High histone variant H3.3 content in mouse prospermatogonia suggests a role in epigenetic reformatting

Histone variants can incorporate into the nucleosome outside of S-phase. Some are known to play important roles in mammalian germ cell development, this cell lineage being characterized by long phases of quiescence, a protracted meiotic phase, and genome-wide epigenetic reformatting events. The best known example of such an event is the global-scale erasure of DNA methylation in sexually indifferent primordial germ cells, then its re-establishment in fetal prospermatogonia and growing oocytes. Histone H3 and its post-translationally modified forms provide important waypoints in the establishment of epigenetic states. Using mass spectrometry and immunoblotting, we show that the H3.3 replacement variant is present at an unusually high amount in mouse prospermatogonia at the peak stage of global DNA methylation re-establishment. We speculate that H3.3 facilitates this process through achieving a greater level of accessibility of chromatin modifiers to DNA.     

Mechanism of Na‐Ion Storage in Hard Carbon Anodes Revealed by Heteroatom Doping

Hard carbon is the leading candidate anode for commercialization of Na‐ion batteries. Hard carbon has a unique local atomic structure, which is composed of nanodomains of layered rumpled sheets that have short‐range local order resembling graphene within each layer, but complete disorder along the c‐axis between layers. A primary challenge holding back the development of Na‐ion batteries is that a complete understanding of the structure–capacity correlations of Na‐ion storage in hard carbon has remained elusive. This article presents two key discoveries: first, the characteristics of hard carbons structure can be modified systematically by heteroatom doping, and second, that these structural changes greatly affect Na‐ion storage properties, which reveals the mechanisms for Na storage in hard carbon. Specifically, via P or S doping, the interlayer spacing is dilated, which extends the low‐voltage plateau capacity, while increasing the defect concentrations with P or B doping leads to higher sloping sodiation capacity. The combined experimental studies and first principles calculations reveal that it is the Na‐ion‐defect binding that corresponds to the sloping capacity, while the Na intercalation between graphenic layers causes the low‐potential plateau capacity. The understanding suggests a new design principle of hard carbon anode: more reversibly binding defects and dilated turbostratic domains, given that the specific surface area is maintained low.     

HIV-1 Tat Activates Neuronal Ryanodine Receptors with Rapid Induction of the Unfolded Protein Response and Mitochondrial Hyperpolarization

Neurologic disease caused by human immunodeficiency virus type 1 (HIV-1) is ultimately refractory to highly active antiretroviral therapy (HAART) because of failure of complete virus eradication in the central nervous system (CNS), and disruption of normal neural signaling events by virally induced chronic neuroinflammation. We have previously reported that HIV-1 Tat can induce mitochondrial hyperpolarization in cortical neurons, thus compromising the ability of the neuron to buffer calcium and sustain energy production for normal synaptic communication. In this report, we demonstrate that Tat induces rapid loss of ER calcium mediated by the ryanodine receptor (RyR), followed by the unfolded protein response (UPR) and pathologic dilatation of the ER in cortical neurons in vitro. RyR antagonism attenuated both Tat-mediated mitochondrial hyperpolarization and UPR induction. Delivery of Tat to murine CNS in vivo also leads to long-lasting pathologic ER dilatation and mitochondrial morphologic abnormalities. Finally, we performed ultrastructural studies that demonstrated mitochondria with abnormal morphology and dilated endoplasmic reticulum (ER) in brain tissue of patients with HIV-1 inflammation and neurodegeneration. Collectively, these data suggest that abnormal RyR signaling mediates the neuronal UPR with failure of mitochondrial energy metabolism, and is a critical locus for the neuropathogenesis of HIV-1 in the CNS.     

Seawater requirement for the production of lipoxazolidinones by marine actinomycete strain NPS8920

A novel marine actinomycete strain NPS8920 produces a new class of 4-oxazolidinone antibiotics lipoxazolidinone A, B and C. Lipoxazolidinone A possesses good potency (1-2 microg/mL) against drug-resistant pathogens methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VRE). Strain NPS8920 exhibits different morphologies in both agar and submerged cultures. The ability of strain NPS8920 to sporulate on saline-based agar media but not on deionized water-based agar medium supported that strain NPS8920 is a marine actinomycete. While strain NPS8920 does not require seawater for growth, the production of lipoxazolidinones by strain NPS8920 can only be detected in the seawater-based media. The optimal production of lipoxazolidinones was observed in the natural seawater-based medium. Strain NPS8920 produced 10-20% of lipoxazolidinones in the synthetic sea salt Instant Ocean-based medium and no production in the sodium chloride-based and deionized water-based media.     

