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The content, distribution of individual species, and the fatty acid composition of phospholipids (PL) from ewe's and goat's milk were analyzed. The binding of enterotoxigenic and uropathogenic Escherichia coli strains to PL and the inhibition of bacterial hemagglutination by PL were addressed using high performance thin-layer chromatography-overlay assays and microtiter plates, respectively. Ovine and caprine milk contained more PL than bovine milk but less than human milk. The profile of individual PL was similar, including sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol in both ovine and caprine milk. Regarding the fatty acid composition, a high content of long-chain fatty acids (more than C16) and unsaturated fatty acids, with C18:1 as the most abundant was found in ovine and caprine milk PL. Ovine milk has longer and less saturated fatty acids while caprine milk has shorter and more saturated ones. Neither the adhesion of any bacterial strains assayed to the individual PL from ovine or caprine milk nor the inhibition of bacterial hemagglutination by PL were observed. These are important constituents of the milk fat globule membrane, but it seems that they do not play a role in the defence of new-borns against bacteria if the results obtained are taken into account. [ABSTRACT FROM AUTHOR]

The symbiovar mediterranense of Sinorhizobium meliloti was initially found in Phaseolus vulgaris nodules in Tunisia and in an eastern location of Lanzarote (Canary Islands). Here we show that the symbiovar mediterranense of S. meliloti also nodulates P. vulgaris in two western locations of this Island. The analyses of the symbiotic nodA and nodC genes reveal the complexity of the symbiovar mediterranense which encompasses strains belonging to several phylogenetic lineages and clusters. The comparison of the nodA and nodC phylogenies showed that the nodC was the most resolutive phylogenetic marker for the delineation of Sinorhizobium symbiovars. Considering that the similarity of this gene within several symbiovars, particularly mediterranense, is around 95 %, the cut-off value for their differentiation should be lower. Considering that a nodC gene cut-off similarity value of around 92 % is accepted for the genus Bradyrhizobium and that the symbiovar concept is identical in all rhizobial genera, we propose to apply this value for symbiovars delineation within all these genera. Therefore, using this cut-off value for the nodC gene analysis of Sinorhizobium symbiovars, we propose to merge the symbiovars aegeanense and fredii into the single symbiovar fredii and to define four novel symbiovars with the names asiaense, culleni, sudanense and tunisiaense., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

Vessel co-option (VCO) is a non-angiogenic mechanism of vascularization that has been associated to anti-angiogenic therapy. In VCO, cancer cells hijack the pre-existing blood vessels and use them to obtain oxygen and nutrients and invade adjacent tissue. Multiple primary tumors and metastases undergo VCO in highly vascularized tissues such as the lungs, liver or brain. VCO has been associated with a worse prognosis. The cellular and molecular mechanisms that undergo VCO are poorly understood. Recent studies have demonstrated that co-opted vessels show a quiescent phenotype in contrast to angiogenic tumor blood vessels. On the other hand, it is believed that during VCO, cancer cells are adhered to basement membrane from pre-existing blood vessels by using integrins, show enhanced motility and a mesenchymal phenotype. Other components of the tumor microenvironment (TME) such as extracellular matrix, immune cells or extracellular vesicles play important roles in vessel co-option maintenance. There are no strategies to inhibit VCO, and thus, to eliminate resistance to anti-angiogenic therapy. This review summarizes all the molecular mechanisms involved in vessel co-option analyzing the possible therapeutic strategies to inhibit this process.

Cajanus cajan L. (guandul) is commonly cultivated in Dominican Republic where this legume is a subsistence crop. Here we identified through MALDI-TOF MS several rhizobial strains nodulating C. cajan in two Dominican locations as Bradyrhizobium yuanmingense. The phylogenetic analysis of recA and glnII housekeeping genes showed that these strains belong to a wide cluster together with the type strain of B. yuanmingense and other C. cajan nodulating strains previously isolated in Dominican Republic. The comparison of genomes from strains representative of different lineages within this cluster support the existence of several genospecies within B. yuanmingense, which is the major microsymbiont of C. cajan in Dominican Republic where it is also nodulated by Bradyrhizobium cajani and Bradyrhizobium pachyrhizi. The analysis of the symbiotic nodC gene showed that the C. cajan nodulating strains from the B. yuanmingense complex belong to two clusters with less than 90% similarity between them. The strains from these two clusters showed nodC gene similarity values lower than 90% with respect to the remaining Bradyrhizobium symbiovars and then they correspond to two new symbiovars for which we propose the names americaense and caribense. The results of the nodC gene analysis also showed that C. cajan is nodulated by the symbiovar tropici, which has been found by first time in this work within the species Bradyrhizobium pachyrhizi. These results confirmed the high promiscuity degree of C. cajan, which is also nodulated by the symbiovar cajani of Bradyrhizobium cajani in Dominican Republic., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier GmbH. All rights reserved.)

