Rapid sex determination of a wild passerine species using loop‐mediated isothermal amplification (LAMP)

Many bird species are sexually monomorphic and cannot be sexed based on phenotypic traits. Rapid sex determination is often a necessary component of avian studies focusing on behavior, ecology, evolution, and conservation. While PCR‐based methods are the most common technique for molecularly sexing birds in the laboratory, a simpler, faster, and cheaper method has emerged, which can be used in the laboratory, but importantly also in the field. Herein, we used loop‐mediated isothermal amplification (LAMP) for rapid sex determination of blood samples from juvenile European blackcaps, Sylvia atricapilla, sampled in the wild. We designed LAMP primers unique to S. atricapilla based on the sex chromosome‐specific gene, chromo‐helicase‐DNA‐binding protein (CHD), optimized the primers for laboratory and field application, and then used them to test a subset of wild‐caught juvenile blackcaps of unknown gender at the time of capture. Sex determination results were fast and accurate. The advantages of this technique are that it allows researchers to identify the sex of individual birds within hours of sampling and eliminates the need for direct access to a laboratory if implemented at a remote field site. This work adds to the increasing list of available LAMP primers for different bird species and is a new addition within the Passeriformes order.     

Residues involved in the pore‐forming activity of the Clostridium perfringens iota toxin

Clostridium perfringens iota toxin is a binary toxin that is organized into enzyme (Ia) and binding (Ib) components. Ib forms channels in lipid bilayers and mediates the transport of Ia into the target cells. Here we show that Ib residues 334–359 contain a conserved pattern of alternating hydrophobic and hydrophilic residues forming two amphipathic β‐strands involved in membrane insertion and channel formation. This stretch of amino acids shows remarkable structural and functional analogies with the β‐pore‐forming domain of C. perfringens epsilon toxin. Several mutations within the two amphipathic β‐strands affected pore formation, single‐channel conductance and ion selectivity (S339E‐S341E, Q345H N346E) confirming their involvement in channel formation. F454 of Ib corresponds to the Φ‐clamp F427 of anthrax protective antigen and F428 of C2II binary toxins. The mutation F454A resulted in a loss of cytotoxicity and strong increase in single‐channel conductance (500 pS as compared with 85 pS in 1 M KCl) with a slight decrease in cation selectivity, indicating that the Φ‐clamp is highly conserved and crucial for binary toxin activity. In contrast, the mutants Q367D, N430D, L443E had no or only minor effects on Ib properties, while T360I, T360A and T360W caused a dramatic effect on ion selectivity and single‐channel conductance, indicating gross disturbance of the oligomer structure. This suggests that, at least in the iota toxin family, T360 has a structural role in the pore organization. Moreover, introduction of charged residues within the channel (S339E‐S341E) or in the vestibule (Q367D, N430D and L443E) had virtually no effect on chloroquine or Ia binding, whereas F454A, T360I, T360A and T360W strongly decreased the chloroquine and Ia affinity to Ib. These results support that distinct residues within the vestibule interact with chloroquine and Ia or are responsible for channel structure, while the channel lining amino acids play a less important role.     

A new species of Schoutedenichia Jadin & Vercammen-Grandjean, 1954 from Madagascar and a re-description of S. dutoiti (Radford, 1948) from South Africa (Acariformes: Trombiculidae)

A new chigger mite species, Schoutedenichia microcebi n. sp. is described from the grey mouse lemur Microcebus murinus (J.F. Miller) from Madagascar. The new species is closely related to S. dutoiti (Radford, 1948), a species described from a single specimen collected on a rodent in South Africa. Examination of the holotype and new material on S. dutoiti from South Africa enabled us to re-describe this species and provide new data on its hosts and geographical distribution.

     

Caenorhabditis elegans BAF-1 and its kinase VRK-1 participate directly in post-mitotic nuclear envelope assembly

Barrier-to-autointegration factor (BAF) is an essential, highly conserved, metazoan protein. BAF interacts with LEM (LAP2, emerin, MAN1) domain-carrying proteins of the inner nuclear membrane. We analyzed the in vivo function of BAF in Caenorhabditis elegans embryos using both RNA interference and a temperature-sensitive baf-1 gene mutation and found that BAF is directly involved in nuclear envelope (NE) formation. NE defects were observed independent of and before the chromatin organization phenotype previously reported in BAF-depleted worms and flies. We identified vaccinia-related kinase (VRK) as a regulator of BAF phosphorylation and localization. VRK localizes both to the NE and chromatin in a cell-cycle-dependent manner. Depletion of VRK results in several mitotic defects, including impaired NE formation and BAF delocalization. We propose that phosphorylation of BAF by VRK plays an essential regulatory role in the association of BAF with chromatin and nuclear membrane proteins during NE formation.     

