Long-lasting inhibition of EGFR autophosphorylation in A549 tumor cells by intracellular accumulation of non-covalent inhibitors

In the present study, a small set of reversible or irreversible 4-anilinoquinazoline EGFR inhibitors was tested in A549 cells at early (1 h) and late (8 h) time points after inhibitor removal from culture medium. A combination of assays was employed to explain the observed long-lasting inhibition of EGFR autophosphorylation. We found that EGFR inhibition at 8 h can be due, besides to the covalent interaction of the inhibitor with Cys797, as for PD168393 (2) and its prodrug 4, to the intracellular accumulation of non-covalent inhibitors by means of an active cell uptake, as for 5 and 6. Compounds 5–6 showed similar potency and duration of inhibition of EGFR autophosphorylation as the covalent inhibitor 2, while being devoid of reactive groups forming covalent bonds with protein thiols.     

FLT3 Mutations in Early T-Cell Precursor ALL Characterize a Stem Cell Like Leukemia and Imply the Clinical Use of Tyrosine Kinase Inhibitors

Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) has been identified as high-risk subgroup of acute T-lymphoblastic leukemia (T-ALL) with a high rate of FLT3-mutations in adults. To unravel the underlying pathomechanisms and the clinical course we assessed molecular alterations and clinical characteristics in a large cohort of ETP-ALL (n = 68) in comparison to non-ETP T-ALL adult patients. Interestingly, we found a high rate of FLT3-mutations in ETP-ALL samples (n = 24, 35%). Furthermore, FLT3 mutated ETP-ALL was characterized by a specific immunophenotype (CD2+/CD5-/CD13+/CD33-), a distinct gene expression pattern (aberrant expression of IGFBP7, WT1, GATA3) and mutational status (absence of NOTCH1 mutations and a low frequency, 21%, of clonal TCR rearrangements). The observed low GATA3 expression and high WT1 expression in combination with lack of NOTCH1 mutations and a low rate of TCR rearrangements point to a leukemic transformation at the pluripotent prothymocyte stage in FLT3 mutated ETP-ALL. The clinical outcome in ETP-ALL patients was poor, but encouraging in those patients with allogeneic stem cell transplantation (3-year OS: 74%). To further explore the efficacy of targeted therapies, we demonstrate that T-ALL cell lines transfected with FLT3 expression constructs were particularly sensitive to tyrosine kinase inhibitors. In conclusion, FLT3 mutated ETP-ALL defines a molecular distinct stem cell like leukemic subtype. These data warrant clinical studies with the implementation of FLT3 inhibitors in addition to early allogeneic stem cell transplantation for this high risk subgroup.     

Impact of Aldosterone Antagonists on Sudden Cardiac Death Prevention in Heart Failure and Post-Myocardial Infarction Patients: A Systematic Review and Meta-Analysis of Randomized Controlled Trials

Background and Objectives

Sudden cardiac death (SCD) is a severe burden of modern medicine. Aldosterone antagonist is publicized as effective in reducing mortality in patients with heart failure (HF) or post myocardial infarction (MI). Our study aimed to assess the efficacy of AAs on mortality including SCD, hospitalization admission and several common adverse effects.

Methods

We searched Embase, PubMed, Web of Science, Cochrane library and clinicaltrial.gov for randomized controlled trials (RCTs) assigning AAs in patients with HF or post MI through May 2015. The comparator included standard medication or placebo, or both. Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines were followed. Event rates were compared using a random effects model. Prospective RCTs of AAs with durations of at least 8 weeks were selected if they included at least one of the following outcomes: SCD, all-cause/cardiovascular mortality, all-cause/cardiovascular hospitalization and common side effects (hyperkalemia, renal function degradation and gynecomastia).

Results

Data from 19,333 patients enrolled in 25 trials were included. In patients with HF, this treatment significantly reduced the risk of SCD by 19% (RR 0.81; 95% CI, 0.67–0.98; p = 0.03); all-cause mortality by 19% (RR 0.81; 95% CI, 0.74–0.88, p<0.00001) and cardiovascular death by 21% (RR 0.79; 95% CI, 0.70–0.89, p<0.00001). In patients with post-MI, the matching reduced risks were 20% (RR 0.80; 95% CI, 0.66–0.98; p = 0.03), 15% (RR 0.85; 95% CI, 0.76–0.95, p = 0.003) and 17% (RR 0.83; 95% CI, 0.74–0.94, p = 0.003), respectively. Concerning both subgroups, the relative risks respectively decreased by 19% (RR 0.81; 95% CI, 0.71–0.92; p = 0.002) for SCD, 18% (RR 0.82; 95% CI, 0.77–0.88, p < 0.0001) for all-cause mortality and 20% (RR 0.80; 95% CI, 0.74–0.87, p < 0.0001) for cardiovascular mortality in patients treated with AAs. As well, hospitalizations were significantly reduced, while common adverse effects were significantly increased.

Conclusion

Aldosterone antagonists appear to be effective in reducing SCD and other mortality events, compared with placebo or standard medication in patients with HF and/or after a MI.     

Early Indicators of Fatal Leptospirosis during the 2010 Epidemic in Puerto Rico

Background

Leptospirosis is a potentially fatal bacterial zoonosis that is endemic throughout the tropics and may be misdiagnosed as dengue. Delayed hospital admission of leptospirosis patients is associated with increased mortality.

