bluuebunny/crossref_metadata_2025 huggingface dataset:
Pyrosequencing of 16S rRNA genes in fecal samples reveals high diversity of hindgut microflora in horses and potential links to chronic laminitis
Authors: Steelman Samantha M , Chowdhary Bhanu P , Dowd Scot , Suchodolski Jan , Janečka Jan E
Published: November 2012

Background

The nutrition and health of horses is closely tied to their gastrointestinal microflora. Gut bacteria break down plant structural carbohydrates and produce volatile fatty acids, which are a major source of energy for horses. Bacterial communities are also essential for maintaining gut homeostasis and have been hypothesized to contribute to various diseases including laminitis. We performed pyrosequencing of 16S rRNA bacterial genes isolated from fecal material to characterize hindgut bacterial communities in healthy horses and those with chronic laminitis.

Results

Fecal samples were collected from 10 normal horses and 8 horses with chronic laminitis. Genomic DNA was extracted and the V4-V5 segment of the 16S rRNA gene was PCR amplified and sequenced on the 454 platform generating a mean of 2,425 reads per sample after quality trimming. The bacterial communities were dominated by Firmicutes (69.21% control, 56.72% laminitis) and Verrucomicrobia (18.13% control, 27.63% laminitis), followed by Bacteroidetes, Proteobacteria, and Spirochaetes. We observed more OTUs per individual in the laminitis group than the control group (419.6 and 355.2, respectively, P = 0.019) along with a difference in the abundance of two unassigned Clostridiales genera (P = 0.03 and P = 0.01). The most abundant bacteria were Streptococcus spp., Clostridium spp., and Treponema spp.; along with unassigned genera from Subdivision 5 of Verrucomicrobia, Ruminococcaceae, and Clostridiaceae, which together constituted ~ 80% of all OTUs. There was a high level of individual variation across all taxonomic ranks.

Conclusions

Our exploration of the equine fecal microflora revealed higher bacterial diversity in horses with chronic laminitis and identification of two Clostridiales genera that differed in abundance from control horses. There was large individual variation in bacterial communities that was not explained in our study. The core hindgut microflora was dominated by Streptococcus spp., several cellulytic genera, and a large proportion of uncharacterized OTUs that warrant further investigation regarding their function. Our data provide a foundation for future investigations of hindgut bacterial factors that may influence the development and progression of chronic laminitis.
Community member perspectives from transgender women and men who have sex with men on pre-exposure prophylaxis as an HIV prevention strategy: implications for implementation
Authors: Galindo Gabriel R. , Garrett-Walker J. J. , Hazelton Patrick , Lane Tim , Steward Wayne T. , Morin Stephen F. , Arnold Emily A.
Published: November 2012

Background

An international randomized clinical trial (RCT) on pre-exposure prophylaxis (PrEP) as an human immunodeficiency virus (HIV)-prevention intervention found that taken on a daily basis, PrEP was safe and effective among men who have sex with men (MSM) and male-to-female transgender women. Within the context of the HIV epidemic in the United States (US), MSM and transgender women are the most appropriate groups to target for PrEP implementation at the population level; however, their perspectives on evidenced-based biomedical research and the results of this large trial remain virtually unknown. In this study, we examined the acceptability of individual daily use of PrEP and assessed potential barriers to community uptake.

Methods

We conducted semi-structured interviews with an ethnoracially diverse sample of thirty HIV-negative and unknown status MSM (n = 24) and transgender women (n = 6) in three California metropolitan areas. Given the burden of disease among ethnoracial minorities in the US, we purposefully oversampled for these groups. Thematic coding and analysis of data was conducted utilizing an approach rooted in grounded theory.

Results

While participants expressed general interest in PrEP availability, results demonstrate: a lack of community awareness and confusion about PrEP; reservations about PrEP utilization, even when informed of efficacious RCT results; and concerns regarding equity and the manner in which a PrEP intervention could be packaged and marketed in their communities.

Conclusions

In order to effectively reduce HIV health disparities at the population level, PrEP implementation must take into account the uptake concerns of those groups who would actually access and use this biomedical intervention as a prevention strategy. Recommendations addressing these concerns are provided.
An array CGH based genomic instability index (G2I) is predictive of clinical outcome in breast cancer and reveals a subset of tumors without lymph node involvement but with poor prognosis
Authors: Bonnet Françoise , Guedj Mickael , Jones Natalie , Sfar Sana , Brouste Véronique , Elarouci Nabila , Banneau Guillaume , Orsetti Béatrice , Primois Charlotte , de Lara Christine Tunon , Debled Marc , de Mascarel Isabelle , Theillet Charles , Sévenet Nicolas , de Reynies Aurélien , MacGrogan Gaëtan , Longy Michel
Published: November 2012

Background

Despite entering complete remission after primary treatment, a substantial proportion of patients with early stage breast cancer will develop metastases. Prediction of such an outcome remains challenging despite the clinical use of several prognostic parameters. Several reports indicate that genomic instability, as reflected in specific chromosomal aneuploidies and variations in DNA content, influences clinical outcome but no precise definition of this parameter has yet been clearly established.

Methods

To explore the prognostic value of genomic alterations present in primary tumors, we performed a comparative genomic hybridization study on BAC arrays with a panel of breast carcinomas from 45 patients with metastatic relapse and 95 others, matched for age and axillary node involvement, without any recurrence after at least 11 years of follow-up. Array-CGH data was used to establish a two-parameter index representative of the global level of aneusomy by chromosomal arm, and of the number of breakpoints throughout the genome.

