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Novel N-substituted azaindoles have been discovered as PIM1 inhibitors. X-ray structures have played a significant role in orienting the chemistry effort in the initial phase of hit confirmation. Disclosure of an unconventional binding mode for 1 and 2, as demonstrated by X-ray crystallography, is presented and was an important factor in selecting and advancing a lead series., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

N-Substituted azaindoles have been discovered as pan-PIM kinase inhibitors. Initial SAR, early ADME and PK/PD data of a series of compounds is described and led to the identification of promising pan-PIM inhibitors which validated our interest in the 7-azaindole scaffold and led us to pursue the identification of a clinical candidate., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Tumor cells display fundamental changes in metabolism and nutrient uptake in order to utilize additional nutrient sources to meet their enhanced bioenergetic requirements. Glutamine (Gln) is one such nutrient that is rapidly taken up by tumor cells to fulfill this increased metabolic demand. A vital step in the catabolism of glutamine is its conversion to glutamate by the mitochondrial enzyme glutaminase (GLS). This study has identified GLS a potential therapeutic target in breast cancer, specifically in the basal subtype that exhibits a deregulated glutaminolysis pathway. Using inducible shRNA mediated gene knockdown, we discovered that loss of GLS function in triple-negative breast cancer (TNBC) cell lines with a deregulated glutaminolysis pathway led to profound tumor growth inhibition in vitro and in vivo. GLS knockdown had no effect on growth and metabolite levels in non-TNBC cell lines. We rescued the anti-tumor effect of GLS knockdown using shRNA resistant cDNAs encoding both GLS isoforms and by addition of an α-ketoglutarate (αKG) analog thus confirming the critical role of GLS in TNBC. Pharmacological inhibition of GLS with the small molecule inhibitor CB-839 reduced cell growth and led to a decrease in mammalian target of rapamycin (mTOR) activity and an increase in the stress response pathway driven by activating transcription factor 4 (ATF4). Finally, we found that GLS inhibition synergizes with mTOR inhibition, which introduces the possibility of a novel therapeutic strategy for TNBC. Our study revealed that GLS is essential for the survival of TNBC with a deregulated glutaminolysis pathway. The synergistic activity of GLS and mTOR inhibitors in TNBC cell lines suggests therapeutic potential of this combination for the treatment of vulnerable subpopulations of TNBC.

The association of Comamonas kerstersii with peritonitis resulting from the presence of perforated appendix has previously been described by our research team. In the present study, we describe the isolation of this microorganism from two forms of unusual presentations of C. kerstersii infection not previously described in the literature: localized intra-abdominal infection (psoas abscess) and pelvic peritonitis.

South American Camelids have an increasing relevance in local economies, worldwide. These animals are bred for their meat, fur and as companion and therapy animals. Thus, their sanitary status should be well-established. According to the OIE (World Organization for Animal Health), respiratory infections mainly produced by Pasteurella spp. have been reported for camelids. It has been stated that this microorganism causes a mild disease, although many authors report it is an important cause of mortality among alpacas. Nevertheless, the incidence of infection by Pasteurella spp. in camelids still needs to be investigated. The aim of the present study was to analyze the occurrence of nasopharyngeal colonization of Lama glama by respiratory bacteria, and to assess the usefulness of serological tests for clinical diagnosis. The colonization was studied by culture techniques carried out with material taken by nasopharyngeal swabs. Bacterial isolates were first phenotypically characterized and then identified by MALDI/TOF-MS. The presence of specific serum antibodies was studied by ELISA and Western blot. In the present work Pasteurella spp. was not found. Nevertheless, we report for the first time, the colonization of L. glama by bacteria of the Acinetobacter lwoffii , at a reliable level in 19.4% of the animals. Acinetobacter species are found in different environmental sources, as well as vegetables, animals, and humans, and their role in infections has recently gained relevance. The results presented herein contribute to a better understanding of the respiratory microbiota in camelids, and increase the knowledge about environmental distribution of Acinetobacter non- baumanii species. Given that these respiratory bacteria might be the cause of infection among cattle, and even humans, this report highlights the need for further research.

Objectives: Patterns of antimicrobial susceptibility in Actinomyces and related genera are very limited in the literature. Data of predominant susceptibility profiles could contribute to the establishment of an accurate empirical treatment., Methods: A total of 113 isolates from clinical samples were included in this study. Each isolate was identified using phenotypic methods and MALDI-TOF/MS. When discrepancies were observed, 16S rRNA gene sequencing was performed. The minimum inhibitory concentrations (MICs) of nine antimicrobial agents (penicillin, ceftriaxone, linezolid, tetracycline, clindamycin, erythromycin, ciprofloxacin, levofloxacin and vancomycin) were tested against the species Actinotignum schaalii (n=23), Actinomyces turicensis (n=18), Actinomyces europaeus (n=13), Actinomyces naeslundii/Actinomyces viscosus group (n=12), Actinomyces urogenitalis (n=11), Actinomyces radingae (n=11), Actinomyces neuii (n=9), Actinomyces odontolyticus (n=8), Bifidobacterium scardovii (n=3), Actinomyces graevenitzii (n=2), Alloscardovia omnicolens (n=2) and Varibaculum cambriense (n=1)., Results: All of the isolates were susceptible to penicillin, ceftriaxone, vancomycin and linezolid. Almost all of the A. urogenitalis isolates (8/11) were resistant to clindamycin and showed susceptibility to erythromycin, suggesting an L-phenotype, however no determinants of clindamycin resistance (lnu and lsa genes) were detected by PCR. High MIC values to quinolones were observed in 54/113 isolates (47.8%). All of the A. urogenitalis isolates were highly resistant to ciprofloxacin and levofloxacin., Conclusions: These data highlight the importance of ongoing surveillance to provide relevant information for empirical management of infections caused by these organisms., (Copyright © 2017 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.)