Methodology/Principal Findings

During a concurrent dengue/leptospirosis epidemic in Puerto Rico in 2010, suspected dengue patients that tested dengue-negative were tested for leptospirosis. Fatal and non-fatal hospitalized leptospirosis patients were matched 1:1–3 by age. Records from all medical visits were evaluated for factors associated with fatal outcome. Among 175 leptospirosis patients identified (4.7 per 100,000 residents), 26 (15%) were fatal. Most patients were older males and had illness onset during the rainy season. Fatal case patients first sought medical care earlier than non-fatal control patients (2.5 vs. 5 days post-illness onset [DPO], p < 0.01), but less frequently first sought care at a hospital (52.4% vs. 92.2%, p < 0.01). Although fatal cases were more often diagnosed with leptospirosis at first medical visit (43.9% vs. 9.6%, p = 0.01), they were admitted to the hospital no earlier than non-fatal controls (4.5 vs. 6 DPO, p = 0.31). Cases less often developed fever (p = 0.03), but more often developed jaundice, edema, leg pain, hemoptysis, and had a seizure (p ≤ 0.03). Multivariable analysis of laboratory values from first medical visit associated with fatal outcome included increased white blood cell (WBC) count with increased creatinine (p = 0.001), and decreased bicarbonate with either increased WBC count, increased creatinine, or decreased platelet count (p < 0.001).

Conclusions/Significance

Patients with fatal leptospirosis sought care earlier, but were not admitted for care any earlier than non-fatal patients. Combinations of routine laboratory values predictive of fatal outcome should be considered in admission decision-making for patients with suspected leptospirosis.     

Impact of grazing,resource availability and light on prokaryotic growth and diversity in the oligotrophic surface global ocean

The impact of grazing, resource competition and light on prokaryotic growth and taxonomic composition in subtropical and tropical surface waters were studied through 10 microcosm experiments conducted between 30°N and 30°S in the Atlantic, Pacific and Indian oceans. Under natural sunlight conditions, significant changes in taxonomic composition were only observed after the reduction of grazing by sample filtration in combination with a decrease in resource competition by sample dilution. Sunlight exposure significantly reduced prokaryote growth (11 ± 6%) and community richness (14 ± 4%) compared to continuous darkness but did not significantly change community composition. The largest growth inhibition after sunlight exposure occurred at locations showing deep mixed layers. The reduction of grazing had an expected and significant positive effect on growth, but caused a significant decrease in community richness (16 ± 6%), suggesting that the coexistence of many different OTUs is partly promoted by the presence of predators. Dilution of the grazer-free prokaryotic community significantly enhanced growth at the level of community, but consistently and sharply reduced the abundance of Prochlorococcus and SAR11 populations. The decline of these oligotrophic bacterial taxa following an increase in resource availability is consistent with their high specialization for exploiting the limited resources available in the oligotrophic warm ocean.     

Deterministic Somatic Cell Reprogramming Involves Continuous Transcriptional Changes Governed by Myc and Epigenetic-Driven Modules

1. Download high-res image (195KB)
2. Download full-size image

     

An Escherichia coli mutant producing a truncated inactive form of GlgC synthesizes glycogen: further evidences for the occurrence of various important sources of ADPglucose in enterobacteria

AC70R1-504 Escherichia coli mutants possess a glgC* gene with a nucleotide change resulting in a premature stop codon that renders a truncated, inactive form of GlgC. Cells over-expressing the wild type glgC, but not those over-expressing the AC70R1-504 glgC*, accumulated high ADPglucose and glycogen levels. AC70R1-504 mutants accumulated glycogen, whereas DeltaglgCAP deletion mutants lacking the whole glycogen biosynthetic machinery displayed a glycogen-less phenotype. AC70R1-504 cells with enhanced glycogen synthase activity accumulated high glycogen levels. By contrast, AC70R1-504 cells with high ADPG hydrolase activity accumulated low glycogen. These data further confirm that enterobacteria possess various sources of ADPglucose linked to glycogen biosynthesis.     

Escherichia coli AspP activity is enhanced by macromolecular crowding and by both glucose-1,6-bisphosphate and nucleotide-sugars

Escherichia coli ADP-sugar pyrophosphatase (AspP) is a "Nudix" hydrolase that catalyzes the hydrolytic breakdown of ADP-glucose linked to glycogen biosynthesis. Moderate increases of AspP activity in the cell are accompanied by significant reductions of the glycogen content. In vitro analyses showed that AspP activity is strongly enhanced by macromolecular crowding and by both glucose-1,6-bisphosphate and nucleotide-sugars, providing a first set of indicative evidences that AspP is a highly regulated enzyme. To our knowledge, AspP is the sole bacterial enzyme described to date which is activated by both G1,6P(2) and nucleotide-sugars.     

The metallo-beta-lactamase GOB is a mono-Zn(II) enzyme with a novel active site

Metallo-beta-lactamases (MbetaLs) are zinc-dependent enzymes able to hydrolyze and inactivate most beta-lactam antibiotics. The large diversity of active site structures and metal content among MbetaLs from different sources has limited the design of a pan-MbetaL inhibitor. Here we report the biochemical and biophysical characterization of a novel MbetaL, GOB-18, from a clinical isolate of a Gram-negative opportunistic pathogen, Elizabethkingia meningoseptica. Different spectroscopic techniques, three-dimensional modeling, and mutagenesis experiments, reveal that the Zn(II) ion is bound to Asp120, His121, His263, and a solvent molecule, i.e. in the canonical Zn2 site of dinuclear MbetaLs. Contrasting all other related MbetaLs, GOB-18 is fully active against a broad range of beta-lactam substrates using a single Zn(II) ion in this site. These data further enlarge the structural diversity of MbetaLs.