The Atad5 RFC-like complex is the major unloader of proliferating cell nuclear antigen in Xenopus egg extracts.

Proliferating cell nuclear antigen (PCNA) is a homo-trimeric clamp complex that serves as the molecular hub for various DNA transactions, including DNA synthesis and post-replicative mismatch repair. Its timely loading and unloading are critical for genome stability. PCNA loading is catalyzed by Replication factor C (RFC) and the Ctf18 RFC-like complex (Ctf18-RLC), and its unloading is catalyzed by Atad5/Elg1-RLC.

Loop-mediated isothermal amplification (LAMP) assays for detection of the New Guinea fruit fly Bactrocera trivialis (Drew) (Diptera: Tephritidae).

The cue-lure-responding New Guinea fruit fly, Bactrocera trivialis, poses a biosecurity risk to neighbouring countries, e.g., Australia.

DDK: The Outsourced Kinase of Chromosome Maintenance.

The maintenance of genomic stability during the mitotic cell-cycle not only demands that the DNA is duplicated and repaired with high fidelity, but that following DNA replication the chromatin composition is perpetuated and that the duplicated chromatids remain tethered until their anaphase segregation. The coordination of these processes during S phase is achieved by both cyclin-dependent kinase, CDK, and Dbf4-dependent kinase, DDK. CDK orchestrates the activation of DDK at the G1-to-S transition, acting as the 'global' regulator of S phase and cell-cycle progression, whilst 'local' control of the initiation of DNA replication and repair and their coordination with the re-formation of local chromatin environments and the establishment of chromatid cohesion are delegated to DDK.

The role of DDK and Treslin-MTBP in coordinating replication licensing and pre-initiation complex formation.

Treslin/Ticrr is required for the initiation of DNA replication and binds to MTBP (Mdm2 Binding Protein). Here, we show that in egg extract, MTBP forms an elongated tetramer with Treslin containing two molecules of each protein. Immunodepletion and add-back experiments show that Treslin-MTBP is rate limiting for replication initiation.

Origin of Charge Trapping in TiO/Reduced Graphene Oxide Photocatalytic Composites: Insights from Theory.

Composites of titanium dioxide (TiO) and reduced graphene oxide (RGO) have proven to be much more effective photocatalysts than TiO alone. However, little attention has been paid so far to the chemical structure of TiO/RGO interfaces and to the role that the unavoidable residual oxygen functional groups of RGO play in the photocatalytic mechanism. In this work, we develop models of TiO rutile (110)/RGO interfaces by including a variety of oxygen functional groups known to be present in RGO.

Histone H4K20 methylation mediated chromatin compaction threshold ensures genome integrity by limiting DNA replication licensing.

The decompaction and re-establishment of chromatin organization immediately after mitosis is essential for genome regulation. Mechanisms underlying chromatin structure control in daughter cells are not fully understood. Here we show that a chromatin compaction threshold in cells exiting mitosis ensures genome integrity by limiting replication licensing in G1 phase.

The High-Affinity Interaction between ORC and DNA that Is Required for Replication Licensing Is Inhibited by 2-Arylquinolin-4-Amines.

In late mitosis and G, origins of DNA replication must be "licensed" for use in the upcoming S phase by being encircled by double hexamers of the minichromosome maintenance proteins MCM2-7. A "licensing checkpoint" delays cells in G until sufficient origins have been licensed, but this checkpoint is lost in cancer cells. Inhibition of licensing can therefore kill cancer cells while only delaying normal cells in G.

Screening mitochondrial DNA sequence variation as an alternative method for tracking established and outbreak populations of Queensland fruit fly at the species southern range limit.

Understanding the relationship between incursions of insect pests and established populations is critical to implementing effective control. Studies of genetic variation can provide powerful tools to examine potential invasion pathways and longevity of individual pest outbreaks. The major fruit fly pest in eastern Australia, Queensland fruit fly (Froggatt), has been subject to significant long-term quarantine and population reduction control measures in the major horticulture production areas of southeastern Australia, at the species southern range limit.