Results

Application of appropriate thresholds allowed us to distinguish three classes of tumors highly associated with metastatic relapse. This index used with the same thresholds on a published set of tumors confirms its prognostic significance with a hazard ratio of 3.24 [95CI: 1.76-5.96] p = 6.7x10-5 for the bad prognostic group with respect to the intermediate group. The high prognostic value of this genomic index is related to its ability to individualize a specific group of breast cancers, mainly luminal type and axillary node negative, showing very high genetic instability and poor outcome. Indirect transcriptomic validation was obtained on independent data sets.

Conclusion

Accurate evaluation of genetic instability in breast cancers by a genomic instability index (G2I) helps individualizing specific tumors with previously unexpected very poor prognosis.
Assisting role redesign: a qualitative evaluation of the implementation of a podiatry assistant role to a community health setting utilising a traineeship approach
Authors: Moran Anna M , Nancarrow Susan A , Wiseman Leah , Maher Kerryn , Boyce Rosalie A , Borthwick Alan M , Murphy Karen
Published: November 2012

Background

Increasing demands for podiatry combined with workforce shortages due to attrition, part‐time working practices and rural healthcare shortages means that in some geographic areas in Australia there are insufficient professionals to meet service demand. Although podiatry assistants have been introduced to help relieve workforce shortages there has been little evaluation of their impact on patient, staff and/or service outcomes. This research explores the processes and outcomes of a ‘trainee’ approach to introducing a podiatry assistant (PA) role to a community setting in the Australian Capital Territory (ACT) Government Health Service Directorate.

Method

A qualitative methodology was employed involving interviews and focus groups with service managers, qualified practitioners, the assistant, service users and consumer representatives. Perspectives of the implementation process; the traineeship approach; the underlying mechanisms that help or hinder the implementation process; and the perceived impact of the role were explored. Data were analysed using the Richie and Spencer Framework approach.

Results

Although the impact of the PA role had not been measured at the time of the evaluation, the implementation of the PA traineeship was considered a success in terms of enabling the transfer of a basic foot‐care service from nursing back to podiatry; releasing Enrolled Nurses (ENs) from foot‐care duties; an increase in the number of treatments delivered by the podiatry service; and high levels of stakeholder satisfaction with the role. It was perceived that the transfer of the basic foot‐care role from nursing to podiatry through the use of a PA impacted on communication and feedback loops between the PA and the podiatry service; the nursing‐podiatry relationship; clinical governance around the foot‐care service; and continuity of care for clients through the podiatry service. The traineeship was considered successful in terms of producing a PA whose skills were shaped by and directly met the needs of the practitioners with whom they worked. However, the resource intensiveness of the traineeship model was acknowledged by most who participated in the programme.

Conclusions

This research has demonstrated that the implementation of a PA using a traineeship approach requires good coordination and communication with a number of agencies and staff and substantial resources to support training and supervision. There are added benefits of the new role to the podiatry service in terms of regaining control over podiatric services which was perceived to improve clinical governance and patient pathways.
Drug-likeness analysis of traditional Chinese medicines: 1. property distributions of drug-like compounds, non-drug-like compounds and natural compounds from traditional Chinese medicines
Authors: Shen Mingyun , Tian Sheng , Li Youyong , Li Qian , Xu Xiaojie , Wang Junmei , Hou Tingjun
Published: November 2012

Background

In this work, we analyzed and compared the distribution profiles of a wide variety of molecular properties for three compound classes: drug-like compounds in MDL Drug Data Report (MDDR), non-drug-like compounds in Available Chemical Directory (ACD), and natural compounds in Traditional Chinese Medicine Compound Database (TCMCD).

Results

The comparison of the property distributions suggests that, when all compounds in MDDR, ACD and TCMCD with molecular weight lower than 600 were used, MDDR and ACD are substantially different while TCMCD is much more similar to MDDR than ACD. However, when the three subsets of ACD, MDDR and TCMCD with similar molecular weight distributions were examined, the distribution profiles of the representative physicochemical properties for MDDR and ACD do not differ significantly anymore, suggesting that after the dependence of molecular weight is removed drug-like and non-drug-like molecules cannot be effectively distinguished by simple property-based filters; however, the distribution profiles of several physicochemical properties for TCMCD are obviously different from those for MDDR and ACD. Then, the performance of each molecular property on predicting drug-likeness was evaluated. No single molecular property shows good performance to discriminate between drug-like and non-drug-like molecules. Compared with the other descriptors, fractional negative accessible surface area (FASA-) performs the best. Finally, a PCA-based scheme was used to visually characterize the spatial distributions of the three classes of compounds with similar molecular weight distributions.

Conclusion

If FASA- was used as a drug-likeness filter, more than 80% molecules in TCMCD were predicted to be drug-like. Moreover, the principal component plots show that natural compounds in TCMCD have different and even more diverse distributions than either drug-like compounds in MDDR or non-drug-like compounds in ACD.
Comprehensive analysis of differentially expressed rice actin depolymerizing factor gene family and heterologous overexpression of OsADF3 confers Arabidopsis Thaliana drought tolerance
Authors: Huang Ya-Chen , Huang Wen-Lii , Hong Chwan-Yang , Lur Hur-Shen , Chang Men-Chi
Published: November 2012

Background

Actin depolymerizing factors (ADFs) are small actin-binding proteins. Many higher-plant ADFs has been known to involve in plant growth, development and pathogen defense. However, in rice the temporal and spatial expression of OsADF gene family and their relationship with abiotic stresses tolerance is still unknown.

