Key mutations stabilize antigen‐binding conformation during affinity maturation of a broadly neutralizing influenza antibody lineage

Affinity maturation, the process in which somatic hypermutation and positive selection generate antibodies with increasing affinity for an antigen, is pivotal in acquired humoral immunity. We have studied the mechanism of affinity gain in a human B‐cell lineage in which two main maturation pathways, diverging from a common ancestor, lead to three mature antibodies that neutralize a broad range of H1 influenza viruses. Previous work showed that increased affinity in the mature antibodies derives primarily from stabilization of the CDR H3 loop in the antigen‐binding conformation. We have now used molecular dynamics simulations and existing crystal structures to identify potentially key maturation mutations, and we have characterized their effects on the CDR H3 loop and on antigen binding using further simulations and experimental affinity measurements, respectively. In the two maturation pathways, different contacts between light and heavy chains stabilize the CDR H3 loop. As few as two single‐site mutations in each pathway can confer substantial loop stability, but none of them confers experimentally detectable stability on its own. Our results support models of the germinal center reaction in which two or more mutations can occur without concomitant selection and show how divergent pathways have yielded functionally equivalent antibodies. Proteins 2014; 83:771–780. © 2014 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.     

Reduced alphabet for protein folding prediction

What are the key building blocks that would have been needed to construct complex protein folds? This is an important issue for understanding protein folding mechanism and guiding de novo protein design. Twenty naturally occurring amino acids and eight secondary structures consist of a 28‐letter alphabet to determine folding kinetics and mechanism. Here we predict folding kinetic rates of proteins from many reduced alphabets. We find that a reduced alphabet of 10 letters achieves good correlation with folding rates, close to the one achieved by full 28‐letter alphabet. Many other reduced alphabets are not significantly correlated to folding rates. The finding suggests that not all amino acids and secondary structures are equally important for protein folding. The foldable sequence of a protein could be designed using at least 10 folding units, which can either promote or inhibit protein folding. Reducing alphabet cardinality without losing key folding kinetic information opens the door to potentially faster machine learning and data mining applications in protein structure prediction, sequence alignment and protein design. Proteins 2015; 83:631–639. © 2015 Wiley Periodicals, Inc.     

Detection of Xeljanz Enantiomers in Diethyl Amine Active Pharmaceutical Ingredients and Tablets

A high‐performance liquid chromatography (HPLC) method was established to detect Xeljanz enantiomers in active pharmaceutical ingredients (APIs) and tablets. The separation was achieved on a Chiralpak IC column using a mobile phase of hexane‐ethanol‐diethylamine (65:35:0.1, v/v). The detection wavelength was 289 nm. The peak areas and the enantiomer concentrations in the range of 0.15–2.25 μg?mL^?1 were in high linearity, with correlation coefficients higher than 0.999. The recoveries were 86.44% at the concentrations of 7.5, 18.75, and 37.5 μg?mL^?1. The limit of detection (LOD) and limit of quantification (LOQ) were 0.042 and 0.14 μg?mL^?1, respectively. This HPLC method is suitable for detecting the enantiomers of Xeljanz in its APIs and tablets. Chirality 27:235–238, 2015. © 2014 Wiley Periodicals, Inc.     

Speciation with gene flow in whiptail lizards from a Neotropical xeric biome

Two main hypotheses have been proposed to explain the diversification of the Caatinga biota. The riverine barrier hypothesis (RBH) claims that the São Francisco River (SFR) is a major biogeographic barrier to gene flow. The Pleistocene climatic fluctuation hypothesis (PCH) states that gene flow, geographic genetic structure and demographic signatures on endemic Caatinga taxa were influenced by Quaternary climate fluctuation cycles. Herein, we analyse genetic diversity and structure, phylogeographic history, and diversification of a widespread Caatinga lizard (Cnemidophorus ocellifer) based on large geographical sampling for multiple loci to test the predictions derived from the RBH and PCH. We inferred two well‐delimited lineages (Northeast and Southwest) that have diverged along the Cerrado–Caatinga border during the Mid‐Late Miocene (6–14 Ma) despite the presence of gene flow. We reject both major hypotheses proposed to explain diversification in the Caatinga. Surprisingly, our results revealed a striking complex diversification pattern where the Northeast lineage originated as a founder effect from a few individuals located along the edge of the Southwest lineage that eventually expanded throughout the Caatinga. The Southwest lineage is more diverse, older and associated with the Cerrado–Caatinga boundaries. Finally, we suggest that C. ocellifer from the Caatinga is composed of two distinct species. Our data support speciation in the presence of gene flow and highlight the role of environmental gradients in the diversification process.     

MicroRNA dysregulation in rhabdomyosarcoma: a new player enters the game

Rhabdomyosarcoma (RMS) is the most common of the soft tissue sarcomas with resultant high morbidity, frequently occuring in paediatric patients and young adults. While the molecular basis of RMS has received considerable attention, exact mechanisms underlying its development and metastasis remain unclear. MicroRNAs (miRNAs) are endogenously expressed small non‐coding RNAs that negatively regulate gene expression via translational inhibition or mRNA degradation. Deregulated expression of miRNA has been implicated in initiation, progression, and metastasis of RMS. miRNAs have emerged as key regulators of several physiological and pathophysiological processes and have opened new avenues for diagnosis and treatment of RMS. This review summarizes deregulation and functional roles of miRNAs in RMS and their potential applications for diagnosis, prognosis and treatment of this malignancy. As a rapidly evolving field in basic and translational medicine, it is hopeful that miRNA research will ultimately improve management of RMS.