This study aimed to isolate lactic acid bacteria (LAB) from the digestive tract, meat and slime of edible snails (Helix lucorum, Helix aspersa and Eobania vermiculata) and investigate their antagonistic activity against Penicillium expansum. They were then characterized for their probiotic potential. Among 900 bacterial isolates, 47 LAB exhibiting anti-P. expansum activity were identified through matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as Levilactobacillus brevis (25), Lactococcus lactis (3), Enterococcus faecium (12), Enterococcus faecalis (4), Enterococcus casseliflavus (1), and Enterococcus mundtii (2). Sixty-two percent of the strains were tolerant to 100 mg/L of lysozyme. Seventy two percent of the isolates were able to survive at pH 3 and most of them tolerate 2.5% bile salt concentration. Moreover, 23% of the strains displayed bile salt hydrolase activity. Interestingly, all strains were biofilm strong producers. However, their auto- and co-aggregation properties were time and pH dependent with high aggregative potentiality at pH 4.5 after 24 h. Remarkably, 48.94% of the strains showed high affinity to chloroform. The safety assessment revealed that the 47 LAB had no hemolytic activity and 64% of them lacked mucin degradation activity. All isolated strains were susceptible to gentamycin, streptomycin, tetracycline, chloramphenicol, and trimethoprim-sulfamethoxazole. Overall, 43 LAB strains showed inhibitory activity against a broad spectrum of pathogenic Gram-positive and gram-negative bacteria, fungi, and yeast. Our findings suggest that L. brevis (EVM12 and EVM14) and Ent. faecium HAS34 strains could be potential candidates for probiotics with interesting antibacterial and anti-P. expansum activities., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Arthropod vectors and parasites are identified morphologically or, more recently, by molecular methods. Both methods are time consuming and require expertise and, in the case of molecular methods, specific devices. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) identification of bacteria has meant a major change in clinical microbiology laboratories because of its simplicity, speed and specificity, and its capacity to identify microorganisms, in some cases, directly from the sample (urine cultures, blood cultures). Recently, MALDI-TOF MS has been shown as useful for the identification of some parasites. On the other hand, the identification of vector arthropods and the control of their populations is essential for the control of diseases transmitted by arthropods, and in this aspect, it is crucial to have fast, simple and reliable methods for their identification. Ticks are blood-sucking arthropods with a worldwide distribution, that behave as efficient vectors of a wide group of human and animal pathogens, including bacteria, protozoa, viruses, and even helminths. They are capable of parasitizing numerous species of mammals, birds and reptiles. They constitute the second group of vectors of human diseases, after mosquitoes. MALDI-TOF MS has been shown as useful for the identification of different tick species, such as Ixodes , Rhipicephalus and Amblyomma . Some studies even suggest the possibility of being able to determine, through MALDI-TOF MS, if the arthropod is a carrier of certain microorganisms. Regarding mosquitoes, the main group of vector arthropods, the possibility of using MALDI-TOF MS for the identification of different species of Aedes and Anopheles has also been demonstrated. In this review, we address the possibilities of this technology for the identification of parasites and arthropod vectors, its characteristics, advantages and possible limitations.

Vaccinium myrtillus is a dwarf shrub of the Ericaceae family with a Palearctic distribution, associated with temperate and cold humid climates. It is widespread on the European continent; on the Iberian Peninsula it is located on Atlantic climate mountains and glacial relicts. In Portugal, we find scattered and interesting populations; however, the majority of them are threatened by climate change and wildfires. Given that, the objective of this study is to determine the rhizospheric and root bacterial communities of this plant in the southernmost regions, and, consequently, its potential range and ability to be used as a biofertilizer. In this work, metabarcoding of 16S rRNA gene showed that the endophytic bacterial diversity is dependent on the plant and selected by it according to the observed alpha and beta diversity. Moreover, a culturomic approach allowed 142 different strains to be isolated, some of them being putative new species. Additionally, some strains belonging to the genera Bacillus, Paenibacillus, Pseudomonas, Paraburkholderia , and Caballeronia showed significant potential to be applied as multifunctional biofertilizers since they present good plant growth-promoting (PGP) mechanisms, high colonization capacities, and an increase in vegetative parameters in blueberry and tomato plants.

Six actinobacterial strains isolated from diverse legume tissues collected in various locations in Spain were characterized to determine their taxonomic status. Using 16S rRNA gene sequencing, the strains were primarily identified as members of the genus Micromonospora with more than 99 % similarity. Digital DNA-DNA hybridization values and average nucleotide identities between the six strains and the nearest type strains confirmed that each strain represented a novel species. Genome sequences were analysed to infer their metabolic profiles, their potential to produce secondary metabolites and plant growth promoting features. Chemotaxonomic and physiological studies were carried out to complete the phenotypic characterization and to distinguish the new Micromonospora species. The genomic and phenotypic characterization of the Micromonospora strains strongly support their classification as representatives of new species with the following names: Micromonospora alfalfae sp. nov., Micromonospora cabrerizensis sp. nov., Micromonospora foliorum sp. nov., Micromonospora hortensis sp. nov., Micromonospora salmantinae sp. nov. and Micromonospora trifolii sp. nov., with the type strains MED01 T , LAH09 T , PSH25 T , NIE111 T , PSH03 T and NIE79 T , respectively.

