bluuebunny/crossref_metadata_2025 huggingface dataset:
Parallel evolution of genome structure and transcriptional landscape in the Epsilonproteobacteria
Authors: Porcelli Ida , Reuter Mark , Pearson Bruce M , Wilhelm Thomas , van Vliet Arnoud HM
Published: September 2013

Background

Gene reshuffling, point mutations and horizontal gene transfer contribute to bacterial genome variation, but require the genome to rewire its transcriptional circuitry to ensure that inserted, mutated or reshuffled genes are transcribed at appropriate levels. The genomes of Epsilonproteobacteria display very low synteny, due to high levels of reshuffling and reorganisation of gene order, but still share a significant number of gene orthologs allowing comparison. Here we present the primary transcriptome of the pathogenic Epsilonproteobacterium Campylobacter jejuni, and have used this for comparative and predictive transcriptomics in the Epsilonproteobacteria.

Results

Differential RNA-sequencing using 454 sequencing technology was used to determine the primary transcriptome of C. jejuni NCTC 11168, which consists of 992 transcription start sites (TSS), which included 29 putative non-coding and stable RNAs, 266 intragenic (internal) TSS, and 206 antisense TSS. Several previously unknown features were identified in the C. jejuni transcriptional landscape, like leaderless mRNAs and potential leader peptides upstream of amino acid biosynthesis genes. A cross-species comparison of the primary transcriptomes of C. jejuni and the related Epsilonproteobacterium Helicobacter pylori highlighted a lack of conservation of operon organisation, position of intragenic and antisense promoters or leaderless mRNAs. Predictive comparisons using 40 other Epsilonproteobacterial genomes suggests that this lack of conservation of transcriptional features is common to all Epsilonproteobacterial genomes, and is associated with the absence of genome synteny in this subdivision of the Proteobacteria.

Conclusions

Both the genomes and transcriptomes of Epsilonproteobacteria are highly variable, both at the genome level by combining and division of multicistronic operons, but also on the gene level by generation or deletion of promoter sequences and 5′ untranslated regions. Regulatory features may have evolved after these species split from a common ancestor, with transcriptome rewiring compensating for changes introduced by genomic reshuffling and horizontal gene transfer.
Perturbation ofBrachypodium distachyon CELLULOSE SYNTHASE A4or7results in abnormal cell walls
Authors: Handakumbura Pubudu P , Matos Dominick A , Osmont Karen S , Harrington Michael J , Heo Kyuyoung , Kafle Kabindra , Kim Seong H , Baskin Tobias I , Hazen Samuel P
Published: September 2013

Background

Cellulose is an integral component of the plant cell wall and accounts for approximately forty percent of total plant biomass but understanding its mechanism of synthesis remains elusive. CELLULOSE SYNTHASE A (CESA) proteins function as catalytic subunits of a rosette-shaped complex that synthesizes cellulose at the plasma membrane. Arabidopsis thaliana and rice (Oryza sativa) secondary wall CESA loss-of-function mutants have weak stems and irregular or thin cell walls.

Results

Here, we identify candidates for secondary wall CESAs in Brachypodium distachyon as having similar amino acid sequence and expression to those characterized in A. thaliana, namely CESA4/7/8. To functionally characterize BdCESA4 and BdCESA7, we generated loss-of-function mutants using artificial microRNA constructs, specifically targeting each gene driven by a maize (Zea mays) ubiquitin promoter. Presence of the transgenes reduced BdCESA4 and BdCESA7 transcript abundance, as well as stem area, cell wall thickness of xylem and fibers, and the amount of crystalline cellulose in the cell wall.

Conclusion

These results suggest BdCESA4 and BdCESA7 play a key role in B. distachyon secondary cell wall biosynthesis.
Identification and functional characterization of a rice NAC gene involved in the regulation of leaf senescence
Authors: Zhou Yong , Huang Weifeng , Liu Li , Chen Taiyu , Zhou Fei , Lin Yongjun
Published: September 2013

Background

As the final stage of leaf development, leaf senescence may cause the decline of photosynthesis and gradual reduction of carbon assimilation, which makes it a possible limiting factor for crop yield. NACs are plant-specific transcription factors and some NACs have been confirmed to play important roles in regulating leaf senescence.

Results

In this study, we reported a member of the NAC transcription factor family named OsNAP whose expression is associated with leaf senescence, and investigated its preliminary function during the process of leaf senescence. The results of qRT-PCR showed that the OsNAP transcripts were accumulated gradually in response to leaf senescence and treatment with methyl jasmonic acid (MeJA). A subcellular localization assay indicated that OsNAP is a nuclear-localized protein. Yeast one-hybrid experiments indicated that OsNAP can bind the NAC recognition site (NACRS)-like sequence. OsNAP-overexpressing transgenic plants displayed an accelerated leaf senescence phenotype at the grain-filling stage, which might be caused by the elevated JA levels and the increased expression of the JA biosynthesis-related genes LOX2 and AOC1, and showed enhanced tolerance ability to MeJA treatment at the seedling stage. Nevertheless, the leaf senescence process was delayed in OsNAP RNAi transgenic plants with a dramatic drop in JA levels and with decreased expression levels of the JA biosynthesis-related genes AOS2, AOC1 and OPR7.

