICG and FA allowed the assessment of treatment response. C. neoformans to infect almost all ocular structures and the critical role of multimodal imaging in baseline evaluation and in the follow-up of patients. These two cases reveal the potential of  Cryptococcus neoformans and Cryptococcus gattii that can affect both immunocompromised and immunocompetent subjects. C. neoformans is a major cause of meningitis in HIV-infected patients with advanced immunosuppression and is associated with high morbidity and mortality in resource-limited settings [C. neoformans spreads hematogenously from the lungs to the central nervous system causing fungal meningitis. Ocular manifestations are frequently observed during cryptococcal meningitis in AIDS patients. Papilledema, visual loss, and cranial nerve palsy are the most frequent ocular findings [Cryptococcosis is an invasive mycosis mainly caused by two species: settings . C. neoffindings . Intraocfindings .We report two cases of cryptococcal intraocular involvement in two AIDS patients with concomitant cryptococcal meningitis. Fluorescein (FA) and indocyanine green (ICG) angiographies as well as optical coherence tomography (OCT) findings are discussed.3. He was diagnosed with cryptococcal meningitis 9 months before. He received a treatment with liposomal amphotericin B and flucytosine followed by fluconazole secondary prophylaxis. After two months, he suffered from a relapse after voluntarily stopping his prophylaxis and the same treatment was given.A 25-year-old man from Sub-Saharan Africa presented at our ophthalmology department for itchy red right eye since 3 weeks. The patient was infected with HIV following vertical transmission and diagnosed with HIV infection at the age of 7. The nadir CD4 count was 67/mmAt presentation day in our clinic, the patient had again voluntarily stopped both antiretroviral treatment (elvitegravir/cobicistat/emtricitabine/tenofovir) and fluconazole 2 months before.Visual acuity was 1/10 in the right eye and counting fingers in the left eye (LE); intraocular pressure was normal. Anterior chamber examination revealed bilateral conjunctival hyperemia, along with granulomatous keratic precipitates. Both eyes' fundus displayed mild vitritis along with multiple yellowish subretinal lesions through the posterior pole and midperiphery, with a well-demarcated large deep macular lesion on the LE . Late-phC. neoformans grew on multiples blood cultures with positive cryptococcal antigen (CRAG). A lumbar puncture demonstrated cerebrospinal fluid (CSF) lymphocytosis, hypoglycorrhachia, a positive CRAG, and C. neoformans culture. Vitreal culture was not performed. Computed tomography of the brain did not reveal any abnormalities, including signs of cerebral toxoplasmosis. Pulmonary tuberculosis was ruled out: both sputum and bronchoalveolar lavage direct examination, PCR, and culture were negative for M. tuberculosis. Blood culture was also negative for Mycobacteria sp. Physical examination was notable for papular verrucous skin lesions on the face. A skin biopsy was performed and PAS, Mucicarmine, and Grocott stainings revealed also the presence of Cryptococcus. Peripheral blood HIV viral load was 36400 copies/ml, and CD4+ T cells count was 61/mm3 .The patient was admitted, and laboratory investigations were performed. 3. The patient was still taking fluconazole secondary prophylaxis. Visual acuity was, respectively, 10/10 and 4/10 and there was no sign of inflammation on the slit lamp. The eye fundus examination demonstrated multiple bilateral peripheral inactive lesions and a macular scar on the LE.A diagnosis of systemic and ocular cryptococcal infection was thus made, and combination therapy with liposomal amphotericin B (250 mg/24 h) and flucytosine (1750 mg/6 h) was restarted for a total of 14 days. The patient was discharged with fluconazole 800 mg along with antiretroviral therapy. At 10-month follow-up, peripheral blood HIV viral load was undetectable (<20 copies/ml) and CD4 count was 144/mmLate-phase FA showed regression of the vasculitis, remaining late diffusion of the optic nerve, and staining of the macular and peripheral cicatricial retinal lesions . Late-phSD-OCT in the left eye showed a retrofoveolar scar with disruption of the retinal pigment epithelium and a loss of macular inner retinal structure .C. neoformans. Brain MRI did not reveal signs of cerebral toxoplasmosis. Pulmonary tuberculosis was ruled out: both sputum and bronchoalveolar lavage direct examination, PCR, and culture were negative for M. tuberculosis. Peripheral blood HIV-1 viral load was 800,000 copies/ml, and CD4+ T cell count was 12/mm3. On the third day of hospitalization, combination therapy by liposomal amphotericin B (350 mg/24 h) and