Cryptococcus neoformans, a fungal pathogen that causes life-threatening fungal meningoencephalitis. We analyse 230 signature-tagged mutant strains for 114 putative phosphatases under 30 distinct in vitro growth conditions, revealing at least one function for 60 of these proteins. Large-scale virulence and infectivity assays using insect and mouse models indicate roles in pathogenicity for 31 phosphatases involved in various processes such as thermotolerance, melanin and capsule production, stress responses, O-mannosylation, or retromer function. Notably, phosphatases Xpp1, Ssu72, Siw14, and Sit4 promote blood-brain barrier adhesion and crossing by C. neoformans. Together with our previous systematic studies of transcription factors and kinases, our results provide comprehensive insight into the pathobiological signalling circuitry of C. neoformans.Phosphatases, together with kinases and transcription factors, are key components in cellular signalling networks. Here, we present a systematic functional analysis of the phosphatases in  Cryptococcus neoformans, revealing roles in virulence, stress responses, O-mannosylation, retromer function and other processes.Phosphatases are key components in cellular signalling networks. Here, the authors present a systematic functional analysis of phosphatases of the fungal pathogen  When an environmental cue occurs, an organism receives the signal through a sensor , which subsequently activates downstream effectors to counteract the incoming stress. Upon resolution of or adaptation to the cue, the signalling pathway is generally desensitized in a timely manner in order to be reactivated later. This process of desensitization is governed by phosphorylation and dephosphorylation, events modulated by kinases and phosphatases, respectively, which constitute key post-translational modifications that switch signalling components on or off. Thus, orchestrated regulation of kinases and phosphatases in signalling pathways are critical to maintaining cellular homoeostasis2, recent large-scale functional analyses of fungal kinases and transcription factors (TFs) provide even more comprehensive insights into how fungal signalling pathways modulate infection and virulence. Indeed, 178 TFs and 183 kinases were identified in Cryptococcus neoformans, which causes life-threatening meningoencephalitis mainly in immunocompromised patients and is responsible for more than 180,000 deaths annually3; of the 155 and 129, respectively, functionally characterized in vitro and in vivo5, 45 TFs and 63 kinases are involved in its pathogenicity. Nevertheless, the regulation of these signalling components and their coordination in pathogenicity remain elusive.Fungal pathogens also utilize these complex signalling pathways to respond and adapt to dramatic environmental changes occurring during infection, colonization, proliferation, and dissemination to various tissues within a host. Although several key signalling pathways governing fungal pathogenicity have been elucidated in past decades6. However, recent systematic functional phosphatome data in several fungal pathogens indicate that phosphatases play critical roles in maintaining cellular homoeostasis by controlling growth and cell cycle, differentiation, stress response, and metabolism. In ascomycete fungi, genome-wide analyses of phosphatases have identified 32 protein phosphatases and functionally characterized 24 of them in Aspergillus fumigatus, the majority of which contribute to stress response, iron assimilation, and toxin production and resistance7. Furthermore, in the wheat scab fungus Fusarium graminearum, Yun et al. identified 82 phosphatase genes, disrupted 71 of them, and functionally analysed these mutants across 15 phenotypic traits , finding that 25 of these phosphatases are involved in virulence of the phytopathogenic fungus8. Among phosphatases, calcineurin is a well-established drug target and a globally conserved fungal virulence factor9.To better understand TF and kinase networks, co-analysis of the phosphatase networks as signalling counterparts is essential. Historically, phosphatases have received less attention than kinases, probably because kinases, which possess high substrate specificity, are generally considered to be better drug targets than substrate-promiscuous phosphatasesC. neoformans phosphatase network and interplay with its kinase and TF networks to better understand its pathobiological signalling. To this end, we constructed a high-quality library of 219 signature-tagged gene-deletion mutant strains representing 109 phosphatases out of 139 putative phosphatases identified in C. neoformans, in addition to 11 signature-tagged mutants representing six phosphatases that we previously constructed11. Using a total of 230 signature-tagged mutants representing 114 phosphatases, we analysed their phenotypic traits under 30 distinct in vitro conditions and performed a large-scale virulence assay using two model host systems (insect and murine). This entire phosphatase phenome data set is freely available to the public through the C. neoformans Phosphatase Phenome Database (http://phosphatase.cryptococcus.org).The goal of this study was to systematically analyse the functions of the C. neoformans (H99 strain) genome database (http://fungidb.org/fungidb) and validated the presence of phosphatase-related domains using protein sequence analysis and classification (Interpro). Through these analyses, we retrieved a total of 139 putative phosphatase genes in C. neoformans . Third, compared to other non-pathogenic and pathogenic yeasts, C. neoformans contains a similar number of putative phosphatases . In addition, we previously deleted two phosphatase genes with kinase domains (OXK1 and FBP26) for construction of the kinase mutant library5 and one phosphatase gene with a DNA-binding domain (APN2) for the TF mutant library4. Besides the 11 signature-tagged mutant strains  that we previously constructed, we performed a large-scale homologous recombination-based gene deletion using nourseothricin-resistance markers containing a series of unique oligonucleotide signature tags that we previously employed for construction of the TF and kinase mutant libraries5 (Supplementary Data Cryptococcus neoformans Phosphatase Phenome Database (http://phosphatase.cryptococcus.org) we constructed for this study. For the remaining 25 phosphatase genes, we failed to obtain any viable transformants or obtained only potential aneuploid mutants possessing both wild-type and mutant alleles after repeated attempts, suggesting these genes may be essential (Supplementary Data Cryptococcus neoformans Phenome Gateway Database (http://www.cryptococcus.org/), in which every TFs, kinases, and phosphatases studied5 are linked to the most widely used fungal genome database “FungiDB” (https://fungidb.org/fungidb/) to maximize connectivity between research data.To obtain comprehensive insights into the C. neoformans phosphatases deleted in this and previous studies, we examined phenotypic traits under 30 distinct in vitro conditions: growth at different temperatures , mating efficiency, virulence factor production , stress responses , and antifungal drug susceptibility  after 3, 7, 14, and 21 days post-infection (dpi) with strain H99S using the nCounter gene expression profile  with 139 novel phosphatase probes. The in vivo expression levels of each phosphatase were normalized to average in vivo expression levels of eight housekeeping genes  and compared to those under basal growth conditions [yeast extract-peptone-dextrose (YPD) medium at 30 °C]  a virulence assay using the insect larval model system Galleria mellonella and (2) signature-tagged mutagenesis (STM)-based lung and brain infectivity assays using a murine inhalation model. Both methods have been successfully used for identifying virulence-related genes from a large-scale gene-deletion mutant set in previous studies24.To identify phosphatases required for the pathogenicity of CNA1, TPS2, CAC1, PTP2, VPS29, PPH3, GDA1, YVH1, SSU72, PHS1, SIW14, DBR1, PSR1, YND1, INP5201, INP5202, HAD1, SIT4, PPG1, GUA1, NEM1, FBP26, and OCA101. STM-based murine lung and brain infectivity revealed 24 infectivity-related phosphatase genes, TPS2, CNA1, VPS29, PHS1, SIW14, CAC1, SSU72, PPG1, INP5201, CCR4, DBR1, GUA1, YMR1, NEM1, OCA1, PTP2, FBP26, PSR1, MRE11, CDC1, XPP1, SDP102, SDP101, and OCA101 11, Had1 16, and Ptp2 (phosphotyrosine phosphatase)10 have previously been reported as requisite for C. neoformans virulence, further corroborating our findings.Using the insect-killing virulence assay, we identified 23 phosphatase genes involved in virulence Fig. : CNA1, TC. neoformans pathogenicity: membrane integrity , DNA damage response , melanin production , cell wall integrity , and growth at 37 °C  . Similarly, although Inp5201 is paralogous to Inp5202, we failed to generate inp5201Δ inp5202Δ double mutants (data not shown), likely because the inp5201Δ mutant alone showed severe growth defects even at 30 °C  showed reduced capsule production  also showed reduced virulence , the latter of which likely resulted from defective melanin production  complex, which contains Vps29, Vps35, and Vps26, and a membrane-deforming sorting nexin (SNX) complex, which contains Vps5 and Vps1732.Among the newly identified virulence-related phosphatases in this study, Vps29 (CNAG_00182) is a putative component of the retromer complex, first discovered in C. neoformans. In the C. neoformans genome, we identified all of the remaining retromer component genes encoding proteins orthologous to Vps35 (CNAG_01837), Vps26 (CNAG_01426), Vps5 (CNAG_01315), and Vps17 (CNAG_00508), suggesting that the retromer complex is evolutionarily conserved in this fungal pathogen. We deleted each gene in the H99 strain and performed phenotypic analyses  of C. neoformans cells were obtained by hydrazinolysis, labelled with 2-aminobenzoic acid (2-AA), and then analysed using HPLC with fluorescence detection , with minor O-glycan species containing xylose (X1M2–X1M4) and a minor α1,2-mannotriose (M3*) species , indicating that the two proteins may have a synthetic lethal relationship in C. neoformans. Because both gda1Δ and ynd1Δ mutants exhibited reduced virulence in the insect killing assay . However, because the phosphatase mutants administered through intranasal inhalation pass through the lungs first, those reaching the brain were less equally distributed than in pooled input mutants, which made direct comparison of lung and brain STM scores difficult. Therefore, to further address the role of these phosphatases in brain infection, we monitored the ability of the pathogenicity-related phosphatase mutants, except those showing reduced growth at 37 °C, to traverse the BBB. Among these, five mutants  showed significantly reduced ability