Euterpe oleracea Mart oil (EOO) and β-cyclodextrin (β-CD) or hydroxypropyl-β-cyclodextrin (HP-β-CD) by either kneading (KND) or slurry (SL). Complexes were analyzed by molecular modeling, Fourier-transform infrared spectroscopy, scanning electron microscopy, powder X-ray diffraction, thermogravimetry analysis and differential scanning calorimetry. The antibacterial activity was expressed as Minimum Inhibitory Concentration (MIC), and the antibiotic resistance modulatory activity as subinhibitory concentration (MIC/8) against Escherichia coli, Streptomyces aureus, Pseudomonas aeruginosa and Enterococcus faecalis. Inclusion complexes with β-CD and HP-β-CD were confirmed, and efficiency was proven by an interaction energy between oleic acid and β-CD of −41.28 ± 0.57 kJ/mol. MIC values revealed higher antibacterial activity of complexes compared to the isolated oil. The modulatory response of EOO and EOO-β-CD prepared by KND as well as of EOO-β-CD and EOO-HP-β-CD prepared by SL showed a synergistic effect with ampicillin against E. coli, whereas it was not significant with the other drugs tested, maintaining the biological response of antibiotics. The antimicrobial response exhibited by the complexes is of great significance because it subsidizes studies for the development of new pharmaceutical forms.The development of inclusion complexes is used to encapsulate nonpolar compounds and improve their physicochemical characteristics. This study aims to develop complexes made up of  Copaifera multijuga, commonly used as an anti-inflammatory [Ocimum basilicum, applied as an antihyperalgesic [Allium sativum oil, displaying antioxidant and antimicrobial activities [Euterpe oleracea oil [Vegetable oils are a target of studies due to their many pharmacological applications. According to Martins da Silva et al. , the cheammatory , the essralgesic , and thetivities  similar acea oil ,6, are aE. oleracea Mart oil (EOO), popularly known as açai oil, is largely produced in Brazil. Rich in long-chain unsaturated fatty acids, its major constituent is oleic acid [eic acid ,8, whicheic acid  represeneic acid .Staphylococcus aureus [Recently, Marques et al. , who phys aureus , antipros aureus , and an s aureus . The abss aureus .Despite the wide range of promising pharmacological properties, oils are poorly used by the pharmaceutical industry due to their low solubility. To solve this problem, drugs may be complexed with different compounds that are often able to improve their physicochemical properties. Among these, cyclodextrins (CD) are the most frequently used ,18 folloCD have been used as a pharmaceutical excipient able to increase the solubility of drugs. These cyclic oligosaccharides also exhibit stable physicochemical characteristics capable of protecting host molecules from degradation in the gastrointestinal tract and against oxidation, provide photo and thermal stability, and reduce physiological effects, undesirable organoleptic characteristics, drug volatility and toxicity ,23,24,25In order to obtain oils with suitable physicochemical characteristics and to improve their functions and pharmacological applications, formulations containing inclusion complexes are being developed, where the product is chemically stable because its constituents are protected by reducing the exposure to environmental and microbiological factors. In addition to these benefits, the formation of inclusion complexes with EOO may improve their solubility, promote oxidative stability against photosensitivity or temperature effects, and especially prevent hydrolytic rancidity.E. oleracea Mart oil with β-cyclodextrin and hydroxypropyl-β-cyclodextrin prepared by kneading and slurry, in order to improve oil physicochemical properties, solubility, stability and antimicrobial activity. The formation of inclusion complexes and the interaction between oleic acid (the main oil component) and cyclodextrins was investigated by Fourier-transform infrared spectroscopy, scanning electron microscopy, powder X-ray diffraction, thermogravimetric analysis, and differential scanning calorimetry. The antibacterial and modulatory activity of the inclusion complexes was tested against standard target bacterial strains using the microdilution test.This work aims to develop inclusion complexes of The fatty acid profile determined using gas chromatography  shows thG) of the β-CD:oleic acid and HP-β-CD:oleic acid systems being −41.28 ± 0.57 and 25.15 ± 0.44 kJ/mol, respectively.Apolar ligands of appropriate size tend to replace water molecules bound to cyclodextrin hydroxyl groups in solution, forming more stable and less stressed systems called inclusion complexes ,28,29,30The formation of inclusion complexes is directly influenced by Van der Waals forces due to the lipophilic nature of cyclodextrin cavity . Since βNot all molecules in a compound form inclusion complexes, and EOO consists primarily of fatty acids. According to Leclercq , β-CD arThe formation of inclusion complexes is influenced by CD-linked substituents and, our findings are in accordance with and Arima . Hydroph−1 that can be assigned to C–H stretching vibrations, C=O stretching vibrations, C=C asymmetric deformation and C–O stretching vibrations, respectively, while the β-CD spectrum , Enterococcus faecalis (≥1024 µg/mL) and Escherichia coli (512 µg/mL), while both EOO-HP-β-CD complexes obtained by KND and SL exhibited their highest efficacy against E. coli (MIC = 341 µg/mL) and the lowest against Pseudomonas aeruginosa (MIC = 512 µg/mL). However, the best performance was obtained with complexes prepared using β-CD as a carrier, especially the EOO-β-CD one obtained by SL, whose MIC values against E. faecalis, P. aeruginosa, S. aureus, and E. coli were as low as 341, 384, 384, and 256 µg/mL, respectively. Nonetheless, the EOO-β-CD complex prepared by KND was the most effective against S. aureus, with a MIC value of 256 µg/mL.It can be seen in There is a growing number of studies on the discovery of new antimicrobial drugs as well as the improvement of existing formulations ,45,46,47E. coli, Ciprofloxacin (CIP 4 µg/mL) or PIP + TAZ at the same dosage against P. aeruginosa, and Vancomycin (VAN 16 µg/mL) or CIP 4 µg/mL against S. aureus and E. faecalis.With this aim in mind, a drug modulation assay was performed on EOO or its inclusion complexes with antibiotics commonly prescribed in clinical practice for the treatment of nosocomial and hospital diseases, in order to establish possible synergistic or antagonistic effects. Two antibiotics suggested by CLSI  were useE. coli  oil (EOO) was obtained from Amazon Oil . This company reports that the açai fruits were collected in the Brazilian Amazon rainforest. The EOO, obtained from the fruit pulp, was extracted through a cold pressing method without the addition of solvents or preservatives. The selected fruits were washed and immersed in water at 45 °C for 60 min and processed with the aid of a stainless-steel stripper. The açai pulp was dried at a temperature of 60 °C and subjected to mechanical pressing  at a temperature of 40 °C. β-cyclodextrin (β-CD), hydroxypropyl-β-cyclodextrin (HP-β-CD) and resazurin were purchased from Sigma-Aldrich  and Brain Heart Infusion (BHI) broth from Kasvi . The ATCC bacterial strains were donated by the Microbiology Laboratory of the Federal University of Campina Grande . All experiments were performed using purified water (<1.3 μS) obtained by reverse osmosis, and the reagents were of analytical grade.The −1, FID detector temperature of 250 °C, injector split ratio of 1:100 and temperature of 250 °C, injection volume of 1 μL. The temperature was initially kept at 80 °C for 4 min and subsequently increased to 205 °C at 4 °C/min. Peaks of individual FAMEs were identified by comparison of retention times with those of standard solutions. The results were expressed as relative percentages of total fatty acids.EOO fatty acids composition was determined after their conversion into methyl esters (FAMEs) by saponification with 0.10 M KOH in methanol and esterification with 0.12 M HCl in methanol . FAMEs w−1) as it was its major component, while the molecular weights of β-CD and HP-β-CD were assumed to be 1134.98 and 1396 g·mol−1, respectively, as proposed by Pinheiro et al. [Inclusion complexes of EOO with either β-cyclodextrin (EOO-β-CD) or hydroxypropyl-β-cyclodextrin (EOO-HP-β-CD) were prepared by two methods, namely kneading (KND) and slurry (SL), using the molar ratio of 1:1 (EOO:CD). The molecular weight of EOO was considered the same as oleic acid  ratio. Samples were then agitated at 400 rpm with a magnetic stirrer for 36 h. Mixtures were kept in a hot air circulating oven at 70 °C for 24 h and then conditioned in a desiccator until complete drying.The SL method was developed as described by Quintans-Júnior et al. . ComplexTo determine the interaction energy between oleic acid, i.e., the main EOO component, and β-CD or HP-β-CD, two molecular models were tested using the UCSF-Chimera software  , one witMolecular dynamics were performed with the GROMACS program   after en−1 with 20 scans and 4 cm−1 resolution.Inclusion complexes were characterized by Fourier transform infrared (FTIR) spectroscopy using a spectrometer, model IR Prestige-21 , according to the Attenuated Total Reflectance (ATR) modality. Samples were read in the wavenumber region from 4000–700 cm® Scanning Electron Microscope model TM-3000 Tabletop  with an accelerated voltage of 20 kV and 100× magnification. Crystalline characterization was performed by PXRD on powdered samples using a D2 Phaser diffractometer  equipped with a Lynxeye detector and operating with CuKα1 radiation nickel filter (λ = 1.54 Å), 0.02° pitch, with measurement in the angular range of 3–45°, and 30 kV voltage and 10 mA current intensity.