Reversal of DDK-Mediated MCM Phosphorylation by Rif1-PP1 Regulates Replication Initiation and Replisome Stability Independently of ATR/Chk1.

Dbf4-dependent kinases (DDKs) are required for the initiation of DNA replication, their essential targets being the MCM2-7 proteins. We show that, in Xenopus laevis egg extracts and human cells, hyper-phosphorylation of DNA-bound Mcm4, but not phosphorylation of Mcm2, correlates with DNA replication. These phosphorylations are differentially affected by the DDK inhibitors PHA-767491 and XL413.

Xenopus Mcm10 is a CDK-substrate required for replication fork stability.

During S phase, following activation of the S phase CDKs and the DBF4-dependent kinases (DDK), double hexamers of Mcm2-7 at licensed replication origins are activated to form the core replicative helicase. Mcm10 is one of several proteins that have been implicated from work in yeasts to play a role in forming a mature replisome during the initiation process. Mcm10 has also been proposed to play a role in promoting replisome stability after initiation has taken place.

Cell Cycle Synchronization in Xenopus Egg Extracts.

Many important discoveries in cell cycle research have been made using cell-free extracts prepared from the eggs of the South African clawed frog Xenopus laevis. These extracts efficiently support the key nuclear functions of the eukaryotic cell cycle in vitro under apparently the same controls that exist in vivo. The Xenopus cell-free system is therefore uniquely suited to the study of the mechanisms, dynamics and integration of cell cycle regulated processes at a biochemical level.

Xenopus Cdc7 executes its essential function early in S phase and is counteracted by checkpoint-regulated protein phosphatase 1.

The initiation of DNA replication requires two protein kinases: cyclin-dependent kinase (Cdk) and Cdc7. Although S phase Cdk activity has been intensively studied, relatively little is known about how Cdc7 regulates progression through S phase. We have used a Cdc7 inhibitor, PHA-767491, to dissect the role of Cdc7 in Xenopus egg extracts.

The Geminin and Idas coiled coils preferentially form a heterodimer that inhibits Geminin function in DNA replication licensing.

Geminin is an important regulator of proliferation and differentiation in metazoans, which predominantly inhibits the DNA replication licensing factor Cdt1, preventing genome over-replication. We show that Geminin preferentially forms stable coiled-coil heterodimers with its homologue, Idas. In contrast to Idas-Geminin heterodimers, Idas homodimers are thermodynamically unstable and are unlikely to exist as a stable macromolecule under physiological conditions.

Monitoring intracellular calcium ion dynamics in hair cell populations with Fluo-4 AM.

We optimized Fluo-4 AM loading of chicken cochlea to report hair-bundle Ca(2+) signals in populations of hair cells. The bundle Ca(2+) signal reported the physiological state of the bundle and cell; extruding cells had very high bundle Fluo-4 fluorescence, cells with intact bundles and tip links had intermediate fluorescence, and damaged cells with broken tip links had low fluorescence. Moreover, Fluo-4 fluorescence in the bundle correlated with Ca(2+) entry through transduction channels; mechanically activating transduction channels increased the Fluo-4 signal, while breaking tip links with Ca(2+) chelators or blocking Ca(2+) entry through transduction channels each caused bundle and cell-body Fluo-4 fluorescence to decrease.

Gadigaleyrodes froggatti, a new genus and species of whitefly (Hemiptera: Aleyrodidae) from Australia.

A new monotypic genus of whiteflies (Aleyrodidae), Gadigaleyrodes gen.n., is described and illustrated for G.

Probing the cochlear amplifier by immobilizing molecular motors of sensory hair cells.

The location and longitudinal extent of cochlear biomechanical amplification has been an open question. In this issue of Neuron, Fisher et al. (2012) demonstrate that sound-induced vibration is amplified over a short region-about one wavelength-prior to the response peak.

Localization of PDZD7 to the stereocilia ankle-link associates this scaffolding protein with the Usher syndrome protein network.

