the next frontier in medicine

PM003/2016publishersversionpublishe

2,2'-(2,2'-oxybis(ethane-2,1-diyl)bis(oxy))bis(N-(2,2'-bithiophen-5-ylmethylene)aniline)

We would like to thank Xunta de Galiza (Spain) for project 09CSA043383PR and the University of Vigo, Vicou for projects INOU UVIGO/VICOU/K914-122P64702/2009 and UVIGO/VICOU/K912-122P64702/2009. Thanks to the FCT-MCTES/FEDER (Portugal) through national projects POCI/QUI/55519/2004 and PDTC/QUI/66250/2006. B. P thanks FCT/Portugal for the PhD Grant SFRH/BD/27786/2006. C.L. and J.L. thank Xunta de Galicia for the Isidro Parga Pondal Research Program.A new flexible fluorescent compound L derived from 1,5-bis(2-aminophenoxy)-3-oxapentane (A) has been synthesized by classical Schiff-base reaction between (A) and 2,2 ́-bithiophene carbaldehyde (B). The same synthesis was reproduced by a green methodology using an ultrasonication reaction in a classical sonication bath. The structure of the new compound was confirmed by elemental analysis, IR, 1H-NMR, MALDI-TOF-MS and EI-MS-spectra, UV-vis and fluorescence emission spectroscopy.publishersversionpublishe

Identification of genomic loci associated with genotypic and phenotypic variation among Pseudomonas aeruginosa clinical isolates from pneumonia

In this work, a genotype-phenotype survey of a highly diversified Pseudomonas aeruginosa collection was conducted, aiming to detail pathogen-associated scenarios that clinicians face nowadays. Genetic relation based on RAPD-PCR of 705 isolates, retrieved from 424 patients and several clinical contexts, reported an almost isolate-specific molecular-pattern. Pneumonia-associated isolates HB13 and HB15, clustered in the same RAPD-PCR group, were selected to evaluate the genomic background underlying their contrasting antibiotic resistance and virulence. The HB13 genome harbors antibiotic-inactivating enzymes-coding genes (e.g. aac(3)-Ia, arr, blaVIM-2) and single-nucleotide variations (SNVs) in antibiotic targets, likely accounting for its pan-resistance, whereas HB15 susceptibility correlated to predicted dysfunctional alleles. Isolate HB13 showed the unprecedented rhl-cluster absence and variations in other pathogen competitiveness contributors. Conversely, HB15 genome comprises exoenzyme-coding genes and SNVs linked to increased virulence. Secretome analysis identified signatures features with unknown function as potential novel pathogenic (e.g. a MATE-protein in HB13, a protease in HB15) and antibiotic resistance (a HlyD-like secretion protein in HB13) determinants. Detection of active prophages, proteases (including protease IV and alkaline metalloproteinase), a porin and a peptidase in HB15 highlights the secreted arsenal likely essential for its virulent behavior. The presented phenotype-genome association will contribute to the current knowledge on Pseudomonas aeruginosa pathogenomics.This work was supported by the strategic programme UID/BIA/0050/2013 (POCI-01-0145-FEDER-007569) funded by national funds through FCT I.P., by ERDF through the COMPETE2020 - Programa Operacional Competitividade e Internacionalização (POCI) and through a PhD grant (SFRH/BD/98558/2013) attributed to C.S.M. The facility for Biological Mass Spectrometry Isabel Moura was funded by Proteomass Scientific Society. H.M.S. is funded by the FCT 2015 Investigator Program (IF/00007/2015)

Biofilm formation of multidrug-resistant MRSA strains isolated from different types of human infections

