Generation of expression vectors for high-throughput functional analysis of target genes in <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

An immediate challenge in the post-genomic era is to assign a biological functions to proteins unraveled by genome analysis. This report is based on studies conducted using Schizosaccharomyces pombe, a simple model organism, and presents various vector systems as tools for high-throughput functional analysis of human genes. We constructed S. pombe expression vectors for efficient cloning of genes via the Gateway system. We modified the pREP and pSLF series vectors, which are widely used for gene expression in S. pombe. The vectors constructed have a uniform backbone of S. pombe autonomously replicating sequence (ARS) elements with different selective markers, namely, urw4 ⁺ and Saccharomyces cerevisiae LEU2 complementing leul. These vectors contain 3 different strengths of the inducible promoter nmtl, which affect the expression levels of the cloned open reading frames (ORFs). Further, target proteins can be fused with an N-terminal or C-terminal tag such as triple hemagglutinin (3× HA), enhanced green fluorescent protein (EGFP), or Discosoma red fluorescent protein (DsRed). We tested the feasibility of the constructed vectors by using 3 human genes, namely, RAB18, SCC-112, and PTEN. Proper expression of tagged RAB18 was confirmed by western blot analysis. Further, localization of RAB18, SCC112, and PTEN was demonstrated. The constructed vectors can be utilized for high-throughput functional analysis of heterologous genes.     

Genome-Wide Screen of Genes Required for Caffeine Tolerance in Fission Yeast

Background

An excess of caffeine is cytotoxic to all eukaryotic cell types. We aim to study how cells become tolerant to a toxic dose of this drug, and the relationship between caffeine and oxidative stress pathways.

Methodology/Principal Findings

We searched for Schizosaccharomyces pombe mutants with inhibited growth on caffeine-containing plates. We screened a collection of 2,700 haploid mutant cells, of which 98 were sensitive to caffeine. The genes mutated in these sensitive clones were involved in a number of cellular roles including the H₂O₂-induced Pap1 and Sty1 stress pathways, the integrity and calcineurin pathways, cell morphology and chromatin remodeling. We have investigated the role of the oxidative stress pathways in sensing and promoting survival to caffeine. The Pap1 and the Sty1 pathways are both required for normal tolerance to caffeine, but only the Sty1 pathway is activated by the drug. Cells lacking Pap1 are sensitive to caffeine due to the decreased expression of the efflux pump Hba2. Indeed, ?hba2 cells are sensitive to caffeine, and constitutive activation of the Pap1 pathway enhances resistance to caffeine in an Hba2-dependent manner.

Conclusions/Significance

With our caffeine-sensitive, genome-wide screen of an S. pombe deletion collection, we have demonstrated the importance of some oxidative stress pathway components on wild-type tolerance to the drug.     

DAB1 and Reelin effects on amyloid precursor protein and ApoE receptor 2 trafficking and processing

Numerous cytoplasmic adaptor proteins, including JIP1, FE65, and X11alpha, affect amyloid precursor protein (APP) processing and Abeta production. Dab1 is another adaptor protein that interacts with APP as well as with members of the apoE receptor family. We examined the effect of Dab1 on APP and apoEr2 processing in transfected cells and primary neurons. Dab1 interacted with APP and apoEr2 and increased levels of their secreted extracellular domains and their cytoplasmic C-terminal fragments. These effects depended on the NPXY domains of APP and apoEr2 and on the phosphotyrosine binding domain of Dab1 but did not depend on phosphorylation of Dab1. Dab1 decreased the levels of APP beta-C-terminal fragment and secreted Abeta. Full-length Dab1 or its phosphotyrosine binding domain alone increased surface levels of APP, as determined by surface protein biotinylation and live cell staining. A ligand for apoEr2, the extracellular matrix protein Reelin, significantly increased the interaction of apoEr2 with Dab1. Surprisingly, we also found that Reelin treatment significantly increased the interaction of APP and Dab1. Moreover, Reelin treatment increased cleavage of APP and apoEr2 and decreased production of the beta-C-terminal fragment of APP and Abeta. Together, these data suggest that Dab1 alters trafficking and processing of APP and apoEr2, and this effect is influenced by extracellular ligands.     

Taxonomic revision of Dictyochales (Dictyochophyceae) based on morphological,ultrastructural, biochemical and molecular data

The taxonomy of the siliceous members of Dictyochales has been based exclusively on the structure of their silica skeletons. In this study, other morphological, ultrastructural, pigment and molecular characters, in addition to the silica skeletons, were used in the systematics of the siliceous members of this group. As very little is known about the ultrastructure of Dictyocha octonaria, cells of both the skeleton‐bearing and naked forms were also studied. A cladistic analysis based on morphological data and a molecular phylogeny based on nuclear coded small subunit ribosomal DNA retrieved a well‐supported monophyletic Dictyochales. D. octonaria and D. speculum were resolved together with strong support. There was no support for a clade for the three species currently placed in Dictyocha; D. fibula is clearly distinct from the other two Dictyocha taxa, D. speculum and D. octonaria. It is highly likely there are two or three undescribed species within D. octonaria/D. speculum clade and two undescribed genera within the Dictyochales, based on the positions of sequences from uncultured eukaryotes present in GenBank. These findings necessitate a taxonomic revision of the three siliceous, skeleton‐bearing species. Because D. fibula is the type species of Dictyocha, we propose that D. octonaria and D. speculum be reassigned respectively to the genus Octactis as O. octonaria Hovasse and O. speculum (Ehrenberg) F. H. Chang, J. M. Grieve & J. E. Sutherland, comb. nov.     