flucytosine (100 mg/kg/24 h) was initiated and was stopped after 18 days because of acute kidney failure possibly related to liposomal amphotericin B and thrombocytopenia possibly related to flucytosine. Secondary prophylaxis by fluconazole 400 mg was initiated. Nevertheless, on the 30th day of hospitalization, fever relapsed accompanied with headaches. Lumbar puncture analysis revealed positive Indian ink examination but negative C. neoformans culture. Consequently, liposomal amphotericin B treatment was reinitiated for probable relapse of cryptococcal meningitis. On the fifth day of hospitalization, the patient, complaining of floaters, was referred to the ophthalmology department. The visual acuity was 10/10 on both eyes. Slit lamp examination revealed few cells in the anterior chamber with no keratic precipitates or synechia. Fundus examination showed mild vitritis in both eyes associated to bilaterally multiple dot and blot hemorrhages, cotton wool spots, multiples deep yellowish lesions, and large whitish peripheral retinal areas , toxoplasma chorioretinitis, syphilitic chorioretinitis, lymphoma , 5. Cryplymphoma . Other flymphoma –7. The mlymphoma . Previoulymphoma . DissemiInterestingly, in both of our cases, chorioretinitis manifests during the course of treatment for cryptococcal meningitis. In the first case, the patient had already received months of treatment and chorioretinitis manifests in the context of a relapse. Similarly, in the second case, ocular inflammation occurs in the setting of a presumed relapse or clinical failure. Similar cases have been reported previously. Carney et al. reported cases of cryptococcal choroiditis in which choroiditis manifests 10 days after initiation of amphotericin B . The autC. neoformans infection, as well as reports describing multimodal imaging findings. Most cases were reported in patients with concomitant immunosuppression. Previous cases were reported in patients with advanced AIDS [C. neoformans to infect all ocular structures including the optic nerve, retinal vessel, and choroidal stroma, leading, respectively, to papillitis, vasculitis, and choroiditis in immunocompromised patients. This involvement is found in some systemic granulomatous diseases, leading to the most frequent differential diagnoses of tuberculosis and sarcoidosis, but also syphilis, which remains the main masquerade syndrome in uveitis patients [Simultaneous involvement of all the different eye structures  has rarely been reported in the course of disseminated ced AIDS –10 or wiced AIDS . As descced AIDS , 9, EDI-patients . We exclpatients .On the other hand, limited involvement of the eye presenting as uveitis with concomitant cryptococcal meningitis has been reported in nonimmunocompromised patients , 15.C. neoformans to infect all ocular structures mimicking other ocular diseases and this leads us to consider Cryptococcus as “a good masquerader” like syphilis and tuberculosis.In conclusion, these two cases reveal the potential of


Francisella, comprising a dataset of 63 genomes and encompassing clinical as well as environmental isolates from distinct geographic locations. To determine the evolutionary relationship within the genus, we performed phylogenetic whole-genome studies utilizing the average nucleotide identity, average amino acid identity, core genes and non-recombinant loci markers. Based on the analyses, the phylogenetic trees obtained identified two distinct clades, A and B and a diverse cluster designated C. The sizes of the pan-, core-, cloud-, and shell-genomes of Francisella were estimated and compared to those of two other facultative intracellular pathogens, Legionella and Piscirickettsia. Francisella had the smallest core-genome, 692 genes, compared to 886 and 1,732 genes for Legionella and Piscirickettsia respectively, while the pan-genome of Legionella was more than twice the size of that of the other two genera. Also, the composition of the Francisella Type VI secretion system (T6SS) was analyzed. Distinct differences in the gene content of the T6SS were identified. In silico approaches performed to identify putative substrates of these systems revealed potential effectors targeting the cell wall, inner membrane, cellular nucleic acids as well as proteins, thus constituting attractive targets for site-directed mutagenesis. The comparative analysis performed here provides a comprehensive basis for the assessment of the phylogenomic relationship of members of the genus Francisella and for the identification of putative T6SS virulence traits.Pan-genome analysis is a powerful method to explore genomic heterogeneity and diversity of bacterial species. Here we present a pan-genome analysis of the genus  Francisella belongs to the