Human TopBP1 participates in cyclin E/CDK2 activation and preinitiation complex assembly during G1/S transition

Human TopBP1 with eight BRCA1 C terminus domains has been mainly reported to be involved in DNA damage response pathways. Here we show that TopBP1 is also required for G(1) to S progression in a normal cell cycle. TopBP1 deficiency inhibited cells from entering S phase by up-regulating p21 and p27, resulting in down-regulation of cyclin E/CDK2. Although co-depletion of p21 and p27 with TopBP1 restored the cyclin E/CDK2 kinase activity, however, cells remained arrested at the G(1)/S boundary, showing defective chromatin-loading of replication components. Based on these results, we suggest a dual role of TopBP1 necessary for the G(1)/S transition: one for activating cyclin E/CDK2 kinase and the other for loading replication components onto chromatin to initiate DNA synthesis.     

Determination of beraprost in human plasma by a high-performance liquid chromatography-tandem mass spectrometry

A simple, rapid, sensitive and specific liquid chromatography-tandem mass spectrometry method was developed and validated for quantification of beraprost, a stable, orally active prostacyclin analogue with vasodilatory, antiplatelet and cytoprotective effects. The analyte and internal standard, indomethacin, were extracted by solid-phase extraction using OASIS HLB cartridge. The chromatographic separation was performed on a C18 column with a mobile of 0.1% formic acid-methanol (30:70, v/v). The highest daughter ion of deprotonated analyte was quantitated in negative ionization by multiple reactions monitoring with a mass spectrometer. The mass transitions m/z 397>269 and m/z 356>312 were used to measure beraprost and internal standard, respectively. The assay exhibited a linear range from 0.02 to 2 ng/mL for beraprost in human plasma. The lower limit of quantitation was 20 pg/mL with a relative standard deviation of less than 20%. The method was validated with respect to linearity, sensitivity, specificity, recovery, accuracy and precision. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic study.     

Proteomic patterns for classification of ovarian cancer and CTCL serum samples utilizing peak pairs indicative of post-translational modifications

Proteomic patterns as a potential diagnostic technology has been well established for several cancer conditions and other diseases. The use of machine learning techniques such as decision trees, neural networks, genetic algorithms, and other methods has been the basis for pattern determination. Cancer is known to involve signaling pathways that are regulated through PTM of proteins. These modifications are also detectable with high confidence using high-resolution MS. We generated data using a prOTOF mass spectrometer on two sets of patient samples: ovarian cancer and cutaneous t-cell lymphoma (CTCL) with matched normal samples for each disease. Using the knowledge of mass shifts caused by common modifications, we built models using peak pairs and compared this to a conventional technique using individual peaks. The results for each disease showed that a small number of peak pairs gave classification equal to or better than the conventional technique that used multiple individual peaks. This simple peak picking technique could be used to guide identification of important peak pairs involved in the disease process.     

Both metaxin and Tom20 together with two mitochondria-specific motifs support mitochondrial targeting of dual-targeting AtSufE1

Plant cells have two endosymbiotic organelles, chloroplasts, and mitochondria. These organelles perform specific functions that depend on organelle-specific proteins. The majority of chloroplast and mitochondrial proteins are specifically imported by the transit peptide and presequence, respectively. However, a significant number of proteins are also dually targeted to these two organelles. Currently, it is not fully understood how proteins are dually targeted to both chloroplasts and mitochondria. In this study, the mechanism underlying mitochondrial targeting of dual targeting AtSufE1 in Arabidopsis was elucidated. The N-terminal fragment containing 80 residues of AtSufE1 (AtSufE1N80) was sufficient to confer dual targeting of reporter protein, AtSufE1N80:GFP, in protoplasts. Two sequence motifs, two arginine residues at 15^th and 21^st positions, and amino acid (aa) sequence motif AKTLLLRPLK from the 31^st to 40^th aa position, were responsible for targeting to mitochondria a portion of reporter proteins amid the chloroplast targeting. The sequence motif PSEVPFRRT from the 41^st to 50^th aa position constitutes a common motif for targeting to both chloroplasts and mitochondria. For mitochondrial import of AtSufE1:N80, Metaxin played a critical role. In addition, BiFC and protein pull-down experiments showed that AtSufE1N80 specifically interacts with import receptors, Metaxin and Tom20. The interaction of AtSufE1N80 with Metaxin was required for the interaction with Tom20. Based on these results, we propose that mitochondrial targeting of dual-targeting AtSufE1 is mediated by both mitochondria-specific and common sequence motifs in the signal sequence through the interaction with import receptors, Metaxin and Tom20, in a successive manner.     