Essential role of EGFR in cardioprotection and signaling responses to A1 adenosine receptors and ischemic preconditioning

Transactivation of epidermal growth factor receptor (EGFR) may contribute to specific protective responses (e.g. mediated by δ-opioid, bradykinin, or muscarinic receptors). No studies have assessed EGFR involvement in cardioprotection mediated by adenosine receptors (ARs), and the role of EGFR in ischemic preconditioning (IPC) is unclear. We tested EGFR, matrix metalloproteinase (MMP), and heparin-binding EGF (HB-EGF) dependencies of functional protection via A(1)AR agonism or IPC. Pretreatment of mouse hearts with 100 nM of A(1)AR agonist 2-chloro-N(6)-cyclopentyladenosine (CCPA) or IPC (3 × 1.5-min ischemia/2-min reperfusion) substantially improved recovery from 25-min ischemia, reducing left ventricular diastolic dysfunction up to 50% and nearly doubling pressure development and positive change in pressure over time (+dP/dt). Benefit with both CCPA and IPC was eliminated by inhibitors of EGFR tyrosine kinase (0.3 μM AG1478), MMP (0.3 μM GM6001), or HB-EGF ligand (0.3 ng/ml CRM197), none of which independently altered postischemic outcome. Phosphorylation of myocardial EGFR, Erk1/2, and Akt increased two- to threefold during A(1)AR agonism, with responses blocked by AG1478, GM6001, and CRM197. Studies in HL-1 myocytes confirm A(1)AR-dependent Erk1/2 phosphorylation is negated by AG1478 or GM6001, and reduced with CRM197 (as was Akt activation). These data collectively reveal that A(1)AR- and IPC-mediated functional protection is entirely EGFR and MMP dependent, potentially involving the HB-EGF ligand. Myocardial survival kinase activation (Erk1/2, Akt) by A(1)AR agonism is similarly MMP/HB-EGF/EGFR dependent. Thus MMP-mediated EGFR activation appears essential to cardiac protection and signaling via A(1)ARs and preconditioning.     

Next‐generation sequencing,FISH mapping and synteny‐based modeling reveal mechanisms of decreasing dysploidy in Cucumis

In the large Cucurbitaceae genus Cucumis, cucumber (C. sativus) is the only species with 2n = 2x = 14 chromosomes. The majority of the remaining species, including melon (C. melo) and the sister species of cucumber, C. hystrix, have 2n = 2x = 24 chromosomes, implying a reduction from n = 12 to n = 7. To understand the underlying mechanisms, we investigated chromosome synteny among cucumber, C. hystrix and melon using integrated and complementary approaches. We identified 14 inversions and a C. hystrix lineage‐specific reciprocal inversion between C. hystrix and melon. The results reveal the location and orientation of 53 C. hystrix syntenic blocks on the seven cucumber chromosomes, and allow us to infer at least 59 chromosome rearrangement events that led to the seven cucumber chromosomes, including five fusions, four translocations, and 50 inversions. The 12 inferred chromosomes (AK1–AK12) of an ancestor similar to melon and C. hystrix had strikingly different evolutionary fates, with cucumber chromosome C1 apparently resulting from insertion of chromosome AK12 into the centromeric region of translocated AK2/AK8, cucumber chromosome C3 originating from a Robertsonian‐like translocation between AK4 and AK6, and cucumber chromosome C5 originating from fusion of AK9 and AK10. Chromosomes C2, C4 and C6 were the result of complex reshuffling of syntenic blocks from three (AK3, AK5 and AK11), three (AK5, AK7 and AK8) and five (AK2, AK3, AK5, AK8 and AK11) ancestral chromosomes, respectively, through 33 fusion, translocation and inversion events. Previous results (Huang, S., Li, R., Zhang, Z. et al., 2009 , Nat. Genet. 41, 1275–1281; Li, D., Cuevas, H.E., Yang, L., Li, Y., Garcia‐Mas, J., Zalapa, J., Staub, J.E., Luan, F., Reddy, U., He, X., Gong, Z., Weng, Y. 2011a, BMC Genomics, 12, 396) showing that cucumber C7 stayed largely intact during the entire evolution of Cucumis are supported. Results from this study allow a fine‐scale understanding of the mechanisms of dysploid chromosome reduction that has not been achieved previously.     

