Lanthanide-binding tags as versatile protein coexpression probes

Comprehensive proteomic analyses require new methodologies to accelerate the correlation of gene sequence with protein function. Key tools for such efforts include biophysical probes that integrate into the covalent architecture of proteins. Lanthanide-binding tags (LBTs) are expressible, multitasking fusion partners that are optimized to bind lanthanide ions and have several desirable attributes, which include long-lived luminescence, excellent X-ray scattering power for phase determination, and magnetic properties to facilitate NMR spectroscopic structure elucidation. Herein, we present peptide sequences with a 40-fold higher affinity for Tb(3+) ions and significantly brighter luminescence intensity compared with existing peptides. Incorporation of an LBT onto ubiquitin as a prototype fusion protein allows the use of powerful protein-visualization techniques, which include rapid luminescence detection of LBT-tagged proteins in SDS-PAGE gels, as well as determination of protein concentrations in complex mixtures. The LBT strategy is a new alternative for expressing fluorescent fusion proteins by routine molecular biological techniques.     

Comparison of the Chemical and Oxidative Characteristics of Particulate Matter (PM) Collected by Different Methods: Filters,Impactors, and BioSamplers

In this study, we compare the chemical and oxidative characteristics of concentration-enriched PM_2.5 samples simultaneously collected by a filter, a Nano-Micro-Orifice Uniform Deposition Impactor, and a BioSampler. Gravimetric measurements showed considerable agreement in particulate matter (PM) collection efficiency for all three samplers. Accordingly, samples from the three collectors exhibited similar chemical compositions. The mass fractions of their inorganic ions, labile and nonlabile, were comparable. Moreover, the organic carbon (OC) content of the BioSampler slurry was similar to that of the filter, while water-soluble OC levels of the filter and impactor samples were close to a 100% agreement. Lastly, linear regression analyses demonstrated that the water-soluble elements existed in similar proportions for the filter and impactor samples. Their respective total components were also in very good agreement. By contrast, the recoverable elements from the BioSampler slurry, determined by high-resolution magnetic sector inductively coupled plasma mass spectrometry, were in good agreement with the water-soluble elements of the filter and impactor samples but not their corresponding total components. In spite of the overall agreement among the samples on their chemical composition, findings from a macrophage reactive oxygen species (ROS), a dithiothreitol (DTT), and a dihydroxybenzoate (DHBA) assay revealed that the oxidative potential of aqueous extracts of the filter and impactor substrates was similar yet substantially lower than that of the BioSampler slurry. However, filtering of the BioSampler slurry, i.e., removal of insoluble PM components, attenuated its ROS activity to about the same level as that of the water extracts of the filter and impactor samples. These findings first indicate that insoluble PM species are potentially redox active, and second that particle collection by the BioSampler, which circumvents the need for PM extraction, constitutes a viable alternative for collecting concentrated particles for characterization of the oxidative properties of PM.     

An Inter-Comparison of Two Black Carbon Aerosol Instruments and a Semi-Continuous Elemental Carbon Instrument in the Urban Environment

Aerosol absorption coefficients were obtained using two versions of the Magee Scientific Aethalometer and a Particle Soot Absorption Photometer (PSAP) in Riverside, California during July and August of 2005. These measurements were subsequently compared to each other and to hourly elemental carbon (EC) mass concentrations as determined by a Sunset Labs semi-continuous OCEC analyzer. Measurements from all four instruments were shown to be highly correlated (R ² = 0.83 to 0.92). Differences between absorption values measured by the PSAP and the Aethalometer were found to be dominated by differences in the filter media used by the respective instruments. Comparison of optical and thermal measurements revealed that the specific attenuation cross section (σ _ATN ) of light absorbing carbon (LAC) varied as a function of the time of the day, most notably during weekdays. Minimum σ _ATN values were observed during morning rush hour when EC concentrations were at their greatest and maxima were seen in the late afternoon. These variations correlated with changes in the OC/EC ratio and the Angstrom exponent for absorption, which is consistent with changes in the mixing state of elemental carbon associated with secondary aerosol condensation on primary EC particles.     

Correlation of the Extent of Fjord-Region Oxidation with DNA Binding and Mutagenicity of the Enantiomeric 11,12-Dihydrodiols of Dibenzo[a,l]pyrene

In vitro studies on the hepatic biotransformation of the enantiomeric trans-11,12-dihydrodiols of dibenzo[a,l]pyrene (DB[a,l]P) using microsomal fractions of animals pretreated with Aroclor 1254 revealed that the formation of fjord-region dihydrodiol epoxides strongly depends on the absolute configuration of the substrate. Both the (-)-11R,12R- and the (+)-11S,12S-enantiomer are converted diastereoselectively to the (-)- and (+)-anti-dihydrodiol epoxide, respectively, by either rat or mouse liver microsomes. Fjord-region oxidation occurs to greatest extent on incubation of the (-)-11R,12R-dihydrodiol with preparations from rats. This finding is in line with the differences seen for the two enantiomers on the total DNA binding under identical activation conditions as well as on the mutagenic activity in Chinese hamster V79 cells using the postmitochondrial hepatic fraction of Aroclor 1254-treated rats as metabolizing system.     

