Posaconazole prophylaxis has proven highly effective in preventing invasive fungal infections, despite relatively low serum concentrations. However, high tissue levels of this agent have been ...reported in treated patients. We therefore hypothesized that the intracellular levels of antifungal agents are an important factor in determining the success of fungal prophylaxis. To examine the effect of host cell-associated antifungals on the growth of medically important molds, we exposed cells to antifungal agents and removed the extracellular drug prior to infection. Epithelial cells loaded with posaconazole and its parent molecule itraconazole, but not other antifungals, were able to inhibit fungal growth for at least 48 h and were protected from damage caused by infection. Cell-associated posaconazole levels were 40- to 50-fold higher than extracellular levels, and the drug was predominantly detected in cellular membranes. Fungistatic levels of posaconazole persisted within epithelial cells for up to 48 h. Therefore, the concentration of posaconazole in mammalian host cell membranes mediates its efficacy in prophylactic regimens and likely explains the observed discrepancy between serum antifungal levels and efficacy.
The anti-apoptotic protein MCL-1 is a key regulator of cancer cell survival and a known resistance factor for small-molecule BCL-2 family inhibitors such as ABT-263 (navitoclax), making it an ...attractive therapeutic target. However, directly inhibiting this target requires the disruption of high-affinity protein-protein interactions, and therefore designing small molecules potent enough to inhibit MCL-1 in cells has proven extremely challenging. Here, we describe a series of indole-2-carboxylic acids, exemplified by the compound A-1210477, that bind to MCL-1 selectively and with sufficient affinity to disrupt MCL-1-BIM complexes in living cells. A-1210477 induces the hallmarks of intrinsic apoptosis and demonstrates single agent killing of multiple myeloma and non-small cell lung cancer cell lines demonstrated to be MCL-1 dependent by BH3 profiling or siRNA rescue experiments. As predicted, A-1210477 synergizes with the BCL-2/BCL-XL inhibitor navitoclax to kill a variety of cancer cell lines. This work represents the first description of small-molecule MCL-1 inhibitors with sufficient potency to induce clear on-target cellular activity. It also demonstrates the utility of these molecules as chemical tools for dissecting the basic biology of MCL-1 and the promise of small-molecule MCL-1 inhibitors as potential therapeutics for the treatment of cancer.
Optimization of KDM6B (JMJD3) HTS hit 12 led to the identification of 3-((furan-2-ylmethyl)amino)pyridine-4-carboxylic acid 34 and 3-(((3-methylthiophen-2-yl)methyl)amino)pyridine-4-carboxylic ...acid 39 that are inhibitors of the KDM4 (JMJD2) family of histone lysine demethylases. Compounds 34 and 39 possess activity, IC50 ≤ 100 nM, in KDM4 family biochemical (RFMS) assays with ≥50-fold selectivity against KDM6B and activity in a mechanistic KDM4C cell imaging assay (IC50 = 6–8 μM). Compounds 34 and 39 are also potent inhibitors of KDM5C (JARID1C) (RFMS IC50 = 100–125 nM).
Candida albicans and Aspergillus fumigatus occur ubiquitously in nature; C. albicans is part of the natural flora of most healthy individuals, and A. fumigatus is commonly found in soil, plant ...debris, and indoor air. Neither fungus poses a threat to healthy individuals, but each can cause fatal infections in immunocompromised patients. The use of tumor necrosis factor (TNF) antagonists for the treatment of rheumatoid arthritis and other autoimmune diseases has been associated with an increased incidence of opportunistic infections, including infections with both of these fungi. Because the use of TNF antagonists is expected to increase in the future, understanding the role that TNF plays and the effect of its antagonism on host defense against infections with these fungi is critical for reducing the associated morbidity and mortality.
Quantitative culture, quantitative PCR and the galactomannan enzyme immunoassay (EIA) were compared for their ability to determine the pulmonary fungal burden in a murine model of invasive ...aspergillosis. Quantitative culture of specimens containing hyphae under-represented the absolute fungal burden in established infection when compared with the two other methods. The best correlation was observed between the two non-culture methods. Higher variability was observed with the galactomannan EIA when compared with quantitative PCR. Collectively, these data suggest that quantitative PCR is the preferred method for determination of the pulmonary fungal burden in experimental aspergillosis.