Distribution of candidate division JS1 and other Bacteria in tidal sediments of the German Wadden Sea using targeted 16S rRNA gene PCR-DGGE

The bacterial candidate division JS1 dominates a number of 16S rRNA gene libraries from deep subseafloor sediments, yet its distribution in shallow, subsurface sediments has still to be fully documented. Sediment cores (down to 5.5 m) from Wadden Sea tidal flats (Neuharlingersieler Nacken and Gr?ninger Plate) were screened for JS1 16S rRNA genes using targeted PCR-denaturing gradient gel electrophoresis (DGGE), which also detects some other important Bacteria. Bacterial subpopulations at both sites were dominated by Gammaproteobacteria in the upper sediment layers (down to 2 m) and in deeper layers by members of the Chloroflexi. The deeper layers of Neuharlingersieler Nacken consisted of grey mud with low sulphate (0.1-10 mM), elevated total organic carbon (TOC) ( approximately 1-2%) and JS1 sequences were abundant. In contrast, the deeper sandy layers of Gr?ninger Plate, despite also having reduced sulphate concentrations, had lower TOC (<0.6%) with few detectable JS1 sequences. Results indicated that JS1 prefers muddy, shallow, subsurface sediments with reduced sulphate, whereas Chloroflexi may out-compete JS1 in shallow, sandy, subsurface sediments. Bacterial population changes at both sites ( approximately 2 m) were confirmed by cluster analysis of DGGE profiles, which correlated with increased recalcitrance of the organic matter. This study extends the biogeographical range of JS1. The presence of JS1 and Chloroflexi in Wadden Sea sediments demonstrates that subsurface tidal flats contain similar prokaryotic populations to those found in the deeper subseafloor biosphere.     

Conserved nucleotides in an RNA essential for hepatitis B virus replication show distinct mobility patterns

The number of regulatory RNAs with identified non-canonical structures is increasing, and structural transitions often play a role in their biological function. This stimulates interest in internal motions of RNA, which can underlie structural transitions. Heteronuclear NMR relaxation measurements, which are commonly used to study internal motion, only report on local motions of few sites within the molecule. Here we have studied a 27-nt segment of the human hepatitis B virus (HBV) pregenomic RNA, which is essential for viral replication. We combined heteronuclear relaxation with the new off-resonance ROESY technique, which reports on internal motions of H,H contacts. Using off-resonance ROESY, we could for the first time detect motion of through-space H,H contacts, such as in intra-residue base-ribose contacts or inter-nucleotide contacts, both essential for NMR structure determination. Motions in non-canonical structure elements were found primarily on the sub-nanosecond timescale. Different patterns of mobility were observed among several mobile nucleotides. The most mobile nucleotides are highly conserved among different HBV strains, suggesting that their mobility patterns may be necessary for the RNA’s biological function.     

Semen-derived amyloid fibrils drastically enhance HIV infection

Sexual intercourse is the major route of HIV transmission. To identify endogenous factors that affect the efficiency of sexual viral transmission, we screened a complex peptide/protein library derived from human semen. We show that naturally occurring fragments of the abundant semen marker prostatic acidic phosphatase (PAP) form amyloid fibrils. These fibrils, termed Semen-derived Enhancer of Virus Infection (SEVI), capture HIV virions and promote their attachment to target cells, thereby enhancing the infectious virus titer by several orders of magnitude. Physiological concentrations of SEVI amplified HIV infection of T cells, macrophages, ex vivo human tonsillar tissues, and transgenic rats in vivo, as well as trans-HIV infection of T cells by dendritic or epithelial cells. Amyloidogenic PAP fragments are abundant in seminal fluid and boost semen-mediated enhancement of HIV infection. Thus, they may play an important role in sexual transmission of HIV and could represent new targets for its prevention.     

INTERMEDIATE CLEAVAGE PEPTIDASE55 Modifies Enzyme Amino Termini and Alters Protein Stability in Arabidopsis Mitochondria