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the main pathogens causing chronic infections, mainly due to its capacity to form biofilms. However, the mechanisms underlying the biofilm formation of MRSA strains from different types of human infections are not fully understood. MRSA strains isolated from distinct human infections were characterized aiming to determine their biofilm-forming capacity, the biofilm resistance to conventional antibiotics and the prevalence of biofilm-related genes, including, icaA, icaB, icaC, icaD, fnbA, fnbB, clfA, clfB, cna, eno, ebpS, fib and bbp. Eighty-three clinical MRSA strains recovered from bacteremia episodes, osteomyelitis and diabetic foot ulcers were used. The biofilm-forming capacity was evaluated by the microtiter biofilm assay and the biofilm structure was analyzed via confocal scanning laser microscopy. The antimicrobial susceptibility of 24-h-old biofilms was assessed against three antibiotics and the biomass reduction was measured. The metabolic activity of biofilms was evaluated by the XTT assay. The presence of biofilm-related genes was investigated by whole-genome sequencing and by PCR. Despite different intensities, all strains showed the capacity to form biofilms. Most strains had also a large number of biofilm-related genes. However, strains isolated from osteomyelitis showed a lower capacity to form biofilms and also a lower prevalence of biofilm-associated genes. There was a significant reduction in the biofilm biomass of some strains tested against antibiotics. Our results provide important information on the biofilm-forming capacity of clinical MRSA strains, which may be essential to understand the influence of different types of infections on biofilm production and chronic infections.This work was funded by the R&D Project CAREBIO2: Comparative assessment of antimicrobial resistance in environmental biofilms through proteomics—towards innovative thera-nostic biomarkers, with reference NORTE-01-0145-FEDER-030101 and PTDC/SAU-INF/30101/2017, financed by the European Regional Development Fund (ERDF) through the Northern Regional Operational Program (NORTE 2020) and the Foundation for Science and Technology (FCT). This work was supported by the Associate Laboratory for Green Chemistry—LAQV which is financed by national funds from FCT/MCTES (UIDB/50006/2020 and UIDP/50006/2020). Vanessa Silva is grateful to FCT (Fundação para a Ciência e a Tecnologia) for financial support through the PhD grant SFRH/BD/137947/2018.info:eu-repo/semantics/publishedVersio

Genetic characterization of methicillin-resistant staphylococcus aureus isolates from human bloodstream infections: detection of mlsb resistance

In this study we aimed to characterize antimicrobial resistance in methicillin-resistant Staphylococcus aureus (MRSA) isolated from bloodstream infections as well as the associated genetic lineages of the isolates. Sixteen MRSA isolates were recovered from bacteremia samples from inpatients between 2016 and 2019. The antimicrobial susceptibility of these isolates was tested by the Kirby–Bauer disk diffusion method against 14 antimicrobial agents. To determine the macrolide–lincosamide–streptogramin B (MLSB) resistance phenotype of the isolates, erythromycin-resistant isolates were assessed by double-disk diffusion (D-test). The resistance and virulence genes were screened by polymerase chain reaction (PCR). All isolates were characterized by multilocus sequence typing (MLST), spa typing, staphylococcal chromosomal cassette mec (SCCmec) typing, and accessory gene regulator (agr) typing. Isolates showed resistance to cefoxitin, penicillin, ciprofloxacin, erythromycin, fusidic acid, clindamycin, and aminoglycosides, confirmed by the presence of the blaZ, ermA, ermC, mphC, msrA/B, aac(6’)-Ie-aph(2’’)-Ia, and ant(4’)-Ia genes. Three isolates were Panton–Valentine-leukocidin-positive. Most strains (n = 12) presented an inducible MLSB phenotype. The isolates were ascribed to eight spa-types (t747, t002, t020, t1084, t008, t10682, t18526, and t1370) and four MLSTs (ST22, ST5, ST105, and ST8). Overall, most (n = 12) MRSA isolates had a multidrug-resistance profile with inducible MLSB phenotypes and belonged to epidemic MRSA clones.info:eu-repo/semantics/publishedVersio

Clonal Diversity and Antimicrobial Resistance of Methicillin-Resistant Staphylococcus pseudintermedius Isolated from Canine Pyoderma

