For both wound healing and the formation of a fibrotic lesion, circulating monocytes enter the tissue and differentiate into fibroblast-like cells called fibrocytes and pro-fibrotic M2a macrophages, which together with fibroblasts form scar tissue. Monocytes can also differentiate into classically activated M1 macrophages and alternatively activated M2 macrophages. The proteases thrombin, which is activated during blood clotting, and tryptase, which is released by activated mast cells, potentiate fibroblast proliferation and fibrocyte differentiation, but their effect on macrophages is unknown. Here we report that thrombin, tryptase, and the protease trypsin bias human macrophage differentiation towards a pro-fibrotic M2a phenotype expressing high levels of galectin-3 from unpolarized monocytes, or from M1 and M2 macrophages, and that these effects appear to operate through protease-activated receptors. These results suggest that proteases can initiate scar tissue formation by affecting fibroblasts, fibrocytes, and macrophages. The failure of wounds to heal properly constitutes a major medical problem, with both acute and chronic wounds consuming treatment time and resources , 2. The One of the events preceding scar tissue formation in a healing wound is the clotting cascade, in which the protease thrombin cleaves fibrinogen to fibrin. Thrombin activity is upregulated immediately after wounding  and in fIn addition to differentiating into fibrocytes, monocytes can differentiate into classically-activated M1 macrophages or alternatively-activated M2 macrophages . M1 macrHuman M1, M2a, and Mreg macrophages, while morphologically similar, display different surface markers and secrete different cytokines . CD163 iIn this report, we show that trypsin, tryptase and thrombin bias populations of human monocytes, M1 macrophages, or M2 regulatory macrophages towards an M2a phenotype, suggesting an additional mechanism whereby blood clotting, and/or mast cell degranulation, releases and/or activates extracellular proteases to induce and/or potentiate wound healing and fibrosis.Tosyl phenylalanyl chloromethyl ketone (TPCK)-treated bovine trypsin  and human thrombin  were resuspended following the manufacturer’s instructions. Tryptase purified from human mast cells  was mixed with 15 kDa heparin from porcine stomach (Sigma) in a 1:10 molar ratio of tryptase to heparin immediately after thawing .Human blood was collected from volunteers who gave written consent and with specific approval from the Texas A&M University human subjects Institutional Review Board. PBMC were isolated and cultured as previously described  to diffeImages of cells were obtained with a Nikon D1X SLR camera  or a 10 MP USB camera  imager on a Nikon diaphot inverted tissue culture microscope (Nikon) with a 10x lens. The image analysis program CellProfiler  was usedNormal adult dermal fibroblasts  and PBMC  were cultured in 96 well plates in 200 μl SFM for 5 days. The media was removed, plates were dried, and were fixed and stained using Masson’s trichrome kit , following the manufacturer’s directions, but omitting the staining with Weigert’s hematoxylin. The plates were air dried, and staining intensity was measured by absorbance at 455 and 700 nm using a Synergy MX plate reader .Statistics were performed using Prism . Differences were assessed by two-tailed unpaired and two-tailed paired t-tests, where indicated. Paired t-tests were used to assess significance in highly variable experiments. Significance was defined by p<0.05.To determine if trypsin, tryptase, or thrombin influence the differentiation of monocytes into macrophages, we co-incubated PBMC and proteases for five days, and examined the expression of CCR7, CD163, CD206 and fibronectin on the macrophages. We used physiological concentrations of proteases that we previously observed to potentiate fibrocyte differentiation , 46. CCRTo determine if protease treatment changed the staining intensity of the macrophage marker immunohistochemistry, stained slides were further analyzed by image analysis that identified cells as macrophages, fibrocytes, or other cells and then measured the mean staining intensity of each cell . CompariTo determine if proteases also affect extracellular cytokine accumulation by cultured macrophages, conditioned media from human PBMC cultured for five days with 12.5 ng/ml of trypsin, tryptase, or thrombin were assayed by ELISA for IL-4, IL-13, IL-10, and IL-12. Trypsin, but not tryptase or thrombin, is predicted to cleave IL-4, IL-13, IL-10, and IL-12 (ExPASy peptide cutter). Compared to conditioned media from untreated control cells, trypsin, tryptase, and thrombin increased IL-4 and IL-10 accumulation in PBMC conditioned media, and tryptase increased IL-13 accumulation . IL-4 anM2a macrophages can differentiate not only from unpolarized monocytes, but also from other macrophage subsets . M2 macrTo determine if proteases also affect extracellular cytokine accumulation by cultured M2 macrophages, conditioned media from M2 macrophages cultured for two days with trypsin, tryptase, or thrombin were assayed by ELISA for IL-4, IL-13, IL-10, and IL-12. Tryptase and thrombin increased IL-4 accumulation . TrypsinM1 macrophages are associated with inflammatory immune responses to pathogens like bacteria and viruses . To deteTo determine if proteases also affect extracellular cytokine accumulation by cultured M1 macrophages, conditioned media from human M1 macrophages cultured for two days with trypsin, tryptase, or thrombin were assayed by ELISA for IL-4, IL-13, IL-10, and IL-12. Tryptase and thrombin increased IL-4 accumulation, while trypsin increased IL-13 accumulation , no protProteases induce fibrocyte differentiation through activation of protease-activated receptors -1 and -2 (PARs -1 and -2) . To deteTryptase and thrombin increased both CD163 and CD206 staining, while trypsin did not increase either. PAR-1, but not PAR-2, inhibitor returned CD163 and CD206 staining to control levels in the presence of thrombin. PAR-2, but not PAR-1, inhibitor returned CD163 and CD206 staining to control levels in the presence of tryptase. PAR1 and PAR2 agonist did not increase either CD163 or CD206 staining .Tryptase and thrombin decreased CCR7 staining, while both PAR-1 and PAR-2 inhibitors returned CCR7 staining to control levels in the presence of tryptase and thrombin. Neither trypsin, PAR-1 agonist, or PAR-2 agonist effected CCR7 staining .For fibrocytes, no treatment increased or decreased CCR7 or CD163 staining. Tryptase and thrombin increased CD206 staining, along with PAR-1 and PAR-2 agonists. PAR-1, but not PAR-2, inhibitor returned CD206 staining to control levels in the presence of thrombin. PAR-2, but not PAR-1, inhibitor returned CD206 to control levels in the presence of tryptase . Both trGalectin-3 potentiates fibrocyte differentiation, and galectin-3 binding protein inhibits fibrocyte differentiation . To deteCollagen is the primary protein component of scar tissue, and is secreted by both fibrocytes and fibroblasts . ProteasIn this report we show that physiological levels of trypsin, tryptase, and thrombin, in addition to acting as pro-fibrotic signals to fibrocytes  and fibrPhysiological levels of tryptase and thrombin  bias macM2a macrophages secrete increased IL-4 and IL-13. IL-4 and IL-13 potentiates fibrocyte differentiation  and collMast cell degranulation lowers the number of M2 macrophages in the local environment without increasing CD163 staining, as we observe . ActivatS1 Data(PZF)Click here for additional data file.S2 Data(PZF)Click here for additional data file.S3 Data(PZF)Click here for additional data file.


Multiple-type human papillomaviruses (HPV) infection presents a greater risk for persistence in asymptomatic individuals and may accelerate cancer development. To extend the scope of HPV types defined by probe-based assays, multiplexing deep sequencing of HPV L1, coupled with an HPV-QUEST genotyping server and a bioinformatic pipeline, was established and applied to survey the diversity of HPV genotypes among a subset of healthy men from the HPV in Men (HIM) Multinational Study. Twenty-one HPV genotypes (12 high-risk and 9 low-risk) were detected in the genital area from 18 asymptomatic individuals. A single HPV type, either HPV16, HPV6b or HPV83, was detected in 7 individuals, while coinfection by 2 to 5 high-risk and/or low-risk genotypes was identified in the other 11 participants. In two individuals studied for over one year, HPV16 persisted, while fluctuations of coinfecting genotypes occurred. HPV L1 regions were generally identical between query and reference sequences, although nonsynonymous and synonymous nucleotide polymorphisms of HPV16, 18, 31, 35h, 59, 70, 73, cand85, 6b, 62, 81, 83, cand89 or JEB2 L1 genotypes, mostly unidentified by linear array, were evident. Deep sequencing coupled with HPV-QUEST provides efficient and unambiguous classification of HPV genotypes in multiple-type HPV infection in host ecosystems. Human papillomaviruses (HPV) are globally ubiquitous DNA viruses found in mucosal and cutaneous anatomical sites and comprised by more than a hundred genotypes. Low-risk HPV genotypes are associated with genital warts, while high-risk HPV genotypes, in particular HPV16, cause a number of cancers in men and women ,4,5,6,7.Studies of multiple-type HPV infection by conventional probe-based assays are hampered by the limited scope of type-specific probes and/or cross-hybridization by probes for related HPV genomes ,28,29,30The study included 22 samples from 18 men from the United States (n = 15), Brazil (n = 1), or Mexico (n = 2), representing a subset of healthy males from the HPV in Men (HIM) Multinational Study recruited between 2005 and 2008 to examine the HPV prevalence in asymptomatic males ,35,36,37Nested PCR with PGMY  followedStandard operating procedures were followed at every step of PCR amplifications to prevent cross-contamination, and no template controls were always negative.Gel-purified amplicons were sequenced from adaptor A using a Genome Sequencer FLX  with Titanium chemistry (Roche) according to the manufacturer’s protocol in the University of Florida Interdisciplinary Center for Biotechnology Research . Multiplex deep sequencing was performed on HPV L1 libraries pooled at equal molar ratio at the bead recovery step, a critical modification as pooling samples at the DNA level produced skewed sequencing output (data not shown). The first 14 samples were pooled in one region of an 8-region PicoTiter plate to obtain a median (QR) of 1,562  raw reads per sample. To increase the sequence depth and accelerate the turnaround time in the ICBR Sequencing Core Facility, the second set of 11 samples, including 8 samples from 4 participants, 2 control cell lines, and 1 HPV16 molecular clone were sequenced in two regions of a 4-region PicoTiter plate to generate a median (QR) of 26,193  raw reads per sample . All rawThe bioinformatic pipeline for analysis of L1 deep sequences is outlined in To assess deep sequencing errors, 25,928 quality L1 deep sequences from a molecular clone of