The taxonomic status of two Gordonia strains, designated BEN371 and CON9 T , isolated from stable foams on activated sludge plants was the subject of a polyphasic study which also included the type strains of Gordonia species and three authenticated Gordonia amarae strains recovered from such foams. Phylogenetic analyses of 16S rRNA gene sequences showed that these isolates formed a compact cluster suggesting a well-supported lineage together with a second branch containing the G. amarae strains. A phylogenomic tree based on sequences of 92 core genes extracted from whole genome sequences of the isolates, the G. amarae strains and Gordonia type strains confirmed the assignment of the isolates and the G. amarae strains to separate but closely associated lineages. Average nucleotide index (ANI) and digital DNA-DNA hybridisation (dDDH) similarities showed that BEN371 and CON9 T belonged to the same species and had chemotaxonomic and morphological features consistent with their assignment to the genus Gordonia . The isolates and the G. amarae strains were distinguished using a range of phenotypic features and by low ANI and dDDH values of 84.2 and 27.0 %, respectively. These data supplemented with associated genome characteristics show that BEN371 and CON9 T represent a novel species of the genus Gordonia . The name proposed for members of this taxon is Gordonia pseudamarae sp. nov. with isolate CON9 T (=DSM 43602 T =JCM 35249 T ) as the type strain.

Renal tubulo-interstitial fibrosis is characterized by the excessive accumulation of extracellular matrix (ECM) in the tubular interstitium during chronic kidney disease. The main source of ECM proteins are emerging and proliferating myofibroblasts. The sources of myofibroblasts in the renal tubular interstitium have been studied during decades, in which the epithelial contribution of the myofibroblast population through the epithelial-to-mesenchymal (EMT) process was assumed to be the major mechanism. However, it is now accepted that the EMT contribution is very limited and other mechanisms such as the proliferation of local resident fibroblasts or the transdifferentiation of endothelial cells seem to be more relevant. Activin receptor-like kinase 1 (ALK1) is a type I receptor which belongs to the transforming growth factor beta (TGF- β ) superfamily, with a key role in tissue fibrosis and production of ECM by myofibroblast. Predominantly expressed in endothelial cells, ALK1 also plays an important role in angiogenesis and vessel maturation, but the relation of these processes with kidney fibrosis is not fully understood. We show that after 3 days of unilateral ureteral obstruction (UUO), ALK1 heterozygous mice ( Alk1 +/- ) display lower levels of kidney fibrosis associated to a lower number of myofibroblasts. Moreover, Alk1 +/- mice have a lower degree of vascular rarefaction, showing improved peritubular microvasculature after UUO. All these data suggest an important role of ALK1 in regulating vascular rarefaction and emergence of myofibroblasts., Competing Interests: 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., (Copyright © 2022 Martínez-Salgado, Sánchez-Juanes, López-Hernández and Muñoz-Félix.)

The original type strains of Agrobacterium radiobacter and Agrobacterium tumefaciens recorded in the eighth edition of Bergey's Manual of Determinative Bacteriology published in 1974 were NCIB 9042 T and ATCC 4720 T , respectively. However, in the list of the valid names of bacteria compiled in 1980, both strains were changed, A. radiobacter NCIB 9042 T to ATCC 19358 T and A. tumefaciens ATCC 4720 T to ATCC 23308 T . These changes were unjustified, particularly in the case of A. tumefaciens whose type strain was replaced by another strain from the same collection, although the original type strain ATCC 4720 T was never lost and it is currently available in several culture collections. Therefore, we request that the type strain of A. tumefaciens be corrected from ATCC 23308 T to ATCC 4720 T .

Acute kidney injury (AKI) is a serious syndrome with increasing incidence and health consequences, and high mortality rate among critically ill patients. Acute kidney injury lacks a unified definition, has ambiguous semantic boundaries, and relies on defective diagnosis. This, in part, is due to the absence of biomarkers substratifying AKI patients into pathophysiological categories based on which prognosis can be assigned and clinical treatment differentiated. For instance, AKI involving acute tubular necrosis (ATN) is expected to have a worse prognosis than prerenal, purely hemodynamic AKI. However, no biomarker has been unambiguously associated with tubular cell death or is able to provide etiological distinction. We used a cell-based system to identify TCP1-eta in the culture medium as a noninvasive marker of damaged renal tubular cells. In rat models of AKI, TCP1-eta was increased in the urine co-relating with renal cortical tubule damage. When kidneys from ATN rats were perfused in situ with Krebs-dextran solution, a portion of the urinary TCP1-eta protein content excreted into urine disappeared, and another portion remained within the urine. These results indicated that TCP1-eta was secreted by tubule cells and was not fully reabsorbed by the damaged tubules, both effects contributing to the increased urinary excretion. Urinary TCP1-eta is found in many etiologically heterogeneous AKI patients, and is statistically higher in patients partially recovered from severe AKI. In conclusion, urinary TCP1-eta poses a potential, substratifying biomarker of renal cortical damage associated with bad prognosis., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Several artisanal cheeses are elaborated in European countries, being commonly curdled with rennets of animal origin. However, in some Spanish regions some cheeses of type "Torta" are elaborated using Cynara cardunculus L. rennets. Two of these cheeses, "Torta del Casar" and "Torta de Trujillo", are elaborated in Cáceres province with ewe's raw milk and matured over at least 60 days without starters. In this work, we identified the lactic acid bacteria present in these cheeses using MALDI-TOF MS and pheS gene analyses, which showed they belong to the species Lactobacillus curvatus , Lactobacillus diolivorans , Lactobacillus paracasei , Lactobacillus plantarum , Lactobacillus rhamnosus , Lactococcus lactis and Leuconostoc mesenteroides . The pheS gene analysis also allowed the identification of the subspecies La. plantarum subsp. plantarum , La. paracasei subsp. paracasei and Le. mesenteroides subsp. jonggajibkimchii . Low similarity values were found in this gene for some currently accepted subspecies of Lc. lactis and for the two subspecies of La. plantarum, and values near to 100% for the subspecies of Le. mesenteroides and La. paracasei . These results, which were confirmed by the calculated ANIb and dDDH values of their whole genomes, showed the need to revise the taxonomic status of these species and their subspecies.

