Live cell imaging of X chromosome reactivation during somatic cell reprogramming

Generation of induced pluripotent stem cells (iPSCs) with naive pluripotency is important for their applications in regenerative medicine. In female iPSCs, acquisition of naive pluripotency is coupled to X chromosome reactivation (XCR) during somatic cell reprogramming, and live cell monitoring of XCR is potentially useful for analyzing how iPSCs acquire naive pluripotency. Here we generated female mouse embryonic stem cells (ESCs) that carry the enhanced green fluorescent protein (EGFP) and humanized Kusabira-Orange (hKO) genes inserted into an intergenic site near either the Syap1 or Taf1 gene on both X chromosomes. The ESC clones, which initially expressed both EGFP and hKO, inactivated one of the fluorescent protein genes upon differentiation, indicating that the EGFP and hKO genes are subject to X chromosome inactivation (XCI). When the derived somatic cells carrying the EGFP gene on the inactive X chromosome (Xi) were reprogrammed into iPSCs, the EGFP gene on the Xi was reactivated when pluripotency marker genes were induced. Thus, the fluorescent protein genes inserted into an intergenic locus on both X chromosomes enable live cell monitoring of XCI during ESC differentiation and XCR during reprogramming. This is the first study that succeeded live cell imaging of XCR during reprogramming.     

Determination of ZD9583, a thromboxane receptor antagonist, in human plasma and urine by liquid chromatography atmospheric pressure chemical ionization tandem mass spectrometry

A liquid chromatography–atmospheric pressure chemical ionization tandem mass spectrometry (LC–APCI-MS–MS) method is described for the determination of a thromboxane receptor antagonist (4Z)-6-((2S,4S,5R)-2-(1-(2-cyano-4-methylphenoxy)-1-methylethyl)-4-(3-pyridyl)-3-dioxan-5-yl)hex-4-enoic acid (ZD9583, I) in human plasma and urine. Proteins in plasma and urine samples are precipitated using acidified acetonitrile. The resulting supernatant is chromatographed on a C₈ reversed-phase chromatography column. Following the diversion of the solvent front from the mass spectrometer by a switching valve, the column eluate is passed on to the mass spectrometer via a heated nebulizer interface where the analyte is detected by multiple reaction monitoring (MRM). The method has a chromatographic run time of less than 2 min, a linear calibration curve with a range of 1–500 ng ml⁻¹ and intra- and inter-day precision estimates of less than 10% over the calibration range.     

Laminin-1 induces endocytosis of 67KDa laminin receptor and protects Neuroscreen-1 cells against death induced by serum withdrawal

Although the function of laminin in the basement membrane is known, the function of soluble “neuronal” laminin is unknown. Since laminin is neuroprotective, we determined whether the soluble laminin-1 induces signaling for neuroprotection via its 67KDa laminin-1 receptor (67LR). Treatment of Neuroscreen-1 (NS-1) cells with laminin-1 or YIGSR peptide, which corresponds to a sequence in laminin-1 β1 chain that binds to 67LR, induced a decrease in the cell-surface expression of 67LR and caused its internalization. Furthermore, intracellular cAMP-elevating agents, dibutyryl-cAMP, forskolin, and rolipram, also induced this internalization. Both soluble laminin-1 and YIGSR induced a sustained elevation of intracellular cAMP under defined conditions, suggesting a causal role of cAMP in the endocytosis of 67LR. This endocytosis was not observed in cells deficient in protein kinase A (PKA) nor in cells treated with either SQ 22536, an inhibitor for adenylyl cyclase, or ESI-09, an inhibitor for the exchange protein directly activated by cAMP (Epac). In addition, when internalization occurred in NS-1 cells, 67LR and adenylyl cyclase were localized in early endosomes. Under conditions in which endocytosis had occurred, both laminin-1 and YIGSR protected NS-1 cells from cell death induced by serum withdrawal. However, under conditions in which endocytosis did not occur, neither laminin-1 nor YIGSR protected these cells. Conceivably, the binding of laminin-1 to 67LR causes initial signaling through PKA and Epac, which causes the internalization of 67LR, along with signaling enzymes, such as adenylyl cyclase, into early endosomes. This causes sustained signaling for protection against cell death induced by serum withdrawal.     