HPV16 were aligned to an HPV16 reference sequence (GI│333031) using MUSCLE in Geneious package, followed by BioEdit . Errors were evaluated using an in-house code. Overall sequencing error rate was 0.46%  with 0.09%, 0.03%, 0.08% and 0.26% for transitions, transversions, and insertions or deletions (indels), respectively. After correction of indels, rate of misincorporation was 0.12% , which is 18 times lower than 2%, the maximum dissimilarity within an HPV genotype ,53,54.Quality sequences were clustered at 3% pairwise distance  PV Specific Blastn [Consensus sequences were queried in HPV-QUEST, a custom HPV-genotyping server which collected 150 cutaneous and mucosal HPV reference sequences containing the L1 region obtained from National Center for Biotechnology Information (NCBI) Genebank , Los Alac Blastn  was usedc Blastn  was applHPV genotype infection and diversity were investigated in cross-section DNA samples from 18 asymptomatic participants. A total of 21 (12 high-risk and 9 low-risk) HPV genotypes, representing 9 species  were detected . Seven iA majority  of the cohort had exclusively high- or low-risk genotypes, including seven individuals  and S17) with high-risk HPVs16, 51, 52, 59, 70 and 73 genotypes, and five  with low-risk HPVs6b, 81, 83, 87 or cand89 genotypes . In contResults of the NGS/HPV-QUEST typing were concordant with LA genotyping for 10 high- or low-risk genotypes  in 8 individuals  . While tGenotypes defined by PaVE PV Specific Blastn were virtually identical to those classified by HPV-QUEST with exactly the same local identities to the same reference sequences used in both databases. Minor differences between the two genotyping tools, including HPVJEB2 typed by HPV-QUEST and classified as HPV72 by PaVE PV Specific Blastn, were due to different reference sequences used by the two programs . HPVJEB2Across the study group, L1 regions from a variety of genotypes were identical to reference sequences at the nucleotide level . In contrast, a number of L1 variants displayed nucleotide differences from the corresponding reference sequences in more than 77% (14 of 18) of individuals . Both syLongitudinal changes of HPV genotypes were evaluated in S15 and S16 based on the ability to generate amplicon libraries from a series of three samples collected at six-month intervals  . Similar+ HNSCC are emerging as a separate biological entity among all HNSCC. HPV-positive HNSCC are less likely to harbor p53 mutations, are more sensitive to radio/chemotherapy, and have a lower risk of recurrence and a better chance of survival than HPV-negative HNSCC [Much recent attention has highlighted the necessity of HPV genotyping in clinical pathology. An increasing line of evidence implicates HPV viral diversity and genotypes as substantial factors in cancer grade, response to therapy, recurrence, and a patient’s survival in HPV-related cancers, e.g., cervical cancers (CC) and head and neck squamous cell carcinomas (HNSCC) ,18,30,64ve HNSCC ,16,17. Pve HNSCC . 10-LiPA25 [10 for SPF10-LiPA25, and GP+ for HPV DNA chip and GP+ reverse line blot, resulting in large differences in sensitivity of detecting HPV infection and individual genotypes among the tests [HPV-genotyping methods vary considerably across laboratories, resulting in substantial disagreement among studies ,66,67. H0-LiPA25 ,69, HPV 0-LiPA25 , and GP+0-LiPA25  detect a0-LiPA25 ,64,72,73he tests ,27,28. With the rapid revolution in technology, NGS applied to HPV genotyping provides high sensitivity in capturing even low frequency types, reflecting unprecedented coverage of viral copies and almost no limit in the number of types detected in a sample by querying reference sequence databases ,74,75,76In this study, we applied nested PCR using PGMY, followed by GP+ primers, to generate HPV L1 amplicon libraries since PGMY/GP+ provided greater sensitivity and broader coverage of HPV genotypes compared to MY alone, PGMY alone, GP+ alone, or MY/GP+ ,47. OtheHPV genotyping of deep sequences relied mainly on querying to NCBI Blastn ,75. NCBISequencing allowed the identification of signature polymorphisms among L1 genotypes in more than 75% of individuals in this exploratory study with relatively small sample size, providing a framework to apply to future studies of HPV clearance, persistence, and super-infection by variants of the same HPV genotypes in a larger cohort. In addition, other regions of the HPV genome that may provide more robust molecular information can be incorporated into a deep sequencing platform to track genotypes over time or evaluate HPV gene expression . A high In the current study, deep sequencing was applied to define the range of HPV genotypes in asymptomatic individuals. Our data clearly demonstrate that healthy men can carry a range of high-risk and/or low-risk types HPV genotypes, including the vaccine types. Infection by multiple-type high-risk HPV, alone or in combination with low-risk genotypes, is a significant clinical issue for prognosis or treatment for a variety of HPV cancers ,16,17,18


Recently a new product category, derived from Antarctic krill, has been launched on the omega-3 nutriceutical market. Antarctic krill oil is marketed as demonstrating a greater ease of absorption due to higher phospholipid content, as being sourced through sustainable fisheries and being free of toxins and pollutants; however, limited data is available on the latter component. Persistent Organic Pollutants (POP) encompass a range of toxic, man-made contaminants that accumulate preferentially in marine ecosystems and in the lipid reserves of organisms. Extraction and concentration of fish oils therefore represents an inherent nutritional-toxicological conflict. This study aimed to provide the first quantitative comparison of the nutritional (EPA and DHA) versus the toxicological profiles of Antarctic krill oil products, relative to various fish oil categories available on the Australian market. Krill oil products were found to adhere closely to EPA and DHA manufacturer specifications and overall were ranked as containing intermediate levels of POP contaminants when compared to the other products analysed. Monitoring of the pollutant content of fish and krill oil products will become increasingly important with expanding regulatory specifications for chemical thresholds.Fish oil dietary supplements and complementary medicines are pitched to play a role of increasing strategic importance in meeting daily requirements of essential nutrients, such as long-chain (≥C Since early observations that Greenland Eskimos who subsisted on large amounts of fish suffered low levels of cardiovascular disease related mortality, epidemiological and experimental evidence has confidently shown the protective role of sufficient ω3 LC-PUFA intake against cardiovascular disease and certain types of cancer, e.g., [Fish are a nutrient-dense food source. The role of marine-derived, long-chain (LC) , a Euphausiid crustacean forming the basis of the Antarctic food web, has been marketed since 2002 and has recently become broadly available in Australia [A new product category has been launched on the omega-3 nutriceutical market and is currently gaining significant market share. A nutriceutical oil derived from Antarctic krill  krill oil; (ii) enriched (in terms of EPA + DHA) fish oil; (iii) nutriceutical formulations containing fish oil; and (iv) standard or budget grade 18:12 (EPA + DHA) fish oil . Productc.a. 0.03 g) oil samples were used for lipid analyses. Individual capsule or liquid oil samples were cut open and dissolved in CHCl3. A known aliquot of total lipid  was transferred into separate vials and made up to 1.5 mL of CHCl3.Pre-weighed  to produce fatty acid methyl esters (FAME). After cooling the mixture and addition of 1 mL of water, FAME were extracted (3×) with 4:1 hexane/dichloromethane. A C19 FAME internal injection standard was added prior to analysis by gas chromatography (GC) using a GC (Agilent Technologies 7890A) equipped with a Supelco Equity™-1 fused silica capillary column  [An aliquot of the total lipid extract was trans-methylated by addition of MeOH/HCl/CHClickness) . GC-massickness) . The relFor lipid class and FA profiling, commercial (Nuchek) and laboratory standards  of known composition were routinely analysed to both confirm component identifications and ensure data quality.Oil samples were analysed for chlorobenzenes (hexa- and penta-chlorobenzene); chlorinated pesticides; hexachlorocyclohexanes ; the dichlorodiphenyltrichloroethane (DDT) group ; toxaphene ; polychlorinated cyclodienes  and the individual compounds mirex and trifluralin. In addition, samples were analysed for the polychlorinated biphenyl (PCB) congeners, 18, 28, 31, 33, 37, 47, 52, 66, 74, 77, 81, 99, 101, 105, 114, 118, 122, 123, 126, 128, 138, 141, 149, 153, 156, 157, 167, 169, 170, 180, 183, 187, 189, 194, 206 and 209 (IUPAC numbers) and polychlorinated dibenzo-p-dioxin/furan (PCDD/F) congeners; 2,3,7,8-TCDD, 1,2,3,7,8-PeCDD, 1,2,3,4,7,8-HxCDD, 1,2,3,6,7,8-HxCDD, 1,2,3,7,8,9-HxCDD, 1,2,3,4,6,7,8-HpCDD, OCDD, 2,3,7,8-TCDF, 1,2,3,7,8/1,2,3,4,8-PeCDF, 2,3,4,7,8-PeCDF, 1,2,3,4,7,8/1,2,3,4,7,9-HxCDF, 1,2,3,6,7,8-HxCDF, 1,2,3,7,8,9-HxCDF, 2,3,4,6,7,8-HxCDF, 1,2,3,4,6,7,8-HpCDF, 1,2,3,4,7,8, 9-HpCDF and OCDF.2SO4, activated silica and potassium silicate; second column, single use Fluid Management Systems (FMS) silica column; third column, single use FMS activated carbon column). The sample portion containing PCDD/Fs and non-ortho PCBs was eluted from column 3 with toluene, reduced and exchanged to hexane before undergoing further clean-up by sulphuric acid coated silica column followed by potassium hydroxide coated silica column.The extraction and clean-up methods for POP have previously been described in full . In brieSamples for PCB and chlorinated pesticide analysis were extracted on a cold-column and cleaned by gel permeation chromatography, alumina and silica gel columns.13C-labelled isomers were used as internal standard for each group. Additionally, the recovery rates of the added internal standard compounds were determined.The isomer identification and quantification was carried out with HRGC/HRMS using a Hewlett-Packard 5890II (1990–2003) or 6890N (2003–2006) gas chromatograph coupled to an AutoSpec mass spectrometer . Resolution of mass spectrometer was >10,000 with electron ionization mass spectrometry in the selected ion monitoring mode (GC/EI-HRMS-SIM). Two SIM values were monitored for each isomer group. The added 13C-labelled isomer; (ii) the isotope ratio of the two monitored masses was within +20% of the theoretical value; (iii) the signal/noise was >3/1 for quantification; (iv) the recovery of the added 13C-labelled internal standards was within 40% to 120% and thereby in agreement with EU and US guidelines and official methods; (v) prior to each new series of samples the blank values of the complete clean-up and quantification procedures were determined. Clean-up of samples only commenced when a sufficiently low blank value was obtained. At least once a year the laboratory participates in an international