Conclusions

These results suggest that OsNAP acts as a positive regulator of leaf senescence and this regulation may occur via the JA pathway.
Upregulation of M3 muscarinic receptor inhibits cardiac hypertrophy induced by angiotensin II
Authors: Liu Yan , Wang Shu , Wang Chao , Song Haoxin , Han Hongmei , Hang Pengzhou , Jiang Yanan , Wei Lanlan , Huo Rong , Sun Lihua , Gao Xu , Lu Yanjie , Du Zhimin
Published: September 2013

Background

M3 muscarinic acetylcholine receptor (M3-mAChR) is stably expressed in the myocardium, but its pathophysiological role remains largely undefined. This study aimed to investigate the role of M3-mAChR in cardiac hypertrophy induced by angiotensin II (Ang II) and elucidate the underlying mechanisms.

Methods

Cardiac-specific M3-mAChR overexpression transgenic (TG) mice and rat H9c2 cardiomyoblasts with ectopic expression of M3-mAChR were established. Models of cardiac hypertrophy were induced by transverse aortic constriction (TAC) or Ang II infusion in the mice in vivo, and by isoproterenol (ISO) or Ang II treatment of H9c2 cells in vitro. Cardiac hypertrophy was evaluated by electrocardiography (ECG) measurement, hemodynamic measurement and histological analysis. mRNA and protein expression were detected by real-time RT-PCR and Western blot analysis.

Results

M3-mAChR was upregulated in hypertrophic heart, while M2-mAChR expression did not change significantly. M3-mAChR overexpression significantly attenuated the increased expression of atrial natriuretic peptide and β-myosin heavy chain induced by Ang II both in vivo and in vitro. In addition, M3-mAChR overexpression downregulated AT1 receptor expression and inhibited the activation of MAPK signaling in the heart.

Conclusion

The upregulation of M3-mAChR during myocardial hypertrophy could relieve the hypertrophic response provoked by Ang II, and the mechanism may involve the inhibition of MAPK signaling through the downregulation of AT1 receptor.
Brachydactyly E: isolated or as a feature of a syndrome
Authors: Pereda Arrate , Garin Intza , Garcia-Barcina Maria , Gener Blanca , Beristain Elena , Ibañez Ane Miren , Perez de Nanclares Guiomar
Published: September 2013

Brachydactyly (BD) refers to the shortening of the hands, feet or both. There are different types of BD; among them, type E (BDE) is a rare type that can present as an isolated feature or as part of more complex syndromes, such as: pseudohypopthyroidism (PHP), hypertension with BD or Bilginturan BD (HTNB), BD with mental retardation (BDMR) or BDE with short stature, PTHLH type. Each syndrome has characteristic patterns of skeletal involvement. However, brachydactyly is not a constant feature and shows a high degree of phenotypic variability. In addition, there are other syndromes that can be misdiagnosed as brachydactyly type E, some of which will also be discussed. The objective of this review is to describe some of the syndromes in which BDE is present, focusing on clinical, biochemical and genetic characteristics as features of differential diagnoses, with the aim of establishing an algorithm for their differential diagnosis. As in our experience many of these patients are recruited at Endocrinology and/or Pediatric Endocrinology Services due to their short stature, we have focused the algorithm in those steps that could mainly help these professionals.
Triterpenoid resinous metabolites from the genus Boswellia: pharmacological activities and potential species-identifying properties
Authors: Zhang Yuxin , Ning Zhangchi , Lu Cheng , Zhao Siyu , Wang Jianfen , Liu Baoqin , Xu Xuegong , Liu Yuanyan
Published: September 2013

The resinous metabolites commonly known as frankincense or olibanum are produced by trees of the genus Boswellia and have attracted increasing popularity in Western countries in the last decade for their various pharmacological activities. This review described the pharmacological specific details mainly on anti-inflammatory, anti-carcinogenic, anti-bacterial and apoptosis-regulating activities of individual triterpenoid together with the relevant mechanism. In addition, species-characterizing triterpenic markers with the methods for their detection, bioavailability, safety and other significant properties were reviewed for further research.
Clostridium thermocellum transcriptomic profiles after exposure to furfural or heat stress
Authors: Wilson Charlotte M , Yang Shihui , Rodriguez Miguel , Ma Qin , Johnson Courtney M , Dice Lezlee , Xu Ying , Brown Steven D
Published: September 2013

Background

The thermophilic anaerobe Clostridium thermocellum is a candidate consolidated bioprocessing (CBP) biocatalyst for cellulosic ethanol production. It is capable of both cellulose solubilization and its fermentation to produce lignocellulosic ethanol. Intolerance to stresses routinely encountered during industrial fermentations may hinder the commercial development of this organism. A previous C. thermocellum ethanol stress study showed that the largest transcriptomic response was in genes and proteins related to nitrogen uptake and metabolism.

