Development of a high-throughput in vitro assay to identify selective inhibitors for human ALDH1A1

The human aldehyde dehydrogenase (ALDH) superfamily consists of at least 19 enzymes that metabolize endogenous and exogenous aldehydes. Currently, there are no commercially available inhibitors that target ALDH1A1 but have little to no effect on the structurally and functionally similar ALDH2. Here we present the first human ALDH1A1 structure, as the apo-enzyme and in complex with its cofactor NADH to a resolution of 1.75 and 2.1Å, respectfully. Structural comparisons of the cofactor binding sites in ALDH1A1 with other closely related ALDH enzymes illustrate a high degree of similarity. In order to minimize discovery of compounds that inhibit both isoenzymes by interfering with their conserved cofactor binding sites, this study reports the use of an in vitro, NAD(+)-independent, esterase-based high-throughput screen (HTS) of 64,000 compounds to discover novel, selective inhibitors of ALDH1A1. We describe 256 hits that alter the esterase activity of ALDH1A1. The effects on aldehyde oxidation of 67 compounds were further analyzed, with 30 selectively inhibiting ALDH1A1 compared to ALDH2 and ALDH3A1. One compound inhibited ALDH1A1 and ALDH2, while another inhibited ALDH1A1, ALDH2, and the more distantly related ALDH3A1. The results presented here indicate that this in vitro enzyme activity screening protocol successfully identified ALDH1A1 inhibitors with a high degree of isoenzyme selectivity. The compounds identified via this screen plus the screening methodology itself represent a starting point for the development of highly potent and selective inhibitors of ALDH1A1 that may be utilized to better understand the role of this enzyme in both normal and disease states

Characterization of Two Distinct Structural Classes of Selective Aldehyde Dehydrogenase 1A1 Inhibitors.

Aldehyde dehydrogenases (ALDH) catalyze the irreversible oxidation of aldehydes to their corresponding carboxylic acid. Alterations in ALDH1A1 activity are associated with such diverse diseases as cancer, Parkinson?s disease, obesity, and cataracts. Inhibitors of ALDH1A1 could aid in illuminating the role of this enzyme in disease processes. However, there are no commercially available selective inhibitors for ALDH1A1. Here we characterize two distinct chemical classes of inhibitors that are selective for human ALDH1A1 compared to eight other ALDH isoenzymes. The prototypical members of each structural class, CM026 and CM037, exhibit submicromolar inhibition constants but have different mechanisms of inhibition. The crystal structures of these compounds bound to ALDH1A1 demonstrate that they bind within the aldehyde binding pocket of ALDH1A1 and exploit the presence of a unique glycine residue to achieve their selectivity. These two novel and selective ALDH1A1 inhibitors may serve as chemical tools to better understand the contributions of ALDH1A1 to normal biology and to disease states

Superconducting Diamond on Silicon Nitride for Device Applications

Chemical vapour deposition (CVD) grown nanocrystalline diamond is an attractive material for the fabrication of devices. For some device architectures, optimisation of its growth on silicon nitride is essential. Here, the effects of three pre-growth surface treatments, often employed as cleaning methods of silicon nitride, were investigated. Such treatments provide control over the surface charge of the substrate through modification of the surface functionality, allowing for the optimisation of electrostatic diamond seeding densities. Zeta potential measurements and X-ray photoelectron spectroscopy (XPS) were used to analyse the silicon nitride surface following each treatment. Exposing silicon nitride to an oxygen plasma offered optimal surface conditions for the electrostatic self-assembly of a hydrogen-terminated diamond nanoparticle monolayer. The subsequent growth of boron-doped nanocrystalline diamond thin films on modified silicon nitride substrates under CVD conditions produced coalesced films for oxygen plasma and solvent treatments, whilst pin-holing of the diamond film was observed following RCA-1 treatment. The sharpest superconducting transition was observed for diamond grown on oxygen plasma treated silicon nitride, demonstrating it to be of the least structural disorder. Modifications to the substrate surface optimise the seeding and growth processes for the fabrication of diamond on silicon nitride devices

Do Cross-National Differences in the Costs of Children Generate Cross-National Differences in Fertility Rates?

Parity-specific probabilities of having a next birth are estimated from national fertility data and are compared with nation-specific costs of having children as measured by time-budget data, by attitude data from the International Social Survey Program, and by panel data on labor earnings and standard of living changes following a birth. We focus on five countries (the US, West Germany, Denmark, Italy, and the United Kingdom), whose fertility rates span the observed fertility range in the contemporary industrialized world and whose social welfare and family policies span the conceptual space of standard welfare-state typologies. Definitive conclusions are difficult because of the multiple dimensions on which child costs can be measured, the possibility that child costs affect both the quantum and the tempo of fertility, the relatively small fertility differences across industrialized nations, and the inherent small-N problem resulting from nation-level comparisons. Empirical analysis, however, supports the assertion that institutionally driven child costs affect the fertility patterns of industrialized nations.

Do Cross-National Differences in the Costs of Children Generate Cross-National Differences in Fertility Rates?

Parity-specific probabilities of having a next birth are estimated from national fertility data and are compared with nation-specific costs of having children as measured by time-budget data, by attitude data from the International Social Survey Program, and by panel data on labor earnings and standard of living changes following a birth. We focus on five countries (the US, West Germany, Denmark, Italy, and the United Kingdom), whose fertility rates span the observed fertility range in the contemporary industrialized world and whose social welfare and family policies span the conceptual space of standard welfare-state typologies. Definitive conclusions are difficult because of the multiple dimensions on which child costs can be measured, the possibility that child costs affect both the quantum and the tempo of fertility, the relatively small fertility differences across industrialized nations, and the inherent small-N problem resulting from nation-level comparisons. Empirical analysis, however, supports the assertion that institutionally driven child costs affect the fertility patterns of industrialized nations

A common cardiac sodium channel variant associated with sudden infant death in African Americans, SCN5A S1103Y.

