Ribosome-binding antibiotics increase bacterial longevity and growth efficiency.

Antibiotics, by definition, reduce bacterial growth rates in optimal culture conditions; however, the real-world environments bacteria inhabit see rapid growth punctuated by periods of low nutrient availability. How antibiotics mediate population decline during these periods is poorly understood. Bacteria cannot optimize for all environmental conditions because a growth-longevity tradeoff predicts faster growth results in faster population decline, and since bacteriostatic antibiotics slow growth, they should also mediate longevity. We quantify how antibiotics, their targets, and resistance mechanisms influence longevity using populations of Escherichia coli and, as the tradeoff predicts, populations are maintained for longer if they encounter ribosome-binding antibiotics doxycycline and erythromycin, a finding that is not observed using antibiotics with alternative cellular targets. This tradeoff also predicts resistance mechanisms that increase growth rates during antibiotic treatment could be detrimental during nutrient stresses, and indeed, we find resistance by ribosomal protection removes benefits to longevity provided by doxycycline. We therefore liken ribosomal protection to a "Trojan horse" because it provides protection from an antibiotic but, during nutrient stresses, it promotes the demise of the bacteria. Seeking mechanisms to support these observations, we show doxycycline promotes efficient metabolism and reduces the concentration of reactive oxygen species. Seeking generality, we sought another mechanism that affects longevity and we found the number of doxycycline targets, namely, the ribosomal RNA operons, mediates growth and longevity even without antibiotics. We conclude that slow growth, as observed during antibiotic treatment, can help bacteria overcome later periods of nutrient stress.

Fluorescent probe for the identification of potent inhibitors of the macrophage infectivity potentiator (Mip) protein of Burkholderia pseudomallei.

The macrophage infectivity potentiator (Mip) protein belongs to the immunophilin superfamily. This class of enzymes catalyzes the interconversion between the cis and trans configuration of proline-containing peptide bonds. Mip has been shown to be important for the virulence of a wide range of pathogenic microorganisms, including the Gram-negative bacterium Burkholderia pseudomallei. Small molecules derived from the natural product rapamycin, lacking its immunosuppression-inducing moiety, inhibit Mip's peptidyl-prolyl cis-trans isomerase (PPIase) activity and lead to a reduction in pathogen load in vitro. Here, a fluorescence polarization assay (FPA) to enable the screening and effective development of BpMip inhibitors was established. A fluorescent probe was prepared, derived from previous pipecolic scaffold Mip inhibitors labeled with fluorescein. This probe showed moderate affinity for BpMip and enabled a highly robust FPA suitable for screening large compound libraries with medium- to high-throughput (Z factor ∼ 0.89) to identify potent new inhibitors. The FPA results are consistent with data from the protease-coupled PPIase assay. Analysis of the temperature dependence of the probe's binding highlighted that BpMip's ligand binding is driven by enthalpic rather than entropic effects. This has considerable consequences for the use of low-temperature kinetic assays.

The synthesis of 4,6-diaryl-2-pyridones and their bioactivation in CYP1 expressing breast cancer cells.

As part of a programme to develop anticancer prodrugs which are activated by cytochrome P450 (CYP)1B1, a library of 4,6-diaryl-2-pyridones was synthesised in yields of 6-60% from the corresponding chalcones. A number of these derivatives showed promising antiproliferative activities in human breast cancer cell lines which express CYP1B1 and CYP1A1, while showing little toxicity towards a non-tumour breast cell line with no CYP expression. Metabolism studies provided evidence supporting the involvement of CYP1 enzymes in the bioactivation of these compounds.

The Synthesis of Chalcones as Anticancer Prodrugs and their Bioactivation in CYP1 Expressing Breast Cancer Cells.