Results

In this study, C. thermocellum was grown to mid-exponential phase and treated with furfural or heat to a final concentration of 3 g.L-1 or 68°C respectively to investigate general and specific physiological and regulatory stress responses. Samples were taken at 10, 30, 60 and 120 min post-shock, and from untreated control fermentations, for transcriptomic analyses and fermentation product determinations and compared to a published dataset from an ethanol stress study. Urea uptake genes were induced following furfural stress, but not to the same extent as ethanol stress and transcription from these genes was largely unaffected by heat stress. The largest transcriptomic response to furfural stress was genes for sulfate transporter subunits and enzymes in the sulfate assimilatory pathway, although these genes were also affected late in the heat and ethanol stress responses. Lactate production was higher in furfural treated culture, although the lactate dehydrogenase gene was not differentially expressed under this condition. Other redox related genes such as a copy of the rex gene, a bifunctional acetaldehyde-CoA/alcohol dehydrogenase and adjacent genes did show lower expression after furfural stress compared to the control, heat and ethanol fermentation profiles. Heat stress induced expression from chaperone related genes and overlap was observed with the responses to the other stresses. This study suggests the involvement of C. thermocellum genes with functions in oxidative stress protection, electron transfer, detoxification, sulfur and nitrogen acquisition, and DNA repair mechanisms in its stress responses and the use of different regulatory networks to coordinate and control adaptation.

Conclusions

This study has identified C. thermocellum gene regulatory motifs and aspects of physiology and gene regulation for further study. The nexus between future systems biology studies and recently developed genetic tools for C. thermocellum offers the potential for more rapid strain development and for broader insights into this organism’s physiology and regulation.
Gut Pharmacomicrobiomics: the tip of an iceberg of complex interactions between drugs and gut-associated microbes
Authors: Saad Rama , Rizkallah Mariam R , Aziz Ramy K
Published: September 2013

The influence of resident gut microbes on xenobiotic metabolism has been investigated at different levels throughout the past five decades. However, with the advance in sequencing and pyrotagging technologies, addressing the influence of microbes on xenobiotics had to evolve from assessing direct metabolic effects on toxins and botanicals by conventional culture-based techniques to elucidating the role of community composition on drugs metabolic profiles through DNA sequence-based phylogeny and metagenomics. Following the completion of the Human Genome Project, the rapid, substantial growth of the Human Microbiome Project (HMP) opens new horizons for studying how microbiome compositional and functional variations affect drug action, fate, and toxicity (pharmacomicrobiomics), notably in the human gut. The HMP continues to characterize the microbial communities associated with the human gut, determine whether there is a common gut microbiome profile shared among healthy humans, and investigate the effect of its alterations on health. Here, we offer a glimpse into the known effects of the gut microbiota on xenobiotic metabolism, with emphasis on cases where microbiome variations lead to different therapeutic outcomes. We discuss a few examples representing how the microbiome interacts with human metabolic enzymes in the liver and intestine. In addition, we attempt to envisage a roadmap for the future implications of the HMP on therapeutics and personalized medicine.
Role of HGF in epithelial–stromal cell interactions during progression from benign breast disease to ductal carcinoma in situ
Authors: Casbas-Hernandez Patricia , D’Arcy Monica , Roman-Perez Erick , Brauer Heather Ann , McNaughton Kirk , Miller Samantha M , Chhetri Raghav K , Oldenburg Amy L , Fleming Jodie M , Amos Keith D , Makowski Liza , Troester Melissa A
Published: September 2013

Introduction

Basal-like and luminal breast cancers have distinct stromal–epithelial interactions, which play a role in progression to invasive cancer. However, little is known about how stromal–epithelial interactions evolve in benign and pre-invasive lesions.

Methods

To study epithelial–stromal interactions in basal-like breast cancer progression, we cocultured reduction mammoplasty fibroblasts with the isogenic MCF10 series of cell lines (representing benign/normal, atypical hyperplasia, and ductal carcinoma in situ). We used gene expression microarrays to identify pathways induced by coculture in premalignant cells (MCF10DCIS) compared with normal and benign cells (MCF10A and MCF10AT1). Relevant pathways were then evaluated in vivo for associations with basal-like subtype and were targeted in vitro to evaluate effects on morphogenesis.

Results

Our results show that premalignant MCF10DCIS cells express characteristic gene expression patterns of invasive basal-like microenvironments. Furthermore, while hepatocyte growth factor (HGF) secretion is upregulated (relative to normal, MCF10A levels) when fibroblasts are cocultured with either atypical (MCF10AT1) or premalignant (MCF10DCIS) cells, only MCF10DCIS cells upregulated the HGF receptor MET. In three-dimensional cultures, upregulation of HGF/MET in MCF10DCIS cells induced morphological changes suggestive of invasive potential, and these changes were reversed by antibody-based blocking of HGF signaling. These results are relevant to in vivo progression because high expression of a novel MCF10DCIS-derived HGF signature was correlated with the basal-like subtype, with approximately 86% of basal-like cancers highly expressing the HGF signature, and because high expression of HGF signature was associated with poor survival.

Conclusions

Coordinated and complementary changes in HGF/MET expression occur in epithelium and stroma during progression of pre-invasive basal-like lesions. These results suggest that targeting stroma-derived HGF signaling in early carcinogenesis may block progression of basal-like precursor lesions.
Local and systemic innate immune response to secondary human peritonitis
Authors: Riché Florence , Gayat Etienne , Collet Corinne , Matéo Joaquim , Laisné Marie-Josèphe , Launay Jean-Marie , Valleur Patrice , Payen Didier , Cholley Bernard P
Published: September 2013

Introduction

Our aim was to describe inflammatory cytokines response in the peritoneum and plasma of patients with peritonitis. We also tested the hypothesis that scenarios associated with worse outcome would result in different cytokine release patterns. Therefore, we compared cytokine responses according to the occurrence of septic shock, mortality, type of peritonitis and peritoneal microbiology.