Precursor proteins containing mitochondrial peptide signals are cleaved after import by a mitochondrial processing peptidase. In yeast (Saccharomyces cerevisiae) and human (Homo sapiens), INTERMEDIATE CLEAVAGE PEPTIDASE55 (ICP55) plays a role in stabilizing mitochondrial proteins by the removal of single amino acids from mitochondrial processing peptidase-processed proteins. We have investigated the role of a metallopeptidase (At1g09300) from Arabidopsis (Arabidopsis thaliana) that has sequence similarity to yeast ICP55. We identified this protein in mitochondria by mass spectrometry and have studied its function in a transfer DNA insertion line (icp55). Monitoring of amino-terminal peptides showed that Arabidopsis ICP55 was responsible for the removal of single amino acids, and its action explained the −3 arginine processing motif of a number of mitochondrial proteins. ICP55 also removed single amino acids from mitochondrial proteins known to be cleaved at nonconserved arginine sites, a subset of mitochondrial proteins specific to plants. Faster mitochondrial protein degradation rates not only for ICP55 cleaved protein but also for some non-ICP55 cleaved proteins were observed in Arabidopsis mitochondrial samples isolated from icp55 than from the wild type, indicating that a complicated protease degradation network has been affected. The lower protein stability of isolated mitochondria and the lack of processing of target proteins in icp55 were complemented by transformation with the full-length ICP55. Analysis of in vitro degradation rates and protein turnover rates in vivo of specific proteins indicated that serine hydroxymethyltransferase was affected in icp55. The maturation of serine hydroxymethyltransferase by ICP55 is unusual, as it involves breaking an amino-terminal diserine that is not known as an ICP55 substrate in other organisms and that is typically considered a sequence that stabilizes rather than destabilizes a protein.Plant mitochondria provide energy production through respiration. Most mitochondrial proteins responsible for the machinery of respiration and metabolism are synthesized in the cytosol and imported into mitochondria. After import, N-terminal presequences containing targeting signals are cleaved from many proteins by the mitochondrial processing peptidase (MPP; Sjoling and Glaser, 1998; Zhang and Glaser, 2002), and the mitochondrial presequences themselves are then degraded by presequence peptidases (Ståhl et al., 2002; Moberg et al., 2003; Bhushan et al., 2005) and oligopeptidase (Kmiec et al., 2013). The specificity of the MPP cutting sites has been analyzed by comparison of the experimentally determined N-terminal sequences of mature proteins with the amino acid sequences of the precursor proteins. From this analysis, the most frequently observed MPP cleavage sites are referred to as −2 from an Arg (the −2R cleavage group) and −3 from an Arg (the −3R cleavage group) within the presequence (Zhang et al., 2001; Huang et al., 2009). However, despite the clear presence of both groups in experimental data, the −3R motif did not fit the high-resolution structure of MPP, which revealed −2R as the only probable cleavage site (Taylor et al., 2001). In yeast (Saccharomyces cerevisiae), there are also a group of mitochondrial proteins with a −10R motif that are now known to be first cleaved by MPP and then by OCTAPEPTIDYL AMINOPEPTIDASE1 (Oct1; Isaya et al., 1991; Vögtle et al., 2011). In plants, a group of mitochondrial proteins with nonconserved Arg cleavage sites has been reported (Huang et al., 2009). For many years, it has remained unclear why mitochondrial proteins from plants and yeast differed in the cleavage motif and what other proteases might be involved in these processes in plants.The identification of INTERMEDIATE CLEAVAGE PEPTIDASE55 (P40005.1) in yeast mitochondria solved the long-standing problem of apparent −2R and −3R cleavage sites by MPP (Vögtle et al., 2009). In yeast, ICP55 removes one residue from the mature protein after cleavage by MPP, leading to the Arg residue in the presequence being −3 from the position of the final N terminus of the mature protein. Therefore, the −3R group of proteins undergo a two-step cleavage, first by MPP and then a single amino acid removal by ICP55. In yeast, ICP55 cleaves exclusively after a Tyr, Leu, or Phe, leaving the first residue of the mature protein to typically be Ser, Ala, or Thr (Vögtle et al., 2009). Our analysis of plant mitochondrial presequence cleavage motifs indicated that the major −3R group (55%–58%) in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa; Huang et al., 2009) had a very similar motif to that cleaved by ICP55 in yeast (Vögtle et al., 2009). ICP55 belongs to the M24 metallopeptidase peptidase family and is critical for mitochondrial protein stability in yeast (Vögtle et al., 2009). The stability, abundance, and turnover of mitochondrial proteins in yeast are also determined by other mitochondrial proteases (Brandner et al., 2005). Plant mitochondria also contain many other proteases, such as filamentation temperature sensitive H protein, long undivided filaments protein, caseinolytic protease, and degradation of periplasmic protein classes (Gibala et al., 2009; Rigas et al., 2009; Kmiec et al., 2012; Kwasniak et al., 2012; Solheim et al., 2012). Metallopeptidase and other protease classes could be part of a complex network controlling protein stability and, thus, the rate of protein turnover in plant mitochondria (van Wijk, 2015).We have compiled lists of Arabidopsis mitochondrial proteins using organelle isolation, fractionation, and proteomic analysis (Heazlewood et al., 2004; Taylor et al., 2011). Our in-depth analysis of mitochondrial matrix proteins has identified a protein with unknown function encoded by the gene At1g09300. This protein has some similarity to yeast ICP55 ({"type":"entrez-protein","attrs":{"text":"P40051.1","term_id":"731481","term_text":"P40051.1"}}P40051.1) and has been suggested to be an ICP55-like protein in plants based on sequence comparisons (Kwasniak et al., 2012). In this study, we have examined the role of this ICP55-like protein (At1g09300) in the cleavage of plant mitochondrial proteins using peptide mass spectrometry (MS) to compare the wild type with a transfer DNA (T-DNA) insertion line, icp55. The plant mitochondrial ICP55-like protein is not only responsible for −3R group protein cleavage, as observed in yeast, but also the cleavage of non-R group proteins that are only found in plant mitochondria, to our knowledge. The lack of ICP55 alters mitochondrial protein stability, as indicated by an analysis of protein degradation rates in isolated mitochondria. We also show that serine hydromethyltransferase (SHMT) is processed by ICP55, a degradation product of SHMT is stabilized in vitro in the absence of ICP55, and SHMT turns over more rapidly in vivo in icp55. The maturation of SHMT by cleavage of a diserine represents a new substrate class for ICP55 and appears to destabilize rather than stabilize this enzyme.