γ-subclass of Proteobacteria, but shows no close relationship to other human pathogens  assembly database and the Joint Genome Institute (JGI) Genome Portal. These huge sequence datasets offer not only the possibility to understand the functional and evolutionary repertoire of bacterial genera, but also open up possibilities for developing therapies and engineering applications. The objective of this study was to elucidate the core- and pan-genome features of the Francisella genus to shed light on its diversity and characteristics, as well as to identify putative T6SS substrates in silico. Our analysis identifies conceptual and technical approaches that may be used for studies of pathogenicity, especially related to secretion systems.For the genus Francisella bacteria were downloaded from the NCBI assembly database1 and used for analysis. Allofrancisella guangzhouensis, a species previously considered to be a member of the genus  and Piscirickettsia (19 complete genomes), to allow comparisons to be made between the three genera. Only one representative of highly related species (ANI ≥ 99.5%) was used for further analysis of the pan-genome, phylogenomic analysis etc.All 62 publicly available (January 2018) whole genome sequences of he genus , was als program , using tFrancisella core- and pan-genome size was assessed in a manner similar to that previously reported, using iterative and combinatorial approaches  and KEGG (Kyoto Encyclopedia of Genes and Genomes) annotations.Curve fitting of the pan-genome was performed using a power-law regression based on Heaps’ law [y = Aescribed . The samF. tularensis strains, we used multiple approaches. First, we used the UBCG  approach, by utilizing its pipeline and default parameters2  alignment program4 using default parameters. Each of the UBCG genes were aligned separately, before being concatenated into a single alignment. A highly resolved maximum likelihood tree was obtained using FastTree5 and visualized using the iTOL server.6 A bootstrap analysis was performed to determine the reliability of the branches obtained.For whole-genome phylogenetic analysis of closely related ameters2 . First, We also constructed a marker-based phylogenetic tree, by using the GET_PHYLOMARKERS software package in the default mode , and wit7 (F. novicida strain U112 (accession number NZ_CP009633) to search a database containing all of the 26 representative Francisella species. To generate blast hits, we set the minimal sequence identify to 25% and the sequence coverage to 30%, while the rest of the parameters were kept as default. Using the same parameters, we also tested the FPI cluster of U112 against the bacterial domain in the NCBI gene bank database to look for the presence of FPI homologous genes in other bacterial genera. To estimate the G + C contents for the FPI cluster and for the whole genome, the following formula was used: (G + C)/(A + T + G + C) ∗ 100%. We also analyzed the amino acid composition of the FPI proteins (encoded by pdpA to anmK) and compared it with the amino acid composition of the rest of the genome. The first was calculated using the concatenated all FPI proteins only, while the second was calculated using the concatenated all protein sequences after excluding the FPI proteins. The genomes included in the analysis, in addition to F. novicida U112, were F. tularensis subsp. tularensis SCHU S4 (NZ_CP010290), F. cf. novicida Fx1 (NC_017450), and F. tularensis subsp. holarctica LVS (NZ_CP009694).Comparative analyses of FPI/T6SS clusters were performed using the MultiGeneBlast program with default parameters7 . This pr8 predicts T6SS effectors using a two-layer SVM-based ensemble model with optimized parameters -annotated with respect to their predicted biological process, molecular function or cellular component, using the PANNZER2 (Protein annotation with Z-scoRE) server  server . We alsoFrancisella genus, we used the sequence for the effector Ssp6 (SMDB11_4673) of Serratia marcescens Db10 as query against the NR database (set as Francisella group) using the PSI-BLASTP program with default parameters. To specifically search for MIX effectors -BLASTP with four iterations and other parameters kept as default. As queries, we used representative sequences for each of the following five classes of MIX effectors: MIX I - Vibrio parahaemolyticus VP1388 (accession: NP_797767), MIX II - Proteus mirabilis IdsD (accession SPY42138), MIX III - Burkholderia thailandensis BTH_I2691 (accession ABC38088), MIX IV - Vibrio cholerae VCA0020 (accession NP_232421) and MIX V – V. parahaemolyticus VPA1263 (accession NP_800773). Each query generated a set of identified hit protein sequences, which we used in a multiple sequence alignment analysis to identify