The Application of CRISPR/Cas Technology to Efficiently Model Complex Cancer Genomes in Stem Cells

CRISPR/Cas gene editing technologies have emerged as powerful tools in the study of oncogenic transformation. The system's specificity, versatility, and ease of implementation allow researchers to identify important molecular markers and pathways which grant cancers stem cell like properties. This technology has already been applied to researching specific cancers, but has seen restricted therapeutic applications due to inherent ethical and technical limitations. Active development and adaptation of the CRISPR/Cas system has produced new methods to take advantage of both non‐homologous end joining and homologous recombination repair mechanisms in attempts to remedy these limitations and improve the versatility of gene edits that can be created. Nonetheless, until issues with specificity and in vivo efficiency are resolved, utilization of CRISPR/Cas systems would be best employed in the modeling and study of various cancer genes. While it may have potential therapeutic applications to targeted cancer therapies in the future, presently CRISPR/Cas is a remarkable technique that can be utilized for easy and efficient gene editing when it comes to cancer research. J. Cell. Biochem. 119: 134–140, 2018. © 2017 Wiley Periodicals, Inc.     

Seasonal patterns in rainforest litterfall: Detecting endogenous and environmental influences from long‐term sampling

Tropical rainforests play an important role in the storage and cycling of global terrestrial carbon. In the carbon cycle, net primary productivity of forests is linked to soil respiration through the production and decomposition of forest litter. Climate seasonality appears to influence the production of litter although there is considerable variability within and across forests that makes accurate estimates challenging. We explored the effects of climate seasonality on litterfall dynamics in a lowland humid rainforest over a 7‐year period from 2007 to 2013, including an El Niño/La Niña cycle in 2010/2011. Litterfall was sampled fortnightly in 24 traps of 0.50 m diameter within a 1‐ha forest plot. Total mean litterfall was 10.48 ± 1.32 (±SD, dry weight) Mg ha^?1 year^?1 and seasonal in distribution. The different components of litterfall were divided into L_Leaf (63.5%), L_Wood (27.7%) and L_{FF[flowers & fruit]} (8.8%), which all demonstrated seasonal dynamics. Peak falls in L_Leaf and L_Wood were highly predictable, coinciding with maximum daily temperatures and 1 and 2 months prior to maximum monthly rainfall. The El Niño/La Niña cycle coincided with elevated local winter temperatures and peak falls of L_Leaf and L_Wood. Importantly, we establish how sampling length and generalized additive models eliminate the requirement for extensive within‐site sampling when the intention is to describe dynamics in litterfall patterns. Further, a greater understanding of seasonal cycles in litterfall allows us to distinguish between endogenous controls and environmental factors, such as El Niño events, which may have significant impacts on biochemical cycles.     

Connecting domination contracts

In Black Rights/White Wrongs, Charles Mills continues his critique of contemporary American political philosophy for ignoring issues of racial oppression, and in particular for ignoring the way that liberal social contracts rest on underlying domination contracts. In this commentary, I will discuss some of the new research inspired by Mills’ account of domination contracts, including recent accounts of the “capability contract” and the “species contract”, and explore how they relate to Mills’ own work on the “racial contract”. While this new research on diverse domination contracts confirms the richness of Mills’ analysis of the social contract tradition, it may also require some revisions to his own preferred vision of how we theorize racial justice.     

Cellular Localization, Expression, and Structure of the Nuclear Dot Protein 52

Nuclear dots containing PML and Sp100 proteins (NDs) play a role in the development of acute promyelocytic leukemia, are modified after infection with various viruses, and are autoimmunogenic in patients with primary biliary cirrhosis (PBC). PML and Sp100 gene expression is strongly enhanced by interferons (IFN). Based on immunostaining with a monoclonal antibody (mAb C8A2), a third protein, nuclear dot protein 52 (NDP52), was recently localized in NDs. Here we analyzed the cellular localization, expression, and structure of NDP52 in more detail. Our NDP52-specific sera revealed mainly cytoplasmic staining but no ND pattern, neither in untreated nor in IFN-treated cells. Cells transfected with NDP52 expression vectors showed exclusively cytoplasmic staining. In subcellular fractionation experiments, NDP52 was found in cytoplasmic and nuclear fractions. Unlike as described for Sp100 and PML, NDP52 mRNA and protein levels were only marginally enhanced by IFN γ and not enhanced at all by IFN β. NDP52 homodimerization but no heterodimerization with Sp100 or PML could be demonstrated. None of the 93 PBC sera tested contained autoantibodies against NDP52. Finally, mAb C8A2 reacted not only with NDP52 but also with a conformation-dependent epitope on the Sp100 protein. These data imply that NDP52 forms homodimers but no heterodimers with Sp100 and PML, lacks autoantigenicity in PBC, localizes mainly in the cytoplasm, and is associated with the nucleus, but not with NDs. Finally, unlike Sp100 and PML, NDP52 expression is neither markedly enhanced nor localization detectably altered by type I and II IFNs.     