Results

Here we reported the first comprehensive gene expression profile analysis of OsADF gene family. The OsADF genes showed distinct and overlapping gene expression patterns at different growth stages, tissues and abiotic stresses. We also demonstrated that both OsADF1 and OsADF3 proteins were localized in the nucleus. OsADF1 and OsADF3 were preferentially expressed in vascular tissues. Under ABA or abiotic stress treatments, OsADF3::GUS activity was enhanced in lateral roots and root tips. Ectopically overexpressed OsADF3 conferred the mannitol- and drought-stress tolerance of transgenic Arabidopsis seedlings by increasing germination rate, primary root length and survival. Several drought-tolerance responsive genes (RD22, ABF4, DREB2A, RD29A, PIP1; 4 and PIP2; 6) were upregulated in transgenic Arabidopsis under drought stress.

Conclusions

These results suggested that OsADF gene family may participate in plant abiotic stresses response or tolerance and would facilitate functional validation of other OsADF genes.
The effect of 12 weeks Anethum graveolens (dill) on metabolic markers in patients with metabolic syndrome; a randomized double blind controlled trial
Authors: Mansouri Masoume , Nayebi Neda , keshtkar Abasali , Hasani-Ranjbar Shirin , Taheri Eghbal , Larijani Bagher
Published: November 2012

Background

The clustering of metabolic abnormalities defined as metabolic syndrome is now both a public health and a clinical problem .While interest in herbal medicine has greatly increased, lack of human evidence to support efficacies shown in animals does exist. This clinical trial study designed to investigate whether herbal medicine, Anethum graveolens (dill) extract, could improve metabolic components in patients with metabolic syndrome.

Methods

A double-blind, randomized, placebo-controlled trial using a parallel design was conducted. 24 subjects who had metabolic syndrome diagnostic criteria (update of ATP III) were randomly assigned to either dill extract (n = 12) or placebo (n = 12) for 3 months.

Results

Across lipid component of metabolic syndrome, no significant differences in triglyceride (TG) concentration and high density lipoprotein cholesterol were seen between the two groups. However TG improved significantly from baseline (257.0 vs. 201.5p = 0.01) with dill treatment but such a significant effect was not observed in placebo group. Moreover, no significant differences in waist circumference, blood pressure and fasting blood sugar were seen between two groups after 3 months follow up period.

Conclusion

In this small clinical trial in patients with metabolic syndrome, 12 weeks of dill extract treatment had a beneficial effect in terms of reducing TG from baseline. However dill treatment was not associated with a significant improvement in metabolic syndrome related markers compared to control group. Larger studies might be required to prove the efficacy and safety of long-term administration of dill to resolve metabolic syndrome components.
Pathway choice in DNA double strand break repair: observations of a balancing act
Authors: Brandsma Inger , Gent Dik C
Published: November 2012

Proper repair of DNA double strand breaks (DSBs) is vital for the preservation of genomic integrity. There are two main pathways that repair DSBs, Homologous recombination (HR) and Non-homologous end-joining (NHEJ). HR is restricted to the S and G2 phases of the cell cycle due to the requirement for the sister chromatid as a template, while NHEJ is active throughout the cell cycle and does not rely on a template. The balance between both pathways is essential for genome stability and numerous assays have been developed to measure the efficiency of the two pathways. Several proteins are known to affect the balance between HR and NHEJ and the complexity of the break also plays a role. In this review we describe several repair assays to determine the efficiencies of both pathways. We discuss how disturbance of the balance between HR and NHEJ can lead to disease, but also how it can be exploited for cancer treatment.
Association of a BMP5 microsatellite with knee osteoarthritis: case-control study
Authors: Rodriguez-Fontenla Cristina , Carr Andrew , Gomez-Reino Juan J , Tsezou Aspasia , Loughlin John , Gonzalez Antonio
Published: November 2012

Introduction

We aimed to explore the involvement of a multiallelic functional polymorphism in knee osteoarthritis (OA) susceptibility as a prototype of possible genetic factors escaping GWAS detection.

Methods

OA patients and controls from three European populations (Greece, Spain and the UK) adding up to 1003 patients (716 women, 287 men) that had undergone total knee joint replacement (TKR) due to severe primary OA and 1543 controls (758 women, 785 men) lacking clinical signs or symptoms of OA were genotyped for the D6S1276 microsatellite in intron 1 of BMP5. Genotype and mutiallelic trend tests were used to compare cases and controls.

Results

Significant association was found between the microsatellite and knee OA in women (P from 3.1 x10-4 to 4.1 x10-4 depending on the test), but not in men. Three of the alleles showed significant differences between patients and controls, one of them of increased risk and two of protection. The gender association and the allele direction of change were very concordant with those previously reported for hip OA.

Conclusions

We have found association of knee OA in women with the D6S1276 functional microsatellite that modifies in cis the expression of BMP5 making this a sounder OA genetic factor and extending its involvement to other joints. This result also shows the interest of analysing other multiallelic polymorphisms.
Role of predisposition, injury, response and organ failure in the prognosis of patients with acute-on-chronic liver failure: a prospective cohort study
Authors: Jalan Rajiv , Stadlbauer Vanessa , Sen Sambit , Cheshire Lisa , Chang Yu-Mei , Mookerjee Rajeshwar P
Published: November 2012

Introduction

Acute deterioration of cirrhosis is associated with high mortality rates particularly in the patients who develop organ failure (OF), a condition that is referred to as acute-on-chronic liver failure (ACLF), which is currently not completely defined. This study aimed to determine the role of predisposing factors, the nature of the precipitating illness and inflammatory response in the progression to OF according to the PIRO (predisposition, injury, response, organ failure) concept to define the risk of in-hospital mortality.