Methods

Peritoneal and plasma cytokines (interleukin (IL) 1, tumor necrosis factor α (TNFα), IL-6, IL-10, and interferon γ (IFNγ)) were measured in 66 patients with secondary peritonitis.

Results

The concentration ratio between peritoneal fluid and plasma cytokines varied from 5 (2 to 21) (IFNγ) to 1310 (145 to 3888) (IL-1). There was no correlation between plasma and peritoneal fluid concentration of any cytokine. In the plasma, TNFα, IL-6, IFNγ and IL-10 were higher in patients with shock versus no shock and in nonsurvivors versus survivors (P ≤0.03). There was no differential plasma release for any cytokine between community-acquired and postoperative peritonitis. The presence of anaerobes or Enterococcus species in peritoneal fluid was associated with higher plasma TNFα: 50 (37 to 106) versus 38 (29 to 66) and 45 (36 to 87) versus 39 (27 to 67) pg/ml, respectively (P = 0.02). In the peritoneal compartment, occurrence of shock did not result in any difference in peritoneal cytokines. Peritoneal IL-10 was higher in patients who survived (1505 (450 to 3130) versus 102 (9 to 710) pg/ml; P = 0.04). The presence of anaerobes and Enterococcus species was associated with higher peritoneal IFNγ: 2 (1 to 6) versus 10 (5 to 28) and 7 (2 to 39) versus 2 (1 to 6), P = 0.01 and 0.05, respectively).

Conclusions

Peritonitis triggers an acute systemic and peritoneal innate immune response with a simultaneous release of pro and anti-inflammatory cytokines. Higher levels of all cytokines were observed in the plasma of patients with the most severe conditions (shock, non-survivors), but this difference was not reflected in their peritoneal fluid. There was always a large gradient in cytokine concentration between peritoneal and plasma compartments highlighting the importance of compartmentalization of innate immune response in peritonitis.
On the design of clone-based haplotyping
Authors: Lo Christine , Liu Rui , Lee Jehyuk , Robasky Kimberly , Byrne Susan , Lucchesi Carolina , Aach John , Church George , Bafna Vineet , Zhang Kun
Published: September 2013

Background

Haplotypes are important for assessing genealogy and disease susceptibility of individual genomes, but are difficult to obtain with routine sequencing approaches. Experimental haplotype reconstruction based on assembling fragments of individual chromosomes is promising, but with variable yields due to incompletely understood parameter choices.

Results

We parameterize the clone-based haplotyping problem in order to provide theoretical and empirical assessments of the impact of different parameters on haplotype assembly. We confirm the intuition that long clones help link together heterozygous variants and thus improve haplotype length. Furthermore, given the length of the clones, we address how to choose the other parameters, including number of pools, clone coverage and sequencing coverage, so as to maximize haplotype length. We model the problem theoretically and show empirically the benefits of using larger clones with moderate number of pools and sequencing coverage. In particular, using 140 kb BAC clones, we construct haplotypes for a personal genome and assemble haplotypes with N50 values greater than 2.6 Mb. These assembled haplotypes are longer and at least as accurate as haplotypes of existing clone-based strategies, whether in vivo or in vitro.

Conclusions

Our results provide practical guidelines for the development and design of clone-based methods to achieve long range, high-resolution and accurate haplotypes.
Elevated Periimplantation Uterine Natural Killer Cell Density in Human Endometrium Is Associated With Impaired Corticosteroid Signaling in Decidualizing Stromal Cells
Authors: Kuroda Keiji , Venkatakrishnan Radha , James Sean , Šućurović Sandra , Mulac-Jericevic Biserka , Lucas Emma S. , Takeda Satoru , Shmygol Anatoly , Brosens Jan J. , Quenby Siobhan
Published: September 2013

Background:

Decidualizing human endometrial stromal cells (HESCs) profoundly up-regulate 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1), the enzyme that converts inert cortisone to active cortisol. We postulated that the induction of a cortisol gradient upon decidualization of the periimplantation endometrium may impact on the uterine natural killer (uNK) cell population and on local expression of corticosteroid-dependent target genes.

Methods:

Midluteal endometrial biopsies (n = 55) were processed for uNK cell (CD56) analysis and primary HESC cultures. The cultures remained either untreated or were decidualized for 4 or 8 days. A tissue microarray was constructed from endometria with normal (n = 18) and elevated uNK cell (n = 18) scores. An abnormal uNK cell test was defined as greater than 5% CD56+ cells in the subluminal stroma.

Results:

Increased uNK cell density was associated with lower endometrial expression of 11βHSD1 and mineralocorticoid receptor (MR) but not glucocorticoid receptor in vivo. Elevated uNK cell density also corresponded to impaired induction of key decidual markers (11βHSD1, prolactin, and insulin-like growth factor binding protein-1) and MR-dependent enzymes (dehydrogenase/reductase member 3 and retinol saturase) in differentiating HESC cultures. Increased uNK cell density in vivo was not associated with increased in vitro expression of either IL-15 or IL-11, two cytokines implicated in uNK cell regulation.