the conserved sequence and predict putative signal peptides. Furthermore, by using the MultiGeneBlast program, the chromosomal location of the identified hits as well as the upstream and downstream ORFs were analyzed for the 26 representative complete genome sequence data set.To specifically search for homologs of T6SS-dependent, ion-selective pore-forming effectors  within tffectors  within tFrancisella genomes  and OrthoANI (93.9%), thus questioning their species belonging  were derived from only two species, P. salmonis and P. litoralis, and showed ANI values ranging from 95.7–99.9% (data not shown).In the genus Francisella, we used our down-selected 26 genomes, from 14 Francisella species, and the binomial mixture model of Snipen and collaborators  are composed of genes absent from one or more of the strains . The latborators  and Tettborators . We obseborators . Thus, sn-genome .Francisella genomes  and for Piscirickettsia to 44.2% (1323 vs. 2995)  . Further72 genes . To assi72 genes . We also72 genes .Francisella, however, the differences within the 16S rRNA sequence are very few  sh clade A .Francisella genus into two distinct clades, with clade A comprising F. tularensis, F. novicida, F. hispaniensis, and F. persica, and clade B containing F. philomiragia and F. noatunensis. Our comprehensive phylogenetic analysis also confirmed this bifurcation of Francisella into two clades, with the addition of a third clade, clade C. Notably, in the UBCG analysis, the F. persica ATCC VR331 and F. opportunistica sp. nov. MA067296 showed a common ancestor and were closer to clade A, while in the selected marker-based trees, these species are more disparate  is a cluster of 16–19 genes, present in most of the Francisella genomes that have been sequenced to date. Although 16 FPI genes are highly conserved, 2–3 genes are absent or interrupted by stop codons in some strains  and F. halioticida DSM23729, for which only the genes encoding IglA and IglB, i.e., the T6SS sheath proteins, were detected. The F. tularensis subsp. holarctica LVS, subsp. mediasiatica FSC147, and subsp. tularensis SCHU S4 all have two copies of the FPI as shown in F. endociliophora FSC1006 and F. salina sp. nov. TX077308, have a single FPI copy with three or more of the FPI genes missing or inactivated .The  strains . Intrigugle copy . We founme, ∼32% . Moreoveme, ∼32% . In suppme, ∼32% . In contme, ∼32% . Similarida U112 . To searctivated . InteresFrancisella genus. The first is characterized by the presence of an intact FPI cluster and includes, e.g., F. hispaniensis FSC454, F. tularensis subsp. mediasiatica FSC147 and F. tularensis subsp. tularensis SCHU S4, F. novicida U112, F. novicida PA107858, F. novicida D9876, F. novicida AZ067470, F. cf. novicida 3523, and F. cf. novicida Fx1. Most of the species belonging to this group clustered to clade A in the phylogenetic tree analysis. The second group is characterized by the presence of an FPI cluster, which lacks both the pdpC and pdpE genes. This group included, e.g., all strains of F. philomiragia and F. noatunensis, and F. noatunensis subsp. orientalis, F. frigiditurris sp. nov. CA971460, F. opportunistica sp. nov. MA067296, F. uliginis sp. nov. TX077310, F. salina sp. nov. TX077308, F. novicida AL972214, and F. endociliophora FSC1006, the latter being unique in that its FPI also lacks pdpD, anmK, and iglI, and exhibits gene rearrangements  outside of the genus exist(s), and then primarily in species closely related to Francisella, such as Piscirickettsia sp., Cysteiniphilum sp., Fangia hongkongensis, and Pseudofrancisella aestuarii. Taken together, our comparative analysis of the FPI gene cluster demonstrates that the FPI genes are highly similar within the genus, but share low similarities with T6SS genes of other bacterial species.We also searched the NCBI database for FPI homologs present outside of the genus onserved , 2012. Ionserved . Also hoFrancisella FPI promotes pathogenesis. So far, a few putative effectors encoded within the FPI have been identified by the use of different reporter assays  analysis, constitute members of the glycosyl hydrolases family 18. Four putative effectors had protein domains of no characterized function, i.e., DUF1338, DUF2147, DUF4124, and DUF4440 , and in some mammalian pathogenic-species of Francisella (Clade A). The first putative effector (MIX-I) is a conserved hypothetical protein (locus tag: “FTT_1768c”), functionally predicted to be a Chitinase/glycoside hydrolase family 18 protein and also identified as a putative effector in the Bastion6 machine learning based predictor for T6SS effectors . Remarkably, the DUF3568 domain-containing protein originally reported as F. tularensis Virulence Determinant protein  has been suggested