Single-cell profiling methods have had a profound impact on the understanding of cellular heterogeneity. While genomes and transcriptomes can be explored at the single-cell level, single-cell profiling of proteomes is not yet established. Here we describe new single-molecule protein sequencing and identification technologies alongside innovations in mass spectrometry that will eventually enable broad sequence coverage in single-cell profiling. These technologies will in turn facilitate biological discovery and open new avenues for ultrasensitive disease diagnostics.
Abstract. Hydraulic capacitance and water storage form a critical buffer against cavitation and loss of conductivity within the xylem system. Withdrawal from water storage in leaves, branches, stems, and roots significantly impacts sap flow, stomatal conductance, and transpiration. Storage quantities differ based on soil water availability, tree size, wood anatomy and density, drought tolerance, and hydraulic strategy (anisohydric or isohydric). However, the majority of studies focus on the measurement of storage in conifers or tropical tree species. We demonstrate a novel methodology using frequency domain reflectometry (FDR) to make continuous, direct measurements of wood water content in two hardwood species in a forest in Michigan. We present results of a two month study comparing the water storage dynamics between a mature red oak and red maple, two species with differing wood densities, hydraulic architecture, and hydraulic strategy. We also include results pertaining to the use of different probe lengths to sample water content only within the active sapwood and over the entire conductive sapwood and the outer portion of heartwood in red oak. Both species studied exhibited diurnal cycles of storage that aligned well with the dynamics of sap flux. Red maple, a diffuse porous, relatively isohydric species showed a strong dependence on stored water during both wet and dry periods. Red oak, a ring porous relatively anisohydric species, was less reliant on storage, and did not demonstrate a dependence on soil water potential. Comparison between long and short FDR probes in the oak revealed that oaks may utilize water stored in the innermost layers of the xylem when soil moisture conditions are limiting. We found the FDR probes to be a reliable, functional means for continuous automated measurement of wood water content in hardwoods at a fast time scale. Application of FDR technology for the measurement of tree water storage will benefit forest ecologists as well as the modeling community as we improve our understanding and simulations of plant hydrodynamic processes on a large scale.
C-terminal peptide thioesters are an essential component of the native chemical ligation approach for the preparation of fully- or semi-synthetic proteins. However, efficient generation of C-terminal thioesters via Fmoc solid phase peptide synthesis remains a challenge. The recent N-acylurea approach to thioester synthesis relies on deactivation of one amine of 3,4-diaminobenzoic acid (Dbz) during Fmoc-SPPS. Here, we demonstrate that this approach results in the formation of side products by over-acylation of Dbz, particularly when applied to Gly-rich sequences. We find that orthogonal allyloxycarbonyl (Alloc) protection of a single Dbz amine eliminates these side products. We introduce a protected Fmoc-Dbz(Alloc) base resin that may be directly used for synthesis with most C-terminal amino acids. Following synthesis, quantitative removal of the Alloc group allows conversion to the active N-acyl-benzimidazolinone (Nbz) species, which may be purified and converted in situ to thioester under ligation conditions. This method is compatible with automated preparation of peptide-Nbz conjugates. We demonstrate that Dbz protection improves synthetic purity of Gly-rich peptide sequences derived from histone H4, as well as a 44-residue peptide from histone H3.
Nucleosomes contain ∼146 bp of DNA wrapped around a histone protein octamer that controls DNA accessibility to transcription and repair complexes. Posttranslational modification (PTM) of histone proteins regulates nucleosome function. To date, only modest changes in nucleosome structure have been directly attributed to histone PTMs. Histone residue H3(T118) is located near the nucleosome dyad and can be phosphorylated. This PTM destabilizes nucleosomes and is implicated in the regulation of transcription and repair. Here, we report gel electrophoretic mobility, sucrose gradient sedimentation, thermal disassembly, micrococcal nuclease digestion and atomic force microscopy measurements of two DNA–histone complexes that are structurally distinct from nucleosomes. We find that H3(T118ph) facilitates the formation of a nucleosome duplex with two DNA molecules wrapped around two histone octamers, and an altosome complex that contains one DNA molecule wrapped around two histone octamers. The nucleosome duplex complex forms within short ∼150 bp DNA molecules, whereas altosomes require at least ∼250 bp of DNA and form repeatedly along 3000 bp DNA molecules. These results are the first report of a histone PTM significantly altering the nucleosome structure.
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