Efficient removal of toxic phthalate by immobilized serine-type aldehyde-tagged esterase G

Esterase G (EstG) from dibutyl phthalate (DBP)-degrading Sphingobium sp. SM42 was immobilized on amine-functionalized supports through aldehyde tag technology. Two different sulfatase motif tags, either LCTPSR (cysteine-type) or MSAPAR (serine-type), each of which is recognized by a specific formylglycine generating enzyme (FGE), were fused to the C-terminus of EstG. The cysteine-specific FGE was derived from Pseudomonas putida KT2440 while Klebsiella sp. SLS5 provided serine-specific FGE. The EstG with serine-type aldehyde tag showed a greater immobilization yield and higher specific activity by 4.8-fold and 1.8-fold, respectively. The immobilized EstG retained over 90% of its original activity after seven cycles of usage, and exhibited significantly improved thermostability by retaining 66% activity after 1 h incubation at 60 °C. Additionally, nearly 100% and over 30% of the DBP in 10 mM and 100 mM solutions, respectively, was degraded by the immobilized EstG within 18 h.     

Selective modification of alkyne-linked peptides and proteins by cyclometalated gold(III) (C^N) complex-mediated alkynylation

Alkyne is a useful functionality incorporated in proteins for site-selective bioconjugation reactions. Although effective bioconjugation reactions such as copper(I)-catalyzed and/or copper-free 1,3-dipolar cycloadditions of alkynes and azides are the most common approaches, the development of new alkyne-based bioconjugation reactions is still an ongoing interest in chemical biology. In this work, a new approach has been developed for selective modification of alkyne-linked peptides and proteins through the formation of arylacetylenes by a cross-coupling reaction of 6-membered ring cyclometalated gold(III) (C^N) complexes (HC^N = 2-arylpyridines) with terminal alkynes. Screening of the reaction conditions with a series of cyclometalated gold(III) complexes with phenylacetylene gave an excellent yield (up to 82%) by conducting the reaction in slightly alkaline aqueous conditions. The reaction scope was expanded to various alkynes, including alkyne-linked peptides to achieve up to >99% conversion. Using fluorescent dansyl (1l) and BODIPY (1m)-linked gold(III) complexes, alkyne-linked lysozyme has been selectively modified.     

Cyanobacterial peptides as a prototype for the design of cathepsin D inhibitors

Cathepsin D (Cath D) is overexpressed and secreted in a number of solid tumors and involved in the progress of tumor invasion, proliferation, metastasis, and apoptosis. Inhibition of Cath D is regarded as an attractive pathway for the development of novel anticancer drugs. Our previous studies revealed that tasiamide B, a cyanobacterial peptide that contained a statine‐like unit, exhibited good inhibition against Cath D and other aspartic proteases. Using this natural product as prototype, we designed and synthesized three new analogs, which bear isophthalic acid fragment at the N‐terminus and isobutyl amine ( 1 ), cyclopropyl amine ( 2 ), or 3‐methoxybenzyl amine ( 3 ) moiety at the C‐terminus. Enzymatic assays revealed that all these three compounds showed moderate‐to‐good inhibition against Cath D, with IC₅₀s of 15, 884, and 353 nM, respectively. Notably, compound 1 showed extreme selectivity for Cath D with 576‐fold over Cath E and 554‐fold over BACE1, which could be a valuable template for the design of highly potent and selective Cath D inhibitors. Additionally, compound 1 showed moderated activity against HeLa cell lines with IC₅₀ of 41.8 μM. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Early environment influences later performance in fishes

Conditions fish encounter during embryogenesis and early life history can leave lasting effects not only on morphology, but also on growth rate, life‐history and behavioural traits. The ecology of offspring can be affected by conditions experienced by their parents and mother in particular. This review summarizes such early impacts and their ecological influences for a variety of teleost species, but with special reference to salmonids. Growth and adult body size, sex ratio, egg size, lifespan and tendency to migrate can all be affected by early influences. Mechanisms behind such phenotypically plastic impacts are not well known, but epigenetic change appears to be one central mechanism. The thermal regime during development and incubation is particularly important, but also early food consumption and intraspecific density can all be responsible for later life‐history variation. For behavioural traits, early experiences with effects on brain, sensory development and cognition appear essential. This may also influence boldness and other social behaviours such as mate choice. At the end of the review, several issues and questions for future studies are given.     

Posttranslational Regulation of Human DNA Polymerase ι

Human DNA polymerases (pols) η and ι are Y-family DNA polymerase paralogs that facilitate translesion synthesis past damaged DNA. Both polη and polι can be monoubiquitinated in vivo. Polη has been shown to be ubiquitinated at one primary site. When this site is unavailable, three nearby lysines may become ubiquitinated. In contrast, mass spectrometry analysis of monoubiquitinated polι revealed that it is ubiquitinated at over 27 unique sites. Many of these sites are localized in different functional domains of the protein, including the catalytic polymerase domain, the proliferating cell nuclear antigen-interacting region, the Rev1-interacting region, and its ubiquitin binding motifs UBM1 and UBM2. Polι monoubiquitination remains unchanged after cells are exposed to DNA-damaging agents such as UV light (generating UV photoproducts), ethyl methanesulfonate (generating alkylation damage), mitomycin C (generating interstrand cross-links), or potassium bromate (generating direct oxidative DNA damage). However, when exposed to naphthoquinones, such as menadione and plumbagin, which cause indirect oxidative damage through mitochondrial dysfunction, polι becomes transiently polyubiquitinated via Lys¹¹- and Lys⁴⁸-linked chains of ubiquitin and subsequently targeted for degradation. Polyubiquitination does not occur as a direct result of the perturbation of the redox cycle as no polyubiquitination was observed after treatment with rotenone or antimycin A, which both inhibit mitochondrial electron transport. Interestingly, polyubiquitination was observed after the inhibition of the lysine acetyltransferase KATB3/p300. We hypothesize that the formation of polyubiquitination chains attached to polι occurs via the interplay between lysine acetylation and ubiquitination of ubiquitin itself at Lys¹¹ and Lys⁴⁸ rather than oxidative damage per se.     

