Alternative Usage Research Articles

TET2 Exon 2 Skipping Confers Sensitivity to AraC and Is an Independent Favorable Prognostic Factor in AML Patients Treated with Intensive Chemotherapy

The nucleoside analogue cytarabine (AraC) has served as the backbone of acute myeloid leukemia (AML) treatment for nearly forty years. About one-third of expressed genes are abnormally spliced in AML yet alternative exon usage (AEU) plays a role in the plasticity of tumor cells and may influence the response to treatment. Here the exon expression profiles of the erythroleukemia K562 cell line were compared to that of its AraC-resistant variant K562/AraC through Affymetrix HTA2 exon arrays. 5140 exon events harbored by 2583 genes distinguished the 2 cell lines. Among these, the skipping of TET2 exon 2 was identified in K562 cells sensitive to AraC whereas TET2 gene expression remained unchanged at the whole transcript level. The results were confirmed by exon-specific RTPCR (ESPCR). Microarray analysis did not evidenced any significant change in mRNA splicing for the 10 remaining exons of the TET2 gene.TET2 is a dioxygenase that catalyzes the conversion of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) and promote DNA demethylation. TET2 somatic mutations occur in about 25% AML, distributing across the whole coding sequence without obvious hot spots. These mutations decrease TET2 enzymatic activity by truncating the protein or affecting its catalytic activity. TET2 exon 2 is spliced in a mutually exclusive manner with exon 1 yet it is used as an alternative promoter (https://fasterdb.lyon.unicancer.fr/). However, TET2 exon 2 is not translated into protein and its role in TET2 regulation is still unknown. Having found that AraC sensitive cells harbor the spliced TET2 isoform, we investigated whether or not skipping of TET2 exon 2 correlate with disease outcome in AML patients treated with AraC-based intensive chemotherapy (AraC-IC).The discovery cohort included 106 consecutive AML patients treated with AraC-IC (median age 57.91, 64 males). RNA was extracted from bone marrow MNCs and assayed for TET2 exon 2 skipping through real-time quantitative ESRTPCR (qESRTPCR) amplification of E1E3 (spliced) and E2E3 (unspliced) TET2 isoforms. TET2 exon 2 skipping was quantified by calculating the ratio E1E3/E2E3. For statistical analysis, the ratio E1E3/E2E3 was dichotomized using median value as the cutoff value. Skipping of TET2 exon 2 was associated with a significantly lower response rate: 65% vs 92%, p = 0.001 but with a significantly lower relapse rate: 39% vs 85%: p<10-4. Univariate analysis (27 months median follow-up) showed that in addition to age and cytogenetic risk, TET2 exon 2 skipping significantly influenced DFS; the higher the level of E1E3 isoform expression, the better the outcome [HR=0.27 (95% confidence interval (CI):0.13-0.53), p<10-4]. The favorable effect of TET2 exon 2 skipping on DFS remained in the 49 patients with normal karyotype: HR=0.31 (CI: 0.12-0.84), p=0.022. Multivariate analysis showed that age [HR=1.83 (CI:1.03-3.24), p=0.039], cytogenetics [HR=3.03 (CI:1.55-5.96), p=0.001], and TET2 exon 2 skipping [HR=0.38 (CI:0.19-0.77), p=0.007] were independent prognostic factors for DFS. The validation cohort included 103 patients with normal karyotype treated with AraC-IC (median age 61.12, 63 males). Skipping of TET2 exon 2 was associated with a lower response rate: 87.5% vs 91.1% (NS) and a significantly lower relapse rate: 3% vs 66%: p<10-4. Univariate analysis showed that in addition to age, FLT3 internal tandem duplication (FLT3-ITD), nucleophosmin (NPM1) exon-12 gene mutation, TET2 exon 2 skipping significantly influenced DFS, EFS, and OS. In multivariate analysis, TET2 exon 2 skipping was the sole prognostic factor for DFS [HR=0.07 (CI: 0.02-0.3), p<10-4], EFS [HR=0.32 (CI: 0.14-0.74), p=0.008], and OS [HR=0.42 (CI: 0.19-0.90), p=0.025]. As an additional control, the prognostic effect of TET2 exon 2 skipping was evaluated in a series of 36 AML unfit for IC and treated with hydroxyurea, azacitidine, decitabine, low dose AraC, or supportive care only. TET2 exon 2 skipping possessed no significant impact on the response rate, the relapse rate, OS, DFS, and EFS in this population.In conclusion TET2 exon 2 skipping is associated with a low relapse rate and possesses a strong favorable prognostic impact in AML treated with AraC-CTI. The mechanism linking this alternative exon usage with resistances to AraC are currently investigated while our results suggest that the determination of TET2 exon 2 splicing status might assist risk stratification. DisclosuresNicolini:Novartis: Consultancy.