Phaseolus vulgaris is a legume indigenous to America which is currently cultivated in Europe including countries located at the Southeast of this continent, such as Croatia, where several local landraces are cultivated, most of them of Andean origin. In this work we identify at species and symbiovar levels several fast-growing strains able to form effective symbiosis with P. vulgaris in different Croatian soils. The identification at species level based on MALDI-TOF MS and core gene sequence analysis showed that most of these strains belong to the species R. leguminosarum, R. hidalgonense and R. pisi. In addition, several strains belong to putative new species phylogenetically close to R. ecuadorense and R. sophoriradicis. All Croatian strains belong to the symbiovar phaseoli and harbour the α and γ nodC alleles typical for American strains of this symbiovar. Nevertheless, most of Croatian strains harboured the γ nodC gene allele supporting its Andean origin since it is also dominant in other European countries, where Andean cultivars of P. vulgaris are traditionally cultivated, as occurs in Spain. The only strains harbouring the α nodC allele belong to R. hidalgonense and R. pisi, this last only containing the symbiovars viciae and trifolii to date. This is the first report about the presence in Europe of the species R. hidalgonense, the nodulation of P. vulgaris by R. pisi and the existence of the symbiovar phaseoli within this species. These results significantly increase the knowledge of the biogeography of Rhizobium-P. vulgaris symbiosis., (Copyright © 2019. Published by Elsevier GmbH.)

Introduction: We have developed a MALDI-TOF-mediated phenotypic method, which determines antibiotic susceptibility (AS) from positive blood cultures (BCs) in 2h. We developed a software for process automation. We report results on Escherichia coli-positive BCs with cefotaxime (CTX) and ciprofloxacin (CIP)., Methods: We studied CIP and CTX activity in 18 and 17 real E. coli-positive BCs, and in 56 and 45 spiked BCs, respectively. Positive BCs were incubated for 2h without any antibiotics, and with 2mg/l and 4mg/l of CIP and CTX. The extraction was performed using ethanol/formic acid. Spectra were processed with specifically developed software which compares the peaks' intensity and the size of specific peaks., Results: The set cut-off point was a 3-fold decrease in the summation of all peaks and/or the 5382m/z peak value (ribosomal protein L34). In simulated BCs, the correlation of CIP 2mg/l and 4mg/l with Etest ® was 94.6% and 98.2%, respectively; for CTX 2mg/l and 4mg/l, this correlation was 95.6%. In real BCs, the correlations were 100% for CIP (2mg/l and 4mg/l) and 88.2% and 94.1% for CTX 2mg/l and 4mg/l, respectively. Resistant isolates were always correctly classified., Conclusion: This method provides accurate, fast and inexpensive AS information. The method can be automated, making it easier to implement in a microbiology laboratory routine., (Copyright © 2018 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.)

Phaseolus vulgaris and Lens culinaris are two legumes with different distribution centers that were introduced in Spain at different times, but in some regions L. culinaris has been traditionally cultivated and P. vulgaris did not. Here we analysed the rhizobia isolated from nodules of these two legumes in one of these regions. MALDI-TOF MS analysis showed that all isolated strains matched with Rhizobium laguerreae and the phylogenetic analysis of rrs, atpD and recA genes confirmed these results. The phylogenetic analysis of these core genes allowed the differentiation of several groups within R. laguerreae and unexpectedly, strains with housekeeping genes identical to that of the type strain of R. laguerreae presented some differences in the rrs gene. In some strains this gene contains an intervening sequence (IVS) identical to that found in Rhizobium strains nodulating several legumes in different geographical locations. The atpD, recA and nodC genes of all isolated strains clustered with those of strains nodulating L. culinaris in its distribution centers, but not with those nodulating P. vulgaris in theirs. Therefore, all these strains belong to the symbiovar viciae, including those isolated from P. vulgaris, which in the studied region established effective symbiosis with the common endosymbiont of L. culinaris, instead to with its common endosymbiont, the symbiovar phaseoli. These results are particularly interesting for biogeography studies, because they showed that, due its high promiscuity degree, P. vulgaris is able to establish symbiosis with local symbiovars well established in the soil after centuries of cultivation with other legumes., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Urine is a biological fluid that can be collected noninvasively in relatively large quantities which can be used for the search of biomarkers of disease, both diseases of the urological tract and systemic diseases. One of the most important aspects in proteomic studies is sample treatment before further analysis. Methods of preparation of a urine sample depend on the techniques that will be used later for separation and identification of the proteins. Also, urine preparation should be as simple as possible to increase reproducibility. Normal urine has a much diluted protein concentration with a high-salt content, which interferes with proteomic analysis. Thus, an initial step in the handling of urine sample should be to concentrate and eliminate salts. As range of protein concentrations in urine spans several orders of magnitude, effective proteomic analyses require either removal of most abundant protein or enrichment of the less abundant ones. In this chapter, we discuss the aspects related to the collection and treatment of urine for proteomic studies.

