Critical Role for Inflammatory Macrophages in Driving Antigen-dependent Th17 Cell Responses?

Peer reviewedPublisher PD

Engineering enzymes for noncanonical amino acid synthesis

The standard proteinogenic amino acids grant access to a myriad of chemistries that harmonize to create life. Outside of these twenty canonical protein building blocks are countless noncanonical amino acids (ncAAs), either found in nature or created by man. Interest in ncAAs has grown as research has unveiled their importance as precursors to natural products and pharmaceuticals, biological probes, and more. Despite their broad applications, synthesis of ncAAs remains a challenge, as poor stereoselectivity and low functional-group compatibility stymie effective preparative routes. The use of enzymes has emerged as a versatile approach to prepare ncAAs, and nature's enzymes can be engineered to synthesize ncAAs more efficiently and expand the amino acid alphabet. In this tutorial review, we briefly outline different enzyme engineering strategies and then discuss examples where engineering has generated new ‘ncAA synthases’ for efficient, environmentally benign production of a wide and growing collection of valuable ncAAs

Engineering enzymes for noncanonical amino acid synthesis

The standard proteinogenic amino acids grant access to a myriad of chemistries that harmonize to create life. Outside of these twenty canonical protein building blocks are countless noncanonical amino acids (ncAAs), either found in nature or created by man. Interest in ncAAs has grown as research has unveiled their importance as precursors to natural products and pharmaceuticals, biological probes, and more. Despite their broad applications, synthesis of ncAAs remains a challenge, as poor stereoselectivity and low functional-group compatibility stymie effective preparative routes. The use of enzymes has emerged as a versatile approach to prepare ncAAs, and nature's enzymes can be engineered to synthesize ncAAs more efficiently and expand the amino acid alphabet. In this tutorial review, we briefly outline different enzyme engineering strategies and then discuss examples where engineering has generated new ‘ncAA synthases’ for efficient, environmentally benign production of a wide and growing collection of valuable ncAAs

M. tuberculosis Ser/Thr Protein Kinase D Phosphorylates an Anti-Anti–Sigma Factor Homolog

Receptor Ser/Thr protein kinases are candidates for sensors that govern developmental changes and disease processes of Mycobacterium tuberculosis (Mtb), but the functions of these kinases are not established. Here, we show that Mtb protein kinase (Pkn) D overexpression alters transcription of numerous bacterial genes, including Rv0516c, a putative anti-anti–sigma factor, and genes regulated by sigma factor F. The PknD kinase domain directly phosphorylated Rv0516c, but no other sigma factor regulator, in vitro. In contrast, the purified PknB and PknE kinase domains phosphorylated distinct sigma regulators. Rather than modifying a consensus site, PknD phosphorylated Rv0516c in vitro and in vivo on Thr2 in a unique N-terminal extension. This phosphorylation inhibited Rv0516c binding in vitro to a homologous anti-anti–sigma factor, Rv2638. These results support a model in which signals transmitted through PknD alter the transcriptional program of Mtb by stimulating phosphorylation of a sigma factor regulator at an unprecedented control site

A critical role for suppressor of cytokine signalling 3 in promoting M1 macrophage activation and function in vitro and in vivo

Funded by Medical Research Council. Grant Number: 74804 NHS Grampian Endowments Research Trust. Grant Number: 12/16 Kidney Research UK. Grant Number: RP1/2012 Cunningham Trust. Grant Number: ACC/KWF/CT08/03Peer reviewedPublisher PD

Advanced Hyperspectral Analysis of Sediment Core Samples from the Chew Bahir Basin, Ethiopian Rift, in the Spectral Range from 0.25 to 17 µm:Support for Climate Proxy Interpretation

Establishing robust environmental proxies at newly investigated terrestrial sedimentary archives is a challenge, because straightforward climate reconstructions can be hampered by the complex relationship between climate parameters and sediment composition, proxy preservation or (in)sufficient sample material. We present a minimally invasive hyperspectral bidirectional reflectance analysis on discrete samples in the wavelength range from 0.25 to 17 mu m on 35 lacustrine sediment core samples from the Chew Bahir Basin, southern Ethiopia for climate proxy studies. We identified and used absorption bands at 2.2 mu m (Al-OH), at 2.3 mu m (Mg-OH), at 1.16 mu m (analcime), and at 3.98 mu m (calcite) for quantitative spectral analysis. The band depth ratios at 2.3/2.2 mu m in the spectra correlate with variations in the potassium content of the sediment samples, which also reflect periods of increased Al-to-Mg substitution in clay minerals during drier climatic episodes. During these episodes of drier conditions, absorption bands diagnostic of the presence of analcime and calcite support this interpretation, with analcime indicating the driest conditions. These results could be compared to qualitative analysis of other characteristic spectral properties in the spectral range between 0.25 and 17 mu m. The results of the hyperspectral measurements complement previous sedimentological and geochemical analyses, allowing us in particular to resolve more finely the processes of weathering in the catchment and low-temperature authigenic processes in the sediment. This enables us to better understand environmental changes in the habitat of early humans

Scalable continuous evolution for the generation of diverse enzyme variants encompassing promiscuous activities

Enzyme orthologs sharing identical primary functions can have different promiscuous activities. While it is possible to mine this natural diversity to obtain useful biocatalysts, generating comparably rich ortholog diversity is difficult, as it is the product of deep evolutionary processes occurring in a multitude of separate species and populations. Here, we take a first step in recapitulating the depth and scale of natural ortholog evolution on laboratory timescales. Using a continuous directed evolution platform called OrthoRep, we rapidly evolve the Thermotoga maritima tryptophan synthase β-subunit (TmTrpB) through multi-mutation pathways in many independent replicates, selecting only on TmTrpB’s primary activity of synthesizing L-tryptophan from indole and L-serine. We find that the resulting sequence-diverse TmTrpB variants span a range of substrate profiles useful in industrial biocatalysis and suggest that the depth and scale of evolution that OrthoRep affords will be generally valuable in enzyme engineering and the evolution of biomolecular functions