[EN] The emergence of methicillin-resistant Staphylococcus pseudintermedius (MRSP) antimicrobial resistance and epidemic genetic lineages is posing a challenge in veterinary medicine due to the limited therapeutical options. MRSP has been identified as an important canine pyoderma pathogen. Thus, we aimed to characterize the antimicrobial resistance and clonal lineages of MRSP isolated from canine cutaneous pyoderma. Thirty-one MRSP isolates recovered from pyoderma were further characterized. The antimicrobial susceptibility testing of the isolates was performed by the KirbyBauer disc diffusion method against 14 antimicrobial agents. The presence of antimicrobial and virulence genes was carried out by PCR. Multilocus sequence typing was performed in all isolates. All strains had a multidrug-resistant profile showing resistance mainly to penicillin, macrolides and lincosamides, aminoglycosides, tetracycline and trimethoprim-sulfamethoxazole, which was encoded by the blaZ, ermB, msr(A/B), aac(6′ )-Ie-aph(2′′ )-Ia, aph(3′ )-IIIa, ant(4′ )-Ia, tetM, tetK and dfrG genes. All isolates harbored the lukS-I/lukF-I virulence factors. Isolates were ascribed to nine previously described sequence types (STs): ST123, ST339, ST727, ST71, ST537, ST45, ST1029, ST118 and ST1468; and to five STs first described in this study: ST2024, ST2025, ST2026, ST2027 and ST2028. In this study, most isolates belonged to ST123 (n = 16), which belongs to CC71 and is the most common clone in Europe. All isolates were multidrug-resistant, which may impose a serious threat to animal healthSIThis research was funded by the Ministerio de Ciencia, Innovación y Universidades (Spain, Project RTI2018-098267-R-C33) and the Junta de Castilla y León (Consejería de Educación, Spain, Project LE018P20). This work was funded by the R&D Project CAREBIO2: Comparative assessment of antimicrobial resistance in environmental biofilms through proteomics—towards innovative thera- nostic biomarkers, with reference NORTE-01-0145-FEDER-030101 and PTDC/SAU-INF/30101/2017, financed by the European Regional Development Fund (ERDF) through the Northern Regional Operational Program (NORTE 2020) and the Foundation for Science and Technology (FCT). This work was supported by the Associate Laboratory for Green Chemistry-LAQV, which is financed by national funds from FCT/MCTES (UIDB/50006/2020 and UIDP/50006/2020). Vanessa Silva is grateful to FCT (Fundação para a Ciência e a Tecnologia) for financial support through the PhD grant SFRH/BD/137947/201

Absolute quantitative proteomics using the total protein approach to identify novel clinical immunohistochemical markers in renal neoplasms

PM003/2016).) .. This project utilized the University of Pittsburgh Hillman Cancer Center shared resource facilities (Cancer Genomics Facility and The Health Science Tissue Bank) supported in part by award P30CA047904 (Dr. W. LaFramboise and R. Dhir).Background: Renal neoplasms encompass a variety of malignant and benign tumors, including many with shared characteristics. The diagnosis of these renal neoplasms remains challenging with currently available tools. In this work, we demonstrate the total protein approach (TPA) based on high-resolution mass spectrometry (MS) as a tool to improve the accuracy of renal neoplasm diagnosis. Methods: Frozen tissue biopsies of human renal tissues [clear cell renal cell carcinoma (n = 7), papillary renal cell carcinoma (n = 5), chromophobe renal cell carcinoma (n = 5), and renal oncocytoma (n = 5)] were collected for proteome analysis. Normal adjacent renal tissue (NAT, n = 5) was used as a control. Proteins were extracted and digested using trypsin, and the digested proteomes were analyzed by label-free high-resolution MS (nanoLC-ESI-HR-MS/MS). Quantitative analysis was performed by comparison between protein abundances of tumors and NAT specimens, and the label-free and standard-free TPA was used to obtain absolute protein concentrations. Results: A total of 205 differentially expressed proteins with the potential to distinguish the renal neoplasms were found. Of these proteins, a TPA-based panel of 24, including known and new biomarkers, was selected as the best candidates to differentiate the neoplasms. As proof of concept, the diagnostic potential of PLIN2, TUBB3, LAMP1, and HK1 was validated using semi-quantitative immunohistochemistry with a total of 128 samples assessed on tissue micro-arrays. Conclusions: We demonstrate the utility of combining high-resolution MS and the TPA as potential new diagnostic tool in the pathology of renal neoplasms. A similar TPA approach may be implemented in any cancer study with solid biopsies.publishersversionpublishe