β-CD, HP-β-CD, EOO-β-CD and EOO-HP-β-CD were then analyzed by both Scanning Electron Microscopy (SEM) and Powder X-Ray Diffraction (PXRD). After mounting samples onto aluminum foil supports (stubs), they were covered with carbon tape, coated with a thin layer of gold and examined with a HITACHI−1) and heating rate of 10 °C·min−1. To obtain the TG/DTG profiles, the equipment was calibrated with calcium oxalate monohydrate, according to Santos Costa et al. [Thermogravimetric (TG) analysis was performed on a TGA-50/50H analyzer  in the temperature range from 25–600 °C, using platinum crucibles containing approximately 3 mg of samples, under a dynamic nitrogen atmosphere  as described by Aguiar et al. [For the Differential Scanning Calorimetry (DSC) analysis, a sample of approximately 5–10 mg was put into a hermetically-closed aluminum crucible and analyzed with a calorimeter, model DSC-60 (Shimadzu), under a nitrogen atmosphere at a flow rate of 50·mLEnterococcus faecalis (ATCC 29212) and Staphylococcus aureus (ATCC 25932) were selected as Gram-positive standard bacterial strains, whereas Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli (ATCC 25922) as Gram-negative ones.The broth microdilution method was used to quantify the bactericidal activity of inclusion complexes expressed as Minimum Inhibitory Concentration (MIC). Strains were replicated in Brain Heart Infusion (BHI), and after 24 h the tests were performed. Initially, each strain was diluted in saline solution at turbidity equivalent to 0.5 on the McFarlane scale. The inoculum was added in a test tube containing 10% BHI in a 1:9 (v/v) ratio, thereby obtaining the bacterial suspension. Microdilution was then performed in 96-well plates, in which all wells received 100 μL of bacterial suspension. Samples (100 μL) of each substance  obtained either by KND or SL were applied to the first well of each plate column, as well as control compounds, namely penicillin, ampicillin, piperacillin-tazobactam and dimethylsulfoxide (DMSO), and a serial microdilution was carried out to the penultimate well. −1 in the first well and decreased to 8 μg·mL−1 in the penultimate one, while the last well received the bacterial suspension. After incubation of plates for 24 h at 37 °C, 20 μL of 0.01 (w/w) resazurin were added in each well, and turbidity was visually assessed after 1 h at room temperature. Tests were performed in triplicate and repeated three times.The concentration of substances was 1024 μg·mL−1 was considered for a final volume of 100 μL per well. After inoculation of each well with 100 μL of bacterial suspension, a serial microdilution was performed with 100 μL of the solution of standard antibiotic for each strain up to the penultimate well. The final 100 μL were discarded from this microdilution. The initial concentration of antibiotics was adopted according to the microbial susceptibility and sensitivity provided in the CLSI manual [The Drug Modulation Test was performed according to Oliveira et al.  in orderI manual . The lasp < 0.05 were considered statistically significant. The antimicrobial study was expressed as means and standard deviation, and the median was considered for modulatory activity. The results were analyzed by two-dimensional analysis of variance (ANOVA) using Graph Pad Prism 6.0 . Values of Euterpe oleracea oil (EOO) and cyclodextrin inclusion complexes. The results of molecular modeling, FT-IR, SEM, PXRD, TG and DSC analyses revealed that EOO was able to successfully form inclusion complexes, especially with β-cyclodextrin (β-CD), confirming the expectations arisen by the estimated interaction energy between oleic acid and β-CD (ΔG = −41.28 ± 0.57 kJ/mol). Accordingly, FT-IR showed displacements in wavenumbers and a variation in peak intensity characteristic of cyclodextrin and EOO. The morphological and crystalline characterizations showed variations in the form, appearance, and amorphous profile of the complexes compared to cyclodextrins alone. As for thermal analysis, temperature variations in mass losses and enthalpy variations between EOO, cyclodextrins and inclusion complexes confirmed the formation of inclusion complexes. Minimum Inhibitory Concentration (MIC) results showed that the inclusion complexes presented antibacterial efficiency in Gram-positive and negative strains, being considerably better compared to pure EOO. The MIC values of the complexes obtained with β-CD were lower than those of EOO and complexes with hydroxypropyl-β-cyclodextrin (HP-β-CD). Notably, β-CD inclusion complexes showed better responses regarding complexation and biological activity than those prepared using HP-β-CD as a carrier. EOO and most of its complexes showed a synergistic effect on ampicillin against E. coli. In addition, inclusion complexes provided physicochemical stability to the compounds and showed no loss in EOO biological efficiency, hence supporting its therapeutic applications. The antimicrobial response exhibited by the complexes was of great significance because it subsidizes studies for the development of new drugs and pharmaceutical forms.This is the first study that evaluates the antibacterial and modular activity of


Recently, single-cell RNA-seq technologies have been rapidly updated, leading to a revolution in biology. We previously developed Microwell-seq, a cost-effective and high-throughput single cell RNA sequencing(scRNA-seq) method with a very simple device. Most cDNA libraries are sequenced using an expensive Illumina platform. Here, we present the first report showing combined Microwell-seq and BGI MGISEQ2000, a less expensive sequencing platform, to profile the whole transcriptome of 11,883 individual mouse adult adrenal gland cells and identify 18 transcriptionally distinct clusters. Moreover, we performed a single-cell comparative analysis of human and mouse adult adrenal glands to reveal the conserved genetic networks in these mammalian systems. These results provide new insights into the sophisticated adrenal gland hierarchy and provide a benchmark, low-cost strategy for high-throughput single-cell RNA study. Cells are the basic unit of life, and cells within a tissue exhibit high heterogeneity. Single-cell RNA-sequencing (scRNA-seq) has become a benchmark method for dissecting cell heterogeneity, unraveling cell status, and identifying cell types . Highly expressed markers, Lyz2 and Fcer1g, defined C3 cells as macrophages. C4 was defined by stromal cells according to the Mgp gene and the gene expression of collagens such as Col1a2 and Col3a1 . Other vascular endothelial cell markers  were also expressed in the C9 cluster. C10 was defined by Cd209a high dendritic cell by marker genes, including Cd209a, Cd74, H2-Ab1, H2-Eb1 and H2-DMa. C11 cells expressed high levels of proliferation marker genes, including Ube2c, Birc5, Cdc20, C1qc, Ctsc, C1qb, Ctss and C1qa. C12 was identified by the high expression of Igkc, Cd79b and Ighm, which suggests it contained B cells. C13 cells highly expressed chromaffin cell marker genes, including Chgb, Chga, Npy, Scg2 and Dlk1. C14 was defined by myeloid cells because of the expression of Lyz1, Ear2, Fn1, Lyz2 and H2-DMb1. C15 cells showed high levels of known smooth muscle markers, namely Acta2, Myl9, Tpm2, Myh11, Mylk and Tpm1 . In addition, the C16 cells highly expressed myeloid markers, including Cd209f, Pf4, Cd209d, Lyv1 and Cd209b, which differed from those expressed in C14 . The human adrenal gland was divided into 15 cell clusters that exhibited highly specific gene expression patterns . Then, we mapped the t-SNE map clustered by the activity of regulons Fig. a-b. EachThrough the GO analysis, we found different signaling pathways for the 12 homologous TF-TF modulator modules . The C57BL/6 mice were raised at the Experimental Animal Center of Zhejiang University. All procedures were approved by the Ethics Committee of the Experimental Animal Center of Zhejiang University. One batch of human adrenal glands was extracted from a volunteer at the First Affiliated Hospital of Zhejiang University School of Medicine. The donor signed an informed consent document. All procedures were approved by the Medical Ethics Committee of Zhejiang University Medical College.5 cells/ml in cold DPBS containing 2 mM EDTA for further Microwell-seq.Mouse and human adrenal gland tissues were put into DMEM plus 10% FBS immediately after resection and minced using scissors into ~ 1-mm pieces on ice. The tissue pieces were transferred to a 15-ml centrifuge tube, rinsed twice with cold DPBS and suspended in 5 ml of a solution containing dissociation enzymes. The mouse adrenal glands were treated with collagen IV (37 °C for 30 min), while the human adrenal glands were treated with collagen IV (37 °C for 90 min). During dissociation, the tissue pieces were pipetted up and down gently several times until no tissue fragments were visible. The dissociated cells were centrifuged at 300×g for 5 min at 4 °C and then resuspended in 3 ml of cold DPBS. After passing through a 40-μm strainer (Biologix), the cells were washed twice, centrifuged at 300×g for 5 min at 4 °C, and resuspended at a density of 1 × 10All adrenal gland cell suspensions were treated to obtain cDNA libraries according to the standard Microwell-seq protocol (Han et al., RNA-seq data preprocessing, alignment, quality filtering and acquisition of expression matrix cellular barcodes and unique molecular indices were achieved in read 1. We discarded the read pairs within which the base quality of any one base of the barcode was below 10, since sequencing errors and low quality may produce spurious barcodes. After tagging the cellular barcode and molecular barcode, we excluded read 1 and only used read 2 as input for the alignment. We used STAR-2.5.1b with default parameters for mapping (Dobin et al., github.com/ggjlab/mca_data_analysis.We used Seurat software (Satija et al., To make the cross-species transcriptome comparable, we used the homology correspondence values from dmodENCODE (Celniker et al., We used SCENIC (Aibar et al., AUCell provided the activity of the regulons across the cells. To see whether there are conserved regulons active in specific cell types in the humans and mice, we created the “binarized regulon activity matrix”, with the coordinates of the matrix that correspond to active regulons in a given pseudocell containing a “1” value, and “0” is attributed to all the others. Then, we used the SCENIC R package (Aibar et al., Additional file 1: Figure S1. Representative gene expression in the mouse adrenal glands. Figure S2. The different expression patterns of the two stromal cell clusters. Figure S3. The different expression patterns of the two myeloid cell clusters. Figure S4. Comparison of BGI MGISEQ2000 and Illumina HiSeq platforms based on the t-SNE and heat maps. Figure S5. Mapping the human adult adrenal gland atlas by Microwell-seq. Figure S6. The signaling pathways of several representative TF modules.Additional file 2: Table S1. Detailed information on the seven sequencing data sets.Additional file 3: Table S2. Markers of mouse adrenal gland cells by a BGI platform.Additional file 4: Table S3. Markers of mouse adrenal gland cells by an Illumina platform.Additional file 5: Table S4. Differentially expressed genes detected in the human adrenal gland.Additional file 6: Table S5. A list of redefined large cell clusters.Additional file 7: Table S6. Human-mouse 12 TF modules.