Objective: To evaluate the performance of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) by using 190 Catalase-negative Gram-Positive Cocci (GPC) clinical isolates., Methods: All isolates were identified by conventional phenotypic tests following the proposed scheme by Ruoff and Christensen and MALDI-TOF MS (Bruker Daltonics, BD, Bremen, Germany). Two different extraction methods (direct transfer formic acid method on spot and ethanol formic acid extraction method) and different cut-offs for genus/specie level identification were used. The score cut-offs recommended by the manufacturer (≥ 2.000 for species-level, 1.700 to 1.999 for genus level and <1.700 no reliable identification) and lower cut-off scores (≥1.500 for genus level, ≥ 1.700 for species-level and score <1.500 no reliable identification) were considered for identification. A minimum difference of 10% between the top and next closest score was required for a different genus or species. MALDI-TOF MS identification was considered correct when the result obtained from MS database agreed with the phenotypic identification result. When both methods gave discordant results, the 16S rDNA or sodA genes sequencing was considered as the gold standard identification method. The results obtained by MS concordant with genes sequencing, although discordant with conventional phenotyping, were considered correct. MS results discordant with 16S or sod A identification were considered incorrect., Results: Using the score cut-offs recommended by the manufacturer, 97.37% and 81.05% were correctly identified to genus and species level, respectively. On the other hand, using lower cut-off scores for identification, 97.89% and 94.21% isolates were correctly identified to genus and species level respectively by MALDI-TOF MS and no significant differences between the results obtained with two extraction methods were obtained., Conclusion: The results obtained suggest that MALDI-TOF MS has the potential of being an accurate tool for Catalase-negative GPC identification even for those species with difficult diagnosis as Helcococcus , Abiotrophia , Granulicatella , among others. Nevertheless, expansion of the library, especially including more strains with different spectra on the same species might overcome potential "intraspecies" variability problems. Moreover, a decrease of the identification scores for species and genus-level identification must be considered since it may improve the MALDI-TOF MS accuracy.

Acinetobacter johnsonii rarely causes human infections. While most A. johnsonii isolates are susceptible to virtually all antibiotics, strains harboring a variety of β-lactamases have recently been described. An A. johnsonii Aj2199 clinical strain recovered from a hospital in Buenos Aires produces PER-2 and OXA-58. We decided to delve into its genome by obtaining the whole genome sequence of the Aj2199 strain. Genome comparison studies on Aj2199 revealed 240 unique genes and a close relation to strain WJ10621, isolated from the urine of a patient in China. Genomic analysis showed evidence of horizontal genetic transfer (HGT) events. Forty-five insertion sequences and two intact prophages were found in addition to several resistance determinants such as blaPER-2, blaOXA-58, blaTEM-1, strA, strB, ereA, sul1, aacC2 and a new variant of blaOXA-211, called blaOXA-498. In particular, blaPER-2 and blaTEM-1 are present within the typical contexts previously described in the Enterobacteriaceae family. These results suggest that A. johnsonii actively acquires exogenous DNA from other bacterial species and concomitantly becomes a reservoir of resistance genes., Competing Interests: The authors have declared that no competing interests exist.

We report here an exceptional pattern of atypical lentiginous melanocytic proliferation within an adenoma, leading to focal lamina propria infiltration and pulmonary metastasis, which was considered as primary colonic mucosal melanoma (MM) in a Caucasian patient. Such case illustrates the diagnosis criteria required to differentiate primary MM from colonic metastasis of melanoma, including the absence of past history of other primary melanoma, a unique colonic and abdominal lesion with predominant features of in situ lentiginous MM and a very focal and unique invasive area without other digestive tract or abdominal localization. This tumor displayed a KIT exon 11 mutation leading to a unique combination of p.I571M and p.D572G deleterious amino acid changes. Such pattern also favors the diagnosis as KIT appears as a master oncogenic player in MM oncogenesis., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) to identify 396 Nonfermenting Gram-Negative Bacilli clinical isolates was evaluated in comparison with conventional phenotypic tests and/or molecular methods. MALDI-TOF MS identified to species level 256 isolates and to genus or complex level 112 isolates. It identified 29 genera including uncommon species., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Empedobacter (formerly Wautersiella) falsenii comb. nov. strain Wf282 was isolated from a cervical neck abscess sample from an 18-year-old female patient. The isolate was resistant to many antibiotics, including meropenem and colistin. The total DNA from the multidrug-resistant E. falsenii comb. nov. Wf282 clinical isolate was sequenced., (Copyright © 2015 Traglia et al.)

Acinetobacter sp. strain A47, which has been recovered from several soft tissue samples from a patient undergoing reconstructive surgery due to a traumatic amputation, was categorized as a taxonomically unique bacterial strain. The molecular analysis based on three housekeeping protein-coding genes (16S rRNA, rpoB, and gyrB) showed that strain A47 does not belong to any of the hitherto known taxa and may represent a previously undescribed Acinetobacter species., (Copyright © 2015 Traglia et al.)

A taxonomically unique bacterial strain, Acinetobacter sp. A47, has been recovered from several soft tissue samples from a patient undergoing reconstructive surgery owing to a traumatic amputation. The results of 16S rRNA, rpoB, and gyrB gene comparative sequence analyses showed that A47 does not belong to any of the hitherto-known taxa and may represent an as-yet-unknown Acinetobacter species. The recognition of this novel organism contributes to our knowledge of the taxonomic complexity underlying infections caused by Acinetobacter., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Postchemotherapy relapse presents a major unmet medical need in acute myeloid leukemia (AML), where treatment options are limited. CD25 is a leukemic stem cell marker and a conspicuous prognostic marker for overall/relapse-free survival in AML. Rare occurrence of genetic alterations among PIM family members imposes a substantial hurdle in formulating a compelling patient stratification strategy for the clinical development of selective PIM inhibitors in cancer. Here we show that CD25, a bona fide STAT5 regulated gene, is a mechanistically relevant predictive biomarker for sensitivity to PIM kinase inhibitors. Alone or in combination with tyrosine kinase inhibitors, PIM inhibitors can suppress STAT5 activation and significantly shorten the half-life of MYC to achieve substantial growth inhibition of high CD25-expressing AML cells. Our results highlight the importance of STAT5 and MYC in rendering cancer cells sensitive to PIM inhibitors. Because the presence of a CD25-positive subpopulation in leukemic blasts correlates with poor overall or relapse-free survival, our data suggest that a combination of PIM inhibitors with chemotherapy and tyrosine kinase inhibitors could improve long-term therapeutic outcomes in CD25-positive AML., (© 2014 by The American Society of Hematology.)