Conclusions:

Elevated levels of uNK cells in the stroma underlying the surface epithelium are associated with inadequate cortisol biosynthesis by resident decidualizing cells and suboptimal induction of key MR-dependent enzymes involved in lipid biogenesis and the retinoid transport pathway. Our observations suggest that uNK cell testing identifies those women at risk of reproductive failure due to relative uterine cortisol deficiency.
Artisans and the New Science of Politics in Early Modern Europe
Authors: Corteguera Luis R.
Published: September 2013

How did artisanal methods and practices inform the new science of politics attributed to Machiavelli and elaborated by others? Historians of science have noted how artisans’ direct observation of nature and knowledge acquired from hands-on experience informed their methods of training and habits of scientific work that became the basis for a new philosophy of science. Likewise, proponents and critics of the new political science ascribed to it characteristics similar to artisanal methods of scientific work: firsthand experience, often described as realism, derived from objective analysis stripped of illusions; and a practical aim, characterized by utilitarian action devoid of scruples. This article examines the relationship between the “art” of politics and the artisanal epistemology of science, the value of artisans’ skills and expertise in politics, and the implications of these issues for our understanding of the political ideas and actions of early modern artisans.
Seizure Outcomes After Corpus Callosotomy for Drop Attacks
Authors: Bower Regina S. , Wirrell Elaine , Nwojo Macaulay , Wetjen Nicholas M. , Marsh W. Richard , Meyer Fredric B.
Published: September 2013

BACKGROUND:

Medically intractable epilepsy involving drop attacks can be difficult to manage and negatively affect quality of life. Most studies investigating the effect of corpus callosotomy (CC) on seizures have been limited, focusing on the pediatric population or drop seizures alone, with little attention to other factors influencing seizure outcome.

OBJECTIVE:

To assess seizure outcomes after CC in adults and children.

METHODS:

Retrospective analysis was performed on all patients who underwent CC (anterior two thirds, 1- or 2-stage complete) at our institution between 1990 and 2011. Change in frequency after CC was assessed for drop seizures and other seizure types. Multiple factors were evaluated for impact on seizure outcome.

RESULTS:

Fifty patients met inclusion criteria. The median age was 1.5 years at seizure onset and 17 years at time of surgery. Anterior two-thirds CC was performed in 28 patients, 1-stage complete in 17, and 2-stage complete in 5. All 3 groups experienced a significant decrease in drop seizures (P < .001, P < .001, and P = .020, respectively), with 40% experiencing complete resolution, and 64% dropping at least 1 frequency category. Other seizure types significantly decreased in anterior two-thirds CC and 1-stage complete (P = .0035, P = .001, respectively). Younger age at surgery correlated with better seizure outcomes (P = .043).

CONCLUSION:

CC for medically refractory generalizing epilepsy is effective for both drop seizures and other seizure types. CC should be considered soon after a patient has been deemed medically refractory because earlier age at surgery results in lower risk and better outcome.
Gprk2 adjusts Fog signaling to organize cell movements in Drosophila gastrulation
Authors: Fuse Naoyuki , Yu Fengwei , Hirose Susumu
Published: September 2013

Gastrulation of Drosophila melanogaster proceeds through sequential cell movements: ventral mesodermal (VM) cells are induced by secreted Fog protein to constrict their apical surfaces to form the ventral furrow, and subsequently lateral mesodermal (LM) cells involute toward the furrow. How these cell movements are organized remains elusive. Here, we observed that LM cells extended apical protrusions and then underwent accelerated involution movement, confirming that VM and LM cells display distinct cell morphologies and movements. In a mutant for the GPCR kinase Gprk2, apical constriction was expanded to all mesodermal cells and the involution movement was abolished. In addition, the mesodermal cells halted apical constriction prematurely in accordance with the aberrant accumulation of Myosin II. Epistasis analyses revealed that the Gprk2 mutant phenotypes were dependent on the fog gene. Overexpression of Gprk2 suppressed the effects of excess Cta, a downstream component of Fog signaling. Based on these findings, we propose that Gprk2 attenuates and tunes Fog-Cta signaling to prevent apical constriction in LM cells and to support appropriate apical constriction in VM cells. Thus, the two distinct cell movements in mesoderm invagination are not predetermined, but rather are organized by the adjustment of cell signaling.
Insulin and Target of rapamycin signaling orchestrate the development of ovarian niche-stem cell units in Drosophila
Authors: Gancz Dana , Gilboa Lilach
Published: September 2013

Tissue-specific stem cells and their niches are organized into functional units that respond to external cues in order to maintain organ homeostasis. Insulin and Target of rapamycin (Tor) signaling mediate external cues that control adult niches and stem cells. Whether these pathways play a role in the establishment of niche-stem cell units during organogenesis has been little explored. We show that during larval development both Insulin-like receptor (InR) and Tor participate in the establishment of ovarian niches and germline stem cells (GSCs) in Drosophila melanogaster. Tor and InR are required cell-autonomously for the proliferation of precursors for both somatic niches and GSCs. These pathways also promote the formation of terminal filaments (part of the somatic niche). Significantly, InR, but not Tor, signaling non-autonomously promotes primordial germ cell (PGC) differentiation. Somatic attenuation of the pathway retards PGC differentiation, whereas its activation results in their precocious differentiation. We also show that InR-mediated PGC differentiation is independent of somatic ecdysone signaling, but that further differentiation into cysts requires an ecdysone input. These results demonstrate that Tor and InR signaling actively participate in the formation of ovarian niches and stem cells by affecting both cell numbers and differentiation. The dual influence of Tor and InR on both somatic cells and PGCs ensures that these two cell populations develop coordinately. Our work further identifies a novel step in the regulation of germ cell differentiation by demonstrating that following bag of marbles expression, cyst formation requires an additional hormonal input.
Nkx genes are essential for maintenance of ventricular identity
Authors: Targoff Kimara L. , Colombo Sophie , George Vanessa , Schell Thomas , Kim Seok-Hyung , Solnica-Krezel Lilianna , Yelon Deborah
Published: September 2013