Methionine aminopeptidase-2 (MetAP2) processes N-terminal methionine from nascent cellular proteins. Inhibition of MetAP2 has been shown to block angiogenesis and suppress tumor growth in preclinical ...tumor models. However, the biological role of MetAP2 in cancer is not well understood. We examined the effect of three distinct chemical classes of MetAP2 inhibitors on the growth of a panel of human cancer cells in vitro. All MetAP2 inhibitors caused inhibition of tumor cell growth in both anchorage-dependent and, particularly, in anchorage-independent manner. These data prompted us to examine the possible roles of MetAP2 in cancers. Ectopic expression of MetAP2 in NIH-3T3 cells caused transformation, evidenced by the formation of foci in monolayer culture and growth of large colonies in soft agar. Overexpression of MetAP2 in an immortalized bronchial epithelial cell line NL20 accelerated growth. These phenotypes induced by the overexpression of MetAP2 were reversed by the treatment with MetAP2 inhibitors, indicating that the catalytic function of MetAP2 was essential. Accordingly, overexpression of a catalytically inactive MetAP2 resulted in growth retardation of HT1080 tumor cells, suggesting a dominant-negative role of the inactive MetAP2 mutant. Finally, we analysed the expression of MetAP2 in patient cancer samples by immunohistochemistry. Moderate-to-high staining was identified in the majority of breast, colon, lung, ovarian and prostate carcinomas examined. These data suggest that MetAP2 plays an important role in tumor cell growth and may contribute to tumorigenesis.
Background: Post‐mortem examinations of adults who were apparently healthy but died suddenly and unexpectedly sometimes reveal no morphological abnormalities to explain their deaths. The frequency of ...such unexplained deaths in relation to other causes of sudden cardiac death is not known. Aim: To estimate the frequency of sudden unexpected cardiac or unexplained death in England. Design: Prospective survey using a stratified random sample of 83 of the 132 H.M. Coroner's jurisdictions in England. Methods: Consecutive White Caucasians, aged 16–64 years, with no medical history of cardiac disease, seen alive within 12 h of death, on whom autopsy found either a cardiac or no identifiable cause of death, were included. The coroner's officer sent a copy of the post‐mortem report and a completed case registration form to the investigators, with tissue samples. Results: Sixty‐seven (81%) coroners participated, each maintaining prospective surveillance for 4 months. Of 692 ascertained cases, case registration forms were received for 650 (94%), post‐mortem reports for 682 (99%), blood samples for 569 (82%), myocardial slices for 517 (75%) and whole hearts for 47 (7%). In cases with myocardial tissue, death was ascribed to ischaemic heart disease in 465 (82.4%). In 43.1% the ischaemia was acute, in 19.1% there was myocardial scarring but no acute ischaemia, and 20.2% had coronary atheroma only. Death was due to left ventricular hypertrophy in 32 (5.7%), to other cardiac causes in 30 (5.3%) and in 23 (4.1%) there was no clear cause. Those with cardiac causes were 81% male, median ages 55.9 (male) and 56.6 (female) years. The 23 unexplained deaths were 57% female, median ages 40.5 (male) and 54.9 (female) years. The estimated annual frequency of sudden unexpected death due to cardiac or unidentified causes, in English adults of employment age, was 11/100 000 (3481 annual deaths). Discussion: In 4.1% of sudden unexpected deaths under 65 years, no cause was found. Until it becomes accepted practice to identify these cases by a name, such as Sudden Adult Death Syndrome (SADS), it will not be possible to study their aetiology systematically.