In recent years, MALDI-TOF Mass Spectrometry (MS) method has emerged as a promising and a reliable tool for bacteria identification. In this study we compared Bruker MALDI-TOF MS and conventional phenotypic methods to identify a collection of 333 Gram-positive clinical isolates comprising 22 genera and 60 species. 16S rRNA sequencing was the reference molecular technique, and rpoB gene sequecing was used as a secondary gene target when 16Sr RNA did not allow species identification of Corynebacterium spp. We also investigate if score cut-offs values of ≥ 1,5 and ≥ 1,7 were accurate for genus and species-level identification using the Bruker system. Identification at species level was obtained for 92,49% of Gram-positive rods by MALDI-TOF MS compared to 85,89% by phenotypic method. Our data validates the score ≥ 1,5 for genus level and ≥ 1,7 for species-level identification in a large and diverse collection of Gram-positive rods. The present study has proved the accuracy of MALDI-TOF MS as an identification method in Gram-positive rods compared to currently used methods in routine laboratories.

Inhibitors of JAK2 kinase are emerging as an important treatment modality for myeloproliferative neoplasms (MPN). However, similar to other kinase inhibitors, resistance to JAK2 inhibitors may eventually emerge through a variety of mechanisms. Effective drug combination is one way to enhance therapeutic efficacy and combat resistance against JAK2 inhibitors. To identify potential combination partners for JAK2 compounds in MPN cell lines, we performed pooled shRNA screen targeting 5,000 genes in the presence or absence of JAK2 blockade. One of the top hits identified was MYC, an oncogenic transcription factor that is difficult to inhibit directly, but could be targeted by modulation of upstream regulatory elements such as kinases. We demonstrate herein that PIM kinase inhibitors efficiently suppress MYC protein levels in MPN cell lines. Importantly, overexpression of MYC restores the viability of PIM inhibitor-treated cells, revealing causal relationship between MYC down-regulation and cell growth inhibition by PIM compounds. Combination of various PIM inhibitors with a JAK2 inhibitor results in significant synergistic growth inhibition of multiple MPN cancer cell lines and induction of apoptosis. Mechanistic studies revealed strong downregulation of phosphorylated forms of S6 and 4EBP1 by JAK2/PIM inhibitor combination treatment. Finally, such combination was effective in eradicating in vitro JAK2 inhibitor-resistant MPN clones, where MYC is consistently up-regulated. These findings demonstrate that simultaneous suppression of JAK2 and PIM kinase activity by small molecule inhibitors is more effective than either agent alone in suppressing MPN cell growth. Our data suggest that JAK2 and PIM combination might warrant further investigation for the treatment of JAK2-driven hematologic malignancies.

Actinobaculum schaalii may be a more common urinary tract pathogen than previously described. Here we report four cases of A. schaalii UTIs and we also propose a simple identification scheme to be used in the conventional microbiology laboratory based on standard biochemical tests.

Sixty-four colistin-resistant Klebsiella pneumoniae isolates recovered from clinical specimens from 57 patients admitted to Hospital de Clinicas Jose de San Martin during the period 2010-2012 were studied to describe the microbiological and epidemiological characteristics and factors associated with the emergence of colistin-resistance. Fifty-four colistin-susceptible K. pneumoniae isolates from the same period were also included in the study. The genetic relatedness among the isolates was studied by a PCR assay. Fifty percent of the resistant isolates were KPC-2 producers, 45.3% were ESBL producers and 4.7% only showed resistance to aminopenicilins. All KPC-producers (resistant and susceptible to colistin) were genotipically indistinguishable except for one, whereas the presence of 7 clonal types, which were different from the ones identified in the colistin-susceptible isolates, were detected among ESBL producers. The previous use of colistin was the main factor associated with the acquisition of resistance, and in the case of non-KPC producers the stay in ICU was another significant factor observed. Colistin resistance emerged in our hospital in the year 2010, reaching 3% in nosocomial isolates and maintaining this rate in successive years, due to the selection of resistant subpopulations in the epidemic clonal type in KPC-producers and due to the dispersion of colistin-resistant clonal types in non-KPC producing-isolates.

The case of a 52-year-old female patient with a history of critical aortic stenosis, hypothyroidism and splenectomy as treatment for her Hodgkin's lymphoma is herein presented. In April 2011, the patient was admitted to the cardiology service due to global heart failure, fever and poor response to diuretic and vasodilator therapy. A transesophageal echocardiogram showed images compatible with vegetations in the aortic, pulmonary, and mitral valves. A diagnosis of infective endocarditis was made. Growth of gram-negative coccobacilli was observed in two blood culture sets. The microorganism was finally identified as Bordetella holmesii. The patient was treated with ceftriaxone 1 g every 12 hours for 28 days with favorable outcome.

Brewing industry by-products are important animal feedstuff alternatives for local swine producers in Córdoba, Argentina. The high content of nutrients makes these by-products vulnerable to bacterial and fungal contamination. The objectives of the present study were (1) to determine the presence of Aspergillus section Flavi in brewer's grain used to feed pigs and (2) to evaluate the incidence of aflatoxin B(1) in the substrate. Total fungal count of most samples exceeded the levels proposed as feed quality limits, and most Aspergillus section Flavi strains found were able to produce high amounts of AFB(1) in vitro. However, the incidence of AFB(1) was low. The presence of contamination by aflatoxicogenic species in feedstuff might affect the productivity of swine producers and indirectly represents a public health issue.

Matrix-associated laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a rapid and simple microbial identification method. Previous reports using the Biotyper system suggested that this technique requires a preliminary extraction step to identify Gram-positive rods (GPRs), a technical issue that may limit the routine use of this technique to identify pathogenic GPRs in the clinical setting. We tested the accuracy of the MALDI-TOF MS Andromas strategy to identify a set of 659 GPR isolates representing 16 bacterial genera and 72 species by the direct colony method. This bacterial collection included 40 C. diphtheriae, 13 C. pseudotuberculosis, 19 C. ulcerans, and 270 other Corynebacterium isolates, 32 L. monocytogenes and 24 other Listeria isolates, 46 Nocardia, 75 Actinomyces, 18 Actinobaculum, 11 Propionibacterium acnes, 18 Propionibacterium avidum, 30 Lactobacillus, 21 Bacillus, 2 Rhodococcus equi, 2 Erysipelothrix rhusiopathiae, and 38 other GPR isolates, all identified by reference techniques. Totals of 98.5% and 1.2% of non-Listeria GPR isolates were identified to the species or genus level, respectively. Except for L. grayi isolates that were identified to the species level, all other Listeria isolates were identified to the genus level because of highly similar spectra. These data demonstrate that rapid identification of pathogenic GPRs can be obtained without an extraction step by MALDI-TOF mass spectrometry.