Expanding the Symbiodinium (Dinophyceae,Suessiales) Toolkit Through Protoplast Technology

Dinoflagellates within the genus Symbiodinium are photosymbionts of many tropical reef invertebrates, including corals, making them central to the health of coral reefs. Symbiodinium have therefore gained significant research attention, though studies have been constrained by technical limitations. In particular, the generation of viable cells with their cell walls removed (termed protoplasts) has enabled a wide range of experimental techniques for bacteria, fungi, plants, and algae such as ultrastructure studies, virus infection studies, patch clamping, genetic transformation, and protoplast fusion. However, previous studies have struggled to remove the cell walls from armored dinoflagellates, potentially due to the internal placement of their cell walls. Here, we produce the first Symbiodinium protoplasts from three genetically and physiologically distinct strains via incubation with cellulase and osmotic agents. Digestion of the cell walls was verified by a lack of Calcofluor White fluorescence signal and by cell swelling in hypotonic culture medium. Fused protoplasts were also observed, motivating future investigation into intra‐ and inter‐specific somatic hybridization of Symbiodinium. Following digestion and transfer to regeneration medium, protoplasts remained photosynthetically active, regrew cell walls, regained motility, and entered exponential growth. Generation of Symbiodinium protoplasts opens exciting, new avenues for researching these crucial symbiotic dinoflagellates, including genetic modification.     

Epigenetic silencing of TIMP4 in heart failure

Tissue inhibitor of matrix metalloprotease 4 (TIMP4) is endogenously one of the key modulators of matrix metalloprotease 9 (MMP9) and we have reported earlier that cardiac specific TIMP4 instigates contractility and helps in differentiation of cardiac progenitor cells. Although studies show that the expression of TIMP4 goes down in heart failure but the mechanism is unknown. This study aims to determine the mechanism of silencing of TIMP4 in heart failure progression created by aorta‐vena cava (AV) fistula. We hypothesize that there is epigenetic silencing of TIMP4 in heart failure. To validate this hypothesis, we created heart failure model by creating AV fistula in C57BL/6 mice and looked into the promoter methylation (methylation specific PCR, high resolution melting, methylation sensitive restriction enzyme and Na bisulphite treatment followed by sequencing), histone modification (ChIP assay) and microRNAs that regulate TIMP4 (mir122a) and MMP9 (mir29b and mir455‐5p). The physiological parameters in terms of cardiac function after AV fistula were assessed by echocardiography. We observed that there are 7 CpG islands in the TIMP4 promoter which get methylated during the progression of heart failure which leads to its epigenetic silencing. In addition, the up‐regulated levels of mir122a in part, contribute to regulation of TIMP4. Consequently, MMP9 gets up‐regulated and leads to cardiac remodeling. This is a novel report to explain the epigenetic silencing of TIMP4 in heart failure.     

ADP-ribosylation of nucleolar proteins in HeLa tumor cells

ADP-ribosylation reactions in nucleoli of exponentially growing HeLa cells were studied. Isolated nuclei or nucleoli were labeled with ³²P-NAD; then the nucleolar proteins were analyzed by 1-dimensional and 2-dimensional polyacrylamide gel electrophoresis (PAGE) and modified proteins were detected by autoradiography. The labeled nucleolar proteins were also chromatographically fractionated on DEAE-cellulose. Electrophoretic analysis of total nucleolar and chromatographically purified proteins revealed that besides nuclear ADP-ribosyltransferase and histones two characteristic nucleolar phosphoproteins numatrin/B23 and nucleolin/C23 were modified by ADP-ribosylation.     

Effects of water discharge on fledging time,growth, and survival of piping plovers on the Missouri River

River flow management and modification is a global issue, and its effects on river-dependent organisms are pervasive. Flow modification can directly affect avian species through mortality or habitat loss, but less is known about indirect and sublethal effects of flow modification on reproductive output in these species. Young birds are more vulnerable to predation between hatching and fledging than after flight is achieved, but tradeoffs must be made to balance growth and survival. Predation pressure appears to be a significant factor affecting the time to fledging in altricial birds, but less is known about this threat for precocial birds. Birds reaching fledging earlier should have greater rates of survival to migration because their predator escape repertoire includes flight at an earlier age. We evaluated the effect of varying outflows from the Gavins Point Dam on the growth, age at fledging, and survival of piping plover (Charadrius melodus) chicks on the Missouri River (2006–2009). The study was characterized by 2 relatively high flow years (2006 and 2009) and 2 relatively low flow years (2007 and 2008). We used success rate in recapturing chicks in capture–mark–recapture models as an index for fledging. We attempted to recapture all chicks (n = 1,099) by hand every 3–4 days throughout the season to acquire morphological measurements. Models indicated that as flows from the dam increased, age at fledging increased. We also found that increasing flows were associated with decreasing daily survival rates (β_flow = −2.401, 95% CI: −4.351 to −0.452). Flow was also negatively related to chick mass gain, but we found less evidence for an effect on wing-chord length. Increased flows covered wet-substrate foraging habitat, and likely affected plover reproductive output directly through chick survival and indirectly through decreased growth and increased fledging times. © The Wildlife Society, 2013