Significant Asia‐Europe divergence in the middle spotted woodpecker (Aves,Picidae)

Population divergence could be strongly affected by species’ ecology and might not be a direct response to climate‐driven environmental change. We tested this in the middle spotted woodpecker (Dendrocoptes medius), a non‐migratory, low‐dispersal habitat specialist associated with old deciduous forests of the Western Palearctic. We present the first phylogeographic study of this species integrating genetic data (three mitochondrial loci, one autosomal and one Z‐linked intron) with species distribution modelling. Based on this species’ ecology, we predicted that the middle spotted woodpecker could have colonized its current range from multiple Last Glacial Maximum (LGM) refugia and that strongly structured populations could be expected. Indeed, we discovered a strong genetic divergence between Asian and European populations, with a split estimated at around one million of years ago. This was surprising given only slight intraspecific variation in plumage and morphology. Although there was no significant phylogeographic structure within the Asian and European groups, we cannot exclude the possibility of multiple refugia within either group during the LGM. This has to be further investigated with more extensive geographic sampling and larger number of variable independently evolving markers. Future studies should also investigate potential differences in vocalizations and ecology between the two groups. Lineages showing similar level of genetic differentiation including woodpeckers are often treated as species‐level taxa. Comparison of our results with the phylogeographic history of other woodpeckers, suggests that sympatric species with similar life‐histories might have idiosyncratic phylogeographic patterns probably resulting from different ecological requirements or historic stochasticity.     

Perfect Narrowband Absorber Based on Patterned Graphene-Silica Multilayer Hyperbolic Metamaterials

Plasmonics - Graphene-based hyperbolic metamaterials are well known for their optical anisotropy, high absorption of electromagnetic radiation, and low energy loss. We proposed a novel multilayer...     

Differential RNA interference: replacement of endogenous with recombinant low density lipoprotein receptor-related protein (LRP)

The interpretation of experiments involving the overexpression of a recombinant cDNA is often hampered by the interference of mRNA expression from the endogenous gene locus. Unless cell lines from naturally occurring mutations or knockout mice are available, difficult and time-consuming gene targeting techniques are required to inhibit endogenous gene expression. Using a method we refer to as "differential RNA interference" we demonstrate that RNA interference can be used to selectively suppress endogenous gene expression without affecting the expression of a co-transfected recombinant version of the same protein. Functional analyses of recombinant low density lipoprotein receptor-related protein (LRP) to study its involvement in lipid metabolism have been shown to be extremely difficult due to its large cDNA and the unavailability of suitable LRP-deficient cell lines. We constructed an expression vector containing the full-length coding sequence of human LRP fused to EGFP and a vector expressing small hairpin RNA directed against the 3'-untranslated region of the wild-type human LRP mRNA (LRP-shRNA). When overexpressed, EGFP-tagged LRP colocalizes with endogenous LRP and stimulates the uptake of LRP ligands. Overexpression of LRP-shRNA vectors significantly inhibits LRP expression, as judged by quantitative RT-PCR, Western blot and immunofluorescence analysis, and it dramatically decreases receptor-associated protein (RAP) uptake. Finally, co-transfection of EGFP-LRP and LRP-shRNA vectors demonstrates selective inhibition of endogenous LRP expression without affecting simultaneous expression of recombinant LRP protein. Thus, utilization of "differential RNA interference" provides a new experimental approach to selectively study the function of any recombinant protein in any given cell line without interference of endogenous protein expression.     

Sulfate reduction and microbial processes of the methane cycle in the sediments of the Sevastopol Bay

The rates of microbial processes of sulfate reduction and of the methane cycle were measured in the bottom sediments of the Sevastopol basin, where seeps of gaseous methane have been previously found. Typically for marine environments, sulfate reduction played the major role in the terminal phase of decomposition of organic matter (OM) in reduced sediments of this area. The rate of this process depended on the amount of available OM. The rate of methanogenesis in the sediments increased with depth, peaking in the subsurface horizons, where decreased sulfate concentration was detected in the pore water. The highest rates of sulfate-dependent anaerobic methane oxidation were found close to the methane-sulfate transition zone as is typical of most investigated marine sediments. The data on the carbon isotopic composition of gaseous methane from the seeps and dissolved CH₄ from the bottom sediments, as well as on the rates of microbial methanogenesis and methane oxidation indicate that the activity of the methane seeps results from accumulation of biogenic methane in the cavities of the underlying geological structures with subsequent periodic release of methane bubbles into the water column.     

