Bacterial lipolytic enzymes were originally classified into eight different families defined by Arpigny and Jaeger (families I-VIII). Recently, the discovery of new lipolytic enzymes allowed for ...extending the original classification to fourteen families (I-XIV). We previously reported that G. thermodenitrificans EstGtA2 (access no. AEN92268) belonged to a novel group of bacterial lipolytic enzymes. Here we propose a 15.sup.th family (family XV) and suggest criteria for the assignation of protein sequences to the N' subfamily. Five selected salt bridges, hallmarks of the N' subfamily (E3/R54, E12/R37, E66/R140, D124/K178 and D205/R220) were disrupted in EstGtA2 using a combinatorial alanine-scanning approach. A set of 14 (R/Kright arrowA) mutants was produced, including five single, three double, three triple and three quadruple mutants. Despite a high tolerance to non-conservative mutations for folding, all the alanine substitutions were destabilizing (decreasing T.sub.m by 5 to 14°C). A particular combination of four substitutions exceeded this tolerance and prevents the correct folding of EstGtA2, leading to enzyme inactivation. Although other mutants remain active at low temperatures, the accumulation of more than two mutations had a dramatic impact on EstGtA2 activity at high temperatures suggesting an important role of these conserved salt bridge-forming residues in thermostability of lipolytic enzymes from the N' subfamily. We also identified a particular interloop salt bridge in EstGtA2 (D194/H222), located at position i -2 and i -4 residues from the catalytic Asp and His respectively which is conserved in other related bacterial lipolytic enzymes (families IV and XIII) with high tolerance to mutations and charge reversal. We investigated the role of residue identity at position 222 in controlling stability-pH dependence in EstGtA2. The introduction of a His to Arg mutation led to increase thermostability under alkaline pH. Our results suggest primary targets for optimization of EstGtA2 for specific biotechnological purposes.
There are several lipid binding sites on serum albumins. The aim of this study was to examine the binding of bovine serum albumin (BSA) to cholesterol (Chol), ...1,2-dioleoyl-3-(trimethylammonium)propane (DOTAP), (dioctadecyldimethyl)ammonium bromide (DDAB), and dioleoylphosphatidylethanolamine (DOPE), at physiological conditions, using constant protein concentration and various lipid contents. Fourier transform infrared (FTIR), circular dichroism (CD) and fluorescence spectroscopic methods were used to analyze the lipid binding mode, the binding constant, and the effects of lipid complexation on BSA stability and conformation. Structural analysis showed that lipids bind BSA via both hydrophilic and hydrophobic contacts with overall binding constants of K Chol = (1.12 ± 0.40) × 103 M−1, K DDAB = (1.50 ± 0.50) × 103 M−1, K DOTAP = (2.45 ± 0.80) × 103 M−1, and K DOPE = (1.35 ± 0.60) × 103 M−1. The numbers of bound lipid (n) were 1.1 (cholesterol), 1.28 (DDAB), 1.02 (DOPE), and 1.21 (DOTAP) in these lipid−BSA complexes. DDAB and DOTAP induced major alterations of BSA conformation, causing a partial protein unfolding, while cholesterol and DOPE stabilized protein secondary structure.
Marine fisheries have a long history of exploitation, with the potential to shape many life history traits, particularly those that are heritable, such as age and size at maturity. Here, we analyze ...43 marine fish stocks, using prewhitened cross-correlations, to examine the relationship between past levels of fishing mortality (F) and resulting shifts in age structure. Significant cross-correlations between F and age structure were evident in 88% of stocks examined. For 37% of stocks, changes in age structure followed changes in F, whereas in 39% of cases age structure preceded changes in F. Sensitivity and response time varied widely, likely a result of management, past exploitation, and life history. The efficacy of our method was tested empirically by simulations, revealing that certain time-series properties (e.g., high variance and low strength correlation) have potential to obscure underlying associations. Methods that allow for the identification of sensitivity and response time to pressures such as fishing contribute to the development of stock-specific management measures that can protect, or rebuild, age structure.
Abstract
Size-selective harvesting is expected to reduce the average age and weight of commercially exploited fishes. The loss of larger, older fish has been hypothesized to negatively affect metrics ...of population viability, such as spawning behaviour, recruitment, and adult survival. Most studies to date have focussed on individual stocks. Here, we examine trends in average age and weight at broad taxonomic and temporal scales, using subsets of data compiled on 95 marine fish stocks. Following moderate declines between 1960 and 1990, we find that the average age has generally increased since 2000, such that 71% of 69 stocks are currently above their long-term average. However, the size of the oldest individuals has generally declined over time; the average weight is currently below average in 75% of 55 stocks. A temporal decline in the mean weight of the youngest constituents within 49 stocks is most evident in the Clupeiformes. Our results indicate that recovery of age structure need not be accompanied by recovery of weights-at-age, evidenced in part by a decline in the size of the oldest individuals within populations. Further study into the drivers of these patterns, and the consequences of declining weights-at-age for population viability, is warranted.
LHS 1140 is a nearby mid-M dwarf known to host a temperate rocky super-Earth (LHS 1140 b) on a 24.737-day orbit. Based on photometric observations by MEarth and Spitzer as well as Doppler ...spectroscopy from the High Accuracy Radial velocity Planet Searcher, we report the discovery of an additional transiting rocky companion (LHS 1140 c) with a mass of 1.81 0.39 M⊕ and a radius of 1.282 0.024 R⊕ on a tighter, 3.77795-day orbit. We also obtain more precise estimates for the mass and radius of LHS 1140 b, which are 6.98 0.89 M⊕ and 1.727 0.032 R⊕. The mean densities of planets b and c are 7.5 1.0 g cm−3 and 4.7 1.1 g cm−3, respectively, both consistent with the Earth's ratio of iron to magnesium silicate. The orbital eccentricities of LHS 1140 b and c are consistent with circular orbits and constrained to be below 0.06 and 0.31, respectively, with 90% confidence. Because the orbits of the two planets are coplanar and because we know from previous analyses of Kepler data that compact systems of small planets orbiting M dwarfs are commonplace, a search for more transiting planets in the LHS 1140 system could be fruitful. LHS 1140 c is one of the few known nearby terrestrial planets whose atmosphere could be studied with the upcoming James Webb Space Telescope.