laboratory inter-calibration exercise.The following quantification conditions were fulfilled for all data presented: (i) the retention time of the native compound was within three seconds of the corresponding Various regulatory bodies and food authorities have assessed the levels of chemicals that are safe for human consumption, based upon observed affect levels in animal models. The tolerable daily intake (TDI) refers to a threshold of a chemical which does not appear to carry an appreciable risk. In the current study we have used a variety of sources for our reference TDIs, namely Health Canada and the US EPA, The World Health Organisation and the International Panel on Chemical Safety (IPCS) as well as peer-reviewed literature.et al.’s 2005 re-evaluation of TEQ values [Toxicity equivalency factors express the toxicity of similar acting, planar, dioxin, furan and certain PCBs relative to the most potent congener 2,3,7,8-TCDD which is assigned a value of 1.0. The TEQ values applied in the current study refer to Van den Berg Q values .vs. 1500 mg observed) and DHA levels in Nature’s Way Kidsmart (133 mg specified vs. 95 mg observed) and Blackmores Pregnancy and Breastfeeding Gold (125 mg specified vs. 85 mg observed). It is noted, that for pure oil capsules it is possible to compensate for EPA and DHA batch variability through marginal capsule volume adjustments. This is however, less readily achievable for formulations, such as the latter two products, and uncontrollable for liquid formulations. A listing of all FA present at >0.5% of the total FA in each product analysed is shown in The majority of categories and brands of seafood oil supplements matched or exceeded manufacturer EPA and DHA specifications, with the exception of three brands which fell slightly below (~10%–30%) the manufacturer specifications . These rNone of the categories or products analysed in the current study, at their highest recommended dosage, came close to fulfilling tolerable daily intake (TDI) levels for any single analyte . DespiteMegaptera novaeangliae), found that the profiles of these predators closely mirrored the profiles of their principal prey, Antarctic krill. In the case of the krill oil products analysed in the current study, however, only trace (440 pg/g lipid or daily dose) levels were quantified in the Swisse krill oil brand which may indicate purification through the manufacturing process.Hexachlorocyclohexane (HCH) congeners did not feature prominently in any product profiles, possibly reflecting the slightly lower lipophilicity of this compound group. The DDT group included the highest concentration of any single compound, with 13 ng/g lipid p,p’-DDE detected in Nature’s Own 1000 mg standard fish oil product, equalling a maximum daily dose 120 ng of p,p’-DDE. Notably, only one krill oil formulation (Swisse) showed detectable levels of ∑DDT. p,p’-DDE has repeatedly been found to be one of the dominant congeners accumulating in Antarctic krill and their predators ,36,37,38Detectable levels of chlordanes were observed in only three products, namely Bio-Organics Super Liquid fish Oil (30 ng per maximum daily dose), Bioceuticals Omegasure fish oil (4.6 ng per maximum daily dose) and Nature’s Way Kidsmart (0.1 ng per daily dose). Similarly, endosulfan-I was only detected at trace levels (390 pg/g lipid) in BioOrganics Super Liquid fish oil.Toxaphene structures were not quantified in five of the eleven products due to loss of the analytes during clean-up. However, notable quantities were detected in Cenovis 1000 mg  and Bioceuticals Omegasure fish oil . Only trace levels of toxaphene were quantified in Nature’s Way Kidsmart and Blackmores Pregnancy and Breastfeeding formulation. These congeners were undetectable in Blackmores Joint formula and BioOrganics Super Liquid fish oil.Chlorobenzenes (penta- and hexa-) were quantified in eight of eleven products at levels ranging from 27–9900 pg/maximum daily dose. Antarctic krill products carried the highest levels of chlorobenzene contamination for both penta- and hexa- congeners. The higher levels of particularly HCB, in Antarctic krill oil is not surprising as this has repeatedly been shown to be the compound dominating POP profiles of the Antarctic sea-ice ecosystem food-web ,33,34. TPolychlorinated biphenyls (PCBs) were detected in all products at cumulative levels ranging from 0.01% TDI (Blackmores Joint formula) to 0.94% TDI (Blackmores 1000 mg). Krill oil products were at the lower end of the spectrum (0.034% and 0.015% TDI for Swisse and Norkrill krill oil respectively), as is expected, given the manufacturing applications of these compounds and the lower historical usage in the southern hemisphere.Dioxins and furans encompass a class of compounds which are not intentionally produced, but originate primarily through the manufacture of other chlorinated chemicals or combustion processes. Whilst the highest detected levels of any single dioxin or furan congener was 73.0 pg/g lipid of octachlorodibenzodioxin (OCDD) found in Bioceuticals Omegasure fish oil, only the krill oil products contained multiple detectable congeners. This is surprising given the low vapour pressure of dioxins and furans which predict long range atmospheric transport in association with particles. This in turn lowers their potential for effective transport to the Antarctic. Toxicity equivalencies (TEQ) are available for dioxins, furans and a sub-set of planar PCBs, and are calculated based upon their common mode of action. The single highest TEQ for any product analysed was obtained for Blackmores 1000 mg standard fish oil product which yielded a TEQ of 5.6 TEQ or 4.7% of the 120 TEQ TDI. Swisse Krill oil, however, also featured among the top five highest ranking TEQ products. Dioxins, furans and planar PCBs are among the POP compounds most effeciently removed by common fish oil cleaning processes . This fiThis study compared a range of readily available fish and krill oil dietary supplements for both their favourable long-chain omega-3 composition and content, as well as their persistent organic pollutant profiles. All products and categories adhered closely to manufacturer specifications and none exceeded chemical guideline thresholds. When krill oil was compared across categories to other fish oil products and formulations, it was the most expensive oil per 500 mg DHA + EPA and adhered to manufacturer EPA and DHA specifications. The two krill oil products were ranked as intermediate in terms of their levels of POP contaminants when compared overall to the remaining omega-3 nutriceutical products selected for this study, with distinct chemical profiles reflecting their geographical region of origin. This study is the first to provide quantitative evaluation of toxicological profiles of Antarctic krill products, an emerging nutriceutical category. It hereby balances consumer information with regard to marketing of krill oil on the basis of product chemical purity. Ongoing monitoring of the pollutant content of fish and krill oil products will become increasingly important as food authorities seek regulatory overview of this rapidly expanding industry.


Traumatic injury to the central nervous system (CNS) or the peripheral nervous system (PNS) triggers a cascade of events which culminate in a robust inflammatory reaction. The role played by inflammation in the course of degeneration and regeneration is not completely elucidated. While, in peripheral nerves, the inflammatory response is assumed to be essential for normal progression of Wallerian degeneration and regeneration, CNS trauma inflammation is often associated with poor recovery. In this review, we discuss key mechanisms that trigger the inflammatory reaction after nervous system trauma, emphasizing how inflammations in both CNS and PNS differ from each other, in terms of magnitude, cell types involved, and effector molecules. Knowledge of the precise mechanisms that elicit and maintain inflammation after CNS and PNS tissue trauma and their effect on axon degeneration and regeneration is crucial for the identification of possible pharmacological drugs that can positively affect the tissue regenerative capacity.  Wallerian degeneration (WD) is a multicomplex phenomenon occurring in the distal portion of injured nerves –3. The tβ, PDGF, EGF, BDNF, and oncomodulin) are secreted by neural and inflammatory cells in order to promote axon elongation . Wi. Wi26]. ongation , 34, 35.ongation . ConcomiContrary to previous belief, the CNS does not constitute an immunoprivileged system, since it shares many commonalities with other systems. Therefore, the immune response that follows a SCI has received increasing attention in recent decades, and it is becoming clear that inflammation exerts an important effect on the progress of degeneration and regeneration following a traumatic lesion . Immediaα, IL-33, uric acid, thioredoxin, and high mobility group box 1, which are present in the lesioned tissue milieu [Following an injury to the CNS, neutrophils can reach the injured parenchyma by activated endothelial cell signaling and/or by secreted molecules derived from microglia , 39. Mice milieu –42.β2 integrins  that interact with ICAM-1, expressed by endothelial cells, through chemokine receptors. Once neutrophils are arrested on the endothelial surface, they can transmigrate to the tissue through the endothelial cell junctions by interacting with PECAM-1, ICAM-1, VE-cadherin, JAMs, and CD99 [Neutrophils are the first cell line to respond to tissue damage. In mechanical injuries, neutrophils are recruited within a few hours after tissue damage, peaking at 1 to 3 days post-injury (dpi). The bactericidal function of these cells is well described in infections , but theand CD99 .