to traverse the BBB , probably due to the low glucose concentration (0.1% glucose). Therefore, it is unlikely that Cryptococcus replication in the bottom well complicated our data.The most lethal damage conferred by BBB Fig. . We receion Fig. , suggest2): PDR802, FZC31, and GAT20139. Therefore, we next addressed whether these genes, in addition to known brain infection-related genes [inositol transporter genes (ITR1a and ITR3c) and MPR1]41, were induced by HMC in xpp1Δ, ssu72Δ, siw14Δ, and sit4Δ mutants. Among these, SSU72 deletion most markedly reduced HMC-mediated MPR1 induction , RNA processing (Ssu72), and purine metabolism (Xpp1-Met3) were critical for BBB crossing by C. neoformans. Collectively, these findings indicate that C. neoformans utilizes complex signalling networks for brain infection.To obtain insight into the integrated signalling networks governing brain infection by ses Fig. 39. We foC. neoformans with those available in other plant and animal pathogenic fungi65, we found several core fungal pathogenicity-related phosphatases  have been shown to be required for A. fumigatus virulence55. Upon comparison with pathogenicity-related phosphatases in F. graminearum, eight phosphatases have been shown to be required for the virulence of both animal and plant fungal pathogens: Sit4, Yvh1, Sdp2/Msg5 (an Sdp101 orthologue), Ptp2, Ac1 (a Cac1 orthologue), Tps2, Inp53 (an Inp5201 orthologue), and Ppg1. Sit4 is involved in the TOR pathway, Cac1 is involved in the cAMP pathway, and Ppg1 and Yvh168 are involved in cell growth, nutrient sensing, and the stress response in fungal pathogens69. The Cna1 and Had1-mediated calcineurin pathway, the Tps2-mediated trehalose pathway, and the Msg5-mediated Mpk1/Slt2 MAPK pathway are all required for maintaining cell wall integrity. Ptp2, which is a major negative feedback regulator of the HOG pathway, is involved in adaptation and the stress response in fungal pathogens. Inp51, Inp52, and Inp53 are involved in phosphoinositide signalling, which controls vesicle trafficking, the actin cytoskeleton, and cell wall integrity71. Based on these data, phosphatases and signalling pathways involved in cell growth, nutrient sensing, cell wall integrity, the stress response, and phosphoinositide signalling appear to play pivotal roles in general fungal pathogenicity. Notably, however, deletion of PPH3 reduces C. neoformans and F. graminearum virulence but enhances C. albicans virulence42. In contrast, deletion of PTC2 and PTC3 reduces C. albicans and F. graminearum virulence, respectively, but does not reduce C. neoformans virulence72. Thus, some phosphatases may play differential roles in controlling the virulence of various fungal pathogens.By comparing the virulence data of phosphatase mutants in ses Fig. . In the C. neoformans, including protein phosphatases, lipid/nucleoside/carbohydrate phosphatases, and pyrophosphatases, and constructed a high-quality library of 219 signature-tagged gene-deletion mutant strains representing 109 phosphatases for functional analysis. By incorporating 11 additional signature-tagged mutants representing Six phosphatases that we previously constructed11, we systematically analysed the in vitro and in vivo phenotypic traits of a total of 230 signature-tagged mutant strains representing 114 phosphatases . Under 30 distinct in vitro growth conditions , 60 out of 114 mutants (53%) exhibited at least one phenotypic trait. Among the remaining 54 mutants that did not show a phenotypic trait under these conditions, none possessed infectivity and virulence defects, suggesting that these 54 phosphatases may not be involved in the pathobiology of C. neoformans. Among these, however, functionally redundant phosphatases could exist, and evident phenotypes may only result from their combined mutation. In particular, our in vivo expression profiling revealed that the following phosphatase genes were markedly upregulated during host infection: CDC1, GDA1, GEP4, NDX2, NDX3, PTP3, APH4, HAD9, YMR1, INM1, PPU1, DPP101, and AKP1  are known to be essential in S. cerevisiae, S. pombe, or C. albicans:  are likely to be core essential fungal phosphatases because they are reported to be essential in S. cerevisiae and S. pombe. Out of 15 reported essential phosphatase genes in S. cerevisiae, we successfully deleted PHS1, CYR1 (Cac1 ortholog), CDC1, CDC14, SSU72, and DUT101 in C. neoformans. PHS1, CDC1, and DUT101 are also essential in S. pombe, suggesting that they are functionally divergent between ascomycete and basidiomycete fungi or have a synthetic lethal relationship with other functionally redundant phosphatases in C. neoformans. We have described similar findings in our previous analysis of kinase mutant collections in C. neoformans5. Therefore, it seems evident that some kinase/phosphatase-mediated signalling networks are functionally divergent among fungi.Our systematic analysis unveiled both evolutionarily conserved and distinct sets of phosphatases in PTP2, CAC1, CNA1, CCR4, and HAD1 in the virulence of C. neoformans16. Furthermore, in examining phosphatase mutants in a large deletion mutant analysis24, Liu et al. demonstrated that 4 of 14 mutants  show reduced infectivity in the lungs24, consistent with our findings in cac1Δ, sdp102Δ, and mre11Δ mutants. Notably, Madhani’s and our studies indicate that the yvh1Δ mutant is as infective as the wild-type strain as quantified by lung STM analysis, but, in our study, the yvh1Δ mutant exhibited attenuated virulence in the insect-killing model. Another deviation from prior research is the secreted acid phosphatase APH1, which was reported by Lev et al. to contribute to the virulence of C. neoformans18, but we found that deletion of APH1 did not significantly contribute to virulence in either the insect-killing assay or murine infection model. We attribute this discrepancy to the fact that Lev et al. incubated infected G. mellonella at 30 °C; because when we incubated insect hosts at 37 °C, the impact of thermotolerance at mammalian temperatures may have offset any advantages seen at lower temperatures18. For the remaining 23 phosphatases reported here, we are the first to describe their role in the pathogenicity of C. neoformans.Here, we unravelled 31 pathogenicity-related phosphatases that affect a variety of biological processes, including growth, virulence factor production, stress response, carbohydrate metabolism, cell signalling, protein sorting, and vesicular trafficking. Some of these findings are corroborated by previous research, including the role of C. neoformans.In this study, we found that a majority of the 31 pathogenicity-related phosphatases were involved in regulation of three major cryptococcal virulence factors: thermotolerance, capsule, and melanin. Fourteen of them  promote thermotolerance at 37 °C. In addition, 13 phosphatases  are involved in melanin production. Finally, capsule production was altered by deletion of six phosphatases: Cac1, Sdp102, Ptp2, Ynd1, Psr1, and Inp5201. Therefore, a total of 20 phosphatases are involved in regulation of any of the three major virulence factors, and 12 of them  are involved in modulating more than two virulence factors. The remaining 11 phosphatases  were unrelated to thermotolerance or capsule/melanin synthesis. Among these, Sdp101 and Inp5202 are paralogous to Sdp102 and Inp5201, respectively, but we failed to obtain corresponding double mutants, suggesting that Sdp101/Sdp102 and Inp5201/Inp5202 may have a synthetic lethal relationship and play redundant roles in C. neoformans pathogenicity. However, the ymr1Δ, cdc1Δ, and had1Δ mutants showed increased susceptibility to cell membrane disruption, and this instability may have contributed to reduced infectivity or virulence relative to WT. The had1Δ mutant also exhibited increased susceptibility to cell wall-destabilizing agents, which is in good agreement with recent reports showing that Had1 is a potential target of calcineurin, which plays a role in regulating cell wall integrity74. Had1 was also shown to be required for virulence in a murine model of systemic cryptococcosis16. YMR1 encodes a phosphatidylinositol 3-phosphate (PI3P) phosphatase involved in vacuolar protein sorting pathways that functionally overlaps with other lipid phosphatases in S. cerevisiae. Therefore, deletion of YMR1 does not generate evident phenotypes in the yeast model76. Cdc1 is a putative mannose-ethanolamine phosphatase phosphodiesterase involved in both cell division cycle and GPI-anchor remodelling78. The role of Ymr1 and Cdc1 in vacuolar protein sorting and GPI remodelling, respectively, may contribute to membrane stability in C. neoformans. In S. cerevisiae, Pph3 is a catalytic subunit of the protein phosphatase PP4 complex and governs recovery from the DNA damage checkpoint80. Pph3 likely plays a similar role in C. neoformans because the only in vitro phenotype observed in the pph3Δ mutant was increased susceptibility to DNA damage by methyl methane sulfonate (MMS). Given our previous demonstration that the DNA damage response pathway is critical for the virulence of C. neoformans82, we hypothesize the role of Pph3 in the DNA damage response likewise contributes to virulence.The pathobiological functions of Ymr1, Cdc1, Had1, and Pph3 remain unclear because mutants of these genes do not demonstrate clear phenotypic traits related to C. neoformans TFs and kinases involved in brain infection processes, including BBB adhesion and crossing39. Here we found that the following pathogenicity-related phosphatases are also involved in BBB crossing of C. neoformans: Xpp1, Sit4, Ssu72, Siw14, and Gda1. In S. cerevisiae, Ppx1 (the orthologue of Xpp1) is a metal-dependent cytosolic expolyphosphatase and generates inorganic phosphate by cleaving polyphosphate that serves as a major phosphate reservoir and is required for long-term cell survival, gene regulation, cell motility, and stress responses83. The functions of Sit4 and Ssu72 are likely related to the target of rapamycin (TOR) pathway involved in nutrient-sensing. In S. cerevisiae, Sit4 is regulated by the nutrient-sensing TOR pathway84 and controls nitrogen catabolite repression genes through the GATA TF Gln385. Prior to transcriptional termination, Ssu72 dephosphorylates the C-terminal domain of the RNA polymerase II, which is degraded through the TOR-signalling pathway in response to rapamycin86. Accordingly, our sit4Δ and ssu72Δ mutants indeed exhibited highly increased susceptibility to rapamycin , and its deletion increases IP7 and decreases IP6 levels87. Because deletion of the inositol pentakisphosphate kinase IPK1 decreases IP6 levels, abolishing the virulence of C. neoformans88, it is likely that Siw14 could promote BBB crossing and virulence of C. neoformans through modulation of the inositol signalling pathway. Finally, Gda1 