To investigate the therapeutic role of a novel telomere-directed inhibitor, 6-thio-2'-deoxyguanosine (THIO) in gliomas both
and
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A panel of human and mouse glioma cell lines was used to test ...therapeutic efficacy of THIO using cell viability assays, flow cytometric analyses, and immunofluorescence. Integrated analyses of RNA sequencing and reverse-phase protein array data revealed the potential antitumor mechanisms of THIO. Four patient-derived xenografts (PDX), two patient-derived organoids (PDO), and two xenografts of human glioma cell lines were used to further investigate the therapeutic efficacy of THIO.
THIO was effective in the majority of human and mouse glioma cell lines with no obvious toxicity against normal astrocytes. THIO as a monotherapy demonstrated efficacy in three glioma cell lines that had acquired resistance to temozolomide. In addition, THIO showed efficacy in four human glioma cell lines grown as neurospheres by inducing apoptotic cell death. Mechanistically, THIO induced telomeric DNA damage not only in glioma cell lines but also in PDX tumor specimens. Integrated computational analyses of transcriptomic and proteomic data indicated that THIO significantly inhibited cell invasion, stem cell, and proliferation pathways while triggering DNA damage and apoptosis. Importantly, THIO significantly decreased tumor proliferation in two PDO models and reduced the tumor size of a glioblastoma xenograft and a PDX model.
The current study established the therapeutic role of THIO in primary and recurrent gliomas and revealed the acute induction of telomeric DNA damage as a primary antitumor mechanism of THIO in gliomas.
A novel gene encoding an esterase from Geobacillus thermodenitrificans strain CMB-A2 was cloned, sequenced and functionally expressed in Escherichia coli M15. Sequence analysis revealed an open ...reading frame of 747 bp corresponding to a polypeptide of 249 amino acid residues (named EstGtA2). After purification, a specific activity of 2.58 U mg⁻¹ was detected using p-NP caprylate (C8) at 50°C and pH 8.0 (optimal conditions). The enzyme catalyses the hydrolysis of triglycerides (tributyrin) and a variety of p-nitrophenyl esters with different fatty acyl chain length (C4-C16). The enzyme has potential for various industrial applications since it is characterized by its activity under a wide range of pH, from 25 to 65°C. Using Geobacillus stearothermophilus Est30 esterase structure as template, a model of EstGtA2 was built using ESyPred3D. Analysis of this structural model allowed identifying putative sequence features that control EstGtA2 enzymatic properties. Based on sequence properties, multiple sequence comparisons and phylogenetic analyses, this enzyme appears to belong to a new family of carboxylesterases.
We report that a shorter Debye length and, as a consequence, decreased colloidal stability are required for the molecular interaction of folic acid-modified Au nanoparticles (Au NPs) to occur on a ...surface-bound receptor, human dihydrofolate reductase (hDHFR). The interaction measured using surface plasmon resonance (SPR) sensing was optimal in a phosphate buffer at pH 6 and ionic strength exceeding 300 mM. Under these conditions, the aggregation constant of the Au NPs was approximately 10(4) M(-1) s(-1) and the Debye length was below 1 nm, on the same length scale as the size of the folate anion (approximately 0.8 nm). Longer Debye lengths led to poorer SPR responses, revealing a reduced affinity of the folic acid-modified Au NPs for hDHFR. While high colloidal stability of Au NPs is desired in most applications, these conditions may hinder molecular interactions due to Debye lengths exceeding the size of the ligand and thus preventing close interactions with the surface-bound molecular receptor.
Despite considerable interest and investigations on cationic lipid–DNA complexes, reports on lipid–RNA interaction are very limited. In contrast to lipid–DNA complexes where lipid binding induces ...partial B to A and B to C conformational changes, lipid–tRNA complexation preserves tRNA folded state. This study is the first attempt to investigate the binding of cationic lipid with transfer RNA and the effect of lipid complexation on tRNA aggregation and condensation. We examine the interaction of tRNA with cholesterol (Chol), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dioctadecyldimethylammoniumbromide (DDAB) and dioleoylphosphatidylethanolamine (DOPE), at physiological condition, using constant tRNA concentration and various lipid contents. FTIR, UV-visible, CD spectroscopic methods and atomic force microscopy (AFM) were used to analyze lipid binding site, the binding constant and the effects of lipid interaction on tRNA stability, conformation and condensation. Structural analysis showed lipid–tRNA interactions with G–C and A–U base pairs as well as the backbone phosphate group with overall binding constants of KChol = 5.94 (± 0.8) × 104 M–1, KDDAB = 8.33 (± 0.90) × 105 M–1, KDOTAP = 1.05 (± 0.30) × 105 M–1 and KDOPE = 2.75 (± 0.50) × 104 M–1. The order of stability of lipid–tRNA complexation is DDAB > DOTAP > Chol > DOPE. Hydrophobic interactions between lipid aliphatic tails and tRNA were observed. RNA remains in A-family structure, while biopolymer aggregation and condensation occurred at high lipid concentrations.
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