Methods

A total of 477 patients admitted with acute deterioration of cirrhosis following a defined precipitant over a 5.5-year period were prospectively studied. Baseline clinical, demographic and biochemical data were recorded for all patients and extended serial data from the group that progressed to OF were analysed to define the role of PIRO in determining in-hospital mortality.

Results

One hundred and fifty-nine (33%) patients developed OF, of whom 93 patients died (58%) compared with 25/318 (8%) deaths in the non-OF group (P < 0.0001). Progression to OF was associated with more severe underlying liver disease and inflammation. In the OF group, previous hospitalisation (P of PIRO); severity of inflammation and lack of its resolution (R of PIRO); and severity of organ failure (O of PIRO) were associated with significantly greater risk of death. In the patients who recovered from OF, mortality at three years was almost universal.

Conclusions

The results of this prospective study shows that the occurrence of OF alters the natural history of cirrhosis. A classification based on the PIRO concept may allow categorization of patients into distinct pathophysiologic and prognostic groups and allow a multidimensional definition of ACLF.
Computational discovery of regulatory elements in a continuous expression space
Authors: Lajoie Mathieu , Gascuel Olivier , Lefort Vincent , Bréhélin Laurent
Published: November 2012

Approaches for regulatory element discovery from gene expression data usually rely on clustering algorithms to partition the data into clusters of co-expressed genes. Gene regulatory sequences are then mined to find overrepresented motifs in each cluster. However, this ad hoc partition rarely fits the biological reality. We propose a novel method called RED2 that avoids data clustering by estimating motif densities locally around each gene. We show that RED2 detects numerous motifs not detected by clustering-based approaches, and that most of these correspond to characterized motifs. RED2 can be accessed online through a user-friendly interface.
Mutant KRAS Codon 12 and 13 Alleles in Patients With Metastatic Colorectal Cancer: Assessment As Prognostic and Predictive Biomarkers of Response to Panitumumab
Authors: Peeters Marc , Douillard Jean-Yves , Van Cutsem Eric , Siena Salvatore , Zhang Kathy , Williams Richard , Wiezorek Jeffrey
Published: November 2012

Purpose

Panitumumab, a fully human monoclonal antibody targeting the epidermal growth factor receptor (EGFR), has demonstrated significant improvements in progression-free survival (PFS) in patients with wild-type KRAS metastatic colorectal cancer (mCRC) in studies 20050203 (first line), 20050181 (second line), and 20020408 (monotherapy). Mutations in KRAS codons 12 and 13 are recognized biomarkers that predict lack of response to anti-EGFR antibody therapies. This retrospective analysis of three randomized phase III studies assessed the prognostic and predictive impact of individual mutant KRAS codon 12 and 13 alleles.

Patients and Methods

Patients were randomly assigned 1:1 to FOLFOX4 (infusional fluorouracil, leucovorin, and oxaliplatin) in study 20050203, FOLFIRI (fluorouracil, leucovorin, and irinotecan) in study 20050181, or best supportive care in study 20020408 with or without panitumumab 6.0 mg/kg once every 2 weeks. In all, 441 (20050203), 486 (20050181), and 126 (20020408) patients with mutant KRAS codon 12 or 13 alleles were included in the analysis.

Results

No mutant KRAS allele in patients treated on the control arm emerged as a consistent prognostic factor for PFS or overall survival (OS). In addition, no mutant KRAS allele was consistently identified as a predictive factor for PFS or OS in patients receiving panitumumab treatment. Significant interactions for individual mutant KRAS alleles were observed only in study 20050203 with G13D negatively and G12V positively associated with OS in the panitumumab-containing arm. Pooled analysis indicated that only G12A was associated with a negative predictive effect on OS.

Conclusion

In this retrospective analysis, results across three treatment regimens suggest that patients with mutant KRAS codon 12 or 13 mCRC tumors are unlikely to benefit from panitumumab therapy. Currently, panitumumab therapy should be limited to patients with wild-type KRAS mCRC.
IGF-I 3′ Untranslated Region: Strain-Specific Polymorphisms and Motifs Regulating IGF-I in Osteoblasts
Authors: Smith Spenser S. , Kessler Catherine B. , Shenoy Vikram , Rosen Clifford J. , Delany Anne M.
Published: November 2012