β, IL-6, and tumor necrosis factor alpha (TNF-α) and chemokines such as macrophage inflammatory protein-1 (MIP-1), macrophage chemoattractant protein-1 (MCP-1), and IL-8 [γ/CCL9, KC/Gro-α/CXCL1, G-CSF, and MCP1/CCL2), which suggests an attempt to compensate for the lack of neutrophils by increasing the recruitment of macrophages to the injury site. Despite the elevated expression of the macrophage chemoattractants, the treatment did not increase the number of microglia/macrophages within the injured spinal cord [When activated, neutrophils secrete large amounts of proteolytic enzymes such as elastase and metalloproteinases (MMPs). These enzymes are able to act in different extracellular matrix substrates and the endothelial cells, damaging the endothelium and thus facilitating leukocyte migration through the injured tissue , 43, 44.and IL-8 . By inhiand IL-8  found a and IL-8  depletedand IL-8 , neutropand IL-8 . Stirlinand IL-8  also obsnal cord . Interesnal cord .2+-binding protein, acts as a neurotrophin and participates in inflammation-induced regeneration [Inflammation induced in nerves has been shown to increase the nerve regenerative capacity by enabling axons to grow and sprout collateral branches in response to neurotrophins; this is clear in the context of the optic nerve injury model. Oncomodulin (Ocm), a small Caneration , 47. Aftneration , 47. In neration . In spithi/CX3CR1low and Ly6Clow/CX3CR1hi refer to pro- and anti-inflammatory monocytes, respectively. These cells respond to a large variety of chemokines such as MCP1  and MCP3  and share the same surface receptor CCR2, inducing Ly6Chi recruitment, while Ly6Clow responds to CX3CL1, also known as fractalkine [α, which in turn activates a macrophage/microglia program inducing the release of molecules that kill neurons and oligodendrocytes, exacerbating tissue damage. These effects can be attenuated by administration of TNFR1, which sequesters TNF-α, reducing its availability to bind to TNF receptors [Microglia are SNC resident glial cells that have several distinct receptors such as cytokines and chemokines on their cell surface, which enable them to recognize different stimuli such as abnormal or unusual concentrations of molecules . Any dictalkine . In a stctalkine . Macrophctalkine . Macropheceptors . Furthereceptors . In a cleceptors  depletedγ (IFN-γ) activates macrophages and induces the production of proinflammatory cytokines  and cytotoxic mediators, while T helper 2 cytokine IL-4 drives the macrophage polarization to an anti-inflammatory phenotype by inhibiting the production of proinflammatory cytokines , increasing MHCII, and reducing the respiratory burst [γ and TLR signaling, and “alternatively” activated macrophages (M2), responsible for IL-4 and IL-13 signaling. The markers CD16, CD32, CD86, MHCII, and iNOS refer to M1 macrophages (proinflammatory), while Arginase-1 (mouse), CD163, CD204, CD206, YM1, and Fizz1 refer to M2 macrophages (anti-inflammatory) [Studies unrelated to the nervous system revealed that macrophages could be polarized into pro- and anti-inflammatory phenotypes based on the specific pathway that is activated. It is well established that T helper 1 cytokine (Th1) interferon-ry burst . These fmmatory) . After aAfter SCI, both populations are present in the injured tissue parenchyma from 3 to 7 days after injury . HoweverAfter SCI, there is a marked increase in the expression of CX3CR1 by macrophages. Importantly, the deficiency of this receptor improved neurological recovery and tissue sparing after SCI by modulating the microglia/macrophage phenotype, diminishing their neurotoxic effects . Of inteAs already mentioned, Yin and collaborators  showed tα and IL-1α, although only TNF-α proteins are found in detectable levels in the uninjured nerve [α, IL-1α, IL-1β, MCP-1, MIP-1, IL-10, TGF-β, and galectin-3 in a time-dependent manner [β and TNF-α are produced after TLRs-NFKb activation [ in vivo functional role of TLRs was elegantly demonstrated by Boivin et al. Taking advantage of TLR-2 and TLR-4 knockout mice, they observed impaired WD and axon regeneration in these deficient animals after sciatic nerve injury [Normally, uninjured peripheral nerves are composed of resident macrophages, fibroblasts, and Schwann cells, the PNS-wrapping glia. Taking into consideration all nucleated nonneuronal cells that are present in a naïve tissue, Schwann cells outnumber resident macrophages by about 10 times , 58. Mored nerve , 60. Cont manner , which st manner , 64. Thet manner . Schwannt manner , 67 and t manner . Althougt manner . For exat manner . Also, stivation . These fe injury . On the β. After nerve injury, IL-1β reaches maximum levels at 24 hours [β induces myelin collapse through a complex cascade involving phospholipase A2 (PLA2) and lysophosphatidylcholine (LPC) activation in Schwann cells [α, IL-1α, and MCP-1 can also increase PLA2 expression in both Schwann cells and macrophages, emphasizing the notion that all compact myelin that surrounds severed axons will be fully fragmented into ovoids by, at least, a sustained expression of PLA2-related factors . In addrefer to . Anotherrefer to , who dem In vitro experiments suggest that the half-life of NMNAT2 is less than 4 hours in transected neurites [Nerve injury-induced inflammation is a precisely orchestrated multicomplex reaction that involves a multitude of inflammatory mediators and cells , 8, 78. neurites . Therefo in vivo dynamics of macrophage polarization after nerve injury is still a matter of intense debate. Ydens and colleagues [ in vitro procedures, during in vivo situations macrophages are exposed to all types of stimuli that are disease/injury- and tissue-specific, probably leading to complex phenotypes and mixed populations of cells [Nerve injury induces recruitment, accumulation, proliferation, and activation of macrophages , 9, 84 . Althouglleagues  demonstrlleagues –91. Theslleagues , 93. Altof cells . Therefoof cells . Severalof cells . In addiof cells .Inflammation is a prerequisite for successful nerve regeneration in the PNS, as it serves, for example, to eradicate harmful myelin from the nerve microenvironment. However, coordination of pro- and anti-inflammatory signals during WD is crucial and must be tightly controlled to ensure successful axon regeneration.β and TNF-α) in the Gal-3 ko injured nerves [ in vitro phagocytic activity, compared to their WT counterparts [ in vitro proliferation of Schwann cells [Of particular interest to our laboratory is the role played by galectin-3 after PNS injury , 20. Orid nerves . Moreoved nerves , althougterparts , 98, Galterparts . For exann cells  and incrnn cells . The preAfter a traumatic lesion, the axon becomes separated into two segments: a proximal segment that remains in contact with the cell soma and a distal segment that becomes separated from the neuron cell body. The distal nerve stump undergoes a cascade of events called WD , 105, whβ, and its production regulates the generation of nerve-growth factor (NGF) by fibroblasts and Schwann cells [While the distal stump axon disintegrates and provides a permissive microenvironment for regeneration, it also generates signals that target the neuronal cell body, resulting in its change from transmitting to a growth-promoting phenotype. These changes reflect variations in the metabolic activity of neurons, which, as a result, start to produce substances that are important for axonal elongation, initiating the process of axon regeneration from the proximal stump . But, honn cells . Anothernn cells . After inn cells  and grownn cells , 128, amnn cells . Indeed,nn cells . Althougnn cells  and autonn cells .Nowadays it seems clear that the CNS and PNS respond differently to traumatic injuries . For exa


BRCA1 and BRCA2 genes, based on studies in western populations. This current study assessed potential predictive factors for BRCA mutation probability, in an Asian population.The National Comprehensive Cancer Network (NCCN) has proposed guidelines for the genetic testing of the A total of 359 breast cancer patients, who presented with either a family history (FH) of breast and/or ovarian cancer or early onset breast cancer, were accrued at the National Cancer Center Singapore (NCCS). The relationships between clinico-pathological features and mutational status were calculated using the Chi-squared test and binary logistic regression analysis.BRCA1 and/or BRCA2. BRCA1 mutations were more likely to be found in ER-negative than ER-positive breast cancer patients (P=0.01). Moreover, ER-negative patients with BRCA mutations were diagnosed at an earlier age . Similarly, triple-negative breast cancer (TNBC) patients were more likely to have BRCA1 mutations (P=0.001) and that these patients were diagnosed at a relatively younger age than non-TNBC patients . Our analysis has confirmed that ER-negative status, TNBC status and a FH of hereditary breast and ovarian cancer (HBOC) are strong factors predicting the likelihood of having BRCA mutations.Of 359 patients, 45 (12.5%) had deleterious or damaging missense mutations in BRCA genetic testing, particularly younger patients (<40 years) or those with a strong FH of HBOC, in Asian patients.Our study provides evidence that TNBC or ER-negative patients may benefit from  BRCA1 and BRCA2, which include specific criteria on the age at diagnosis of the patients and family members; the occurrence of breast, ovarian, pancreatic or prostate cancer in close relatives; and the diagnosis of triple-negative breast cancer (TNBC) [BRCA1 and BRCA2 mutations of up to 30% and 17% respectively [The National Comprehensive Cancer Network (NCCN) has recommended various guidelines for the genetic testing of ectively , with yoectively . Most ofBRCA1 and BRCA2 genes [Next-generation sequencing (NGS) techniques enable the mutation screening of a larger set of samples in parallel, in a cost effective and accurate manner ,7. RecenA2 genes ,9. NGS tA2 genes ,11.BRCA mutations in an Asian clinic-based population, using next-generation sequencing and Sanger sequencing. In addition, we evaluated the predictive value of ER-, PR- and HER2- receptor status, age at diagnosis, FH, and histological type for determining the likelihood of mutations in the BRCA1 and BRCA2 genes.Here, we studied 359 breast cancer patients to determine the prevalence of Peripheral blood samples were obtained from 359 breast cancer patients attending a risk assessment clinic at the National Cancer Centre Singapore (NCCS). Subjects were eligible if they had a FH of breast and/or ovarian cancer in first- and/or second-degree relatives (n = 176), or if they had early-onset breast cancer in the absence of FH (≤40 years of age) (n = 183). Patients were accrued from 2002 till 2013. Samples from two earlier studies  were also included in this current study ,13. Of tBRCA1 and BRCA2 genes, to predict damaging mutations and to identify driver/passenger mutations. Frameshift and nonsense mutations were considered to be deleterious.BRCA1 and BRCA2 genes was performed as described previously [Sanger sequencing of the eviously , using tMore recent DNA samples were sequenced by next-generation sequencing, either by SureSelect capture  followed by sequencing on the Illumina MiSeq platform, or SeqCap EZ capture  with sequencing on the Illumina HiSeq platform.For samples sequenced by NGS, reads were aligned to the UCSC human reference genome (hg 19) using the BWA aligner (version 0.5.6). Variant calling was done using the GATK Unified Genotyper , and CRIhttp://research.nhgri.nih.gov/bic/), HGMD (http://www.hgmd.org/) and ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/) databases, and were regarded as ‘pathogenic’ if classified as such in two or more databases. All deleterious or pathogenic mutations detected were confirmed by re-sequencing the samples by conventional Sanger sequencing, as described above.All mutations identified from Sanger sequencing or NGS were annotated using the ANNOVAR tool, which provides tools such as SIFT, PolyPhen- II HDIV, PolyPhen—II HVAR, LRT and Mutation Taster to predict the effect of amino acid substitution for each missense mutation. Every missense mutation was scored as damaging or benign with each of the five prediction tools. If the missense mutation was scored as damaging by three or more of the prediction tools, the mutation was classified as a ‘Damaging’ mutation and if less than three, the mutation was classified as ‘Benign’. BRCA1 and SALSA MLPA P045-BRCA2 