Positively Selected Sites in Cetacean Myoglobins Contribute to Protein Stability

Since divergence ∼50 Ma ago from their terrestrial ancestors, cetaceans underwent a series of adaptations such as a ∼10–20 fold increase in myoglobin (Mb) concentration in skeletal muscle, critical for increasing oxygen storage capacity and prolonging dive time. Whereas the O₂-binding affinity of Mbs is not significantly different among mammals (with typical oxygenation constants of ∼0.8–1.2 µM⁻¹), folding stabilities of cetacean Mbs are ∼2–4 kcal/mol higher than for terrestrial Mbs. Using ancestral sequence reconstruction, maximum likelihood and Bayesian tests to describe the evolution of cetacean Mbs, and experimentally calibrated computation of stability effects of mutations, we observe accelerated evolution in cetaceans and identify seven positively selected sites in Mb. Overall, these sites contribute to Mb stabilization with a conditional probability of 0.8. We observe a correlation between Mb folding stability and protein abundance, suggesting that a selection pressure for stability acts proportionally to higher expression. We also identify a major divergence event leading to the common ancestor of whales, during which major stabilization occurred. Most of the positively selected sites that occur later act against other destabilizing mutations to maintain stability across the clade, except for the shallow divers, where late stability relaxation occurs, probably due to the shorter aerobic dive limits of these species. The three main positively selected sites 66, 5, and 35 undergo changes that favor hydrophobic folding, structural integrity, and intra-helical hydrogen bonds.     

A cross-linked anti-TNF-α aptamer for neutralization of TNF-α-induced cutaneous Shwartzman phenomenon: A simple and novel approach for improving aptamers' affinity and efficiency

To increase the efficiency of aptamers to their targets, a simple and novel method has been developed based on aptamer oligomerization. To this purpose, previously anti-human TNF-α aptamer named T1–T4 was trimerized through a trimethyl aconitate core for neutralization of in vitro and in vivo of TNF-α. At first, 54 mer T1–T4 aptamers with 5′-NH₂ groups were covalently coupled to three ester residues in the trimethyl aconitate. In vitro activity of novel anti-TNF-α aptamer and its dissociation constant (K_d) was done using the L929 cell cytotoxicity assay. In vivo anti-TNF-α activity of new oligomerized aptamer was assessed in a mouse model of cutaneous Shwartzman. Anchoring of three T1–T4 aptamers to trimethyl aconitate substituent results in formation of the 162 mer fragment, which was well revealed by gel electrophoresis. In vitro study indicated that the trimerization of T1–T4 aptamer significantly improved its anti-TNF-α activity compared to non-modified aptamers (P < 0.0001) from 40% to 60%. The determination of K_d showed that trimerization could effectively enhance K_d of aptamer from 67 nM to 36 nM. In vivo study showed that trimer aptamer markedly reduced mean scar size from 15.2 ± 1.2 mm to 1.6 ± 0.1 mm (P < 0.0001), which prevent the formation of skin lesions. In vitro and in vivo studies indicate that trimerization of anti-TNF-α aptamer with a novel approach could improve the anti-TNF-α activity and therapeutic efficacy. According to our findings, a new anti-TNF-α aptamer described here could be considered an appropriate therapeutic agent in treating several inflammatory diseases.     

Antimicrobial therapeutic enhancement of levofloxacin via conjugation to a cell‐penetrating peptide: An efficient sonochemical catalytic process

Herein, we make an effort to enhance the antimicrobial activity of levofloxacin (LVX) antibiotic via conjugation to a cell‐penetrating peptide (CPP) including Cys‐Gly‐Ala‐Phe‐Pro‐His‐Arg. For this purpose, cysteine is used as a linker between the LVX and CPP chain, and two heterogeneous nanoscale catalytic systems are employed as the substantial alternatives for traditional peptide coupling reagents like N,N,N′,N′‐tetramethyl‐O‐(benzotriazol‐1‐yl)uronium tetrafluoroborate (TBTU). Briefly, it has been found out that the antimicrobial potency of the synthesized CPP‐LVX conjugate (on the gram‐positive and gram‐negative bacteria) is noticeably enhanced (~20% more). It has been revealed via zone of inhibition (ZOI) and optical density (OD) evaluations. As a convenient method for making this type of the effective conjugations, ultrasound waves (with a specific frequency and power density) activate the catalytic sites of the heterogeneous nanoparticles. Through this synergistic effect, peptide/amide bond is formed during a short time (10 min), and high reaction yield (>90%) is obtained under mild conditions. Moreover, as a simple purification process, the catalyst nanoparticles are collected and separated through their high magnetic property.