Reduced IGF-I is associated with low bone mass in humans and mice. C3H/He/J (C3H) mice have higher skeletal IGF-I and greater bone mass than C57BL/6J (B6). We hypothesized that strain-related genotypic differences in Igf1 affected skeletal function. The Igf1 coding region is nonpolymorphic, but its 3′ untranslated region (UTR) is polymorphic between C3H and B6. Luciferase-Igf1 3′ UTR reporter constructs showed that these polymorphic regions did not affect UTR function. IGF-I splice variants give rise to a common mature IGF-I peptide, but different E peptides. We identified two splice products, exon 4+6 (Ea) and exon 4+5+6 (Eb, mechano-growth factor) and found that their abundance was unchanged during osteoblastic differentiation. The Igf1 3′ UTR encoded by exon 6 contains alternative polyadenylation sites. Proximal site use produces a short 3′ UTR of approximately 195 bases, whereas distal site usage results in an approximately 6300-base UTR. Although Igf1 mRNA levels did not change during osteoblastic differentiation, distal polyadenylation site usage was increased in B6 cells but not in C3H. The resulting long Igf1 RNA isoform is less stable and has decreased translation efficiency, which may be one mechanism contributing to decreased IGF-I in B6 vs. C3H mice. Although the long UTR contains a conserved [GU]18 repeat, which is a positive regulator of UTR activity, it is also targeted by negative regulators, miR-29 and miR-365. These microRNAs are increased in B6 and C3H cells during osteoblastic differentiation. Differential expression of the long Igf1 3′ UTR isoform may be a possible mechanism for enhanced IGF-I regulation in B6 vs. C3H mice.
Reactivation of the Silenced Thyroid Hormone Receptor β Gene Expression Delays Thyroid Tumor Progression
Authors: Kim Won Gu , Zhu Xuguang , Kim Dong Wook , Zhang Lisa , Kebebew Electron , Cheng Sheue-yann
Published: November 2012

That a knock-in mouse harboring a dominant-negative thyroid hormone receptor (TR)-β (Thrb) mutation develops metastatic thyroid cancer strongly suggests the involvement of TRβ in carcinogenesis. Epigenetic silencing of the THRB gene is common in human cancers. The aim of the present study was to determine how DNA methylation affected the expression of the THRB gene in differentiated thyroid cancer (DTC) and how reexpression of the THRB gene attenuated the cancer phenotypes. We used methylation-specific PCR to examine the expression and promoter methylation of the THRB gene in DTC tissues. Thyroid cancer cells with hypermethylated THRB were treated with the demethylating agents 5′-aza-2′-deoxycytidine (5′-aza-CdR) and zebularine to evaluate their impact on the cancer cell phenotypes. THRB mRNA expression in DTC was 90% lower than in normal controls, and this decrease was associated with a higher tumor/lymph node staging. The promoter methylation level of the THRB gene had a significant negative correlation with the expression level of the THRB gene. Treatment of FTC-236 cells with 5′-aza-CdR or zebularine induced reexpression of the THRB gene and inhibited cell proliferation and migration. FTC-236 cells stably expressing TRβ exhibited lower cell proliferation and migration through inhibition of β-catenin signaling pathways compared with FTC-236 without TRβ. 5′-Aza-CdR also led to suppression of tumor growth in an in vivo xenograft model using FTC-236 cells consistent with the cell-based studies. These finding indicate that TRβ is a tumor suppressor and could be tested as a potential therapeutic target.
Estradiol Antagonism of Glucocorticoid-Induced GILZ Expression in Human Uterine Epithelial Cells and Murine Uterus
Authors: Whirledge Shannon , Cidlowski John A.
Published: November 2012

Sex hormone signaling regulates a variety of functions in the uterine endometrium essential for embryo implantation and immunity. Epithelial cells of the uterine endometrium are the target of the coordinated actions of estradiol (E2) and progesterone. However, little information exists regarding the interplay of estrogens with glucocorticoids in this tissue. Using the human uterine epithelial cell line ECC1, E2 was found to antagonize induction of the glucocorticoid-induced leucine zipper (GILZ) gene expression, which is associated with several of the immune-related functions of glucocorticoids. Interestingly, E2 antagonizes glucocorticoid regulated nascent RNA GILZ expression within 1 h of hormone treatment. Repression of glucocorticoid-induced GILZ expression requires the estrogen receptor (ER), because both treatment with the ER-antagonist ICI 182,780 and small interfering RNA knockdown of ERα block E2’s ability to repress GILZ gene expression. Antagonism of glucocorticoid-induced GILZ expression may not be unique to ERα, as the ERβ agonist Liquiritigenin is also able to antagonize glucocorticoid signaling. Transcriptional regulation appears to be at the level of promoter binding. Both the glucocorticoid receptor and ERα are recruited to regions of the GILZ promoter containing glucocorticoid response elements and the transcriptional start site. Glucocorticoid receptor binding to these regions in the presence of dexamethasone decreases with E2 treatment. GILZ gene expression was also found to be repressed in the whole mouse uterus treated with a combination of dexamethasone and E2. Regulation of the antiinflammatory gene GILZ by glucocorticoids and E2 suggests cross talk between the immune modulating functions of glucocorticoids and the reproductive actions of estradiol signaling.
Differential Effects of Acute and Chronic Estrogen Treatment on Thermogenic and Metabolic Pathways in Ovariectomized Sheep
Authors: Clarke Scott D. , Clarke Iain J. , Rao Alexandra , Evans Roger G. , Henry Belinda A.
Published: November 2012