test kits, and validated using the MLPA P087 and P077 confirmation kits , respectively. The MLPA analyses were done by DNA fragment analysis on the ABI 3130 Genetic Analyzer and comparative analysis of samples using the Coffalyser freeware v.131123.1303 .All DNA samples were screened for large genomic rearrangements by MLPA using the SALSA MLPA P002-C2 BRCA mutation status. Binary logistic regression analysis was used to estimate the predictive effects of the significantly associated factors for predicting the probability of BRCA mutations. P-values of <0.05 were considered statistically significant.Statistical analysis was done using SPSS version 18.0.2 . The non-parametric test, i.e., Mann-Whitney U-test was used to compare the median age of the carriers and non-carriers. The Fisher’s exact test was used to determine significant associations between clinico-pathologic features and the BRCA1 and BRCA2 are listed in Deleterious mutations detected in BRCA1 and/or the BRCA2 genes. One patient (case 79) had two deleterious mutations, a BRCA2 nonsense  and a BRCA1 splice-site error (IVS7-15del10) .Of 359 patients, 45 (12.5%) had deleterious or damaging missense mutations in the 15del10)  Tables. BRCA1 mutations, including one frameshift, one nonsense and one large genomic rearrangement  although not statistically significant.BRCA mutation carriers were more likely to have a FH of HBOC than non-carriers (39.4% vs 9.2%). Conversely, BRCA carriers were less likely to have early-onset breast cancer in the absence of FH as compared to non-carriers (21.2% vs 54%).Among 359 patients, 43 (12%) had a FH of HBOC, 132 (37%) had a FH of breast cancer, 1 (0.3%) had a FH of ovarian cancer and 183 (50.9%) were early-onset breast cancer patients without a FH . BRCA muBRCA mutations and none of the medullary cases had BRCA mutations. In patients with IDC, the percentage of BRCA mutation carriers was higher at 57.6%, as compared to other histological types of breast cancer.The most common histological type of breast cancer in our study was infiltrating ductal carcinoma (IDC), at 72.2%, followed by infiltrating lobular carcinoma (ILC) (3.3%) and medullary cancer types (3.3%) . Only 1 BRCA mutation carriers, were likely to be ER-Negative than non-carriers (50% vs 25.9%). All BRCA mutation carriers with known Her2 status had HER2 negative tumors. Of 206 patients with known ER, PR and HER2 status, 13.6% were TNBC patients. Among our 28 TNBC patients, eight (40%) were BRCA mutation carriers.The percentages of ER-positive and ER-negative patients were 72% (202/281) and 28% (79/281) respectively. BRCA1 mutation carriers  were more likely to have BRCA1 mutations than ER-positive patients (5/202) .There was a significant association of ER-negativity with  = 0.01, ; howeverBRCA1 carriers and TNBC status  than non-TNBC patients (5/178) .Similarly there was a strong association between = 0.001, . TNBC paBRCA mutations was significantly younger than for ER-positive patients . When stratified by BRCA1 and BRCA2 mutational status independently, age at diagnosis for ER-negative patients with BRCA1 and BRCA2 mutations was significantly younger than for ER-positive patients  (The median age at diagnosis for ER-positive and ER-negative patients was 40 years and 39 years respectively . In addictively) .vs 40) . When stratified by BRCA1 or BRCA2 mutational status independently, age at diagnosis for TNBC patients with BRCA1 and BRCA2 mutations was significantly younger than for non-TNBC patients .The median age at diagnosis for TNBC patients was younger than for non-TNBC patients although not statistically significant (38 vs 40) . The medvs 11.8%, P = 0.035). However, there was no statistical difference between the TNBC and non-TNBC patients for patients with other FH.Furthermore, the percentage of TNBC patients with a FH of HBOC was higher than for non-TNBC , ER-negative status and a FH of HBOC were the strongest predictors for BRCA1 or BRCA2 mutations. The likelihood of patients with HBOC having BRCA1/2 mutations was higher than for other patients . The odds of having a BRCA1 or BRCA2 mutation in ER-positive patients was 0.390 times  less than ER-negative patients . Neither of the beta coefficients of both factors exceeded the absolute constant value (1.890), indicating that a single factor was insufficient to predict the mutation status. Both ER negative status and a FH of HBOC are required to predict the likelihood of having a BRCA1/2 mutation.BRCA1/2 mutations. However, no statistical significance was found (data not shown).Similar analyses were performed to investigate the potential contribution of PR status, as a predictive factor, to predict the likelihood of having BRCA1 and BRCA2 mutations carriers in TNBC patients . The odds of TNBC patients being diagnosed with BRCA1/2 mutations was 4.651 higher compared to non-TNBC patients . Similarly, neither of the beta coefficients of both factors exceeded the absolute constant value (2.835), indicating that a single factor was insufficient to predict the mutation status. Thus, both TNBC status and a FH of HBOC are required to predict the probability of having BRCA1/2 mutations.A similar analysis was done to evaluate the potential predictive factors for patients . The anaBRCA mutations as well as damaging missense mutations , with an increase in ER-positive breast cancers with increasing age [BRCA1 or BRCA2 mutations were significantly younger than ER-positive patients.Patients with sing age . Our datBRCA1 and BRCA2 mutations, respectively. A higher frequency of BRCA1 mutations (20.9%) as compared to BRCA2 mutations (3.6%) was also observed in another study on TNBC patients from Malaysia [et al has reported that among TNBC patients, the proportion of BRCA1 and BRCA2 carriers ranged from 9 to 100% and 2 to 12%, respectively [In our cohort, 14% (28/206) of our patients were TNBC, of which approximately 21.4% (6/28) and 7.1% (2/28) had Malaysia . A literectively .BRCA1 mutation carriers, 37.5% (6/16) were TNBC patients; whilst among our BRCA2 carriers, 10.5% (2/19) were TNBC patients. This frequency of BRCA mutations in our TNBC patients is slightly lower than that reported by Peshkin et al (2010) of between 42% to 100% and 14% to 35%, for BRCA1 and BRCA2 mutations respectively [Among our ectively .BRCA1/2 mutations was 3.164 , highlighting the importance of FH when estimating BRCA mutations prevalence. This is consistent with another study from the US that reported that TNBC patients with a FH of breast cancer or ovarian cancer had a higher probability of having BRCA mutations as compared to those without any FH of breast cancer or ovarian cancer  [Our logistic regression analyses indicated that the odds ratio of TNBC patients with HBOC having ctively) .BRCA mutation testing. Recently, a Korean study demonstrated that TNBC patients are more likely to be diagnosed at a younger age than non-TNBC patients in the cohort (42 vs 44.1) although the association was not statistically significant [vs 34.6 for BRCA1 and 51.5 vs 44.0 for BRCA2). Our data, however, are in contrast to these findings. We showed that the median age at diagnosis for TNBC patients with either BRCA1 or BRCA2 is younger than for non-TNBC patients (38 vs 46 for BRCA1 and 38.5 vs 48 for BRCA2), suggesting that BRCA1- and BRCA2-associated breast cancer is most likely early-onset. A study from Malaysia showed that TNBC patients aged below 35 years had a higher prevalence of BRCA1 and BRCA2 mutations compared to non-TNBC patients (28% vs 9.9%) [The NCCN guidelines have proposed the inclusion of TNBC patients aged 60 years or younger for nificant . Neverthvs 9.9%) . HoweverBRCA mutations was higher compared to non-TNBC patients, and the inclusion of additional criteria like a FH of HBOC may increase the probability of identifying BRCA1/2 mutations. A study from Malaysia showed an improvement in the sensitivity and specificity of the Manchester scoring system with the combination of negative ER status, FH and TNBC status [Collectively, and confirming previous findings in western populations, our results showed that the likelihood of TNBC patients being diagnosed with C status .BRCA mutation carriers in our cohort, are ER-negative. We also found that 29% (8/28) of TNBC patients are BRCA mutation carriers, with the majority being BRCA1 mutation carriers. In addition, we have shown that our TNBC patients with either BRCA1 or BRCA2 mutations were diagnosed at an earlier age. The discovery of the predictive factors, ER-negative status, TNBC status and HBOC, in our study, warrants confirmation in additional Asian populations.In conclusion, our data showed that almost half of our S1 Fig# Filtration criteria is explained in the methods section.* Computational algorithms used were SIFT, Polyphen-II HDIV, Polyphen-II HVAR, LRT and Mutational Taster; (TIF)Click here for additional data file.S2 FigA) Gel photo of PCR products obtained from the amplification of a 563bp-target region from the sample FH42 and control cDNA template; and a sequencing chromatogram of the 272bp-band observed from FH42. (B and C) Changes in mRNA sequence brought about by the deletion of 291bp in FH42 and its corresponding amino acid sequence.(TIF)Click here for additional data file.S1 TableC, Chinese; I, Indian; B, Burmese; M, Malay; BC, Breast Cancer; OC, Ovarian Cancer; PC, Pancreatic Cancer; IDC, Invasive Ductal Carcinoma; D&L, Mixed Ductal and lobular; ILC, Invasive Lobular Carcinoma; HR, Hormone Receptor; ER, Estrogen Receptor; PR, Progesterone Receptor; TNBC, Triple Negative Breast Cancer; Unk, Unknown; Fs, Frameshift; Del, Deletion of exon; Dup, Duplication of exon; N, Nonsense; SE, Splice-site Error; Ref, References.(XLSX)Click here for additional data file.S2 TableC, Chinese; I, Indian; B, Burmese; M, Malay; BC, Breast Cancer; OC, Ovarian Cancer; PC, Pancreatic Cancer; IDC, Invasive Ductal Carcinoma; D&L, Mixed Ductal and lobular; ILC, Invasive Lobular Carcinoma; HR, Hormone Receptor; ER, Estrogen Receptor; PR, Progesterone Receptor; TNBC, Triple Negative Breast Cancer; Unk, Unknown; Fs, Frameshift; Del, Deletion of exon; Dup, Duplication of exon; N, Nonsense; SE, Splice-site Error; Ref, References.(XLSX)Click here for additional data file.S3 TableCa, Cancer; C, Chinese; M, Malay; BC, Breast Cancer; OC, Ovarian Cancer; IDC, Invasive Ductal Carcinoma; ILC, Invasive Lobular Carcinoma; DCIS, Ductal Carcinoma in situ; HR, Hormone Receptor; ER, Estrogen Receptor; PR, Progesterone Receptor; TNBC, Triple Negative Breast Cancer; Unk, Unknown; Ref, References; OS, Overall Scores for the prediction of damaging mutations; D, Damaging; P, Potential damaging; N, Neutral.(XLSX)Click here for additional data file.S4 Table* P-values that were statistically significant are indicated in bold; NS: Not Significant.(XLSX)Click here for additional data file.S5 Table* P-values that were statistically significant are indicated in bold; NS: Not Significant.(XLSX)Click here for additional data file.S6 Table* P-values that were statistically significant are indicated in bold; NS: Not Significant.(XLSX)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.