is a Golgi membrane-bound apyrase that modulates O-mannosylation in yeast35. We previously reported that the extended structure of O-glycans by mannose addition in the Golgi is required for cell integrity and full pathogenicity of C. neoformans38. Based on these observations, we conclude that phosphate utilization, TOR-mediated nutrient sensing, inositol-signalling, and O-mannosylation are critical for BBB crossing and brain infection.We recently identified a group of C. neoformans, C. albicans, and A. fumigatus. Sit4, Yvh1, Sdp101, Ptp2, Cac1, Tps2, Inp5201, and Ppg1 are required for virulence of both human and plant fungal pathogens, thus could be targets for the development of broad-spectrum antifungal drugs. Tps2 is a particularly promising antifungal drug target because the trehalose pathway is missing in humans89. Recently, the structure of the N-terminal domain of C. albicans Tps2 has been resolved90 and provides a structural basis for the design of Tps2-specific antifungal agents. Siw14, Had1, and Oca1/101 could also be good targets for cryptococcal treatment because there are no evident orthologues of these phosphatases in humans. Recently, the structure of the S. cerevisiae inositol phosphatase Siw14 has also been resolved91, whereas structural information for Had1 and Oca1/Oca101 is not yet available.The 31 pathogenicity-related phosphatases that were identified here and in previous studies could be potential antifungal drug targets, especially Sit4, Cna1, and Tps2, which are required for virulence of the major human fungal pathogens 92, FK506 inhibition of the calcineurin complex95, and inhibition of dual-specificity phosphatases by several small molecules96. An example of the latter strategy includes ceramide and its derivatives, which are bioactive sphingolipid molecules that activate PP2A by preventing SET inhibitor binding to PP2A98. To increase the specificity of a drug that targets a phosphatase, both the catalytic and regulatory subunits of the phosphatase complex should be considered, such as was considered with the drug FK506, which targets both the catalytic and regulatory subunits of the calcineurin complex via FKBP1295. Based on BLASTP analysis using annotated information from the Saccharomyces Genome Database , we found 26 putative phosphatase regulatory subunit orthologues in C. neoformans  of Yonsei University. The Yonsei University IACUC approved all vertebrate studies.C. neoformans serotype A H99S strain background through homologous recombination using gene-disruption cassettes containing the nourseothricin-resistance marker  using NAT-split marker/double joint PCR (DJ-PCR) strategies99 (see Supplementary Data NAT marker was amplified by PCR with primers M13Fe (M13 forward extended) and M13Re (M13 reverse extended) from a pNAT-STM plasmid containing the NAT gene with each unique, signature-tagged sequence. After amplification of the 5′- and 3′-flanking regions and NAT marker in the first round of PCR to generate template DNA, the second round of PCR constructed 5′- and 3′-regions of the NAT-split gene-disruption cassette with primer pairs L1/NSL and R2/NSR, respectively. Biolistic transformation introduced the NAT gene-disruption cassettes by incubating the H99S strain in 50 ml YPD medium for 16 h at 30 °C, followed by spin-down, re-suspension in 5 ml distilled water, application on YPD agar medium containing 1 M sorbitol, and further incubation for 3 h at 30 °C. Gene-disruption cassettes were then combined with 600 μg of 0.6-μm gold microcarrier beads  and introduced into cells using a particle delivery system . After 4 h incubation at 30 °C for recovery of cell membrane integrity, cells were scraped and spread on YPD agar medium containing nourseothricin (100 μg/ml). Diagnostic PCR identified NAT-positive transformants. Southern blot analysis confirmed the genotype of each screened transformant, and we constructed at least two independent mutant strains for each phosphatase gene.We constructed phosphatase and retromer mutant strains in the 39, we performed an nCounter gene expression analysis (NanoString) to quantify in vivo expression levels of the 139 phosphatases. Total RNA samples were obtained from 6-week-old female A/J mice infected with 5 × 105 cells through nasal inhalation. Each group of three mice was sacrificed 3, 7, 14, or 21 dpi, and the lungs, brain, spleen, and kidneys were recovered and lyophilized. Dried organs were homogenized, and total RNA was extracted with a commercial RNA extraction kit . Samples containing 10 ng of total RNA isolated from C. neoformans grown under in vitro basal conditions  or 10 μg of total RNA isolated from C. neoformans-infected mouse tissues were reacted with the custom-designed probe code set according to the manufacturer’s standard protocol of the nCounter multiplex platform 23. Scanning was performed by digital analyser through high resolution (600 fields) option and normalized by nSolver software (provided by NanoString). A total of eight housekeeping genes  were used for expression normalization100. Normalized data were transformed to log2 scores to express fold-change and subjected to clustering using one minus Pearson correlation with average linkage by Morpheus .Using previously reported RNA samplesC. neoformans was grown for 16 h at 30 °C, serially diluted 10-fold (1–104), and spotted on YPD agar media containing the following chemical agents to induce environmental stress as described previously5: osmotic stress (sorbitol) and cation/salt stresses (NaCl and KCl) under either glucose-rich (YPD) or glucose-starved  conditions; oxidative stress ; toxic heavy metal stress [cadmium sulfate (CdSO4)]; genotoxic stress (methyl methanesulphonate and hydroxyurea); membrane destabilizing stress [sodium dodecyl sulfate (SDS)]; cell wall destabilizing stress ; ER stress [tunicamycin and dithiothreitol (DTT)]; and antifungal drug susceptibility . Cells were incubated at 30 °C for 1–5 days and photographed daily. To examine the growth of C. neoformans strains at different temperatures, we spotted serially diluted cells on YPD agar medium, incubated them at 25, 37, and 39 °C, and photographed the cultures daily. To quantitatively examine the growth rate of phosphatase mutants, the WT strain (H99S) and phosphatase mutants were incubated at 30 °C overnight and sub-cultured into fresh liquid YPD medium [optical density at 600 nm (OD600) = 0.2]. Cells were than incubated at 30 or 37 °C in a multi-channel bioreactor , and OD600nm was automatically measured for 40–90 h.To analyse the susceptibility of each phosphatase mutant to in vitro stress conditions, MATα phosphatase mutant constructed in the H99S strain and MATa KN99a strain were cultured in YPD medium for 16 h at 30 °C, washed twice with phosphate-buffered saline (PBS), mixed at equal concentrations (107 cells/ml), spotted on V8 mating media (pH 5), and incubated at 25 °C in the dark for 7–14 days. Filamentous growth was observed and photographed weekly.To examine unilateral mating efficiency, each serotype A 8 cells/ml, and 50 µl of the cell suspension was injected into microhaematocrit capillary tubes . Capillary tubes were placed vertically for 10 days to pack cells by gravity. Packed cell volume ratio  was measured, and the relative packed cell volume of each mutant was calculated by normalizing each ratio with packed cell volume ratio of wild-type H99S strain. Statistical differences in relative packed cell volume ratios were determined by one-way analysis of variance (ANOVA) with Bonferroni’s multiple comparison test in Prism 8 . To measure capsule production efficiency in other media, 5 μl of each culture was spotted on Littman’s agar medium101 and FBS agar medium , incubated at 37 °C for 2 days, scraped, and resuspended with distilled water. Resuspended cells were stained by India ink  and observed by differential interference contrast (DIC) microscopy . Capsule thickness was measured by subtracting the cell diameter from capsule diameter . For quantitative measurement of capsule thickness, 50 cells were measured for the H99S strain and each phosphatase mutant. To examine melanin production efficiency, each phosphatase mutant was cultured for 16 h in YPD medium at 30 °C, washed with PBS, and spotted (3 μl) on Niger seed, dopamine, or epinephrine media  containing 0.1% or 0.2% glucose. Spotted cells were incubated at 37 °C and photographed after 1–3 days. For the phosphatase mutants growth-defective at 37 °C, melanin and capsule production efficiency were examined at 30 °C. To examine urease production, each phosphatase mutant was cultured at 30 °C for 16 h, washed with PBS, and inoculated (106 cells) onto liquid Christensen’s media in a 10-ml medical tube , then incubated at 30 °C in a shaking incubator for 1–3 days and photographed daily.To test capsule production efficiency, each mutant was cultured at 30 °C, spotted onto Dulbecco’s modified Eagle’s (DME) agar medium, and incubated at 37 °C for 2 days, after which cells were scraped, washed with distilled water, fixed with 10% formalin, and washed again with distilled water. Fixed cells were adjusted to a concentration of 3 × 10LAC1, HOB1, MBS1, and BZP4), the H99S strain and phosphatase mutants  were incubated in liquid YPD medium for 16 h at 30 °C and sub-cultured into fresh liquid YPD medium (OD600 = 0.2). When the cells reached the early logarithmic phase (OD600 = 0.6–0.8), half of the cell culture was sampled to prepare a basal sample. The remaining cell culture was washed three times with PBS and incubated in nutrient-starvation conditions (YNB medium with ammonium sulfate without glucose) for 2 h, then immediately pelleted with liquid nitrogen and lyophilized. To measure the expression level of BBB crossing-related genes , the H99S strain and phosphatase mutants  were incubated in YPD broth at 30 °C for 16 h, sub-cultured into 50 ml of fresh YPD broth, and further incubated until OD600 reached 0.8. The culture was then separated into two 25-ml tubes, centrifuged, and washed three times with sterile distilled water. One tube was kept in liquid nitrogen to monitor basal expression levels, and the other tube was resuspended with an equal volume of RPMI1640 medium containing 10% FBS. After 3 h incubation at 37 °C in a CO2 incubator with 120 rpm horizontal shaking, cells were centrifuged and lyophilized overnight. Total RNA was extracted from each sample using a commercial RNA extraction kit  and cDNA was synthesized using RTase . Quantitative reverse transcription-PCR (qRT-PCR) was performed with target gene-specific primer pairs listed in Supplementary Data To measure the expression level of known melanin-regulating genes (O-linked glycans from cwMPs was conducted as described previously102. The O-linked oligosaccharides were released from the purified cwMPs by modified hydrazinolysis. Dried