Pteroptyx maipo. The circular genome of 16,127 bp has a base composition of A (44.22%), C (11.60%), G (8.23%) and T (35.96%). Our sequence is similar to other Metazoa, which contains 13 protein-coding genes. All 13 protein-coding genes were initiated by the ATN  codon. Nine protein-coding genes stopped with TAA or TAG codon and the other four genes have an incomplete termination codon, a single T. We sequenced the mitochondrial genome of fireflies to analyze phylogenetic relationships and deduce the evolution of their flashing signals.We report the complete mitochondrial genome of firefly,  Pteroptyx following revisions by Ballantyne and McLean  in 13 July 2013, and were stored in Natural History Museum, Huazhong Agricultural University, Wuhan, Hubei, China (its accession no. PM2011052301). Specific primers were designed based on these conserved regions sequences. The PCR reaction was carried out with LA Taq polymerase for 35 cycles at 94 °C for 30 s, and annealed at 50 °C for 30 s, followed by extension at 72 °C for 1 min per 1 kb. Sequences were assembled using the software DNAstar v7.1  and adjusted manually to generate the complete sequence of mitochondrial DNA.P. maipo (GenBank MF686051) has 16,127 bp and has a base composition of A (44.22%), C (11.59%), G (8.23%), and T (35.96%). Similar to other Metazoa, our sequence contains 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes and a non-coding AT-rich region, which represents a typical insect mitochondrial genome  (with 1000 bootstrap replicates) to construct a Neighbour-Joining tree .P. maipo is most closely related to Luciola Substriata (recently identified as Sclerotia flavida by Ballantyne et al. The result shows P. maipo provides an important molecular framework for further phylogenetic analyses of fireflies. These data are essential for comprehensive understanding of the role of sexual and natural selection in the evolution of firefly flashing signals.In conclusion, the complete mitochondrial genome sequence of


Water-pipe smoking (WPS) is becoming the most popular form of tobacco use among the youth, especially in the Middle East, replacing cigarettes rapidly and becoming a major risk of tobacco addiction worldwide. Smoke from WPS contains similar toxins as those present in cigarette smoke and is linked directly with different types of cancers including lung and head and neck (HN) carcinomas. However, the underlying molecular pathways and/or target genes responsible for the carcinogenic process are still unknown. In this study, human normal oral epithelial (HNOE) cells, NanoString PanCancer Pathways panel of 770 gene transcripts and quantitative real-time polymerase chain reaction (qRT-PCR) analysis were applied to discover differentially expressed genes (DEG) modulated by WPS. In silico analysis was performed to analyze the impact of these genes in HN cancer patient’s biology and outcome. We found that WPS can induce the epithelial–mesenchymal transition  of HNOE cells. More significantly, our analysis of NanoString revealed 23 genes deregulated under the effect of WPS, responsible for the modulation of cell cycle, proliferation, migration/invasion, apoptosis, signal transduction, and inflammatory response. Further analysis was performed using qRT-PCR of HNOE WPS-exposed and unexposed cells supported the reliability of our NanoString data. Moreover, we demonstrate those DEG to be upregulated in cancer compared with normal tissue. Using the Kaplan–Meier analysis, we observed a significant association between WPS-deregulated genes and relapse-free survival/overall survival in HN cancer patients. Our findings imply that WPS can modulate EMT as well as a set of genes that are directly involved in human HN carcinogenesis, thereby affecting HN cancer patients’ survival. Tobacco smoking is the most common preventable risk factor for several non-communicable diseases, such as, cardiovascular, lung, diabetes as well as cancer, and can be considered lethal ,2. WidesSmoke emanating from WPS includes toxins resembling those found in cigarettes, such as carbon monoxide (CO), hydrocarbons, and carcinogenic polycyclic aromatic volatile aldehydes ,11. In cTo date, various studies have confirmed the association between WPS and several types of human cancers, including lung, esophageal, oral, and pancreatic carcinomas ,24,25. CA standard smoking protocol (Aleppo Method) was used as described previously by our group ,27. The 2 in air. The cells were cultured in KSFM with 5  mg/100 mL of bovine pituitary extract (BPE) , and 100 µg/mL penicillin–streptomycin. Cells were treated either with 100 μg/mL of WPS in PBS or KSFM solution for 48 h.Two human normal oral epithelial (NOE) cell lines established in our laboratory  were useTotal RNA was extracted from cells using RNeasy Mini Kit spin columns (Qiagen). First strand cDNA was synthesized using 5X All-In-On MasterMix  per manufacturer’s protocol. Reverse transcriptase real-time polymerase chain reaction (RT-PCR) was carried out on 96-well plates using iTaq Universal SYBR Green Supermix (BioRad). Concentrations for each sample were measured using the NanoDrop ND-100 spectrophotometer 119  and Qubit . The primer sequences were designed using Primer ExpressTM Software v3.0.1  .Gene expression was assessed using the NanoString PanCancer Pathway Panel  consisting of probes for 770 genes implicated in carcinogenic pathways, curated from The Cancer Genome Atlas (TCGA) data. All RCC files (direct outputs/raw data from NanoString runs) were normalized using nSolver analysis software  according to the manufacturer’s protocols . In brief, a normalization factor was calculated by obtaining the geometric mean of the controls used for each sample and applied to the raw counts of the nCounter output data to eliminate variability that was unrelated to the samples. The resulting data were normalized again with the geometric mean of the housekeeping genes. Normalized data were log2-transformed and exported to Microsoft Excel for analysis.The in silico approach used in our study helped us to support and confirm our findings  were discovered by NanoString analysis as previously described). The large, publicly available database Oncomine TM consists of approximately 65 gene expression datasets and was used to explore the differential expression of our genes to compare HN cancer with respective normal tissues as well as clinico-pathological parameters. From this database we used Toruner, Ginos, Cromer, Ye, Peng, Sengupta, Estilo, Kuriakose, and TCGA datasets to evaluate mRNA expression of the discovered DEGs in normal versus malignant patient samples. In addition, TCGA Head and Neck dataset (270 patients) was used to evaluate the differences in the DNA copy numbers between smoker head and neck cancer patients compared with non-smokers with head and neck cancer. In brief, the parameters were set and the program generated the expression levels per dataset; analysis was performed, and we finally selected genes that were statistically relevant to our study. Moreover, we used a cohort of 500 HN squamous cell carcinomas (HNSCC) samples from the Pan-cancer RNA-seq dataset of the Kaplan–Meier plotter database to analyze the patients’ clinical outcome.https://string-db.org/) tool was used to investigate the network and interaction between the different WPS deregulated genes as well as biological function. This is a biological database and web resource of known and predicted protein–protein interactions. Briefly, we uploaded the obtained gene list and the software imported protein association knowledge from databases of physical interaction and databases of curated biological pathway knowledge; the program utilizes computational predictions to generate maps and connections between different proteins. We used this tool to highlight the importance of the potential connectivity network of our genes that need to be considered for the full understanding of the biological phenomena.The Search Tool for the Retrieval of Interacting Genes (STRING v9.1) .In vitro assays were all performed in triplicates of at least three independent experiments. Results were shown as means ± SEM. Student’s t-test was used to evaluate the statistical significance. Statistical analyses were performed using nSolver and GraphPad Prism (version 8.4.3) analysis software. Overall survival and relapse-free survival (RFS) were performed using the Kaplan–Meier survival analysis and a In order to study the effect of WPS on human HN carcinogenesis, we examined the outcome of WPS on two human normal oral epithelial, 2N and 11N, which were established in our lab . Our datSimilar to our study, we previously examined the expression of E-cadherin and focal adhesion kinase (FAK) proteins in cancer cells exposed to WPS . Our datCCL5, C1R, MMP9, IL-1B, CCL4, MASP2, OXER1, TLR3, STAT1, PPP1R12B, MX1, MX2, CCL21, IL-3, TLR9, HSH2D, CCR4, IFNγ, LT-β, IFIT1, TGF-β2, ALOX5, and LIMK1 (p < 0.05).Next, gene expression was applied on both cell lines, using the NanoString PanCancer Pathway Panel consisting of probes for 770 genes implicated in carcinogenic pathways; our data showed that out of these genes, 23 were found to be differentially expressed in WPS-exposed versus unexposed 2N and 11N cells: IL-IB, CCL5, C1R, MMP9, LIMK1, CCR4, MASP2, OXER1, TLR3, STAT1, PPP1R12B, MX2, CCL21, IL-3, TLR9, HSH2D, CCL4, IFNγ, LT-β, IFIT1, TGF-β2, ALOX5 and MX1) upregulated genes by a factor ranging from 1.58 to 3.8 folds (p < 0.05)  .Based on the molecular pathways of carcinogenesis, these 23 deregulated genes are directly involved in the modulation of cell cycle, proliferation, migration, invasion, apoptosis, angiogenesis, signal transduction, and inflammatory response .Using the Oncomine database, we herein initially evaluated the mRNA expression levels of the DEGs discovered in normal tissue versus head and neck tumor samples.IFIT1, (p = 2.76 × 10−8) and ALOX5 (p = 0.0019) genes were high in oral squamous cell carcinoma (OSCC) compared to normal squamous cells , STAT1 (p = 9.25 × 10−11), MX2 (p = 0.22 × 10−8), LT-β (p = 2.43 × 10−6), C1R (p = 0.005), CCL5 (p = 2.28 × 10−9), MMP9 (p = 7.07 × 10−26), ALOX5 (p = 0.050) and CCL4 (p = 3.20 × 10−15) genes were upregulated in HNSCC compared with the normal buccal mucosa (IL-1B (p = 0.002), STAT1 (p = 0.001) and IFNγ (p = 3.46 × 10−4) genes were high in HNSCC compared with normal uvula tissue (IL-1B (p = 2.67 × 10−6), TGF-β2 (p = 2.23 × 10−4), and MMP9 (p = 0.01) genes to be upregulated in tongue squamous cell carcinoma (SCC) compared with normal tongue tissue (IL-1B (−10p = 4.52 × 10), IFIT1 (p = 1.27 × 10−17), TGF-β2 (p = 2.23 × 10−4), STAT1 (p = 7.18 × 10−20), MX2 (p = 5.11 × 10−11), OXER1 , LT-β (p = 0.004), CCL21 , C1R (p = 0.005), CCR4 (p = 2.50 × 10−5), HSH2D (p = 2.47 × 10−7), MASP2 (p = 5.16 × 10−5), PPP1R12B (p = 4.10 × 10−4), CCL5 , MX1 (p = 1.70 × 10−9), and IFNγ (p = 6.82 × 10−13) genes to be over expressed in OSCC compared with normal oral cavity (IFIT1 (p = 5.38 × 10−5), CCR4 (p = 0.046), and CCL4 (p = 2.79 × 10−8) genes were increased in nasopharyngeal carcinoma compared with normal nasopharynx (STAT1 (p = 1.07 × 10−12), MX2 (p = 4.53 × 10−5), and MX1 (p = 1.31 × 10−10) genes to be highly expressed in tongue SCC compared with normal tongue tissue (IL-3 expression in Kuriakose dataset (20 patient samples) was predominant in lip and OSCC  (OXER1 (p = 1.38 × 10−9) expression using TCGA dataset is highly upregulated in HNSCC (364 patient samples) (Using the Toruner dataset (20 patient samples), we found that the expression of us cells . The Ginl mucosa . Moreovea tissue . The date tissue . In addil cavity . Senguptopharynx . Estilo e tissue . While I= 0.009) . Additiosamples) .CCL5, C1R, MMP9, IL-1B, CCL4, OXER1, TLR3, STAT1, PPP1R12B, MX1, MX2, HSH2D, IFNγ, LT-β, IFIT1, and TGF-β2 (p ≤ 0.05)  of the Oncomine database. Interestingly, our results confirmed that, of the 22 genes, 16 were upregulated in smoking HN cancer patients compared to those who had never smoked. These genes include  ≤ 0.05) .Subsequently, we explored whether the DEGs induced by WPS in oral epithelial cells could have an impact on the prognosis of HN cancer patients. To asses this point, we analyzed the association between the DEGs mRNA expression and patient’s outcome, relapse-free survival (RFS), or overall survival (OS), using a large HNSCC cohort (n = 500 patients) from the Kaplan–Meier plotter database.ALOX5 (p = 0.0091), IFNγ (p = 0.054), C1R (p = 0.0028), CCL4 (p = 0.001), MASP2 (p = 0.041), PPP1R12B (p = 0.031), TGF-β2 (p = 0.014), and CCL21 (p = 0.0062) correlates positively with poor RFS (TLR9 (p = 0.027), IFIT1 (p = 0.021) and IL-3 (p = 0.0031) correlates positively with poor OS  chemokine binding receptors , Table .This investigation, to the best of our knowledge, is the first cancer genes profiling study on the effects of WPS exposure on human normal oral epithelial cells. Previously, our group has revealed that WPS can play an important role in the initiation and progression of human oral cancer, which represents the majority of HN cancer cases . In thisCCL5, CCL21, CCL4, and CCR4) belong to the family of chemokines. Elevated CCL5 levels are significantly associated with oral cancer progression [CCL5 association with oral cancer progression upon WPS intake. Moreover, CCL5 is capable of upregulating the release of MMP-9 [CCL5 enhances oral cancer cell migration through the increase in MMP-9 production [MMP9 is observed in oral cancer [ALOX5 acts as a mediator of invasion via MMP-9 induction [ALOX5 expression is known to be involved in carcinogenesis [ALOX5 genotype was found to be linked with asthma and poorer lung function [ALOX5 with poor asthma controls [ALOX5 with RFS; we herein show for the first time that ALOX5 is associated with shorter RFS.Of the nine differentially expressed genes, four -positive tumors has been previously reported [OXER1 expression and HN as well as oral cancers.Similar to l cancer . It has ogenesis . A recenl cancer . Likewisn cancer . On the nd CCL21 , therebynd CCL21 . Additio outcome . In thisN cancer ,50. In tN cancer ,52,53. Ccurrence . Moreove tissues . Howeverreported . Since Hreported ,57,58, wTLR3, TLR9, C1R and MASP-2) of them are involved in innate immune system. We reveal that TLR3 and TLR9 are upregulated in WPS-exposed oral epithelial cells. It has been demonstrated that HN cancer cell lines as well as OSCC tissue samples express TLR3 thereby enhancing the expression levels NF-κB and its regulated oncogene, c-myc, thus inciting cellular proliferation and migration, which is significantly associated with poorly differentiated tumor cells and perineural invasion [TLR9 in HNSCC as well OSCC was found to promote tumor cell invasion, proliferation as well as migration by enhancing MMP-2 expression [C1R is known to regulate the complement pathway of the innate immune system, in this study, we found upregulated CIR expression in HNSCC. This is in concordance with a previous study that correlated the expression of C1R in cutaneous SCC (cSCC) with tumor progression, cell proliferation, and migration [C1R has not been studied in cancer; however, it is associated with tumor progression and migration [MASP-2 gene, which is upregulated in oral epithelial cells exposed to WPS. MASP-2 produced in hepatocytes is involved in innate response, and its promoter is regulated by cytokines (interleukins and TGF-β) or transcription factor (STAT) [MASP-2 expression and cancer [MASP-2 expression significantly correlates with late clinical stage and nodal metastasis, thus indicating its role in cancer progression and aggressive tumor behavior in esophageal SCC [MASP-2 is significantly associated with recurrence and poor survival of colorectal as well as ovarian cancers [Moreover, in this study, of the 23 genes, 4  ; our stud cancer ,72,73; Mgeal SCC . MoreoveIL-1B, is elevated in HNSCC including oral cancer [IL-1B in tobacco and betel quid-mediated OSCC; IL-1B promotes proliferation of dysplasia of oral cells, thus triggering oncogenic cytokines as promoters of tumor aggressiveness [IL-3, is a selective growth factor that stimulates tumor angiogenesis [IL-3 in COPD as well as cigarette smoking is not well defined, IL-3 levels were previously detected in SCC [IL-3 in oral epithelial cells under the effect of WPS, thus indicating its role in oral cancer progression. Although data have shown strong correlation between IL-3 and poor survival in acute myeloid leukemia [IL-3 in OSCC is scarce. Interestingly, a recent study by Almeida et al. (2019) showed an association between IL-4, IL-6, IL-8, IL-10, IL-12, and IL-13 and poor survival in OSCC [IL-3 to significantly correlate with poor overall survival, which requires further investigation. On the other hand, transforming growth factor (TGF), a cytokine, is involved in promoting cellular invasion as well as angiogenesis in OSCC cells [TGF expression was significantly associated with shorter OS, RFS, and DFS in patients with OSCC [IFNγ. In oral epithelial cells, a loss of IFNγ expression may be caused by the IFN-γ promoter methylation as a plausible underlying mechanism for oral cancer progression [IFNγ levels are associated with poor prognosis in HNSCC including oral [LT-β, was also identified in this study; LT-β has been shown to correlate with human oral cancer [STAT1 has a dual role in HNSCC [STAT1 in OSCC as well as HNSCC, as reported previously [STAT1 in the pathogenesis of HNSCC.The pro-inflammatory cytokine, l cancer ,75. Furtsiveness . On the ogenesis . Howeverd in SCC . We hereleukemia , researc in OSCC . In thisCC cells ,82. Our CC cells  as well CC cells . Similarith OSCC . The othgression . Furthering oral ,88. The l cancer , and addl cancer ,91. Whilin HNSCC ,93,94, weviously , suggestIFIT-1, in WPS-exposed HNOE cells. In this context, a previous study showed that over-expression of IFIT1 in OSCC cells promote tumor growth and metastasis by activating EGFR signaling [IFIT1 expression with T-stage, lymph node metastasis, lymphovascular, perineural invasion, as well as poor overall survival in OSCC patients [IFIT1 expression in OSCC cells enhance resistance to several therapeutic agents  [MXI along with its paralogue MX2. MX1 has a contradictory role in cancer; in one study, MX1 is upregulated in OSCC [MX1, indicating its possible oncogenic role in oral cancer.On the other hand, we report the upregulation of an interferon-stimulated gene, ignaling . Researcpatients . Moreoveiplatin) ,97, thus in OSCC , but nev in OSCC . HoweverHSH2D and PPP1R12B) in our cohort that were not previously reported, as possible players in OSCC or HNSCC. Traditionally, HSH2D has a role in T-cell activation and is a downstream target of CD28 costimulatory signaling pathway [HSH2D activation in HNSCC. Moreover, PPP1R12B, also known as MYPT2, is a subunit of MYPT [MYPT2 is implicated in cancer, MYPT is found to be involved in cancer [MYPT2 in HNSCC. We found PPP1R12B to be associated with poor RFS. However, studies have shown MYPT to correlate significantly with drug resistance and poor prognosis in human carcinomas [PPP1R12B in drug resistance and poor prognosis in OSCC or HNSCC.Interestingly, we discovered two genes ( pathway . A previ pathway ; thus, w of MYPT . Althougn cancer ,104, indrcinomas ,104, thuAn earlier study by our group show clearly that WPS stimulate cell invasion of human breast cancer cells . While sWe reveal for the first time, that WPS can induce EMT in human normal oral epithelial cells, which is accompanied by the deregulation of a set of genes related to oncogenesis. Thus, WPS can promote HN cancer initiation and/or progression mainly due to its effect on key regulatory genes of carcinogenesis that have a direct impact on HN cancer patients’ outcome. Nevertheless, further studies are needed to elucidate the expression of different proteins involved in EMT as well as to understand the full mechanism by which WPS can induce HN carcinogenesis.