Comprehensive Analysis of Aberrant RNA Splicing in Myelodysplastic Syndromes

IntroductionSplicing factor (SF) mutations represent a novel class of driver mutations highly prevalent in myelodysplastic syndromes (MDS), where four genes, including SF3B1, SRSF2, U2AF1, and ZRSR2, are most frequently affected. SF3B1 and SRSF2 mutations show prominent specificity to RARS/RCMD-RS and CMML subtypes, respectively. Although abnormal RNA splicing is thought to play a central role in the pathogenic mechanism of mutated SFs, little is known about exact gene targets, whose abnormal splicing is implicated in the pathogenesis of MDS or about the molecular mechanism that explains the unique subtype specificity of SF mutations, especially to those subtypes characterized by increased ring sideroblasts.MethodsTo address these issues, comprehensive analysis of abnormal RNA splicing was performed for a total of 336 MDS patients with different SF mutations. High-quality RNA was extracted from bone marrow mononuclear cells (BM/MNCs) and/or CD34+ cells and subjected to high-throughput sequencing, followed by exhaustive detection of splicing junctions for all relevant reads. Aberrant splicing events associated with different SF mutations were explored by comparing observed splicing junctions between samples with and without mutations. To specifically determine the role of SF3B1 mutations in ring sideroblast formation, CD34+ bone marrow cells from 13 patients with or without SF3B1 mutations were differentiated in vitro into erythroid cells. RNA sequencing was performed on cells recovered on day 7 and day 14 and differentially spliced genes in erythroid cells between SF3B1-mutated and unmutated samples were investigated.ResultsSF3B1, SRSF2, U2AF1, and ZRSR2 were mutated in 28%, 18%, 5%, and 7% of the patients, respectively. First, we compared SF3B1-mutated samples with those without known SF mutations. RNA sequencing of CD34+ cells revealed 230 splicing events significantly enriched in SF3B1-mutated cases, of which 90% (n = 206) were caused by misrecognition of 3′ splice sites. A similar result was obtained in the experiment for BM/MNCs, where 177 (83%) out of 206 splicing events significantly enriched in SF3B1-mutated samples were caused by misrecognition of 3′ splice sites. These observations were in accordance with the known function of SF3B1 in branch point recognition in the U2 snRNP complex. In both BM/MNCs and CD34+ cells, approximately 70% of the unusual 3′ splice sites were located from 5 to 25 bases downstream from the authentic junctions. The bases immediately upstream to these 3′ splice sites were more often pyrimidines, which was not accordance with the general rule: the bases next to 3′ splice sites are purines, especially guanines. About 50% of these altered 3′ splice sites resulted in frameshift, indicating that SF3B1 mutations cause deleterious effects in many genes simultaneously. Next, to explore the genes whose abnormal splicing is responsible for increased ring sideroblast formation, RNA sequencing was carried out for erythroid progenitor cells differentiated in vitro from CD34+ cells from MDS patients with or without SF3B1 mutations. We found that a total of 146 altered 3′ splice sites were significantly associated with SF3B1 mutations, of which 87 were overlapped to the aberrant splice sites shown to be enriched in SF3B1 mutated primary MDS specimens. These splice sites were found in genes involved in heme biosynthesis, cell cycle progression, and DNA repair and their consequence was mostly deleterious due to aberrant frameshifts. Abnormal splicing events associated with other SF mutations were also identified. Among these, the most common abnormalities associated with mutated SRSF2 and U2AF1 were alternative exon usage. Misrecognition of 3′ splice sites was also common in U2AF1-mutated cases. ZRSR2 mutations were associated with retentions of U12 introns, which is consistent with the known role of ZRSR2 as an essential component of the minor (U12-type) spliceosome.ConclusionSF mutations were associated with characteristic abnormal splicing changes in primary MDS samples as well as in vitro cultured cells. Our results provide insights into the pathogenic role of SF mutations in MDS. DisclosuresNo relevant conflicts of interest to declare.