Establishment of specific characteristics of each embryonic cardiac chamber is crucial for development of a fully functional adult heart. Despite the importance of defining and maintaining unique features in ventricular and atrial cardiomyocytes, the regulatory mechanisms guiding these processes are poorly understood. Here, we show that the homeodomain transcription factors Nkx2.5 and Nkx2.7 are necessary to sustain ventricular chamber attributes through repression of atrial chamber identity. Mutation of nkx2.5 in zebrafish yields embryos with diminutive ventricular and bulbous atrial chambers. These chamber deformities emerge gradually during development, with a severe collapse in the number of ventricular cardiomyocytes and an accumulation of excess atrial cardiomyocytes as the heart matures. Removal of nkx2.7 function from nkx2.5 mutants exacerbates the loss of ventricular cells and the gain of atrial cells. Moreover, in these Nkx-deficient embryos, expression of vmhc, a ventricular gene, fades, whereas expression of amhc, an atrial gene, expands. Cell-labeling experiments suggest that ventricular cardiomyocytes can transform into atrial cardiomyocytes in the absence of Nkx gene function. Through suggestion of transdifferentiation from ventricular to atrial fate, our data reveal a pivotal role for Nkx genes in maintaining ventricular identity and highlight remarkable plasticity in differentiated myocardium. Thus, our results are relevant to the etiologies of fetal and neonatal cardiac pathology and could direct future innovations in cardiac regenerative medicine.
Eph/Ephrin signalling maintains eye field segregation from adjacent neural plate territories during forebrain morphogenesis
Authors: Cavodeassi Florencia , Ivanovitch Kenzo , Wilson Stephen W.
Published: September 2013

During forebrain morphogenesis, there is extensive reorganisation of the cells destined to form the eyes, telencephalon and diencephalon. Little is known about the molecular mechanisms that regulate region-specific behaviours and that maintain the coherence of cell populations undergoing specific morphogenetic processes. In this study, we show that the activity of the Eph/Ephrin signalling pathway maintains segregation between the prospective eyes and adjacent regions of the anterior neural plate during the early stages of forebrain morphogenesis in zebrafish. Several Ephrins and Ephs are expressed in complementary domains in the prospective forebrain and combinatorial abrogation of their activity results in incomplete segregation of the eyes and telencephalon and in defective evagination of the optic vesicles. Conversely, expression of exogenous Ephs or Ephrins in regions of the prospective forebrain where they are not usually expressed changes the adhesion properties of the cells, resulting in segregation to the wrong domain without changing their regional fate. The failure of eye morphogenesis in rx3 mutants is accompanied by a loss of complementary expression of Ephs and Ephrins, suggesting that this pathway is activated downstream of the regional fate specification machinery to establish boundaries between domains undergoing different programmes of morphogenesis.
Wnt/β-catenin signaling directs the regional expansion of first and second heart field-derived ventricular cardiomyocytes
Authors: Buikema Jan Willem , Mady Ahmed S. , Mittal Nikhil V. , Atmanli Ayhan , Caron Leslie , Doevendans Pieter A. , Sluijter Joost P. G. , Domian Ibrahim J.
Published: September 2013

In mammals, cardiac development proceeds from the formation of the linear heart tube, through complex looping and septation, all the while increasing in mass to provide the oxygen delivery demands of embryonic growth. The developing heart must orchestrate regional differences in cardiomyocyte proliferation to control cardiac morphogenesis. During ventricular wall formation, the compact myocardium proliferates more vigorously than the trabecular myocardium, but the mechanisms controlling such regional differences among cardiomyocyte populations are not understood. Control of definitive cardiomyocyte proliferation is of great importance for application to regenerative cell-based therapies. We have used murine and human pluripotent stem cell systems to demonstrate that, during in vitro cellular differentiation, early ventricular cardiac myocytes display a robust proliferative response to β-catenin-mediated signaling and conversely accelerate differentiation in response to inhibition of this pathway. Using gain- and loss-of-function murine genetic models, we show that β-catenin controls ventricular myocyte proliferation during development and the perinatal period. We further demonstrate that the differential activation of the Wnt/β-catenin signaling pathway accounts for the observed differences in the proliferation rates of the compact versus the trabecular myocardium during normal cardiac development. Collectively, these results provide a mechanistic explanation for the differences in localized proliferation rates of cardiac myocytes and point to a practical method for the generation of the large numbers of stem cell-derived cardiac myocytes necessary for clinical applications.
Growth factor-mediated mesodermal cell guidance and skeletogenesis during sea urchin gastrulation
Authors: Adomako-Ankomah Ashrifia , Ettensohn Charles A.
Published: September 2013

