Performance evaluation of existing de novo sequencing algorithms

Two methods have been developed for protein identification from tandem mass spectra: database searching and de novo sequencing. De novo sequencing identifies peptide directly from tandem mass spectra. Among many proposed algorithms, we evaluated the performance of the five de novo sequencing algorithms, AUDENS, Lutefisk, NovoHMM, PepNovo, and PEAKS. Our evaluation methods are based on calculation of relative sequence distance (RSD), algorithm sensitivity, and spectrum quality. We found that de novo sequencing algorithms have different performance in analyzing QSTAR and LCQ mass spectrometer data, but in general, perform better in analyzing QSTAR data than LCQ data. For the QSTAR data, the performance order of the five algorithms is PEAKS > Lutefisk, PepNovo > AUDENS, NovoHMM. The performance of PEAKS, Lutefisk, and PepNovo strongly depends on the spectrum quality and increases with an increase of spectrum quality. However, AUDENS and NovoHMM are not sensitive to the spectrum quality. Compared with other four algorithms, PEAKS has the best sensitivity and also has the best performance in the entire range of spectrum quality. For the LCQ data, the performance order is NovoHMM > PepNovo, PEAKS > Lutefisk > AUDENS. NovoHMM has the best sensitivity, and its performance is the best in the entire range of spectrum quality. But the overall performance of NovoHMM is not significantly different from the performance of PEAKS and PepNovo. AUDENS does not give a good performance in analyzing either QSTAR and LCQ data.     

The oribatid mite genus Macrogena (Acari,Oribatida, Ceratozetidae), with description of two new species from New Zealand

Two new species of oribatid mites of the genus Macrogena (Oribatida, Ceratozetidae) are described from alpine soils of the South Island of New Zealand. Macrogena brevisensilla sp. n. and Macrogena abbreviata sp. n. differ from all species of this genus by the tridactylous legs and by the comparatively short interlamellar setae, respectively. New generic diagnosis and an identification key to the known species of Macrogena are provided.     

Positions of the cytoplasmic end of BK α S0 helix relative to S1–S6 and of β1 TM1 and TM2 relative to S0–S6

The large-conductance, voltage- and Ca²⁺-gated K⁺ (BK) channel consists of four α subunits, which form a voltage- and Ca²⁺-gated channel, and up to four modulatory β subunits. The β1 subunit is expressed in smooth muscle, where it slows BK channel kinetics and shifts the conductance–voltage (G-V) curve to the left at [Ca²⁺] > 2 µM. In addition to the six transmembrane (TM) helices, S1–S6, conserved in all voltage-dependent K⁺ channels, BK α has a unique seventh TM helix, S0, which may contribute to the unusual rightward shift in the G-V curve of BK α in the absence of β1 and to a leftward shift in its presence. Such a role is supported by the close proximity of S0 to S3 and S4 in the voltage-sensing domain. Furthermore, on the extracellular side of the membrane, one of the two TM helices of β1, TM2, is adjacent to S0. We have now analyzed induced disulfide bond formation between substituted Cys residues on the cytoplasmic side of the membrane. There, in contrast, S0 is closest to the S2–S3 loop, from which position it is displaced on the addition of β1. The cytoplasmic ends of β1 TM1 and TM2 are adjacent and are located between the S2–S3 loop of one α subunit and S1 of a neighboring α subunit and are not adjacent to S0; i.e., S0 and TM2 have different trajectories through the membrane. In the absence of β1, 70% of disulfide bonding of W43C (S0) and L175C (S2–S3) has no effect on V₅₀ for activation, implying that the cytoplasmic end of S0 and the S2–S3 loop move in concert, if at all, during activation. Otherwise, linking them together in one state would obstruct the transition to the other state, which would certainly change V₅₀.     

Sliding of a 43S ribosomal complex from the recognized AUG codon triggered by a delay in eIF2-bound GTP hydrolysis

During eukaryotic translation initiation, 43S ribosomal complex scans mRNA leader unless an AUG codon in an appropriate context is found. Establishing the stable codon–anticodon base-pairing traps the ribosome on the initiator codon and triggers structural rearrangements, which lead to P_i release from the eIF2-bound GTP. It is generally accepted that AUG recognition by the scanning 43S complex sets the final point in the process of start codon selection, while latter stages do not contribute to this process. Here we use translation reconstitution approach and kinetic toe-printing assay to show that after the 48S complex is formed on an AUG codon, in case GTP hydrolysis is impaired, the ribosomal subunit is capable to resume scanning and slides downstream to the next AUG. In contrast to leaky scanning, this sliding is not limited to AUGs in poor nucleotide contexts and occurs after a relatively long pause at the recognized AUG. Thus, recognition of an AUG per se does not inevitably lead to this codon being selected for initiation of protein synthesis. Instead, it is eIF5-induced GTP hydrolysis and P_i release that irreversibly trap the 48S complex, and this complex is further stabilized by eIF5B and 60S joining.     