Aflatoxin (highly toxic and carcinogenic secondary metabolites produced by fungi) contamination is a serious problem worldwide. Modern agriculture and animal production systems need to use high-quality and mycotoxin-free feedstuffs. The use of microorganisms to preserve food has gained importance in recent years due to the demand for reduced use of chemical preservatives by consumers. Lactic acid bacteria are known to produce various antimicrobial compounds that are considered to be important in the biopreservation of food and feed. Lactobacillus rhamnosus L60 and Lactobacillus fermentum L23 are producers of secondary metabolites, such as organic acids, bacteriocins and, in the case of L60, hydrogen peroxide. The antifungal activity of lactobacilli strains was determined by coculture with Aspergillus section Flavi strains by two qualitative and one quantitative methods. Both L23 and L60 completely inhibited the fungal growth of all aflatoxicogenic strains assayed. Aflatoxin B (1) production was reduced 95.7-99.8% with L60 and 27.5-100% with L23. Statistical analysis of the data revealed the influence of L60 and L23 on growth parameters and aflatoxin B (1) production. These results are important given that these aflatoxicogenic fungi are natural contaminants of feed used for animal production, and could be effectively controlled by Lactobacillus L60 and L23 strains with probiotic properties., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Objective: To assess the feasibility and actual performance of ultrasound control in verification of the correct positioning of a nasogastric tube in pre-hospital settings., Study Type: Prospective, observational, single-centre study., Patients and Methods: Correct positioning of nasogastric tubes in patients intubated in a pre-hospital setting was verified by ultrasound and routinely compared with the results of two pre-hospital tests, namely a test involving insufflation of air through a syringe coupled with epigastric auscultation and a test involving aspiration of gastric fluid with a syringe. Routine x-ray control was carried out and compared with the pre-hospital results., Results: Ninety-six patients were included. Mean age was 52 years (median: 53.5 years, SD: 23 years). In 83% of the patients (n=80), the nasogastric tube was located by ultrasound immediately during the insertion procedure. The mean times to ultrasound confirmation of correct positioning of the nasogastric tube were 7s (median: 2s; SD: 16s) and 19s for the syringe tests (median 19s, SD: 5s). Eight ultrasound control tests were negative. Location coupled with insufflation of air through a syringe allowed detection of the nasogastric tube in the stomach but without providing confirmation of the actual gastric position. The pre-hospital ultrasound results were confirmed by subsequent radiological controls at the hospital., Conclusion: The ultrasound test performed in our study to verify correct positioning of a nasogastric tube is feasible in a pre-hospital setting. This technique is rapid and non-irradiating and is more sensitive and specific than the syringe tests commonly used in pre-hospital settings, and it may be performed in place of the latter tests., (Copyright © 2012 Société française d’anesthésie et de réanimation (Sfar). Published by Elsevier SAS. All rights reserved.)

The aims of this study were to evaluate the in vitro activity of extended-spectrum cephalosporins (ESC) in non-inducible AmpC enterobacteria throµgh phenotypic and genotypic characterization of the mechanisms of resistance (ESBL, plasmid-mediated AmpC and KPC) and to evaluate the interpretation criteria proposed by the existing recommendations and the new breakpoints established by the CLSI and the EUCAST. Susceptibility tests and PCR multiplex for b/aSHV and b/aCTX-M and amplification using specific primers was performed. One hundred sixty nine resistant isolates: K/ebsie//a pneumoniae (95), Escherichia co/i (55), and Proteus mirabi/is (19) were recovered. ESC resistance was 56.2 %, 32.6%, and 11.2 %, respectively. ESBL was detected in 152 (90 %) isolates, plasmid-mediated AmpC in 12 (7 %) and KPC in 5 (3 %). The CLSI 2009 recommendations and the breakpoints sµggested by the CLSI 2010 and the EUCAST for ceftriaxone were efficacious to detect ESBL, whereas the different breakpoints for ceftazidime presented discrepancies. The CLSI 2010 breakpoints only detected 55 % of the ESBL-producing isolates due to the endemic presence of CTX-M ESBLs in our country. Regarding the plasmid-mediated AmpC producers, the recommendations of the CLSI 2010 and the EUCAST 2010 proved to be more efficient than the old ones.

The mammalian gastric and oral mucosa may be colonized by mixed Helicobacter and Campylobacter species, respectively, in individual animals. To better characterize the presence and distribution of Helicobacter and Campylobacter among marine mammals, we used PCR and 16S rDNA sequence analysis to examine gastric and oral samples from ten dolphins (Tursiops gephyreus), one killer whale (Orcinus orca), one false killer whale (Pseudorca crassidens), and three wild La Plata river dolphins (Pontoporia blainvillei). Helicobacter spp. DNA was widely distributed in gastric and oral samples from both captive and wild cetaceans. Phylogenetic analysis demonstrated two Helicobacter sequence clusters, one closely related to H. cetorum, a species isolated from dolphins and whales in North America. The second related cluster was to sequences obtained from dolphins in Australia and to gastric non-H. pylori helicobacters, and may represent a novel taxonomic group. Dental plaque sequences from four dolphins formed a third cluster within the Campylobacter genus that likely represents a novel species isolated from marine mammals. Identification of identical Helicobacter spp. DNA sequences from dental plaque, saliva and gastric fluids from the same hosts, suggests that the oral cavity may be involved in transmission. These results demonstrate that Helicobacter and Campylobacter species are commonly distributed in marine mammals, and identify taxonomic clusters that may represent novel species., (Copyright © 2011 Elsevier B.V. All rights reserved.)