BACKGROUND: Although the expression levels of many P450s differ between tumour and corresponding normal tissue, CYP1B1 is one of the few CYP subfamilies which is significantly and consistently overexpressed in tumours. CYP1B1 has been shown to be active within tumours and is capable of metabolising a structurally diverse range of anticancer drugs. Because of this, and its role in the activation of procarcinogens, CYP1B1 is seen as an important target for anticancer drug development. OBJECTIVE: To synthesise a series of chalcone derivatives based on the chemopreventative agent DMU-135 and investigate their antiproliferative activities in human breast cancer cell lines which express CYP1B1 and CYP1A1. METHOD: A series of chalcones were synthesised in yields of 43-94% using the Claisen-Schmidt condensation reaction. These were screened using a MTT assay against a panel of breast cancer cell lines which have been characterised for CYP1 expression. RESULT: A number of derivatives showed promising antiproliferative activities in human breast cancer cell lines which express CYP1B1 and CYP1A1, while showing significantly lower toxicity towards a non-tumour breast cell line with no CYP expression. Experiments using the CYP1 inhibitors acacetin and α-naphthoflavone provided supporting evidence for the involvement of CYP1 enzymes in the bioactivation of these compounds. CONCLUSION: Chalcones show promise as anticancer agents with evidence suggesting that CYP1 activation of these compounds may be involved.

Benefits of polidocanol endovenous microfoam (Varithena®) compared with physician-compounded foams.

OBJECTIVE: To compare foam bubble size and bubble size distribution, stability, and degradation rate of commercially available polidocanol endovenous microfoam (Varithena®) and physician-compounded foams using a number of laboratory tests. METHODS: Foam properties of polidocanol endovenous microfoam and physician-compounded foams were measured and compared using a glass-plate method and a Sympatec QICPIC image analysis method to measure bubble size and bubble size distribution, Turbiscan™ LAB for foam half time and drainage and a novel biomimetic vein model to measure foam stability. Physician-compounded foams composed of polidocanol and room air, CO2, or mixtures of oxygen and carbon dioxide (O2:CO2) were generated by different methods. RESULTS: Polidocanol endovenous microfoam was found to have a narrow bubble size distribution with no large (>500 µm) bubbles. Physician-compounded foams made with the Tessari method had broader bubble size distribution and large bubbles, which have an impact on foam stability. Polidocanol endovenous microfoam had a lower degradation rate than any physician-compounded foams, including foams made using room air (p < 0.035). The same result was obtained at different liquid to gas ratios (1:4 and 1:7) for physician-compounded foams. In all tests performed, CO2 foams were the least stable and different O2:CO2 mixtures had intermediate performance. In the biomimetic vein model, polidocanol endovenous microfoam had the slowest degradation rate and longest calculated dwell time, which represents the length of time the foam is in contact with the vein, almost twice that of physician-compounded foams using room air and eight times better than physician-compounded foams prepared using equivalent gas mixes. CONCLUSION: Bubble size, bubble size distribution and stability of various sclerosing foam formulations show that polidocanol endovenous microfoam results in better overall performance compared with physician-compounded foams. Polidocanol endovenous microfoam offers better stability and cohesive properties in a biomimetic vein model compared to physician-compounded foams. Polidocanol endovenous microfoam, which is indicated in the United States for treatment of great saphenous vein system incompetence, provides clinicians with a consistent product with enhanced handling properties.

The role of clinically-relevant parameters on the cohesiveness of sclerosing foams in a biomimetic vein model.