Human Immunodeficiency Virus Type 1 Nucleocapsid p1 Confers ESCRT Pathway Dependence

To facilitate the release of infectious progeny virions, human immunodeficiency virus type 1 (HIV-1) exploits the Endosomal Sorting Complex Required for Transport (ESCRT) pathway by engaging Tsg101 and ALIX through late assembly (L) domains in the C-terminal p6 domain of Gag. However, the L domains in p6 are known to be dispensable for efficient particle production by certain HIV-1 Gag constructs that have the nucleocapsid (NC) domain replaced by a foreign dimerization domain to substitute for the assembly function of NC. We now show that one such L domain-independent HIV-1 Gag construct (termed Z_WT) that has NC-p1-p6 replaced by a leucine zipper domain is resistant to dominant-negative inhibitors of the ESCRT pathway that block HIV-1 particle production. However, Z_WT became dependent on the presence of an L domain when NC-p1-p6 was restored to its C terminus. Furthermore, when the NC domain was replaced by a leucine zipper, the p1-p6 region, but not p6 alone, conferred sensitivity to inhibition of the ESCRT pathway. In an authentic HIV-1 Gag context, the effect of an inhibitor of the ESCRT pathway on particle production could be alleviated by deleting a portion of the NC domain together with p1. Together, these results indicate that the ESCRT pathway dependence of HIV-1 budding is determined, at least in part, by the NC-p1 region of Gag.Human immunodeficiency virus type 1 (HIV-1) and other retroviruses hijack the cellular Endosomal Sorting Complex Required for Transport (ESCRT) pathway to promote the detachment of virions from the cell surface and from each other (3, 21, 42, 44, 47). The ESCRT pathway was initially identified based on its requirement for the sorting of ubiquitinated cargo into multivesicular bodies (MVB) (50, 51). During MVB biogenesis, the ESCRT pathway drives the membrane deformation and fission events required for the inward vesiculation of the limiting membrane of this organelle (26, 29, 50, 51). More recently, it emerged that the ESCRT pathway is also essential for the normal abscission of daughter cells during the final stage of cell division (10, 43). Most of the components of the ESCRT pathway are involved in the formation of four heteromeric protein complexes termed ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III. Additional components include ALIX, which interacts both with ESCRT-I and ESCRT-III, and the AAA ATPase Vps4, which mediates the disassembly of ESCRT-III (29, 42).The deformation and scission of endocytic membranes is thought to be mediated by ESCRT-III, which, together with Vps4, constitutes the most conserved element of the pathway (23, 26, 42). Indeed, it was recently shown that purified yeast ESCRT-III induces membrane deformation (52), and in another study three subunits of yeast ESCRT-III were sufficient to promote the formation of intralumenal vesicles in an in vitro assay (61). In mammals, ESCRT-III is formed by the charged MVB proteins (CHMPs), which are structurally related and tightly regulated through autoinhibition (2, 33, 46, 53, 62). The removal of an inhibitory C-terminal domain induces polymerization and association with endosomal membranes and converts CHMPs into potent inhibitors of retroviral budding (34, 46, 53, 60, 62). Alternatively, CHMPs can be converted into strong inhibitors of the ESCRT pathway and of HIV-1 budding through the addition of a bulky tag such as green fluorescent protein (GFP) or red fluorescent protein (RFP) (27, 36, 39, 54). Retroviral budding in general is also strongly inhibited by catalytically inactive Vps4 (22, 41, 55), or upon Vsp4B depletion (31), confirming the crucial role of ESCRT-III.Retroviruses engage the ESCRT pathway through the activity of so-called late assembly (L) domains in Gag. In the case of HIV-1, the primary L domain maps to a conserved PTAP motif in the C-terminal p6 domain of Gag (24, 28) and interacts with the ESCRT-I component Tsg101 (15, 22, 40, 58). HIV-1 p6 also harbors an auxiliary L domain of the LYPx_nL type, which interacts with the V domain of ALIX (20, 35, 39, 54, 59, 63). Interestingly, Tsg101 binding site mutants of HIV-1 can be fully rescued through the overexpression of ALIX, and this rescue depends on the ALIX binding site in p6 (20, 56). In contrast, the overexpression of a specific splice variant of the ubiquitin ligase Nedd4-2 has been shown to rescue the release and infectivity of HIV-1 mutants lacking all known L domains in p6 (12, 57). Nedd4 family ubiquitin ligases had previously been implicated in the function of PPxY-type L domains, which also depend on an intact ESCRT pathway for function (4, 32, 38). However, HIV-1 Gag lacks PPxY motifs, and the WW domains of Nedd4-2, which mediate its interaction with PPxY motifs, are dispensable for the rescue of HIV-1 L domain mutants (57).ALIX also interacts with the nucleocapsid (NC) region of HIV-1 Gag (18, 49), which is located upstream of p6 and the p1 spacer peptide. ALIX binds HIV-1 NC via its Bro1 domain, and the capacity to interact with NC and to stimulate the release of a minimal HIV-1 Gag construct is shared among widely divergent Bro1 domain proteins (48). Based on these findings and the observation that certain mutations in NC cause a phenotype that resembles that of L domain mutants, it has been proposed that NC cooperates with p6 to recruit the machinery required for normal HIV-1 budding (18, 49).NC also plays a role in Gag polyprotein multimerization, and this function of NC depends on its RNA-binding activity (5-8). It has been proposed that the role of the NC-nucleic acid interaction during assembly is to promote the formation of Gag dimers (37), and HIV-1 assembly in the absence of NC can indeed be efficiently rescued by leucine zipper dimerization domains (65). Surprisingly, in this setting the L domains in p6 also became dispensable, since particle production remained efficient even when the entire NC-p1-p6 region of HIV-1 Gag was replaced by a leucine zipper (1, 65). These findings raised the possibility that the reliance of wild-type (WT) HIV-1 Gag on a functional ESCRT pathway is, at least in part, specified by NC-p1-p6. However, it also remained possible that the chimeric Gag constructs engaged the ESCRT pathway in an alternative manner.In the present report, we provide evidence supporting the first of those two possibilities. Particle production became independent of ESCRT when the entire NC-p1-p6 region was replaced by a leucine zipper, and reversion to ESCRT dependence was shown to occur as a result of restoration of p1-p6 but not of p6 alone. Furthermore, although the deletion of p1 alone had little effect in an authentic HIV-1 Gag context, the additional removal of a portion of NC improved particle production in the presence of an inhibitor of the ESCRT pathway. Together, these data imply that the NC-p1 region plays an important role in the ESCRT-dependence of HIV-1 particle production.     