cwMPs (50 μg) were resuspended in hydrazine monohydrate and incubated at 60 °C for 4 h. After cooling and desiccation of the reactant, the pellets were dissolved in NaHCO3, mixed with (CH3CO)2O, and incubated on ice for 30 min. O-glycans were purified by Dowex 50WX8-400 resins  and labelled with 2-AA. The purified O-glycan was analysed using HPLC on a TSKgel Amide-80 column  with 90% solvent A (2% acetic acid and 1% tetrahydrofuran in acetonitrile) and 10% solvent B . After sample injection, the proportion of solvent B increased to 90% over 60 min at a flow rate of 1.0 ml/min and then O-glycans were detected with a fluorescence detector  at excitation and emission wavelengths of 360 and 425 nm, respectively. Data were analysed by using chromatography software .Analysis of G. mellonella caterpillars  in the final larval instar with a body weight of 200–300 mg, arriving within 7 days from the day of shipment, were used for in vivo insect virulence assays. Each phosphatase mutant and wild-type H99S strain was incubated at 30 °C overnight, pelleted, washed three times with PBS, and resuspended in PBS at a concentration of 106 cells/ml. Four thousand C. neoformans cells per larvae were injected into the second-to-last prolegs with a 100-µl syringe equipped with a 10-µl needle and repeating dispenser . Negative control G. mellonella received PBS only. Infected larvae were placed in Petri dishes in a humidified container, incubated at 37 °C, and monitored daily. Larvae were considered dead when they turned black and showed no movement upon touching. Larvae that pupated during the experiment were censored for statistical analysis. Survival curves were illustrated using Prism 8  and analysed with a log-rank (Mantel–Cox) test. We examined two independent strains for each phosphatase mutant.At least 15 NAT selection markers was cultured for 16 h at 30 °C. The ste50∆ (STM#282) and ire1∆ (STM#169) mutants were used as virulent and avirulent control strains, respectively, as described previously104. Mutants and control strains were pelleted, washed three times with PBS, resuspended in PBS, and then pooled at equal numbers (5 × 105 cells). Seven-week-old female A/J mice  anaesthetized with an intraperitoneal injection of Avertin  were infected with 5 × 105 cells of pooled mutants (in 50 µl PBS) through intranasal inhalation. To prepare the input phosphatase genomic DNA library, 200 µl of pooled strains were spread on YPD media containing 100 µg/ml chloramphenicol, incubated at 30 °C for 3 days, and collected by scraping. The infected mice were sacrificed with an overdose of Avertin 14 dpi. Lungs and brains of infected mice were recovered and homogenized with 5 ml of PBS. Homogenized tissues were then spread on YPD medium containing 100 µg/ml chloramphenicol, incubated at 30 °C for 3 days, and collected by scraping. Genomic DNA was extracted from collected input and output cells using the cetyl trimethylammonium bromide (CTAB) method103. Quantitative PCR was performed with tag-specific primers output−input)−.A set of phosphatase mutants with the 41 unique signature-tagged 107. Briefly, 5 × 104 hCMEC/D3 cells in EGM-2 medium  were seeded on collagen -coated 8-μm porous membranes  for the BBB-crossing assay or 12-well plates (BD Biosciences) for the BBB-adhesion assay. The day after seeding, medium was refreshed with EGM-2 medium supplemented with 2.5% human serum and further incubated for 4 days. A day before C. neoformans inoculation, the medium was replaced with 0.5 × diluted EGM-2 medium, and the cells were maintained at 37 °C and 5% CO2. The integrity of tight junctions between cells was confirmed by verifying that the trans-endothelial electrical resistance (TEER) measured ~200 Ω/cm2, as assed by an epithelial volt/ohm meter .Human brain microvascular endothelial cells  were cultured as follows based on the reported methods5 cells of C. neoformans WT (H99), mpr1∆ mutant, and phosphatase deletion mutants were added to 500 µl of PBS and inoculated onto the porous membranes. After 24 h incubation at 37 °C in 5% CO2, the number of yeast cells passing through the porous membrane was measured by counting CFU. Tight junction integrity was measured by TEER as described above before and after inoculation of yeast cells. The BBB migration ratio was calculated by dividing the output CFU of each tested strain by that of WT. For the BBB-adhesion assay, 5 × 105 yeast cells in 100 µl of PBS were inoculated onto a monolayer of hCMEC/D3 cells grown in a 12-well plate and incubated for 24 h at 37 °C in 5% CO2. Following incubation, cultures were washed three times with PBS, incubated with sterile distilled water for 30 min at 37 °C to burst the host cells, and collected for CFU quantification. The BBB-adhesion ratio was calculated by dividing the adhered CFU of each test strain by that of WT C. neoformans.For the BBB-crossing assay, 5 × 10Cryptococcus neoformans Phosphatase Phenome Database (http://phosphatase.cryptococcus.org) and Cryptococcus neoformans Phenome Gateway Database (http://www.cryptococcus.org/) to facilitate public access to the phenomic and genomic data for the C. neoformans phosphatase mutant library. The database was built under the same software development environment as described in our two previous publications5. Genome sequences and annotations of the H99S strain were obtained from the standardized genome data warehouse in the Comparative Fungal Genomics Platform Database 108. Except reported phosphatases, those first functionally characterized by this study were named based on the published gene nomenclature rules for C. neoformans109  and mutants was calculated by one-way ANOVA with Bonferroni’s multiple comparisons test.Statistical analyses were performed with GraphPad Prism version 8. For the capsule production assay and expression analysis, ANOVA with Bonferroni’s multiple comparison test was used. For the insect-killing assay, the log-rank (Mantel–Cox) test was used for statistical analysis. For the murine STM analysis, the statistical significance between Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Supplementary Data 7Supplementary Data 8Supplementary Data 9Supplementary Data 10Reporting Summary


Concurrent type 1 diabetes mellitus (T1DM) and idiopathic nephrotic syndrome is rare, and most previously reported cases were in children. We report the case of an adult woman who developed T1DM and minimal change nephrotic syndrome (MCNS) nearly simultaneously.A 24-year-old woman had first presented to another hospital with nausea, vomiting, and fatigue. She was diagnosed with diabetic ketoacidosis and T1DM on the basis of her hyperglycemia, ketoacidosis, and positive anti-glutamic acid decarboxylase antibody test result. Rapid infusion of normal saline and insulin administration alleviated hyperglycemia and ketoacidosis. Two weeks after admission, however, she developed nephrotic syndrome (NS) with rapidly decreasing urine volume. She was referred to our hospital with a diagnosis of acute kidney injury. Although she temporarily required dialysis and high doses of insulin, within 1 month NS and acute kidney injury had been alleviated by oral prednisolone and low-density lipoprotein apheresis. Renal biopsy showed minor glomerular abnormalities without diabetic nephropathy, so we diagnosed her with MCNS. Seven weeks after the discharge, NS relapsed, and cyclosporine was added to prednisolone. However, NS relapsed twice within the next 4 months, so we started her on rituximab. At 6 months after initiating rituximab therapy, she remained in complete remission.Her mother also had T1DM but not MCNS. The patient had HLA-DRB1*09:01/09:01, DQB1*03:03/03:03, and her mother had HLA-DRB1*04:05/09:01, DQB1*03:03/04:01.Concurrent T1DM and MCNS is rare and their coexistence might be coincidental. Alternatively, they might have been caused by an underlying, unidentified genetic predisposition. Previous reports and our patient’s findings suggest that specific HLA alleles and haplotypes or a Th1/Th2 imbalance might be associated with T1DM and MCNS that occurred nearly simultaneously. Type 1 diabetes mellitus (T1DM) and idiopathic nephrotic syndrome are more common in children than in adults. The incidence of T1DM in Japanese children < 14 years of age is 1.4–2.25/100,000 , which iSome case reports described children who had developed idiopathic nephrotic syndrome soon after being diagnosed with T1DM, whereas others, conversely, had developed T1DM soon after being diagnosed with idiopathic nephrotic syndrome –11. Most3], 8 mmol/L; anion gap, 21.3 mmol/L), and hemoglobin A1c (12.7%). The anti-glutamic acid decarboxylase antibody test result was 708 U/mL, and she was diagnosed with diabetic ketoacidosis and T1DM.A 24-year-old Japanese woman was admitted to another hospital with nausea, vomiting, and general fatigue. She had a 2-month history of strongly feeling thirsty, and she had lost 5 kg during that period. She had no history of infectious diseases such as bronchitis before feeling thirsty. Her only medical history was seasonal allergic rhinitis, but her family history included T1DM. Her mother was diagnosed with T1DM at 29 years of age. Blood tests in the present patient showed a high blood glucose level (340 mg/dL), ketoacidosis  after explaining the prognosis of frequently relapsing nephrotic syndrome. Serum CD19/CD20 levels decreased to 0.1/0.0% after 1 month, and we discontinued cyclosporine. PSL was tapered from 30 mg/day to 5 mg/day over 6 months, and hemoglobin A1c remained at 7.1–7.9% with insulin treatment. At 6 months after initiating rituximab, she remained in complete remission without any reported side effects.Seven weeks later, the patient had a relapse of nephrotic syndrome, and cyclosporine 1.5 mg/kg (75 mg/day) was added to PSL. However, NS relapsed twice within the next 4 months, so we started her on a single dose of rituximab  administration might cause a drastic shift in the Th1 response to a Th2 response. Second, insulin lispro may have triggered an allergic reaction causing MCNS because she developed an itchy eruption shortly after the first insulin lispro injection and had a high level of serum IgE. No previous reports, however, have demonstrated the simultaneous onset of T1DM and MCNS, and due to a Th1/Th2 imbalance, and we did not measure the Th1/Th2 ratios and serum cytokine levels that were secreted by Th1 and Th2.In summary, we report the rare case of an adult woman with acute kidney injury that resulted from MCNS, which had developed soon after she was diagnosed with diabetic ketoacidosis that was caused by T1DM. The mechanism by which T1DM and MCNS occurred concurrently remains unclear, although some previous reports and our case indicate that genetic factors  or a Th1/Th2 imbalance might be associated with the onset of these two diseases.Additional file 1.