MALDI-TOF mass spectrometry is now a routine resource in Clinical Microbiology, because of its speed and reliability in the identification of microorganisms. Its performance in the identification of bacteria and yeasts is perfectly contrasted. The identification of mycobacteria and moulds is more complex, due to the heterogeneity of spectra within each species. The methodology is somewhat more complex, and expanding the size of species libraries, and the number of spectra of each species, will be crucial to achieve greater efficiency. Direct identification from blood cultures has been implemented, since its contribution to the management of severe patients is evident, but its application to other samples is more complex. Chromogenic media have also contributed to the rapid diagnosis in both bacteria and yeast, since they accelerate the diagnosis, facilitate the detection of mixed cultures and allow rapid diagnosis of resistant species., (Copyright © 2017 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.)

The species Seliberia stellata was described in 1963 and the name validly published in 1980. Its type strain, INMI N-9(T), was deposited in the VKM collection by one of the authors reporting its 5S rRNA gene sequence. Based on the analysis of this sequence, the currently distributed strains VKM B-1340 and CECT 7960 are not the original type strain of Seliberia stellata. A 16S rRNA gene sequence analysis of strain CECT 7960 had previously shown that this strain belongs to the species Bradyrhizobium betae, and this result was confirmed in the present paper by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS analysis for both CECT 7960 and VKM B-1340. Therefore, we propose that the Judicial Commission consider the following. (1) That the organism currently deposited as VKM B-1340 and CECT 7960 be recognized as a member of the species Bradyrhizobium betae. (2) That the organism deposited as VKM B-1340 and CECT 7960 does not represent the type strain of the species Seliberia stellata. (3) To place the species name Seliberia stellataAristovskaya and Parinkina 1963 (Approved Lists 1980) on the list of rejected names if a suitable replacement strain, or a neotype, cannot be found within two years of publication of this Request (Rule 18c). (4) To place the genus name SeliberiaAristovskaya and Parinkina 1963 (Approved Lists 1980) on the list of rejected names (Recommendation 20d) if a suitable replacement type strain or a neotype for the type species of the genus SeliberiaAristovskaya and Parinkina 1963 (Approved Lists 1980) is not identified as indicated in point (3).

Nephrotoxicity limits the therapeutic efficacy of the antineoplastic drug cisplatin. Due to dosage adjustment and appropriate monitoring, most therapeutic courses with cisplatin produce no or minimal kidney damage. However, we studied whether even sub-nephrotoxic dosage of cisplatin poses a potential risk for the kidneys by predisposing to acute kidney injury (AKI), specifically by lowering the toxicity threshold for a second nephrotoxin. With this purpose rats were treated with a single sub-nephrotoxic dosage of cisplatin (3mg/kg, i.p.) and after two days, with a sub-nephrotoxic regime of gentamicin (50mg/kg/day, during 6 days, i.p.). Control groups received only one of the drugs or the vehicle. Renal function and renal histology were monitored throughout the experiment. Cisplatin treatment did not cause any relevant functional or histological alterations in the kidneys. Rats treated with cisplatin and gentamicin, but not those under single treatments, developed an overt renal failure characterized by both renal dysfunction and massive tubular necrosis. In addition, the urinary excretion of fumarylacetoacetase was increased in cisplatin-treated animals at subtoxic doses, which might be exploited as a cisplatin-induced predisposition marker. In fact, the urinary level of fumarylacetoacetase prior to the second nephrotoxin correlated with the level of AKI triggered by gentamicin in predisposed animals., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Chicha is a drink prepared in several Andean countries from Inca's times by maize fermentation. Currently this fermentation is carried out in familiar artesanal "chicherías" that make one of the most known types of chicha, the "chicha de jora". In this study we isolate and identify the yeasts mainly responsible of the fermentation process in this type of chicha in 10 traditional "chicherías" in Cusco region in Peru. We applied by first time MALDI-TOF MS analysis for the identification of yeast of non-clinic origin and the results showed that all of yeast strains isolated belong to the species Saccharomyces cerevisiae. These results agree with those obtained after the analysis of the D1/D2 and 5.8S-ITS regions. However the chicha strains have a phenotypic profile that differed in more than 40% as compared to that of current S. cerevisiae strains. To the best of our knowledge this is the first report concerning the yeasts involved in chicha fermentation., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

