ModifiComb, a new proteomic tool for mapping substoichiometric post-translational modifications, finding novel types of modifications, and fingerprinting complex protein mixtures

A major challenge in proteomics is to fully identify and characterize the post-translational modification (PTM) patterns present at any given time in cells, tissues, and organisms. Here we present a fast and reliable method ("ModifiComb") for mapping hundreds types of PTMs at a time, including novel and unexpected PTMs. The high mass accuracy of Fourier transform mass spectrometry provides in many cases unique elemental composition of the PTM through the difference DeltaM between the molecular masses of the modified and unmodified peptides, whereas the retention time difference DeltaRT between their elution in reversed-phase liquid chromatography provides an additional dimension for PTM identification. Abundant sequence information obtained with complementary fragmentation techniques using ion-neutral collisions and electron capture often locates the modification to a single residue. The (DeltaM, DeltaRT) maps are representative of the proteome and its overall modification state and may be used for database-independent organism identification, comparative proteomic studies, and biomarker discovery. Examples of newly found modifications include +12.000 Da (+C atom) incorporation into proline residues of peptides from proline-rich proteins found in human saliva. This modification is hypothesized to increase the known activity of the peptide.     

Kinetics of actin unfolding induced by guanidine hydrochloride.

The kinetics of actin unfolding induced by guanidine hydrochloride has been studied. On the basis of obtained experimental data a new kinetic pathway of actin unfolding was proposed. We have shown that the transition from native to inactivated actin induced by guanidine hydrochloride (GdnHCl) passes through essential unfolding of the protein. This means that inactivated actin should be considered as the off-pathway species rather than an intermediate conformation between native and completely unfolded states of actin, as has been assumed earlier. The rate constants of the transitions that give rise to the inactivated actin were determined. At 1.0-2.0 M GdnHCl the value of the rate constant of the transition from native to essentially unfolded actin exceeds that of the following step of inactivated actin formation. It leads to the accumulation of essentially unfolded macromolecules early in the unfolding process, which in turn causes the minimum in the time dependencies of tryptophan fluorescence intensity, parameter A, characterizing the intrinsic fluorescence spectrum position, and tryptophan fluorescence anisotropy.     

Molecular characterization of geminivirus-derived small RNAs in different plant species

DNA geminiviruses are thought to be targets of RNA silencing. Here, we characterize small interfering (si) RNAs—the hallmarks of silencing—associated with Cabbage leaf curl begomovirus in Arabidopsis and African cassava mosaic begomovirus in Nicotiana benthamiana and cassava. We detected 21, 22 and 24 nt siRNAs of both polarities, derived from both the coding and the intergenic regions of these geminiviruses. Genetic evidence showed that all the 24 nt and a substantial fraction of the 22 nt viral siRNAs are generated by the dicer-like proteins DCL3 and DCL2, respectively. The viral siRNAs were 5′ end phosphorylated, as shown by phosphatase treatments, and methylated at the 3′-nucleotide, as shown by HEN1 miRNA methylase-dependent resistance to β-elimination. Similar modifications were found in all types of endogenous and transgene-derived siRNAs tested, but not in a major fraction of siRNAs from a cytoplasmic RNA tobamovirus. We conclude that several distinct silencing pathways are involved in DNA virus-plant interactions.     

The Effects of Permafrost Thaw on Soil Hydrologic, Thermal, and Carbon Dynamics in an Alaskan Peatland

Recent warming at high-latitudes has accelerated permafrost thaw in northern peatlands, and thaw can have profound effects on local hydrology and ecosystem carbon balance. To assess the impact of permafrost thaw on soil organic carbon (OC) dynamics, we measured soil hydrologic and thermal dynamics and soil OC stocks across a collapse-scar bog chronosequence in interior Alaska. We observed dramatic changes in the distribution of soil water associated with thawing of ice-rich frozen peat. The impoundment of warm water in collapse-scar bogs initiated talik formation and the lateral expansion of bogs over time. On average, Permafrost Plateaus stored 137 ± 37 kg C m⁻², whereas OC storage in Young Bogs and Old Bogs averaged 84 ± 13 kg C m⁻². Based on our reconstructions, the accumulation of OC in near-surface bog peat continued for nearly 1,000 years following permafrost thaw, at which point accumulation rates slowed. Rapid decomposition of thawed forest peat reduced deep OC stocks by nearly half during the first 100 years following thaw. Using a simple mass-balance model, we show that accumulation rates at the bog surface were not sufficient to balance deep OC losses, resulting in a net loss of OC from the entire peat column. An uncertainty analysis also revealed that the magnitude and timing of soil OC loss from thawed forest peat depends substantially on variation in OC input rates to bog peat and variation in decay constants for shallow and deep OC stocks. These findings suggest that permafrost thaw and the subsequent release of OC from thawed peat will likely reduce the strength of northern permafrost-affected peatlands as a carbon dioxide sink, and consequently, will likely accelerate rates of atmospheric warming.     