Escherichia coli (STEC) is an important foodborne pathogen. The increasing incidence of non-O157 STEC has posed a great risk to public health. Besides the Shiga toxin (Stx), the adherence factor, intimin, coded by eae gene plays a critical role in STEC pathogenesis. In this study, we investigated the prevalence and polymorphisms of eae gene in non-O157 STEC strains isolated from different sources in China. Among 735 non-O157 STEC strains, eae was present in 70 (9.5%) strains. Eighteen different eae genotypes were identified in 62 eae-positive STEC strains with the nucleotide identities ranging from 86.01% to 99.97%. Among which, seven genotypes were newly identified in this study. The eighteen eae genotypes can be categorized into five eae subtypes, namely β1, γ1, ε1, ζ3 and θ. Associations between eae subtypes/genotypes and serotypes as well as origins of strains were observed in this study. Strains belonging to serotypes O26:H11, O103:H2, O111:H8 are associated with particular eae subtypes, i.e., β1, ε1, θ, respectively. Most strains from diarrheal patients  carried eae-β1 subtype, while most isolates from cattle  carried eae-ζ3 subtype. This study demonstrated a genetic diversity of eae gene in non-O157 STEC strains from different sources in China.Shiga toxin-producing  Escherichia coli (STEC) is a group of food-borne pathogens that can cause non-bloody diarrhea, hemorrhagic colitis (HC), and the fatal hemolytic uremic syndrome (HUS) in humans1. It has been estimated that there are more than 470 STEC serotypes, among which O157:H7 serotype is usually associated with more severe clinical outcomes3. However, non-O157 STEC strains such as O26, O45, O103, O111, O121, and O145 (referred to as the ‘top six’ non-O157 STEC) have been increasingly recognized to cause food poisoning, bloody diarrhea, HUS, and other gastrointestinal illnesses in recent years5. Domestic and wild animals, including cattle, sheep and goats are considered to be the most important reservoirs of STEC6. Human infections mainly occur through ingestion of contaminated food or water, exposure to the environment or direct contact with animals7.Shiga toxin-producing 8. However, Stx alone is insufficient to cause severe illness without the adherence of bacteria to gut epithelial cells9. Multiplex genes that enable STEC strains to attach, colonize, produce and secrete toxin proteins have been identified9. In a subset of STEC strains, intimin plays a critical role in intestinal colonization, which is encoded by the eae gene that resides on the locus of enterocyte effacement (LEE) pathogenicity island. The LEE island encodes a type III secretion system that is responsible for the attaching and effacing (A/E) lesions on intestinal epithelia10. A/E lesions is characterized by the local effacement of microvilli, the tight attachment of bacteria to the eukaryotic surface and the subsequent reorganization of filamentous actin to pedestal-like structures11. Intimin is also an important virulence factor of other bacteria, such as enteropathogenic E. coli (EPEC), E. albertii, and Citrobacter rodentium12.Shiga toxin (Stx) is considered to be the primary virulence factor of STEC that is responsible for immunopathologies such as HC and HUSeae gene is about 2800 nucleotides. The N-terminal of intimin from different sources is highly conserved, while the C-terminal where cellular binding activity is highly variable13. Based on the difference of the C-terminal, at least 30 intimin subtypes have been defined, namely, α1, α2, α8, β1, β2, β3, γ1, γ2, ε1, ε2, ε3, ε4, ξ, z, z3, η, η2, θ, τ, ι1, ι2, κ, λ, μ, ν, υ, ο, π, ρ, and σ14. Intimin subtypes are correlated with host specificity and tissue tropism15. Intimin subtype β1 appears to be the most frequent among atypical EPEC strains from diarrheal patients and animals in China16. Several studies have shown the association between serotypes and specific intimin subtypes17. For example, O157:H7 and O145:H28 serotypes are associated with the eae-γ1 subtype, whereas O26:H11 often carries eae-β1, O103:H2 and O121:H19 harbor eae-ε, and O111:H8 harbors eae-θ subtype18. These serotypes were most frequently reported in global dysentery and HUS cases caused by STEC5. Thus, eae subtyping would be a valuable tool for risk assessment and prediction of disease outcome. However, there are limited studies of intimin characteristics among other non-O157 serotypes. In this study, we investigated the prevalence of eae gene and analyzed eae subtypes and polymorphisms in non-O157 STEC strains isolated from diarrheal patients, healthy carriers, animals, and raw meats in China.The full length of eae was present in 70 (9.5%) strains, which were isolated from cattle (n = 41), yak (n = 2), raw beef (n = 3), raw mutton (n = 1), and diarrheal patients (n = 9). Fourteen eae-positive strains were identified from unknown sources. All strains recovered from goat, pig, plateau pika, marmot, Tibetan antelope, water, and healthy carriers were eae-negative  were identified in each eae subtype to represent the diversity within a subtype. Except θ, the other four subtypes contained 2 to 8 genotypes respectively  in this study are unique comparing with all available eae sequences in the database (accessed 25/7/2019).The complete eae genotypes  contained 22, 8 and 8 strains respectively, and 10 genotypes contained only one strain, while the rest contained two to five strains , followed by O26:H11 , and O177: HNT .eae genotypes. Each eae genotype contained one to three different serotypes, with the exception of serotypes O5:HNT and O84:H2. Strains of O5:HNT serotype were assigned to β1/GT7, β1/GT8 or ε1/GT10 eae genotype, while O84:H2 strains carried eae ζ3/GT1, ζ3/GT2, or ζ3/GT3 genotypes  were detected among the 62 STEC strains.Overall, 43 strains harbored eae-β1 harboring strains, 10 strains carried stx1a, nine carried stx2c and the other possessed stx2a, or stx2f subtypes. The strains with eae-ε1 subtype carried diverse stx subtypes: two strains contained stx1a, two strains carried stx2c, one carried stx2a, and one harbored both stx1a and stx2c. Two of eae-γ1 strains carried stx1a and one harbored both stx1a and stx2a. All the 24 eae-ζ3 and 5 eae-θ containing strains possessed stx1a  are unique. Furthermore, all cattle-derived strains belonged to five unique eae genotypes, i.e. β1/GT5, β1 /GT6, ε1/GT2, ζ3/GT2, and ζ3/GT3  were due to the new combinations of previously known alleles. An eae genotype was corresponding to one or more STs and vice versa (Table The 62 strains were typed into 18 sequence types (STs) with 5 novel STs named as N1-N5. One new ST (strain STEC430) was resulted from a novel allele in A minimum spanning tree was constructed with the STs from this study and those of O157 and ‘top six’ non-O157 STEC from MLST database Fig. . Most STeae is an important virulence factor in many STEC strains, which plays a critical role in intestinal colonization. Previous studies revealed that most clinical STEC strains possessed eae20. Moreover, the presence of eae was significantly associated with a higher risk for HUS development21. STEC O157 isolates were strongly associated with the simultaneous presence of both stx2 and eae, forming the basis of why STEC O157 predominates in patients with HUS, when compared with non-O157 strains22. However, a subsequent study reported that eae was detected in the majority (52.5%) of non-O157 STEC strains in England23. A recent investigation of STEC infections in the south east of England revealed that 76% of non-O157 HUS-associated STEC isolates possessed eae gene24. These data indicated that the presence of eae is strongly related with disease severity irrespective of seropathotypes. In this study, we observed a lower prevalence of eae (29.0%) among non-O157 STEC strains from diarrheal patients, which might partially be due to limitation of the current study where the source of 14 eae-positive isolates is unavailable. On the other hand, the low prevalence of eae in diarrheal patients could account for the less severe disease in this study. Among the nine eae-positive isolates from diarrheal patients, only one was both stx1a and stx2a positive, while the rest all harbored stx1a only  have been reported to be associated with more virulent STEC and thus posing greater health risk28. In a previous study, the eae subtypes of STEC strains recovered from children with HUS in Uruguay, include γ1, γ2 and β129, highlighting the clinical significance of these eae subtypes. In this study, we found that β1 and ζ3 were the most prevalent eae subtypes, which were detected in strains from different sources. Notably, eae-β1 was the most predominant subtype among strains from diarrheal patients30.The eae subtypes in STEC strains, particularly predominant serotypes. Strains belonging to serotypes O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28 are associated with particular eae subtypes, i.e., γ1, β1, ε, θ, and γ1, respectively32. Consistent with previous studies, we found that all eight O26:H11 strains  in this study carried the eae-β1 subtype, all three O111 strains harbored the eae-θ subtype, and one O103 strain possessed the eae-ε1 subtype. However, the origin of three O111 strains and one O103 strain were unavailable. Notably, the three O111 strains share identical eae sequences with the outbreak strain O111:H8 in the United States33.The eae sequence of strain STEC801 (ONT:H7) isolated from a diarrheal patient was identical to that of strain Sakai, which caused a notorious outbreak in Japan in 199634. Comparison with STs observed in human non-O157 STEC infections gives an indication of the potential risk for different eae subtype STEC. Strains of the three STs  in this study had the same O serogroups with ‘top six’ non-O157 STEC . The three eae subtypes were θ, β1 and ε1, respectively. These results indicated that determination of eae subtypes could be used as a valuable tool in combinations with serotypes, and other virulence factors in risk assessment and prediction of severe disease outcomes.Studies have indicated association between serotypes and 36. The stx2a gene is most often present in eae positive STEC strains and has consistently been associated with HUS37. In this study, 6 out of 62 isolates carried stx2a subtypes, which were isolated from diarrheal patients, yak and other unknown sources. Notably, the 6 stx2a isolates harbored eae-β1, eae-ε1 and eae-γ1 subtypes, all of which were associated with high virulence, thus they were likely to have high pathogenic potential to humans.Shiga toxin subtypes have been found to differ in toxin potency. Strains that carry Stx2a (with or without Stx2c) are often associated with severe symptoms such as HC and HUSeae, the plasmid-carried enterohemolysin gene (ehxA) also plays an important role in STEC pathogenicity and frequently associated with diarrheal disease and HUS38. In a previous study, we described the presence and genetic diversity of the ehxA gene in 434 non-O157 STEC isolates. The ehxA gene was positive in 138 (31.8%) isolates, and 15 (10.9%) ehxA-positive isolates harbored eae, which were grouped into ehxA group I (n = 2) and group II (n = 13). All strains from diarrheal patients belonged to ehxA group II and most of those strains harbored eae. Thus, ehxA group II and eae-positive strains were clinically related39.Besides intimin gene eae genotypes and host sources. Most strains from diarrheal patients  carried eae-β1 subtype, and most isolates from cattle  carried eae-ζ3 subtype. However, the prevalence of eae subtypes in a specific source varied significantly among studies. For instance, Tostes et al.28 reported the predominance of eae subtypes λ/ γ1 and β in cattle isolates, and λ/ γ1 in human isolates. Similarly, Blanco et al.39 reported eae-ζ as most frequent subtype in E. coli isolates from sheep in Spain. Whereas, Aktan et al.40 identified eae-β and eae-γ as the most frequent subtypes among E. coli from sheep in England and Wales. The distribution of eae subtypes in the same source may vary among different regions.In this study, we observed the association between eae gene among non-O157 STEC strains from a wide variety of sources in China. Among 735 non-O157 STEC strains, eae was present in 70 (9.5%) strains isolated from diarrheal patients, animals, raw meats and other unknown sources. Five eae subtypes and 18 different eae genotypes were identified, suggesting the high diversity of eae among different sources. To our knowledge, this is the first study investigating the prevalence of eae subtypes among non-O157 STEC strains from a wide range of sources. Our study suggests associations between eae subtypes and serotypes as well as host sources. Furthermore, it can be inferred from this study that the determination of eae subtype could be considered together with seropathotypes and other virulence factors in risk assessment of STEC infections.In conclusion, the current study reports the prevalence and subtype of The current study and all experimental protocols were approved by the ethic committee of the National Institute for Communicable Diseases Control and Prevention, China CDC, with the number ICDC-2017006. This work was part of STEC surveillance program conducted in China, the informed permission has been obtained from patients and the owners of animals to use fecal samples and conduct relevant studies. All methods used in this study were performed in accordance with the relevant guidelines and regulations.43. Of the 735 STEC strains, 608, 60, 35, and 1 were isolated from animals, raw meats, humans and environmental water, respectively. The sources of 31 strains were unavailable  in eleven geographical regions in China. Some strains were reported in our previous studiesle Table . All streae gene among all 735 non-O157 STEC strains were screened using PCR method with primer eae-F (5′-TCAATGCAGTTCCGTTATCAGTT-3′) and eae-R (5′-GTAAAGTCCGTTACCCCAACCTG-3′)44.The presence of et al.45, and further confirmed by using E. coli antisera, O1–O188 . The entire coding sequence of fliC was amplified by PCR with the primers fliC-F (5′-ATGGCACAAGTCATTAATACCCAAC-3′) and fliC-R (5′-CTAACCCTGCAGCAGAGACA-3′) as reported by Fields et al. and then sequenced with Sanger sequencing46. The sequences were analyzed with the SerotypeFinder database (https://cge.cbs.dtu.dk/services/SerotypeFinder/) to determine H types47.The O serogroup of each isolate was initially screened by an O-genotyping PCR method designed by Iguchi stx1 subtypes  and stx2 subtypes (stx2a to stx2h) were determined by a PCR-based subtyping protocol in combination with a phylogeny scheme as described previously49.The eae gene was obtained by PCR using previously described method16. Two additional primer designed in this study were used for sequencing: eaeR3-A (5′-TCCATGTGTATTTTCCATTGCC-3′) and eaeR3-B (5′-TATATTTCCCATCACCTCCAC-3′). All PCR products were visualized by agarose gel electrophoresis and purified by using a QIAquick PCR purification kit , and then sequenced using BigDye™ Terminator V3.1 Cycle Sequencing kit .The complete eae gene was checked and assembled by SeqMan II . The eae subtypes reference sequences were downloaded from GenBank. The MEGA 7 software (www.megasoftware.net)50 was used to align the complete eae sequences obtained in this study and the reference eae sequences. A Neighbor-Joining tree was constructed with maximum composite likelihood model. Bootstrap analyses were performed  to estimate the stability and genetic distances were calculated by the maximum composite likelihood method. A novel subtype was defined by a cutoff value of 95% nucleotide sequence identity as described previously51.Each of the sequenced ~3.2 kb LEE region that contained the complete adk, fumC, gyrB, icdF, mdh, purA, and recA) were amplified by PCR and sequenced according to the scheme provided by the E. coli MLST website . Each locus was assigned an allele number by comparing sequences against the E. coli MLST database. The allelic profile of seven housekeeping genes was used to generate a specific sequence type (ST) for each isolate. STs of human STEC O157 and the ‘top six’ non-O157 serogroups were downloaded from the E. coli MLST database for comparison. A minimum spanning tree was generated with BioNumerics software version 4.0 .Seven housekeeping genes (eae sequences obtained in this study were submitted to GenBank (Acc. MK761151–MK761168).The 18 diverse Supplementary Information.