Genus Bradyrhizobium includes slow growing bacteria able to nodulate different legumes as well as species isolated from plant tumours. The slow growth presented by the members of this genus and the phylogenetic closeness of most of its species difficults their identification. In the present work we applied for the first time Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS) to the analysis of Bradyrhizobium species after the extension of MALDI Biotyper 2.0 database with the currently valid species of this genus. With this methodology it was possible to identify strains belonging to phylogenetically closely related species of genus Bradyrhizobium allowing the discrimination among species with rrs gene identities higher than 99%. The application of MALDI-TOF MS to strains isolated from nodules of different Lupinus species in diverse geographical locations allowed their correct identification when comparing with the results of rrs gene and ITS analyses. The nodulation of Lupinus gredensis, an endemic species of the west of Spain, by B. canariense supports the European origin of this species., (Copyright © 2013. Published by Elsevier GmbH.)

Background: Recently, bacterial identification by MALDI-TOF MS has acquired a high relevance in terms of speed and reliability. Conventional mycological identification has some disadvantages: it is frequently slow, reliability is sometimes low, and an extensive experience is required. The risk population for fungal infections, and therefore their clinical significance has progressively increased in recent years., Methods: 153 yeast and mould clinical isolates were analyzed by MALDI-TOF MS and conventional identification. When both methods were discrepant to the genus or species level, ITS-2 sequencing was performed. Results. The correlation in yeasts identification between conventional identification methods and MALDI-TOF MS was extremely high (99.2% to the species level and 100% to the genus level). The only discrepancy was checked by ITS-2 sequencing and confirmed the MALDI-TOF identification. The correlation in moulds identification was more heterogeneous. 68.7% of the isolates showed correlation at least to the genus level and 40.6% to the species level. Therefore, the correlation between conventional identification and MALDI-TOF MS in fungal identification was, in whole, 87% to the species level, and 93.5% to the genus level., Conclusions: Identification of fungi by MALDI-TOF MS is reliable and shows potential advantages over conventional identification methods.

Urine is a suitable biological fluid to look for markers of physiological and pathological processes, including renal and nonrenal diseases. In addition, it is an optimal body sample for diagnosis, because it is easily obtained without invasive procedures and can be sampled in large quantities at almost any time. Rats are frequently used as a model to study human diseases, and rat urine has been analyzed to search for disease biomarkers. The normal human urinary proteome has been studied extensively, but the normal rat urinary proteome has not been studied in such depth. In light of this, we were prompted to analyze the normal rat urinary proteome using three complementary proteomics platforms: SDS-PAGE separation, followed by LC-ESI-MS/MS; 2DE, followed by MALDI-TOF-TOF and 2D-liquid chromatography-chromatofocusing, followed by LC-ESI-Q-TOF. A total of 366 unique proteins were identified, of which only 5.2% of unique proteins were identified jointly by the three proteomics platforms used. This suggests that simultaneous proteomics techniques provide complementary and nonredundant information. Our analysis affords the most extensive rat urinary protein database currently available and this may be useful in the study of renal physiology and in the search for biomarkers related to renal and nonrenal diseases., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Aim of the Study: MALDI-TOF mass spectrometry (MS) is becoming a major resource in the Clinical Microbiology laboratory. Results on some groups of microorganisms are still controversial. We have studied the reliability of MALDI-TOF MS for the identification of anaerobic clinical isolates was studied compared to conventional biochemical methods, with rRNA 16S sequencing being used as a reference when discrepancies arose., Material and Methods: A total of 126 anaerobic bacteria clinical isolates were studied by using API20A kits (bioMérieux, Marcy l'Étoile, France) and MALDI-TOF MS (Autoflex II, Bruker Daltonics, Germany), and using the data library BioTyper 2.0 (Bruker Daltonics, Germany). When discrepancies arose, or MALDI-TOF MS was not able to identify any microorganism, rRNA 16S sequencing was used as the reference standard., Results: The biochemical method and MALDI-TOF MS agreed in identifying 60.9% of isolates at species level, and 20.3% of isolates at genus level. Among the 48 discrepancies observed, rRNA 16S sequencing supported MALDI-TOF MS identification, at species level, in 32 isolates (66.7%), and in 8 isolates (16.7%) at genus level. rRNA 16S sequencing supported biochemical identification in only two isolates (4.2%) at species level, and in 26 isolates (54.2%) at genus level. The eight isolates for which MALDI-TOF MS did not manage to identify, or the identification obtained was rejected by sequencing, belonged to species that are still not added to the BioTyper II data library., Conclusions: Results obtained in this study show that, overall, MALDI-TOF MS identification of anaerobic bacteria is more reliable than identification obtained by conventional biochemical methods (24% more correct identifications at species level). The number of major errors (incorrect identification at the genus level) is also 2.5-times lower. Moreover, all the major errors obtained by MALDI-TOF MS were due to the absence of some species in the data library. Thus, when data libraries are more complete, reliability differences between both methods will probably be even higher., (Copyright © 2011 Elsevier España, S.L. All rights reserved.)