We have recently reported on the development of a biomimetic vein model to measure the performance of sclerosing foams. In this study we employed the model to compare the commercially-available Varithena(®) (polidocanol injectable foam) 1% varicose vein treatment (referred to as polidocanol endovenous microfoam, or PEM) with physician compounded foams (PCFs) made using different foam generation methods (Double Syringe System and Tessari methods) and different foam formulations [liquid to gas ratios of 1:3 or 1:7; gas mixtures composed of 100% CO2, various CO2:O2 mixtures and room air (RA)]. PCFs produced using the DSS method had longer dwell times (DTs) (range 0.54-2.21 s/cm in the 4 mm diameter vein model) than those of the corresponding PCFs produced by the Tessari technique (range 0.29-0.94 s/cm). PEM had the longest DT indicating the best cohesive stability of any of the foams produced (2.92 s/cm). Other biomimetic model variables investigated included effect of vessel size, delayed injection and rate of plug formation (injection speed). When comparing the 4 and 10 mm vessel diameters, the DTs seen in the 10 mm vessel were higher than those observed for the 4 mm vessel, as the vein angle had been reduced to 5° to allow for foam plug formation. PCF foam performance was in the order RA > CO2:O2 (35:65) ≅ CO2:O2 (65:35) > CO2; PEM had a longer DT than all PCFs (22.10 s/cm) except that for RA made by DSS which was similar but more variable. The effect of delayed injection was also investigated and the DT for PEM remained the longest of all foams with the lowest percentage deviation with respect to the mean values, indicating a consistent foam performance. When considering rate of plug formation, PEM consistently produced the longest DTs and this was possible even at low plug expansion rates (mean 29.5 mm/s, minimum 20.9 mm/s). The developed vein model has therefore demonstrated that PEM consistently displays higher foam stability and cohesiveness when compared to PCFs, over a range of clinically-relevant operational variables.

The effect of ultrasound-related stimuli on cell viability in microfluidic channels.

BACKGROUND: In ultrasonic micro-devices, contrast agent micro-bubbles are known to initiate cavitation and streaming local to cells, potentially compromising cell viability. Here we investigate the effects of US alone by omitting contrast agent and monitoring cell viability under moderate-to-extreme ultrasound-related stimuli. RESULTS: Suspended H9c2 cardiac myoblasts were exposed to ultrasonic fields within a glass micro-capillary and their viability monitored under different US-related stimuli. An optimal injection flow rate of 2.6 mL/h was identified in which, high viability was maintained (~95%) and no mechanical stress towards cells was evident. This flow rate also allowed sufficient exposure of cells to US in order to induce bioeffects (~5 sec), whilst providing economical sample collection and processing times. Although the transducer temperature increased from ambient 23°C to 54°C at the maximum experimental voltage (29 Vpp), computational fluid dynamic simulations and controls (absence of US) revealed that the cell medium temperature did not exceed 34°C in the pressure nodal plane. Cells exposed to US amplitudes ranging from 0-29 Vpp, at a fixed frequency sweep period (tsw = 0.05 sec), revealed that viability was minimally affected up to ~15 Vpp. There was a ~17% reduction in viability at 21 Vpp, corresponding to the onset of Rayleigh-like streaming and a ~60% reduction at 29 Vpp, corresponding to increased streaming velocity or the potential onset of cavitation. At a fixed amplitude (29 Vpp) but with varying frequency sweep period (tsw = 0.02-0.50 sec), cell viability remained relatively constant at tsw ≥ 0.08 sec, whilst viability reduced at tsw < 0.08 sec and minimum viability recorded at tsw = 0.05 sec. CONCLUSION: The absence of CA has enabled us to investigate the effect of US alone on cell viability. Moderate-to-extreme US-related stimuli of cells have allowed us to discriminate between stimuli that maintain high viability and stimuli that significantly reduce cell viability. Results from this study may be of potential interest to researchers in the field of US-induced intracellular drug delivery and ultrasonic manipulation of biological cells.

A novel biomimetic analysis system for quantitative characterisation of sclerosing foams used for the treatment of varicose veins.

A novel analysis system for the quantification of sclerosing foam properties under clinically relevant conditions was developed with the purpose of establishing a robust methodology for comparative characterisation of different foam formulations and production strategies. The developed biomimetic-inspired model comprised of 4 or 10 mm inner diameter polytetrafluoroethylene tubing, filled with a blood substitute and fixed to a platform with an adjustable inclination angle. Sclerosing foams were produced by mixing polidocanol with either atmospheric air or 100 % CO2, using a double-syringe system method. Individual foams were injected into the tube, while videos were captured simultaneously. Videos were then transferred to an in-house computational foam analysis system (CFAS) which performed a sequence of semi-automated operations, allowing quantitative characterisation of sclerosing foam dynamic behaviour. Using CFAS, degradation rates of different foams were measured and the effect of gas composition, liquid sclerosant concentration and time delay between foam production and injection were evaluated.