The incidence of invasive fungal infections (IFIs) has recently increased, and early and accurate diagnosis of IFIs is important for the rational selection of antifungal drugs with high efficacy. We developed a method for rapid and accurate clinical diagnosis of IFIs and provide a reference for personalized drug treatment.We designed and screened fungal internal transcribed spacer regions with universal primers and designed 8 TaqMan detection probes to establish a multi-channel real-time fluorescent polymerase chain reaction (PCR) melting curve analysis (MCA) method. The sensitivity, specificity, and reproducibility of this method were investigated using standard fungal strains and clinical isolates. Candidemia was detected using the MCA method.Candida albicans were 0.05 pg/μL and 66.50 °C; Candida glabrata were 0.1 pg/μL and 66.25 °C; Candida tropicalis were 0.1 pg/μL and 60.15 °C; Candida krusei were 0.1 pg/μL and 72.15 °C; Candida parapsilosis were 0.2 pg/μL and 63.10 °C; Candida guilliermondii were 0.1 pg/μL and 61.84 °C; Cryptococcus neoformans were 0.1 pg/μL and 65.50 °C; Aspergillus flavus were 0.05 pg/μL and 71.50 °C; Aspergillus terreus, Aspergillus fumigatus, and Aspergillus niger were 0.05 pg/μL and 76.80 °C. Analytical specificity was evaluated using 13 clinical pathogens including Streptococcus pneumoniae, Staphylococcus aureus, and Haemophilus influenzae, etc. No false-positive results were obtained for any of these samples. The MCA method can detect and identify different candidemia simulations. The limit detection concentration of C albicans was 44 cfu/mL, C glabrata was 73 cfu/mL, C tropicalis was 29 cfu/mL, C parapsilosis was 21 cfu/mL, C krusei was 71 cfu/mL, and C guilliermondii was 53 cfu/mL.The limit of detection and assay cut-off (melting temperature [Tm]) for Candida, Cryptococcus, and Aspergillus and identified Candida at the species level. Our method can facilitate early and accurate clinical diagnosis and personalized medication regimens.The multi-channel real-time fluorescence PCR melting curve analysis displayed high sensitivity and specificity in detecting various clinically invasive fungi. Furthermore, it simultaneously detected the genera  IFIs primarily occur in patients with severe underlying diseases, malignant tumors, and other severe diseases compromising immune function and in those undergoing organ transplantation. The clinical manifestations of IFIs are often non-specific and easily masked by primary underlying diseases. Early diagnosis is difficult, often resulting in delayed diagnosis, misdiagnosis, and delayed treatment. Furthermore, IFIs have a poor prognosis and are associated with high mortality, with approximately 1.4 million deaths worldwide each year. Sun et al reported that the total mortality rate with IFIs is 13.4%, and Barnes reported that the mortality rates associated with invasive candidiasis (IC) and invasive aspergillosis are 36% to 63% and 70%, respectively.Invasive fungal infections (IFIs) have increased with the widespread use of broad-spectrum antibiotics, immunosuppressive agents, antineoplastic drugs, and in-depth development of organ transplantation, and various invasive diagnostic techniques. Traditional methods have a low positive detection rate and poor sensitivity and do not yield a rapid positive clinical diagnosis. Serological analysis primarily includes the 1,3-β-D-glucan test (G test), galactomannan test (GM test), and latex agglutination test. Although these methods potentially provide a basis for the early diagnosis of IFIs, they can be influenced by various factors, leading to false-positive findings. Furthermore, they do not accurately detect fungal species. Molecular biology technologies based on PCR have been widely used to detect IFIs. Fluorescence PCR melting curve analysis (MCA) is an emerging detection method for identifying fungal species without sequencing. MCA has a high sensitivity, throughput, speed, and accuracy,,18 and it is less expensive; thus, it is applicable for detecting fungal infections.,20 This method is based on the principle that different double-stranded DNA molecules have different Tm values, and changes in the shape of the melting curve can be monitored using fluorescent dyes or probes to rapidly and accurately detect and identify various fungi. The TaqMan probe melting curve analysis uses the TaqMan probe rather than the SYBR Green dye to specifically bind to the target DNA. By analyzing the melting curve and Tm value of specific binding products, fungal detection at the species level can be achieved. Furthermore, TaqMan probes can link fluorescent reporter groups of different wavelengths, and thus completely harness the advantages of multi-channel fluorescent probe PCR for simultaneous detection of various fungi. For example, Alonso et al performed real-time fluorescent probe PCR melting curve analysis to detect and identify Aspergillus, and Valero et al also detected and identified Mucor, Rhizopus, and endemic diseased-related fungi and established a corresponding melting curve library. In this study, we used a combination of PCR amplification technology and multi-channel real-time fluorescent probe melting curve analysis for closed-tube detection and species identification of common IFI-causing pathogens . This study was conducted to develop a method for rapid and accurate clinical diagnosis of IFIs and provide a reference for personalized drug treatment.Laboratory detection of IFIs primarily involves traditional detection methods including direct microscopy, culture, and histopathology.22.1Candida albicans (ATCC 10230), Candida glabrata (BNCC 337348), Candida tropicalis (BNCC 337310), Candida parapsilosis (BNCC 337317), Candida krusei (ATCC 6258), Candida guilliermondii (ATCC 6260), Cryptococcus neoformans (BNCC 337347), Aspergillus flavus (HB-CICC 71005), Aspergillus terreus (HB-CICC 70504), Aspergillus niger (01018094), and Aspergillus fumigatus (01010287). The following clinical isolates were used: C albicans, C glabrata, C tropicalis, C parapsilosis, C krusei, C guilliermondii, Streptococcus pneumoniae, Streptococcus salivarius, Staphylococcus aureus, Staphylococcus epidermidis, Klebsiella pneumoniae, Haemophilus influenzae, Escherichia coli, Pseudomonas maltophilia, Pseudomonas aeruginosa, Nocardia amarae, Legionella sp., Bordetella pertussis, and Corynebacterium diphtheriae .The strains used in this study were subjected to sequencing analysis with specific primers . The following fungal strains were used Table : Candida2.2Blood was collected from patients who visited the First People's Hospital for a health checkup, with no clinical symptoms of sepsis and inflammatory markers.2.3g, 2–3 days). Pretreatment: 200 μL aliquots of the culture was centrifuged , and then the pellet was treated with 600 μL of 1.2 M sorbitol and 10 U Lyticase , thoroughly mixed, incubated at 30 °C for 30 minutes to disrupt the fungal cell wall, and centrifuged , and the supernatant was discarded. DNA extraction: Genomic DNA of the fungi was extracted using the magnetic bead method with the fungal DNA extraction kit  in accordance with the manufacturer's instructions. DNA purity and concentration were measured using the Nano-300 spectrophotometer . DNA samples were diluted to 1 ng/μL, 10, 1, 0.1, 0.05, and 0.01 pg/μL.Strain preparation: Each fungal strain was inoculated on potato glucose liquid medium  and cultured  database. Based on the highly conserved/relatively specific segment of the ITS region of the strain, primer 5.0 software was used to design universal primers (ITSF1/ITSR1)/specific probes (CaP1/CgP1/CtP1/CkP1/CpP1/CguP1/CnP1/A. P1), and the probes were labeled with different fluorophores  in the fungal genome is a conserved, species-specific 18S rDNA–ITS1–5.8S rDNA–ITS2–28S rDNA tandem repeat. The ITS sequences of 2.5PCR amplification and MCA were performed using SLAN-96S . The PCR was carried out with a reaction mixture of volume 25 μL comprising 2.5 μL of 10× PCR buffer, 5 μL of 5× probe buffer, 0.2 mM dN(U)TPs, 1 U of Hot Start Taq polymerase, 0.2 U of UDG, 0.4 μM ITSF1, 0.04 μM ITSR1, 0.16 μM primers, and sterile water. The PCR program comprised a UDG enzyme reaction for 2 minutes at 50 °C, followed by initial denaturation for 3 minutes at 95 °C, 60 cycles for 30 seconds at 95 °C, 30 seconds at 56 °C, and 30 seconds at 72 °C, and termination for 2 minutes at 95 °C. The amplification products for MCA were cooled for 2 minutes at 40 °C and heated to 85 °C to monitor fluorescence at 0.06 °C/s. Thereafter, 1 ng/μL of DNA each reference fungal strain was analyzed in 10 independent runs and the mean melting temperature (Tm) and standard deviation (SD) values were determined for each melting domain.2.6The genomic DNA samples of each fungal strain were diluted to 10, 1, 0.1, 0.05, and 0.01 pg/μL, and then 4 μL of each sample was analyzed in 20 independent runs and the limit of detection was determined. The genomic DNA of the clinical isolates was extracted using the DNA Extraction kit  in accordance with the manufacturer's instructions, and 100 pg/μL of each sample was analyzed to evaluate the specificity of the analysis.2.73, 102, 101 cfu/mL for each of simulated candidemia sample. The diluted sample (400 μL) was added into the pathogen lysis tubes (L)  and treated with 10 U Lyticase, thoroughly mixed, and incubated at 30 °C for 30 minutes. DNA extraction: simulated candidemia samples’ DNA was extracted according to the manufacturer's instructions of the QIAmp UCP Pathogen kit .Candidemia’ clinical samples were detected using the MCA method. Clinical sample preparation: blood samples were drawn into a 2-mL anticoagulation tube . The 6 model clinical isolates of fungal strains were counted, and inoculated blood samples later, in order to obtain the fungal cell number of 106 cfu/mL of each fungal suspension was used as the positive control and healthy human blood as the negative control. The MCA was performed with DNA samples from the positive control, negative control, and different concentrations of simulated candidemia samples. The research was approved by the local Bioethics Committee of Hangzhou First People's Hospital.Furthermore, 1033.1Candida reference strains, 1 Cryptococcus reference strain, and 4 Aspergillus reference strains revealed reproducible melting peaks for each species. Most strains displayed a single peak  together with the shape of the melt curve distinguished the 6 Candida spp., 1 Cryptococcus sp., and 1 Aspergillus sp.  displayed specific melting curve and Tm value, whereas the negative control did not show a specific response. The MCA method can detect simulated candidemia with different concentrations