Anther developmental defects in Arabidopsis thaliana male-sterile mutants

 We identified Arabidopsis thaliana sterility mutants by screening T-DNA and EMS-mutagenized lines and characterized several male-sterile mutants with defects specific for different anther processes. Approximately 44 and 855 sterile mutants were uncovered from the T-DNA and EMS screens, respectively. Several mutants were studied in detail with defects that included the establishment of anther morphology, microspore production, pollen differentiation, and anther dehiscence. Both non-dehiscencing and late-dehiscencing mutants were identified. In addition, pollenless mutants were observed with either apparent meiotic defects and/or abnormalities in cell layers surrounding the locules. Two mutant alleles were identified for the POLLENLESS3 locus which have defects in functional microspore production that lead to the degeneration of cells within the anther locules. pollenless3–1 contains a T-DNA insertion that co-segregates with the mutant phenotype and pollenless3–2 has a large deletion in the POLLENLESS3 gene. The POLLENLESS3 gene has no known counterparts in the GenBank, but encodes a protein containing putative nuclear localization and protein-protein interaction motifs. The POLLENLESS3 gene was shown recently to be the same as MS5, a previously described Arabidopsis thaliana male-sterility mutant. Three genes were identified in the POLLENLESS3 genomic region: GENEY, POLLENLESS3, and β9-TUBULIN. The segment of the Arabidopsis thaliana genome containing the POLLENLESS3 and β9-TUBULIN genes is duplicated and present on a different chromosome. Analysis of the POLLENLESS3 expression pattern determined that the 1.3-kb POLLENLESS3 mRNA is localized specifically within meiotic cells in the anther locules and that POLLENLESS3 mRNA is present only during late meiosis. Received: 15 October 1998 / Revision accepted: 19 November 1998     

Universal Strategy with Structural and Chemical Crosslinking Interface for Efficient and Stable Perovskite Solar Cells

Due to the limited interface contact and weak interfacial interaction, planar heterojunction perovskite solar cells (PSCs) have space for further improvement. Herein, a structural and chemical crosslinking interface is proposed and constructed by introducing an extra layer, which blends tin dioxide (SnO₂) nanoparticles with chloride salts. Since the incorporated materials can be dissolved during the fabrication of perovskite, the quality of perovskite films is improved, leading to larger grain size and reduced trap-state density. Also, more chloride ions at the SnO₂/perovskite interface are observed and the interaction between Cl⁻ and Sn⁴⁺ is confirmed. It results in more pronounced n-type SnO₂ with better conductivity and deeper conduction bands, leading to preferable energy level alignment between SnO₂ and perovskite. Consequently, the open-circuit voltage and fill factor of the devices increase, and target cells present better stability, retaining 98% of initial efficiencies after >10 000 h storage in dry air (≈5% relative humidity) and maintaining 85.50% of the initial efficiency after 1000 h of operation under light. This strategy enables the achievement of 25.28% efficiency with a low bandgap (1.53 eV) perovskite composition, and it is confirmed to be universal when other related materials are utilized.     

To treat or not to treat: a quantitative review of the effect of biofouling and control methods in shellfish aquaculture to evaluate the necessity of removal

The global growth of farmed shellfish production has resulted in considerable research investigating how biofouling compromises farm productivity. Shellfish fitness can be compared between fouled stock and stock which has undergone treatment. As treatment options are often harsh, they may deleteriously affect stock. The projected impact of biofouling may therefore be confounded by the impact of treatments. Given the substantial cost of fouling removal, some have questioned the necessity of biofouling mitigation strategies. Meta-analysis revealed that biofouling typically reduces shellfish fitness. However, the fitness of treated stock was often lower or equal to fouled control stock, indicating that many common antifouling (AF) strategies are ineffective at enhancing farm productivity. Overall, caution and diligence are required to successfully implement biofouling mitigation strategies. The need remains for increased passive prevention approaches and novel AF strategies suitable for shellfish culture, such as strategic siting of bivalve farms in areas of low biofouling larval supply.     

Biochemical and spectroscopic studies of human melanotransferrin (MTf): electron-paramagnetic resonance evidence for a difference between the iron-binding site of MTf and other transferrins

Melanotransferrin (MTf) is a member of the transferrin (Tf) family of iron (Fe)-binding proteins that was first identified as a cell-surface marker of melanoma. Although MTf has a high-affinity Fe-binding site that is practically identical to that of serum Tf, the protein does not play an essential role in Fe homeostasis and its precise molecular function remains unclear. A Zn(II)-binding motif, distinct from the Fe-binding site, has been proposed in human MTf based on computer modelling studies. However, little is known concerning the interaction of its proposed binding site(s) with metals and the consequences in terms of MTf conformation. For the first time, biochemical and spectroscopic techniques have been used in this study to characterise metal ion-binding to recombinant MTf. Initially, the binding of Fe to MTf was examined using 6M urea gel electrophoresis. Although four different iron-loaded forms were observed with serum Tf, only two forms were found with MTf, the apo-form and the N-monoferric holo-protein, suggesting a single high-affinity site. The presence of a single Fe(III)-binding site was also supported by EPR results which indicated that the Fe(III)-binding characteristics of MTf were unique, but somewhat comparable to the N-lobes of human serum Tf and chicken ovo-Tf. Circular dichroism (CD) analysis indicated that, as for Tf, no changes in secondary structure could be observed upon Fe(III)-binding. The ability of MTf to bind Zn(II) was also investigated using CD which demonstrated that the single high-affinity Fe-binding site was distinct from a potential Zn(II)-binding site.     

Discovery of a potent and selective aurora kinase inhibitor

This communication describes the discovery of a novel series of Aurora kinase inhibitors. Key SAR and critical binding elements are discussed. Some of the more advanced analogues potently inhibit cellular proliferation and induce phenotypes consistent with Aurora kinase inhibition. In particular, compound 21 (SNS-314) is a potent and selective Aurora kinase inhibitor that exhibits significant activity in pre-clinical in vivo tumor models.