A Streamlined CRISPR Pipeline to Reliably Generate Zebrafish Frameshifting Alleles

Over the past few years, the cost of directed mutagenesis in zebrafish has plummeted, opening the doors for many researchers to mutate their genes of interest.1 Most recently, CRISPR-Cas-based mutagenesis has taken center stage, with significant advances in zebrafish mutagenesis occurring almost every month. In this system, a short guide RNA (gRNA) directs Cas9 nuclease to cleave specific genomic target sites (Fig. 1A).2 Because it is simple to construct the targeting portion of this system,2 it is possible to target many genes with CRISPR on a limited budget and timescale.1 However, researchers often need to do significant troubleshooting and protocol building before generating mutants with CRISPR, which can slow progress. In this study, we present a detailed start-to-finish guide to CRISPR mutagenesis in zebrafish (Supplementary Data, http://molgen.osu.edu/amacher/resources; Supplementary Data are available online at www.liebertpub.com/zeb), intended to provide a simple method for generating strong loss of function alleles. We have used this pipeline to create mutations in 13 genes to date and provide an example of the pipeline using six1a CRISPR. FIG. 1. Mutagenesis pipeline with time estimates. (A) Illustration of a Cas9-gRNA complex bound to target DNA.12 (B) Example of CRISPR target site design for six1a (NCBI Gene ID 494168), with the target sequence downstream of potential alternative start sites ... We provide guidelines to identify CRISPR targets sites most likely to cause loss of protein function, two protocols to generate gRNA,2,3 and a pipeline to identify and confirm mutations (Fig. 1B–G). CRISPR target sites are selected at locations that cannot be bypassed by alternative start codon usage or alternative first exon usage and are 5′ to regions predicted to be important for protein function. After injection of gRNA and nuclear localized zebrafish codon-optimized cas9 (nls-zCas9-nls) mRNA,4 mutagenesis is quantitatively assayed using high-resolution melt analysis (HRMA) on polymerase chain reaction (PCR) products spanning the CRISPR target. Successful mutagenesis in the injected generation (F0) results in a mixture of WT and mutant PCR products that melt at lower temperatures than uninjected WT controls (Fig. 1C).5 HRMA can also speed up founder recovery in the first outcrossed generation (F1) (Fig. 1D) because each heterozygous lesion produces a characteristic melt curve.5 We recognize that HRMA machines typically cost $10–25K, but costs can be mitigated by sharing one machine between several laboratories or by pairing an existing quantitative PCR machine with online HRMA software6; alternative approaches have also been developed to identify lesions.7–10 The insertions and deletions of genomic DNA (indels) caused by CRISPR mutagenesis result in overlapping sequence reads in heterozygotes, and the mutant read can easily be extracted from heterozygous sequencing by subtracting WT sequence (Fig. 1E). The HRMA-identified potential mutants (putants) found in F1 generation embryos are confirmed on F1 adults (Fig. 1F), and sequencing from F2 generation homozygotes confirms the F1 heterozygous sequencing (Fig. 1G). This pipeline can be completed using 5 weeks labor, spread over 5–7 months (Fig. 1), and during this time, a single researcher can construct many CRISPR mutants in parallel. The CRISPR-Cas technology has provided an abundance of options for genome modification.11 In this protocol, we do not explore every possible CRISPR application, but focus on a single purpose: to frameshift target genes at locations where small (<60 bp) indels are likely to cause loss of protein function. Although our protocol is written with simple indel construction in mind, we expect that it will provide zebrafish researchers with a good starting point to explore the vast array of CRISPR-Cas applications.