A257T linker region mutant of T7 helicase-primase protein is defective in DNA loading and rescued by T7 DNA polymerase

The helicase and primase activities of the hexameric ring-shaped T7 gp4 protein reside in two separate domains connected by a linker region. This linker region is part of the subunit interface between monomers, and point mutations in this region have deleterious effects on the helicase functions. One such linker region mutant, A257T, is analogous to the A359T mutant of the homologous human mitochondrial DNA helicase Twinkle, which is linked to diseases such as progressive external opthalmoplegia. Electron microscopy studies show that A257T gp4 is normal in forming rings with dTTP, but the rings do not assemble efficiently on the DNA. Therefore, A257T, unlike the WT gp4, does not preassemble on the unwinding DNA substrate with dTTP without Mg(II), and its DNA unwinding activity in ensemble assays is slow and limited by the DNA loading rate. Single molecule assays measured a 45 times slower rate of A257T loading on DNA compared with WT gp4. Interestingly, once loaded, A257T has almost WT-like translocation and DNA unwinding activities. Strikingly, A257T preassembles stably on the DNA in the presence of T7 DNA polymerase, which restores the ensemble unwinding activity of A257T to ～75% of WT, and the rescue does not require DNA synthesis. The DNA loading rate of A257T, however, remains slow even in the presence of the polymerase, which explains why A257T does not support T7 phage growth. Similar types of defects in the related human mitochondrial DNA helicase may be responsible for inefficient DNA replication leading to the disease states.     

Differentiation of repetitive DNA sites and sex chromosome systems reveal closely related group in Parodontidae (Actinopterygii: Characiformes)

Parodon and Apareiodon lack sufficiently consistent morphological traits to be considered a monophyletic group in Parodontidae. Species within this family are either sex-homomorphic or sex-heteromorphic (i.e., lacking a differentiated sex chromosome system, ZZ/ZW or ZZ/ZW(1)W(2)). In this study, a DNA fragment from the heterochromatin segment of the W chromosome of Apareiodon ibitiensis (named WAp) was microdissected and used for in situ mapping of nine Parodontidae species. The species were also characterized using a satellite DNA probe (pPh2004). The species were phylogenetically clustered according to 17 characters, which were examined by both classical and molecular cytogenetic techniques. Given the present results, the single ZZ/ZW sex chromosome system seems to have been derived from a paracentric inversion of a terminal WAp site onto the proximal regions of the short arms of a metacentric chromosome pair, followed by WAp site amplification. We reason that these events restrained recombination and favored differentiation of the W chromosome in some species. Moreover, co-hybridization experiments targeting the WAp and pPh2004 repetitive DNA sites of A. affinis suggest that the ZZ/ZW(1)W(2) sex chromosomes of this species may have arisen from a translocation between the proto-sex chromosome and an autosome. Our phylogenetic analysis corroborates the hypothesis of sex chromosome differentiation and establishes groups of closely related species. The phylogenetic reorganization in response to these new data supports the presence of internal monophyletic groups within Parodontidae.     

Dissection of the karyopherin alpha nuclear localization signal (NLS)-binding groove: functional requirements for NLS binding

Classical protein import, mediated by the binding of a classical nuclear localization signal (NLS) to the NLS receptor, karyopherin/importin alpha, is the most well studied nuclear transport process. Classical NLSs are either monopartite sequences that contain a single cluster of basic amino acids (Lys/Arg) or bipartite sequences that contain two clusters of basic residues separated by an unconserved linker region. We have created mutations in conserved residues in each of the three NLS-binding sites/regions in Saccharomyces cerevisiae karyopherin alpha (SRP1). For each mutant we have analyzed binding to both a monopartite and a bipartite NLS cargo in vitro. We have also expressed each karyopherin alpha mutant in vivo as the only cellular copy of the NLS receptor and examined the impact on cell growth and import of both monopartite and bipartite NLS-containing cargoes. Our results reveal the functional significance of specific residues within karyopherin alpha for NLS cargo binding. A karyopherin alpha variant with a mutation in the major NLS-binding site exhibits decreased binding to both monopartite and bipartite NLS cargoes, and this protein is not functional in vivo. However, we also find that a karyopherin alpha variant with a mutation in the minor NLS-binding site, which shows decreased binding only to bipartite NLS-containing cargoes, is also not functional in vivo. This suggests that the cell is dependent on the function of at least one bipartite NLS cargo that is imported into the nucleus by karyopherin alpha. Our experiments also reveal functional importance for the linker-binding region. This study provides insight into how changes in binding to cellular NLS sequences could impact cellular function. In addition, this work has led to the creation of conditional alleles of karyopherin alpha with well characterized defects in NLS binding that will be useful for identifying and characterizing novel NLS cargoes.     

Current approaches to native woodland restoration in Scotland

Summary

A large number of projects have recently been initiated in Scotland aiming to restore native woodland, which are being undertaken by a variety of organisations, often in partnership, with environmental NGOs playing a leading role. The objectives, constraints and methodologies of these projects are critically reviewed, partly through a questionnaire survey. Most aim to restore ‘natural’ woodland, but the lack of appropriate reference ecosystems and uncertainty about the characteristics of the original forest hinder the development of precise objectives, and consequently the criteria for success are poorly defined. Most projects face major practical constraints, particularly browsing by herbivores and invasion by exotic species, indicating that they will require long-term management interventions. Most woodlands are isolated from other woodlands, which threatens their long-term viability, restricting colonisation by woodland organisms. Greater reference to ecological theory in practical restoration projects such as these would enable objectives to be defined with more precision, encourage a greater emphasis on ecological processes rather than community composition, and improve management plans through use of predictive tools. In particular, the integration of woodlands into habitat networks, increasing ecological connectivity between woodland fragments, is considered essential to ensure success in the long term.