Synthesis of functionalized fluorescent silver nanoparticles and their toxicological effect in aquatic environments (Goldfish) and HEPG2 cells

Silver nanoparticles, AgNPs, are widely used in our daily life, mostly due to their antibacterial, antiviral, and antifungal properties. However, their potential toxicity remains unclear. In order to unravel this issue, emissive AgNPs were first synthetized using an inexpensive photochemical method, and then their permeation was assessed in vivo in goldfish and in vitro in human hepatoma cells (HepG2). In addition, the oxidative stress caused by AgNPs was assessed in enzymes such as glutathione-S-transferase (GST), catalase (CAT), and in lipid peroxidation (LPO). This study demonstrates that the smallest sized AgNPs@ 3 promote the largest changes in gold fish livers, whereas AgNPs@ 1 were found to be toxic in HEPG2 cells depending on both the size and functionalized/stabilizer ligand

Engineered Nanostructured Materials for Ofloxacin Delivery

Antibiotic resistance is emerging as a growing worldwide problem and finding solutions to this issue is becoming a new challenge for scientists. As the development of new drugs slowed down, advances in nanotechnology offer great opportunities, with the possibility of designing new systems for carrying, delivery and administration of drugs already in use. Engineered combinations of the synthetic, broad-spectrum antibiotic ofloxacin, rarely studied in this field, with different types of silver, mesoporous silica-based and Pluronic/silica-based nanoparticles have been explored. The nanocarriers as silver core@silica mesoporous (AgMSNPs) and dye-doped silica nanoparticles functionalized with ofloxacin were synthesized and their antibacterial properties studied against S. aureus and E. coli. The best antibacterial results were obtained for the AgMSNPs nanosystem@ofloxacin for the strain S. aureus ATCC 25923, with MIC and MBC values of 5 and 25 μg/mL, proving the efficacy and synergetic effect of the antibiotic and the Ag core of the nanoparticles

Inventory and review of the Mio–Pleistocene São Jorge flora (Madeira Island, Portugal): palaeoecological and biogeographical implications

The occurrence of plant fossils on Madeira Island has been known since the mid-nineteenth century. Charles Lyell and George Hartung discovered a leaf bed rich in Lauraceae and fern fossils at S~ao Jorge in 1854. The determinations were controversial but a full review was never performed. Here we propose possible geological settings for the fossiliferous outcrop, and present an inventory and a systematic review of the surviving specimens of the S~ao Jorge macroflora. The S~ao Jorge leaf bed no longer outcrops due to a landslide in 1865. It was possible to establish the two alternative volcano stratigraphical settings in the sedimentary intercalations from the Middle Volcanic Complex, ranging in age from 7 to 1.8 Ma. The descriptions of Heer (1857), Bunbury (1859) and Hartung & Mayer (1864) are reviewed based on 82 surviving specimens. From the initial 37 taxa, we recognize only 20: Osmunda sp., Pteridium aquilinum, Asplenium cf. onopteris, aff. Asplenium, cf. Polystichum, cf. Davallia, Woodwardia radicans, Filicopsida gen. et sp. indet. 1 and 2, Ocotea foetens, Salix sp., Erica arborea, cf. Vaccinium, Rubus sp, cf. Myrtus, Magnoliopsida gen. et sp. indet. 1 to 3, Liliopsida gen. et sp. indet. 1. Magnoliopsida gen. et sp. indet. 4 is based on one previously undescribed flower or fruit. The floristic composition of the S~ao Jorge fossils resembles the current floristic association of temperate stink laurel (Ocotea foetens) forest, suggesting a warm and humid palaeoclimate and indicating that laurel forests were present in Macaronesia at least since the Gelasian, a time when the palaeotropical geofloral elements were almost extinct in Europe.info:eu-repo/semantics/publishedVersio