HTLV-1-infected CD4+ T-cells display alternative exon usages that culminate in adult T-cell leukemia

BackgroundReprogramming cellular gene transcription sustains HTLV-1 viral persistence that ultimately leads to the development of adult T-cell leukemia/lymphoma (ATLL). We hypothesized that besides these quantitative transcriptional effects, HTLV-1 qualitatively modifies the pattern of cellular gene expression.ResultsExon expression analysis shows that patients’ untransformed and malignant HTLV-1+ CD4+ T-cells exhibit multiple alternate exon usage (AEU) events. These affect either transcriptionally modified or unmodified genes, culminate in ATLL, and unveil new functional pathways involved in cancer and cell cycle. Unsupervised hierarchical clustering of array data permitted to isolate exon expression patterns of 3977 exons that discriminate uninfected, infected, and transformed CD4+ T-cells. Furthermore, untransformed infected CD4+ clones and ATLL samples shared 486 exon modifications distributed in 320 genes, thereby indicating a role of AEUs in HTLV-1 leukemogenesis. Exposing cells to splicing modulators revealed that Sudemycin E reduces cell viability of HTLV-1 transformed cells without affecting primary control CD4+ cells and HTLV-1 negative cell lines, suggesting that the huge excess of AEU might provide news targets for treating ATLL.ConclusionsTaken together, these data reveal that HTLV-1 significantly modifies the structure of cellular transcripts and unmask new putative leukemogenic pathways and possible therapeutic targets.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-014-0119-3) contains supplementary material, which is available to authorized users.

The multidrug resistance 1 gene Abcb1 in brain and placenta: comparative analysis in human and guinea pig.

The Multidrug Resistance 1 (MDR1; alternatively ABCB1) gene product P-glycoprotein (P-gp), an ATP binding cassette transporter, extrudes multiple endogenous and exogenous substrates from the cell, playing an important role in normal physiology and xenobiotic distribution and bioavailability. To date, the predominant animal models used to investigate the role of P-gp have been the mouse and rat, which have two distinct genes, Abcb1a and Abcb1b. In contrast, the human has a single gene, ABCB1, for which only a single isoform has been validated. We and others have previously shown important differences between Abcb1a and Abcb1b, limiting the extrapolation from rodent findings to the human. Since the guinea pig has a relatively long gestation, hemomonochorial placentation and neuroanatomically mature offspring, it is more similar to the human, and may provide a more comparable model for investigating the regulation of P-gp in the brain and placenta, however, to date, the Abcb1 gene in the guinea pig remains to be characterized. The placenta and fetal brain are barrier sites that express P-gp and that play a critical role of protection of the fetus and the fetal brain from maternally administered drugs and other xenobiotics. Using RNA sequencing (RNA-seq), reverse transcription-polymerase chain reaction (RT-PCR) and quantitative PCR (QPCR) to sequence the expressed isoforms of guinea pig Abcb1, we demonstrate that like the human, the guinea pig genome contains one gene for Abcb1 but that it is expressed as at least three different isoforms via alternative splicing and alternate exon usage. Further, we demonstrate that these isoforms are more closely related to human than to rat or mouse isoforms. This striking, overall similarity and evolutionary relatedness between guinea pig Abcb1 and human ABCB1 indicate that the guinea pig represents a relevant animal model for investigating the function and regulation of P-gp in the placenta and brain.