of fungal strain. The limit detection concentration of C albicans was 44 cfu/mL, C glabrata was 73 cfu/mL, C tropicalis was 29 cfu/mL, C parapsilosis was 21 cfu/mL, C krusei was 71 cfu/mL, and C guilliermondii was 53 cfu/mL TPs from 0.20 to 0.24 μM, and the annealing temperature from 50 to 60 °C to optimize the system and finally determine the optimal PCR system and conditions.As an emerging detection method, fluorescence PCR melting curve analysis is useful for diagnosing IFIs. with the former showing higher sensitivity. During fungal identification, based on specific melting curves and Tm values, this method distinguished C albicans, C glabrata, C tropicalis, C krusei, C parapsilosis, C neoformans, and A flavus. However, it was difficult to distinguish and identify A fumigatus, A terreus, and A niger using this method because of the similarity of their melting curves and Tm values, which was likely related to the specificity of the A.P1 probe. With respect to the entire probe for >25 nucleotides, a certain degree of conservation among fungal genera and species specificity is required, which would adjust the probe position and Tm values for interaction with the appropriate probe sequence, such that it can be completely combined with the corresponding sequences and simultaneously distinguish fungi. Furthermore, the development of a single probe with greater specificity to identify four Aspergillus spp. is even more challenging. The A.P1 probe detects Aspergillus at the genus level; however, it cannot identify A fumigatus, A terreus, and A niger. This issue can be resolved by designing and synthesizing a single A fumigatus probe, A flavus probe, A terreus probe, and A niger probe to rapidly identify clinically common Aspergillus spp.The multi-channel real-time fluorescence PCR melting curve analysis is a closed system, thus effectively preventing false-positive results caused by external influences. This method has high sensitivity and specificity and displays a lower limit of detection of 0.5 to 1 pg/mL during MCA than the method devised by Nemcova et al, To date, more than 17 different Candida species have been identified as pathogens of IC, and C albicans is the leading and most widely studied pathogen. However, in recent years, the proportion of non-C albicans has increased rapidly. The antibacterial activity of antibacterial drugs varies with strains, and the choice of treatment drugs is also different; early identification of the pathogenic species of IC is essential for early clinical medication. In this study, we detected IC using the MCA method; 6 clinically isolated fungal strains were used to simulate candidemia samples, which included 6 positive controls, 1 negative control, and 3 concentration gradients of candidemia samples, and then the MCA was performed. All positive controls were positive, whereas the negative control showed no reaction. The results showed that there was no positive effect of healthy human blood on the established MCA detection system, and each clinical isolate of Candida strain (106 cfu/mL) could be detected successfully. Furthermore, the MCA method can detect simulated candidemia with different concentrations of fungal strains, and the lowest detection concentration of C glabrata and C krusei was 70 to 80 cfu/mL, C albicans and C guilliermondii was 40 to 50 cfu/mL, C tropicalis and C parapsilosis was 20 to 30 cfu/mL . However, there was a melting curve peak of C parapsilosis blood sample at Tm of around 76 °C, which was that of Aspergillus. That is, there was contamination by Aspergillus in the detection process. Therefore, the test results of C parapsilosis should be deliberated.Invasive candidiasis (IC) is an important type of IFIs, and it has become a major threat to public health.In summary, compared with the gold standard culture method, the MCA method can detect candidemia samples and yield results within 4 to 6 hours, and its sensitivity was 20 to 80 cfu/mL, indicating that the method is fast, accurate, and sensitive. However, our study had some limitations. The clinical sample is a simulated candidemia sample, and not a clinical sample, and we plan to collect samples from patients with invasive candidiasis for verification. Multi-channel real-time fluorescence PCR melting curve analysis is rapid and accurate compared with the traditional culture methods. This method has a high sensitivity and specificity and can simultaneously detect various common invasive fungi, invasive candidiasis especially, yielding detection results within 4 to 6 hours and facilitating early diagnosis of clinical IFIs. In the future, multi-channel real-time fluorescent PCR melting curve analysis may be combined with other detection methods (G and GM test), to potentially improve the early diagnosis rate of IFIs and providing a reference for clinical rationalization of antifungal treatment, reducing misdiagnosis, improving prognosis, and reducing mortality during clinical diagnosis and treatment.Conceptualization: Xianjun Wang, Qiong Chen, Huali Yin, Xiaozi Wen, Shenghai Wu.Data curation: Xiaozi Wen, Qiong Chen, Huali Yin, Xianjun Wang.Formal analysis: Xiaozi Wen, Qiong Chen, Huali Yin, Xianjun Wang.Funding acquisition: Xianjun WangInvestigation: Xiaozi Wen, Qiong Chen, Huali Yin, Xianjun Wang, Shenghai Wu.Methodology: Xiaozi Wen, Qiong Chen, Huali Yin, Xianjun Wang.Project administration: Qiong Chen, Huali Yin, Xiaozi Wen.Resources: Qiong Chen, Huali Yin, Xianjun Wang, Shenghai Wu.Software: Qiong Chen, Huali Yin, Xianjun Wang, Shenghai Wu.Supervision: Qiong Chen, Huali Yin, Xianjun Wang.Validation: Xiaozi Wen, Qiong Chen, Huali Yin, Xianjun Wang.Visualization: Xiaozi Wen, Qiong Chen, Huali Yin, Xianjun Wang.Writing – original draft: Xiaozi Wen, Qiong Chen, Huali Yin, Xianjun Wang.Writing – review & editing: Xiaozi Wen, Qiong Chen, Xianjun Wang.


The aim of this study was to describe the pharmacokinetics of ceftolozane-tazobactam in plasma and cerebrospinal fluid (CSF) of infected critically ill patients. In a prospective observational study, critically ill patients (≥18 years) with an indwelling external ventricular drain received a single intravenous dose of 3.0 g ceftolozane-tazobactam. Serial plasma and CSF samples were collected for measurement of unbound ceftolozane and tazobactam concentration by liquid chromatography. fAUC0-inf) in the CSF and plasma were 30 (19 to 128) h·mg/liter and 323 (183 to 414) h·mg/liter, respectively. For tazobactam, these values were 5.6 (2 to 24) h·mg/liter and 52 (36 to 80) h·mg/liter, respectively. Mean ± standard deviation (SD) CSF penetration ratios were 0.2 ± 0.2 and 0.2 ± 0.26 for ceftolozane and tazobactam, respectively. With the regimen of 3.0 g every 8 h, a probability of target attainment (PTA) of ≥0.9 for 40% fT>MIC in the CSF was possible only when MICs were ≤0.25 mg/liter. The CSF cumulative fractional response for Pseudomonas aeruginosa-susceptible MIC distribution was 73%. The tazobactam PTA for the minimal suggested exposure of 20% fT>1 mg/liter was 12%. The current maximal dose of ceftolozane-tazobactam (3.0 g every 8 h) does not provide adequate CSF exposure for treatment of Gram-negative meningitis or ventriculitis unless the MIC for the causative pathogen is very low (≤0.25 mg/liter).The aim of this study was to describe the pharmacokinetics of ceftolozane-tazobactam in plasma and cerebrospinal fluid (CSF) of infected critically ill patients. In a prospective observational study, critically ill patients (≥18 years) with an indwelling external ventricular drain received a single intravenous dose of 3.0 g ceftolozane-tazobactam. Serial plasma and CSF samples were collected for measurement of unbound ceftolozane and tazobactam concentration by liquid chromatography. Unbound concentration-time data were modeled in R using Pmetrics. Dosing simulations were performed using the final model. A three-compartment model adequately described the data from 10 patients. For ceftolozane, the median (interquartile range [IQR]) area under the unbound concentration-time curve from time zero to infinity ( Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter aerogenes) exhibit relatively high MICs within the susceptibility range  in the treatment of critically ill patients with an indwelling external ventricular drain (EVD).The study enrolled 10 patients; plasma) and body weight on volumes of distribution of the central compartment (cV) and the CSF compartment (VCSF) were the only covariates that improved model fit  for both ceftolozane and tazobactam. CLplasma was linearly related to creatinine clearance normalized to the study population average as CLplasma = CL × (CLCRurinary/129), where CL is the typical value of clearance and CLCRurinary is measured urinary creatinine clearance. Similarly, for both drugs, cV and VCSF were linearly related to body weight (wt) normalized by the study population average as cV = cV × (wt/74) and VCSFcompartment = VCSF × (wt/74), where cV and VCSF are typical values. Residual error or uncertainty associated with the assay (standard deviation [SD] of observations/concentrations) was best described by first-degree polynomial functions: SD = 0.5 × [Obs] + 0.01 and error = 0.5 × [Obs] + 0.1 for plasma and CSF observations (concentrations) [Obs]. Residual unexplained source of variability (error) was described by error = (SD2 + 0.8)0.5. Parameter estimates for the final models are given in n = 1,000) are given in The schematic representation of the final structural model is given in the supplemental material . Creatinine clearance on plasma clearance (CLfAUC0–inf) in the CSF was 30 (19 to 128) h·mg/liter, more than 10-fold lower than the corresponding plasma value of 323 (183 to 414) h·mg/liter. For tazobactam, the median (IQR) fAUC0–inf in the CSF was 5.6 (2 to 24) h·mg/liter compared to a ∼10-fold higher value in plasma, 52 (36 to 80) h·mg/liter. The CSF exposure for those patients with confirmed/suspected CNS infection compared to those with other sites of infection appeared higher for both ceftolozane and tazobactam . The CSF penetration ratios estimated from the CSF-to-plasma fAUC0–inf ratio were highly variable but comparable for ceftolozane and tazobactam, with a mean ± SD of 0.2 ± 0.2 and 0.2 ± 0.26, respectively .Relative to plasma, the overall exposure in the CSF was low for both ceftolozane and tazobactam . For cefzobactam . Unpaireectively . Similarnfection  and C; hfT>MIC targets for ceftolozane in the CSF, unlike the high probability for plasma exposure (fT>MIC targets was attained in the CSF only when MICs were ≤0.25 mg/liter. The more conservative target of 100% fT>MIC was not attained even for the lowest MIC