Summary
Filamentation and adherence to host cells are critical virulence factors of Candida
albicans. Multiple filamentation regulatory pathways have been discovered in
C. albicans using ...Saccharomyces cerevisiae as a model. In S. cerevisiae,
these pathways converge on Flo11p, which functions as a downstream effector of filamentation
and also mediates cell–cell adherence (flocculation). In C. albicans,
such effector(s) have not yet been identified. Here, we demonstrate that the cell
surface protein Als1p is an effector of filamentation in C. albicans. We show
that Als1p expression is controlled by the transcription factor Efg1p, which is known
to be a key regulator of filamentation in C. albicans. Further, disruption
of ALS1 inhibited filamentation, and autonomous expression of Als1p restored
filamentation in an efg1 homozygous null mutant. Thus, Als1p functions as
a downstream effector of the EFG1 filamentation pathway. In addition, we found
that Als1p mediates both flocculation and adherence of C. albicans to endothelial
cells in vitro. As a cell surface glycoprotein that mediates filamentation
and adherence, Als1p has both structural and functional similarity to S. cerevisiae
Flo11p. Consistent with our in vitro results, Als1p was required for both normal filamentation and virulence in the mouse model of haematogenously disseminated candidiasis.
Medium energy ion scattering, using 100keVH+ incident ions, has been used to investigate the growth of epitaxial films, up to thicknesses of ~200Å, of V2O3 on both Pd(111) and Au(111). Scattered-ion ...energy spectra provide a measure of the average film thickness and the variations in this thickness, and show that, with suitable annealing, the crystalline quality is good. Plots of the scattering yield as a function of scattering angle, so-called blocking curves, have been measured for two different incidence directions and have been used to determine the surface structure. Specifically, scattering simulations for a range of different model structures show poor agreement with experiment for half-metal (….V′O3V) and vanadyl (….V′O3V=O) terminations, with and without surface interlayer relaxations. However, good agreement with experiment is found for the modified oxygen-termination structure, first proposed by Kresse et al., in which a subsurface V half-metal layer is moved up into the outermost V buckled metal layer to produce a VO2 overlayer on the underlying V2O3, with an associated layer structure of ….O3VV′′V ′O3. This result is consistent with the predictions of thermodynamic equilibrium at the surface under the surface preparation conditions, but is at variance with the conclusions of earlier studies of this system that have favoured the vanadyl termination. The results of these previous studies are re-evaluated in the light of the new result.
► V2O3(0001) grown epitaxially on Au(111) and Pd(111). ► Medium energy ion scattering (MEIS) shows crystalline quality is good. ► MEIS blocking curves plus VEGAS simulations used to investigate surface structure. ► MEIS data indicate the surface termination is VO2-like, and not (1×1)-vanadyl. ► results consistent with theoretical calculations of equilibrium surface termination.
Aims: To characterise the development of noise induced damage to hearing. Methods: Hearing and noise exposure were prospectively monitored among a cohort of newly enrolled construction industry ...apprentices and a comparison group of graduate students, using standard pure tone audiometry and distortion product otoacoustic emissions (DPOAEs). A total of 328 subjects (632 ears) were monitored annually an average of 3.4 times. In parallel to these measures, noise exposure and hearing protection device (HPD) use were extensively monitored during construction work tasks. Recreational/non-occupational exposures also were queried and monitored in subgroups of subjects. Trade specific mean exposure Leq levels, with and without accounting for the variable use of hearing protection in each trade, were calculated and used to group subjects by trade specific exposure level. Mixed effects models were used to estimate the change in hearing outcomes over time for each exposure group. Results: Small but significant exposure related changes in DPOAEs over time were observed, especially at 4 kHz with stimulus levels (L1) between 50 and 75 dB, with less clear but similar patterns observed at 3 kHz. After controlling for covariates, the high exposure group had annual changes in 4 kHz emissions of about 0.5 dB per year. Pure tone audiometric thresholds displayed only slight trends towards increased threshold levels with increasing exposure groups. Some unexpected results were observed, including an apparent increase in DPOAEs among controls over time, and improvement in behavioural thresholds among controls at 6 kHz only. Conclusions: Results indicate that construction apprentices in their first three years of work, with average noise exposures under 90 dBA, have measurable losses of hearing function. Despite numerous challenges in using DPOAEs for hearing surveillance in an industrial setting, they appear somewhat more sensitive to these early changes than is evident with standard pure tone audiometry.
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