Generation of macro- and microplastic databases by high-throughput FTIR analysis with microplate readers.

FTIR spectral identification is today's gold standard analytical procedure for plastic pollution material characterization. High-throughput FTIR techniques have been advanced for small microplastics (10-500 µm) but less so for large microplastics (500-5 mm) and macroplastics (> 5 mm). These larger plastics are typically analyzed using ATR, which is highly manual and can sometimes destroy particles of interest. Furthermore, spectral libraries are often inadequate due to the limited variety of reference materials and spectral collection modes, resulting from expensive spectral data collection. We advance a new high-throughput technique to remedy these problems using FTIR microplate readers for measuring large particles (> 500 µm). We created a new reference database of over 6000 spectra for transmission, ATR, and reflection spectral collection modes with over 600 plastic, organic, and mineral reference materials relevant to plastic pollution research. We also streamline future analysis in microplate readers by creating a new particle holder for transmission measurements using off-the-shelf parts and fabricating a nonplastic 96-well microplate for storing particles. We determined that particles should be presented to microplate readers as thin as possible due to thick particles causing poor-quality spectra and identifications. We validated the new database using Open Specy and demonstrated that additional transmission and reflection spectra reference data were needed in spectral libraries.

Inhibitors of the NAD(+)-Dependent Protein Desuccinylase and Demalonylase Sirt5.

NAD(+)-dependent histone deacetylases (sirtuins) play important roles in epigenetic regulation but also through nonhistone substrates for other key cellular events and have been linked to the pathogenesis of cancer, neurodegeneration, and metabolic diseases. The subtype Sirt5 has been shown recently to act as a desuccinylating and demalonylating enzyme. We have established an assay for biochemical testing of Sirt5 using a small labeled succinylated lysine derivative. We present a comparative study on the profiling of several established sirtuin inhibitors on Sirt1-3 as well as Sirt5 and also present initial results on a screening for new compounds that block Sirt5. Thiobarbiturates were identified as new Sirt5 inhibitors in the low micromolar range, which are selective over Sirt3 that can be found in the same cell compartment as Sirt5.

From cosubstrate similarity to inhibitor diversity--inhibitors of ADP-ribosyltransferases from kinase inhibitor screening.

ADP-ribosyltransferases (ADP-RTs) use NAD(+) to transfer an ADP-ribosyl group to target proteins. Although some ADP-RTs are bacterial toxins only few inhibitors are known. Here we present the development of fluorescence-based assays and a focussed library screening using kinase inhibitors as a new approach towards inhibitors of ADP-RTs. Different screening setups were established using surrogate small molecule substrates or the quantitation of the cofactor NAD(+). Proof-of-principle screening experiments were performed using a kinase inhibitor library in order to target the NAD(+) binding pockets. This led to the discovery of structurally different lead inhibitors for the mono-ADP-ribosyltransferases Mosquitocidal toxin (MTX) from Bacillus sphaericus SSII-1, C3bot toxin from Clostridium botulinum and CDTa from Clostridium difficile. The interaction of the inhibitors with the toxin proteins was analyzed by means of docking and binding free energy calculations. Binding at the nicotinamide subpocket, which shows a significant difference in the three enzymes, is used to explain the selectivity of the identified inhibitors and offers an opportunity for further development of potent and selective inhibitors.

reCAPTCHA: human-based character recognition via Web security measures.

CAPTCHAs (Completely Automated Public Turing test to tell Computers and Humans Apart) are widespread security measures on the World Wide Web that prevent automated programs from abusing online services. They do so by asking humans to perform a task that computers cannot yet perform, such as deciphering distorted characters. Our research explored whether such human effort can be channeled into a useful purpose: helping to digitize old printed material by asking users to decipher scanned words from books that computerized optical character recognition failed to recognize. We showed that this method can transcribe text with a word accuracy exceeding 99%, matching the guarantee of professional human transcribers. Our apparatus is deployed in more than 40,000 Web sites and has transcribed over 440 million words.