in the EUCAST distribution (0.032 mg/liter). A 24-h continuous infusion of 9.0 g with an initial 3.0-g loading did not improve the % fT>MIC in the CSF, resulting in a similar rate of target attainment with the intermittent regimen. Similar to ceftolozane, the probability of attaining previously proposed exposure targets for tazobactam was also extremely low in the CSF  and that lipid solubility is a major determinant of antibiotic penetration across the blood-brain barrier (BBB) via passive diffusion , 11.It is worth noting that in the present study, half of the participants had presumed or confirmed ventriculitis; this is distinct from meningitis patients , where CSF concentrations of beta-lactam antibiotics are more likely to be increased because inflamed meninges open the tight junctions, which likely enable increased passive drug diffusion into CSF . Given c0–inf, unlike most previous studies of other beta-lactam antibiotics, where either concentration ratio or total AUC ratio was used. Penetration ratios estimated with fAUC for other beta-lactams appear consistent with our findings, for example, 0.26 ± 0.12 for cefotaxime  and tazobactam (0.2 ± 0.26) from unbound plasma and CSF AUCfotaxime . The usefACU0–inf in those patients with presumed/confirmed CNS infections , fosfomycin (4 g q6h), and rifampin (600 mg q6h). The ceftolozane-tazobactam MIC was 3 mg/liter (Etest), for which, according to our dosing simulations  was nearly zero for the simulated dosing regimens. Similarly, the probability of attaining 100% fT>4 mg/liter was zero, which means that the constant concentration of 4 mg/liter used during in vitro susceptibility testing is not achieved in vivo. In other words, in vitro susceptibility results may not relate to in vivo susceptibility within the CSF. Therefore, ceftolozane-tazobactam MIC values should be carefully interpreted in the context that in vitro susceptibility does not guarantee clinical outcomes in CNS infections in particular.Of note, simulated tazobactam exposures in CSF were extremely low even after a tazobactam dose of 1.5 g q8h . Althougro study  examininThe interpretation of the current data is also subjected to the limitations of this study. An important limitation is that the pharmacokinetics (PK) in the CSF could be variably affected by the presence of EVD. In shunt CSF, flow rates are variable between patients and within a patient and may not relate to the amount of CSF produced . This woin vitro MIC testing; therefore, the susceptibility of even low-MIC pathogens causing CSF infection remains questionable. Future research should study potential solutions, including whether higher doses can safely be administered via intravenous infusion to achieve adequate CSF exposure, whether alternative routes of administration, such as intrathecal/intraventricular administration, are required, or if synergistic/additive combination with other antibiotics enables the use of standard ceftolozane-tazobactam doses of known safety in the treatment of CNS infections.In conclusion, the current maximal dose of ceftolozane-tazobactam (3.0 g q8h) does not provide adequate CSF exposure of ceftolozane unless the MIC for the causative pathogen is very low (≤0.25 mg/liter). However, tazobactam exposures remain far lower than the fixed 4-mg/liter concentration used for This prospective observational pharmacokinetic study was conducted at the University of Queensland Centre for Clinical Research, Brisbane, Australia. Patients were recruited from the quaternary referral intensive care unit (ICU) of the Royal Brisbane and Women’s Hospital (RBWH). The University of Queensland (2017000698) and the RBWH (HREC/17/QRBW/117) human research ethics committees granted ethics approval.Adult patients (≥18 years) admitted to the ICU of the RBWH were enrolled if they had an indwelling external ventricular drain (EVD) and informed consent was obtained. Patients were excluded if they had a history of allergy to penicillins and/or cephalosporins, they were receiving renal replacement therapy or had estimated creatinine clearance of <10 ml/min, and were receiving piperacillin-tazobactam or had received it in the 7 days before the time of screening.All patients received a single dose of 3.0 g ceftolozane-tazobactam (2:1 ratio) administered via intravenous infusion over 1 h. Arterial blood samples (1 to 2 ml) were collected first, prior to dose administration, and serially after the dose at 15 min, 45 min, 75 min (after 15 min of line flushing), 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, and 24 h. CSF samples (0.5 to 1 ml) were simultaneously collected from the EVD prior to dose administration at 2 h, 4 h, 6 h, 8 h, and 24 h. The drip chamber of the EVD system was emptied about 30 min before the first CSF sample collection and after each sample collection. In addition, the volume of CSF drainage during the 8 h of sampling was recorded.The Research Electronic Data Capture (REDCap) web application was used to develop an electronic case report form and collect clinical data. These data included patient demographics; physical examination, including vital signs; ICU and hospital admission and discharge dates and times; Acute Physiology and Chronic Health Evaluation II (APACHE II) score; sequential organ failure assessment (SOFA) score at ICU admission; presence of mechanical ventilation; renal function markers (serum creatinine concentration and urinary creatinine clearance); liver laboratory test results ; medication list on days of sampling; antibiotic data, including type, dose, dosing interval, duration of infusion, and other antibiotics administered on the day of sampling; and infection data .18 Ultra IBD, 100- by 2.1-mm, 3-μm column  operated at room temperature. Mobile phase A was 0.1% (vol/vol) formic acid in 10 mM ammonium formate, and mobile phase B was 100% acetonitrile with 0.1% (vol/vol) formic acid. The mobile phase was delivered with a gradient from 15% to 50% B at a flow rate of 0.3 ml/min for a 5-min run time and produced a backpressure of approximately 2,800 lb/in2. Ceftolozane was monitored by positive mode electrospray at multiple reaction monitoring (MRM) of 667.00→199.15. Labeled cefazolin was monitored in positive mode at 457.85→326.05. Tazobactam and sulbactam were monitored by negative-mode electrospray at MRMs of 299.20→138.00 and 232.20→140.00, respectively. The method met U.S. FDA validation criteria; between-run and within-run precision and accuracy were within 10%.An ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed on a Shimadzu Nexera2 UHPLC system coupled to a Shimadzu 8050 triple-quadrupole mass spectrometer  for unbound ceftolozane and tazobactam measurement. Test samples were assayed alongside matrix-matched calibrators and quality controls, and batches were subjected to acceptance criteria . Sample preparation involved isolation of free (non-protein-bound) fractions in plasma and CSF by ultrafiltration at 37°C using Centrifree devices  prior to analysis. Sample (10 μl) was spiked with phosphate-buffered saline (pH 7.4) and internal standard (sulbactam and stable isotope-labeled cefazolin) and treated with acetonitrile. The stationary phase was performed in a C2 + λ2)0.5 and error = SD × γ, where λ and γ are process noises associated with the observations. In addition, error associated with assay was modeled as a second-degree polynomial function of the SD of measurements.Pmetrics version 1.5.2, a package for R, was used for pharmacometric analysis based on the nonparametric adaptive grid algorithm (NPAG). Two- and three-compartment structural models were initially tested with linear elimination from the central compartments and linear and/or Michaelis-Menten distribution from the central to a peripheral/CSF compartment(s). An additive or multiplicative error model available within Pmetrics was tested with each of the structural models. The error models are based on the standard deviation (SD) of observations that take the form error = 2/(SD)2]/N − Σ(predicted-observed)/SD/N, where N is the number of observations/predictions), and objective functions, including the log likelihood ratio (LLR) test for the nested models, Akaike information criterion (AIC), and Bayesian information criterion (BIC). The LLR chi-square test was used for statistical comparison of nested models .Diagnostic plots included observed versus population or individual predicted concentrations, visual predictive check plots, and normalized prediction distribution errors (NPDE) versus time or observation plots. Statistical comparison of modes was based on the regression coefficient of observed versus predicted concentrations, bias [defined as the mean weighted error of predicted minus observed concentrations, i.e., Σ(predicted − observed/SD)/n = 1,000) were performed in R. The simulator in Pmetrics draws random samples repeatedly from a joint parameter probability distribution from NPAG. From each sampled set of parameter values, observations are calculated based on a predefined template of the input, covariates, and output measurement times. Simulations were performed for ceftolozane-tazobactam (2:1 ratio) regimens of 3.0-g intermittent infusion (over 1 h) q8h, 4.5 g q8h, 3.0-g loading dose over 1 h followed by 9.0-g continuous infusion over 24 h, and 4.5-g loading dose followed by 13.5-g continuous infusion over 24 h. Dosing simulations were performed up to steady state from 0 to 144 h.Monte Carlo dosing simulation  (fT>2 mg/liter (fT>4 mg/liter to assess exposure relative to the in vitro susceptibility testing condition of fixed 4-mg/liter tazobactam concentration (The probability of target attainment (PTA) was estimated primarily for ceftolozane PK/pharmacodynamic (PD) exposure targets of 40% fT>MIC , 20 and g/liter)  and 50% mg/liter  and 100%ntration . PTA wasP. aeruginosa EUCAST MIC distributions (accessed 27 May 2020) within the susceptible category (≤4 mg/liter). A CFR value of ≥85% was considered acceptable. The following equation was used for CFR calculation.i is MIC category ranging from 0.125 to 4 mg/liter, PTAi is the PTA at each MIC category, and iF is the fraction of the bacterial population at each MIC category.Cumulative fractional response (CFR) was estimated for ceftolozane, using t test comparison of CSF penetration ratio between patients with CNS infection versus those without.In addition to inbuilt tools within Pmetrics for R, GraphPad Prism  was used to generate plots from simulation outputs and perform Welch’s