We describe a case of 17-year- old man native of Dominican Republic, with Hodgkin's lymphoma, who presented soft espontaneous draining nodules. In the clinical samples grew Burkholderia pseudomallei; the etiological agent of melioidosis. He received antimicrobial treatment with imipenem and amoxicillin/clavulanic with very good clinical evolution of the infectious process. Melioidosis diagnosis could be underestimated due to the low incidence of Burkholderia pseudomallei in our continent. The definitive diagnosis depends of the isolation and identification in the clinical sample.

The effect of mixtures of antioxidants butylated hydroxyanisol (BHA) and propyl paraben (PP) on lag phase, growth rate and ochratoxin A (OTA) production by four Aspergillus section Nigri strains was evaluated on peanut meal extract agar (PMEA) under different water activities (a(w)). The antioxidant mixtures used were: BHA + PP (mM), M1 (0.5 + 0.5), M2 (1.0 + 0.5), M3 (2.5 + 0.5), M4 (0.5 + 1.0), M5 (1.0 + 1.0), M6 (2.5 + 1.0), M7 (5.0 + 2.5) and M8 (10 + 2.5). The mixture M8 completely suppressed mycelial growth for all strains. A significant stimulation in OTA production was observed with mixtures M1 to M5 mainly at the highest a(w); whereas M6, M7 and M8 completely inhibited OTA production in all strains assayed; except M6 in A. carbonarius strain (RCP G). These results could enable a future intervention strategy to minimize OTA contamination.

We describe structure-based optimization of a series of novel 2,4-diaminopyrimidine MK2 inhibitors. Co-crystal structures (see accompanying Letter) demonstrated a unique inhibitor binding mode. Resulting inhibitors had IC(50) values as low as 19nM and moderate selectivity against a kinase panel. Compounds 15, 31a, and 31b inhibit TNFalpha production in peripheral human monocytes., (Copyright 2009 Elsevier Ltd. All rights reserved.)

This study was carried out to determine the efficacy of the phenolic antioxidant propyl paraben (PP) under different interacting water activity (a(W)) and temperature regimes on lag phase, growth rate and Ochratoxin A production by Aspergillus section Nigri strains. In this experiment six Aspergillus section Nigri strains were used. Peanut meal extract agar (PMEA) was prepared at 2%. The a(W) of the medium was adjusted to 0.995, 0.980 and 0.930, PP levels of 1, 5, 10 and 20 mmol/L were added to the basic medium. Plates were inoculated and incubated for 30 days at 18 and 25 degrees C. Lag phase (h) and radial growth rates (mm/day) were calculated. In control treatments, the lag phase increased and the growth rate decreased as a(W) reduced in all assayed strains. At all a(W) levels, when antioxidant concentrations increased the growth rate decreased. At 5, 10 and 20 mmol/L of PP the strains were not able to reach the exponential phase and completely inhibited fungal growth and OTA production regardless of a(W) used in all the evaluated strains.

There is ample evidence that Staphylococcus aureus capsular polysaccharide (CP) promotes virulence. Loss of capsule expression, however, may lead to S. aureus persistence in a chronically infected host. This study was conducted to determine the relative prevalence of nonencapsulated S. aureus in patients with chronic and acute osteomyelitis. Only 76/118 (64%) S. aureus isolates from patients with osteomyelitis expressed CP, whereas all 50 isolates from blood cultures of patients with infections other than osteoarticular infections expressed CP (P = 0.0001). A significantly higher prevalence of nonencapsulated S. aureus was found in patients with chronic osteomyelitis (53%) than in those with acute osteomyelitis (21%) (P = 0.0046). S. aureus isolates obtained from multiple specimens from five of six patients with chronic osteomyelitis exhibited phenotypic (expression of CP, alpha-hemolysin, beta-hemolysin, slime, and the small-colony variant phenotype) and/or genotypic (pulsed-field gel electrophoresis and spa typing) differences. Nonencapsulated S. aureus was recovered from at least one specimen from each chronic osteomyelitis patient. Fourteen isolates obtained from two patients with acute osteomyelitis were indistinguishable from each other within each group, and all produced CP5. In conclusion, we demonstrated that nonencapsulated S. aureus is more frequently isolated from patients with chronic osteomyelitis than from those with acute osteomyelitis, suggesting that loss of CP expression may be advantageous to S. aureus during chronic infection. Our findings on multiple S. aureus isolates from individual patients allow us to suggest that selection of nonencapsulated S. aureus is likely to have occurred in the patient during long-term bone infection.

Ochratoxin A (OTA) is one of the most important mycotoxins of worldwide concern for human health. The aims of this study were to evaluate the effects of water activity, temperature, incubation time and their interactions on growth, lag phase and OTA production by two strains belonging to A. niger aggregate in irradiated corn grains. This study shows that OTA production on corn grains occurs within a more restricted range of water activity and temperature than fungal growth. This point is important in order to maintain the appropriate conditions in stored corn that prevent toxigenic fungal growth and thus OTA production. Irradiated corn grains were re-hydrated to 0.910-0.995 of a(W) with sterile distillate water. The temperatures assayed were 15, 25 and 30 degrees C. Growth assessment was made every day during the incubation period (21 days) to calculate the growth rate, lag phase and the OTA production at 7, 14 and 21 days. Both strains showed optimum growth at 30 degrees C at 0.951 a(W). Ochratoxin A production occurred over a range of temperatures (15-30 degrees C) with optimum production at 25 degrees C depending on a(W) assayed. Maximum OTA production was obtained for both strains at 0.973 a(W). The results obtained suggest that the storage of corn grains at water activities lower than 0.951 and 15 degrees C should prevent these fungal species growth and OTA production during 21 days approximately.

The aims of this study were to evaluate the effects of water activity, temperature, incubation time and their interactions on lag phase, growth rate and ochratoxin A (OTA) production by strains belonging to the Aspergillus niger aggregate on irradiated peanut seeds. In the temperature and water activity range assayed, the optimal conditions of growth for RCP42 and RCP176 strains on irradiated peanut seeds were 0.995 a(w) and 30 degrees C being the growth rates of 12.4 and 14.6 mm/day, respectively. The maximum OTA production occurred at 0.973 a(w) and 25 degrees C for both strains assayed; whereas the minimum OTA production was obtained at 0.951 a(w) and 15 degrees C, at 14 and 21 days of incubation for RCP42 and RCP176 strains, respectively. The amount of OTA accumulated during 21 days assayed by both strains varied from 6.5 to 460 microg/g and from 10 to 210 microg/g with mean levels of 119.2 and 97.5 microg/g for RCP42 and RCP176 strains, respectively. The variance analysis (ANOVA) revealed that OTA concentration produced by RCP42 strain was significantly (p<0.0001) greater than that produced by RCP176 strain. If the strains with which the experiments were carried out were representative of the Aspergillus niger aggregate toxigenic species and the water activity in peanut seeds stored at 0.910 or lower was maintained, OTA production would be reduced during at least 21 days at variable temperatures.