Macrophages are central to both innate and adaptive immunity. With few exceptions, macrophages are the first cells that sense trouble and respond to disturbances in almost all tissues and organs. They sense their environment, inhibit or kill pathogens, take up apoptotic and necrotic cells, heal tissue damage, and present antigens to T cells. Although the origins (yolk sac versus monocyte-derived) and phenotypes  of macrophages vary between tissues, they have many receptors in common that are specific to one or a few molecular species. Here, we review the expression and function of almost 200 key macrophage receptors that help the macrophages sense what is going on, including pathogen-derived molecules, the state of the surrounding tissue cells, apoptotic and necrotic cell death, antibodies and immune complexes, altered self molecules, extracellular matrix components, and cytokines, including chemokines. IL12 and iNOS, and become M1 macrophages (S1P)], lipoproteins , antibodies , complement , cytokines , and pathogens . Dendritic cells, another key sensor of pathogens, act in concert with the macrophages in initiating an adaptive immune response. Dendritic cells activate the adaptive immune system by antigen presentation to naive T cells in the context of co-activating receptors. These T cells become licensed to traffic to the infected or inflamed tissue . Retinoic acid induces the transcription factor GATA6, which in turn controls a number of effector molecules , NOD-like receptors (NLR), RNA and DNA receptors, C-type lectins, scavenger receptors, selected cytokine receptors, TNF receptor superfamily members, Fc receptors, selected G-protein-coupled receptors, and integrins]. Cross-platform comparison of gene expression profiles between microarray  and RNA-Seq (GSE62826) was done on normalized data provided by the authors on the GEO site. The normalized values from different platforms were scaled by log2 transformation for comparison through generation of heat maps , where red and green indicate higher and lower expression levels, respectively , microglia  data se data se  from macrophages that engage apoptotic cells. TGF-β and IL10 have anti-inflammatory functions. Depending on the receptor triggered (EP1-EP4), PGE2 can have pro- and anti-inflammatory functions, modulate pain sensation, and can activate mast cells . Tyro3, Axl, and Mer bind PtdSer through GAS6 (gene name Gas6) or protein S (gene name Pros1). Mertk, the gene encoding Mer tyrosine kinase, distinguishes macrophages from dendritic cells and has been proposed as a universal mouse macrophage marker  can detect CD47 on apoptotic lymphocytes. The results presented in Figure Mer and CD11b, can be downregulated, a process that limits further signaling and uptake of apoptotic cells.There are several recognition systems for PtdSer Table . Routinest cells . See Refst cells , 39 for grin see  recognize marker  when useMacrophages sense when tissue cells are distressed by events, such as ischemia , reperfusion (pro-inflammatory), tissue necrosis (pro-inflammatory), and altered self induced by enzymatic modification resulting in (lipo) protein citrullination , oxidatiRAGE (see below) also recognizes apoptotic cells and triggers pro-inflammatory mechanisms. The IL33 receptor ST2 detects IL33 released by necrotic cells. CD24 on macrophages binds HMGB1, which in turn binds necrotic cells. Mincle (Clec4e) and CLEC9A are two C-type lectins (listed below) involved in the uptake of necrotic cells. TLRs (see below) can recognize the nuclear protein HMGB1, the Sin3A-Associated Protein SAP130, the heat shock protein HSP90, DNA, and urate crystals, all of which can be exposed on or released from necrotic cells.Necrotic cells are opsonized by complement factors that are recognized through complement receptors , 45 TabB. The scTLR (NLR (RIG-I like receptors (RLR)  , recepto)   .Toll-like receptors Table  are a fandosomes , 54. MosTLR3, 7, 8, and 9 are expressed include ER, endosomes, lysosomes, and endolysosomes. An excellent review of TLR expression and function is found in Ref. , a common adaptor required for the activation of caspase-1 by several inflammasomes, and is comprised of a pyrin domain and a CARD domain. Linear ubiquitination via the assembly complex LUBAC is known to enhance ASC function in the activation of NLRP3-dependent inflammasome .The best-studied inflammasome is anchored by ammasome –57. The , MARCH7 . NLRP3 iammasome . The NLRNLRC4 inflammasome activation  is the NLR that defined the inflammasome  of Bacillus anthracis. Reminiscent of the requirement of a cleavage/activation process for caspase-1, IL1β, and IL18 function, a maturation cleavage of the N-terminal of murine NLRP1 is also required for its activation by LT , and TM7 microbiota  exhibits at least two functions as an inhibitor of the non-canonical and the canonical NFkB pathway. This was confirmed by in vivo studies of colitis-associated colon carcinoma models . Single-stranded (ss) viral RNA often forms stem–loop structures that can be detected by TLR7  are double-stranded (ds)DNA viruses, where the dsDNA is recognized by TLR7 see  and RIG-ee RIG-I . ssDNA , MDA5 (Ifih1), and LGP2 (Dhx58). RIG-I detects many ssRNA viruses, including influenza virus, hepatitis C, Dengue, yellow fever, west Nile, and Ebola. The RIG-I family receptors require MAVS as an adapter protein and IRF3 or IRF7 as downstream transcription factors, resulting in the elaboration of IFN-α and β.These receptors are expressed in macrophages, epithelial cells, and other cells. The immune response starts in infected cells, where the viruses replicate in the cytoplasm. The intracellular viral RNA is detected by the TLR9  , which produces cGMP that in turn activates STING. STING is also involved in detecting herpes virus. Other sensors for intracellular DNA are DAI , Aim2 (see under NLRs), DDX41 (Ddx41), DHX9 (Dhx9), IFI16 , and DHX36 (Dhx36). DAI senses dsDNA. The results in Figure Aim2 expression was only detected in bone marrow-derived macrophage (BMDM), peritoneal macrophages, and microglia.Single-stranded DNA is recognized by TLR9 see . DNA-derTmem173) . Humans DCIR and MICL are inhibitory, because their cytoplasmic tails contain ITIM domains. Dectin-2, Mincle, and DCAR are coupled to an ITAM-domain containing adaptor. Dectin-2 is the prototypic receptor of the Dectin-2 subfamily of C-type lectins that also contains CLECSF8, DCIR and Mincle and DCIR2, 3, and 4, DCAR, and DCAR1 in mice only . CD36 is an important receptor for diacyl glycerides, oxidized LDL, and the malaria parasites. CD36 is important for ROS and cytokine production by macrophages. There are no mammalian members of the SR-C subfamily. SR-D is CD68 , an intracellular receptor of unknown function. SR-E1 is Lox1 (Loxin), which detects oxidized LDL. Dectin-1 is SR-E2. The mannose receptor MR is SR-E3 and the asialoglycoprotein receptor is SR-E4. SR-F1 is SCARF1, Ced1 in Caenorhabditis elegans, and SR-F2 is SCARF2. The only known SR-G is CXCL16, a receptor for oxidized LDL and a chemokine that binds CXCR6. SR-H1 is FEEL-1, also known as Stabilin-1 or Clever-1, SR-I is CD163, the macrophage receptor for hemoglobin–haptoglobin complexes. In bovine and ovine species, this subfamily has many more members. SR-J is RAGE, a seven-transmembrane receptor with multiple immunoglobulin domains that sees glycated proteins and advanced glycation end products. SR-L1 is also known as LRP1, and SR-L2 as LRP-2.CD207 (Langerin), MRC1 (CD206), CLEC7A, and CD209 (DC-SIGN) are also unclassified. CD206 is prominently expressed on M2 macrophages. C1q interacts with the Aβ protein relevant in Alzheimer’s disease. The results in Figure Some macrophage scavenger receptors are currently unclassified. Galectin-3-binding protein is a secreted product of M1 macrophages that binds galectin-3 and other cell surface ligands. Another class of macrophage receptors sense products of adaptive immune cells. These receptors can be considered secondary amplifiers, because they do not detect non-self or altered self, but rather amplify the macrophage response. Since most cytokines are produced by activated T cells that require antigen presentation and an ongoing immune response, the cytokine receptors on macrophages sense the nature of that ongoing immune response   (The TNF receptor superfamily (Tnfrsf) –91 bind Tnfrsf) . These cTnfrsf members are well known for controlling responses of T and B cells, dendritic cells, NK, and NKT cells, as well as inflammatory activity in structural cells, such as fibroblasts, epithelial cells, and osteoclasts. This has resulted in clinical targeting of many of the molecules for autoimmune and inflammatory diseases , Tnfrsf1b (TNFR2), Tnfrsf11a (RANK), and Tnfrsf3 (LTβR) are highly expressed by most tissue macrophages. Tnfrsf5 (CD40), Tnfrsf12a (Fn14), Tnfrsf14 (HVEM), Tnfrsf21 (DR6) are also highly expressed in some macrophage populations, but variably expressed in others at moderate levels. TNFR2 is largely pro-inflammatory, whereas TNFR1 can display both pro-inflammatory and anti-inflammatory (apoptosis-inducing) activities. Since TNF is a product of macrophages, various reports have suggested both positive and negative effects of TNF on macrophages correlating with these functions of its receptors  that only binds BAFF was low in all macrophages, but Tnfrsf13b (TACI) and Tnfrsf17 (BCMA) that bind BAFF and APRIL were high in intestinal, adipose, and peritoneal macrophages and adipose macrophages and microglia, respectively. BAFF can be made by macrophages in soluble and membrane form, and membrane BAFF may (reverse) signal to promote inflammatory mediators , Tnfrsf25 (DR3), Tnfrsf4 (OX40), Tnfrsf18 (GITR), Tnfrsf8 (CD30), and Tnfrsf7 (CD27) showed low levels of transcript expression in most tissue macrophages, as did the decoy receptor for RANK (Tnfrsf11b – OPG). 4-1BB, DR3, OX40, GITR, CD30, and CD27 are best known as regulators of conventional T cells as well as Treg cells, NK, or NKT cells. Most have rarely been found on the surface of macrophages, although several studies have seen DR3 and GITR on macrophages/foam cells associated with atherosclerotic plaques, or in joints associated with RA  was low/absent in most macrophages, not surprising given its role outside the immune system in formation of tissues, such as sweat glands. Tnfrsf19 (TROY) is primarily expressed in the nervous system and has been suggested to regulate axon regeneration, but may also contribute to hair follicle formation. TROY was also low in most tissue macrophages and BMDMs. One prior report had suggested that it could be expressed in microglia in MS patients (patients  but thisTnfrsf10b (TRAILR2) and Tnfrsf6 (Fas), and the mouse decoy receptors for TRAIL (Tnfrsf22 and 23 – mDcTRAILR1 and R2) showed low levels of expression in most macrophages, except for Fas in peritoneal macrophages and mDcTRAILR1 in BMDM, lung, liver, and peritoneal cells. Fas is likely to promote apoptosis of macrophages in some cases to limit autoimmunity or inflammation (IL1 and IL18 or other activities (Tnfrsf21 (DR6) is another death receptor that has been reported to regulate neuronal death and may also limit T and B cell activation, but whether it has other functions is not clear. It was highly expressed in microglia, peritoneal, spleen, and lung macrophages and BMDM  (Fce receptor). They vary in their affinity (high to low) and in their downstream signaling effects (activating and inhibitory). Fc receptors are very highly expressed and are of key importance for clearing antibody-opsonized pathogens and antibody-opsonized necrotic cells. In the mouse, most Fc receptors signal through a common adaptor protein called Fc receptor γ chain or CD23.Fc receptors Table  are receand III) , 129 andFcgr1, the gene encoding the CD64 Fc γ receptor, is specific for macrophages and has been