Plants primed with 500 mg L−1 nitrogen possessed a higher photosynthetic rate, relative water content, electrolyte leakage and lipid peroxidation under water deficit conditions, compared to control plants. The induction of water deficit tolerance was supported with the activation of antioxidant defense system, induced by the accumulation of nitric oxide in leaves and roots of rice plants. We originally demonstrated the accumulation of nitric oxide in leaves of rice plants. The elevated nitrogen priming can be used to enhance water deficit tolerance in irrigated paddy fields, instead of nitric oxide donors.Under water deficit conditions, the essential macronutrient nitrogen becomes limited as a result of reduced dissolved nitrogen and root nitrogen uptake. An elevated nitrogen level might be able to mitigate these effects, integrated with the idea of using nitric oxide as abiotic stress tolerant inducers. In this study, we evaluated the potential of using elevated nitrogen priming prior to water shortage to mitigate plant stress through nitric oxide accumulation. We grew rice plants in 300 mg L Water deficit conditions cause widespread crop loss globally due to abiotic stress. By the end of the 21st century, drought and water deficit stress may account for more than 70 percent of crop loss below optimal productivity . DroughtUnder water deficit conditions, the loss of transpiration and cell turgor pressure lead to a reduction in uptake and transport of nitrogen, silicon, magnesium and calcium and other important nutrients . Water dNitric oxide (NO) has been proposed as a regulator of the antioxidant defense system during stress conditions ,9,10,11.m), which limits photosynthetic capacity and N-use efficiency [Long-term application of high amounts of chemical N fertilizer lessened the water and nutrient use efficiency as well as inducing environmental problems and sensitivity to insects and diseases ,18. Howeficiency . Some reficiency . AlthougWe therefore hypothesized that if we increased the nitrogen concentration in the fertilizer for only a short-period of time (priming), NO accumulation would be activated. Thus, the antioxidant defense system would also be activated. An addition of nitrogen could delay protein and chlorophyll degradation during a short period of water scarcity. In this study we aim to identify the appropriate level of N for priming treatment prior to stress episodes and to assess the mechanisms of nitrogen induced water deficit tolerance.−1 and 1000 mg L−1 N) maintained higher photosynthetic activity (−1 N promoted rice plant growth as indicated by increasing leaf area under water deficit conditions (−1 N). Elevated nitrogen priming failed to alter the net photosynthetic rate or leaf area of rice plants under well-watered conditions. Low N level priming had lower photosynthetic activity and growth under well-watered conditions  on photosynthesis and growth have not been investigated. Rice plants primed with an elevated N level  had significantly higher electrolyte leakage  plants  under well-watered conditions  plants c. In con control a,b. Howenditions b.−1 N but supplied with a NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) (500D + cPTIO)  accumulation occurred under the PEG-induced water deficit conditions for elevated nitrogen primed plant roots and leaves (500D), and plants without elevated nitrogen priming but supplied with a nitric oxide donor, sodium nitroprusside (SNP) (300D + SNP) . In cont+ cPTIO) .2O2 content and staining showed low ROS levels in roots and leaves in plants without PEG treatment (control condition)  a,b. The ndition) , while tndition) . The cPTndition) .−1 N prior to water deficit conditions maintained higher photosynthetic rates  priming benefits the tolerance of rice plants, excessive N (1000 mg L−1-N) priming leads to stress symptoms, like decreasing photosynthetic activity , wheat (Triticum aestivum L.) seedlings and peanuts (Arachis hypogaea L.) [Redox imbalance under water deficit conditions is accountable for significant plant cell damage. The antioxidant induction system is a viable approach to alleviate cellular damage and a higher tolerability of plants to water deficit conditions ,32,33. Wmed rice a. Perhap leakage c and MDA leakage d, which  leakage a,c. Antigaea L.) ,36,37,382O2 accumulation, electrolyte leakage and MDA content. Using NO donors, SNP as a seed priming solution and foliar spray has previously induced rice drought tolerance in rice [−1 N leaves and roots accumulated higher endogenous NO and released NO, which was inhibited by the NO scavenger, cPTIO (−1 N) plants supplied with 1 mM SNP  had less O2•− and H2O2 further suggesting a relationship between elevated N priming and increased NO (2•−) [The antioxidant defense system that controls ROS status in plant cells has also been reported to be regulated by the level of reactive nitrogen species (RNS) from both endogenous production and exogenous application ,35,36,39 in rice . Because in rice . The 500r, cPTIO b–d. The 1 mM SNP b,d. In cfication a,b. We m with NO ,41. Addieased NO a,b. ThisNO (2•−) , limits NO (2•−) . MoreoveNO (2•−) ,42. TherOryza sativa L. subspecies indica cv. Pathumthani 1) were germinated on moist germination paper for 10 d at 28 °C in the dark. Seedlings were transplanted into 2-L pots filled with 1:1 vermiculite: perlite. The seedlings were grown in the greenhouse with day lengths of 11–12 h and daily average temperatures of 35 ± 4/24 ± 7 °C day/night and 700–1500 µmol m−2 s−1 mid-day photosynthetically active radiation (PAR). Plants were fertilized every other day with , P 20 mg L−1, K 75 mg L−1, Ca 25 mg L−1, Mg 17 mg L−1, S 55 mg L−1, Fe 3.30 mg L−1, Mn 0.50 mg L−1, Zn 0.05 mg L−1, Mo 0.01 mg L−1, Cu 0.02 mg L−1) for 10 weeks until the plants were one week before panicle initiation. Then, different concentrations of nitrogen (N) fertilizer were applied by using N 100 mg L−1 , P 20 mg L−1, K 75 mg L−1, Ca 25 mg L−1, Mg 17 mg L−1, S 55 mg L−1, Fe 3.30 mg L−1, Mn 0.50 mg L−1, Zn 0.05 mg L−1, Mo 0.01mg L−1, Cu 0.02 mg L−1 as base fertilizer and added ammonium nitrate (NH4NO3) to reach target concentrations of 300, 500 and 1000 mg L−1 N and used double volume of the field capacity of the pots to wash the previous nitrogen fertilizer. Water deficit stress was applied by withholding water for ~7 d when visual stress symptoms  appeared (10–15% relative soil water content) [Seeds of Thai rice (−2 s−1 light intensity and 380 ± 10 μmol mol−1 CO2 surrounding the leaf (Ca) at 32 ± 2 °C. To calculate instantaneous transpiration, the transpiration rate was divided by the net photosynthetic rate.Net photosynthetic rate and stomatal conductance were measured with the photosynthesis system ADC LCi-SD . The measurements were conducted under 900 ± 50 μmol mAfter one-day post recovery, all remaining leaves were removed and all surface dust was wiped away prior to measuring the area with an LI-3100C area meter  in square centimeters.RWC was determined according to Gao et al. 2019 . Leaves 2 leaves from each treatment into a 50-mL-tube with 20 mL distilled deionized water, the tubes were shaken and electro-conductivity (iEC) was immediately measured with a pH/cond meter . After soaking the leaves for 12 h, the second conductivity was measured (fEC). Then, the leaves were boiled for 1 h and the total electro-conductivity was measured (tEC). The electrolyte leakage was calculated as follows:The electrolyte leakage measurement was adjusted based on the method described previously by Cai et al. 2015 . After pw/v). The homogenate was centrifuged at 15,000× g for 20 min at 4 °C. The supernatant was collected for SOD, CAT and APX activity assays.Enzymes were extracted by the modified method of Umnajkitikorn et al. 2013 . The froSOD activity was assayed based on the method described by Beauchamp and Fridovich 1971  and Vaid2O2 in 50 mM potassium phosphate buffer (pH 7.0), 0.1 mM disodium-EDTA and 10 µL of extracts. The reactions were carried in Nunc 96 well UV transparent plate . The absorbance was measured at 240 nm with a UV/VIS spectrophotometer . The enzyme activity was defined as the amount of H2O2 decomposed min−1 mg protein−1. The molar coefficient of H2O2 (E) is 39.4 M−1 cm−1) at 240 nm.CAT activity was assayed based on the method of Sunohara and Matsumoto 2004 . One hun2O2 and 20 µL of extracts. The subsequent decrease in ascorbic acid was observed at 290 nm (E = 2.8 mM−1 cm−1) with a UV/VIS spectrophotometer . The enzyme activity was defined as the amount of ascorbic acid (ASA) which decomposed min−1 mg protein−1.APX activity was assayed based on the method of Sunohara and Matsumoto 2004 . One hunThe Bradford assay  was usedOryza sativa subspecies indica cv. Pathumthani 1) were germinated and grown for 5 d at 25 °C in darkness. Seedlings were transplanted into 9-cm pots filled with vermiculite 4 plants/pot and grown in the growth room with 12 h/12 h light/dark cycles and 25/20 °C day/night cycles for 3 weeks with fertilizer containing 300 mg L−1 N. Then, the plants were randomly divided into 6 groups and transferred to glass tubes with the following treatments: (1) 300 mg L−1 N (300 C); (2) 500 mg L−1 a day and switched back to the 300 mg L−1 (500 C); (3) 300 mg L−1 N + gradually increased polyethylene glycol (PEG) 6000 (300 D); (4) 500 mg L−1 a day and switched back to the 300 mg L−1 + gradually increased PEG (500 D); (5) 300 mg L−1 N + PEG 6000 + nitric oxide donor, 1 mM SNP (300 D + SNP); (6) 500 mg L−1 N + PEG 6000 + 3 mM of Nitric oxide scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) (500 D + cPTIO). After 3 days under each treatment, the seedlings were harvested for further ROS and RNS analyses.Rice seeds . The epifluorescence images were captured with an Eclipse 90i microscope . The intensity of the fluorescent signal from at least 30 pictures of each treatment was quantified by using Image J software .The concentration of NO released from the roots was assessed in leaf and root segments of the seedlings in each treatment after incubation in 7 μM DAF-FM for 2 h. The fluorescence intensity of the DAF-FM solution was measured as described in Fukudome et al. 2016  but adap2O2 was detected in situ according to Fukudome et al. 2019 [w/v) 3,3’-diaminobenzidine (DAB), at pH 7.8. Samples were incubated overnight in the dark. The leaf segments were boiled in 95% ethanol at 90oC to remove chlorophyll. The clear leaf segments were then photographed. The staining areas were calculated by Image J software , in the red channel according to procedure described in [Hal. 2019  and Signal. 