to share structural homology to Bet v1 allergen proteins  in the G + C content of this genome dataset was indicative of a stable boundary delineation within the genus. The distinctly lower G + C content of the FPI suggests that horizontal gene transfer has been a major factor driving the evolution of the FPI of Francisella. Indeed, Nano et al., suggested that the FPI originally had been acquired through horizontal gene transfer from an organism with a lower G + C content , the latter being the lowest value of all 26 Francisella genomes analyzed. Interestingly, in the recent study by Challacombe et al., F. frigiditurris sp. nov. was suggested to be a new member of the genus Francisella   lacked the entire FPI, i.e., F. halioticida DSM23729, (ii) possessed one complete FPI copy or more, e.g., F. hispaniensis FSC454 (1 copy) and F. tularensis subsp. tularensis SCHU S4 (2 copies), (iii) lacked both of pdpC and pdpE, e.g., F. philomiragia, (iv) lacked a functional pdpD gene, i.e., F. persica ATCC VR331 and F. tularensis subsp. holarctica LVS, or (v), lacked all of pdpC, pdpD and pdpE genes (F. endociliophora FSC1006). Similar results were obtained in the previous study by Challacombe et al., which was based on 31 Francisella genomes in total  . All of eviously . Both, tin total . The advreported , but, ineviously . The twol strain . NeverthV. cholerae, a modest number of substrates has been identified for the Francisella T6SS (Brucella-containing vacuoles (BCVs) from the endolysosomal pathway and the establishment of the intracellular replicative niche (L. pneumophila inhibits retromer activity to promote intracellular replication by directly binding to the retromer subunit VPS29 (Francisella, and whether this involves a direct physical interaction with the retromer. To our knowledge, this has not been investigated. Interestingly, this putative effector is highly conserved among the different subspecies of F. tularensis, >99% identity, but is less conserved within the species F. philomiragia and F. noatunensis, 37–52%, that only rarely infect humans, possibly reflecting a difference in function. These candidate genes may therefore constitute interesting targets for designing novel strategies to prevent and control infections with species that belong to this highly diverse and environmentally adapted genus.While the repertoire of effector proteins is quite abundant for some T6SS, e.g., lla T6SS . Naturallla T6SS . The latve niche . Moreoveit VPS29 , therebyit VPS29 . This raFrancisella and for the identification of putative T6SS virulence traits.Collectively, the comparative genomic analysis performed provides a comprehensive basis for the assessment of the phylogenomic relationship of members of the genus All datasets presented in this study are included in the article/RK, AS, and JB designed the study, analyzed the data, and wrote the manuscript. RK performed all experiments.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Microsporidia are obligate intracellular and eukaryotic pathogens that can infect immunocompromised and immunocompetent mammals, including humans. Both innate and adaptive immune systems play important roles against microsporidian infection. The innate immune system can partially eliminate the infection by immune cells, such as gamma delta T cell, natural killer cells (NKs), macrophages and dendritic cells (DCs), and present the pathogens to lymphocytes. The innate immune cells can also prime and enhance the adaptive immune response via surface molecules and secreted cytokines. The adaptive immune system is critical to eliminate microsporidian infection by activating cytotoxic T lymphocyte (CTL) and humoral immune responses, and feedback regulation of the innate immune mechanism. In this review, we will discuss the cellular and molecular responses and functions of innate and adaptive immune systems against microsporidian infection. Anncaliia, Encephalitozoon, Enterocytozoon, Microsporidium, Nosema, Pleistophora, Trachipleistophora, Tubulonosema, Vittaforma) of microsporidia have been reported to be able to infect humans  on the surface , and subnfection  dependinnfection . The RNSnfection . Nonethenfection . Moreovenfection . These fnfection .Encephalitozoon, the expression of IL-10 increased  involved in Toll-like signaling pathway is needed  against nfection , 2012. Tnfection , and CD8nfection . In infenfection . Studiesimmunity . Moreoveimmunity , and is nfection . In Entes needed . TherefoOn the other hand, the differentiation of DCs was found to be inhibited by microsporidia via an IL-6-dependent mechanism so that the parasites can escape from stronger immune defense . In addiE. cuniculi  response , 2012. Tet cells . These fet cells .Apoptosis is a form of