Thousands die each year from sudden infant death syndrome (SIDS). Neither the cause nor basis for varied prevalence in different populations is understood. While 2 cases have been associated with mutations in type Valpha, cardiac voltage-gated sodium channels (SCN5A), the "Back to Sleep" campaign has decreased SIDS prevalence, consistent with a role for environmental influences in disease pathogenesis. Here we studied SCN5A in African Americans. Three of 133 SIDS cases were homozygous for the variant S1103Y. Among controls, 120 of 1,056 were carriers of the heterozygous genotype, which was previously associated with increased risk for arrhythmia in adults. This suggests that infants with 2 copies of S1103Y have a 24-fold increased risk for SIDS. Variant Y1103 channels were found to operate normally under baseline conditions in vitro. As risk factors for SIDS include apnea and respiratory acidosis, Y1103 and wild-type channels were subjected to lowered intracellular pH. Only Y1103 channels gained abnormal function, demonstrating late reopenings suppressible by the drug mexiletine. The variant appeared to confer susceptibility to acidosis-induced arrhythmia, a gene-environment interaction. Overall, homozygous and rare heterozygous SCN5A missense variants were found in approximately 5% of cases. If our findings are replicated, prospective genetic testing of SIDS cases and screening with counseling for at-risk families warrant consideration

Effect of reactor irradiation on Santowax WR : irradiations from 425° F to 800° F at 40% fast neutron fraction

"MIT-334-34."Includes bibliographical referencesM.I.T. DSR Project no. 9819Contract AT(38-1)-33

RETROCAM: A Versatile Optical Imager for Synoptic Studies

We present RETROCAM, an auxiliary CCD camera that can be rapidly inserted into the optical beam of the MDM 2.4m telescope. The speed and ease of reconfiguring the telescope to use the imager and a straightforward user interface permit the camera to be used during the course of other observing programs. This in turn encourages RETROCAM's use for a variety of monitoring projects.Comment: 6 pages, 6 figures, Accepted by A

N,N-diethylaminobenzaldehyde (DEAB) as a substrate and mechanism-based inhibitor for human ALDH isoenzymes

N,N-diethylaminobenzaldehyde (DEAB) is a commonly used "selective" inhibitor of aldehyde dehydrogenase isoenzymes in cancer stem cell biology due to its inclusion as a negative control compound in the widely utilized Aldefluor assay. Recent evidence has accumulated that DEAB is not a selective inhibitory agent when assayed in vitro versus ALDH1, ALDH2 and ALDH3 family members. We sought to determine the selectivity of DEAB toward ALDH1A1, ALDH1A2, ALDH1A3, ALDH1B1, ALDH1L1, ALDH2, ALDH3A1, ALDH4A1 and ALDH5A1 isoenzymes and determine the mechanism by which DEAB exerts its inhibitory action. We found that DEAB is an excellent substrate for ALDH3A1, exhibiting a Vmax/KM that exceeds that of its commonly used substrate, benzaldehyde. DEAB is also a substrate for ALDH1A1, albeit an exceptionally slow one (turnover rate ∼0.03 min(-1)). In contrast, little if any turnover of DEAB was observed when incubated with ALDH1A2, ALDH1A3, ALDH1B1, ALDH2 or ALDH5A1. DEAB was neither a substrate nor an inhibitor for ALDH1L1 or ALDH4A1. Analysis by enzyme kinetics and QTOF mass spectrometry demonstrates that DEAB is an irreversible inhibitor of ALDH1A2 and ALDH2 with apparent bimolecular rate constants of 2900 and 86,000 M(-1) s(-1), respectively. The mechanism of inactivation is consistent with the formation of quinoid-like resonance state following hydride transfer that is stabilized by local structural features that exist in several of the ALDH isoenzymes

Continuous Diffraction of Molecules and Disordered Molecular Crystals

The diffraction pattern of a single non-periodic compact object, such as a molecule, is continuous and is proportional to the square modulus of the Fourier transform of that object. When arrayed in a crystal, the coherent sum of the continuous diffracted wave-fields from all objects gives rise to strong Bragg peaks that modulate the single-object transform. Wilson statistics describe the distribution of continuous diffraction intensities to the same extent that they apply to Bragg diffraction. The continuous diffraction obtained from translationally-disordered molecular crystals consists of the incoherent sum of the wave-fields from the individual rigid units (such as molecules) in the crystal, which is proportional to the incoherent sum of the diffraction from the rigid units in each of their crystallographic orientations. This sum over orientations modifies the statistics in a similar way that crystal twinning modifies the distribution of Bragg intensities. These statistics are applied to determine parameters of continuous diffraction such as its scaling, the beam coherence, and the number of independent wave-fields or object orientations contributing. Continuous diffraction is generally much weaker than Bragg diffraction and may be accompanied by a background that far exceeds the strength of the signal. Instead of just relying upon the smallest measured intensities to guide the subtraction of the background it is shown how all measured values can be utilised to estimate the background, noise, and signal, by employing a modified "noisy Wilson" distribution that explicitly includes the background. Parameters relating to the background and signal quantities can be estimated from the moments of the measured intensities. The analysis method is demonstrated on previously-published continuous diffraction data measured from imperfect crystals of photosystem II.Comment: 34 pages, 11 figures, 2 appendice