Rapid Glucose Depletion Immobilizes Active Myosin V on Stabilized Actin Cables

Polarization of eukaryotic cells requires organelles and protein complexes to be transported to their proper destinations along the cytoskeleton. When nutrients are abundant, budding yeast grows rapidly transporting secretory vesicles for localized growth and actively segregating organelles. This is mediated by myosin Vs transporting cargos along F-actin bundles known as actin cables. Actin cables are dynamic structures regulated by assembly, stabilization, and disassembly. Polarized growth and actin filament dynamics consume energy. For most organisms, glucose is the preferred energy source and generally represses alternative carbon source usage. Thus, upon abrupt glucose depletion, yeast shuts down pathways consuming large amounts of energy, including the vacuolar-ATPase, translation, and phosphoinositide metabolism. Here we show that glucose withdrawal rapidly (<1 min) depletes ATP levels and that the yeast myosin V, Myo2, responds by relocalizing to actin cables, making it the fastest response documented. Myo2 immobilized on cables releases its secretory cargo, defining a new rigor-like state of a myosin V in vivo. Only actively transporting Myo2 can be converted to the rigor-like state. Glucose depletion has differential effects on the actin cytoskeleton, resulting in disassembly of actin patches with concomitant inhibition of endocytosis and strong stabilization of actin cables, thereby revealing a selective and previously unappreciated ATP requirement for actin cable disassembly. A similar response is seen in HeLa cells to ATP depletion. These findings reveal a new fast-acting energy conservation strategy halting growth by immobilizing myosin V in a newly described state on selectively stabilized actin cables.

Sustaining Workforce Effectiveness: Self-Care Practices by Nursing Staff

The paper addresses the health maintenance practices and utilization of complementary and alternative therapies by nurses and nursing students from Canada, Hawaii, and American Samoa. Forecasts of an ongoing nursing shortage coupled with a focus on healthy behavior provided an impetus to describe actual practices of self-care and use of alternative therapies. The study addresses the dearth of knowledge in this area. Nurses and students were asked to anonymously complete a two-part survey that enumerated their perspectives on alternative therapies usage and self-care. Indicators of self-care included sleep, nutrition, exercise, and time. Use of alternative therapies is an integral part of self-care for the nurses and students in the three study regions.

Molecular phylogeny of sequenced Saccharomycetes reveals polyphyly of the alternative yeast codon usage.

The universal genetic code defines the translation of nucleotide triplets, called codons, into amino acids. In many Saccharomycetes a unique alteration of this code affects the translation of the CUG codon, which is normally translated as leucine. Most of the species encoding CUG alternatively as serine belong to the Candida genus and were grouped into a so-called CTG clade. However, the “Candida genus” is not a monophyletic group and several Candida species are known to use the standard CUG translation. The codon identity could have been changed in a single branch, the ancestor of the Candida, or to several branches independently leading to a polyphyletic alternative yeast codon usage (AYCU). In order to resolve the monophyly or polyphyly of the AYCU, we performed a phylogenomics analysis of 26 motor and cytoskeletal proteins from 60 sequenced yeast species. By investigating the CUG codon positions with respect to sequence conservation at the respective alignment positions, we were able to unambiguously assign the standard code or AYCU. Quantitative analysis of the highly conserved leucine and serine alignment positions showed that 61.1% and 17% of the CUG codons coding for leucine and serine, respectively, are at highly conserved positions, whereas only 0.6% and 2.3% of the CUG codons, respectively, are at positions conserved in the respective other amino acid. Plotting the codon usage onto the phylogenetic tree revealed the polyphyly of the AYCU with Pachysolen tannophilus and the CTG clade branching independently within a time span of 30–100 Ma.

Complementary and alternative medicine usage in Scottish children and adolescents during cancer treatment

AimTo determine the prevalence of the use of CAM and spiritual practices in the paediatric oncology population of SE Scotland and to establish both the reasons for their use and the perceived benefits. MethodsA retrospective survey was performed using previously piloted questionnaires. These were distributed to families whose children were <18 years and diagnosed with cancer. Demographic and clinical data were collected, descriptive statistics were used to establish frequencies and univariate associations were established by χ2 test. ResultsOf 169 families approached, 74 (44%) returned completed questionnaires. 41 (55%) families used CAM and 42 (57%) sought spiritual remedies whilst receiving conventional treatment. Higher socioeconomic status was the only factor associated with CAM usage and the most popular therapies were vitamins and minerals (n = 22; 53%), followed by massage (n = 12; 29%) and fish oils (n = 12; 29%). Most families used CAM to reduce stress and, overall, CAM was perceived to be beneficial. ConclusionThe high prevalence of CAM usage in this population highlights the need for physicians to enquire routinely about CAM use and warrants high-quality interventional studies to assess safety and efficacy. SummaryThe use of Complementary and Alternative Medicine (CAM) among paediatric patients during cancer treatment is popular worldwide, yet data from the UK are scarce. This study showed that more than half of this Scottish cohort used CAM and that there was an overall positive perception of the effect that these therapies had on the patients. Also, socio-economically advantaged families might be more likely to use CAM in Scotland.