Usher syndrome is the leading cause of genetic deaf-blindness. Monoallelic mutations in PDZD7 increase the severity of Usher type II syndrome caused by mutations in USH2A and GPR98, which respectively encode usherin and GPR98. PDZ domain-containing 7 protein (PDZD7) is a paralog of the scaffolding proteins harmonin and whirlin, which are implicated in Usher type 1 and type 2 syndromes.

Improved biolistic transfection of hair cells.

Transient transfection of hair cells has proven challenging. Here we describe modifications to the Bio-Rad Helios Gene Gun that, along with an optimized protocol, improve transfection of bullfrog, chick, and mouse hair cells. The increased penetrating power afforded by our method allowed us to transfect mouse hair cells from the basal side, through the basilar membrane; this configuration protects hair bundles from damage during the procedure.

Preparation and use of Xenopus egg extracts to study DNA replication and chromatin associated proteins.

The use of cell-free extracts prepared from eggs of the South African clawed toad, Xenopus laevis, has led to many important discoveries in cell cycle research. These egg extracts recapitulate the key nuclear transitions of the eukaryotic cell cycle in vitro under apparently the same controls that exist in vivo. DNA added to the extract is first assembled into a nucleus and is then efficiently replicated.

Large membrane domains in hair bundles specify spatially constricted radixin activation.

The plasma membrane of vertebrate hair bundles interacts intimately with the bundle cytoskeleton to support mechanotransduction and homeostasis. To determine the membrane composition of bundles, we used lipid mass spectrometry with purified chick vestibular bundles. While the bundle glycerophospholipids and acyl chains resemble those of other endomembranes, bundle ceramide and sphingomyelin nearly exclusively contain short-chain, saturated acyl chains.

Digenic inheritance of deafness caused by 8J allele of myosin-VIIA and mutations in other Usher I genes.

Inherited hearing loss in mice has contributed substantially to our understanding of inner-ear function. We identified a new allele at the Myo7a locus, Myo7a(sh1-8J); genomic characterization indicated that Myo7a(sh1-8J) arose from complex deletion encompassing exons 38-40 and 42-46. Homozygous mutant mice had no detectable auditory brainstem response, displayed highly disorganized hair-cell stereocilia and had no detectable MYO7A protein.

Stereocilia membrane deformation: implications for the gating spring and mechanotransduction channel.

In hair cells, although mechanotransduction channels have been localized to tips of shorter stereocilia of the mechanically sensitive hair bundle, little is known about how force is transmitted to the channel. Here, we use a biophysical model of the membrane-channel complex to analyze the nature of the gating spring compliance and channel arrangement. We use a triangulated surface model and Monte Carlo simulation to compute the deformation of the membrane under the action of tip link force.

Distinct energy metabolism of auditory and vestibular sensory epithelia revealed by quantitative mass spectrometry using MS2 intensity.

Measuring the abundance of many proteins over a broad dynamic range requires accurate quantitation. We show empirically that, in MS experiments, relative quantitation using summed dissociation-product ion-current intensities is accurate, albeit variable from protein to protein, and outperforms spectral counting. By applying intensities to quantify proteins in two complex but related tissues, chick auditory and vestibular sensory epithelia, we find that glycolytic enzymes are enriched threefold in auditory epithelia, whereas enzymes responsible for oxidative phosphorylation are increased at least fourfold in vestibular epithelia.

Reply to "on cochlear impedances and the miscomputation of power gain" by Shera et Al. J. Assoc. Re. Otolaryngol.

Using a scanning laser interferometer, we recently measured the volume velocity of the basilar membrane vibration in the sensitive gerbil cochlea and estimated that the cochlear power gain is ~100 at low sound pressure levels (Ren et al., Nat Commun 2:216-223, 2011a). We thank Shera et al.

Evidence for a mammalian late-G1 phase inhibitor of replication licensing distinct from geminin or Cdk activity.

Pre-replication complexes (pre-RCs) are assembled onto DNA during late mitosis and G1 to license replication origins for use in S phase. In order to prevent re-replication of DNA, licensing must be completely shutdown prior to entry into S phase. While mechanisms preventing re-replication during S phase and mitosis have been elucidated, the means by which cells first prevent licensing during late G1 phase are poorly understood.