2019 . Detacheribed in . Data we2•−) was detected in situ essentially as described by to Fukudome et al. 2019 [w/v) nitro blue tetrazolium (NBT), and 0.05% (v/v) Tween 20, pH 7.8. Treated samples were incubated overnight in the dark, cleared and photographed as described above. The staining areas were calculated by Image J software , in the blue channel according to the procedure described in [Superoxide radical , homogenized at 1500 rpm for 5 min at 4 °C and then centrifuged for 5 min at 16,200× g at 4 °C. The H2O2 was colorimetrically quantified by Amplex Red detection assay (Thermo Fisher Scientific). The measurement was performed on the supernatant as described in Brumbarova et al. 2016 [2O2 concentration from 0–5 µM.The frozen leaves were ground and 5–10 mg of samples were extracted with 1 mL of 20 mM Kal. 2016  with somg for 25 min. For measurements of MDA concentration, 4 mL of 20% trichloroacetic acid containing 0.5% thiobarbituric acid were added to a 1 mL aliquot of the supernatant. The mixture was heated at 95 °C for 30 min, quickly cooled in ice and then centrifuged at 10,000× g for 10 min. The absorbance of the supernatant was measured at 532 nm. The results are shown as MDA content mg–1 FW, using malondialdehyde tetrabutylammonium salt as a standard .The youngest fully expanded leaves were homogenized with 5 mL of 50 mM potassium phosphate buffer pH 7.5 and centrifuged at 20,000× The SPSS 25 statistical package was used for statistical analyses. The experiments were based on a randomized complete block design.Elevated N priming at approximately 60% more than normal N level enhanced water deficit tolerance with NO-triggered antioxidant defense systems. The elevated N priming also supported the sustainability of photosynthetic activity and relative water contentof the leaves, together with the reduction of membrane damage. This approach has potential for in-situ investigation in aerobic rice fields with fertigation systems as a potential mitigating factor for enhancing drought tolerance in rice.


Pseudomonas chilensis strain ABC1, which was isolated from a soil interstitial water sample collected at the University Adolfo Ibañez, Valparaiso, Chile. We assembled PacBio reads into a single closed contig with 209× mean coverage, yielding a 4,035,896-bp sequence with 62% GC content and 3,555 predicted genes.Here, we report the complete genome sequence of  Pseudomonas chilensis strain ABC1, which was isolated from a soil interstitial water sample collected at the University Adolfo Ibañez, Valparaiso, Chile. We assembled PacBio reads into a single closed contig with 209× mean coverage, yielding a 4,035,896-bp sequence with 62% GC content and 3,555 predicted genes.Here, we report the complete genome sequence of  Pseudomonas is a widespread genus of Gram-negative bacteria belonging to the Gammaproteobacteria, inhabiting a great diversity of environments  between 15°C and 30°C. The strain was isolated from soil interstitial water (0 to 5 cm deep) in Valparaiso, Chile , using a liquid preculture on AAM medium  analysisde novo utilizing the PacBio Hierarchical Genome Assembly Process version 3  , including genomic DNA extraction, PCR amplification, purification, 20-kb SMRTbell library construction, and sequencing using the PacBio RS II platform. Sequence reads were filtered and assembled ersion 3  using deCP058349. This project has been submitted under BioProject number PRJNA641577 and BioSample accession number SAMN15356716. The PacBio raw reads are available in the SRA under the accession number SRR12183682.The annotated genome was deposited in GenBank under the accession number


Synthetic CpG oligodeoxynucleotides (ODNs) with defined CpG motifs possess broad immunostimulatory properties that make CpG ODNs suitable as therapeutic interventions in a variety of human disease conditions, including Alzheimer’s disease (AD). Rodent models are often used to preclinically test the effectiveness of CpG ODN therapeutic agents for AD and other disorders. However, the translatability of findings in such models is limited due to the significant difference of the expression of TLR9 between primates and rodents. The squirrel monkey (SQM), a New World non-human primate (NHP), is known to be phylogenetically proximate to humans, and develops extensive age-dependent cerebral amyloid angiopathy (CAA), a key pathological feature of AD. Hence, this model is currently being used to test AD therapeutics. In the present study, we conducted the first examination of Class C CpG ODN’s immunomodulatory role in elderly SQMs. We documented the effectiveness of CpG ODN to trigger an immune response in an aged cohort whose immune system is senescent. The specific immune response patterns detected here closely resembled CpG ODN-induced immunostimulatory patterns observed in prior human studies. Overall, our findings provide critical data regarding the immunomodulatory potential of CpG ODN in this NHP model, allowing for future translational studies of innate immunity stimulation via TLR9 agonists for diverse indications, including AD therapeutics.One means of stimulating the mammalian innate immune system is  The mammalian innate immune system naturally recognizes unmethylated cytosine-phosphate-guanine (CpG) DNA as pathogen-associated molecular patterns (PAMPs) indicative of viruses or bacteria. CpG oligodeoxynucleotides (ODN), as synthetic ODNs with defined CpG motifs, mimic the structure of these pathogenic DNA to incite an innate immune response. The immunomodulatory properties of CpG ODN are enacted through an endosomal pattern recognition receptor (PRR), the Toll-like receptor 9 (TLR9), to propagate a specific downstream innate immune response Krieg, , 2012.Three classes of TLR9 agonists, CpG ODN Class A, B, and C, each pose a distinct biologic activity, due to their unique structures and different cell activations, to produce varying innate immune signaling cascades. Class A CpG ODNs are single CpG motifs of partially phosphorothioated (PS-modified) and phosphodiester backbone bases in a palindromic sequence that induce the production of IFNα by peripheral dendritic cells (pDCs) and indirectly activate NK cells. Class B CpG ODNs are multiple CpG motifs composed of fully PS-modified nucleotides. Unlike Class A CpG ODNs, Class B contain B-cell activators and stimulate pDC maturation. The Class C CpG ODNs, which combine characteristics of the A and B classes, are generally fully PS-modified, double-stranded palindromic motifs that induce strong IFNα production, pDC maturation, and efficient B cell stimulation , small New World NHP known to be phylogenetically proximate to humans, have served an important role in studying pathogenesis of human disease conditions such as AD .Saimiri boliviensis boliviensis). They were selected from our ongoing AD study using SQMs as a model of naturally occurring brain vascular amyloid pathology (at the SQM Breeding Research Resources (SMBRR) located at the University of Texas MD Anderson Cancer Center Michale E. Keeling Center for Comparative Medicine and Research). These monkeys were housed in social groups within two connecting cages that were 4′ wide × 6′ tall × 14′ long.Subject animals consisted of elderly female SQMs (ad libitum access to New World Primate Diet (Purina #5040) and water. In addition, they were fed either fresh fruit or vegetables daily. Specialty foods, such as seeds, peanuts, raisins, yogurt, cereals, frozen juice cups and peanut butter, were distributed daily to them as enrichment. At no time were the subjects ever food- or water-deprived. Subjects were also provided with destructible enrichment manipulanda and different travel/perching materials on a rotating basis to promote the occurrence of typical species behavior.Animals had The study population consisted of aged female SQMs (18–20 years of age). A subset of 10 CpG ODN-injected monkeys was selected for our analyses. All subjects were considered healthy and in their normal social groups at the time they were sampled. A SQM’s average life span is 20 years and maximum life expectancy of <25 years Abee, . With anEndotoxin-free Class C CpG ODN, a 30-mer phosphothioate ODN containing juxtaposed CpG motifs with flanking regions in a self-complimentary palindromic sequence (5′-TCGAACGTTCGAACGTTCGAACGTTCGAAT-3′), was purchased from Integrated DNA Technologies, Inc.Blood samples (2–3 ml) were collected in EDTA coated collection tubes from the femoral vein of study animals which had been injected twice with Class C CpG ODN (2 mg/kg) at two intervals, four to 5 weeks apart. CpG ODN was administered subcutaneously (s.c.) into the interclavicular space. All injections and blood sample collections  occurred in the morning (8–9 AM) before the animals were fed.Hematology was performed on EDTA-preserved blood by using an automated analyzer Advia . The absolute number of lymphocytes and monocytes, as obtained from hematologic analysis, was used to convert the frequency of the lymphocyte population obtained from FACS analysis to calculate the absolute numbers in each of the lymphocyte and monocyte subset populations. The assessment of CpG ODN immunostimulatory activity was first examined in whole blood samples collected prior to injection (baseline/day 0) and on day 1, day 3, and day 7 after CpG ODN administration.in vitro analyses, including viability and functional assays, and flow cytometry characterization of CpG ODN effects on expression of major