Loss of PP6 function interferes with spindle formation and chromosome alignment because of amplified Aurora A activity.

No abstract

Spatial organisation of nuclear compartments is an important regulator of chromatin function, yet the molecular principles that maintain nuclear architecture remain ill-defined. We have used RNA interference to deplete key structural nuclear proteins, the nuclear lamins. In HeLa cells, we show that reduced expression of lamin B1, but not lamin A/C, severely inhibits RNA synthesis – first by RNA polymerase II and later by RNA polymerase I. Declining levels of transcription correlate with different morphological changes in major nuclear compartments, nucleoli and nuclear speckles. Ultimately, nuclear changes linked to the loss of synthetic activity result in expansion of the inter-chromatin domain and corresponding changes in the structure and spatial organisation of chromosome territories, which relocate towards the nuclear periphery. These results show that a lamin B1-containing nucleoskeleton is required to maintain RNA synthesis and that ongoing synthesis is a fundamental determinant of global nuclear architecture in mammalian cells.

Cdc14A and Cdc14B knockout cells with double-strand breaks still arrest in G2, but they fail to repair the damage.

SummaryMutations in the PPP6C catalytic subunit of protein phosphatase 6 (PP6) are drivers for the development of melanoma. Here, we analyse a panel of melanoma-associated mutations in PPP6C and find that these generally compromise assembly of the PP6 holoenzyme and catalytic activity towards a model substrate. Detailed analysis of one mutant, PPP6C-H114Y, in both primary melanoma and engineered cell lines reveals it is destabilized and undergoes increased proteasome-mediated turnover. Global analysis of phosphatase substrates by mass spectrometry identifies the oncogenic kinase Aurora-A as the major PP6 substrate that is dysregulated under these conditions. Accordingly, cells lacking PPP6C or carrying the PPP6C-H114Y allele have elevated Aurora-A kinase activity and display chromosome instability with associated Aurora-A-dependent micronucleation. Chromosomes mis-segregated to these micronuclei are preferentially stained by the DNA damage marker c-H2AX, suggesting that loss of PPP6C promotes both chromosome instability and DNA damage. These findings support the view that formation of micronuclei rather than chromosome instability alone explains how loss of PPP6C, and more generally mitotic spindle and centrosome defects, can act as drivers for genome instability in melanoma and other cancers.

BackgroundProteomics is increasingly becoming an important tool for the study of many different aspects of plant functions, such as investigating the molecular processes underlying in plant physiology, development, differentiation and their interaction with the environments. To investigate the cassava (Manihot esculenta Crantz) proteome, we extracted proteins from somatic embryos, plantlets and tuberous roots of cultivar SC8 and separated them by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).ResultsAnalysis by liquid chromatography-electrospray ionisation-tandem mass spectrometry (LC-ESI-MS/MS) yielded a total of 383 proteins including isoforms, classified into 14 functional groups. The majority of these were carbohydrate and energy metabolism associated proteins (27.2%), followed by those involved in protein biosynthesis (14.4%). Subsequent analysis has revealed that 54, 59, 74 and 102 identified proteins are unique to the somatic embryos, shoots, adventitious roots and tuberous roots, respectively. Some of these proteins may serve as signatures for the physiological and developmental stages of somatic embryos, shoots, adventitious roots and tuberous root. Western blotting results have shown high expression levels of Rubisco in shoots and its absence in the somatic embryos. In addition, high-level expression of α-tubulin was found in tuberous roots, and a low-level one in somatic embryos. This extensive study effectively provides a huge data set of dynamic protein-related information to better understand the molecular basis underlying cassava growth, development, and physiological functions.ConclusionThis work paves the way towards a comprehensive, system-wide analysis of the cassava. Integration with transcriptomics, metabolomics and other large scale "-omics" data with systems biology approaches can open new avenues towards engineering cassava to enhance yields, improve nutritional value and overcome the problem of post-harvest physiological deterioration.

The oligometastatic state is hypothesized to represent an intermediary state of cancer between widely metastatic disease and curable, localized disease. Advancements in radiotherapy have allowed for delivery of high precision, dose escalated treatment known as stereotactic body radiotherapy (SBRT) to targets throughout the body with excellent rates of local control. Recently, the first phase II randomized trial comparing conventional radiotherapy to comprehensive SBRT of oligometastatic disease demonstrated an overall survival and progression free survival advantage. The spine is a common site of metastasis, and a complex site for SBRT given the adjacent spinal cord and the tumor embedded within the bone tissue putting the patient at risk of fracture. Although there are expert spine SBRT guidelines for practice, there are as yet no reported randomized trials that proves superiority as compared to conventional radiation. The use of SBRT in patients with oligometastatic disease and spinal metastases is the focus of this review.

We report a simple, fast, and sensitive approach for visual detection of single-nucleotide polymorphism (SNP) based on hairpin oligonucleotide-functionalized gold nanoparticle (HO-Au-NP) and lateral flow strip biosensor (LFSB). The results presented here expand on prior work ( Mao , X. , Xu , H. , Zeng , Q. , Zeng , L. , and Liu , G. Chem. Commun. 2009 , 3065-3067 .) by providing new approach to prepare HO-Au-NP conjugates with a deoxyadenosine triphosphate (dATP) blocker, which shortens the preparation time of the conjugates from 50 to 8 h and lowers the detection limit 500 times. A hairpin oligonucleotide modified with a thiol at the 5'-end and a biotin at the 3'-end was conjugated with Au-NP through a self-assembling process. Following a blocking step with dATP, the hairpin structure of HO and dATP embed the biotin groups, and make the biotin groups in close proximity to the Au-NP surface, leading to the biotins being "inactive". The strategy of detecting SNP depends on the unique molecular recognition properties of HO to the perfect-matched DNA and single-base-mismatched DNA to generate different quantities of "active" biotin groups on the Au-NP surface. After hybridization reactions, the Au-NPs associated with the activated biotins are captured on the test zone of LFSB via the specific reaction between the activated biotin and preimmobilized streptavidin. Accumulation of Au-NPs produces the characteristic red bands, enabling visual detection of SNP. The preparations of HO-Au-NP conjugates with dATP and the parameters of assay were optimized systematically, and the abilities of detecting SNP were examined in details. The current approach is capable of discriminating as low as 10 pM of perfect-matched DNA and single-base-mismatched DNA within 25 min without instrumentation. Moreover, the approach provides a lower background and higher selectivity compared to the current molecular beacon-based SNP detection. The protocol should facilitate the simple, fast, and cost-effective screening of important SNPs and could readily find wide applications in molecular diagnosis laboratories and in point-of-care testing (field testing).