Demystifying the RAD fad

We are writing in response to the population and phylogenomics meeting review by Andrews & Luikart ( 2014 ) entitled ‘Recent novel approaches for population genomics data analysis’. Restriction‐site‐associated DNA (RAD) sequencing has become a powerful and useful approach in molecular ecology, with several different published methods now available to molecular ecologists, none of which can be considered the best option in all situations. A&L report that the original RAD protocol of Miller et al. ( 2007 ) and Baird et al. ( 2008 ) is superior to all other RAD variants because putative PCR duplicates can be identified (see Baxter et al. 2011 ), thereby reducing the impact of PCR artefacts on allele frequency estimates (Andrews & Luikart 2014 ). In response, we (i) challenge the assertion that the original RAD protocol minimizes the impact of PCR artefacts relative to that of other RAD protocols, (ii) present additional biases in RADseq that are at least as important as PCR artefacts in selecting a RAD protocol and (iii) highlight the strengths and weaknesses of four different approaches to RADseq which are a representative sample of all RAD variants: the original RAD protocol (mbRAD, Miller et al. 2007 ; Baird et al. 2008 ), double digest RAD (ddRAD, Peterson et al. 2012 ), ezRAD (Toonen et al. 2013 ) and 2bRAD (Wang et al. 2012 ). With an understanding of the strengths and weaknesses of different RAD protocols, researchers can make a more informed decision when selecting a RAD protocol.     

The Role of Influenza A Virus Hemagglutinin Residues 226 and 228 in Receptor Specificity and Host Range Restriction

Influenza A viruses can be isolated from a variety of animals, but their range of hosts is restricted. For example, human influenza viruses do not replicate in duck intestine, the major replication site of avian viruses in ducks. Although amino acids at positions 226 and 228 of hemagglutinin (HA) of the H3 subtype are known to be important for this host range restriction, the contributions of specific amino acids at these positions to restriction were not known. Here, we address this issue by generating HAs with site-specific mutations of a human virus that contain different amino acid residues at these positions. We also let ducks select replication-competent viruses from a replication-incompetent virus containing a human virus HA by inoculating animals with 10^10.5 50% egg infectious dose of the latter virus and identified a mutation in the HA. Our results showed that the Ser-to-Gly mutation at position 228, in addition to the Leu-to-Gln mutation at position 226 of the HA of the H3 subtype, is critical for human virus HA to support virus replication in duck intestine.     

Effect of inhibitors of DNA synthesis on the sensitivity of mammalian cells to radiation with different levels of linear energy transfer

Radiosensitivity of Chinese hamster cells increased by 1.71 times in the presence of arabinoside cytosine and hydroxyurea after gamma-irradiation, and no sensitization occurred after irradiation with carbon ions of 6.6 MeV/nuclon (LET, 227 keV/micron). Under a standard set of conditions, the RBE coefficient of carbon ions decreased from 3.09 to 1.78 in the presence of DNA synthesis inhibitors. The possible mechanism of this phenomenon is discussed.     

Estimating the potential for coral adaptation to global warming across the Indo‐West Pacific

The potential of reef‐building corals to adapt to increasing sea‐surface temperatures is often debated but has rarely been comprehensively modeled on a region‐wide scale. We used individual‐based simulations to model adaptation to warming in a coral metapopulation comprising 680 reefs and representing the whole of the Central Indo‐West Pacific. Encouragingly, some reefs—most notably Vietnam, Japan, Taiwan, New Caledonia and the southern half of the Great Barrier Reef—exhibited high capacity for adaptation and, in our model, maintained coral cover even under a rapid “business‐as‐usual” warming scenario throughout the modeled period (200 years). Higher resilience of these reefs was observed under all tested parameter settings except the models prohibiting selection and/or migration during warming. At the same time, the majority of reefs in the region tended to collapse within the first 100 years of warming. The adaptive potential (odds of maintaining high coral cover) of a given reef could be predicted based on two metrics: the reef's present‐day temperature, and the proportion of recruits immigrating from warmer locations. The latter metric explains the most variation in adaptive potential, and significantly correlates with actual coral cover changes observed throughout the region between the 1970s and the early 2000s. These findings will help prioritize coral conservation efforts and plan assisted gene flow interventions to boost the adaptive potential of specific coral populations.