Evaluation of hit chemotypes from high throughput screening identified a novel series of 2,4-disubstituted thieno[2,3-c]pyridines as COT kinase inhibitors. Structural modifications exploring SAR at the 2- and 4-positions resulting in inhibitors with improved enzyme potency and cellular activity are disclosed.

Contamination of foodstuff with mycotoxins such as ochratoxins is a major matter of concern for human and animal health. In Aspergillus species, ochratoxin synthesis depends on several environmental factors. The aims of this work were to evaluate the effect of water activity (0.995-0.85), temperature (15, 25 and 30 degrees C), incubation time (7, 14 and 21 days) and their interactions on OTA production on peanut, maize kernels, dried grapes and coffee beans meal extract agar medium by eight strains of Aspergillus section Nigri isolated from human food in Argentina. The optimum temperature for OTA production was 25 or 30 degrees C depending on the strains assayed, in most cases the highest OTA levels were achieved after 7 days of incubation, whereas this situation occurred at 15 degrees C after 14 days of incubation for only one strain. The maximum OTA level was obtained at earlier growth states when incubation temperature increased. In general, OTA concentration increased as water activity (a(W)) increased with no significant production at 0.85-0.91 a(W) under all temperature levels tested. Production occurred over a range of temperatures (15-30 degrees C) with optimum production at 30 degrees C depending on a(W) assayed. The knowledge of Aspergillus section Nigri ecophysiology is critical in the development and prediction of the risk models of raw material and final product contamination by these species under fluctuating and interacting environmental parameters.

Peanut is an important food commodity in Argentina. Last year Córdoba Province accounted for approximately 96% of the total Argentinian production. Few surveys of peanuts for the natural occurrence of ochratoxins and ochratoxin-producing fungi have been reported. The objectives of this study were to investigate the occurrence of Aspergillus section Nigri and ochratoxin A (OTA) in storage peanuts during a three-month-period. The capacity to produce OTA by Aspergillus section Nigri was also studied. A total of 100 samples were collected from May to July 2004. The frequency of contaminating fungi were determined by surface-disinfection the seeds and plating onto several agar types. Detection of OTA in seed samples was performed using an HPLC method. Strains belonging to Aspergillus section Nigri or Flavi were detected in all seeds samples. From the section Nigri, the species belonging to A. niger aggregate were isolated in 100% of the samples. The main ochratoxigenic specie, A. carbonarius, was present at low levels throughout the study period. OTA was found in 50% of the peanut samples, with mean levels ranging from 5.6 to 130 ng g(-1). The mean value of OTA obtained after the first month of storage (30 ng g(-1)) was significantly higher from those obtained after the second (6.5 ng g(-1)) and third (13 ng g(-1)) month (p<0.0001). One hundred and four (32%) of 322 strains of Aspergillus section Nigri, were OTA producers. The levels of toxin produced ranged from 2 to 24 ng ml(-1) of culture medium (mean level: 12.7 ng ml(-1)). These results indicate that humans and animals being may be frequently exposed to OTA in Argentina through the ingestion of peanut seed and foods based on peanuts. The presence of this toxin in peanuts might be an appropriate focus for future studies to estimate exposure through normal consumption of this commodity. These data are important in formulating guidelines for quality control of peanuts in Argentina.

Non-enterococcal Gram-positive bacteria that are intrinsically vancomycin-resistant have been infrequently isolated in association with serious infections. However, well-documented infections have lately been reported with increasing frequency. Because these organisms may be pathogens, we tested the MICs of 19 antimicrobial agents by the agar dilution method for predicting susceptibility. The activity of these antimicrobial agents was assessed against 28 strains (Lactobacillus rhamnosus, 6; Lactobacillus acidophilus, 1; Lactobacillus casei, 1; Lactobacillus fermentum, 2; Lactobacillus brevis, 1; Lactobacillus plantarum, 1; Weissella confusa, 2; Leuconostoc mesenteroides, 7; Leuconostoc lactis, 4; Pediococcus acidilactici, 2; Pediococcus pentosaceus, 1), isolated from clinical specimens in an Argentinian university hospital from 1997 to 2003. The MICs of penicillin for 67% of the Lactobacillus strains and 100% of the Leuconostoc spp. and Pediococcus spp. strains tested were in the 0.25-2 microg/mL range. Erythromycin was the most active antimicrobial overall. Multiresistance was observed in 2 strains (Lactobacillus rhamnosus, 1; Lactobacillus plantarum, 1).

The aim of this study was to assess the clinical outcomes of self-expandable metallic stents placing followed by laparoscopic resection and primary anastomosis for the treatment of acute colonic obstruction. From January 2003 to December 2004, 14 patients diagnosed with acute and complete colonic obstruction were treated with endoscopic colonic stenting as a bridge to an elective 1-stage laparoscopic resection. Three patients who underwent a successful stent insertion but had an inoperable tumor were excluded from the analyzed data. Ninety-three percent technical and clinical success was achieved. The stent insertion related perforation rate was 7% (1/14). The mean duration of stent insertion was approximately 1 hour and the mean time between the stent insertion and surgery was 6.2 days. Mean operating time was 132 +/- 38 minutes. No cases required conversion to laparotomy and there were no intraoperative complications. One case of anastomotic leakage was observed and treated by laparoscopic drainage and protective ileostomy. Ambulation time after operation was 1.8 +/- 0.6 days and total hospital stay length was 16.4 +/- 5.0 days. During a period of 11 +/- 7 months of follow-up, neither recurrences nor port-site metastases were observed. The management of acute colonic obstruction using endoscopic stent decompression, followed by laparoscopic resection, had good results and can be considered feasible and safe. Larger comparative studies may help to establish this approach.