proposed as a universal mouse macrophage marker  and promotes macrophage survival. It is expressed in all macrophages, highest in microglia and intestinal macrophages.Macrophages and dendritic cells are important sources of many inflammatory chemokines including rs Table  and, thud CX3CR1 , 131. CCAdora 1, 2a, 2b, and 3. Adenosine is a ubiquitous metabolite found in the extracellular space, and macrophages can respond to adenosine through these receptors. ATP is released by apoptotic cells through a pannexin-dependent mechanism, and ATP and ADP are important metabolites influencing macrophages through P2Y receptors. Sphingosine-1-phosphate is produced by platelets and other blood cells and is sensed by macrophages through S1P receptors S1P1-4  is expressed at low levels in all tissue macrophages except microglia. This receptor downregulates inflammatory responses. A2B (Adora2b) is also expressed at low levels, but much higher in adipose tissue macrophages. This is considered a pro-inflammatory receptor.The adenosine receptor A2A (P2Y1 (P2ry1) is an ADP receptor with low expression in all macrophages except microglia, where it does not appear to be expressed. By contrast, the ADP receptor P2Y12 (P2ry12) shows low expression in all macrophages but very high expression in microglia. P2Y12 on platelets is a target for anti-thrombotic treatment. P2Y2 is a receptor for UTP and ATP, which is released from apoptotic cells via pannexin (PANX1) ( (PANX1) .S1pr1 is expressed by all tissue macrophages. S1pr2 is also broadly expressed, with highest levels in BMDM and bone marrow macrophages. S1pr3 and S1pr4 are expressed at low levels. Expression of these GPCRs is shown in Figure The sphigosine-1-phosphate receptor encoded by Macrophages use integrins Table , αβ heteItgb1). Itga1, Itga11, Itga2, Itga2b, Itga3, and Itga7 are expressed at low levels. Itga4 pairs with Itgb1 forming VLA-4, an integrin known to be involved in monocyte recruitment to tissues. Itga4 expression varies widely between tissue macrophages, with highest expression levels seen in large and small peritoneal macrophages. Itga5 pairs with Itgb1 to form a fibronectin receptor that is expressed at moderate levels in all tissue macrophages, highest in thioglycollate-elicited peritoneal macrophages. Itga6/Itgb1 encodes a laminin receptor that is highly expressed in peritoneal macrophages and microglia, but lower in liver, spleen, and adipose tissue macrophages. Itga8 is expressed in all tissue macrophages except peritoneal. Itga9 and 10 are expressed at intermediate to high levels in all macrophages.Many integrin α subunits pair with β1, which is expressed in all macrophages  is expressed at modest levels in most macrophages but not in microglia. Itgam (Mac-1), also known as complement receptor-3, is expressed at intermediate levels in most macrophages but very high in peritoneal and BMDM. Itgax (CD11c) is widely used as a dendritic cell marker, but this integrin is also expressed in all macrophages, most highly in thioglycollate-elicited peritoneal macrophages. Itgad (CD11d) is expressed at very low levels except for spleen macrophages, where it is very high.The integrin subunits Itgb3 to form the vitronectin receptor αVβ3, also known as leukocyte response integrin. Both subunits are expressed across all macrophages. Itgav can also pair with Itgb6 to form αVβ6 integrin, which is involved in TGF-β processing and fibrosis. Itgae and Itga4 pair with Itgb7, a receptor family generally found more in intestinal tissues. Indeed, Itgb7 expression is highest in peritoneal and intestinal macrophages. Itgae is highly specialized and binds to E-cadherin. Its expression is low except in intestinal macrophages, where it is very high  and others are “blind” to certain challenges or inputs. Recent reviews on each of the 12 classes of receptors are cited in the respective subsections for more detailed information. The remarkable heterogeneity in expression of macrophage receptors suggests highly specialized functions of resting tissue macrophages in lung, liver, spleen, intestine, fat tissue, the peritoneal cavity, and the brain (microglia).This review is limited to mRNA expression data, and it is unknown how these expression patterns correlate with protein levels. In macrophages, the correlation between mRNA and protein levels is modest. Also, we consider the 12 receptor classes individually, but they may cooperate or even compete for ligand recognition, which would affect macrophage function. There could be more than one “danger” that affects a tissue, such as infection with multiple pathogens or viral infection and apoptotic cells. How tissue macrophages respond to such complex inputs remains to be studied. We did not analyze infiltrating monocyte-derived macrophages here, which can be a dominant population in inflammatory diseases, infections, autoimmune diseases, and cancer.KL wrote the paper. AP constructed the heat maps and input to the tables. MC wrote and edited the TNFSF part. KR edited the apoptotic cell uptake part. JT wrote and edited the NLR and RLR part.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.


Salmonella enterica and Escherichia coli can inhabit humans and animals from multiple origins. These bacteria are often associated with gastroenteritis in animals, being a frequent cause of resistant zoonotic infections. In fact, bacteria from animals can be transmitted to humans through the food chain and direct contact. In this study, we aimed to assess the antibiotic susceptibility of a collection of S. enterica and E. coli recovered from animals of different sources, performing a genomic comparison of the plasmid-mediated quinolone resistance (PMQR)-producing isolates detected. Antibiotic susceptibility testing revealed a high number of non-wild-type isolates for fluoroquinolones among S. enterica recovered from poultry isolates. In turn, the frequency of non-wild-type E. coli to nalidixic acid and ciprofloxacin was higher in food-producing animals than in companion or zoo animals. Globally, we detected two qnrS1 and two aac(6′)-Ib-cr in E. coli isolates recovered from animals of different origins. The genomic characterization of QnrS1-producing E. coli showed high genomic similarity (O86:H12 and ST2297), although they have been recovered from a healthy turtle dove from a Zoo Park, and from a dog showing symptoms of infection. The qnrS1 gene was encoded in a IncN plasmid, also carrying blaTEM−1−containing Tn3. Isolates harboring aac(6′)-Ib-cr were detected in two captive bottlenose dolphins, within a time span of two years. The additional antibiotic resistance genes of the two aac(6′)-Ib-cr-positive isolates -IIa, and tetA) were enclosed in IncFIA plasmids that differed in a single transposase and 60 single nucleotide variants. The isolates could be assigned to the same genetic sublineage—ST131 fimH30-Rx (O25:H4), confirming clonal spread. PMQR-producing isolates were associated with symptomatic and asymptomatic hosts, which highlight the aptitude of E. coli to act as silent vehicles, allowing the accumulation of antibiotic resistance genes, mobile genetic elements and other relevant pathogenicity determinants. Continuous monitoring of health and sick animals toward the presence of PMQR should be strongly encouraged in order to restrain the clonal spread of these antibiotic resistant strains. Salmonella enterica and Escherichia coli are among the most problematic zoonotic bacteria, causing severe gastroenteritis in animals and humans -Ib-cr, and oqxAB , broilers (n = 33), layers (n = 33), swine (n = 6), and food products of animal origin (n = 5)  in triple sugar iron slopes or SMID plates.This study included 89 ders n = 2, broileE. coli isolates (Table n = 32)], pets  and zoo animals . Samples consisted of swabs from organic fluids and cavities, fecal samples, urine samples, blood cultures, and organs collected during post-mortem examination and submitted for bacteriological analysis. Suspected E. coli colonies obtained in MacConkey agar plates were confirmed by API 20E strips .This study also included 91 S. enterica isolates were serotyped by the slide agglutination method, using the method of Kauffmann-White scheme . For Salmonella spp., the cut-off value used for sulfamethoxazole was that for sulfonamides from Clinical Standards Laboratory Institute (http://clsi.org). MIC50 and MIC90 were calculated as reported elsewhere  if they presented non-wild-type phenotypes against three or more structurally unrelated antibiotics  were determined by agar dilution following standard recommendations, using a panel of 10 antimicrobial compounds: ampicillin, cefotaxime, nalidixic acid, ciprofloxacin, gentamicin, streptomycin, chloramphenicol, tetracycline, sulfamethoxazole, and trimethoprim Table . IsolateqnrA, qnrB, qnrC, qnrD, qnrS, aac(6′)-Ib-cr, and qepA genes, using primers and conditions previously described . PMQR-producing isolates were also characterized regarding the production of β-lactamase-encoding genes and conventional Multilocus sequence typing (MLST), as described elsewhere  were characterized. Genomic DNA was extracted using DNeasy Blood and Tissue Kit  and quantified using Qubit 1.0 Fluorometer . The Nextera XT DNA Sample Preparation Kit  was used to prepare sequencing libraries from 1 ng of genomic DNA, according to the manufacturer's instructions. Paired-end sequencing of 150 bp reads was performed on a MiSeq (Illumina). Sequence reads were then trimmed and filtered according to quality criteria, and assembled de novo using CLC genomics workbench version 8.5.1 . RAST (Rapid Annotation using Subsystem Technology) was used for subsystem annotation of the genomes , being present in all food animals except swine. The remaining Salmonella serotypes were detected in a less extent and were comprised of Salmonella 4,5:i:- (n = 1), Salmonella 6,7,14:-:1,2 (n = 1), Salmonella Bradenburg (n = 1), Salmonella Gallinarum (n = 1), Salmonella Give (n = 1), Salmonella Hadar (n = 1), Salmonella Heidelberg (n = 1), Salmonella IIIa 48:z10:- (n = 1), Salmonella Mbandaka (n = 1), Salmonella Rissen (n = 2), Salmonella Typhimurium (n = 3), and Salmonella Virchow (n = 4).ns EFSA,  and it wS. enterica and E. coli isolates differed with the animal group  and ciprofloxacin (from 64 to 100%) in all groups, they were particularly evident in poultry, and predominant in breeders. S. enterica isolates recovered from other sources , showed higher non-wild-type phenotypes for ampicillin (36%), streptomycin (64%), tetracycline (45%), sulfamethoxazole (36%), and trimethoprim (27%) , tetracycline , sulfamethoxazole  and trimethoprim . Although, no major discrepancies were noticed for E. coli in rates of non-wild-type isolates for the different animal groups, isolates recovered from food animals still presented more non-wild-type phenotypes than zoo or companion animals against nalidixic acid (41%), ciprofloxacin (41%), tetracycline (53%), and chloramphenicol (16%).The frequency of non-wild-type isolates was globally higher for S. enterica isolates from poultry, similar MIC50 and MIC90 values were observed for the majority of the antibiotics tested; major differences (≥3 fold dilutions) were observed for the group “others” for ampicillin (8 and 64 mg/L), tetracycline (4 and 64 mg/L), sulfamethoxazole (128 and >512 mg/L) and trimethoprim (0.25 and 32 mg/L). For E. coli, the most significant differences in MIC50 and MIC90 values were observed for nalidixic acid (4 and 128 mg/L), ampicillin (8 and 64 mg/L), streptomycin (4 and 128 mg/L), tetracycline (2 and 64 mg/L), sulfamethoxazole (16 and >512 mg/L) and trimethoprim (0.5–32 mg/L).In S. enterica only 9.0% (8/89) MDR isolates were detected, for E. coli, MDR was registered in 38.9% (35/90) of the isolates, which were distributed among 16/90 isolates from food-producing animals, 9/90 isolates from companion