Estrogen is protective against weight gain, but the underlying mechanisms are not fully elucidated. We sought to characterize the effects of estrogen on energy expenditure in skeletal muscle and adipose tissue in ovariectomized sheep. Temperature probes were implanted into sc (gluteal) and visceral (retroperitoneal) fat depots and skeletal muscle of the hind limb (vastus lateralis). Food was available from 1100–1600 h to entrain postprandial thermogenesis. We characterized the effects of single (50 μg estradiol benzoate, im) and repeated (25 μg estradiol-17β, iv) injections as well as chronic (3 × 3 cm estradiol-17β implants for 7 d) treatment on heat production. A single injection of estrogen increased heat production in visceral fat and skeletal muscle, without an effect on food intake. Increased heat production in skeletal muscle was sustained by repeated estradiol-17β injections. On the other hand, continuous treatment reduced food intake but had no effect on thermogenesis. To determine possible mechanisms that underpin estradiol-17β-induced heat production, we measured femoral artery blood flow, the expression of uncoupling protein (UCP) mRNA and the phosphorylation of AMP-activated protein kinase and Akt in fat and muscle. There was little effect of either single or repeated injections of estradiol-17β on the expression of UCP1, -2, or -3 mRNA in visceral fat or skeletal muscle. Acute injection of estradiol-17β increased the phosphorylation of AMP-activated protein kinase and Akt in muscle only. Estradiol-17β treatment did not alter femoral artery blood flow. Thus, the stimulatory effect of estradiol-17β on thermogenesis in female sheep is dependent upon a pulsatile pattern of treatment and not constant continuous exposure.
The Calcium Signaling Pathway Regulates Leydig Cell Steroidogenesis through a Transcriptional Cascade Involving the Nuclear Receptor NR4A1 and the Steroidogenic Acute Regulatory Protein
Authors: Abdou Houssein S. , Villeneuve Gabrielle , Tremblay Jacques J.
Published: November 2012

In the gonads and adrenal glands, the transient increase in steroidogenesis after hormonal stimulation requires modulation of steroidogenic acute regulatory protein (Star) expression and activity in a tightly regulated process involving cAMP and Ca2+. In Leydig cells, the cAMP and Ca2+ pathways account for most if not all of LH-induced steroidogenesis. Although the cAMP-activated molecular network has been well characterized in Leydig cells, little is known about the molecular cascade triggered by the Ca2+ signaling pathway and the transcription factors responsible for mediating the genomic response. It is established that LH induces an increase in cytoplasmic Ca2+ from the endoplasmic reticulum primarily through the ryanodine receptors. Previous reports also suggested a role of the Ca2+ signaling pathway in Star expression based on the fact that inhibition of the Ca2+/calmodulin (CaM) protein kinase pathway greatly impaired Star expression in Leydig and adrenal cells. In this study, we used ryanodine receptors and CaM antagonists to show that the increase in intracellular Ca2+ level is an essential modulator of progesterone synthesis through the regulation of Star gene expression in MA-10 Leydig cells. Furthermore, we mapped a Ca2+/CaM-sensitive element in the Star promoter, which led to the identification of the nuclear receptor 4A1 (NR4A1) as a key mediator of the Ca2+/CaM signaling pathway in these cells. These data provide new insights into the Ca2+ molecular pathway essential for steroidogenesis in Leydig cells.
The Microsomal Enzyme 17β-Hydroxysteroid Dehydrogenase 3 Faces the Cytoplasm and Uses NADPH Generated by Glucose-6-Phosphate Dehydrogenase
Authors: Legeza Balázs , Balázs Zoltán , Nashev Lyubomir G. , Odermatt Alex
Published: November 2012

Recent studies proposed a functional coupling between 17β-hydroxysteroid dehydrogenase 3 (17β-HSD3)-dependent testosterone formation and 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1)-mediated interconversion of glucocorticoids through competition for the luminal pyridine nucleotide pool. To test this hypothesis, we used human embryonic kidney-293 cells transfected with 17β-HSD3 and/or 11β-HSD1, in the absence or presence of hexose-6-phosphate dehydrogenase that generates reduced nicotinamide adenine dinucleotide phosphate (NADPH) in the endoplasmic reticulum and determined enzyme activities. As an endogenous cell model, mouse MA-10 Leydig cells were used. 17β-HSD3-dependent reduction of Δ4-androstene-3,17-dione was affected by neither coexpression with 11β-HSD1 nor overexpression or knockdown of hexose-6-phosphate dehydrogenase. In contrast, knockdown of glucose-6-phosphate dehydrogenase decreased 17β-HSD3 activity, indicating dependence on cytoplasmic NADPH. Upon selective permeabilization of the plasma membrane by digitonin, 17β-HSD3 but not 11β-HSD1 was detected by antibodies against C-terminal epitope tags, suggesting a cytoplasmic orientation of 17β-HSD3. The cytoplasmic orientation was confirmed using proteinase K digestion of microsomal preparations and by analysis of glycosylation of wild-type 17β-HSD3 and chimera in which the N-terminal anchor sequences between 17β-HSD3 and 11β-HSD1 were exchanged. In conclusion, the results demonstrate a cytoplasmic orientation of 17β-HSD3 and dependence on glucose-6-phosphate dehydrogenase-generated NADPH, explaining the lack of a direct functional coupling with the luminal 11β-HSD1-mediated glucocorticoid metabolism.
P21-Activated Protein Kinase 1 (Pak1) Mediates the Cross Talk between Insulin and β-Catenin on Proglucagon Gene Expression and Its Ablation Affects Glucose Homeostasis in Male C57BL/6 Mice
Authors: Chiang Yuting Alex , Shao Weijuan , Xu Xiao Xue , Chernoff Jonathan , Jin Tianru
Published: November 2012