Growth factor signaling pathways provide essential cues to mesoderm cells during gastrulation in many metazoans. Recent studies have implicated the VEGF and FGF pathways in providing guidance and differentiation cues to primary mesenchyme cells (PMCs) during sea urchin gastrulation, although the relative contributions of these pathways and the cell behaviors they regulate are not fully understood. Here, we show that FGF and VEGF ligands are expressed in distinct domains in the embryonic ectoderm of Lytechinus variegatus. We find that PMC guidance is specifically disrupted in Lv-vegf3 morphants and these embryos fail to form skeletal elements. By contrast, PMC migration is unaffected in Lv-fgfa morphants, and well-patterned but shortened skeletal elements form. We use a VEGFR inhibitor, axitinib, to show that VEGF signaling is essential not only for the initial phase of PMC migration (subequatorial ring formation), but also for the second phase (migration towards the animal pole). VEGF signaling is not required, however, for PMC fusion. Inhibition of VEGF signaling after the completion of PMC migration causes significant defects in skeletogenesis, selectively blocking the elongation of skeletal rods that support the larval arms, but not rods that form in the dorsal region of the embryo. Nanostring nCounter analysis of ∼100 genes in the PMC gene regulatory network shows a decrease in the expression of many genes with proven or predicted roles in biomineralization in vegf3 morphants. Our studies lead to a better understanding of the roles played by growth factors in sea urchin gastrulation and skeletogenesis.
EphA2 and Src regulate equatorial cell morphogenesis during lens development
Authors: Cheng Catherine , Ansari Moham M. , Cooper Jonathan A. , Gong Xiaohua
Published: September 2013

High refractive index and transparency of the eye lens require uniformly shaped and precisely aligned lens fiber cells. During lens development, equatorial epithelial cells undergo cell-to-cell alignment to form meridional rows of hexagonal cells. The mechanism that controls this morphogenesis from randomly packed cuboidal epithelial cells to highly organized hexagonal fiber cells remains unknown. In Epha2-/- mouse lenses, equatorial epithelial cells fail to form precisely aligned meridional rows; moreover, the lens fulcrum, where the apical tips of elongating epithelial cells constrict to form an anchor point before fiber cell differentiation and elongation at the equator, is disrupted. Phosphorylated Src-Y424 and cortactin-Y466, actin and EphA2 cluster at the vertices of wild-type hexagonal epithelial cells in organized meridional rows. However, phosphorylated Src and phosphorylated cortactin are not detected in disorganized Epha2-/- cells with altered F-actin distribution. E-cadherin junctions, which are normally located at the basal-lateral ends of equatorial epithelial cells and are diminished in newly differentiating fiber cells, become widely distributed in the apical, lateral and basal sides of epithelial cells and persist in differentiating fiber cells in Epha2-/- lenses. Src-/- equatorial epithelial cells also fail to form precisely aligned meridional rows and lens fulcrum. These results indicate that EphA2/Src signaling is essential for the formation of the lens fulcrum. EphA2 also regulates Src/cortactin/F-actin complexes at the vertices of hexagonal equatorial cells for cell-to-cell alignment. This mechanistic information explains how EphA2 mutations lead to disorganized lens cells that subsequently contribute to altered refractive index and cataracts in humans and mice.
Evaluation of Normalization Strategies Used in Real-time Quantitative PCR Experiments in HepaRG Cell Line Studies
Authors: Ceelen Liesbeth , De Craene Jurgen , De Spiegelaere Ward
Published: September 2013

BACKGROUND

The HepaRG cell line is widely used as an alternative for primary human hepatocytes for numerous applications, including drug screening, and is progressively gaining importance as a human-relevant cell source. Consequently, increasing numbers of experiments are being performed with this cell line, including real-time quantitative PCR (RT-qPCR) experiments for gene expression studies.

CONTENT

When RT-qPCR experiments are performed, results are reliable only when attention is paid to several critical aspects, including a proper normalization strategy. Therefore, in 2011 we determined the most optimal reference genes for gene expression studies in the HepaRG cell system, according to the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines. This study additionally provided clear evidence that the use of a single reference gene [glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein S18 (RPS18), or actin, beta (ACTB)] was insufficient for normalization in HepaRG cells. Our screening of relevant studies published after our study suggested that the findings of our study were completely ignored.

In none of the 24 reviewed studies was a proper normalization method used. Only 1 reference gene was included for normalization in 21 out of the 24 reported studies we screened, with RPS18 and GAPDH used most frequently, followed by hypoxanthine phosphoribosyltransferase 1 (HPRT1), glutathione synthetase (GSS) (hGus), β-2 microglobin (B2M), and acidic ribosomal phosphoprotein P0 (36B4). For 2 studies the use of multiple reference genes (2 and 3) was reported, but these had not been prevalidated for expression stability in HepaRG cells. In 1 study, there was no evidence that any reference gene had been used. Current RT-qPCR gene expression studies in HepaRG cells are being performed without adequate consideration or evaluation of reference genes. Such studies can yield erroneous and biologically irrelevant results.
Sleep disorders in children with developmental delay
Authors: Ahmareen Oneza , Neary Elaine , Sharif Farhana
Published: September 2013