The human U5 snRNP-specific 100-kD protein is an RS domain-containing, putative RNA helicase with significant homology to the yeast splicing factor Prp28p.

Through UV-crosslinking experiments, we previously provided evidence suggesting that a U5 snRNP protein with a molecular weight in the 100-kDa range is an ATP-binding protein (Laggerbauer B, Lauber J, Lührmann R, 1996, Nucleic Acid Res 24:868-875). Separation of HeLa U5 snRNP proteins on 2D gels revealed multiple variants with apparent molecular masses of 100 kDa. Subsequent microsequencing of these variants led to the isolation of a cDNA encoding a protein with an N-terminal RS domain and a C-terminal domain that contains all of the conserved motifs characteristic of members of the DEAD-box family of RNA-stimulated ATPases and RNA helicases. Antibodies raised against cDNA-encoded 100-kDa protein specifically recognized native U5-100kD both on immunoblots and in purified HeLa U5 snRNPs or [U4/U6.U5] tri-snRNP complexes, confirming that the bona fide 100-kDa cDNA had been isolated. In vitro phosphorylation studies demonstrated that U5-100kD can serve as a substrate for both Clk/Sty and the U1 snRNP-associated kinase, and further suggested that the multiple U5-100kD variants observed on 2D gels represent differentially phosphorylated forms of the protein. A database homology search revealed a significant degree of homology (60% similarity, 37% identity) between the Saccharomyces cerevisiae splicing factor, Prp28p, which lacks an N-terminal RS domain, and the C-terminal domain of U5-100kD. Consistent with their designation as structural homologues, anti-Prp28 antibodies recognized specifically the human U5-100kD protein on immunoblots. Together with the DEXH-box U5-200kD protein (Lauber J et al., 1996, EMBO J 15:4001-4015), U5-100kD is the second example of a putative RNA helicase that is tightly associated with the U5 snRNP. Given the recent identification of the U5-116kD protein as a homologue of the ribosomal translocase EF-2 (Fabrizio P, Laggerbauer B, Lauber J, Lane WS, Lührmann R, 1997, EMBO J 16:4092-4106), at least three integral U5 snRNP proteins thus potentially facilitate conformational changes in the spliceosome during nuclear pre-mRNA splicing.     

Effects of boron starvation on boron compartmentation, and possibly hormone-mediated elongation growth and apical dominance of pea (Pisum sativum) plants

Two experiments were carried out to study the effects of boron (B) deficiency on 7-day-old pea plants for 6 or 9 days under controlled growth chamber conditions. Growth and apical dominance (AD) of the plants and their B concentration and compartmentation were followed throughout the starvation period. Additionally, auxin (indoleacetic acid, IAA) concentration in the shoot apex and polar transport from it were measured along with the cytokinin (CK) concentration in the shoot apex and the roots. The results demonstrate that during a 6-day B-deficiency period, B concentration in the water-insoluble residue of the roots was very stable and could not easily be reduced. In contrast, B concentration in the cell sap fraction was very sensitive to external B supply. Twelve hours after transferring the plants from B-sufficient to B-deficient solutions, the B concentration in root cell sap declined to half the concentration of the control plants. In addition, B concentration in the new aerial plant parts, which developed after the onset of the B-deficiency treatment, was extremely low. A decline in elongation growth could be observed as soon as about 4 days after the imposition of B deficiency. This preceded the first measurable growth of lateral buds (release from AD). Before the onset of these morphological changes, there was a considerable decline in CK concentration, accompanied by a dramatic decrease in IAA export out of the shoot apex, a decline in IAA concentration in the shoot apex and the roots and a reduced capacity for polar IAA-transport. These changes are discussed as possible reasons for the observed reduction in elongation growth and AD. These hormonal changes themselves are possibly the result of the decreased symplasmic B concentration, which in turn may be responsible for the reduced concentration in apical CKs. A sequence of events, which may be causally related, is suggested to explain the effects of B deficiency on the growth and AD of pea plants.