animals, and 10/90 isolates from zoo animals.While for E. coli isolates: two qnrS1 were detected in isolates recovered from a captive turtle dove (LV46221) and a pet dog (LV46743), and two aac(6′)-Ib-cr were isolated from E. coli recovered from captive bottlenose dolphins (LV36464 and LV27950). The detection of β-lactamase-encoding genes showed the presence of blaTEM−1in isolates LV46221 and LV46743, and blaTEM−1, blaOXA−1, and blaCTX−M−15 in LV36464 and LV27950. No other PMQR- or β-lactamase-encoding genes were identified in the collection of E. coli and S. enterica isolates.Overall, among the 180 isolates studied, we have detected and identified four PMQR determinants in qnrS1-harboring E. coli, LV46221 and LV46743, yielded 200 and 199 contigs (each >200 bp long), which together comprised 4,799,985 bp and 4,801,518 bp, respectively. The average coverage of LV46221 was 135.9, while LV46743 displayed 114.1 fold. The maximum contig length obtained for these genomes was 398,205 bp and 333,601 bp, respectively  and LV46743  presented a similar number of sequences attributed to specific subsystems Figure . GeneralIn silico analysis of the antibiotic resistance genes (90% identity and 40% minimum length) confirmed the presence of a qnrS1, and identified blaTEM−1gene in both isolates .gyrA, gyrB, parC, and parE, which are known to confer high level resistance to fluoroquinolones , IS200_IS605 , IS21 , IS3 , IS4 , IS481 , IS5 , IS6 , ISAs1, ISKra4 , ISL3 , ISNCY , and finally Tn3 . ISAs1 was the most frequent element detected in both isolates, and IS66 was exclusively detected in LV46743 (1.96%). Moreover, in LV46221 we identified 10 prophage regions among which three were questionable but seven were intact. The latter included prophage regions reaching up to 90.6Kb, containing 133 coding sequences  of genes es Table . In turnes Table .E. coli LV46221 and LV46743: 607 and 611 different pathogenic families showed a 93.5% certainty of the isolates being human pathogens. Finally, the virulence factor glutamate decarboxylase (gad) was detected in both isolates, while the increased serum survival factor iss was exclusively identified in LV46221  were recovered for LV36464 and LV27950 with a maximum contig length of 399,998 and 369,918 bp, respectively -Ib-cr and CTX-M-15, were also compared. Their The automated annotation of the genomes showed a total number of coding sequences of 5107 for LV36464 and 5069 for LV27950, excluding 77 and 76 annotated RNA molecules. The distribution of the annotated coding sequences by subsystem showed an identical representation of functions in both isolates Ib-cr, blaOXA−1, blaTEM−1,blaCTX−M−15, catB3, aac(3)-IIa, and tetA. By mapping, the main difference between the plasmids carried by these isolates was the deletion of a 2400 bp sequence that displayed 99.7% homology with the transposase of Tn5403. Moreover, 60 single nucleotide variants have been detected between them. Both plasmids displayed an IncF plasmid from a ST131 E. coli isolate  as its best blast hit. The comparative analysis with JJ2434 showed the absence of two regions of 9329 and 1740 bp that corresponded to deletions of genes coding for unknown functions, replication proteins, endonucleases, transcriptional regulators, and conjugation transfer proteins in LV36464 and LV27950 plasmids. The analysis of the QRDR of genes gyrA (from 67 to 106 aminoacids), gyrB (from 415 to 470 aminoacids), parC (from 47 to 133 aminoacids), and parE (from 450 to 528 aminoacids) revealed the presence of amino acid substitutions in gyrA (S83L and D87N) and parC (S80I and E84V) in both isolates.Globally, in isolates LV36464 and LV27950 seven different acquired antibiotic resistance genes were detected: 1 , IS110 , IS1380 , IS200_IS605 , IS21 , IS3 , IS30 , IS4 , IS481 , IS6 , IS66 , ISAs1 , ISL3  and ISNCY . It is worth mentioning that the worldwide disseminated Tn3 was only represented in the genome of LV36464 (3.39%), and IS92 (1.89%) in LV27950. The specialized annotation of phage and prophages revealed that LV36464 harbored 17 regions: 8 intact, 6 incomplete and 4 questionable. These intact prophage regions ranged between 17.4 and 51.5 Kb, showing different numbers of coding sequences that varied between 24 and 88. In turn, LV27950 harbored 13 prophage regions: it displayed 10 intact regions spanning between 20.6 and 86.1 Kb. Globally, regions from five phages were present in the genomes of both and two were exclusive of each isolate (Table A high number of mobile genetic elements was detected in the draft genomes of these isolates. Both harbored a plasmid (>98% homology) from incompatibility group IncFIA, which according to PlasmidFinder was classified as an IncFIA type 1. LV36464 accommodated an additional IncX plasmid. The distribution of insertion sequences present in LV36464 and LV27950 genomes was also globally similar: ISsat (Table gad). The overall estimation of pathogenicity factors present in the genome of the isolates, using known proteins with recognized involvement in pathogenicity as reference, enabled us to determine that the assembled contigs of LV36464 and LV27950 matched 553 and 544 pathogenic families, which resulted in the estimation of both isolates being human pathogens (93.1 and 93.3%), confirming their zoonotic potential.The detection of virulence factors in the genome of LV27950 revealed the presence of an increased serum survival factor provided by an ISS-encoding gene and a secreted autotransporter toxin denominated at Table . LV36464E. coli. To understand the antibiotic susceptibility background of these specific isolates we have also evaluated the antibiotic susceptibility phenotypes of a collection of S. enterica and E. coli recovered from animals of different origins, in which the isolates were originally included.The prevalence of antibiotic resistance genes in isolates from animal origin has been fairly assessed  were noted for some groups of each species: E. coli isolates for nalidixic acid, ciprofloxacin, ampicillin, streptomycin, tetracycline, sulfamethoxazole and trimethoprim, and S. enterica for ampicillin, cefotaxime, tetracycline, and trimethoprim.The levels of non-wild type phenotypes revealed to be very distinct among es EFSA, . Portugaes EFSA, . DiffereAlthough, PMQR determinants are typically responsible by low level resistance, their presence has been increasingly reported in animals, resulting in an additional effect on the nonsusceptibility of fluoroquinolones -Ib-cr) in E. coli LV46221, LV46743, LV36464, and LV27950 recovered from animals of different origins: a healthy turtle dove from a Zoo Park (2008), a diseased pet dog (2008), a bottlenose dolphin from a Zoo Park showing signs of respiratory infection (2009), and a second but healthy bottlenose dolphin from the same Zoo Park (2011)  -Ib-cr- and blaCTX−M−15-harboring E. coli. In fact, the isolates could be assigned to the same genetic sublineage—ST131 fimH30-Rx, confirming clonal spread. Although, samples have been recovered within a reasonable time span of 2 years, their origin refers to two bottlenose dolphins of the same species held captive in the same Zoo Park. The presence of four chromosomal alterations in the QRDR region of isolates LV36464 and LV27950 was reflected in the high levels of fluoroquinolone MICs, which ranged between 8 and >256 mg/L. All antibiotic resistance genes detected in LV36464 and LV27950  could be traced back to a single multidrug resistance IncFIA plasmid that showed 99.9% of homology with a plasmid submitted this year to Genbank in U.S.A . Although, 60 single nucleotide variants have been detected between the LV36464 and LV27950 plasmids, the main difference consisted of a single deletion that involved part of a transposase-encoding gene. The absence of a set of conjugation transfer proteins (tra genes), among other genes, highlighted the preponderance of clonal spread over horizontal gene transfer in ST131 E. coli , as previously reported -Ib-cr-harboring isolates. The data gathered throughout this study illustrates two scenarios: the presence of the same strain in different hosts inhabiting remote locations and the persistence of a unique strain in a single niche during a long period of time. The strains were each associated with a case of symptomatic infection (LV46743 and LV36464) and with a report of microbiological control of an asymptomatic host (LV46221 and LV27950), which reinforces the ability of E. coli isolates to act as silent vehicles, allowing the accumulation of antibiotic resistance determinants, mobile genetic elements and other relevant pathogenicity determinants . DJD has received research funding from Funda73o para a Cie a Tecnologia . VM was supported by FCT fellowship (grant SFRH/BPD/77486/2011), financed by the European Social Funds (COMPETE-FEDER) and national funds of the Portuguese Ministry of Education and Science (POPH-QREN).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.


DMPK gene. Despite characteristic muscular, cardiac, and neuropsychological symptoms, CTG trinucleotide repeats are unstable both in the somatic and germinal lines, making the age of onset, clinical presentation, and disease severity very variable. A molecular biomarker to stratify patients and to follow disease progression is, thus, an unmet medical need. Looking for a novel biomarker, and given that specific miRNAs have been found to be misregulated in DM1 heart and muscle tissues, we profiled the expression of 175 known serum miRNAs in DM1 samples. The differences detected between patients and controls were less than 2.6 fold for all of them and a selection of six candidate miRNAs, miR-103, miR-107, miR-21, miR-29a, miR-30c, and miR-652 all failed to show consistent differences in serum expression in subsequent validation experiments.Myotonic dystrophy type 1 (DM1) is an autosomal dominant genetic disease caused by expansion of a CTG microsatellite in the 3’ untranslated region of the  Unaffected individuals carry between 5 and 37 CTG repeats whereas DM1 patients carry between 50 and thousands of CTG triplets [Myotonic dystrophy type 1 (DM1) can appear at any time in life and is regarded as the human disease which probably has the most variable clinical presentation, somehow affecting virtually all body systems . Characttriplets . DespiteExpanded RNA transcripts containing CUG repeats are retained in the cell nucleus as insoluble RNA aggregates known as ribonuclear foci . These fmiR-103, miR-107, miR-21, miR-29a, miR-30c, and miR-652) were further investigated but no significant differences between the control and DM1 conditions were found for any of them.Because several miRNAs have been detected to be altered in DM1 cardiac and muscle tissues , 24, 35,32P-labelled cDNA25 probe  of 26 DM1 and 22 healthy individuals  was used for miRNA expression profiling. These panels contain primers for the detection of the 175 most-expressed miRNAs in human serum . Each 38t method  using tht method , 48. ExpA logarithmic transformation (log2) was used to normalise the expression data in the profiling experiment. Expression differences were analysed using the Student t-test and different methods for multiple-testing correction were applied, including Bonferroni, Benjamini-Hochberg , Westfall-Young, and Benjamini-Yekutieli corrections. Cluster software was used for hierarchical clustering analysis of genes and samples. Euclidean distances and the average linkage method were selected, using the normalised expression values of each miRNA to represent clusters.P-value lower than 0.05, of which 24 miRNAs were up- and 11 were downregulated when compared to controls Click here for additional data file.S1 Table(XLS)Click here for additional data file.S2 Table(XLS)Click here for additional data file.S3 Table(XLS)Click here for additional data file.S4 Table(XLS)Click here for additional data file.