Alternative splicing of Wilms tumor suppressor 1 (Wt1) exon 4 results in protein isoforms with different functions

The Wilms tumor suppressor gene Wt1 encodes a zinc finger transcription factor that is essential for development of multiple organs including kidneys, gonads, spleen and heart. In mammals Wt1 comprises 10 exons with two characteristic splicing events: inclusion or skipping of exon 5 and alternative usage of two splice donor sites between exons 9 and 10. Most fish including zebrafish and medaka possess two wt1 paralogs, wt1a and wt1b, both lacking exon 5. Here we have characterized wt1 in guppy, platyfish and the short-lived African killifish Nothobranchius furzeri. All fish except zebrafish show alternative splicing of exon 4 of wt1a but not of wt1b with the wt1a(−exon 4) isoform being the predominant splice variant. With regard to function, Wt1a(+exon 4) showed less dimerization but stimulated transcription more effectively than the Wt1a(−exon 4) isoform. A specific knockdown of wt1a exon 4 in zebrafish was associated with anomalies in kidney development demonstrating a physiological function for Wt1a exon 4. Interestingly, alternative splicing of exon 4 seems to be an early evolutionary event as it is observed in the single wt1 gene of the sturgeon, a species that has not gone through teleost-specific genome duplication.

Restoring degraded landscapes: Assessing the utility of biodiversity offsets for the business sector in Africa

Increasing pressure for the conversion of tropical and sub-tropical wetlands and forests to alternative land usage raises the possibility that biodiversity offsets will increasingly take centre stage in biodiversity conservation planning and ecosystem restoration discourses. This article explores the major discourses on and utility of biodiversity offsets in the African context with a view to identifying and articulating some of the challenges and opportunities evident in attempts to operationalise the concept in practice. The discussion establishes that as intuitively pleasing as they have become in recent years, with potentially large benefits expected to be derived from offset initiatives, several significant hurdles need to be overcome for them to become well established practice in Africa. For instance, some observers have argued strongly that, in practice, land use and wetland mitigation in most countries have come nowhere near achieving the goal of ‘no-net-loss’. There are also enduring questions about the credibility of the formulae used to calculate net-losses and net-gains in biodiversity offset schemes. In the light of these and other outstanding questions, the article concludes that biodiversity offsets may seem simple but are much more complex to design and implement to the extent that they become really convincing as a conservation tool for businesses in Africa.

Multi-mode resource constrained multi-project scheduling and resource portfolio problem

This paper introduces a multi-project problem environment which involves multiple projects with assigned due dates; activities that have alternative resource usage modes; a resource dedication policy that does not allow sharing of resources among projects throughout the planning horizon; and a total budget. Three issues arise when investigating this multi-project environment. First, the total budget should be distributed among different resource types to determine the general resource capacities, which correspond to the total amount for each renewable resource to be dedicated to the projects. With the general resource capacities at hand, the next issue is to determine the amounts of resources to be dedicated to the individual projects. The dedication of resources reduces the scheduling of the projects’ activities to a multi-mode resource constrained project scheduling problem (MRCPSP) for each individual project. Finally, the last issue is the efficient solution of the resulting MRCPSPs. In this paper, this multi-project environment is modeled in an integrated fashion and designated as the resource portfolio problem. A two-phase and a monolithic genetic algorithm are proposed as two solution approaches, each of which employs a new improvement move designated as the combinatorial auction for resource portfolio and the combinatorial auction for resource dedication. A computational study using test problems demonstrated the effectiveness of the solution approach proposed.