programmed cell death that also responses to pathogen infection and plays significant roles in the control of the immune response .Toxoplasma gondii can suppress host apoptosis in order to have more time for proliferation , lysozyme (Lz), human beta defensin 2 (HBD2), human alpha defensin 5 (HD5), and human alpha defensin 1 (HNP1) are capable of inhibiting microsporidian spore germination and reducing enterocytes infection . For exaand HNP1 . Howevered above . It is lAdaptive immunity is broader and more efficient protection against non-self antigens than innate immune system. Adaptive immunity involves T lymphocytes-mediated cellular immunity and B lymphocytes-mediated humoral immunity, which have a tightly regulated interplay with antigen-presenting cells and facilitate the pathogen-specific elimination . When thE. cuniculi infection in vitro. One explanation for the inhibition is that P5/H1 neutralizes sensitive epitopes on E. cuniculi spores so that the growth of the pathogens was suppressed (Humoral immunity is a very effective way to scavenge pathogens from the host . Treatmeppressed . Antibodppressed . In addippressed . Furtherppressed .E. cuniculi are able to induce a strong antibody response. Increase of IgA, IgM, and IgG were detected in infected mice (E. cuniculi infection (Microsporidia like ted mice . Howevernfection , suggestE. intestinalis were shown to have elevated levels of IFN-γ and IL-2 (E. cuniculi, the KLRG1+ T cell subset was the majority of polyfunctional effector CD8+ T cells (+ effector CD8+ T cells against microsporidian infection (T cell-mediated immune response in host is crucial for prevention of infection. CD4+ and CD8+ T cells were found induced in infected mice and rabbits , and werand IL-2 , which aand IL-2 . Howeverand IL-2 . These rand IL-2 . During  T cells . A more nfection . These snfection , suggest–/– mice became more susceptible to E. cuniculi infection (E. cuniculi. And this protection depends on the activity of CD8+ CTLs, which lead to lysis of infected cells by perforin pathway (E. cuniculi infection (E. cuniculi, while lacking of γδ T cell causes down-regulation of CD8+ cell immune response (E. cuniculi (E. cuniculi (Strong CD8+ T cell responses were also observed during microsporidian infection . The CD8nfection . And SCInfection . These r pathway . Moreove pathway . Howevernfection , suggestnfection . It was nfection . The γδ nfection . It was response . In addicuniculi . In micrcuniculi . This sucuniculi . Probablcuniculi . Besidescuniculi . These fIn general, T cell-mediated immune protection is essential for elimination of microsporidian infection. However, microsporidian spores still can remain in some organs of immunocompetent mice and may become a source of infection onset . This raThe innate and adaptive immune systems play vital roles against microsporidian infection. Further understanding of host immune responses against the infection will greatly help with the diagnosis and treatment of microsporidiosis. The innate immune system not only directly resists the infection but also triggers the adaptive immunity. Innate immune cells, such as macrophages, DCs, γδ T cell, and NKs, can control microsporidia to some extent, and also secret cytokines and chemokines to assist both the innate and adaptive immunities against the infection. The cytokine IL-12 and IFN-γ can help to clear the parasite by activating related immune responses. However, early expression of IL-10 is benefit for the pathogen growth by negatively regulating the IFN-gamma expression. Yet the relation between IL-10 and microsporidia remains to be clarified. Besides, it is worth noting that lymphokines can also activate macrophages to kill microsporidia . With thTo completely clear the infection, the host needs to initiate the adaptive immunity. First of all, the CTL can be activated to restrict the proliferation of microsporidia by lysing infected cells via perforin pathway . In addiIn summary, the interactions between microsporidia and host immune systems have been further studied, but are far to be fully elucidated. For example, what are the key host cells and factors that confer the defense effects. And what are the mechanisms that microsporidia modulate host immune responses. Hopefully, genomics and proteomics studies on microsporidia have provided some important clues and candidates to dissect the questions .TL and ZZ contributed to conception and design of the study. TL and YH wrote the first draft of the manuscript. HG, JX, JL, BH, JB, and GP wrote sections of the manuscript. All authors contributed to manuscript revision, read and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer LW declared a shared affiliation with the author BH at the time of review.