Multidrug resistance (MDR), a ubiquitous phenomenon conserved from bacteria to humans, causes serious problems in the treatment of human cancers and infections of bacterial and fungal origin. The development of MDR in yeast is frequently associated with gain-of-function mutations in the Zn(2)Cys(6) transcription factors activating the expression of several plasma membrane exporters. In the aerobic yeast Kluyveromyces lactis the Zn(2)Cys(6) transcription factor KlPdr1p is involved in the control of multidrug resistance. The aim of the present study was to identify the changes in K. lactis proteome of the Klpdr1Δ deletion mutant compared with the wild-type expressing the KlPDR1 gene from a multicopy plasmid. A total of 15 differentially expressed proteins, out of 20 spots with different intensities detected, were identified. In the Klpdr1Δ deletion mutant, the increase in the abundance of proteins involved in carbohydrate metabolism (mainly glycolysis/gluconeogenesis) was observed. Most of the proteins overexpressed in the wild type strain containing the KlPDR1 gene on multicopy plasmid were involved in the stress defence and redox homeostasis. The results indicate a close connection between MDR and oxidative stress response associated with the post-translational mechanisms regulating the levels of active forms of proteins involved in K. lactis MDR., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) is rapidly becoming a new routine resource in Clinical Microbiology laboratories. Its usefulness for bacterial identification is now generally accepted, although there is still some reluctance as regards specific bacterial groups and some other microorganisms, such as moulds. There are other potential applications of this technology in Clinical Microbiology, which are beginning to be developed. A review is presented on the current data on the identification of microorganisms, including the most problematic groups, such as mycobacteria, anaerobic bacteria and moulds. We also analyse its applications for direct sample identification, its impact on pathogenic characteristics of microorganisms, and its potential epidemiological applications. Finally, we review the studies published on its applications for determining antimicrobial susceptibility, and its applications on amplicons, instead of microorganism protein extracts., (Copyright © 2011 Elsevier España, S.L. All rights reserved.)

Ras small GTPases function as transducers of extracellular signals regulating cell survival, growth and differentiation. There are three major ras isoforms: H-, N- and K-Ras. To improve the understanding of H- and N-Ras protein signalling networks, we compared total proteome changes in mouse embryonic fibroblasts knock out for H-ras and/or N-ras, using proteomics tools combining 2DE, semi-quantitative image analysis, in-gel trypsin digestion and mass spectrometry. There are four up-regulated proteins due to the loss of expression of H-Ras (including cyclin-dependent kinase inhibitor 2A) and eight down-regulated (including stress-70 protein, dihydropyrimidinase-related-protein 3, heat shock cognate 71 kDa protein, tropomyosin beta chain, Rho GDP-dissociation inhibitor 1) and six up-regulated proteins (e.g. leukocyte elastase inhibitor A, L-lactate dehydrogenase B chain, c-Myc-responsive protein Rcl, interleukin-1 receptor antagonist protein) due to the loss of expression of both N- and H-Ras. Most of these proteins are related to Ras signalling in one way or another. Changes in expression of some of these proteins were further confirmed by Western blot. This proteomic comparative analysis from loss of function of H- and N-Ras knockout fibroblasts yields interpretable data to elucidate the differential protein expression, and contributes to evaluate the possibilities for physiological and therapeutic targets., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Family Rhizobiaceae includes fast growing bacteria currently arranged into three genera, Rhizobium, Ensifer and Shinella, that contain pathogenic, symbiotic and saprophytic species. The identification of these species is not possible on the basis of physiological or biochemical traits and should be based on sequencing of several genes. Therefore alternative methods are necessary for rapid and reliable identification of members from family Rhizobiaceae. In this work we evaluated the suitability of Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS) for this purpose. Firstly, we evaluated the capability of this methodology to differentiate among species of family Rhizobiaceae including those closely related and then we extended the database of MALDI Biotyper 2.0 including the type strains of 56 species from genera Rhizobium, Ensifer and Shinella. Secondly, we evaluated the identification potential of this methodology by using several strains isolated from different sources previously identified on the basis of their rrs, recA and atpD gene sequences. The 100% of these strains were correctly identified showing that MALDI-TOF MS is an excellent tool for identification of fast growing rhizobia applicable to large populations of isolates in ecological and taxonomic studies.

Background: MALDI-TOF mass spectrometry (MS) is a reliable method for bacteria identification. Some databases used for this purpose lack reference profiles for Brucella species, which is still an important pathogen in wide areas around the world. We report the creation of profiles for MALDI-TOF Biotyper 2.0 database (Bruker Daltonics, Germany) and their usefulness for identifying brucellae from culture plates and blood cultures., Methodology/principal Findings: We created MALDI Biotyper 2.0 profiles for type strains belonging to B. melitensis biotypes 1, 2 and 3; B. abortus biotypes 1, 2, 5 and 9; B. suis, B. canis, B ceti and B. pinnipedialis. Then, 131 clinical isolates grown on plate cultures were used in triplicate to check identification. Identification at genus level was always correct, although in most cases the three replicates reported different identification at species level. Simulated blood cultures were performed with type strains belonging to the main human pathogenic species (B. melitensis, B. abortus, B. suis and B. canis), and studied by MALDI-TOF MS in triplicate. Identification at genus level was always correct., Conclusions/significance: MALDI-TOF MS is reliable for Brucella identification to the genus level from culture plates and directly from blood culture bottles.