ZNF143 is regulated through alternative 3'UTR isoforms.

ZNF143 is a ubiquitously expressed transcription factor conserved in all vertebrates, regulating genes involved in primary metabolism and cell growth. It is therefore crucial to tightly maintain the adequate level of this factor in the cell. Although ZNF143 expression is auto-regulated at the transcriptional level, nothing is known about the post-transcriptional events influencing its expression. In this work, performed in mammalian cells, we show that ZNF143 expresses different 3'-untranslated regions (3'-UTR) asa result of alternative polyadenylation. These 3'UTR isoforms have a diverse impact on the ZNF143 transcript fate. Indeed, we show that the longest isoform, unlike the short one, contains a destabilizingAU-Rich element and is targeted by the miRNA 590-3p. Additionally we observed a correlation between ZNF143 downregulation and miR-590-3p up-regulation in retinoic acid treated teratocarcinoma cells. This strongly suggests that ZNF143 post-transcriptional regulation depends on the long 3'UTR isoform during teratocarcinoma cells differentiation. Finally we evidenced that the alternative polyadenylation site usage is independent of the previously identified ZNF143 transcriptional auto-regulation.

Lost in Transition: Dynamic Enhancer Organization across Naive and Primed Stem Cell States

Pluripotent stem cells transition between distinct naive and primed states that are controlled by overlapping sets of master regulatory transcription factors. In this issue of Cell Stem Cell, Buecker et al. (2014) and Factor et al. (2014) demonstrate that alternate enhancer usage, regulated by state-specific binding partners of master regulators, defines these pluripotent state transitions.

Herbage production and chemical characteristics for bioenergy production by plant functional groups from semi-natural grasslands

The current paper is devoted to biomass yield and proportion, chemical composition and energy yield by functional groups in different semi-natural grassland types. The study was performed for three grassland types in Estonia which were represented by five NATURA 2000 meadows each. The highest yield was obtained from alluvial meadows followed by mesic and wooded meadows. Hence, the largest amount of other herbs was found from alluvial meadows, even if the proportion of this functional group was dominant in wooded meadows. The contribution of the sedges&rushes was the largest in alluvial meadows. The grasses were prevalent in mesic meadows. The legumes were growing in all studied grassland types in small amounts. Nitrogen content in legumes differed significantly from other studied functional groups, but no significant differences between functional groups in other chemical (Cl and S) contents were found. The other herbs had the highest ash content and the lowest calorific value contrary to the sedges&rushes with the lowest ash content and the highest calorific value. The highest area-specific energy potential was calculated for alluvial meadows followed by mesic and wooded meadows. The energy potential depends more on the amount of biomass than the calorific value of particular functional group. Improved knowledge about the functional groups and their chemical content enables to promote and optimise alternative usage of this late harvested mixed biomass from semi-natural grasslands for bioenergy production.

Retracted: Molecular phylogeny of sequenced saccharomycetes reveals polyphyly of the alternative yeast codon usage.

The universal genetic code defines the translation of nucleotide triplets, called codons, into amino acids. In many Saccharomycetes a unique alteration of this code affects the translation of the CUG codon, which is normally translated as leucine. Most of the species encoding CUG alternatively as serine belong to the Candida genus and were grouped into a so-called CTG clade. However, the "Candida genus" is not a monophyletic group and several Candida species are known to use the standard CUG translation. The codon identity could have been changed in a single branch, the ancestor of the Candida, or to several branches independently leading to a polyphyletic alternative yeast codon usage (AYCU). In order to resolve the monophyly or polyphyly of the AYCU, we performed a phylogenomics analysis of 26 motor and cytoskeletal proteins from 60 sequenced yeast species. By investigating the CUG codon positions with respect to sequence conservation at the respective alignment positions we were able to unambiguously assign the standard code or AYCU. Quantitative analysis of the highly conserved leucine and serine alignment positions showed, that 61.1% and 17% of the CUG codons coding for leucine and serine, respectively, are at highly conserved positions, while only 0.6% and 2.3% of the CUG codons, respectively, are at positions conserved in the respective other amino acid. Plotting the codon usage onto the phylogenetic tree revealed the polyphyly of the AYCU with Pachysolen tannophilus and the CTG clade branching independently within a time span of 30 to 100 Mya.