In gut endocrine L cells, the Wnt signaling pathway effector β-catenin (β-cat)/transcription factor 7-like 2 mediates the stimulatory effect of insulin on proglucagon (gcg) expression and glucagon-like peptide-1 (GLP-1) production. In several other cell lineages, insulin is able to stimulate p21-activated protein kinase 1 (Pak1). Here we determined the role of Pak1 in gcg expression and the effect of Pak1 deletion on glucose homeostasis. Insulin stimulated Pak1 activation through increasing its Thr423 phosphorylation in gut gcg-expressing cell lines, associated with increased gcg mRNA levels. This stimulation was attenuated by the Pak inhibitor 2,2′-dihydroxy-1,1′-dinaphthyldisulfide (IPA3) or dominant-negative Pak1. Both insulin and cAMP-promoting agents activated β-cat Ser675 phosphorylation, which was attenuated by IPA3 or protein kinase A inhibition, respectively. Gut gcg levels were reduced in male Pak1−/− mice, associated with impaired glucose tolerance after an ip or oral glucose challenge. These mice had lower circulating active GLP-1 levels after a glucose challenge as well as reduced distal ileum GLP-1 content after insulin treatment. Finally, the Pak1−/− mice exhibited reduced brainstem gcg level and abolished β-cat Ser675 phosphorylation in brain neurons after insulin treatment. We suggest that Pak1 mediates the cross talk between insulin and Wnt signaling pathways on gut and brain gcg expression, and its ablation impairs glucose homeostasis.
The Sphingosine-1-Phosphate Analog FTY720 Reduces Muscle Ceramide Content and Improves Glucose Tolerance in High Fat-Fed Male Mice
Authors: Bruce Clinton R. , Risis Steve , Babb Joanne R. , Yang Christine , Lee-Young Robert S. , Henstridge Darren C. , Febbraio Mark A.
Published: November 2012

FTY720 is a sphingosine-1-phosphate analog that has been shown to inhibit ceramide synthesis in vitro. Because ceramide accumulation in muscle is associated with insulin resistance, we aimed to examine whether FTY720 would prevent muscle ceramide accumulation in high fat-fed mice and subsequently improve glucose homeostasis. Male C57Bl/6 mice were fed either a chow or high fat-diet (HFD) for 6 wk, after which they were treated with vehicle or FTY720 (5 mg/kg) daily for a further 6 wk. The ceramide content of muscle was examined and insulin action was assessed. Whereas the HFD increased muscle ceramide, this was prevented by FTY720 treatment. This was not associated with alterations in the expression of genes involved in sphingolipid metabolism. Interestingly, the effects of FTY720 on lipid metabolism were not limited to ceramide because FTY720 also prevented the HFD-induced increase in diacylglycerol and triacylglycerol in muscle. Furthermore, the increase in CD36 mRNA expression induced by fat feeding was prevented in muscle of FTY720-treated mice. This was associated with an attenuation of the HFD-induced increase in palmitate uptake and esterification. In addition, FTY720 improved glucose homeostasis as demonstrated by a reduction in plasma insulin, an improvement in whole-body glucose tolerance, an increase in insulin-stimulated glucose uptake, and Akt phosphorylation in muscle. In conclusion, FTY720 exerts beneficial effects on muscle lipid metabolism that prevent lipid accumulation and improve glucose tolerance in high fat-fed mice. Thus, FTY720 and other compounds that target sphingosine-1-phosphate signaling may have therapeutic potential in treating insulin resistance.
Fibroblast Growth Factor-19 Action in the Brain Reduces Food Intake and Body Weight and Improves Glucose Tolerance in Male Rats
Authors: Ryan Karen K. , Kohli Rohit , Gutierrez-Aguilar Ruth , Gaitonde Shrawan G. , Woods Stephen C. , Seeley Randy J.
Published: November 2012

Fibroblast growth factor-19 (FGF19) and its rodent ortholog, FGF15, are hormones produced in the distal small intestine and secreted into the circulation after a meal. In addition to controlling the enterohepatic circulation of bile acids, FGF15/19 also regulates systemic lipid and glucose metabolism. In these experiments we investigated the hypothesis that, like other gut-derived postprandial hormones, FGF15/19 can act in the central nervous system to elicit its metabolic effects. We found that FGF-receptors 1 and 4 are present in rat hypothalamus, and that their expression was reduced by up to 60% in high-fat fed rats relative to lean controls. Consistent with a potential role for brain FGF15/19 signaling to regulate energy and glucose homeostasis, and with a previous report that intracerebroventricular (i.c.v.) administration of FGF19 increases energy expenditure, we report that acute i.c.v. FGF19 reduces 24-h food intake and body weight, and acutely improves glucose tolerance. Conversely, i.c.v. administration of an FGF-receptor inhibitor increases food intake and impairs glucose tolerance, suggesting a physiological role for brain FGF receptor signaling. Together, these findings identify the central nervous system as a potentially important target for the beneficial effects of FGF19 in the treatment of obesity and diabetes.
Expression of claudins -2 and -4 and cingulin is coordinated with the start of stratification and differentiation in corneal epithelial cells: retinoic acid reversibly disrupts epithelial barrier
Authors: Ortiz-Melo María Teresa , Sánchez-Guzmán Erika , González-Robles Arturo , Valdés Jesús , Gómez-Flores Eber , Castro-Muñozledo Federico
Published: November 2012

