Protein lysine posttranslational modification by an increasing number of different acyl groups is becoming appreciated as a regulatory mechanism in cellular biology. Sirtuins are class III histone deacylases that use NAD ؉ as a co-substrate during amide bond hydrolysis. Several studies have described the sirtuins as sensors of the NAD ؉ /NADH ratio, but it has not been formally tested for all the mammalian sirtuins in vitro. To address this problem, we first synthesized a wide variety of peptide-based probes, which were used to identify the range of hydrolytic activities of human sirtuins. These probes included aliphatic ⑀-N-acyllysine modifications with hydrocarbon lengths ranging from formyl (C 1 ) to palmitoyl (C 16 ) as well as negatively charged dicarboxyl-derived modifications. In addition to the well established activities of the sirtuins, "long chain" acyllysine modifications were also shown to be prone to hydrolytic cleavage by SIRT1-3 and SIRT6, supporting recent findings. We then tested the ability of NADH, ADP-ribose, and nicotinamide to inhibit these NAD ؉ -dependent deacylase activities of the sirtuins. In the commonly used 7-amino-4-methylcoumarin-coupled fluorescence-based assay, the fluorophore has significant spectral overlap with NADH and therefore cannot be used to measure inhibition by NADH. Therefore, we turned to an HPLC-MS-based assay to directly monitor the conversion of acylated peptides to their deacylated forms. All tested sirtuin deacylase activities showed sensitivity to NADH in this assay. However, the inhibitory concentrations of NADH in these assays are far greater than the predicted concentrations of NADH in cells; therefore, our data indicate that NADH is unlikely to inhibit sirtuins in vivo. These data suggest a re-evaluation of the sirtuins as direct sensors of the NAD ؉ /NADH ratio.

C5-Sugars form a new bio-monomer (trans-2,5-dihydroxy-3-pentenoic acid methyl ester), which can undergo enzymatic copolymerization with E6-HH to form multifunctional polymers.

Alginate is a linear polysaccharide from brown algae consisting of 1,4-linked β-d-mannuronic acid (M) and α-l-guluronic acid (G) arranged in M, G, and mixed MG blocks. Alginate was assumed to be indigestible in humans, but bacteria isolated from fecal samples can utilize alginate. Moreover, genomes of some human gut microbiome–associated bacteria encode putative alginate-degrading enzymes. Here, we genome-mined a polysaccharide lyase family 6 alginate lyase from the gut bacterium Bacteroides cellulosilyticus (BcelPL6). The structure of recombinant BcelPL6 was solved by X-ray crystallography to 1.3 Å resolution, revealing a single-domain, monomeric parallel β-helix containing a 10-step asparagine ladder characteristic of alginate-converting parallel β-helix enzymes. Substitutions of the conserved catalytic site residues Lys-249, Arg-270, and His-271 resulted in activity loss. However, imidazole restored the activity of BcelPL6-H271N to 2.5% that of the native enzyme. Molecular docking oriented tetra-mannuronic acid for syn attack correlated with M specificity. Using biochemical analyses, we found that BcelPL6 initially releases unsaturated oligosaccharides of a degree of polymerization of 2–7 from alginate and polyM, which were further degraded to di- and trisaccharides. Unlike other PL6 members, BcelPL6 had low activity on polyMG and none on polyG. Surprisingly, polyG increased BcelPL6 activity on alginate 7-fold. LC–electrospray ionization–MS quantification of products and lack of activity on NaBH4-reduced octa-mannuronic acid indicated that BcelPL6 is an endolyase that further degrades the oligosaccharide products with an intact reducing end. We anticipate that our results advance predictions of the specificity and mode of action of PL6 enzymes.

Antibacterial surfaces have been researched for more than 30 years and remain highly desirable. In particular, there is an interest in providing antimicrobial properties to commodity plastics, because these, in their native state, are excellent substrates for pathogens to adhere and proliferate on. Therefore, efficient strategies for converting surfaces of commodity plastics into contact‐active antimicrobial surfaces are of significant interest. Many systems have been prepared and tested for their efficacy. Here, the synthetic approaches to such active surfaces are reviewed, with the restriction to only include systems with tested antibacterial properties. The review focuses on the synthetic approach to surface functionalization of the most common materials used and tested for biomedical applications, which effectively has limited the study to quaternary materials. For future developments in the field, it is evident that there is a need for development of simple methods that permit scalable production of active surfaces. Furthermore, in terms of efficacy, there is an outstanding concern of a lack of universal antimicrobial action as well as rapid deactivation of the antibacterial effect through surface fouling.

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Poly­(dimethylsiloxane) (PDMS) is an attractive, versatile, and convenient material for use in biomedical devices that are in direct contact with the user. A crucial component in such a device is its surface in terms of antimicrobial properties preventing infection. Moreover, due to its inherent hydrophobicity, PDMS is rather prone to microbial colonization. Thus, developing an antimicrobial PDMS surface in a simple, large-scale, and applicable manner is an essential step in fully exploiting PDMS in the biomedical device industry. Current chemical modification methods for PDMS surfaces are limited; therefore, we present herein a new method for introducing an atom transfer radical polymerization (ATRP) initiator onto the PDMS surface via the base-catalyzed grafting of [(chloromethyl)­phenylethyl]­trimethoxysilane to the PDMS. The initiator surface was grafted with poly­[2-(dimethylamino)­ethyl methacrylate] (PDMAEMA) brushes via a surface-initiated supplemental activator and reducing agent ATRP (SI-SARA-ATRP). The use of sodium sulfite as a novel reducing agent in SI-SARA-ATRP allowed for polymerization during complete exposure to air. Moreover, a fast and linear growth was observed for the polymer over time, leading to a 400 nm thick polymer layer in a 120 min reaction time. Furthermore, the grafted PDMAEMA was quaternized, using various alkylhalides, in order to study the effect on surface antimicrobial properties. It was shown that antimicrobial activity not only depended highly on the charge density but also on the amphiphilicity of the surface. The fast reaction rate, high oxygen tolerance, increased antimicrobial activity, and the overall robustness and simplicity of the presented method collectively move PDMS closer to its full-scale exploitation in biomedical devices.

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