Propagation of protein glycation damage involves modification of tryptophan residues via reactive oxygen species: inhibition by pyridoxamine

Nonenzymatic modification of proteins is one of the key pathogenic factors in diabetic complications. Uncovering the mechanisms of protein damage caused by glucose is fundamental to understanding this pathogenesis and in the development of new therapies. We investigated whether the mechanism involving reactive oxygen species can propagate protein damage in glycation reactions beyond the classical modifications of lysine and arginine residues. We have demonstrated that glucose can cause specific oxidative modification of tryptophan residues in lysozyme and inhibit lysozyme activity. Furthermore, modification of tryptophan residues was also induced by purified albumin-Amadori, a ribose-derived model glycation intermediate. The AGE inhibitor pyridoxamine (PM) prevented the tryptophan modification, whereas another AGE inhibitor and strong carbonyl scavenger, aminoguanidine, was ineffective. PM specifically inhibited generation of hydroxyl radical from albumin-Amadori and protected tryptophan from oxidation by hydroxyl radical species. We conclude that oxidative degradation of either glucose or the protein-Amadori intermediate causes oxidative modification of protein tryptophan residues via hydroxyl radical and can affect protein function under physiologically relevant conditions. This oxidative stress-induced structural and functional protein damage can be ameliorated by PM via sequestration of catalytic metal ions and scavenging of hydroxyl radical, a mechanism that may contribute to the reported therapeutic effects of PM in the complications of diabetes.     