lymphocyte and monocyte subsets.Blood samples collected from SQMs prior to CpG ODN injection were used for peripheral blood mononuclear cells (PBMCs) isolation. PBMCs were isolated by Ficoll-Hypaque density gradient separation as described previously , CD16 (Clone 3G8), CD14 (Clone M5E2), CD20 (Clone L27), CD123 (Clone 7G3), HLA DR (Clone L243), and appropriate isotype-control antibodies which had been conjugated to either fluoroscein isothiocyanate (FITC), phycoerythrin (PE), peridinin-chlorophyl-protein (PerCP), PE-Cy5, or allophycocyanin (APC), were used for these studies . The Lin1-FITC antibody cocktail containing anti-CD3, CD14, CD16, CD20, as well as antibodies against CD123 marker (PE) and HLA DR (PerCP) were used as part of the identification of plasmacytoid DCs . Phenotypic characterization of lymphocytes and monocytes in peripheral blood from the monkeys was performed by cell surface staining of whole blood samples and flow cytometry analysis as described previously  and analyzed using FlowJO . Logical gating was used to identify specific innate immune cell populations  and the activation or maturation markers expressed by these cells are shown as a percentage of the parent population.To evaluate CD123 expression on T cells, B cells, and monocytes, PBMCs were stimulated with CpG ODN for 30 h at 37°C. PBMCs were harvested and washed with cold PBS without Ca5/well in 96-well round-bottomed plates  with 5 and 50 μg/ml Class C CpG ODN or 5 μg/ml concanavalin A (Con A) in a volume of 200 μl. Cultures were incubated in a humidified 5% CO2 environment at 37°C for 96 h. At the end of the culture period, supernatants were harvested and cells were assayed for viability content by the standard MTT dye reduction assay described previously  were seeded in triplicate wells of 96-well plates  pre-coated with the primary IFNγ or IL12p40 antibody, and the lymphocyte were stimulated with the different mitogens or antigen. After incubation for 30–32 h at 37°C, the cells were removed, and the wells were thoroughly washed with PBS and developed as per the protocol provided by the manufacturer. Purple colored spots representing individual cells secreting IFNγ or IL12p40 were counted by an independent agency  using the KS-ELISPOT automatic system  for the quantitative analysis of the number of IFNγ or IL12p40 spot forming cells (SFC) for 105 input PBMC. Responses were considered positive when the number of SFCs with the test antigen was at least five and was five above the background control values from cells cultured in the medium alone.Freshly-isolated PBMC, as described above, were stimulated with the mitogens CpG ODN  and 5 μg/ml of Con A to determine the numbers of IFNγ- or IL12p40-producing cells by the Enzyme Linked Immuno Spot (ELISPOT) assay using the methodology reported earlier  according to the manufacturers’ protocols. There is 91.4%–98.1% homology between the nucleotide sequences of SQM cytokine genes and published sequences of equivalent human and nonhuman primate genes  and 25 μl of aliquots were used in assay. The 96-well filter plate was blocked with assay buffer for 10 min at room temperature, washed, and 25 μl of standard or control samples were dispersed into appropriate wells. After adding 25 μl of beads to each well, the plate was incubated on a shaker overnight at 4°C. The next day, after washing two times with wash buffer, the plate was incubated with detection antibody for 1 h at room temperature and again incubated with 25 μl of Streptavidin-Phycoerythin for 30 min at room temperature. After washing two times with wash buffer, 150 μl of sheath fluid was added into each well and multianalyte profiling was performed on the Bio-Plex 200 system (Luminex X MAP technology). Calibration microspheres for classification and reporter readings as well as sheath fluid, assay, and wash buffer were also purchased from Bio-Rad . Acquired fluorescence data were analyzed by the Bio-Plex manager 5.0 . All steps of incubations were performed on a shaker. The minimum detectable concentration was calculated by the Multiplex Analyst immunoassay analysis Software from Millipore. The minimum IFNα detectable concentration was 2.9 pg/ml.IFNα cytokine was measured in cell-free PBMC supernatant using MILLIPLEX-Post hoc comparison compared day 0 to days 1, 3, and 7 using a Dunnett’s test. Comparisons between groups without repeated sampling . Symbols representing significance are: ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05; ns, p > 0.05.For statistical analysis, samples were grouped according to treatment of the animals from which samples were obtained. Comparison analyses between samples collected post-treatment , 2Da–De sampling , 3–5 werPost hoc analyses were conducted when appropriate using Dunnett’s test to compare day 0 to days 1, 3, and 7. Eta squared effect sizes were calculated to estimate the magnitude of the effect.Peripheral blood samples from CpG ODN injected monkeys were analyzed by flow cytometry in order to enumerate various cell populations. To study kinetics in the expression of different lymphocyte and monocyte subsets, EDTA blood was collected prior to injection (baseline/day 0) and on day 1, day 3, and day 7 after CpG ODN administration. Phenotyping characterization of monocytes, T cells, B cells, NK cells, and NKT cells was performed using the logical gating strategy shown in F = 33.97, p < 0.0001, η2 = 0.53; Post hoc analysis indicated that there was a significant peak in lymphocytes expression on day 3 that remained significantly different on day 7  = 15.42, p < 0.01, η2 = 0.51), with days 3 and 7 significantly lower than day 0  = 40.02, p < 0.0001, η2 = 0.30), with days 3 and 7 significantly lower than day 0  = 36.28, p < 0.0001, η2 = 0.23), with days 3 and 7 being significantly lower than day 0  = 25.99, p < 0.0001, η2 = 0.61; Post hoc analysis revealed a significant peak in the numbers of monocytes on day 3  = 12.44, p < 0.001, η2 = 0.40), with day 3 significantly decreased compared to day 0  = 6.52, p < 0.01, η2 = 0.22) with day 7 significantly lower than day 0 . We observed a significant overall effect of days on the absolute number of lymphocytes (in vitro) and medium-only control group  = 4.59, p < 0.05, η2 = 0.29), with days 1 and 3 expressing significantly lower amounts of CD123 compared to day 0  = 6.88, p < 0.05, η2 = 0.26), with significantly lower expression on days 1 and 3 compared to day 0  = 9.61, p < 0.01, η2 = 0.37), with expression significantly lower on days 1 and 3 compared to day 0  = 13.6, p < 0.001, η2 = 0.44), with expression significantly higher on days 3 and 7 compared to day 0  = 4.75, p < 0.05, η2 = 0.34), with expression on day 3 significantly less than day 0  as Lin1-HLA DR+ population expressing CD123 marker . We beliol group . The gatWe further examined the viability as well as functional activity of PBMCs exposed to different concentrations of Class C CpG ODN. PBMCs isolated from SQMs were cultured in the presence or absence of CpG ODN (doses of 5 μg/ml or 50 μg/ml) and viability was measured using MTT assay. Mitogen Con A was used as positive control. The results were analyzed using a one-way ANOVA for repeated measures. Where appropriate, a Dunnett’s test was used to compare treatment groups to the medium-only sample. Eta squared was calculated to estimate the magnitude of the effect size.F = 7.08, p < 0.01, η2 = 0.17) with post hoc analysis revealing significantly increased percent viability in PBMCs stimulated with 5 μg/ml of CpG ODN compared to medium-only control cells  for 30–32 h. PBMCs were further subjected to flow cytometry to determine expression frequency of T cells, B cells, and monocytes, as well as the activation status of CD123 expression on those particular cell populations. The results were analyzed using a one-tailed paired t = 14.7, df = 12, p < 0.0001, g = 5.46; t = 9.87, df = 12, p < 0.0001, g = 3.26; t = 2.27, df = 12, p < 0.05, g = 0.89; t = 2.61, df = 12, p < 0.05, g = 1.05; There was a significant increase in the percent of T cells (CD3+) expression  or stimulated with nontoxic doses of class C CpG ODN (5 μg/ml and 50 μg/ml). The numbers of IFNγ- and IL12p40-producing cells were determined by ELISPOT assay. Results were analyzed using a one-tailed paired t = 2.51, df = 10, p < 0.05, g = 1.29) compared to unstimulated PBMCs. Similarly, the numbers of IFNγ-producing cells were significantly higher after stimulation with 50 μg/ml of CpG ODN compared to medium only  is advantageous because of the highly conserved innate immune system among primates  were utilized in the present study due to their unique characteristic of naturally occurring amyloid-related pathology. An important feature of Aβ depositions in SQMs is their species-specific propensity to CAA , myeloid and plasmacytoid, can be isolated from human peripheral blood (PBMCs), and are distinguished by differential expression of the cell surface markers CD11c and CD123. These two populations of DCs are also different in their expression of TLRs, which are involved in their activation  the NYU—Institutional Animal Care and Use Committee and (2) The University of Texas, MD Anderson Cancer Center—Institutional Animal Care and Use Committee. This research was conducted at the AAALAC-I accredited Michale E. Keeling Center for Comparative Medicine and Research, UT MD Anderson Cancer Center, Bastrop, TX, USA (Common SQMs 03-09-02781). All animal experiments were carried out according to the provisions of the Animal Welfare Act, PHS Animal Welfare Policy, and the principles of the NIH Guide for the Care and Use of Laboratory Animals . BN performed experiments . AP conducted background literature search and contributed to manuscript preparation. MR contributed to manuscript preparation. TW contributed to experiment development, data interpretation, and manuscript preparation. HS conceived and supervised the research, contributed to experiment development and data interpretation, and wrote 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.