Although tight junctions (TJ) have been extensively studied in simple epithelial cells, it is still unknown whether their organization is coupled to cell differentiation in stratified epithelia. We studied the expression of TJ in RCE1(5T5) cells, an in vitro model which mimics the sequential steps of rabbit corneal epithelial differentiation. RCE1(5T5) cells expressed TJ components which were assembled once cells constituted differentiated epithelia, as suggested by the increase of transepithelial electrical resistance (TER) which followed a similar kinetic to the expression of the early differentiation marker Pax-6. TJ were functional as indicated by the establishment of an epithelial barrier nonpermeable to ruthenium red or a biotin tracer. In immunostaining experiments, TJ were located at the superficial cells from the suprabasal layers; Western blot and RT-PCR suggested that TJ were composed of claudins (cldn) -1, -2, -4, cingulin (cgn), occludin (ocln) and ZO-1. Semi-quantitative RT-PCR and TER measurements showed that TJ became organized when cells began to form a 3–5 layers stratified epithelium; TER increased once cells reached confluence, with a time course comparable to the raise in the expression of cgn, cldn-2 and -4. Nevertheless, cldn-1, -2, ZO-1 and ocln were present in the cells from the beginning of cultivation, suggesting that TER increases mainly depend on TJ assembly. While EGF increased epithelial barrier strength, retinoic acid disrupted it, increasing paracellular flux about 2-fold; this effect was concentration dependent and completely reversible. Our results suggest that TJ assembly is tightly linked to the expression of corneal epithelial terminal phenotype.
Fluorescence-based alternative splicing reporters for the study of epithelial plasticity in vivo
Authors: Somarelli Jason A. , Schaeffer Daneen , Bosma Reggie , Bonano Vivian I. , Sohn Jang Wook , Kemeny Gabor , Ettyreddy Abhinav , Garcia-Blanco Mariano A.
Published: November 2012

Alternative splicing generates a vast diversity of protein isoforms from a limited number of protein-coding genes, with many of the isoforms possessing unique, and even contrasting, functions. Fluorescence-based splicing reporters have the potential to facilitate studies of alternative splicing at the single-cell level and can provide valuable information on phenotypic transitions in almost real time. Fibroblast growth factor receptor 2 (FGFR2) pre-mRNA is alternatively spliced to form the epithelial-specific and mesenchymal-specific IIIb and IIIc isoforms, respectively, which are useful markers of epithelial–mesenchymal transitions (EMT). We have used our knowledge of FGFR2 splicing regulation to develop a fluorescence-based reporter system to visualize exon IIIc regulation in vitro and in vivo. Here we show the application of this reporter system to the study of EMT in vitro in cell culture and in vivo in transgenic mice harboring these splicing constructs. In explant studies, the reporters revealed that FGFR2 isoform switching is not required for keratinocyte migration during cutaneous wound closure. Our results demonstrate the value of the splicing reporters as tools to study phenotypic transitions and cell fates at single cell resolution. Moreover, our data suggest that keratinocytes migrate efficiently in the absence of a complete EMT.
The microRNA-302-367 cluster suppresses the proliferation of cervical carcinoma cells through the novel target AKT1
Authors: Cai Na , Wang Yi-Dong , Zheng Peng-Sheng
Published: November 2012

The miR-302-367 cluster is specifically expressed in human embryonic stem cells and has been shown to convert human somatic cells into induced pluripotent stem cells. Here, we investigated the role of the miR-302-367 cluster in cervical carcinoma. The cluster was not endogenously expressed in cervical cancer cells, and its ectopic expression did not reprogram the cervical cancer cells to an embryonic stem cell-like state. However, ectopic expression of the miR-302-367 cluster in HeLa and SiHa cervical cancer cells inhibited cell proliferation and tumor formation by blocking the G1/S cell cycle transition. We identified a new cell cycle regulatory pathway in which the miR-302-367 cluster directly down-regulated both cyclin D1 and AKT1 and indirectly up-regulated p27Kip1 and p21Cip1, leading to the suppression of cervical cancer cell proliferation. Our findings suggest that the miR-302-367 cluster may be used as a therapeutic reagent for the treatment of cervical carcinoma.
The treatment of complex tibial shaft fractures by the Ilizarov method
Authors: Foster P. A. L. , Barton S. B. , Jones S. C. E. , Morrison R. J. M. , Britten S.
Published: November 2012

We report on the use of the Ilizarov method to treat 40 consecutive fractures of the tibial shaft (35 AO 42C fractures and five AO 42B3 fractures) in adults. There were 28 men and 12 women with a mean age of 43 years (19 to 81). The series included 19 open fractures (six Gustilo grade 3A and 13 grade 3B) and 21 closed injuries. The mean time from injury to application of definitive Ilizarov frame was eight days (0 to 35) with 36 fractures successfully uniting without the need for any bone-stimulating procedure. The four remaining patients with nonunion healed with a second frame. There were no amputations and no deep infections. None required intervention for malunion. The total time to healing was calculated from date of injury to removal of the frame, with a median of 166 days (mean 187, (87 to 370)). Minor complications included snapped wires in two patients and minor pin-site infections treated with oral antibiotics in nine patients (23%). Clinical scores were available for 32 of the 40 patients at a median of 55 months (mean 62, (26 to 99)) post-injury, with ‘good’ Olerud and Molander ankle scores (median 80, mean 75, (10 to 100)), ‘excellent’ Lysholm knee scores (median 97, mean 88, (29 to 100)), a median Tegner activity score of 4 (mean 4, (0 to 9)) (comparable to ‘moderately heavy labour / cycling and jogging’) and Short Form-12 scores that exceeded the mean of the population as a whole (median physical component score 55 (mean 51, (20 to 64)), median mental component score 57 (mean 53, (21 to 62)). In conclusion, the Ilizarov method is a safe and reliable way of treating complex tibial shaft fractures with a high rate of primary union.