Angiotensin-converting enzyme inhibitors attenuated advanced glycation end products-induced renal tubular hypertrophy via enhancing nitric oxide signaling

Advanced glycation end products (AGE) and angiotensin II were closely correlated with the progression of diabetic nephopathy (DN). Nitric oxide (NO) is a protective mediator of renal tubular hypertrophy in DN. Here, we examined the molecular mechanisms of angiotensin-converting enzyme inhibitor (ACEI) and NO signaling responsible for diminishing AGE-induced renal tubular hypertrophy. In human renal proximal tubular cells, AGE decreased NO production, inducible NOS activity, guanosine 3′,5′-cyclic monophosphate (cGMP) synthesis, and cGMP-dependent protein kinase (PKG) activation. All theses effects of AGE were reversed by treatment with ACEIs (captopril and enalapril), the NO donor S-nitroso-N-acetylpenicillamine (SNAP), and the PKG activator 8-para-chlorophenylthio-cGMPs (8-pCPT-cGMPs). In addition, AGE-enhanced activation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) were clearly reduced by captopril, enalapril, SNAP, and 8-pCPT-cGMPs. The abilities of ACEIs and NO/PKG activation to inhibit AGE-induced hypertrophic growth were verified by the observation that captopril, enalapril, SNAP, and 8-pCPT-cGMPs decreased protein levels of fibronectin, p21 ^Waf1/Cip1, and receptor for AGE. The results of the present study suggest that ACEIs significantly reduced AGE-increased ERK/JNK/p38 MAPK activation and renal tubular hypertrophy partly through enhancement of the NO/PKG pathway.     

ULK1 negatively regulates Wnt signaling by phosphorylating Dishevelled

Wnt signaling pathway plays critical roles in body axes patterning, cell fate specification, cell proliferation, cell migration, stem cell maintenance, cancer development and etc. Deregulation of this pathway can be causative of cancer, metabolic disease and neurodegenerative disease such as Parkinson`s disease. Among the core components of Wnt signaling pathway, we discovered that Dishevelled (Dsh) interacts with ULK1 and is phosphorylated by ULK1. Unexpectedly, the knockdown of ULK1 elicited a marked increase in Wnt/β-catenin signaling. Multiple ULK1 phosphorylation sites existed on Dsh and many of them were located on the PDZ-DEP region. By using evolutionarily well conserved Drosophila Dsh, we found that S239, S247 and S254 in the PDZ-DEP region are involved in phosphorylation of Dsh by ULK1. Among these, S247 and S254 were conserved in human Dsh. When phospho-mimetic mutants (2D and 2E Dsh mutants) of these conserved residues were generated and expressed in the eyes of the fruit flies, the activity of Dsh was significantly decreased compared to wild type Dsh. Through additional alanine scanning, we further identified that S239, S247, S254, S266, S376, S554 and S555 on full length Dsh were phosphorylated by ULK1. In regards to the S266A mutation located in the PDZ domain among these phosphorylated residues, our results suggested that Dsh forms an SDS-resistant high molecular weight complex with β-catenin and TCF in the nucleus in an S266 phosphorylation-dependent manner. Based on these results, we propose that ULK1 plays a pivotal role in the regulation of Wnt/β-catenin signaling pathway by phosphorylating Dsh.     

Flip‐flop organization in the chloroplast genome of Capsosiphon fulvescens (Ulvophyceae,Chlorophyta)

To better understand organelle genome evolution of the ulvophycean green alga Capsosiphon fulvescens, we sequenced and characterized its complete chloroplast genome. The circular chloroplast genome was 111,561 bp in length with 31.3% GC content that contained 108 genes including 77 protein‐coding genes, two copies of rRNA operons, and 27 tRNAs. In this analysis, we found the two types of isoform, called heteroplasmy, were likely caused by a flip‐flop organization. The flip‐flop mechanism may have caused structural variation and gene conversion in the chloroplast genome of C. fulvescens. In a phylogenetic analysis based on all available ulvophycean chloroplast genome data, including a new C. fulvescens genome, we found three major conflicting signals for C. fulvescens and its sister taxon Pseudoneochloris marina within 70 individual genes: (i) monophyly with Ulotrichales, (ii) monophyly with Ulvales, and (iii) monophyly with the clade of Ulotrichales and Ulvales. Although the 70‐gene concatenated phylogeny supported monophyly with Ulvales for both species, these complex phylogenetic signals of individual genes need further investigations using a data‐rich approach (i.e., organelle genome data) from broader taxon sampling.     

Colorimetric biosensor using dual-amplification of enzyme-free reaction through universal hybridization chain reaction system

On-site genetic detection needs to develop a sensitive and straightforward biosensor without special equipment, which can detect various genetic biomarkers. Hybridization chain reaction (HCR) amplifying signal isothermally could be considered as a good candidate for on-site detection. Here, we developed a novel genetic biosensor on the basis of enzyme-free dual-amplification of universal hybridization chain reaction (uHCR) and hemin/G-quadruplex horseradish peroxidase (HRP)-mimicking DNAzyme. The uHCR is the strategy which enables simple design for multiple target detection by the introduction of target-specific trigger hairpin without changing the whole system according to a target change. Also, HRP-mimicking DNAzyme could produce a sensitive and quantitative colorimetric signal with increased stability with a limit of detection (LOD) of 5.67 nM. The universality of the uHCR biosensor was proven by the detection of four different targets (miR-21, miR-125b, KRAS-Q61K, and BRAF-V600E) for cancer diagnosis. The uHCR biosensor showed specificity that could discriminate single-nucleotide polymorphism. Moreover, the uHCR biosensor could detect targets in the diluted serum sample. Overall, the uHCR biosensor demonstrated the potential for field testing with a simple redesign without complicated steps or special equipment using a universal hairpin system and enzyme-free amplification. This strategy could enable stable and sensitive detection of a variety of targets. Therefore, it could be applied to urgent detection of various pathogens, remote diagnosis, and self-screening of diseases.