Calcium Signaling Controls Pathogenic Th17 Cell-Mediated Inflammation by Regulating Mitochondrial Function

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Metal-controlled interdomain cooperativity in parvalbumins

Conformational behavior of five homologous proteins, parvalbumins (PAs) from northern pike (α and β isoforms), Baltic cod, and rat (α and β isoforms), was studied by scanning calorimetry, circular dichroism, and bis-ANS fluorescence. The mechanism of the temperature-induced denaturation of these proteins depends dramatically on both the peculiarities of their amino acid sequences and on their interaction with metal ions. For example, the pike α-PA melting can be described by two successive two-state transitions with mid-temperatures of 90 and 120 °C, suggesting the presence of two thermodynamic domains. The intermediate state populated at the end of the first transition was shown to bind Ca²⁺ ions, and was characterized by the largely preserved secondary structure and increased solvent exposure of hydrophobic groups. Mg²⁺- and Na⁺-loaded forms of pike α-PA demonstrated a single two-state transition. Therefore, the mechanism of the PA thermal denaturation is controlled by metal binding. It ranged from the absence of detectable first-order transition (apo-form of pike PA), to the two-state transition (e.g., Mg²⁺- and Na⁺-loaded forms of pike α-PA), to the more complex mechanisms (Ca²⁺-loaded PAs) involving at least one partially folded intermediate. Analysis of isolated cavities in the protein structures revealed that the interface between the CD and EF subdomains of Ca²⁺-loaded pike α-PA is much more loosely packed compared with PAs manifesting single heat-sorption peak. The impairment of interactions between CD and EF subdomains may cause a loss of structural cooperativity and appearance of two separate thermodynamic domains. One more peculiar feature of pike α-PA is that depending on its interactions with metal ions, it can be an intrinsically disordered protein (apo-form), an ordered protein of mesophilic (Na⁺-bound state), thermophilic (Mg²⁺-form), or even of the hyperthermophilic origin (Ca²⁺-form).     

Influence of the N-terminal domain and divalent cations on self-association and DNA binding by the Saccharomyces cerevisiae TATA binding protein

The localization of a single tryptophan to the N-terminal domain and six tyrosines to the C-terminal domain of TBP allows intrinsic fluorescence to separately report on the structures and dynamics of the full-length TATA binding protein (TBP) of Saccharomyces cerevisiae and its C-terminal DNA binding domain (TBPc) as a function of self-association and DNA binding. TBPc is more compact than the C-terminal domain within the full-length protein. Quenching of the intrinsic fluorescence by DNA and external dynamic quenchers shows that the observed tyrosine fluorescence is due to the four residues surrounding the "DNA binding saddle" of the C-terminal domain. TBP's N-terminal domain unfolds and changes its position relative to the C-terminal domain upon DNA binding. It partially shields the DNA binding saddle in octameric TBP, shifting upon dissociation to monomers to expose the saddle to DNA. Structure-energetic correlations were obtained by comparing the contribution that electrostatic interactions make to DNA binding by TBP and TBPc; DNA binding by TBPc is more hydrophobic than that by TBP, suggesting that the N-terminal domain either interacts with bound DNA directly or screens a part of the C-terminal domain, diminishing its electronegativity. The competition between divalent cations, K+, and DNA is not straightforward. Divalent cations strengthen binding of TBP to DNA and do so more strongly for TBPc. We suggest that divalent cations affect the structure of the bound DNA perhaps by stabilizing its distorted conformation in complexes with TBPc and TBP and that the N-terminal domain mimics the effects of divalent cations. These data support an autoinhibitory mechanism in which competition between the N-terminal domain and DNA for the saddle diminishes the DNA binding affinity of the full-length protein.