Introduction: The methods routinely used for bacterial identification in Clinical Microbiology Laboratory, although miniaturized and automated, are still based on the same basic principles as classical identification methods. Nevertheless, technological advances are emerging which could modify these routine methods. We report a comparative study between conventional identification methods and mass spectrometry MALDI-TOF (MS MALDI-TOF) for bacterial identification in the Clinical Microbiology Laboratory., Methods: We analysed 294 facultative anaerobic and aerobic isolates (65 Gram positives and 229 Gram negatives), obtained from different clinical samples, using conventional microbiological methods (Wider, Fco. Soria Melguizo, Madrid, Spain; Vitek-2, APIStaph, API 20 Strep, API Coryne and API NH, bioMérieux, Marcy L'Etoile, France) and an Autoflex III MS with a MALDI-TOF device (Bruker Daltonics GmbH, Leipzig, Germany). Salmonella isolates were also typed by using specific sera. Isolates identified with a confidence rate <95% were checked by using API systems. Isolates which were not accurately identified by API systems were rejected. MS MALDI-TOF identification is automatically scored by the system software between 1 and 3 points. Isolates with scores <1.5 were classified as unreliable. Correlation between both identifications was classified as correlation at the species level, at the genus level or no correlation., Results: Correlation at the species level in Gram positives was 100%. Correlation in Gram negatives was 87.7% at the species level and 97.7% at the genus level. There was no correlation in 2.2% of Gram negatives studied. Identification failures occurred in the genera Raoultella and Acinetobacter, in Stenotrophomonas maltophilia and in Francisella tularensis., Conclusion: Bacterial clinical isolates identification obtained by MS MALDI-TOF shows excellent correlation with identification obtained by conventional microbiological methods. Moreover, MS MALDI-TOF allows the identification of bacteria from colonies grown on agar culture plates in just a few minutes, with a very simple methodology and hardly any consumable costs, although the financial costs of purchasing the device can be high., (Copyright © 2009 Elsevier España, S.L. All rights reserved.)

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been suggested as a reliable method for bacterial identification from cultures. Direct analysis of clinical samples might increase the usefulness of this method, shortening the time for microorganism identification. We compared conventional methods for the diagnosis of urinary tract infections (UTIs) and identification of the urinary tract pathogens (automated screening, plate cultures, and identification based on biochemical characteristics) and a fast method based on conventional screening and MALDI-TOF MS. For this latter method, 4 ml of urine was centrifuged at a low-revolution setting (2,000 x g) to remove leukocytes and then at high revolutions (15,500 x g) to collect bacteria. The pellet was washed and then applied directly to the MALDI-TOF MS plate. Two hundred sixty urine samples, detected as positive by the screening device (UF-1000i), were processed by culture and MALDI-TOF MS. Twenty samples were positive in the screening device but negative in culture, and all of them were also negative by MALDI-TOF MS. Two-hundred thirty-five samples displayed significant growth of a single morphological type in culture. Two-hundred twenty of them showed bacterial growth of >10(5) CFU/ml. Microorganism identifications in this group were coincident at the species level in 202 cases (91.8%) and at the genus level in 204 cases (92.7%). The most frequent microorganism was Escherichia coli (173 isolates). MALDI-TOF MS identified this microorganism directly from the urine sample in 163 cases (94.2%). Our results show that MALDI-TOF MS allows bacterial identification directly from infected urine in a short time, with high accuracy, and especially when Gram-negative bacteria with high bacterial counts are involved.

Several components of milk fat globule membranes (MFGMs) have been reported to display beneficial health properties and some of them have been implicated in the defense of newborns against pathogens. These observations prompted us to determine the glycosphingolipid content of MFGMs and their interaction with pathogens. A comparative study with whole milk components was also carried out. Milk fat globules and MFGMs were isolated from milk. Gangliosides and neutral glycosphingolipids were obtained from MFGMs and whole milk and their fatty acid contents were determined by gas chromatography-mass spectrometry (GC-MS). MFGMs and whole milk showed similar ganglioside and neutral glycosphingolipid contents, with whole milk having more GM3 and glucosylceramide and less GD3, O-acetyl GD3, O-acetyl GT3, and lactosylceramide. The fatty acid content of gangliosides from both sources showed a similar composition. However, the neutral glycosphingolipid fatty acid content seemed to be quite different. Whole milk had fewer very-long-chain fatty acids (18.1% vs. 46.4% in MFGMs) and more medium-chain and unsaturated C18:1 and C18:2 fatty acids. Milk fat globules, MFGMs, lactosylceramide, and gangliosides GM3 and GD3 were observed to bind enterotoxigenic Escherichia coli strains. Furthermore, bacterial hemagglutination was inhibited by MFGMs and glycosphingolipids.