Developmental disorders in children are common. Associated demanding co-morbidities that include sleep problems are prevalent in this group. In turn, these are linked to daytime performance issues, reduced developmental and academic growth, and considerable familial psychopathology. Behavioral modification measures are and should be the first line approach to management. Medications for sleep disorders have an important role in addition to behavioral modification therapy. This literature review looks into the occurrence and severity of sleep problems in children with developmental disorders. The probable advantages of using medications, mainly focusing on the neurohormone melatonin (a sleep inducer), are also explored.
On the other side: formulaic organizing chunks in spoken and written academic ELF
Authors: Carey Ray
Published: September 2013

An ongoing discussion in ELF research is the ability of ELF speakers to store and retrieve holistic chunks of language, facilitating efficient and fluent production of speech. These questions involve the frequency effects of formulaic chunks of language and their varying degrees of entrenchment for ELF users. In addition, the variable forms in which these chunks may be attested can be treated as approximations of conventional chunks, while serving identical functions. This study addresses these issues by investigating high-frequency organizing chunks in ELF corpora using the Linear Unit Grammar (LUG) framework (Sinclair and Mauranen 2006). Drawing data from the ELFA corpus of spoken academic ELF, the study also considers organizing chunks in written academic ELF from the nascent WrELFA corpus. With ENL comparison data taken from the Michigan Corpus of Academic Spoken English (MICASE), findings are presented on the forms and frequencies of textual and interactive organizing chunks in ELF, with implications for the reality of frequency effects and their connection to distributions of approximated chunks. The lower frequency, organizing chunks showed a higher rate of approximation and number of unique forms, while the higher frequency chunks were primarily attested in conventional forms both in written and spoken ELF.
Non-conformity to ENL norms: a perspective from Chinese English users
Authors: Wang Ying
Published: September 2013

English as a lingua franca (ELF) research poses challenge to mainstream English language teaching (ELT) in terms of its exclusive reference to English as a native language (ENL). On the other hand, some studies have been conducted to show that non-native English speakers (NNESs) have an aspiration for ENL and, in turn, add emphasis to ELT practitioners' effort in helping NNESs to approximate native English speaker (NES) competence. In response to the debate, this paper focuses on Chinese English speakers' perceptions of (non-)conformity to ENL in order to offer insights into the extent to which NNESs are ENL-(in)dependent.

This paper draws on the data contributed by 502 Chinese university students and 267 Chinese professionals through questionnaires and interviews. The questionnaire responses displayed a slightly positive orientation towards instances of non-conformity to ENL norms and revealed the respondents' choice between the formulaic conformity and the functions of non-conformity. The interviews unfolded the tension between exonormative and endonormative orientations to English. The participants' exonormative orientations reflect their belief in the centrality of NESs to norms of English use, their conceptualization of English as a fixed entity, and their aspiration for social advantages that they believe the conformity to ENL norms can bring to them. Their endonormative orientations relate to their acknowledgement of the communicative function of English that diverges from the norms of ENL and their concern for their cultural identity, which they believe conflicts with the conformity to ENL norms. This paper thus demonstrates that the preference for ENL is only one side of NNESs' attitudinal picture. Further, the reasons behind this preference point to the need for change from monolithic English to pluralistic Englishes in mainstream ELT.
Spanish scholars' research article publishing process in English-medium journals: English used as a lingua franca?
Authors: Mur-Dueñas Pilar
Published: September 2013

English has become the language of dissemination of research findings for scholars, especially in certain fields, and this is not an exception for Spanish scholars. They are under increasing pressure to write and publish research articles in international English-medium journals to establish their credentials, get academic promotion and rewards, and also to gain professional recognition. It is the aim of this paper to look at the process of writing research articles by a group of Spanish scholars in the field of finance by exploring the role played by “literacy brokers” (Lillis and Curry 2010): “wordface professionals” (Shashok 2008) (i.e., proofreaders and language consultants, who tend to be external community members) and gatekeepers (i.e., journal editors and reviewers). The study is based on eight text histories which contain the subsequent versions of the authors' manuscripts, the editors' letters, and the referees' reports. Referees and editors frequently point out the non-nativeness of the Spanish scholars' discourse, and encourage them to resort to English native wordface professionals. The analysis reveals the most common type of language revision which their manuscripts go through to comply not only with standard linguistic and discursive uses of the language but also with prevailing Anglo-American rhetorical conventions. Through this analysis an insight is gained into the extent to which English is used as a lingua franca (ELF) in this disciplinary academic community for research publication and dissemination.
Shinjo Matsuri: Community Revitalization through the Construction and Circulation of Decorated Floats
Authors: Kuroishi Izumi
Published: September 2013

Shinjo Matsuri in Yamagata prefecture is a 250-year old summer festival recognized as one of the nationally designated Important Folk and Cultural Properties. Besides the procession of a mikoshi from Tenman shrine, 20 decorated floats circulate through the city for two days, by collaboration between the people in the town and farm areas. Characteristically, as the local lord Tozawa allowed people to design the float as they liked at the beginning of the festival, groups of young people in each area still design, finance and construct the float by themselves without any institution’s nor profession’s help. This paper aims to explain how the construction and the spatial assignment of the float work to sustain the community bond between generations and define its local identity in the whole city. Especially, I would like to discuss how people interpret the idea of furyu in enhancing their well-being in the community by creating the extraordinary socio-symbolical space, and by accommodating the festival to the social change.