An increasing amount of ligand binding data on G protein-coupled receptors (GPCRs) is not compatible with the prediction of the simple mass action law. This may be related to the propensity of most GPCRs, if not all, to oligomerize. Indeed, one of the consequences of receptor oligomerization could be a possible cross-talk between the protomers, which in turn could lead to negative or positive cooperative ligand binding. We prove here that this can be demonstrated experimentally. Saturation, dissociation, and competition binding experiments were performed on vasopressin and oxytocin receptors expressed in Chinese hamster ovary or COS-7 cells. Linear, concave, and convex Scatchard plots were then obtained, depending on the ligand used. Moreover, some competition curves exhibited an increase of the radiotracer binding for low concentrations of competitors, suggesting a cooperative binding process. These data demonstrate that various vasopressin analogs display either positive or negative cooperative binding. Because positive cooperative binding cannot be explained without considering receptor as multivalent, these binding data support the concept of GPCR dimerization process. The results, which are in good accordance with the predictions of previous mathematical models, suggest that binding experiments can be used to probe the existence of receptor dimers.

We report a novel chemical class of potent oxytocin receptor antagonists showing a high degree of selectivity against the closely related vasopressin receptors (V1a, V1b, V2). An initial compound, 7, was shown to be active in an animal model of preterm labor when administered by the intravenous but not by the oral route. Stepwise SAR investigations around the different structural elements revealed one position, the arenesulfonyl moiety, to be amenable to structural changes. Consequently, this position was used to introduce a variety of substituents to improve the physicochemical properties. Some of the resulting analogues were found to be superior to 7 both in terms of potency in vitro and aqueous solubility, which translated into significantly improved efficacy in the animal model after intravenous and oral administration. The best compound, 73, potently inhibited oxytocin-induced uterine contractions in nonpregnant rats and reduced spontaneous uterine contractions in late-term pregnant rats.

New insights into the structure-activity relationship of the peptide hormone oxytocin are presented. Incorporation of the novel cis-prolyl mimic 2,2-dimethyl-1,3-thiazolidine-4-carboxylic acid (pseudoproline, PsiPro) at position 7 of the hormone yielded the analogue [Cys(Psi(Me,Me)pro)]7oxytocin (1) that showed a 92-95% induction of the cis peptide bond conformation between Cys6 and PsiPro7, as determined by one- and two-dimensional NMR spectra in water and in DMSO-d6. The impact of the dimethyl moiety regarding conformation and bioactivity was investigated by the synthesis of the corresponding dihydro compound, [Cys(Psi(H,H)pro)]7oxytocin (2). Biological tests of the uterotonic activity, the pressor activity, and the binding affinity to the rat and human oxytocin receptors were carried out. As a most significant result, no antagonistic activities were found for both the cis-constrained analogue 1 and analogue 2, suggesting that a cis conformation between residues 6 and 7 of the molecule does not result in antagonistic activity. However, the about 10-fold reduction in agonistic activity of 1 as compared to oxytocin is consistent with the reduction of the trans conformation from 90% for oxytocin to 5-8% for compound 1. Compound 1 retained a high binding affinity for the oxytocin receptor, with K(i) values of 8.0 and 1.9 nM for the rat and the human receptor, respectively. The correlation between the biological activities and the cis contents obtained from NMR analysis for compounds 1, 2, and oxytocin leads to the hypothesis that a cis/trans conformational change plays an important role in oxytocin receptor binding and activation.

The peptide oxytocin (OT) antagonist atosiban, approved for tocolytic use in Europe (under the tradename Tractocile), represents an important new therapeutic advance for the treatment of premature labor. This paper presents some new peptide OT antagonists which offer promise as superior tocolytics. The solid phase synthesis is reported of four pairs of L and D-2-naphthylalanine (L/D-2Nal) position-2 modified analogs of the following four oxytocin (OT) antagonists: des-9-glycinamide [1-(beta-mercapto-beta,beta-pentamethylene propionic acid), 2-O-methyltyrosine, 4-threonine]ornithine-vasotocin (desGly-NH(2),d(CH(2))(5)[Tyr(Me)(2),Thr(4)]OVT) (A); the Tyr-NH(2) (9) analog of (A), d(CH(2))(5)[Tyr(Me)(2),Thr(4),Tyr-NH(2) (9)]OVT (B); the Eda(9) analog of (A), d(CH(2))(5)[Tyr(Me)(2),Thr(4),Eda(9)]OVT (C); and the retro COCH(2)Ph(4-0H)(10) modified analog of (C), d(CH(2))(5)[Tyr(Me)(2),Thr(4),Eda(9)<-- COCH(2)Ph(4-0H)(10)]OVT (D). The eight new analogs of A-D are (1) desGly-NH(2),d(CH(2))(5)[D-2Nal(2),Thr(4)]OVT, (2) desGly-NH(2),d(CH(2))(5)[2-Nal(2),Thr(4)]OVT, (3) d(CH(2))(5)[D-2Nal(2),Thr(4),Tyr-NH(2) (9)]OVT, (4) d(CH(2))(5)[2Nal(2),Thr(4),Tyr-NH(2) (9)]OVT, (5) d(CH(2))(5)[D-2Nal(2),Thr(4),Eda(9)]OVT, (6) d(CH(2))(5)[2Nal(2),Thr(4),Eda(9)]OVT, (7) d(CH(2))(5)[D-2Nal(2),Thr(4),Eda(9)<-- COCH(2)Ph(4-0H)(10)]OVT, (8) d(CH(2))(5)[2Nal(2),Thr(4),Eda(9)<-- COCH(2)Ph(4-OH)(10)]OVT. Peptides 1-8 were evaluated for agonistic and antagonistic activities in in vitro and in vivo rat bioassays, in rat OT receptor (rOTR) binding assays and in human OT receptor (hOTR) and human vasopressin (VP) vasopressor (V(1a)) receptor (hV(1a)R) binding assays. Also reported are the hOTR and hV(1a)R affinity data for atosiban and for B. None of the eight peptides exhibit oxytocic or vasopressor agonism. Peptides 1-8 exhibit weak antidiuretic agonism (activities in the range 0.014-0.21 U/mg). Peptides 1-6 exhibit potent in vitro (no Mg(2+)) OT antagonism (anti-OT pA(2) values range from 7.63 to 8.08). Peptides 7 and 8 are weaker OT antagonists. Peptides 1-6 are all OT antagonists in vivo (estimated in vivo anti-OT pA(2) values in the range 6.94-7.23). Peptides 1-8 exhibit vasopressor antagonism, anti-V(1a) pA(2) values in the range 5.1-7.65. Peptides 1-8 exhibit high affinities for the rOTR (K(i) values = 0.3-7.8 nM). Peptides 1-4 and B exhibit surprisingly very high affinities for the hOTR; their K(i) values are 0.17, 0.29, 0.07, 0.14 and 0.59 nM, respectively. Peptides 1-4 and B exhibit respectively 449, 263, 1091, 546 and 129 times greater affinity for the hOTR than atosiban (K(i) = 76.4 nM). Peptides 1-4 exhibit high affinities for the hV(1a)R (K(i)s = 1.1 nM, 1.3 nM, 0.19 nM and 0.54 nM, all higher than the hV1(a)R affinities exhibited by atosiban (K(i) = 5.1 nM) and by B (K(i) = 5.26 nM). Because of their strikingly higher affinities for the hOTR than atosiban, peptides 1-4 and B exhibit gains in anti hOT/anti hV(1a) receptor selectivity compared with atosiban of 93, 64, 39, 56 and 127, respectively. These OT antagonists are thus promising candidates for development as potential new tocolytic agents.