Pusa hispida), 8 hooded seals (Cystophora cristata), 21 harp seals (Pagophilus groenlandicus), and 29 grey seals  were tested for parasites of the Sarcocystidae family including Toxoplasma gondii, Sarcocystis spp., and Neospora spp. using nested PCR followed by Sanger sequencing. Toxoplasma gondii DNA was present in 26% of ringed seals, 63% of hooded seals, 57% of harp seals, and 31% of grey seals. Sarcocystis sp. DNA was found in 9% of ringed seals, 13% of hooded seals, 14% of harp seals, and 4% of grey seals, while N. caninum-like DNA was present in 26% of ringed seals. While it is unclear how pinnipeds may become infected with these protozoans, horizontal transmission is most likely. However, one harp seal pup (4 days old) was PCR-positive for T. gondii, suggesting vertical transmission may also occur. Phylogenetic analysis of the 18S gene region indicates that Sarcocystis sp. in these seals belongs to a unique genotype. Furthermore, this study represents a new host report for T. gondii in harp seals, a new host and geographic report for N. caninum-like parasites in ringed seals, and four new hosts and geographic reports for Sarcocystis sp. These results demonstrate that parasites of the Sarcocystidae family are prevalent in northern and eastern Canadian seals. While the zoonotic potential of Sarcocystis sp. and the N. caninum-like parasite are unclear, consumption of raw or undercooked seal meat or organ tissues pose a risk of T. gondii infection to consumers.Zoonotic parasites of seals that are harvested for food may pose a health risk when seal meat or organ tissues of infected animals are eaten raw or undercooked. In this study, 124 tissue samples from 81 seals, comprising four species, were collected from northern and eastern Canada. Tissues from 23 ringed seals ( •Toxoplasma, Sarcocystis and Neospora.Tissues from ringed, hooded, harp and grey seals in Canada were PCR-positive for •T. gondii to consumers.Raw or undercooked seal meat may pose a risk for zoonotic transmission of •Sarcocystis sp. and the Neospora caninum-like parasite is unknown.The risk for zoonotic transmission of  In addition to subsistence harvests, some seal species are also harvested commercially in a government regulated sustainable harvest in eastern Canada , and seaOdobenus rosmarus), bearded seals (Erignathus barbutus), ringed seals (Pusa hispida), harbour seals (Phoca vitulina), hooded seals (Cystophora cristata), harp seals (Pagophilus groenlandicus), and grey seals . Most of these species are hunted for subsistence purposes by Inuit and others, but the latter three species are commercially hunted, with meat and by-products being sold to restaurants and exported. These pinnipeds have different distributions, abundance and, for some species, seasonal migrations which affect their availability to hunters. They also have different diets which affect their exposure to parasites and thus pose differential zoonotic risks to human consumers.Seven pinniped species are hunted in Canada, including: walrus  and in patients with neurological disorders (18%)  . Howeverrs (18%) .Toxoplasma, Sarcocystis, and Neospora infections in four species of seals that are harvested for food in northern and eastern Canada. Results from this study will aid in evaluating the risk of transmission of these parasites to humans through the consumption of seal meat or organ tissues.The objective of this study was to determine the prevalence of 22.1P. hispida, (n = 19), 12 young-of-the-year (YOY) or juvenile females (10 Age = 0 (YOY), 1 Age = 2, 1 Age = 4) and 7 YOY or juvenile males , were collected by Inuit hunters and sampled in 1993 and 1994 at Salluit, Nunavik, Quebec  , 19 adult females, one YOY male and one YOY female, and hooded seals, C. cristata, (n = 8), adult females only, were shot under scientific permit issued by Fisheries and Oceans Canada and sampled in 2005 from breeding ice floes located west of the Magdalen Islands  in the Gulf of St. Lawrence, Québec. Grey seals, H. grypus, (n = 29), 14 adult females and 15 adult males, were shot under scientific permit and sampled in 2012 from breeding colonies on Saddle Island (45°48′N 63°15′W) and Pictou Island (45°49′N 62°33′W), Nova Scotia.Ringed seals, 75°39′W) . In addiCanine teeth were extracted from lower jaws for age determination of ringed and grey seals only. Thin cross-sections of teeth were made and the number of dentinal annuli were counted with one growth layer group = one year of age. Hooded and grey seals were aged based on total length and sexual maturity (only adults are present on the breeding ice floes). Seals were classified as YOY, juvenile or adult as described in A total of 124 tissue samples were collected from 81 seals and included diaphragm (n = 53), brain (n = 28), heart muscle (n = 20), lung (n = 19) and skeletal muscle (n = 4). Tissue samples were stored at −20 °C.2.2Tissue samples were thawed, and 1 g subsample of each was divided into two 500 mg aliquots which were used for DNA extraction. The cell lysis protocol was adapted from g for 15 min at 12 °C. The supernatant was dispensed into a new 15 ml conical tube. An equal volume of chloroform/isoamyl-alcohol (24:1) was added, and the samples were placed on a revolver for 1 h at RT. Samples were then centrifuged as indicated above. The supernatant was collected and 2 vol of cold 100% ethanol were added to precipitate the DNA. Samples were stored overnight at 4 °C for complete precipitation.To each homogenized tissue sample, 625 μl of 5 M NaCl and 510 μl of cetyl trimethylammonium bromide (CTAB)/NaCl (10% CTAB in 0.7 M NaCl) were added. Samples were incubated at 65 °C for 15 min. An equal volume of phenol/chloroform/isoamyl-alcohol (25:24:1) was added to the sample, followed by a 1.5 h incubation at room temperature (RT) while mixing on a Revolver™ Rotator . The solution was then centrifuged at 3000×g for 20 min at 4 °C. An equal volume of 70% ethanol was added to wash the DNA pellet before centrifugation at 1000×g for 10 min at 4 °C. This step was repeated twice. The pellet was then transferred to a 1.5 ml LoBind tube  and air dried until translucent. The dry pellet was resuspended in 150 μl of EB Elution Buffer  at 50 °C for 4 h. The extracted DNA was stored at −20 °C.The precipitated DNA was pelleted at 3000×2.3Sarcocystis-specific primers were used to confirm Sarcocystis sp. All PCR reactions were performed with a total reaction volume of 25 μl containing 1 × concentration of a 5 × Green GoTaq Reaction Buffer, 2 mM of MgCl2, 200 μM of dNTPs, 0.625 U GoTaq Polymerase , 300 nM of each primer , 1 μl of template DNA, and UltraPure water . The DNA concentration, quantified using Nanodrop , was normalized to 500 ng per reaction. PCR reaction was performed using the Mastercycler Nexus X2 thermocycler  for all samples. Cycling conditions for all samples were: 95 °C for 2 min, 35 cycles of 94 °C for 30 s, 50–68 °C for 30 s, and 72 °C for 60 s, following by a final extension at 72 °C for 10 min, and final hold temperature of 10 °C. The annealing temperatures varied between primers and are listed in T. gondii oocysts kindly donated by Dr. J. P. Dubey, USDA. While positive controls were not available for Sarcocystis sp. or Neospora caninum, T. gondii positive control was used as a negative control for these parasites.The gene regions, primers, and their respective nucleotide sequences that were used in this study are listed in Positive samples as determined by gel electrophoresis were purified using either the QIAquick PCR purification kit or the QIAquick Gel Extraction kit  following manufacturer's instructions.2.4T. gondii, Sarcocystis spp. and Neospora spp., and trimmed to identical lengths of 441bp using BioEdit  as recommended by the manufacturer. Amplified sequence products were purified using Wizard MagneSil green  sequencing reaction clean-up system, and capillary electrophoresis was performed on a 3500 Genetic Analyser . Sequences were assembled, edited and aligned using SeqScape v3 software . Resulting consensus sequences were aligned with representative GenBank 18S sequence data from  BioEdit . Sequenc2.5The evolutionary history was inferred using the Maximum Likelihood method based on the Kimura 2-parameter model . The tre33.1Depending upon the seal species and collection method, different tissues were available for testing, including muscle, brain, heart, lung, and diaphragm . PCR wasToxoplasma gondii, Sarcocystis sp. and N. caninum-like DNA was detected in 40%, 9% and 7% of seals, respectively .ectively . AnalyseSarcocystis sp.-infected animals of known sex were female; however, 97% of the seals harvested from the Magdalen Islands were female and the sex of the seals from Inukjuak was unknown. All animals from the Salluit cohort were <5 years of age; of the 6 N. caninum-like positive animals, 4 (67%) were female and 2 (33%) were male, and 5 of the 6 were YOY and one was Age = 1. For T. gondii-infected animals, the sex was known for 31 of 32 animals, of which 19 (61%) were female and 12 (39%) were male, which included one 4-day-old male harp seal pup (Supplementary Table).All 3.2T. gondii, Sarcocystis sp., and N. caninum-like parasites in the seal populations differed by harvest location  from Salluit but not in ringed seals from Inukjuak, nor in any other seal species.The prevalence of location . Toxoplaocations . Sarcocyl muscle , and thi3.3T. gondii included diaphragm, brain, heart muscle, lung and skeletal muscle, in similar prevalences  of the parasite   . There w3.5Sarcocystis sp.-positive tissues had a single nucleotide polymorphism (SNP) that was distinct from known Sarcocystis spp. reference sequences archived in GenBank , but not ribbon seals (Histriophoca fasciata), in Canada and Alaska, USA   and Atlantic salmon  have recently been identified as T. gondii DNA-positive  pup  in one 4-day-old male harp seal pup (Para0262H). Vertical transmission may also have occurred in five ida) see . Muscle positive . Measuretra) pup . Furtherreported . Transmireported .N. caninum, Sarcocystis spp. and T. gondii and where infected wild and domestic canids and felids contaminate coastal environments (Canis latrans) are known to venture onto ice in coastal environments to scavenge and predate seals (Vulpes vulpes) and wolves (C. lupus) are infected with Sarcocystis spp. and N. caninum , northern fur seals , and California sea lions ] and phocids  ] . Sarcocy, Canada . SarcocyNeospora caninum or N. caninum-like or “Coccidia C″ were reported in otariids  and phocids , and spotted seals  undertake seasonal migrations to more southern waters. The types and numbers of tissues were also limited for some seals. Consequently, it is difficult to fully assess the risk to consumers except to state that these three parasites are present in Canadian pinnipeds and that further data are required to evaluate zoonotic risk.