A Hierarchically Organized Photoelectrode Architecture for Highly Efficient CdS/CdSe‐Sensitized Solar Cells

A form of photoelectrode architecture suitable for inorganic semiconductor solar cells is reported. The developed architecture consists of hierarchically organized TiO₂ nanostructures with several tens of nanometer‐sized particles that have a large surface area and open channels with several hundred‐nanometer‐gaps perpendicular to the substrate. These are tailored by controlling the kinetic energy of the ablated species during pulsed laser deposition (PLD). To fabricate the solar cells, CdS and CdSe inorganic sensitizers are assembled onto the architecture by successive ionic layer adsorption and reaction and polysulfide solution is used as an electrolyte with lead sulfide counter‐electrodes. The inorganic semiconductor solar cells using the developed architecture (PLD‐TiO₂) show high energy conversion efficiencies of 5.57% compared to a conventional mesoporous TiO₂ film(NP‐TiO₂) (3.84%) with an optical mask at 1 sun of illumination. The improved cell performance of PLD‐TiO₂ is attributed to greater light‐harvesting ability, which results in the enhancement of the J_sc value. PLD‐TiO₂ absorbs more CdS/CdSe because of its larger surface area and excellent adhesion properties with fluorine‐doped tin oxide (FTO) substrates. Additionally, due to its unique channel‐shaped architecture, PLD‐TiO₂ has a longer electron lifetime compared to NP‐TiO₂.     

External Validation and Evaluation of Reliability and Validity of the S-ReSC Scoring System to Predict Stone-Free Status after Percutaneous Nephrolithotomy

Objectives

The Seoul National University Renal Stone Complexity (S-ReSC) scoring system was developed to predict the stone-free rate (SFR) after single-tract percutaneous nephrolithotomy (PCNL). This study is an external validation of this scoring system.

Materials and methods

A retrospective review included 327 patients who underwent PCNL at 2 tertiary referral centers. The S-ReSC score was assigned from 1 to 9 based on the number of sites involved. The stone free status was defined as either complete clearance or clinically insignificant residual fragments <4 mm in size at 1 month follow-up imaging. Inter-observer and test-retest reliabilities were evaluated. The statistical performance of the prediction model was assessed by its predictive accuracy, predictive probability, and clinical usefulness.

Results

The overall SFR was 65.4%. SFRs were 83.9%, 47.6%, and 21.4% in low (1–2), intermediate (3–4), and high (5–9) score groups, respectively, with significant differences (P<0.001). Inter-observer and test-retest reliabilities revealed almost perfect agreements. External validation of the S-ReSC scoring system revealed an AUC of 0.731 (95% CI 0.675–0.788). The AUC of 3-titered S-ReSC score groups was 0.691 (95% CI, 0.629–0.753). The calibration plot showed that the predicted probability of SFR had a concordance comparable to that of the observed frequency. The Hosmer–Lemeshow goodness-of-fit statistic revealed an adequate performance of the predictive model (P = 0.10). Inter-observer and test-retest reliability showed a good level of agreement.

Conclusions

The S-ReSC scoring system is useful in predicting the post-PCNL SFR and in describing the complexity of renal stones.     

Systematic Study on Genetic and Epimutational Profile of a Cohort of Amsterdam Criteria-Defined Lynch Syndrome in Singapore

Background

Germline defects of mismatch repair (MMR) genes underlie Lynch Syndrome (LS). We aimed to gain comprehensive genetic and epigenetic profiles of LS families in Singapore, which will facilitate efficient molecular diagnosis of LS in Singapore and the region.

Methods

Fifty nine unrelated families were studied. Mutations in exons, splice-site junctions and promoters of five MMR genes were scanned by high resolution melting assay followed by DNA sequencing, large fragment deletions/duplications and promoter methylation in MLH1, MSH2, MSH6 and PMS2 were evaluated by multiplex ligation-dependent probe amplification. Tumor microsatellite instability (MSI) was assessed with five mononucleotide markers and immunohistochemical staining (IHC) was also performed.

Results

Pathogenic defects, all confined to MLH1 and MSH2, were identified in 17 out of 59 (28.8%) families. The mutational spectrum was highly heterogeneous and 28 novel variants were identified. One recurrent mutation in MLH1 (c.793C>T) was also observed. 92.9% sensitivity for indication of germline mutations conferred by IHC surpassed 64.3% sensitivity by MSI. Furthermore, 15.6% patients with MSS tumors harbored pathogenic mutations.

Conclusions

Among major ethnic groups in Singapore, all pathogenic germline defects were confined to MLH1 and MSH2. Caution should be applied when the Amsterdam criteria and consensus microsatellite marker panel recommended in the revised Bethesda guidelines are applied to the local context. We recommend a screening strategy for the local LS by starting with tumor IHC and the hotspot mutation testing at MLH1 c.793C>T followed by comprehensive mutation scanning in MLH1 and MSH2 prior to proceeding to other MMR genes.