Cultivating an Active Online Counterpublic

While alternative media have long been playing important roles in the politics of protests and resistance in many countries, the Internet has led to the emergence of online alternative media and arguably expanded the reach of such outlets. This article focuses on the audience of Internet alternative media. It examines the factors associated with usage and whether and how usage relates to political participation. Analysis of survey data ( N = 1,018) in Hong Kong shows that, not surprisingly, Internet alternative media usage was driven by preexisting political attitudes and critical views toward the mainstream media. But social media usage could also drive alternative media usage even among people who did not hold congruent preexisting attitudes. Meanwhile, alternative media usage leads to protest participation and support for unconventional protest tactics. The study thus provides empirical evidence regarding how Internet alternative media can facilitate the formation of an active online counterpublic and the role of social media in potentially enlarging the counterpublic.

From better understandings to proactive actions: Housing location and commuting mode choices among university students

Reducing car dependence has become an important public policy issue. This manuscript examines the issue by focusing on university students, who have not been well studied in existing literature. It proposes a confirmatory framework for studies on university students׳ commuting mode and housing choice and their determinants. It also conducts a case study based on this framework to get more insights. This case study shows or re-confirms that when compared to the employees from the same university, university students are more likely to share a residence in exchange for rent affordability, bus proximity and short commute. They are also more likely to jointly determine their housing and mode choices. Transit pass subsidies significantly influence university students’ alternative transportation use. Female students or graduate students are less likely to use alternative transportation. Undergraduate students have a shorter commute and use alternative transportation more. The above provide new implications for integrated housing–transportation planning and group-sensitive policies to increase alternative transportation usage among university students.

Widespread use of non-productive alternative splice sites in Saccharomyces cerevisiae.

Saccharomyces cerevisiae has been used as a model system to investigate the mechanisms of pre-mRNA splicing but only a few examples of alternative splice site usage have been described in this organism. Using RNA-Seq analysis of nonsense-mediated mRNA decay (NMD) mutant strains, we show that many S. cerevisiae intron-containing genes exhibit usage of alternative splice sites, but many transcripts generated by splicing at these sites are non-functional because they introduce premature termination codons, leading to degradation by NMD. Analysis of splicing mutants combined with NMD inactivation revealed the role of specific splicing factors in governing the use of these alternative splice sites and identified novel functions for Prp17p in enhancing the use of branchpoint-proximal upstream 3′ splice sites and for Prp18p in suppressing the usage of a non-canonical AUG 3′-splice site in GCR1. The use of non-productive alternative splice sites can be increased in stress conditions in a promoter-dependent manner, contributing to the down-regulation of genes during stress. These results show that alternative splicing is frequent in S. cerevisiae but masked by RNA degradation and that the use of alternative splice sites in this organism is mostly aimed at controlling transcript levels rather than increasing proteome diversity.

Automated Separation of &lt;em&gt;C. elegans&lt;/em&gt; Variably Colonized by a Bacterial Pathogen

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression of a fluorescent reporter. Here, we highlight an alternative usage of this instrument, for sorting worms according to their degree of colonization by a GFP-expressing pathogen. This new usage allowed us to address the relationship between colonization of the worm intestine and induction of immune responses. While C. elegans immune responses to different pathogens have been documented, it is still unknown what initiates them. The two main possibilities (which are not mutually exclusive) are recognition of pathogen-associated molecular patterns, and detection of damage caused by infection. To differentiate between the two possibilities, exposure to the pathogen must be dissociated from the damage it causes. The wormsorter enabled separation of worms that were extensively-colonized by the Gram-negative pathogen Pseudomonas aeruginosa, with the damage likely caused by pathogen load, from worms that were similarly exposed, but not, or marginally, colonized. These distinct populations were used to assess the relationship between pathogen load and the induction of transcriptional immune responses. The results suggest that the two are dissociated, supporting the possibility of pathogen recognition.