Sure facts, speculations, and open questions about the evolution of transposable element copy number

Transposable elements (TEs) are sequences capable of multiplying in their host's genome. They survive by increasing copy numbers due to transpositions, and natural selection washes them out because hosts with heavier loads of TEs have lower fitness. The available phylogenetic evidence supports the view that TEs have existed in living organisms for hundreds of millions of years. A fundamental question facing the field is how can an equilibrium be attained between transposition and selection which allows these parasitic genetic elements to persist for such a long time period? To answer this question, it is necessary to understand how the rate of TE transposition is controlled and to describe the mechanisms with which natural selection opposes TE accumulation. Perhaps the best models for such a study are copia and gypsy retrotransposons in Drosophila. Their average rate of transposition in nature is between 10^?5 ? 10^?4 transpositions per copy per generation. Unlike nature, transposition rates vary widely, from zero to 10^?2, between laboratory lines. This variability in transposition rate is controlled by host genes. It is probable that in nature TE site heterogeneity is caused by frequent transpositions in rare flies with permissive alleles, and no transpositions happen in the rest of flies. The average rate of TE transposition in nature may thus depend on the frequency of permissive alleles, which is a function of the rate of mutation from restrictive to permissive alleles, the mechanism and the strength of selection opposing TE multiplication, and population size. Thus, evolution of the frequency of permissive alleles of genes controlling transposition must be accounted for to understand evolution of TE copy numbers.     

Expression of humanized anti-Her2/neu single-chain IgG1-like antibody in mammary glands of transgenic mice

A system for production of single-chain antibody in mammary glands of mice was developed on the basis of a hybrid gene constructed from the coding sequence of anti-Her2/neu single-chain antibody inserted into the first exon of the sheep beta-lactoglobulin gene. Lines of transgenic mice were obtained that expressed humanized single-chain anti-Her2/neu IgG1-like antibody in their milk. These antibodies interact with Her2/neu antigen with high affinity (K_d = 0.4 nM). The expression level of the transgene depended on its integration site in the genome but not on the copy number. The transgene had no toxic effect on the mice and was stably inherited, at least for two generations. The results reveal new opportunities of producing single-chain antibodies in the milk of animals.     

Complexation of lipofectamine and cholesterol-modified DNA sequences studied by single-molecule fluorescence techniques

Lipoplex formation for normal and cholesterol-modified oligonucleotides is investigated by fluorescence correlation spectroscopy (FCS). To overcome the problems related to the fitting of autocorrelation curves when fluorescence bursts are present, the baseline fluorescence levels and the fluorescence bursts in the same trace were separately analyzed. This approach was not previously used in FCS studies of lipoplexes and allowed a more detailed characterization of this heterogeneous system. From the baseline levels, the number of free/bound DNA molecules and the presence of tens to hundreds of nanometer-sized lipoplexes were estimated using various mathematical models. Analysis of the fluorescent bursts provided an indication about the sizes of the lipoplexes, the number of DNA molecules in these aggregates, and the relative amount of lipids in each aggregate. An explanation for the higher transfection efficiency previously reported for one of the cholesterol-modified oligonucleotide compounds was found in relation to the formation of large size lipoplexes.     

Single Cells within the Puerto Rico Trench Suggest Hadal Adaptation of Microbial Lineages

Hadal ecosystems are found at a depth of 6,000 m below sea level and below, occupying less than 1% of the total area of the ocean. The microbial communities and metabolic potential in these ecosystems are largely uncharacterized. Here, we present four single amplified genomes (SAGs) obtained from 8,219 m below the sea surface within the hadal ecosystem of the Puerto Rico Trench (PRT). These SAGs are derived from members of deep-sea clades, including the Thaumarchaeota and SAR11 clade, and two are related to previously isolated piezophilic (high-pressure-adapted) microorganisms. In order to identify genes that might play a role in adaptation to deep-sea environments, comparative analyses were performed with genomes from closely related shallow-water microbes. The archaeal SAG possesses genes associated with mixotrophy, including lipoylation and the glycine cleavage pathway. The SAR11 SAG encodes glycolytic enzymes previously reported to be missing from this abundant and cosmopolitan group. The other SAGs, which are related to piezophilic isolates, possess genes that may supplement energy demands through the oxidation of hydrogen or the reduction of nitrous oxide. We found evidence for potential trench-specific gene distributions, as several SAG genes were observed only in a PRT metagenome and not in shallower deep-sea metagenomes. These results illustrate new ecotype features that might perform important roles in the adaptation of microorganisms to life in hadal environments.     

Photobleaching and phototoxicity of KillerRed in tumor spheroids induced by continuous wave and pulsed laser illumination

The purpose of this study was to evaluate photobleaching of the genetically encoded photosensitizer KillerRed in tumor spheroids upon pulsed and continuous wave (CW) laser irradiation and to analyze the mechanisms of cancer cell death after the treatment. We observed the light‐dose dependent mechanism of KillerRed photobleaching over a wide range of fluence rates. Loss of fluorescence was limited to 80% at light doses of 150 J/cm² and more. Based on the bleaching curves, six PDT regimes were applied for irradiation using CW and pulsed regimes at a power density of 160 mW/cm² and light doses of 140 J/cm², 170 J/cm² and 200 J/cm². Irradiation of KillerRed‐expressing spheroids in the pulsed mode (pulse duration 15 ns, pulse repetition rate 10 Hz) induced predominantly apoptotic cell death, while in the case of CW mode the cancer cells underwent necrosis. In general, these results improve our understanding of photobleaching mechanisms in GFP‐like proteins and show the importance of appropriate selection of treatment mode for PDT with KillerRed.

Representative fluorescence image of two KillerRed‐expressing spheroids before and immediately after CW irradiation.