Enterobacter spp. are becoming increasingly frequent nosocomial pathogens with multiple resistance mechanism to beta-lactam antibiotics. We carried out the phenotypic and genotypic characterization of beta-lactamases in 27 Enterobacter spp. (25 Enterobacter cloacae y 2 Enterobacter aerogenes), as well as the ability of different extended spectrum-lactamase (ESBL) screening methods. Resistance to third generation cephalosporins was observed in 15/27 (63%) isolates. Twelve resistant isolates produced high level chromosomal encoded AmpC beta-lactamase; 6 of them were also producers of PER-2. Resistance to third generation cephalosporins in the remaining 3 isolates was due to the presence of ESBLs, PER-2 in 2 cases, and CTX-M-2 in the other. Only CTX-M-2 production was detected with all tested cephalosporins using difusion synergy tests, while cefepime improved ESBLs detection in 7/8 PER-2 producers, 4/8 in the inhibitor approximation test and 7/8 with double disk test using cefepime containing disk with and without clavulanic acid. Dilution method, including cephalosporins with and without the inhibitor detected 1/9 ESBLs producers.

We designed and synthesized new photoactivatable linear vasopressin analogues containing benzophenone photophores. All compounds were monitored and purified using RP-HPLC and characterized by mass spectrometry. Affinity and selectivity were determined in CHO cells expressing either human V(1a), V(1b) or V(2) receptor subtypes. Within the series, compounds 6 (PhCH(2)CO-lBpa-Phe-Gln-Asn-Arg-Pro-Arg-Tyr(3I)-NH(2)) and 9 (PhCH(2)CO-dBpa-Phe-Gln-Asn-Arg-Pro-Arg-Tyr(3I)-NH(2)), containing a benzoylphenylalanine residue (Bpa), were selected and their antagonistic properties determined (K(inact) = 1.87 and 0.35 nM, respectively). The dissociation constant of the most potent candidate (compound 9) was further calculated from saturation experiments using the (125)I derivative (K(d) = 0.07 +/- 0.01 nM). Photolabeling experiments using radioactive compound 9 as a probe were specific and UV-dependent and allowed the identification of two bands at molecular masses around 85-90 kDa and 46 kDa, respectively, as previously described by Phalipou et al., using two photoreactive linear azidopeptide antagonists. The results suggest therefore that compound 9 is a potent new tool for the accurate mapping of the human V(1a) receptor antagonist binding site.

The purpose was to apply oxidative crosslinking reactions to the study of recognition and signaling mechanisms associated to G-protein-coupled receptors. Using a ruthenium chelate, Ru(bipy)(3)(2+), as photosensitizer and visible light irradiation, in the presence of ammonium persulfate, we performed fast and efficient covalent labeling of the B(2) bradykinin receptor by agonist or antagonist ligands possessing a radio-iodinated phenol moiety. The chemical and topographical specificities of these crosslinking experiments were investigated. The strategy could also be applied to the covalent labeling of the B(1) bradykinin receptor, the AT(1) angiotensin II receptor, the V(1a) vasopressin receptor and the oxytocin receptor. Interestingly, we demonstrated the possibility to covalently label the AT(1) and B(2) receptors with functionalized ligands. The potential applications of metal-chelate chemistry to receptor structural and signaling studies through intramolecular or intermolecular crosslinking are presented.

Until recently, pharmacological studies dealing with vasopressin receptor isoforms were severely hampered by the lack of selective agonists or antagonists that recognize the pituitary V(1b) vasopressin receptor. By contrast, many selective vasopressin-related compounds are available for characterization of the vasopressor (V(1a)) or antidiuretic (V(2)) vasopressin receptor subtypes. Recently, SSR149415, a selective nonpeptide molecule, was discovered with nanomolar affinity for mammalian V(1b) receptors and good selectivity for the other vasopressin and oxytocin receptor isoforms. This molecule exhibits potent antagonist properties both in vitro and in vivo. We also designed synthetic peptides derived from [deaminocysteine(1),arginine(8)]vasopressin (dAVP), modified in position 4 by various amino acid residues. Some of these, d[cyclohexylalanine(4)]AVP or d[lysine(4)]AVP, have a high affinity and an excellent selectivity for the human V(1b) receptor subtype. However, they exhibit a mixed V(1b)/V(2) pharmacological profile for the rat vasopressin receptor isoforms. Whatever the species considered, these peptides behave as agonists both in bioassays performed in vitro and in vivo. The d[cyclohexylalanine(4)]AVP was tritiated and represents the first selective radiolabelled ligand available for studying the human V(1b) receptors. The discovery of these new selective V(1b) agonists and V(1b) antagonist allows an accurate pharmacological characterization of all the vasopressin receptor isoforms. As emphasized in this review, attention to the vasopressin and oxytocin receptor species differences is of critical importance in studies with all vasopressin and oxytocin ligands.