Measurement of residence time distribution in microfluidic systems

We present a method for measuring the residence time distribution (RTD) in microfluidic systems. A piezoelectrically actuated sample injector releases approximately 100 nl of tracer liquid into a microchannel of rectangular cross section. The spreading of the tracer pulse in pressure-driven microflows is monitored with fluorescence microscopy measurements. Residence time distributions are determined for single-phase liquid and segmented gas-liquid microflows, with the RTD being significantly narrower for the latter case. The selected flow conditions are relevant to synthesis in microreactors with residence times up to several minutes.     

4‐Biphenylalanine‐ and 3‐Phenyltyrosine‐Derived Hydroxamic Acids as Inhibitors of the JumonjiC‐Domain‐Containing Histone Demethylase KDM4A

Overexpression of the histone lysine demethylase KDM4A, which regulates H3K9 and H3K36 methylation states, has been related to the pathology of several human cancers. We found that a previously reported hydroxamate‐based histone deacetylase (HDAC) inhibitor (SW55) was also able to weakly inhibit this demethylase with an IC₅₀ value of 25.4 μm . Herein we report the synthesis and biochemical evaluations, with two orthogonal in vitro assays, of a series of derivatives of this lead structure. With extensive chemical modifications on the lead structure, also by exploiting the versatility of the radical arylation with aryldiazonium salts, we were able to increase the potency of the derivatives against KDM4A to the low‐micromolar range and, more importantly, to obtain demethylase selectivity with respect to HDACs. Cell‐permeable derivatives clearly showed a demethylase‐inhibition‐dependent antiproliferative effect against HL‐60 human promyelocytic leukemia cells.     

Potent and Selective Non‐hydroxamate Histone Deacetylase 8 Inhibitors

Specific inhibition of histone deacetylase 8 (HDAC8) has been suggested as a promising option for the treatment of neuroblastoma and T‐cell malignancies. A novel class of highly potent and selective HDAC8 inhibitors with a pyrimido[1,2‐c][1,3]benzothiazin‐6‐imine scaffold was studied that is completely different from the traditional concept of HDAC inhibitors comprising a zinc binding group (ZBG), in most cases a hydroxamate group, a spacer, and a capping group that may interact with the surface of the target protein. Although lacking a ZBG, some of the new compounds were shown to have outstanding potency against HDAC8 in the single‐digit nanomolar range. The pyrimido[1,2‐c][1,3]benzothiazin‐6‐imines also inhibited the growth of solid and hematological tumor cells. The small size and beneficial physicochemical properties of the novel HDAC inhibitor class underline the high degree of drug likeness. This and the broad structure–activity relationship suggest great potential for the further development of compounds with the pyrimido[1,2‐c][1,3]benzothiazin‐6‐imine scaffold into innovative and highly effective therapeutic drugs against cancer.     

miR-16-5p Is a Stably-Expressed Housekeeping MicroRNA in Breast Cancer Tissues from Primary Tumors and from Metastatic Sites

For quantitative microRNA analyses in formalin-fixed paraffin-embedded (FFPE) tissue, expression levels have to be normalized to endogenous controls. To investigate the most stably-expressed microRNAs in breast cancer and its surrounding tissue, we used tumor samples from primary tumors and from metastatic sites. MiRNA profiling using TaqMan^® Array Human MicroRNA Cards, enabling quantification of 754 unique human miRNAs, was performed in FFPE specimens from 58 patients with metastatic breast cancer. Forty-two (72%) samples were collected from primary tumors and 16 (28%) from metastases. In a cross-platform analysis of a validation cohort of 32 FFPE samples from patients with early breast cancer genome-wide microRNA expression analysis using SurePrintG3 miRNA (8 × 60 K)^® microarrays from Agilent^® was performed. Eleven microRNAs could be detected in all samples analyzed. Based on NormFinder and geNorm stability values and the high correlation (rho ≥ 0.8) with the median of all measured microRNAs, miR-16-5p, miR-29a-3p, miR-126-3p, and miR-222-3p are suitable single gene housekeeper candidates. In the cross-platform validation, 29 human microRNAs were strongly expressed (mean log2-intensity > 10) and 21 of these microRNAs including miR-16-5p and miR-29a-3p were also stably expressed (CV < 5%). Thus, miR-16-5p and miR-29a-3p are both strong housekeeper candidates. Their Normfinder stability values calculated across the primary tumor and metastases subgroup indicate that miR-29a-3p can be considered as the strongest housekeeper in a cohort with mainly samples from primary tumors, whereas miR-16-5p might perform better in a metastatic sample enriched cohort.     

Scale-up of fluidized-bed combustion - A review

Methods for scaling of fluidized-bed combustors are reviewed. It is found that a general scaling methodology, including simultaneously fluid-dynamic and combustion scaling, cannot be applied in practical scaling tests. Simplifications are needed. The approach followed here is to differentiate between fluid-dynamic scaling, combustion scaling, both related to the basic equations describing the phenomena, and boiler scaling that means scale-up from one boiler size to another, where established design elements can be utilized in the scaling procedure.