Pharmacoproteomic study of the natural product Ebenfuran III in DU-145 prostate cancer cells: the quantitative and temporal interrogation of chemically induced cell death at the protein level.

A naturally occurring benzofuran derivative, Ebenfuran III (Eb III), was investigated for its antiproliferative effects using the DU-145 prostate cell line. Eb III was isolated from Onobrychis ebenoides of the Leguminosae family, a plant endemic in Central and Southern Greece. We have previously reported that Eb III exerts significant cytotoxic effects on certain cancer cell lines. This effect is thought to occur via the isoprenyl moiety at the C-5 position of the molecule. The study aim was to gain a deeper understanding of the pharmacological effect of Eb III on DU-145 cell death at the translational level using a relative quantitative and temporal proteomics approach. Proteins extracted from the cell pellets were subjected to solution phase trypsin proteolysis followed by iTRAQ-labeling. The labeled tryptic peptide extracts were then fractionated using strong cation exchange chromatography and the fractions were analyzed by nanoflow reverse phase ultraperformance liquid chromatography-nanoelectrospray ionization-tandem mass spectrometry analysis using a hybrid QqTOF platform. Using this approach, we compared the expression levels of 1360 proteins analyzed at ≤ 1% global protein false discovery rate (FDR), commonly present in untreated (control, vehicle only) and Eb III-treated cells at the different exposure time points. Through the iterative use of Ingenuity Pathway Analysis with hierarchical clustering of protein expression patterns, followed by bibliographic research, the temporal regulation of the Calpain-1, ERK2, PAR-4, RAB-7, and Bap31 proteins were identified as potential nodes of multipathway convergence to Eb III induced DU-145 cell death. These proteins were further verified with Western blot analysis. This gel-free, quantitative 2DLC-MS/MS proteomics method effectively captured novel modulated proteins in the DU-145 cell line as a response to Eb III treatment. This approach also provided greater insight to the multifocal and combinatorial signaling pathways implicated in Eb III-induced cell death.

Contrast agent-free sonoporation: The use of an ultrasonic standing wave microfluidic system for the delivery of pharmaceutical agents.

Sonoporation is a useful biophysical mechanism for facilitating the transmembrane delivery of therapeutic agents from the extracellular to the intracellular milieu. Conventionally, sonoporation is carried out in the presence of ultrasound contrast agents, which are known to greatly enhance transient poration of biological cell membranes. However, in vivo contrast agents have been observed to induce capillary rupture and haemorrhage due to endothelial cell damage and to greatly increase the potential for cell lysis in vitro. Here, we demonstrate sonoporation of cardiac myoblasts in the absence of contrast agent (CA-free sonoporation) using a low-cost ultrasound-microfluidic device. Within this device an ultrasonic standing wave was generated, allowing control over the position of the cells and the strength of the acoustic radiation forces. Real-time single-cell analysis and retrospective post-sonication analysis of insonated cardiac myoblasts showed that CA-free sonoporation induced transmembrane transfer of fluorescent probes (CMFDA and FITC-dextran) and that different mechanisms potentially contribute to membrane poration in the presence of an ultrasonic wave. Additionally, to the best of our knowledge, we have shown for the first time that sonoporation induces increased cell cytotoxicity as a consequence of CA-free ultrasound-facilitated uptake of pharmaceutical agents (doxorubicin, luteolin, and apigenin). The US-microfluidic device designed here provides an in vitro alternative to expensive and controversial in vivo models used for early stage drug discovery, and drug delivery programs and toxicity measurements.